Bth 204 environmental biotechnology

for Bangalore university Msc biotechnology 2nd sem students.
All the best

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FOR MSC BIOTECHNOLOGY STUDENTS
2014 ONWARDS

Biochemistry scanner
THE IMPRINT
BTH-204: ENVIRONMENTAL BIOTECHNOLOGY

As per Bangalore University (CBCS) Syllabus
2016 Edition

BY: Prof. Balasubramanian Sathyamurthy

Supported By:
Ayesha Siddiqui
Kiran K.S.



THE MATERIALS FROM “THE IMPRINT (BIOCHEMISTRY SCANN ER)” ARE NOT
FOR COMMERCIAL OR BRAND BUILDING. HENCE ONLY ACADEM IC CONTENT
WILL BE PRESENT INSIDE. WE THANK ALL THE CONTRIBUTO RS FOR
ENCOURAGING THIS.
BE GOOD – DO GOOD & HELP OTHERS

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DEDICATIONDEDICATIONDEDICATIONDEDICATION

I dediI dediI dediI dedicate this material to my spiritual guru Shri Raghavendra swamigal, cate this material to my spiritual guru Shri Raghav endra swamigal, cate this material to my spiritual guru Shri Raghav endra swamigal, cate this material to my spiritual guru Shri Raghav endra swamigal,
parents, teachers, well wishers and students who always increase my morale parents, teachers, well wishers and students who always increase my morale parents, teachers, well wishers and students who always increase my morale parents, teachers, well wishers and students who always increase my morale
and confidence to share my and confidence to share my and confidence to share my and confidence to share my knowledge knowledge knowledge knowledge to to to to reach reach reach reach all beneficiariesall beneficiariesall beneficiariesall beneficiaries....

PREFACEPREFACEPREFACEPREFACE

Biochemistry scanner ‘THE IMPRINT’ consists of last ten years solved question
paper of Bangalore University keeping in mind the s yllabus and examination
pattern of the University. The content taken from t he reference books has been
presented in a simple language for better understan ding.

The Author Prof. Balasubramanian Sathyamurthy has 1 5 years of teaching
experience and has taught in 5 Indian Universities including Bangalore
University and more than 20 students has got univer sity ranking under his
guidance.
THE IMPRINT is a genuine effort by the students to help their peers with their
examinations with the strategy that has been succes sfully utilized by them.
These final year M.Sc students have proven their me ttle in university
examinations and are College / University rank hold ers.
This is truly
for the students, by the students. We thank all the contributors for
their valuable suggestion in bringing out this book . We hope this will be
appreciated by the students and teachers alike. Suggestions are welcomed.
For any comments, queries, and suggestions and to g et your free copy write us
at [email protected]
or call 9980494461

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CONTRIBUTORS:
CHETAN ABBUR ANJALI TIWARI
AASHITA SINHA ASHWINI BELLATTI
BHARATH K CHAITHRA
GADIPARTHI VAMSEEKRISHNA KALYAN BANERJEE
KAMALA KISHORE
KIRAN KIRAN H.R
KRUTHI PRABAKAR KRUPA S
LATHA M MAMATA
MADHU PRAKASHHA G D MANJUNATH .B.P
NAYAB RASOOL S NAVYA KUCHARLAPATI
NEHA SHARIFF DIVYA DUBEY
NOOR AYESHA M PAYAL BANERJEE
POONAM PANCHAL PRAVEEN
PRAKASH K J M PRADEEP.R
PURSHOTHAM PUPPALA DEEPTHI
RAGHUNATH REDDY V RAMYA S
RAVI RESHMA
RUBY SHA SALMA H.
SHWETHA B S SHILPI CHOUBEY
SOUMOUNDA DAS SURENDRA N
THUMMALA MANOJ UDAYASHRE. B
DEEPIKA SHARMA

EDITION : 2016
PRINT : Bangalore
CONTACT : [email protected] or 9980494461

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M. SC. BIOTECHNOLOGY – SECOND SEMESTER
BTH-204: ENVIRONMENTAL BIOTECHNOLOGY
4 units (52 hrs)
UNIT: 1 ENVIRONMENT AND MONITORING 8 hrs
Introduction, renewable and non – renewable sources of energy; Environmental
pollution – Water pollution, soil pollution and air pollution – sources and
measurements. Xenobiotic compounds and their source s. Biomagnification,
Bioindicators.
Biomonitoring: Biosensors and Biochips.
UNIT: 2 WATER MANAGEMENT AND WASTE WATER TREATMENT 12 hrs
Water as a scarce natural resource, water management including rain water harvesting.
Waste water characterisitics, waste water treatment- physical, chemical, biological
processes. Aerobic processes: Activated sludge, oxidation ditches, trickling filter,
oxidation ponds. Anaerobic processes; Anaerobic digestion, anaerobic filters, anaerobic
sludge, membrane bioreactors. Reverse osmosis and u ltra filtration. Treatment of
industrial effluents.
UNIT: 3 BIOMINING AND BIODIESEL 4 hrs
Bioleaching of ores to retrieve scarce metals, Bio – mining; Biodiesel production from
Jatropa, Pongamia and Castor.
UNIT: 4 BIOREMEDIATION 8 hrs
Concept and principles, Bioremediation using microb es, In situ and ex situ
bioremediation, biosorption and bioaccumulation of heavy metals; Phytoremediation,
bioremediation of xenobiotics ( heavy metals, pesti cides, oil slicks, plastic).
Bioremediation of soil and water contaminated with hydrocarbons and surfactants,
biofilms.
UNIT: 5 BIOWASTE TREATMENT 12 hrs
Microorganisms involved in the degradation of plant fibre, cell wall, lignin, fungal de –
lignifications and pulping of wood. Pitch problems in pulp and paper processes and
solving by enzymes or fungi. Hemicellulases in pulp bleaching. Solving slime problem in
the pulp and paper industry. Reduction of organochlorine compounds in bleach plant
effluents.

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Solid wastes: Sources and management, waste as a source of energy. Production of oils
and fuels from solid waste, composting, vermiculture, Biogas production, methanol
production from organic wastes, byproducts of sugar industries.
UNIT: 6 GLOBAL ENVIRONMENTAL PROBLEMS 8 hrs
Global warming, ozone depletion, UV – B, green house effect and acid rain, their impact
and management. Biodiversity and its conservation, status of biodiversity, hotspots,
Red data book.

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UNIT: 1 ENVIRONMENT AND MONITORING
Introduction, renewable and non – renewable sources of energy; Environmental
pollution – Water pollution, soil pollution and air pollution – sources and
measurements. Xenobiotic compounds and their source s. Biomagnification,
Bioindicators.
Biomonitoring: Biosensors and Biochips.
INTRODUCTION
Environmental biotechnology deals with far less apparently dramatic topics and, though
their importance, albeit different, may be every bit as great, their direct relevance is far
less readily appreciated by the bulk of the population. Cleaning up contamination and
dealing rationally with wastes is, of course, in everybody’s best interests, but for most
people, this is simply addressing a problem which they would rather have not existed in
the first place.
Even for industry, though the benefits may be noticeable on the balance sheet, the likes
of effluent treatment or pollution control are more of an inevitable obligation than a
primary goal in themselves. In general, such activities are typically funded on a
distinctly limited budget and have traditionally be en viewed as a necessary
inconvenience. This is in no way intended to be disparaging to industry; it simply
represents commercial reality.
In many respects, there is a logical fit between th is thinking and the aims of
environmental biotechnology. For all the media circus surrounding the grand questions
of our age, it is easy to forget that not all forms of biotechnology involve
xenotransplantation, genetic modification, the use of stem cells or cloning. Some of the
potentially most beneficial uses of biological engineering, and which may touch the lives
of the majority of people, however indirectly, involve much simpler approaches. Less
radical and showy, certainly, but powerful tools, j ust the same. Environmental
biotechnology is fundamentally rooted in waste, in its various guises, typically being
concerned with the remediation of contamination caused by previous use, the impact
reduction of current activity or the control of pollution. Thus, the principal aims of this
field are the manufacture of products in environmentally harmonious ways, which allow
for the minimisation of harmful solids, liquids or gaseous outputs or the clean-up of the
residual effects of earlier human occupation.

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The means by which this may be achieved are essenti ally two-fold. Environmental
biotechnologists may enhance or optimise conditions for existing biological systems to
make their activities happen faster or more efficiently, or they resort to some form of
alteration to bring about the desired outcome. The variety of organisms which may play
a part in environmental applications of biotechnology is huge, ranging from microbes
through to trees and all are utilised on one of the same three fundamental bases –
accept, acclimatise or alter. For the vast majority of cases, it is the former approach,
accepting and making use of existing species in their natural, unmodified form, which
predominates.
The Scope:
There are three key points for environmental biotechnology interventions, namely in the
manufacturing process, waste management or pollution control, as shown in Fig.

Accordingly, the range of businesses to which envir onmental biotechnology has
potential relevance is almost limitless. One area where this is most apparent is with
regard to waste. All commercial operations generate waste of one form or another and
for many, a proportion of what is produced is biodegradable. With disposal costs rising
steadily across the world, dealing with refuse cons titutes an increasingly high
contribution to overheads. Thus, there is a clear incentive for all businesses to identify
potentially cost-cutting approaches to waste and employ them where possible. Changes
in legislation throughout Europe, the US and elsewhere, have combined to drive these
issues higher up the political agenda and biological methods of waste treatment have
gained far greater acceptance as a result. For those industries with particularly high
biowaste production, the various available treatmen t biotechnologies can offer
considerable savings.
Manufacturing industries can benefit from the applications of whole organisms or
isolated biocomponents. Compared with conventional chemical processes, microbes and
enzymes typically function at lower temperatures and pressures. The lower energy

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demands this makes leads to reduced costs, but also has clear benefits in terms of both
the environment and workplace safety. Additionally, biotechnology can be of further
commercial significance by converting low-cost organic feedstocks into high value
products or, since enzymatic reactions are more highly specific than their chemical
counterparts, by deriving final substances of high relative purity. Almost inevitably,
manufacturing companies produce wastewaters or effl uents, many of which contain
biodegradable contaminants, in varying degrees. Tho ugh traditional permitted
discharges to sewer or watercourses may be adequate for some, other industries,
particularly those with recalcitrant or highly concentrated effluents, have found
significant benefits to be gained from using biological treatment methods themselves on
site. Though careful monitoring and process control are essential, biotechnology stands
as a particularly cost-effective means of reducing the pollution potential of wastewater,
leading to enhanced public relations, compliance with environmental legislation and
quantifiable cost-savings to the business.
Those involved in processing organic matter, for example, or with drying, printing,
painting or coating processes, may give rise to the release of volatile organic compounds
(VOCs) or odours, both of which represent environmental nuisances, though the former
is more damaging than the latter. For many, it is not possible to avoid producing these
emissions altogether, which leaves treating them to remove the offending contaminants
the only practical solution. Especially for relatively low concentrations of readily water-
soluble VOCs or odorous chemicals, biological technologies can offer an economic and
effective alternative to conventional methods.
The use of biological cleaning agents is another area of potential benefit, especially
where there is a need to remove oils and fats from process equipment, work surfaces or
drains. Aside from typically reducing energy costs, this may also obviate the need for
toxic or dangerous chemical agents. The pharmaceutical and brewing industries, for
example, both have a long history of employing enzy me-based cleaners to remove
organic residues from their process equipment. In addition, the development of effective
biosensors, powerful tools which rely on biochemica l reactions to detect specific
substances, has brought benefits to a wide range of sectors, including the
manufacturing, engineering, chemical, water, food and beverage industries. With their
ability to detect even small amounts of their particular target chemicals, quickly, easily
and accurately, they have been enthusiastically ado pted for a variety of process

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monitoring applications, particularly in respect of pollution assessment and control.
Contaminated land is a growing concern for the construction industry, as it seeks to
balance the need for more houses and offices with w ider social and environmental
goals. The reuse of former industrial sites, many of which occupy prime locations, may
typically have associated planning conditions attached which demand that the land be
cleaned up as part of the development process.
With urban regeneration and the reclamation of ‘brown-field’ sites increasingly favoured
in many countries over the use of virgin land, reme diation has come to play a
significant role and the industry has an ongoing interest in identifying cost-effective
methods of achieving it. Historically, much of this has involved simply digging up the
contaminated soil and removing it to landfill elsewhere.
Bioremediation technologies provide a competitive and sustainable alternative and in
many cases, the lower disturbance allows the overall scheme to make faster progress.
As the previous brief examples show, the range of those which may benefit from the
application of biotechnology is lengthy and includes the chemical, pharmaceutical,
water, waste management and leisure industries, as well as manufacturing, the
military, energy generation, agriculture and horticulture. Clearly, then, this may have
relevance to the viability of these ventures and, as was mentioned at the outset,
biotechnology is an essentially commercial activity.
Environmental biotechnology must compete in a world governed by the best practicable
environmental option (BPEO) and the best available techniques not entailing excessive
cost (BATNEEC). Consequently, the economic aspect will always have a large influence
on the uptake of all initiatives in environmental biotechnology and, most particularly, in
the selection of methods to be used in any given situation.
RENEWABLE SOURCES OF ENERGY
Introduction
Conventional energy sources based on oil, coal, and natural gas have proven to be
highly effective drivers of economic progress, but at the same time damaging to the
environment and to human health. Furthermore, they tend to be cyclical in nature, due
to the effects of oligopoly in production and distribution. These traditional fossil fuel-
based energy sources are facing increasing pressure on a host of environmental fronts,
with perhaps the most serious challenge confronting the future use of coal being the
Kyoto Protocol greenhouse gas (GHG) reduction targets. It is now clear that any effort to

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maintain atmospheric levels of CO 2 below even 550 ppm cannot be based
fundamentally on an oil and coal-powered global eco nomy, barring radical carbon
sequestration efforts.
The potential of renewable energy sources is enormous as they can in principle meet
many times the world’s energy demand. Renewable ene rgy sources such as biomass,
wind, solar, hydropower, and geothermal can provide sustainable energy services,
based on the use of routinely available, indigenous resources. A transition to
renewables-based energy systems is looking increasingly likely as the costs of solar and
wind power systems have dropped substantially in the past 30 years, and continue to
decline, while the price of oil and gas continue to fluctuate. In fact, fossil fuel and
renewable energy prices, social and environmental c osts are heading in opposite
directions. Furthermore, the economic and policy mechanisms needed to support the
widespread dissemination and sustainable markets for renewable energy systems have
also rapidly evolved. It is becoming clear that future growth in the energy sector is
primarily in the new regime of renewable, and to so me extent natural gas-based
systems, and not in conventional oil and coal sources. Financial markets are awakening
to the future growth potential of renewable and other new energy technologies, and this
is a likely harbinger of the economic reality of truly competitive renewable energy
systems.
Renewable energy sources currently supply somewhere between 15 percent and 20
percent of world’s total energy demand. The supply is dominated by traditional biomass,
mostly fuel wood used for cooking and heating, especially in developing countries in
Africa, Asia and Latin America. A major contribution is also obtained from the use of
large hydropower; with nearly 20 percent of the global electricity supply being provided
by this source. New renewable energy sources (solar energy, wind energy, modern bio-
energy, geothermal energy, and small hydropower) are currently contributing about two
percent. A number of scenario studies have investigated the potential contribution of
renewables to global energy supplies, indicating that in the second half of the 21st
century their contribution might range from the present figure of nearly 20 percent to
more than 50 percent with the right policies in place.
Biomass Energy
Biomass is the term used for all organic material originating from plants (including
algae), trees and crops and is essentially the collection and storage of the sun’s energy

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through photosynthesis. Biomass energy, or bioenergy, is the conversion of biomass
into useful forms of energy such as heat, electricity and liquid fuels.
Biomass for bioenergy comes either directly from the land, as dedicated energy crops, or
from residues generated in the processing of crops for food or other products such as
pulp and paper from the wood industry. Another impo rtant contribution is from post
consumer residue streams such as construction and d emolition wood, pallets used in
transportation, and the clean fraction of municipal solid waste (MSW). The biomass to
bioenergy system can be considered as the managemen t of flow of solar generated
materials, food, and fiber in our society. These interrelationships are shown in Figure,
which presents the various resource types and applications, showing the flow of their
harvest and residues to bioenergy applications. Not all biomass is directly used to
produce energy but rather it can be converted into intermediate energy carriers called
biofuels. This includes charcoal (higher energy density solid fuel), ethanol (liquid fuel),
or producer-gas (from gasification of biomass).
Biomass and bioenergy flow chart

Wind Energy
Wind has considerable potential as a global clean energy source, being both widely
available, though diffuse, and producing no pollution during power generation. Wind
energy has been one of humanity’s primary energy so urces for transporting goods,
milling grain, and pumping water for several millennia. From windmills used in China,
India and Persia over 2000 years ago to the generation of electricity in the early 20th
century in Europe and North America wind energy has played an important part in our
recorded history. As industrialization took place in Europe and then in America, wind
power generation declined, first gradually as the use of petroleum and coal, both

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cheaper and more reliable energy sources, became widespread, and then more sharply
as power transmission lines were extended into most rural areas of industrialized
countries. The oil crises of the 70’s, however, triggered renewed interest in wind energy
technology for gridconnected electricity production, water pumping, and power supply
in remote areas, promoting the industry’s rebirth.
Solar Photovoltaic and Solar Thermal Technologies
There are two basic categories of technologies that convert sunlight into useful forms of
energy, aside from biomass-based systems that do this in a broader sense by using
photosynthesis from plants as an intermediate step. First, solar photovoltaic (PV)
modules convert sunlight directly into electricity. Second, solar thermal power systems
use focused solar radiation to produce steam, which is then used to turn a turbine
producing electricity. The following provides a brief overview of these technologies, along
with their current commercial status.
Solar Photovoltaics
Solar PV modules are solid-state semiconductor devices with no moving parts that
convert sunlight into direct-current electricity. The basic principle underlying the
operation of PV modules dates back more than 150 years, but significant development
really began following Bell Labs’ invention of the silicon solar cell in 1954. The first
major application of PV technology was to power satellites in the late 1950s, and this
was an application where simplicity and reliability were paramount and cost was a
secondary concern. Since that time, enormous progre ss has been made in PV
performance and cost reduction, driven at first by the U.S. space program’s needs and
more recently through private/public sector collaborative efforts in the U.S., Europe,
and Japan.
Hydropower
Hydropower is the largest renewable resource used for electricity. It plays an essential
role in many regions of the world with more than 150 countries generating hydroelectric
power. A survey in 1997 by The International Journal on Hydropower Dams found
that hydro supplies at least 50 percent of national electricity production in 63 countries
and at least 90 percent in 23 countries. About 10 countries obtain essentially all their
commercial electricity from hydro, including Norway, several African nations, Bhutan
and Paraguay. There is about 700 GW of hydro capaci ty in operation worldwide,
generating 2600 TWh/year (about 19 percent of the w orld’s electricity production).

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About half of this capacity and generation is in Europe and North America with Europe
the largest at 32 percent of total hydro use and North America at 23 percent of the total.
However, this proportion is declining as Asia and Latin America commission large
amounts of new hydro capacity. Small, mini and micro hydro plants (usually defined as
plants less than 10 MW, 2 MW and 100kW, respectivel y) also play a key role in many
countries for rural electrification. An estimated 300 million people in China, for
example, depend on small hydro.
Small Hydro
Small-scale hydro is mainly ‘run of river,’ so does not involve the construction of large
dams and reservoirs. It also has the capacity to make a more immediate impact on the
replacement of fossil fuels since, unlike other sources of renewable energy, it can
generally produce some electricity on demand (at least at times of the year when an
adequate flow of water is available) with no need for storage or backup systems. It is
also in many cases cost competitive with fossil-fuel power stations, or for remote rural
areas, diesel generated power. Small hydro has a large, and as yet untapped, potential
in many parts of the world. It depends largely on a lready proven and developed
technology with scope for further development and optimization. Least-cost hydro is
generally high-head hydro since the higher the head, the less the flow of water required
for a given power level, and so smaller and less costly equipment is needed. While this
makes mountainous regions very attractive sites they also tend to be in areas of low
population density and thus low electricity demand and long transmission distances
often nullify the low cost advantage. Low-head hydro on the other hand is relatively
common, and also tends to be found in or near conce ntrations of population where
there is a demand for electricity. Unfortunately, the economics also tend to be less
attractive unless there are policy incentives in place to encourage their development.
Geothermal Energy
Geothermal energy, the natural heat within the earth, arises from the ancient heat
remaining in the Earth's core, from friction where continental plates slide beneath each
other, and from the decay of radioactive elements that occur naturally in small amounts
in all rocks. For thousands of years, people have benefited from hot springs and steam
vents, using them for bathing, cooking, and heating. During this century, technological
advances have made it possible and economic to loca te and drill into hydrothermal
reservoirs, pipe the steam or hot water to the surface, and use the heat directly (for

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space heating, aquaculture, and industrial processes) or to convert the heat into
electricity.
The amount of geothermal energy is enormous. Scientists estimate that just 1 percent
of the heat contained in just the uppermost 10 kilo meters of the earth’s crust is
equivalent to 500 times the energy contained in all of the earth's oil and gas resources.
Yet, despite the fact that this heat is present in practically inexhaustible quantities, it is
unevenly distributed, seldom concentrated and often at depths too great to be exploited
industrially and economically. Geothermal energy has been produced commercially for
70 years for both electricity generation and direct use. Its use has increased rapidly
during the last three decades and from 1975 – 1995 the growth rate for electricity
generation worldwide has been about 9 percent per y ear and for direct use of
geothermal energy it has been about 6 percent per year. In 1997 geothermal resources
had been identified in over 80 countries and there were quantified records of
geothermal utilization in at least 46 countries.
NON – RENEWABLE SOURCES OF ENERGY
Introduction
Sufficient, reliable sources of energy are a necessity for industrialized nations. Energy is
used for heating, cooking, transportation and manufacturing. Energy can be generally
classified as non-renewable and renewable. Over 85% of the energy used in the world is
from non-renewable supplies. Most developed nations are dependent on non-renewable
energy sources such as fossil fuels (coal and oil) and nuclear power. These sources are
called non-renewable because they cannot be renewed or regenerated quickly enough to
keep pace with their use. Some sources of energy ar e renewable or potentially
renewable. Examples of renewable energy sources are: solar, geothermal, hydroelectric,
biomass, and wind. Renewable energy sources are mor e commonly by used in
developing nations.
Industrialized societies depend on non-renewable energy sources. Fossil fuels are the
most commonly used types of non-renewable energy. T hey were formed when
incompletely decomposed plant and animal matter was buried in the earth's crust and
converted into carbon-rich material that is useable as fuel. This process occurred over
millions of years. The three main types of fossil fuels are coal, oil, and natural gas. Two
other less-used sources of fossil fuels are oil shales and tar sands.
Coal

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Coal is the most abundant fossil fuel in the world with an estimated reserve of one
trillion metric tons. Most of the world's coal reserves exist in Eastern Europe and Asia,
but the United States also has considerable reserves. Coal formed slowly over millions
of years from the buried remains of ancient swamp plants.
During the formation of coal, carbonaceous matter was first compressed into a spongy
material called "peat," which is about 90% water. As the peat became more deeply
buried, the increased pressure and temperature turned it into coal.
Different types of coal resulted from differences in the pressure and temperature that
prevailed during formation. The softest coal (about 50% carbon), which also has the
lowest energy output, is called lignite. Lignite has the highest water content (about
50%) and relatively low amounts of smog-causing sulfur. With increasing temperature
and pressure, lignite is transformed into bituminous coal (about 85% carbon and 3%
water). Anthracite (almost 100% carbon) is the hardest coal and also produces the
greatest energy when burned. Less than 1% of the coal found in the United States is
anthracite. Most of the coal found in the United States is bituminous. Unfortunately,
bituminous coal has the highest sulfur content of all the coal types. When the coal is
burned, the pollutant sulfur dioxide is released into the atmosphere.
Coal mining creates several environmental problems. Coal is most cheaply mined from
near-surface deposits using strip mining techniques. Strip-mining causes considerable
environmental damage in the forms of erosion and ha bitat destruction. Sub-surface
mining of coal is less damaging to the surface environment , but is much more
hazardous for the miners due to tunnel collapses and gas explosions. Currently, the
world is consuming coal at a rate of about 5 billion metric tons per year. The main use
of coal is for power generation, because it is a relatively inexpensive way to produce
power.
Coal is used to produce over 50% of the electricity in the United States. In addition to
electricity production, coal is sometimes used for heating and cooking in less developed
countries and in rural areas of developed countries. If consumption continues at the
same rate, the current reserves will last for more than 200 years. The burning of coal
results in significant atmospheric pollution.
The sulfur contained in coal forms sulfur dioxide w hen burned. Harmful nitrogen
oxides, heavy metals, and carbon dioxide are also released into the air during coal
burning. The harmful emissions can be reduced by in stalling scrubbers and

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electrostatic precipitators in the smokestacks of power plants. The toxic ash remaining
after coal burning is also an environmental concern and is usually disposed into
landfills.
Oil
Crude oil or liquid petroleum is a fossil fuel that is refined into many different energy
products (e.g., gasoline, diesel fuel, jet fuel, heating oil). Oil forms underground in rock
such as shale, which is rich in organic materials. After the oil forms, it migrates upward
into porous reservoir rock such as sandstone or limestone, where it can become trapped
by an overlying impermeable cap rock.
Wells are drilled into these oil reservoirs to remove the gas and oil. Over 70 percent of
oil fields are found near tectonic plate boundaries, because the conditions there are
conducive to oil formation.
Oil recovery can involve more than one stage. The primary stage involves pumping oil
from reservoirs under the normal reservoir pressure. About 25 percent of the oil in a
reservoir can be removed during this stage. The sec ondary recovery stage involves
injecting hot water into the reservoir around the well. This water forces the remaining
oil toward the area of the well from which it can be recovered. Sometimes a tertiary
method of recovery is used in order to remove as much oil as possible. This involves
pumping steam, carbon dioxide gas or nitrogen gas i nto the reservoir to force the
remaining oil toward the well.
Tertiary recovery is very expensive and can cost up to half of the value of oil removed.
Carbon dioxide used in this method remains sequeste red in the deep reservoir, thus
mitigating its potential greenhouse effect on the atmosphere. The refining process
required to convert crude oil into useable hydrocarbon compounds involves boiling the
crude and separating the gases in a process known as fractional distillation. Besides its
use as a source of energy, oil also industrial chemicals.
Over 50 percent of the world's oil is found in the Middle East; sizeable additional
reserves occur in North America. Most known oil reserves are already being exploited,
and oil is being used at a rate that exceeds the rate of discovery of new sources. If the
consumption rate continues to increase and no significant new sources are found, oil
supplies may be exhausted in another 30 years or so.

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Despite its limited supply, oil is a relatively inexpensive fuel source. It is a preferred fuel
source over coal. An equivalent amount of oil produces more kilowatts of energy than
coal. It also burns cleaner, producing about 50 percent less sulfur dioxide.
Oil, however, does cause environmental problems. Th e burning of oil releases
atmospheric pollutants such as sulfur dioxide, nitrogen oxides, carbon dioxide and
carbon monoxide. These gases are smog-precursors that pollute the air and greenhouse
gases that contribute to global warming. Another environmental issue associated with
the use of oil is the impact of oil drilling. Substantial oil reserves lie under the ocean.
Oil spill accidents involving drilling platforms kill marine organisms and birds. Some
reserves such as those in northern Alaska occur in wilderness areas. The building of
roads, structures and pipelines to support oil recovery operations can severely impact
the wildlife in those natural areas.
Natural gas
Natural gas production is often a by-product of oil recovery, as the two commonly share
underground reservoirs. Natural gas is a mixture of gases, the most common being
methane (CH
4). It also contains some ethane (C 2H5), propane (C 3H8), and butane
(C
4H10). Natural gas is usually not contaminated with sul fur and is therefore the
cleanest burning fossil fuel. After recovery, propane and butane are removed from the
natural gas and made into liquefied petroleum gas (LPG). LPG is shipped in special
pressurized tanks as a fuel source for areas not directly served by natural gas pipelines
(e.g., rural communities). The remaining natural gas is further refined to remove
impurities and water vapor, and then transported in pressurized pipelines. The United
States has over 300,000 miles of natural gas pipelines. Natural gas is highly flammable
and is odorless. The characteristic smell associated with natural gas is actually that of
minute quantities of a smelly sulfur compound (ethyl mercaptan) which is added during
refining to warn consumers of gas leaks.
The use of natural gas is growing rapidly. Besides being a clean burning fuel source,
natural gas is easy and inexpensive to transport on ce pipelines are in place. In
developed countries, natural gas is used primarily for heating, cooking, and powering
vehicles. It is also used in a process for making ammonia fertilizer. Current usage
levels, this supply will last an estimated 100 years. Most of the world's natural gas
reserves are found in Eastern Europe and the Middle East.
Oil shale and tar sands

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Oil shale and tar sands are the least utilized foss il fuel sources. Oil shale is
sedimentary rock with very fine pores that contain kerogen, a carbon-based, waxy
substance. If shale is heated to 490º C, the keroge n vaporizes and can then be
condensed as shale oil, a thick viscous liquid. This shale oil is generally further refined
into usable oil products. Production of shale oil requires large amounts of energy for
mining and processing the shale. Indeed about a half barrel of oil is required to extract
every barrel of shale oil. Oil shale is plentiful, with estimated reserves totaling 3 trillion
barrels of recoverable shale oil. These reserves alone could satisfy the world's oil needs
for about 100 years. Environmental problems associated with oil shale recovery include:
large amounts of water needed for processing, dispo sal of toxic waste water, and
disruption of large areas of surface lands.
Tar sand is a type of sedimentary rock that is impregnated with a very thick crude oil.
This thick crude does not flow easily and thus normal oil recovery methods cannot be
used to mine it. If tar sands are near the surface, they can be mined directly. In order to
extract the oil from deep-seated tar sands, however, steam must be injected into the
reservoir to make the oil flow better and push it toward the recovery well. The energy
cost for producing a barrel of tar sand is similar to that for oil shale. The largest tar-
sand deposit in the world is in Canada and contains enough material (about 500 billion
barrels) to supply the world with oil for about 15 years. However, because of
environmental concerns and high production costs these tar sand fields are not being
fully utilized.
Nuclear power
In most electric power plants, water is heated and converted into steam, which drives a
turbine-generator to produce electricity. Fossil-fueled power plants produce heat by
burning coal, oil, or natural gas. In a nuclear power plant, the fission of uranium
atoms in the reactor provides the heat to produce steam for generating electricity.
Several commercial reactor designs are currently in use in the United States. The most
widely used design consists of a heavy steel pressure vessel surrounding a reactor core.
The reactor core contains the uranium fuel, which is formed into cylindrical ceramic
pellets and sealed in long metal tubes called fuel rods. Thousands of fuel rods form the
reactor core. Heat is produced in a nuclear reactor when neutrons strike uranium
atoms, causing them to split in a continuous chain reaction. Control rods, which are

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made of a material such as boron that absorbs neutrons, are placed among the fuel
assemblies.
When the neutron-absorbing control rods are pulled out of the core, more neutrons
become available for fission and the chain reaction speeds up, producing more heat.
When they are inserted into the core, fewer neutrons are available for fission, and the
chain reaction slows or stops, reducing the heat generated. Heat is removed from the
reactor core area by water flowing through it in a closed pressurized loop. The heat is
transferred to a second water loop through a heat exchanger. The water also serves to
slow down, or "moderate" the neutrons which is necessary for sustaining the fission
reactions. The second loop is kept at a lower pressure, allowing the water to boil and
create steam, which is used to power the turbine-generator and produce electricity.
Originally, nuclear energy was expected to be a clean and cheap source of energy.
Nuclear fission does not produce atmospheric pollution or greenhouse gases and it
proponents expected that nuclear energy would be cheaper and last longer than fossil
fuels. Unfortunately, because of construction cost overruns, poor management, and
numerous regulations, nuclear power ended up being much more expensive than
predicted. The nuclear accidents at Three Mile Isla nd in Pennsylvania and the
Chernobyl Nuclear Plant in the Ukraine raised concerns about the safety of nuclear
power. Furthermore, the problem of safely disposing spent nuclear fuel remains
unresolved. The United States has not built a new nuclear facility in over twenty years,
but with continued energy crises across the country that situation may change.
ENVIRONMENTAL POLLUTION
Pollution has become one of the most frequently talked about of all environmental
problems by the world at large and yet, in many respects, it can often remain one of the
least understood. The word itself has a familiar ring to it and inevitably the concept of
pollution has entered the wider consciousness as a significant part of the burgeoning
‘greening’ of society in general.
The UK Environmental Protection Act (EPA) 1990 statutorily offers the following:
‘Pollution of the environment’ means pollution of the environment due to the release
(into any environmental medium) from any process of substances which are capable of
causing harm to man or any other living organisms supported by the environment.
In essence, then, pollution is the introduction of substances into the environment
which, by virtue of their characteristics, persistence or the quantities involved, are likely

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to be damaging to the health of humans, other anima ls and plants, or otherwise
compromise that environment’s ability to sustain life. It should be obvious that this is
an expressly inclusive definition, encompassing not simply the obviously toxic or
noxious substances, but also other materials which can have a polluting effect under
certain circumstances.
Classifying Pollution
While, as we said earlier, this diverse nature of potential pollutants makes their
systematisation difficult in absolute terms, it is possible to produce functional
classifications on the basis of various characteristics. However, it must be clearly borne
in mind that all such classification is essentially artificial and subjective, and that the
system to be adopted will typically depend on the purpose for which it is ultimately
intended. Despite these limitations, there is considerable value in having some method,
if only as a predictive environmental management to ol, for considerations of likely
pollutant effect.
Classification may, for example, be made on the bas is of the chemical or physical
nature of the substance, its source, the environmen tal pathway used, the target
organism affected or simply its gross effect. Fig.shows one possible example of such a
categorisation system and clearly many others are possible. The consideration of a
pollutant’s properties is a particularly valuable approach when examining real-life
pollution effects, since such an assessment requires both the evaluation of its general
properties and the local environment. This may include factors such as:
Toxicity;
Persistence;
Mobility;
Ease of control;
Bioaccumulation;
Chemistry.
Toxicity
Toxicity represents the potential damage to life and can be both short and long term. It
is related to the concentration of pollutant and the time of exposure to it, though this
relationship is not an easy one. Intrinsically highly toxic substances can kill in a short
time, while less toxic ones require a longer period of exposure to do damage. This much
is fairly straightforward. However, some pollutants which may kill swiftly in high

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concentrations may also have an effect on an organism’s behaviour or its susceptibility
to environmental stress over its lifetime, in the case of low concentration exposure.
Availability also features as an important influence, both in a gross, physical sense and
also in terms of its biological availability to the individual organism, together with
issues of its age and general state of health. Other considerations also play a significant
part in the overall picture of toxicity and we shall return to look at some of them in
greater depth shortly.


Persistence
This is the duration of effect. Environmental persistence is a particularly important
factor in pollution and is often linked to mobility and bioaccumulation.
Highly toxic chemicals which are environmentally unstable and break down rapidly are
less harmful than persistent substances, even though these may be intrinsically less
toxic.
Mobility
The tendency of a pollutant to disperse or dilute is a very important factor in its overall
effect, since this affects concentration. Some pollutants are not readily mobile and tend
to remain in ‘hot-spots’ near to their point of origin. Others spread readily and can
cause widespread contamination, though often the distribution is not uniform. Whether
the pollution is continuous or a single event, and if it arose from a single point or
multiple sources, form important considerations.
Ease of control
Many factors contribute to the overall ease with which any given example of pollution
can be controlled, including the mobility of the pollutant, the nature, extent or duration
of the pollution event and local site-specific considerations. Clearly, control at source is
the most effective method, since it removes the problem at its origin. However, this is
not always possible and in such cases, containment may be the solution, though this
can itself lead to the formation of highly concentrated hot-spots. For some substances,

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the dilute and disperse approach, which is discussed more fully later in this chapter,
may be more appropriate, though the persistence of the polluting substances must
obviously be taken into account when making this decision.
Bioaccumulation
As is widely appreciated, some pollutants, even when present in very small amounts
within the environment, can be taken up by living organisms and become concentrated
in their tissues over time. This tendency of some chemicals to be taken up and then
concentrated by living organisms is a major consideration, since even relatively low
background levels of contamination may accumulate up the food chain.
WATER POLLUTION – SOURCES AND MEASUREMENTS
Introduction:
Any physical, biological, or chemical change in water quality that adversely affects living
organisms or makes water unsuitable for desired use s can be considered pollution.
There are natural sources of water contamination (e .g. poison springs and oil
seeps).Pollution control standards and regulations usually distinguish between point
and nonpoint pollution sources. Point sources: disc harge pollution from specific
locations (e.g. drain pipes, ditches, or sewer outfalls).Nonpoint sources: pollution is
scattered or diffuse, having no specific location where they discharge into a particular
body of water (e.g. runoff from farm fields and feedlots, golf courses, lawns, and
gardens). The ultimate in diffuse, nonpoint pollution is atmospheric deposition of
contaminants carried by air currents and precipitated into watersheds or directly onto
surface waters as rain, snow, or dry particles.
Types and Effects of Water Pollution
Infectious Agents
The most serious water pollutants in terms of human health worldwide are pathogenic
organisms. The main source of these pathogens is from untreated or improperly treated
human wastes. In developed countries, sewage treatm ent plants and other pollution-
control techniques have reduced or eliminated most of the worst sources of pathogens
in inland surface waters. The situation is quite different in less-developed countries.
The United Nations estimates that at least 2.5 billion people in these countries lack
adequate sanitation, and that about half these people also lack access to clean drinking
water. Water quality control personnel usually analyze water for the presence of

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coliform bacteria, any of the types that live in the colon or the intestines of humans and
other animals (e.g. E. coli)
Oxygen-Demanding Wastes
The amount of oxygen dissolved in water is a good indicator of water quality and of the
kinds of life it will support. The addition of certain organic materials, such as sewage,
paper pulp, or food-processing wastes, to water stimulates oxygen consumption by
decomposers. Biochemical oxygen demand (BOD): a sta ndard test of the amount of
dissolved oxygen consumed by aquatic microorganisms over a five-day period. Dissolved
oxygen content (DO): measure dissolved oxygen conte nt directly using an oxygen
electrode. The oxygen decline downstream from point source is called the oxygen sag.
Immediately below the source of pollution, oxygen levels begin to fall as decomposers
metabolize waste materials.
Plant Nutrients and Cultural Eutrophication
Water clarity is affected by sediments, chemicals, and the abundance of plankton
organisms, and is a useful measure of water quality and water pollution.
Oligotrophic: describes rivers and lakes that have clear water a nd low biological
productivity.
Eutrophic: describes waters that are rich in organisms and organic materials. Human
activities can greatly accelerate eutrophication (cultural eutrophication).
High biological productivity of eutrophic systems is often seen in "blooms" of algae or
thick growth of aquatic plants stimulated by elevated phosphorous or nitrogen levels.
Eutrophication also occurs in marine ecosystems, especially in near-shore waters and
partially enclosed bays or estuaries.
Toxic Tides
Red tide: bloom of deadly aquatic microorganisms called dinoflagellates.
Red tides - and other colors, depending on the spec ies involved-have become
increasingly common in slow-moving rivers, brackish lagoons, estuaries, and bays.
One of the most feared of these organisms is Pfiesteria piscicida, an extraordinarily
poisonous dinoflagellate that has recently wiped out hundreds of thousands to millions
of fish every year in polluted rivers and estuaries such as North Carolina's Palmico
Sound.
Under the right conditions, a population explosion can produce a dense bloom of these
cells.

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Inorganic Pollutants
Some toxic inorganic chemicals are released from rocks by weathering, are carried by
runoff into lakes or rivers, or percolate into groundwater aquifers.
Humans can accelerate the rate of release of these inorganic chemicals through the
mining, processing, using, and discarding of minerals.
Metals
Many metals such as mercury, lead, cadmium, and nickel are highly toxic.
A famous case of mercury poisoning occurred in Japa n in the 1950s. Heavy metals
released as a result of human activities also are concentrated by hydrological and
biological processes so that they become hazardous to both natural ecosystems and
human health. Mine drainage and leaching of mining wastes are serious sources of
metal pollution in water.
Nonmetallic salts
Desert soils often contain high concentrations of soluble salts, including toxic selenium
and arsenic. Salts such as sodium chloride that are nontoxic at low concentrations also
can be mobilized by irrigation and concentrated by evaporation, reaching levels that are
toxic for plants and animals. Acids and bases
Acids are released as by-products of industrial processes (e.g. leather tanning, metal
smelting and planting) Coal and oil combustion also leads to formation of atmospheric
sulfuric and nitric acids, which are disseminated by long-range transport processes.
Organic Chemicals
Many chemicals used in the chemical industry to mak e pesticides, plastics,
pharmaceuticals, pigments, and other products we use in everyday life are highly toxic.
The two most important sources of toxic organic che micals in water are improper
disposal of industrial and household wastes and runoff of pesticides.
Many of the toxic organic chemicals (e.g. DDT, Diox ins, and other chlorinated
hydrocarbons) in water are passed through ecosystems and accumulated at high levels
in certain nontarget organisms. Hundreds of millions of toxic hazardous organic wastes
are thought to be stored in dumps, landfills, lagoons, and underground tanks in the
United States.
Sediment
Rivers have always carried sediment to the oceans, but erosion rates in many areas
have been greatly accelerated by human activities. Sources of erosion include forests,

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grazing lands, and urban construction sites. Sedime nt fills lakes and reservoirs,
obstructs shipping channels, clogs hydroelectric turbines, and makes purification of
drinking water more costly. Excess sediment deposits can fill estuaries and smother
aquatic life on coral reefs and shoals near shore.
Sediment can also be beneficial. Mud carried by rivers nourishes floodplain farm fields.
Thermal Pollution and Thermal Shocks
Raising or lowering water temperatures from normal levels can adversely affect water
quality and aquatic life. Humans cause thermal pollution by altering vegetation cover
and runoff patterns, as well as by discharging heated water directly into rivers and
lakes. The cheapest way to remove heat from an industrial facility is to draw cool water
from an ocean, river, lake, or aquifer, run it through a heat-exchanger to extract excess
heat, and dump the heated water back into the original source. In some circumstances,
introducing heated water into a water body is beneficial.
Warming catfish-rearing ponds
Attract fish, birds, and marine mammals that find food and refuge there, especially
during cold weather.
Water Pollution Control
Source Reduction
The cheapest and most effective way to reduce pollution is to avoid producing it or
releasing it to the environment in the first place.
Industry can modify manufacturing processes so fewer wastes are created.
Recycling or reclaiming materials that otherwise might be discarded in the waste stream
also reduces pollution.
Nonpoint Sources and Land Management
Among the greatest remaining challenges in water po llution control are diffuse,
nonpoint pollution sources.
Nonpoint sources have many origins and numerous rou tes by which contaminants
enter ground and surface waters; therefore, it is difficult to identify, monitor, and
control all these sources and routes.
Some main causes of nonpoint pollution include agriculture, urban runoff, construction
sites, and land disposal.
Generally soil conservation methods also help protect water quality.
In urban areas, reducing materials carried away by storm runoff is helpful.

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A good example of watershed management is seen in t he Chesapeake Bay, America's
largest estuary.
Principal objectives of this plan include reducing nutrient loading, pollution prevention
measures, replanting thousands of hectares of seagrasses, and restoring wetlands that
filter out pollutants.
Although progress has been made, the goals of reducing both nitrogen and phosphate
levels by 40 percent and restoring viable fish and shellfish populations are still decades
away
Human Waste Disposal
Human and animal wastes usually create the most ser ious health-related water
pollution problems.
Natural Processes
In poorer countries of the world, most rural people simply go out into the fields and
forests to relieve themselves as they have always done. When population densities are
low, natural processes eliminate wastes quickly. Wh ere intensive agriculture is
practiced, it has long been customary to collect human and animal waste to be spread
on the fields as fertilizer and become a source of disease-causing pathogens in the food
supply.
Until about fifty years ago, most rural American families and quite a few residents of
towns and small cities depended on a pit toilet or "outhouse" for waste disposal from
which the untreated wastes would seep into the ground.
The development of septic tanks and properly constructed drain fields represented a
considerable improvement in public health.
Municipal Sewage Treatment
Over the past 100 years, sanitary engineers have de veloped effective municipal
wastewater treatment systems to protect human health, ecosystem stability, and water
quality.
Primary treatment is the first step in municipal wa ste treatment that physically
separates large solids from the waste stream.
Secondary treatment consists of biological degradat ion of the dissolved organic
compounds.
Tertiary treatment removes plant nutrients, especially nitrates and phosphates, from
the secondary effluent.

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In many American cities, sanitary sewers are connected to storm sewers, which carry
runoff from streets and parking lots which generall y contain a variety of refuse,
fertilizers, pesticides, oils, rubber, tars, lead, and other undesirable chemicals.
Low-Cost Waste Treatment
The municipal sewage systems used in developed coun tries are often too expensive to
build and operate in the developing world where low -cost, low-tech alternatives for
treating wastes are needed.
One option is effluent sewerage, a hybrid between a traditional septic tank and a full
sewer system.
Another alternative is to use natural or artificial wetlands to dispose of wastes.
Wetland waste treatment systems are now operating in many developing countries.
Effluent from these operations can be used to irrigate crops or raise fish for human
consumption if care is taken to first destroy pathogens.
SOIL POLLUTION – SOURCES AND MEASUREMENTS
Addition of substances which adversely affect the quality of soil or its fertility is known
as soil pollution. Generally polluted water also pollute soil. Solid waste is a mixture of
plastics, cloth, glass, metal and organic matter, sewage, sewage sludge, building debris,
generated from households, commercial and industrie s establishments add to soil
pollution. Fly ash, iron and steel slag, medical and industrial wastes disposed on land
are important sources of soil pollution. In addition, fertilizers and pesticides from
agricultural use which reach soil
as run-off and land filling by municipal waste are growing cause of soil pollution. Acid
rain and dry deposition of pollutants on land surface also contribute to soil pollution.
Sources of soil pollution
Plastic bags – Plastic bags made from low density polyethylene (LDPE), is virtually
indestructible, create colossal environmental hazard. The discarded bags block drains
and sewage systems. Leftover food, vegetable waste etc. on which cows and dogs feed
may die due to the choking by plastic bags. Plastic is non biodegradable and burning of
plastic in garbage dumps release highly toxic and p oisonous gases like carbon
monoxide, carbon dioxide, phosgene, dioxine and oth er poisonous chlorinated
compounds.

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Industrial sources – It includes fly ash, chemical residues, metallic and nuclear
wastes. Large number of industrial chemicals, dyes, acids, etc. find their way into the
soil and are known to create many health hazards including cancer.
Agricultural sources – Agricultural chemicals especially fertilizers and pesticides
pollute the soil. Fertilizers in the run off water from these fields can cause
eutrophication in water bodies. Pesticides are highly toxic chemicals which affect
humans and other animals adversely causing respiratory problems, cancer and death.
Control of soil pollution
Indiscriminate disposal of solid waste should be avoided.
To control soil pollution, it is essential to stop the use of plastic bags and instead use
bags of degradable materials like paper and cloth. Sewage should be treated properly
before using as fertilizer and as landfills. The organic matter from domestic, agricultural
and other waste should be segregated and subjected to vermicomposting which
generates useful manure as a by product. The industrial wastes prior to disposal should
be properly treated for removing hazardous materials. Biomedical waste should be
separately collected and incinerated in proper incinerators.
AIR POLLUTION – SOURCES AND MEASUREMENTS
Natural Sources of Air Pollution
There are many natural sources of air quality degradation.
Natural fires release smoke.
Volcanoes spew out ash, acid mists, hydrogen sulfide, and other toxic gases.
Sea spray and decaying vegetation are major sources of reactive sulfur compounds in
the air.
Trees and bushes emit millions of tons of volatile organic compounds.
Pollen, spores, viruses, bacteria, and other small bits of organic material are present in
the air.
Bacterial metabolism of decaying vegetation in swamps and of cellulose in the guts of
termites and ruminant animals is responsible for large methane releases.
Human-Caused Air Pollution
Primary and Secondary Pollutants
Primary pollutants: those released directly from the source into the air in a harmful
form.

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Secondary pollutants: modified to a hazardous form after they enter the air or are
formed by chemical reactions as components of the air mix and interact.
Solar radiation often provides the energy for these reactions.
Fugitive emissions: those that do not go through a smokestack (e.g. dust from soil
erosion, strip mining, rock crushing, and building construction).
Unconventional Pollutants
EPA has authority under the Clean Air Act to set emission standards (regulating the
amount released) for certain unconventional or nonc riteria pollutants that are
considered especially hazardous or toxic.
Examples of these unconventional pollutants include asbestos, benzene, beryllium,
mercury, polychlorinated biphenyls, and vinyl chloride.
Aesthetic degradation: includes any undesireable ch anges in the physical
characteristics or chemistry of the atmosphere (e.g. noise, ordors and light pollution).
Conventional or Criteria Pollutants
Sulfur compounds
Natural sources: evaporation of sea spray, erosion of sulfate-containing dust from arid
soils, fumes from volcanoes and fumaroles, and biogenic emissions of hydrogen sulfide
and organic sulfur-containing compounds.
The predominant form of anthropogenic sulfur is sulfur dioxide from combustion of
sulfur-containing fuel.
Sulfur dioxide is a colorless corrosive gas that is directly damaging to both plants and
animals.
Can be oxidized to sulfur trioxide, which reacts with water vapor or dissolves in water
droplets to form sulfuric acid (major component of acid rain).
Nitrogen compounds
Nitrogen oxides: highly reactive gases formed when nitrogen in fuel or combustion air is
heated to temperatures above 650deg.C in the presence of oxygen, or when bacteria in
soil or water oxidize nitrogen-containing compounds.
Nitrogen oxides combine with water to make nitric acid, which is a major component of
atmospheric acidification.
Excess nitrogen also causes fertilization and eutrophication of inland waters and
coastal seas.
Carbon Oxides

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Carbon dioxide (CO2) is the predominant form of carbon in the air.
Usually considered nontoxic and innocuous, increasing levels of carbon dioxide appears
to be causing a global climate warming.
Burning of fossil fuels is estimated to add between 5 and 5.5 billion tons of carbon to
the atmosphere each year.
Uncertainty exists about where the extra carbons goes.
Carbon monoxide: colorless, odorless, nonirritating but highly toxic gas.
About 90 percent of the carbon monoxide in the air is consumed in photochemical
reactions that produce ozone.
Metals and Halogens
Many toxic metals are mined and used in manufacturi ng processes or occur as trace
elements in fuels, especially coal.
Lead
Worldwide lead emissions amount to about 2 million metric tons per year, or two-thirds
of all metallic pollution.
Most lead is from leaded gasoline.
An estimated 20 percent of all inner-city children suffer some degree of mental
retardation from high environmental lead levels.
Mercury
Two largest sources of atmospheric mercury appear to be coal-burning power plants
and waste incinerators.Other toxic metals of concern are nickel, beryllium, cadmium,
thallium, uranium, cesium, and plutonium.Halogens (fluorine, chlorine, bromine, and
iodine) are highly reactive and generally toxic in their elemental form. About 600 million
tons of highly persistent chlorofluorocarbons (CFCs) are used annually worldwide in
spray propellants, refrigeration compressors, and for foam blowing. CFCs diffuse into
the stratosphere where they release chlorine and fluorine atoms that destroy the ozone
shield that protects the earth from U.V. radiation.
Particulate material
Particulate material: all atmospheric aerosols, whether solid or liquid.
Includes dust, ash, soot, lint, smoke, pollen, spores, algal cells, and many other
suspended materials. Particulates often are the most apparent form of air pollution
since they reduce visibility and leave dirty deposits on windows, painted surfaces, and

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textiles. Respirable particles smaller than 2.5 micrometers are among the most
dangerous of this group because they can be drawn into the lungs.
Volatile organic compounds
Volatile organic compounds (VOCs): organic chemicals that exist as gases in the air.
Plants are the largest source of VOCs. A large numb er of other synthetic organic
chemicals, such as benzene, toluene, formaldehyde, vinyl chloride, phenols, chloroform,
and trichloroethylene, are released into the air by human activities.
These chemicals play an important role in the formation of photochemical oxidants.
Of the 188 air toxics listed in the Clean Air Act, about two-thirds are VOCs and most of
the rest are metal compounds. EPA has identified 33 chemical compounds considered
to be the greatest threat to public health in urban areas.
Photochemical oxidants


Photochemical oxidants: products of secondary atmospheric reactions driven by solar
energy. One of the most important reactions involve s formation of singlet (atomic)
oxygen by splitting nitrogen dioxide (NO
2). Then the atmoic oxygen reacts with another
molecule of O
2 to make ozone (O3).
Ozone formed in the stratosphere provides a valuabl e shield for the biosphere by
absorbing incoming ultraviolet radiation. In ambient air, however, O
3 is a strong
oxidizing reagent and damages vegetation, building materials, and sensitive tissues.
Effects of Air Pollution
Human Health
Heart attacks, respiratory diseases, and lung cancer all are significantly higher in
people who breathe dirty air, compared to matching groups in cleaner

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environments.Conditions are often much worse in other countries than Canada or the
United States. The United Nations estimates that at least 1.3 billion people around the
world live in areas where air is dangerously polluted.
The most common route of exposure to air pollutants is by inhalation, but direct
absorption through the skin or contamination of food and water are also important
pathways. Because they are strong oxidizing agents, sulfates, SO
2, NOx, and O3 act as
irritants that damage delicate tissues in the eyes and respiratory passages.
Carbon monoxide binds to hemoglobin and decreases t he ability of red blood cells to
carry oxygen.
Some important chronic health effects of air pollut ants include bronchitis and
emphysema.
Bronchitis: persistent inflammation of bronchi and bronchioles (large and small
airways in the lung) that cause a painful cough and involuntary muscle spasms that
constrict airways.
Emphysema: an irreversible obstructive lung disease in which airways become
permanently constricted and alveoli are damaged or even destroyed.
Half of all lungs examined at autopsy in the United States have some degree of alveolar
deterioration.
Smoking is undoubtedly the largest cause of obstructive lung disease and preventable
death in the world.
Plant Pathology
In the early days of industrialization, fumes from furnaces, smelters, refineries, and
chemical plants often destroyed vegetation and created desolate, barren landscapes
around mining and manufacturing centers.
Copper-nickel smelter at Sudbury, Ontario, is a notorious example of air pollution
effects on vegetation and ecosystems.
There are two probable ways that air pollutants damage plants.
They can be directly toxic, damaging sensitive cell membranes much as irritants do in
human lungs.
They can act as metabolic regulators or plant hormones and disrupt normal patterns of
growth and development.
Synergistic effects: effects caused following exposure to two factors which together is
more than the sum of exposure to each factor individually.

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Pollutant levels too low to produce visible symptoms of damage may still have important
effects.
Acid Deposition
Acid precipitation: the deposition of wet acidic solutions or dry acidic particles from the
air. By the 1940's, it was known that pollutants, including atmospheric acids, could be
transported long distances by wind currents.
pH and atmospheric acidity
Acidity is described in terms of pH (the negative l ogarithm of the hydrogen ion
concentration in a solution). pH scale ranges from 0 to 14 with 7, the midpoint, being
neutral. Values less than 7 indicate progressively greater acidity, while above 7 are
progressively more alkaline. Normal, unpolluted rain generally has a pH of about 5.6
due to carbonic acid created by CO
2 in the air.
Aquatic effects
Generally, reproduction is the most sensitive stage in fish life cycles.
Eggs and fry of many species are killed when the pH drops to about 5.0.
This level of acidification (pH 5.0) can also disrupt the food chain by killing aquatic
plants, insects, and invertebrates on which fish depend for food.
There are several ways acids kill fish.
Alters body chemistry
Destroys kills and prevents oxygen uptake
Causes bone decalcification
Disrupts muscle contraction.
Acid water leaches toxic metals, such as mercury and aluminum, out of soil and rocks.
Studies in the Adirondack Mountains of New York revealed that about half of the high
altitude lakes are acidified and have no fish.
Much of the western United States has relatively alkaline bedrock and carbonate-rich
soil, which counterbalance acids from the atmosphere.
Sulfates account for about two-thirds of the acid deposition in eastern North America
and most of Europe, while nitrates contribute most of the remaining one-third.
Forest damage
In the early 1980s, disturbing reports appeared of rapid forest declines in both Europe
and North America.

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A 1980 survey on Camel's Hump Mountain in Vermont s howed that seedling
production, tree density, and viability of spruce-fir forests at high elevations had
declined about 50 percent in 15 years.
By 1990, almost all the red spruce, once the dominant species on the upper part of the
mountain, were dead or dying.
European forests also are dying at an alarming rate.
In 1982, German foresters estimated only 8 percent of their forests showed pollution
damage.
By 1983, some 34 percent of the forest was affected.
By 1985, more than 4 million hectares (about half the total) were reported to be in a
state of decline.
Similar damage is reported in Czechoslovakia, Poland, Austria, and Switzerland.
Researchers at the Hubbard Brook Experimental Forest in New Hampshire have shown
that forest soils have become depleted of natural buffering reserves of basic cations
such as calcium and magnesium through years of exposure to acid rain.
Plant pathogens and insect pests may damage trees or attack trees debilitated by air
pollution.
Buildings and monuments
In cities throughout the world, some of the oldest and most glorious buildings and
works of art are being destroyed by air pollution.
Air pollution also damages ordinary buildings and structures by corroding steel in
reinforced concrete in the buildings as well as roads and bridges.
Visibility reduction
Foul air obscuring the skies above industrialized cities has long been recognized as a
problem.
Pollution affects rural areas as well (e.g. Grand Canyon National Park and Shenandoah
National Park).
Air Pollution Control
Moving Pollution to Remote Areas
Among the earliest techniques for improving local air quality was moving pollution
sources to remote locations and/or dispersing emissions with smokestacks.
Particulate Removal

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Filters remove particle physically by trapping them in a porous mesh of cotton cloth,
spun glass fibers, or asbestos-cellulose, which allows air to pass through but holds
back solids. Electrostatic precipitators are the most common particulate controls in
power plants. Fly ash particles pick up an electrostatic surface charge as they pass
between large electrodes in the effluent stream.
Performance depends on particle size and chemistry, strength of the electric field, and
flue gas velocity.
Sulfur Removal
Sulfur removal can be done a variety of ways either by using low-sulfur fuel or by
removing sulfur from effluents.
Fuel switching and fuel cleaning
Switching from soft coal with a high sulfur content to low-sulfur coal can greatly reduce
sulfur emission.
Changing to another fuel, such as natural gas or nu clear energy, can eliminate all
sulfur emissions as well as those of particulates and heavy metals.
Alternative energy sources, such as wind and solar power, are preferable to either fossil
fuel or nuclear power, and are becoming economically competitive.
Coal can be crushed, washed, and gassified to remov e sulfur and metals before
combustion.
Limestone injection and fluidized bed combustion
Sulfur emissions can be reduced as much as 90 percent by mixing crushed limestone
with coal before it is fed into a boiler.
A relatively new technique for burning, called fluidized bed combustion, offers several
advantages in pollution control.
Flue gas desulfurization
Crushed limestone, lime slurry, or alkali can be injected into a stack gas stream to
remove sulfur after combustion.
Sulfur recovery processes
Sulfur can be removed from effluent gases by processes that yield a usable product,
such as elemental sulfur, sulfuric acid, or ammonium sulfate.
Nitrogen Oxide Control
Staged burners, in which the flow of air and fuel are carefully controlled, can reduce
nitrogen oxide formation by as much as 50 percent.

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The approach adopted by U.S. automakers for NO x reductions has been to use selective
catalysts to change pollutants to harmless substances.
Raprenox (rapid removal of nitrogen oxides) is a new technique for removing nitrogen
oxides that was developed by the U.S. Department of Energy Sandia Laboratory in
Livermore, California.
Hydrocarbon Controls
Closed systems that prevent escape of fugitive gases can reduce many hydrocarbon
emissions (e.g. positive crankcase ventilation (PCV) systems in automobiles).
Controls on fugitive losses from valves, pipes, and storage tanks in industry can have a
significant impact on air quality.
Afterburners are often the best method for destroying volatile organic chemicals in
industrial exhaust stacks.
XENOBIOTIC COMPOUNDS AND THEIR SOURCES
The word is derived from the Greek ‘xenos’ meaning foreign. Xenobiotics are compounds
which are not produced by a biological procedure and for which no equivalent exists in
nature. They present a particular hazard if they ar e subject to bioaccumulation
especially so if they are fat soluble since that enables them to be stored in the body fat
of organisms providing an obvious route into the food chain. Despite the fact that these
chemicals are man made, they may still be degraded by micro-organisms if they fit into
one of the following regimes; gratuitous degradation, a process whereby the xenobiot
resembles a natural compound sufficiently closely t hat it is recognised by the
organism’s enzymes and may be used as a food source , or cometabolism where the
xenobiot is degraded again by virtue of being recognized by the organism’s enzymes but
in this case its catabolism does not provide energy and so cannot be the sole carbon
source.
Consequently, cometabolism may be sustained only if a carbon source is supplied to
the organism. The ability of a single compound to be degraded can be affected by the
presence of other contaminants. For example, heavy metals can affect the ability of
organisms to grow, the most susceptible being Gram positive bacteria, then Gram
negative.
Fungi are the most resistant and actinomycetes are somewhere in the middle.
This being the case, model studies predicting the rate of contaminant degradation may
be skewed in the field where the composition of the contamination may invalidate the

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study in that application. Soil micro-organisms in particular are very versatile and may
quickly adapt to a new food source by virtue of the transmission of catabolic plasmids.
Of all soil bacteria, Pseudomonads seem to have the most highly developed ability to
adapt quickly to new carbon sources.
In bacteria, the genes coding for degradative enzymes are often arranged in clusters,
or operons, which usually are carried on a plasmid. This leads to very fast transfer from
one bacterium to another especially in the case of Pseudomonas where many of the
plasmids are self-transmissible. The speed of adaptation is due in part to the exchange
of plasmids but in the case of the archaeans particularly, the pathways they carry,
which may have been latent over thousands of bacter ial generations, owe their
existence to previous exposure over millions of years to an accumulated vast range of
organic molecules. It is suggested that, unless there has been evolutionary pressure to
the contrary, these latent pathways are retained to a large extent requiring little
modification if any to utilise new xenobiotics.
Briefly, the pathways may be expanded by adaptation to the new molecule, or very
much less commonly, wholescale insertion of ‘foreign’ genes may occur by genetic
manipulation. There have been several cases reported where catabolic pathways have
been expanded in the laboratory. Hedlund and Staley (2001) isolated a strain of Vibrio
cyclotrophicus from marine sediments contaminated with creosote. B y supplying the
bacteria with only phenanthrene as a carbon and ene rgy source, the bacteria were
trained to degrade several PAHs although some of these only by cometabolism with a
supplied carbon source.
Endocrine disrupters
To date, there are chemicals, including xenobiotics, which still resist degradation in the
environment. This may be due to a dearth, at the site of contamination, of organisms
able to degrade them fully or worse, microbial activity which changes them in such a
way that they pose a bigger problem than they did previously. One such example is
taken from synthetic oestrogens such as 17α-ethinyloestradiol commonly forming the
active ingredient of the birth control pills, and the natural oestrogens which, of course,
are not xenobiotics. Natural oestrogens are deactivated in humans by glucuronidation,
as shown in Fig., which is a conjugation of the hormone with UDP-glucuronate making
the compound more polar and easily cleared from the blood by the kidneys. It is in this
modified and inactive form that it is excreted into the sewage.

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However, bacteria present in the aerobic secondary treatment in sewage treatment
plants, have the enzyme, β-glucuronidase, which removes this modification thus
reactivating the hormone. As an aside, glucuronidation is not confined to hormones but
is a process used to detoxify a number of drugs, toxins and carcinogens in the liver. The
enzyme catalysing this process is induced in response to prolonged exposure to the
toxin thus imparting increased tolerance or even resistance to the chemical.
Returning to the problem of elevated levels of active hormones in the waterways,
another aspect is that steroids do not occur in bacteria, although they are present in
fungi, and so bacteria lack the necessary pathways to allow complete degradation of
these hormones at a rate compatible with the dwell time in sewage treatment plants.
The consequence has been raised levels of reactivat ed oestrogen and 17 α-
ethinyloestradiol in the waterways leading to distu rbances of the endocrine, or
hormonal, system in fauna downstream from sewage tr eatment plants. Such
disturbances have been monitored by measuring the presence of the protein vitellogenin
(Sole et al. 2001) which is a precursor to egg yolk protein, the results of which have
indicated feminisation of male fish in many species including minnows, trout and
flounders. The source of environmental oestrogens is not confined to outfall from
sewage treatment plants; however, the fate of endocrine disrupters, examples of which
are given in Fig., in sewage treatment plants is the subject of much research (Byrns
2001).

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Many other chemicals, including polyaromatic hydroc arbons (PAHs), dichloro
diphenyltrichloroethane (DDT), alkyl phenols and some detergents may also mimic the
activity of oestrogen. There is general concern as to the ability of some organisms to
accumulate these endocrine disrupters in addition to the alarm being raised as to the
accumulative effects on humans of oestrogen-like activity from a number of xenobiotic
sources.
To date there is no absolute evidence of risk to human health but the Environmental
Agency and Water UK are recommending the monitoring of environmental oestrogens in
sewage treatment outfall. Assays are being develope d further to make these
assessments (Gutendorf and Westendorf 2001) and to predict potential endocrine
disrupter activity of suspected compounds (Takeyoshi et al 2002).
Oestrogen and progesterone are both heat labile. In addition, oestrogen appears to be
susceptible to treatment with ultra-violet light, the effects of which are augmented by
titanium dioxide (Eggins 1999). The oestrogen is degraded completely to carbon dioxide
and water thus presenting a plausible method for water polishing prior to consumption.
Another method for the removal of oestrogens from w ater, in this case involving
Aspergillus, has also been proposed (Ridgeway and Wiseman 1998 ). Sulphation of the
molecule by isolated mammalian enzymes, as a means of hormone inactivation is also
being investigated (Suiko 2000). Taken overall, it seems unlikely that elevated levels of
oestrogen in the waterways will pose a problem to human health in drinking water
although, this does not address the problem affecting hormone-susceptible organisms
living in contaminated water and thus exposed to this potential hazard.
New discoveries

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Almost daily, there are novel bacteria being reported in the literature which have been
shown to have the capacity to degrade certain xenob iots. Presumably the mutation
which occurred during the evolution of the organism conferred an advantage, and
selective pressure maintained that mutation in the DNA, thus producing a novel strain
with an altered phenotype.
Some example of such isolates is described here. Reference has already been made to
some PAHs mimicking oestrogen which earns those che micals the title of ‘endocrine
disrupters’. This is in addition to some being toxic for other reasons and some being
carcinogenic or teratogenic. The PAHs are derived primarily from the petrochemicals
industry and are polycyclic hydrocarbons of three or more rings which include as
members, naphthalene and phenanthrene and historica lly have been associated with
offshore drilling, along with alkylphenols. Several genera of bacteria are now known to
be able to degrade PAHs and recently, a novel strain of Vibrio cyclotrophicus able to
digest naphthalene and phenanthrene, was isolated from creosote-contaminated marine
sediments from Eagle Harbour, Washington, USA.
It would appear that bacteria isolated from the same marine or estuarine environments
may vary quite considerably in their abilities to degrade certain PAHs.
This observation is viewed as indicative of diverse catabolic pathways demonstrated by
these organisms and awaiting our full understanding (Hedlund and Staley 2001).
Polycyclic hydrocarbons (PCBs) are xenobiotics whic h, due to their high level of
halogenation, are substrates for very few pathways normally occurring in nature.
However, a strain of Pseudomonas putida able to degrade PCBs, was isolated recently
from wastewater outflow from a refinery. This was achieved by the bacterium employing
two pathways encoded by two separate operons; the tod pathway employed in toluene
degradation, and the cmt pathway which normally is responsible for the catabolism of
p-cumate which is a substituted toluene. The mutation which allowed this strain to
utilise the cmt pathway was found to be a single base change to the promoter-operator
sequence. This allowed all the enzymes in this path way to be expressed under
conditions where their synthesis would normally be repressed. Thus, the two pathways
could work in conjunction with each other to metabolise PCBs, a relationship described
as mosaic (Ohta et al 2001).

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The pthalates are substituted single-ring phenols and include terephthalic acid and its
isomers, the major chemicals used in manufacture of polyester fibres, films, adhesives,
coatings and plastic bottles.
In a recent analysis of anaerobic sewage sludge, a methanogenic consortium of over 100
bacterial clones was found to have the capability t o digest terephthalate.
Characterisation of these by analysis of their ribosomal DNA sequences revealed that
almost 70% were archaeans most of which had not been previously identified, and that
nearly 90% of the total bacteria comprised two of the novel Archaean species. These two
species are believed to be responsible for the degradation of terephthalic acid (Wu et al
2001). During wastewater treatment, terepthalic acid is usually treated by aerobic
processes. However, this consortium, or others like it provide an anaerobic alternative
which, being methanogenic, may be structured to off set processing costs by the
utilisation of the methane.
BIOMAGNIFICATION
Pollutants that exist in small amounts in the environment (such as certain heavy
metals and organic agents found in pesticides) become concentrated in organisms near
the top of the food chain. In an estuary, for example, microorganisms called plankton
may absorb small amounts of pollutants such as PCBs (polychlorinated biphenyls); fish
that eat lots of plankton might retain the pollutants in their tissues; birds or people that
eat the fish might concentrate the pollutants still more. This process, called
biomagnification, can produce health issues. Some substances that a re capable of
bioaccumulating include PCBs, fluoride, dioxins, boron, DDT, and mercury.
BIOINDICATORS
Bioindicators are organisms, such as lichens,birds and bacteria, that are used to
monitor the health of the environment. The organisms and organism associations are
monitored for changes that may indicate a problem within their ecosystem. The changes
can be chemical, physiological or behavioural. Bioindicators are relevant for Ecological
health. Ecological health can be viewed in terms of ecosystems, whereby structural and
functional characteristics are maintained. Ecological health can be expanded to include
many aspects of human health and well-being. Each organism within an ecosystem has
the ability to report on the health of its environment.
Bioindicators are used to: detect changes in the natural environment, monitor for the
presence of pollution and its effect on the ecosystem in which the organism lives,

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monitor the progress of environmental cleanup and t est substances, like drinking
water, for the presence of contaminants.
Specific physiological and behavioral changes in bioindicators are used to detect
changes in environmental health. The specific chang es differ from organism to
organism. The use of organisms as bioindicators encompasses many areas of science.
Wildlife conservation genetics is an example of how traditional approaches can be
combined with emerging biotechnologies to improve accuracy, and to collect information
not available through conventional methods. Wildlife conservation genetics combines
traditional monitoring of wildlife populations, like raccoons, with the scientific discipline
of genetics, to gain information about the health of ecosystems.
Several biotechology – based methods use microorganisms to test environmental health.
Unlike traditional methods,biotechnology - based methods do not rely on observation
alone but set out to create specific reactions that indicate the presence of a specific
pollutant or an unwanted microorganism. In this way they are similar to traditional
chemical analysis of environmental samples.
In traditional bioassays,a bioindicator organism or organisms association are
introduced to environmental samples, such as soil or water,and researchers observe
any changes that occur as a result of exposure. These methods are based primarily on
observation to detect changes. Bioindicators can be a measure, an index of measures,
or a model that characterizes an ecosystem or one of its critical components. They are
also a method of monitoring or detecting the negative impacts that industrial activity
has on the environment. This information helps develop strategies that will prevent or
lower such effects and make industry more sustainab le. The role of bioindicators in
sustainable development will help ensure that industry leaves the smallest footprint
possible on the environment.
BIOMONITORING: BIOSENSORS AND BIOCHIPS.
A biosensor is a two-component analytical device comprised of a biological recognition
element that outputs a measurable signal to an interfaced transducer (Fig.).

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Biorecognition typically relies on enzymes, whole cells, antibodies, or nucleic acids,
whereas signal transduction exploits electrochemica l (amperometric,
chronoamperometric, potentiometric, field-effect tr ansistors, conductometric,
capacitative), optical (absorbance, reflectance, luminescence, chemiluminescence,
bioluminescence, fluorescence, refractive index, light scattering), piezoelectric (mass
sensitive quartz crystal microbalance), magnetic, or thermal (thermistor, pyroelectric)
interfaces. This wide selection of interchangeable components has resulted in a
similarly wide selection of biosensors focused here toward those related to
environmental monitoring.
The detection of specific analytes of importance to environmental monitoring can be
achieved with great precision using analytical techniques that center around mass
spectrometry (MS), such as gas chromatography (GC)-MS, liquid chromatography (LC)-
MS, liquid chromatography coupled to tandem MS (LC- MS2), ion trap (IT)-MS, and
quadrupole linear ion trap (QqLIT)-MS. With great precision, however, comes significant
time, effort, and expense. Samples must be collected and transported to the obligatory
confinements of the laboratory, and requisite preconcentration and cleanup steps must
be performed prior to the sample being analyzed on an expensive, high-technology
instrument by accompanying trained technical personnel.
Considering that some percentage of the samples collected will be negative, either not
being contaminated or containing the target analyte at concentrations too low to be
detected, the adjusted cost on a per positive sample basis can be extensive. Although
biosensors cannot unequivocally replace the replicate accuracy and reproducibility of
conventional analytical instrumentation, they can complement and supplement their
operation through ease of sample preprocessing, which is often minimal to none, on-site
field portability, simplicity and rapidity of operation, versatility, real-time to near-real-
time monitoring capabilities, and miniaturization that has evolved down to a “lab-on-a-
chip” format. Biosensors can therefore often find their niche as continuous monitors of

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environmental contamination or as bioremediation process monitoring and control tools
to provide informational data on what contaminants are present, where they are
located, and a very sensitive and accurate evaluation of their concentrations in terms of
bioavailability. Bioavailability measurements are central to environmental monitoring as
well as risk assessment because they indicate the biological effect of the chemical,
whether toxic, cytotoxic, genotoxic, mutagenic, carcinogenic, or endocrine disrupting,
rather than mere chemical presence as is achieved with analytical instruments.
Despite their benefits, biosensors remain relatively unused in the environmental
monitoring/bioremediation fields, due primarily to a lack of real-world, real-sample
testing and standardization against conventional analytical techniques. Thus, although
biosensors show significant promise, it is clear that more field validation studies need to
be performed before regulatory agencies and other e nd users will gain sufficient
confidence to adopt their routine use.
Enzyme-based biosensors
The historical foundation of the biosensor rests with the enzyme glucose oxidase and its
immobilization on an oxygen electrode by Leland Clark in the 1960s for blood glucose
sensing. Although the research emphasis of enzyme-based biosensors continues to be
driven by more lucrative medical diagnostics, there has been a predictable application
overlap toward environmental monitoring as well. Enzymes act as organic catalysts,
mediating the reactions that convert substrate into product. Since enzymes are highly
specific for their particular substrate, the simplest and most selective enzyme-based
biosensors merely monitor enzyme activity directly in the presence of the substrate.
Perhaps the most relevant examples are the sulfur/s ulfate-reducing bacterial
cytochrome c3 reductases that reduce heavy metals. Michel et al. (2003) immobilized
cytochrome c3 on a glassy carbon electrode and monitored its re dox activity
amperometrically in the presence of chromate [Cr(VI )] with fair sensitivity (lower
detection limit of 0.2 mg/L) and rapid response (several minutes) (Figure 9.2). When
tested under simulated groundwater conditions, the biosensor did cross-react with
several other metal species, albeit at lower sensit ivities, and was affected by
environmental variables such as pH, temperature, an d dissolved oxygen, thus
exemplifying certain disadvantages common to enzyme -based biosensors. Similarly
operated biosensors for the groundwater contaminant perchlorate using perchlorate
reductase as the recognition enzyme (detection limit of 10 µg/L) (Okeke et al., 2007),

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organophosphate pesticides using parathion hydrolase or organophosphorus hydrolase
as recognition enzymes (detection down to low µM concentrations) (Trojanowicz, 2003),
and environmental estrogens using tyrosinase as the recognition enzyme (detection
down to 1 µM) (Andreescu and Sadik, 2004) have also been designed.
Another type of enzyme biosensor relies on enzyme activation upon interaction with the
target of interest. For example, heavy metals in the form of cofactors—inorganic ions
that bind to and activate the enzyme—can be detecte d based on this integral
association.
Metalloenzymes such as alkaline phosphatase, ascorb ate oxidase, glutamine
synthetase, and carbonic anhydrase require association of a metal ion cofactor with
their active sites for catalytic activity, and can thus be used as recognition elements for
heavy metals. Strong chelating agents are first used to strip the enzyme of all metal ion
cofactors to form the inactive apoenzyme. Upon exposure to the sample, the apoenzyme
binds any metal ions present and is reactivated, and this rate of reactivation can be
related directly to the stoichiometric amount of metal complexed to the enzyme’s active
site. Alkaline phosphatase, for example, can be applied in this regard as a biosensor for
zinc [Zn(II)] or ascorbate oxidase for biosensing copper(II) with detection limits down to
very low part-per-billion levels (Satoh and Iijima, 1995).
However, as various other metals as well as other sample cross-contaminants can act
as cofactors and/or inhibitors of the metalloenzyme, selectivity becomes somewhat
problematic. To enhance selectivity, molecular tech niques such as site-directed
mutagenesis or directed evolution can be used to ge netically engineer or select for
enzymes with superior specificity for the target, as has been accomplished with
carbonic anhydrase and its selective biosensing of Zn(II) (Fierke and Thompson,
2001).Alternatively, and more commonly, target analytes such as heavy metals can also
inhibit enzyme activity, thereby diminishing the conversion of substrate to product. By
monitoring subsequent Michaelis–Menten rate kinetics, a highly sensitive measurement
of target can be obtained, often at picomolar detection limits, with little prerequisite
sample processing. However, selectivity cannot be ascertained since the specificity of
enzymes toward inhibitors is not target specific. Thus, inhibitor-based biosensors detect
the global presence of heavy metals rather than identifying a particular heavy metal ion
in a sample. The standard suite of enzymes used in these biosensors includes oxidases,
urease, alkaline phosphatase, choline esterases, and invertase for the detection of

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arsenic, bismuth, beryllium, zinc, mercury, cadmium , lead, and copper.
Cholinesterases (acetylcholinesterase, butyrlcholinesterase) are other well-applied
biosensor enzymes geared toward the detection of organophosphoros and carbamate
pesticides/insecticides and tyrosinase (polyphenol oxidase) and peroxidase have been
extensively applied for phenols. Other environmentally relevant inhibition enzymes
include acid phosphatase for the detection of arsenic(V) and protein phosphatase 2A for
algal microcystins (both nonselectively). By co-immobilizing several enzymes on the
same transducer, biosensors capable of multiplexed sensing can be achieved.
Additionally, as with the activated enzymes, the activity of inhibitory enzymes can be
enhanced through genetic engineering, as has been a ccomplished with
acetylcholinesterase, where site-directed mutagenesis yielded enzymes with 300-fold
more sensitivity to the organophosphate target dichlorvos (Boubik et al., 2002). An
extensive list of inhibition-based enzyme biosensors as they apply to environmental
monitoring has been provided by Amine et al. (2006).
Biosensors can also incorporate nonenzymatic protei ns or peptides as sensory
elements.
For environmental monitoring, this typically involves metal ion sensing and is
accomplished either through the use of naturally occurring or engineered proteins.
Metallothionein, for example, is a mammalian-derived metal-binding protein that has
been incorporated into an optical biosensor for non discriminatory detection of
cadmium, zinc, or nickel (Wu and Lin, 2004). Glutathione and phytochelatin proteins
are also commonly used. Specificity is broad and interference or inhibition by other
metals or sample constituents is problematic but can be partially addressed through
genetic engineering to add, subtract, or replace amino acids to acquire improved metal-
binding motifs, as has been done with phytochelatin (Bontidean et al., 2003).
Whole cell–based biosensors
Whole-cell biosensors consist of two components: a bioreporter strain that functions as
the detector of toxicity or a specific pollutant, coupled to a signal transducer that
converts the response from the bioreporter to a detectable electric signal. Bioreporters
are typically composed of a promoter (responsible for sensing or interacting with the
target analyte) fused to a reporter gene (responsible for generation of the detectable
biological signal) encoded on a plasmid or genetic construct, which is then transformed
into a cell (Fig.).

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The combination of a bioreporter with appropriate sensing technology in an integrated
biosensor has great potential for environmental con taminant detection, with a
multitude of possible pollutants monitored by various biosensors, depending on the
reporter gene, promoter element, and detection method chosen.
Many types of reporter genes are employed in chemical detection, the most common
being those that use luciferase (luc or lux ) or green fluorescence protein (gfp and its
derivatives) genes. The luc genes were first isolated from Photinus pyralis (firefly) and
encode for luciferase, which catalyzes the two-step conversion of d-luciferin (which
must be added to the reaction) to oxyluciferin, with light emission at 560 nm. Similarly,
lux genes have been isolated from several bacterial sources (e.g., Vibrio spp. and
Photorhabdus luminescens), all of which are encoded on a single operon in the order
luxCDABE. Within this operon, luxAB encodes for the luciferase enzyme, which converts
a long-chain aldehyde substrate (encoded by luxCDE) and a reduced flavin
mononucleotide (FMNH2 ) to FMN and a long-chain carboxylic acid, with light
production at 490 nm.

Biosensors that utilize the entire luxCDABE operon are self-contained, in that there is
no need for substrate addition, although production and recycling of the long-chain
aldehyde substrate requires energy expenditure by the cell. However, for biosensors
that use luxAB, without luxCDE, the aldehyde substrate must be continually added to
the reaction medium (as is also the case for luc-based luminescence), that detect a

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single contaminant. There are numerous examples of bioreporters that detect toxicity,
most of which contain a reporter gene fused to a strong constitutive promoter such that
a decrease in signal indicates a toxic response. Bhattacharyya et al. (2005) utilized two
such bacterial bioreporters (bioluminescent Pseudomonas putida and Escherichia coli ),
along with a naturally bioluminescent Vibrio fischeri (recently reclassified as Aliivibrio
fischeri ), to identify areas of a contaminated groundwater site in southern England that
contained toxic substances. They also used two “cat abolic” bioreporter strains for
monitoring trichloroethylene (TCE) and compared dat a from all of these strains to
chemical data, with the goal of gathering data to a ssess the bioavailability of
contaminants in the groundwater.
The three bacterial toxicity bioreporters differed in their assessment of the toxic
potential for the samples, attributed to each strain being more or less sensitive to the
primary pollutants in each sample, underscoring the benefit of using multiple
bioreporters within biosensors.
Antibody-based biosensors (Immunosensors)
Biosensors that use antibodies as recognition elements (immunosensors) are used
widely as environmental monitors because antibodies are highly specific, versatile, and
bind stably and strongly to target analytes (antigens). This high affinity for target,
however, can also be disadvantageous since the target cannot easily be released from
the antibody after the measurement has been made, resulting in many antibody-based
biosensors being single-use disposable units [although release (regeneration) can be
promoted by the addition of organic solvents or chaotrophic reagents, this requirement
for a supplementary assay step is not optimal]. Additionally, the synthesis of antibodies
and further testing and optimization of their target affinities can be a long, tedious, and
expensive process, cross-reactivity with multiple a nalytes can occur, and
antibody/antigen reactivity can be affected by environmental variables such as pH and
temperature (see Hock, 2000 for a review). Nonethel ess, antibodies can be highly
effective detectors for environmental contaminants, and advancements in techniques
such as phage display for the preparation and selection of recombinant antibodies with
novel binding properties assures their continued environmental application. Perhaps
the best introduction to antibody-based biosensing is the AWACSS (Automated Water
Analyzer Computer Supported System) environmental m onitoring system developed for
remote, unattended, and continuous detection of organic pollutants for water quality

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control [and also its predecessor, the RIANA (River Analyzer)] (Figure 9.6) (Tschmelak et
al., 2005).
AWACSS uses an optical evanescent wave transducer a nd fluorescently labeled
polyclonal antibodies for multiplexed detection of targeted groups of contaminants,
including endocrine disruptors, pesticides, industrial chemicals, pharmaceuticals, and
other priority pollutants, without requisite sample preprocessing. Antibody binding to a
target sample analyte occurs in a short 5-minute pr eincubation step, followed by
microfluidic pumping of the sample over the transducer element, which consists of an
optical waveguide chip impregnated with 32 separate wells of immobilized antigen
derivatives. As the antibody/analyte complexes flow through these wells, only
antibodies with free binding sites can attach to the well surface (in what is referred to
as a binding inhibition assay). Thus, antibodies with both of their binding sites bound
with analyte will not attach to the surface and will pass through the detector. A
semiconductor laser then excites the fluorophore label of bound antibodies, allowing for
their quantification, with high fluorescence signal s indicating low analyte
concentrations and low fluorescence signals indicating high analyte concentrations. A
fiber optic array tied to each well permits separation and identification of signals by the
well, thereby yielding a simultaneous measurement o f up to 32 different sample
contaminants.
The instrument has been used for groundwater, waste water, surface water, and
sediment sample testing with detection limits for most analytes in the ng/L range
within assay times of approximately 18 minutes.
DNA-Based biosensors
The foundation of nucleic acid–based biosensors relies on a transducer capable of
monitoring a change in the nucleic acid’s structure occurring after exposure to a target
chemical. These structural changes are brought on either by the mutagenic nature of
the chemical, resulting in mutations, intercalations, and/or strand breaks, or by the
chemical’s ability to covalently or noncovalently attach to the nucleic acid. Immobilizing
the nucleic acid as a recognition layer on the transducer surface forms the biosensor,
and detection of the chemically induced nucleic acid conformational change is then
typically achieved electrochemically (i.e., a change in the current) or less so through
optical or other means (see Fojta, 2002 for an excellent review).

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Nucleic acid biosensors are generally nonselective and provide an overall indication of a
potentially harmful (genotoxic, carcinogenic, cytotoxic) chemical or chemical mix in the
test environment and, depending on the biosensor format, an estimate of concentration.
A conventional example is illustrated in work by Bagni et al. (2005), where a DNA
biosensor was used to screen soil samples for genotoxic compounds, using benzene,
naphthalene, and anthracene derivates as model targets.
Double-stranded DNA was immobilized on a single-use disposable screen-printed
electrochemical cell operating off a handheld battery-powered potentiostat (Fig).

The AWACSS immunosensor (A) is capable of simultaneous multianalyte detection of up
to 32 different target contaminants. Its detection methodology uses a semiconductor
laser flow cell (B) to excite fluorophore-tagged antibody/target analyte complexes bound
to the surface of a multisensor optical waveguide chip (C). (From Tschmelak et al.,
2005, with permission.)
A 10-µL drop of a preprocessed and preextracted contaminated soil sample was placed
onto the working electrode for 2 minutes, and resulting electrochemical scans, based on
the chemical’s propensity to oxidize DNA guanine residues, were measured. (Adenine
moieties can be similarly redox reactive.) The magnitude of these “guanine peaks” in
relation to a reference electrode was linearly related to their concentration in solution
(i.e., the higher the concentration of the target chemical, the more damage is imposed
on the DNA, and the lower the electrochemical measurement of the oxidation signal).

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UNIT: 2 WATER MANAGEMENT AND WASTE WATER TREATMENT
Water as a scarce natural resource, water managemen t including rain water
harvesting. Waste water characterisitics, waste wat er treatment- physical,
chemical, biological processes. Aerobic processes: Activated sludge, oxidation
ditches, trickling filter, oxidation ponds. Anaerob ic processes; Anaerobic
digestion, anaerobic filters, anaerobic sludge, mem brane bioreactors. Reverse
osmosis and ultra filtration. Treatment of industrial effluents.

WATER AS A SCARCE NATURAL RESOURCE
Water is becoming more and more a scarce and valuab le resource as population and
consumption rise. Many human factors influence the availability of water, including
dams or other engineering, population, and consumer ism - or our water use on an
individual, business, and government levels. Evaluation of these factors, as well as
technology and action to support healthy water supplies, is necessary to gain control of
the situation.
WATER MANAGEMENT
Watershed management and conservation are often more economical and
environmentally sound ways to prevent flood damage and store water for futre use than
building huge dams and reservoirs.
Watershed Management
A watershed is all the land drained by a stream or river. Retaining vegetation and
ground cover in a watershed helps hold back rainwater and lessens downstream floods.
Domestic Conservation
We could probably save as much as half of the water we now use for domestic purposes
without great sacrifice or serious changes in our lifestyles.
Shorter showers, fixing leaks, and using conserving appliances all may forestall the
coming water shortages that many experts predict.
Our largest domestic use of water is toilet flushing. Many new, more efficient methods
of treating sewage waste are being researched.
Recycling and Water
In many developing countries, as much as 70% of all agricultural water is lost to leaks
in irrigation canals, evaporation, or application to areas where plants don't grow. Better
farming techniques could reduce these water losses dramatically.

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Nearly half of all industrial water is used for cooling power plants and industrial
facilities. Better cooling systems could significantly lessen the amount of water used for
this purpose.
The fastest growing water supply in California comes from human waste. Water
recovered from sewage treatment plants is an important part of the state's water
budget.
RAIN WATER HARVESTING
Rain Water Harvesting, is an age-old system of collection of rainwater for future use.
But systematic collection and recharging of ground water, is a recent development and
is gaining importance as one of the most feasible and easy to implement remedy to
restore the hydrological imbalance and prevent a crisis.
Technically speaking, water harvesting means A system that collects rainwater from
where it falls rather than allowing it to drain away. It includes water that is collected
within the boundaries of a property, from roofs and surrounding surfaces. Experts
suggest various ways of harvesting water:
Capturing run-off from rooftops
Capturing run-off from local catchments
Capturing seasonal flood water from local streams
Conserving water through watershed management
Local water harvesting systems developed by local communities and households can
reduce the pressure on the state to provide all the financial resources needed for water
supply. In addition, involving people will give them a sense of ownership and reduce the
burden on government funds.
Need for Water Harvesting
The scarcity of water is a well-known fact. In spite of higher average annual rainfall in
India (1,170 mm, 46 inches) as compared to the global average (800 mm, 32 inches) it
does not have sufficient water. Most of the rain falling on the surface tends to flow away
rapidly, leaving very little for the recharge of groundwater. As a result, most parts of
India experience lack of water even for domestic uses.
Surface water sources fail to meet the rising demands of water supply in urban areas;
groundwater reserves are being tapped and over-expl oited resulting into decline in
groundwater levels and deterioration of groundwater quality. This precarious situation
needs to be rectified by immediately recharging the depleted aquifers. Hence, the need

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for implementation of measures to ensure that rain falling over a region is tapped as
fully as possible through water harvesting, either by recharging it into the groundwater
aquifers or storing it for direct use.
Science of Water Harvesting
In scientific terms, water harvesting refers to collection and storage of rainwater and
also other activities aimed at harvesting surface and groundwater, prevention of losses
through evaporation and seepage and all other hydro logical studies and engineering
inventions, aimed at conservation and efficient uti lization of the limited water
endowment of physiographic unit such as a watershed.
Rain is a primary source of water for all of us. There are two main techniques of
rainwater harvesting:
Storage of rainwater on surface for future use.
Recharge to groundwater.
Directly collected rainwater can be stored for direct use or can be recharged into the
groundwater.
All the secondary sources of water like rivers, lakes and groundwater are entirely
dependent on rain as a primary source.
The term water harvesting is understood to encompas s a wide range of concerns,
including rainwater collection with both rooftop an d surface runoff catchment,
rainwater storage in small tanks and large-scale ar tificial reservoirs, groundwater
recharge, and also protection of water sources against pollution.
The objective of water harvesting in India differs between urban and rural areas. In
urban areas, emphasis is put on increasing groundwater recharge and managing storm
water. On the other hand, in rural areas securing water is more crucial. There the aim
is to provide water for drinking and farming, especially for life-saving irrigation, and to
increase groundwater recharge.
Rooftop / Runoff Rainwater Harvesting for Artificial Recharge to Ground Water
Water harvesting is the deliberate collection and storage of rainwater that runs off on
natural or manmade catchment areas. Catchment inclu des rooftops, compounds, rocky
surface or hill slopes or artificially prepared impervious/ semi-pervious land surface.
The amount of water harvested depends on the freque ncy and intensity of rainfall,
catchment characteristics, water demands and how mu ch runoff occurs and how
quickly or how easy it is for the water to infiltrate through the subsoil and percolate

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down to recharge the aquifers. Moreover, in urban areas, adequate space for surface
storage is not available, water levels are deep enough to accommodate additional
rainwater to recharge the aquifers, rooftop and runoff rainwater harvesting is ideal
solution to solve the water supply problems.
Design Considerations
Three most important components, which need to be e valuated for designing the
rainwater harvesting structure, are:
Hydrogeology of the area including nature and extent of aquifer, soil cover, topography,
depth to water levels and chemical quality of ground water
Area contributing for runoff i.e. how much area and land use pattern, whether
industrial, residential or green belts and general built up pattern of the area
Hydro-meteorological characters like rainfall duration, general pattern and intensity of
rainfall.
Components Of Rain Harvesting System
A rainwater harvesting system comprises components of various stages - transporting
rainwater through pipes or drains, filtration, and storage in tanks for reuse or recharge.
The common components of a rainwater harvesting system involved in these stages are
illustrated here.
Catchments

The catchment of a water harvesting system is the surface which directly receives the
rainfall and provides water to the system. It can be a paved area like a terrace or
courtyard of a building, or an unpaved area like a lawn or open ground. A roof made of
reinforced cement concrete (RCC), galvanised iron or corrugated sheets can also be
used for water harvesting.
COARSE MESH
At the roof to prevent the passage of debris.

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GUTTERS
Channels all around the edge of a sloping roof to collect and transport rainwater to the
storage tank. Gutters can be semi-circular or rectangular and could be made using:
Locally available material such as plain galvanised iron sheet (20 to 22 gauge), folded to
required shapes.
Semi-circular gutters of PVC material can be readily prepared by cutting those pipes
into two equal semi-circular channels.
Bamboo or betel trunks cut vertically in half.
The size of the gutter should be according to the flow during the highest intensity rain.
It is advisable to make them 10 to 15 per cent oversize.
Gutters need to be supported so they do not sag or fall off when loaded with water. The
way in which gutters are fixed depends on the construction of the house; it is possible
to fix iron or timber brackets into the walls, but for houses having wider eaves, some
method of attachment to the rafters is necessary.
Conduits
Conduits are pipelines or drains that carry rainwater from the catchment or rooftop
area to the harvesting system. Conduits can be of any material like polyvinyl chloride
(PVC) or galvanized iron (GI), materials that are commonly available.
First-Flushing
A first flush device is a valve that ensures that runoff from the first spell of rain is
flushed out and does not enter the system. This needs to be done since the first spell of
rain carries a relatively larger amount of pollutants from the air and catchment surface.


FILTER
The filter is used to remove suspended pollutants from rainwater collected over roof. A
filter unit is a chamber filled with filtering media such as fibre, coarse sand and gravel

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layers to remove debris and dirt from water before it enters the storage tank or recharge
structure. Charcoal can be added for additional filtration.

Advantages of rainwater harvesting
To meet the ever increasing demand for water. Water harvesting to recharge the
groundwater enhances the availability of groundwater at specific place and time and
thus assures a continuous and reliable access to groundwater.
To reduce the runoff which chokes storm drains and to avoid flooding of roads.
To reduce groundwater pollution and to improve the quality of groundwater through
dilution when recharged to groundwater thereby providing high quality water, soft and
low in minerals.
Provides self-sufficiency to your water supply and to supplement domestic water
requirement during summer and drought conditions.
It reduces the rate of power consumption for pumping of groundwater. For every 1m
rise in water level, there is a saving of 0.4 KWH of electricity.
Reduces soil erosion in urban areas
The rooftop rainwater harvesting is less expensive, easy to construct, operate and
maintain.
In saline or coastal areas, rainwater provides good quality water and when recharged to
ground water, it reduces salinity and helps in maintaining balance between the fresh-
saline water interfaces.
In Islands, due to limited extent of fresh water aquifers, rainwater harvesting is the
most preferred source of water for domestic use.
In desert, where rainfall is low, rainwater harvesting has been providing relief to people.
WASTE WATER CHARACTERISITICS
Wastewater is mostly water by weight. Other materials make up only a small portion of
wastewater, but can be present in large enough quantities to endanger public health

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and the environment. Because practically anything that can be flushed down a toilet,
drain, or sewer can be found in wastewater, even household sewage contains many
potential pollutants.
The wastewater components that should be of most co ncern to homeowners and
communities are those that have the potential to ca use disease or detrimental
environmental effects.
Organisms
Many different types of organisms live in wastewate r and some are essential
contributors to treatment. A variety of bacteria, protozoa, and worms work to break
down certain carbon-based (organic) pollutants in wastewater by consuming them.
Through this process, organisms turn wastes into carbon dioxide, water, or new cell
growth. Bacteria and other microorganisms are particularly plentiful in wastewater and
accomplish most of the treatment. Most wastewater treatment systems are designed to
rely in large part on biological processes.
Pathogens
Many disease-causing viruses, parasites, and bacteria also are present in wastewater
and enter from almost anywhere in the community. Th ese pathogens often originate
from people and animals who are infected with or are carriers of a disease. For example,
graywater and blackwater from typical homes contain enough pathogens to pose a risk
to public health. Other likely sources in communities include hospitals, schools, farms,
and food processing plants.
Some illnesses from wastewater-related sources are relatively common. Gastroenteritis
can result from a variety of pathogens in wastewater, and cases of illnesses caused by
the parasitic protozoa Giardia lambia and Cryptosporidium are not unusual in the U.S.
Other important wastewater-related diseases include hepatitis A, typhoid, polio,
cholera, and dysentery. Outbreaks of these diseases can occur as a result of drinking
water from wells polluted by wastewater, eating contaminated fish, or recreational
activities in polluted waters. Some illnesses can be spread by animals and insects that
come in contact with wastewater.
Even municipal drinking water sources are not completely immune to health risks from
wastewater pathogens. Drinking water treatment efforts can become overwhelmed when
water resources are heavily polluted by wastewater. For this reason, wastewater
treatment is as important to public health as drinking water treatment.

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Organic Matter
Organic materials are found everywhere in the environment. They are composed of the
carbon-based chemicals that are the building blocks of most living things.
Organic materials in wastewater originate from plants, animals, or synthetic organic
compounds, and enter wastewater in human wastes, pa per products, detergents,
cosmetics, foods, and from agricultural, commercial, and industrial sources.
Organic compounds normally are some combination of carbon, hydrogen, oxygen,
nitrogen, and other elements. Many organics are proteins, carbohydrates, or fats and
are biodegradable, which means they can be consumed and broken down by organisms.
However, even biodegradable materials can cause pollution. In fact, too much organic
matter in wastewater can be devastating to receiving waters.
Large amounts of biodegradable materials are dangerous to lakes, streams, and oceans,
because organisms use dissolved oxygen in the water to break down the wastes. This
can reduce or deplete the supply of oxygen in the water needed by aquatic life, resulting
in fish kills, odors, and overall degradation of water quality. The amount of oxygen
organisms need to break down wastes in wastewater is referred to as the biochemical
oxygen demand (BOD) and is one of the measurements used to assess overall
wastewater strength.
Some organic compounds are more stable than others and cannot be quickly broken
down by organisms, posing an additional challenge for treatment. This is true of many
synthetic organic compounds developed for agriculture and industry.
In addition, certain synthetic organics are highly toxic. Pesticides and herbicides are
toxic to humans, fish, and aquatic plants and often are disposed of improperly in drains
or carried in stormwater. In receiving waters, they kill or contaminate fish, making
them unfit to eat. They also can damage processes in treatment plants. Benzene and
toluene are two toxic organic compounds found in some solvents, pesticides, and other
products. New synthetic organic compounds are being developed all the time, which can
complicate treatment efforts.
Oil and Grease
Fatty organic materials from animals, vegetables, and petroleum also are not quickly
broken down by bacteria and can cause pollution in receiving environments.
When large amounts of oils and greases are discharg ed to receiving waters from
community systems, they increase BOD and they may f loat to the surface and harden,

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causing aesthetically unpleasing conditions. They also can trap trash, plants, and other
materials, causing foul odors, attracting flies and mosquitoes and other disease vectors.
In some cases, too much oil and grease causes septic conditions in ponds and lakes by
preventing oxygen from the atmosphere from reaching the water.
Onsite systems also can be harmed by too much oil and grease, which can clog onsite
system drainfield pipes and soils, adding to the risk of system failure. Excessive grease
also adds to the septic tank scum layer, causing more frequent tank pumping to be
required. Both possibilities can result in significant costs to homeowners. Petroleum-
based waste oils used for motors and industry are considered hazardous waste and
should be collected and disposed of separately from wastewater.
Inorganics
Inorganic minerals, metals, and compounds, such as sodium, potassium, calcium,
magnesium, cadmium, copper, lead, nickel, and zinc are common in wastewater from
both residential and nonresidential sources. They can originate from a variety of
sources in the community including industrial and commercial sources, stormwater,
and inflow and infiltration from cracked pipes and leaky manhole covers. Most
inorganic substances are relatively stable, and cannot be broken down easily by
organisms in wastewater. Large amounts of many inor ganic substances can
contaminate soil and water. Some are toxic to anima ls and humans and may
accumulate in the environment. For this reason, ext ra treatment steps are often
required to remove inorganic materials from industrial wastewater sources.
Heavy metals, for example, which are discharged wit h many types of industrial
wastewaters, are difficult to remove by conventional treatment methods.
Nutrients
Wastewater often contains large amounts of the nutrients nitrogen and phosphorus in
the form of nitrate and phosphate, which promote plant growth. Organisms only require
small amounts of nutrients in biological treatment, so there normally is an excess
available in treated wastewater. In severe cases, excessive nutrients in receiving waters
cause algae and other plants to grow quickly depleting oxygen in the water. Deprived of
oxygen, fish and other aquatic life die, emitting foul odors. Nutrients from wastewater
have also been linked to ocean “red tides” that poison fish and cause illness in humans.
Nitrogen in drinking water may contribute to miscarriages and is the cause of a serious
illness in infants called methemoglobinemia or “blue baby syndrome.”

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Solids
Solid materials in wastewater can consist of organic and/or inorganic materials and
organisms. The solids must be significantly reduced by treatment or they can increase
BOD when discharged to receiving waters and provide places for microorganisms to
escape disinfection. They also can clog soil absorption fields in onsite systems.
Settleable solids
Certain substances, such as sand, grit, and heavier organic and inorganic materials
settle out from the rest of the wastewater stream during the preliminary stages of
treatment. On the bottom of settling tanks and ponds, organic material makes up a
biologically active layer of sludge that aids in treatment.
Suspended solids in wastewater must be treated, or they will clog soil absorption
systems or reduce the effectiveness of disinfection systems.
Dissolved solids
Small particles of certain wastewater materials can dissolve like salt in water. Some
dissolved materials are consumed by microorganisms in wastewater, but others, such
as heavy metals, are difficult to remove by conventional treatment. Excessive amounts
of dissolved solids in wastewater can have adverse effects on the environment.
Gases
Certain gases in wastewater can cause odors, affect treatment, or are potentially
dangerous. Methane gas, for example, is a byproduct of anaerobic biological treatment
and is highly combustible. Special precautions need to be taken near septic tanks,
manholes, treatment plants, and other areas where wastewater gases can collect. The
gases hydrogen sulfide and ammonia can be toxic and pose asphyxiation hazards.
Ammonia as a dissolved gas in wastewater also is dangerous to fish. Both gases emit
odors, which can be a serious nuisance. Unless effectively contained or minimized by
design and location, wastewater odors can affect the mental well-being and quality of
life of residents. In some cases, odors can even lower property values and affect the
local economy.
In addition to the many substances found in wastewater, there are other characteristics
system designers and operators use to evaluate wastewater. For example, the color,
odor, and turbidity of wastewater give clues about the amount and type of pollutants
present and treatment necessary.

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The following are some other important wastewater c haracteristics that can affect
public health and the environment, as well as the design, cost, and effectiveness of
treatment.
Temperature
The best temperatures for wastewater treatment probably range from 77 to 95 degrees
Fahrenheit. In general, biological treatment activity accelerates in warm temperatures
and slows in cool temperatures, but extreme hot or cold can stop treatment processes
altogether. Therefore, some systems are less effective during cold weather and some
may not be appropriate for very cold climates.
Wastewater temperature also affects receiving waters. Hot water, for example, which is
a byproduct of many manufacturing processes, can be a pollutant. When discharged in
large quantities, it can raise the temperature of receiving streams locally and disrupt
the natural balance of aquatic life.
pH
The acidity or alkalinity of wastewater affects both treatment and the environment.
Low pH indicates increasing acidity, while a high pH indicates increasing alkalinity (a
pH of 7 is neutral). The pH of wastewater needs to remain between 6 and 9 to protect
organisms. Acids and other substances that alter pH can inactivate treatment processes
when they enter wastewater from industrial or commercial sources.
Flow
Whether a system serves a single home or an entire community, it must be able to
handle fluctuations in the quantity and quality of wastewater it receives to ensure
proper treatment is provided at all times. Systems that are inadequately designed or
hydraulically overloaded may fail to provide treatm ent and allow the release of
pollutants to the environment. To design systems that are both as safe and as cost-
effective as possible, engineers must estimate the average and maximum (peak) amount
of flows generated by various sources.
Because extreme fluctuations in flow can occur during different times of the day and on
different days of the week, estimates are based on observations of the minimum and
maximum amounts of water used on an hourly, daily, weekly, and seasonal basis. The
possibility of instantaneous peak flow events that result from several or all waterusing
appliances or fixtures being used at once also is taken into account. The number, type,
and efficiency of all water-using fixtures and appliances at the source is factored into

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the estimate (for example, the number and amount of water normally used by faucets,
toilets, and washing machines), as is the number of possible users or units that can
affect the amount of water used (for example, the n umber of residents, bedrooms,
customers, students, patients, seats, or meals served).
WASTE WATER TREATMENT- PHYSICAL, CHEMICAL, BIOLOGIC AL PROCESSES
Physical forces as well as chemical and biological processes drive the treatment of
wastewater. Treatment methods that rely on physical forces are called unit operations.
These include screening, sedimentation, filtration, or flotation. Treatment methods
based on chemical and biological processes are called unit processes. Chemical unit
processes include disinfection, adsorption, or precipitation.
Biological unit processes involve microbial activity, which is responsible for organic
matter degradation and removal of nutrients (Metcalf and Eddy, 1991).
Wastewater treatment comprises the following four steps (Fig.):

Preliminary treatment
The objective of this operation is to remove debris and coarse materials that may clog
equipment in the plant.
The typical sewage treatment sequence normally begi ns with preliminary screening,
with mechanical grids to exclude large material which has been carried along with the
flow. Paper, rags and the like are shredded by a series of rotating blades known as

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comminutors and any grit is removed to protect the pumps and ensure free movement
of the water through the plant.
Primary treatment
Treatment is brought about by physical processes (unit operations) such as screening
and sedimentation.
Primary treatment involves the removal of fine solids by means of settlement and
sedimentation, the aim being to remove as much of the suspended organic solid content
as possible from the water itself and up to a 50% reduction in solid loading is commonly
achieved. At various times, and in many parts of th e world, discharge of primary
effluent direct to the sea has been permissible, but increasing environmental legislation
means that this has now become an increasingly rare option. Throughout the whole
procedure of sewage treatment, the effective reduction of nitrogen and phosphorus
levels is a major concern, since these nutrients may, in high concentration, lead to
eutrophication of the waterways. Primary stages have a removal efficiency of between 5–
15% in respect of these nutrients, but greater reductions are typically required to meet
environmental standards for discharge, thus necessitating the supernatant effluent
produced passing to a secondary treatment phase.
Secondary treatment.
Biological (e.g., activated sludge, trickling filter, oxidation ponds) and chemical (e.g.,
disinfection) unit processes are used to treat wastewater.Nutrient removal also generally
occurs during secondary treatment of wastewater.
This contains the main biological aspect of the regime and involves the two essentially
linked steps of initial bioprocessing and the subsequent removal of solids resulting from
this enhanced biotic activity. Oxidation is the fundamental basis of biological sewage
treatment and it is most commonly achieved in one o f three systems, namely the
percolating filter, activated sludge reactor or, in the warmer regions of the globe,
stabilization ponds. The operational details of the processing differ between these three
methods and will be described in more detail later in this section, though the
fundamental underlying principle is effectively the same. Aerobic bacteria are
encouraged, thriving in the optimised conditions provided, leading to the BOD, nitrogen
and ammonia levels within the effluent being signif icantly reduced. Secondary
settlement in large tanks allows the fine floc particles, principally composed of excess
microbial biomass, to be removed from the increasingly cleaned water. The effluent

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offtake from the biological oxidation phase flows s lowly upwards through the
sedimentation vessels at a rate of no more than 1–2 metres per hour, allowing residual
suspended solids to settle out as sludge. The secondary treatment stage routinely
achieves nutrient reductions of between 30–50%.
Tertiary or advanced treatment
Unit operations and chemical unit processes are use d to further remove BOD,
nutrients, pathogens, and parasites, and sometimes toxic substances.
In some cases, tertiary treatment is required as an advanced final polishing stage to
remove trace organics or to disinfect effluent. Thi s is dictated by watercourse
requirements, chiefly when the receiving waters are either unable to dilute the
secondary effluent sufficiently to achieve the target quality, or are themselves
particularly sensitive to some component aspect of the unmodified influx. Tertiary
treatment can add significantly to the cost of sewage management, not least because it
may involve the use of further sedimentation lagoon s or additional processes like
filtration, microfiltration, reverse osmosis and the chemical precipitation of specific
substances.
AEROBIC PROCESSES:
Aeration
Introducing air into liquid wastes is a well-established technique to reduce pollutant
potential and is often employed as an on-site method to achieve discharge consent
levels, or reduce treatment costs, in a variety of industrial settings. It works by
stimulating resident biomass with an adequate suppl y of oxygen, while keeping
suspended solids in suspension and helping to mix the effluent to optimize treatment
conditions, which also assists in removing the carbon dioxide produced by microbial
activity. In addition, aeration can have a flocculant effect, the extent of which depends
on the nature of the effluent. The systems used fall into one of two broad categories, on
the basis of their operating criteria:
• Diffused air systems.
• Mechanical aeration.
This classification is a useful way to consider the methods in common use, though it
takes account of neither the rate of oxygen transfer, nor the total dissolved oxygen
content, which is occasionally used as an alternative way to define aeration approaches.

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Diffused air systems
The liquid is contained within a vessel of suitable volume, with air being introduced at
the bottom, oxygen diffusing out from the bubbles a s they rise, thus aerating the
effluent. These systems can be categorised on the basis of their bubble size, with the
crudest being coarse open-ended pipes and the most sophisticated being specialized
fine diffusers.
Ultra-fine bubble (UFB) systems maximise the oxygen transfer effect, producing a dense
curtain of very small bubbles, which consequently have a large surface area to volume
ratio to maximise the diffusion. The UFB system is the most expensive, both to install in
the first place and subsequently to run, as it requires comparatively high maintenance
and needs a filtered air supply to avoid air-borne particulates blocking the narrow
diffuser pores. Illustrative UFB aeration results, based on operational data, obtained
from the amelioration of post-anaerobic digestion liquor from a horticultural waste
processing plant, are shown in Table.

Though the comparatively simple approaches which pr oduce large to medium sized
bubbles are the least efficient, they are commonly encountered in use since they offer a
relatively inexpensive solution.
Mechanical aeration systems
In this method, a partly submerged mechanically driven paddle mounted on floats or
attached to a gantry vigorously agitates the liquid, drawing air in from the surface and
the effluent is aerated as the bubbles swirl in the vortex created. Other variants on this
theme are brush aerators, which are commonly used t o provide both aeration and

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mixing in the sewage industry and submerged turbine spargers, which introduce air
beneath an impeller, which again mixes as it aerates. This latter approach, shown in
Fig., can be considered as a hybrid between mechani cal and diffused systems and
though, obviously, represents a higher capital cost, it provides great operational
efficiency.

A major factor in this is that the impeller establishes internal currents within the tank.
As a result the bubbles injected at the bottom, instead of travelling straight up, follow a
typically spiral path, which increases their mean transit time through the body of the
liquid and hence, since their residence period is lengthened, the overall efficacy of
oxygen diffusion increases.
The design of the system and the processing vessel is crucial to avoid problems of
oxygen transfer, liquid stratification and foaming, all of which can be major problems in
operation. The time taken to effect treatment depends on the regime used and the
nature of the effluent.

In this context, Table shows typical oxygen transfer rates for aeration systems at 20 ◦C.
The value of aeration in the treatment process is n ot restricted to promoting the
biological degradation of organic matter, since the addition of oxygen also plays an

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important role in removing a number of substances b y promoting direct chemical
oxidation. This latter route can often help eliminate organic compounds which are
resistant to straightforward biological treatments.
ACTIVATED SLUDGE
Activated sludge is a suspended-growth process that began in England at the turn of
the century. This process has since been adopted worldwide as a secondary biological
treatment for domestic wastewaters. This process consists essentially of an aerobic
treatment that oxidizes organic matter to CO
2 and H2O, NH4, and new cell biomass. Air
is provided by using diffused or mechanical aeration. The microbial cells form flocs that
are allowed to settle in a clarification tank.
Conventional Activated Sludge System
A conventional activated sludge process includes (Fig.) the following:

Aeration tank
Aerobic oxidation of organic matter is carried out in this tank. Primary effluent is
introduced and mixed with return activated sludge (RAS) to form the mixed liquor,
which contains 1500–2500 mg/L of suspended solids. Aeration is provided by
mechanical means. An important characteristic of the activated sludge process is the
recycling of a large portion of the biomass. This makes the mean cell residence time
(i.e., sludge age) much greater than the hydraulic retention time (Sterritt and Lester,
1988). This practice helps maintain a large number of microorganisms that effectively
oxidize organic compounds in a relatively short time. The detention time in the aeration
basin varies between 4 and 8 hours.
Sedimentation tank
This tank is used for the sedimentation of microbial flocs (sludge) produced during the
oxidation phase in the aeration tank. A portion of the sludge in the clarifier is recycled

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back to the aeration basin and the remainder is wasted to maintain a proper F/M (food
to microorganisms ratio).
Mixed Liquor Suspended Solids (MLSS)
The content of the aeration tank in an activated sludge system is called mixed liquor.
The MLSS is the total amount of organic and mineral suspended solids, including
microorganisms, in the mixed liquor. It is determined by filtering an aliquot of mixed
liquor, drying the filter at 105°C, and determining the weight of solids in the sample.
Mixed Liquor Volatile Suspended Solids (MLVSS)
The organic portion of MLSS is represented by MLVSS, which comprises nonmicrobial
organic matter, as well as dead and live microorganisms and cellular debris (Nelson and
Lawrence, 1980). The MLVSS is determined after heat ing of dried filtered samples at
600–650°C, and represents approximately 65–75 percent of MLSS.
Food-to-Microorganism Ratio (F/M)
The food-to-microorganisms (F/M) ratio indicates the organic load into the activated
sludge system and is expressed in kilogram BOD per kilogram of MLSS per day (Curds
and Hawkes, 1983; Nathanson, 1986). It is expressed as:

where Q = flow rate of sewage in million gallons per day (MGD);
BOD = five-day biochemical oxygen demand (mg/L);
MLSS = mixed liquor suspended solids (mg/L);
And V = volume of aeration tank (gallons).
The food-to-microorganism ratio is controlled by the rate of activated sludge wasting.
The higher the wasting rate the higher the F/M ratio. For conventional aeration tanks
the F/M ratio is 0.2–0.5 lb BOD
5/day/lb MLSS but it can be higher (≤1.5) for activated
sludge using high purity oxygen (Hammer, 1986). A l ow F/M ratio means that the
microorganisms in the aeration tank are starved, generally leading to a more efficient
wastewater treatment.
Hydraulic Retention Time (HRT)
Hydraulic retention time is the average time spent by the influent liquid in the aeration
tank of the activated sludge process; it is the reciprocal of the dilution rate D (Sterritt
and Lester, 1988).

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where V = volume of the aeration tank;
Q = flow rate of the influent wastewater into the aeration tank;
and D = dilution rate.
Sludge Age
Sludge age is the mean residence time of microorgan isms in the system. While the
hydraulic retention time may be in the order of hours, the mean cell residence time may
be in the order of days. This parameter is the reciprocal of the microbial growth rate m.
Sludge age is given by the following formula (Hammer, 1986; Curds and Hawkes, 1983):

Where
MLSS = mixed liquor suspended solids (mg/L);
V = volume of aeration tank (L);
SSe = suspended solids in wastewater effluent (mg/L);
Qe = quantity of wastewater effluent (m
3
/day);
SSw = suspended solids in wasted sludge (mg/L);
and Qw = quantity of wasted sludge (m
3
/day).
Sludge age may vary from 5 to 15 days in conventional activated sludge. It varies with
the season of the year and is higher in the winter than in the summer season (U.S. EPA,
1987a).
The important parameters controlling the operation of an activated sludge are organic
loading rates, oxygen supply, and control and operation of the final settling tank. This
tank has two functions: clarification and thickening. For routine operation, one must
measure sludge settleability by determining the sludge volume index (SVI) (Forster and
Johnston, 1987)
Some Modifications of the Conventional Activated Sludge Process
There are several modifications of the conventional activated sludge process
(Nathanson, 1986; U.S. EPA, 1977). These are given in the following subsections.
Extended Aeration System

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This process, used in package treatment plants, has the following features:
The aeration time is much longer (about 30 h) than in conventional systems. The sludge
age is also longer and can be extended to .15 days.
The wastewater influent entering the aeration tank has not been treated by primary
settling.
The system operates at much lower F/M ratio (genera lly, 0.1 lb BOD/day/lb MLSS)
than conventional systems (0.2–0.5 lb BOD/day/lb MLSS).
This system requires less aeration than conventional treatment and is mainly suitable
for small communities that use package treatment
OXIDATION DITCHES
The oxidation ditch consists of an aeration oval channel with one or more rotating
rotors for wastewater aeration. This channel receives screened wastewater and has a
hydraulic retention time of approximately 24 h.

Step Aeration
The primary effluent enters the aeration tank through several points, thus improving its
distribution into the tank and making more efficient use of oxygen. This increases the
treatment capacity of the system.
Contact Stabilization

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After contact of the wastewater with the sludge in a small contact tank for a short
period of time (20–40 min), the mixture flows to a clarifier and the sludge is returned to
a stabilization tank with a retention time of 4–8 h. This system produces less sludge.
Completely Mixed Aerated System
A completely mixed aerated system allows a more uniform aeration of the wastewater in
the aeration tank. This system can sustain shock and toxic loads.
High-Rate Activated Sludge
This system is used for the treatment of highstrength wastes and is operated at much
higher BOD loadings than those encountered in the c onventional activated sludge
process. This results in shorter hydraulic retention periods (i.e., shorter aeration
periods). The system is operated at higher MLSS concentrations.
Pure Oxygen Aeration
The pure oxygen aeration system is based on the principle that the rate of transfer of
pure oxygen is higher than that of atmospheric oxyg en. This results in higher
availability of dissolved oxygen, leading to improved treatment and reduced production
of sludge.
Table summarizes the design and operational characteristics of some activated sludge
processes.

Biology of activated sludge

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There are two main goals of the activated sludge system:
Oxidation of the biodegradable organic matter in the aeration tank (soluble organic
matter is thus converted to new cell mass).
Flocculation, that is, the separation of the newly formed biomass from the treated
effluent.
Survey of Organisms Present in Activated Sludge Flo cs
The activated sludge flocs contain mostly bacterial cells as well as other
microorganisms, and inorganic and organic particles. Floc size varies between < 1 mm
(the size of some bacterial cells) to ≥1000 mm (Parker et al., 1971; U.S. EPA, 1987a).

Fig. illustrates the main microorganisms in the activated sludge microbial community
(Wagner and Amann, 1996). Early studies showed that viable cells in the floc, as
measured by ATP analysis and dehydrogenase activity, would account for 5–20 percent
of the total cells (Weddle and Jenkins, 1971). Some investigators estimated that the
active fraction of bacteria in activated sludge flocs represents only 1–3 percent of total
bacteria (Hanel, 1988).
However, fluorescently labeled oligonucleotide probes show that a higher percentage of
the microbial biomass is metabolically active (Head et al., 1998; Wagner et al., 1993).
Flow cytometry, in conjunction with fluorogenic viability/activity dyes, were also used to
detect the viability and activity of microorganisms in activated sludge flocs. It was found
that 62 percent of the total bacteria were active in the flocs (Ziglio et al., 2002). These
techniques allow the distinction between viable cells, dead cells, and damaged cells. The
development of microsensor technology has enabled t he exploration of the floc
microenvironment.
Microelectrodes are now being used to determine oxygen, pH, redox potential, nitrate,
and ammonia or sulfide microprofiles within activat ed sludge flocs. These

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microelectrodes give information on microbial activity within the floc (Li and Bishop,
2004).
Activated sludge flocs contain a wide range of prok aryotic and eukaryotic
microorganisms, and many of them can be routinely o bserved with regular phase-
contrast microscopy. A color atlas of wastewater organisms is available and should be
consulted to become familiar with the most encountered organisms in activated sludge
or trickling filters (Berk and Gunderson, 1993).
TRICKLING FILTER
The trickling or biological filter system involves a bed, which is formed by a layer of filter
medium held within a containing tank or vessel, often cast from concrete, and equipped
with a rotating dosing device, as shown in a stylised form in Fig.

The filter is designed to permit good drainage and ventilation and in addition
sedimentation and settling tanks are generally associated with the system. Effluent,
which has been mechanically cleaned to remove the l arge particles which might
otherwise clog the interparticulate spaces in the filter bed, flows, or is pumped, into the
rotating spreader, from which it is uniformly distributed across the filter bed. This
dosing process can take place either continuously or intermittently, depending on the
operational requirements of the treatment works. Th e wastewater percolates down
through the filter, picking up oxygen as it travels over the surface of the filter medium.
The aeration can take place naturally by diffusion, or may sometimes be enhanced by
the use of active ventilation fans.
The combination of the available nutrients in the effluent and its enhanced oxygenation
stimulates microbial growth, and a gelatinous biofilm of microorganisms forms on the
filter medium. This biological mass feeds on the organic material in the wastewater
converting it to carbon dioxide, water and microbial biomass. Though the resident

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organisms are in a state of constant growth, ageing and occasional oxygen starvation of
those nearest the substrate leads to death of some of the attached growth, which
loosens and eventually sloughs, passing out of the filter bed as a biological sludge in the
water flow and thence on to the next phase of treatment.
The filter medium itself is of great importance to the success of these systems and in
general the requirements of a good material are that it should be durable and long
lasting, resistant to compaction or crushing in use and resistant to frost damage. A
number of substances have been used for this purpose including clinker, blast-furnace
slag, gravel and crushed rock. A wholly artificial plastic lattice material has also been
developed which has proved successful in some applications, but a clinker and slag mix
is generally said to give some of the best results. The ideal filter bed must provide
adequate depth to guarantee effluent retention time, since this is critical in allowing it
to become sufficiently aerated and to ensure adequate contact between the microbes
and the wastewater for the desired level of pollutant removal. It should also have a large
surface area for biomass attachment, with generous void spaces between the particles
to allow the required biomass growth to take place without any risk of this causing
clogging. Finally, it should have the type of surface which encourages splashing on
dosing, to entrap air and facilitate oxygenation of the bed.
The trickling filters in use at sewage works are squat, typically around 8–10metres
across and between 1–2 metres deep; though these ar e the most familiar form, other
filters of comparatively small footprint but 5 to 20metres in height are used to treat
certain kinds of trade effluents, particularly those of a stronger nature and with a more
heavy organic load than domestic wastewater.
They are of particular relevance in an industrial setting since they can achieve a very
high throughput and residence time, while occupying a relatively small base area of
land. To maximise the treatment efficiency, it is clearly essential that the trickling filter
is properly sized and matched to the required processing demands. The most important
factors in arriving at this are the quality of the effluent itself, its input temperature, the
composition of the filter medium, detail of the surface-dosing arrangements and the
aeration. The wastewater quality has an obvious significance in this respect, since it is
this, combined with the eventual clean-up level required, which effectively defines the
performance parameters of the system. Although in an ideal world, the filter would be
designed around input character, in cases where industrial effluents are co-treated with

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domestic wastewater in sewage works, it is the feed rate which is adjusted to provide a
dilute liquor of given average strength, since the filters themselves are already in
existence.
Hence, in practice, the load is often adjusted to the facility, rather than the other way
about. The input temperature has a profound influence on the thermal relations within
the filter bed, not least because of the high specific heat capacity of water at 4200
J/kg/ ◦C. This can be of particular relevance in industrial reed bed systems, which are
discussed in the following chapter, since warm liqu or can help to overcome the
problems of cold weather in temperate climes. By contrast the external air temperature
appears to have less importance in this respect. The situation within the reaction space
is somewhat complicated by virtue of the nonlinear nature of the effect of temperature
on contaminant removal. Although the speed of chemi cal reactions is well known to
double for every 10 ◦C rise, at 20 ◦C, in-filter biodegradation only represents an increase
of 38% over the rate at 10 ◦C. Below 10 ◦C, the risk of clogging rises significantly, since
the activity of certain key members of the microbial community becomes increasingly
inhibited.
The general properties of the filter media were discussed earlier. In respect of sizing the
system, the porosity and intergranular spaces govern the interrelation between relative
ease of oxygen ingress, wastewater percolation and nutrient to biofilm contact. Clearly,
the rougher, pitted or irregular materials tend to offer the greatest surface area per unit
volume for microbial attachment and hence, all other things being equal, it follows that
the use of such media allows the overall filter dimensions to be smaller. In practice,
however, this is seldom a major deciding factor.
In the main, filter systems use rotational dosing systems to ensure a uniform dispersal
of the effluent, though nozzles, sprays and mechanised carts are not unknown. The feed
must be matched to the medium if the surface aeration effect is to be optimised, but it
must also take account of the fluidity, concentration and quality of the wastewater itself
and the character of the resident biofilm.
Since the biological breakdown of effluents within the filter is brought about by aerobic
organisms, the effectiveness of aeration is of considerable importance. Often adequate
oxygenation is brought about naturally by a combination of the surface effects as the
wastewater is delivered to the filter, diffusion from atmosphere through the filter
medium and an in-filter photosynthetic contribution from algae. Physical air flow due to

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natural thermal currents may also enhance the oxygenation as may the use of external
fans or pumps which are a feature on some industrial units.
OXIDATION PONDS
Treatment of wastewater in ponds is probably the mo st ancient means of waste
treatment known to humans. Oxidation ponds are also called stabilization ponds or
lagoons and serve mostly small rural areas, where land is readily available at relatively
low cost. They are used for secondary treatment of wastewater or as polishing ponds. It
is estimated that there are over 7000 waste stabilization ponds in the United States
(Mara, 2002).
Waste stabilization ponds are classified as facultative, aerobic, anaerobic, aerated, high-
rate aerated, and maturation ponds
Biology of Facultative Ponds
Waste treatment in oxidation ponds is the result of natural biological processes carried
out mainly by bacteria and algae. Waste treatment i s carried out by a mixture of
aerobic, anaerobic, as well as facultative microorganisms. These ponds allow the
accumulation of solids, which are degraded anaerobically at the bottom of the pond.
Many categories of organisms play a role in the treatment process. These include
mainly algae, heterotrophic and autotrophic bacteri a, and zooplankton. The
microbiological processes in facultative ponds are summarized in Fig.

Activity in the Photic Zone
In the photic zone, photosynthesis is carried out by a wide range of algal species (mostly
green algae, euglenophyta, and diatoms), producing from 10 to 66 g algae/m
2
/day
(Edeline, 1988). Chlorophyll a concentration in facultative ponds ranges between 500

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and 2000 mg/L (Mara, 2002). The most common species encountered in oxidation
ponds are Chlamydomonas, Euglena, Chlorella, Scened esmus, Microactinium,
Oscillatoria, and Microcystis (Hawkes, 1983b). The type of predominant algae is
determined by a variety of factors. Motile algae tend to predominate in turbid waters
because they can control their position within the water column to optimally use the
incident light for photosynthesis (Mara, 2002). Diatoms prevail at lower temperatures
than blue-green algae (Edeline, 1988). Algal photosynthesis depends on available
temperature and light. In the presence of high algal numbers, light penetration is
limited to the first 2 ft of the water column. Wind-induced mixing is important for the
maintenance of aerobic conditions within the pond and for providing the exchange of
nutrients and gases between phototrophs and heterotrophs. This exchange is impeded
when the pond becomes stratified, a phenomenon that occurs under warm conditions,
in the absence of natural circulation. Stratification is caused by the establishment of a
temperature difference between the warm upper layer or epilimnion and the lower and
colder layer, the hypolimnion. The zone between the epilimnion and hypolimnion is
called the thermocline and is characterized by a sharp decrease in temperature (Curds
and Hawkes, 1983) (Fig.).
Algae are also involved in nutrient uptake, mainly nitrogen and phosphorus. Some
algae are able to fix nitrogen (e.g., blue-green algae), while most others utilize
ammonium or nitrate. Photosynthesis leads to an increase in pH, particularly if the
treated wastewater has a low alkalinity; this may create conditions for removal of
nutrients. At high pH, phosphorus precipitates as calcium phosphate, and ammonium
ion may be lost as ammonia.
Furthermore, algal photosynthesis produces oxygen, which is used by heterotrophic
microorganisms. Oxygen concentration reaches a peak at mid-afternoon and then
decreases to a minimum during the night. Other phot osynthetic microorganisms in
oxidation ponds are the photosynthetic bacteria that use H
2S as electron donor instead
of H
2O. Therefore, their main role is the removal of H2S, which causes odor problems.
The numbers of both algae and photosynthetic bacteria decrease with increased organic
loading (Houghton and Mara, 1992). Algae are inhibited by ammonia and hydrogen
sulfide, which are produced in waste stabilization ponds, and both inhibit
photosynthesis.

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Heterotrophic Activity
Bacterial heterotrophs are the principal microbial agents responsible for organic matter
degradation in facultative ponds. The role of fungi and of protozoa appears to be less
significant. Heterotrophic activity results in the production of CO
2 and micronutrients
necessary for algal growth. In return, algae provide oxygen that is necessary for aerobic
heterotrophs to oxidize organic matter. Surface reaeration is another source of oxygen
for heterotrophs. Dead bacterial and algal cells and other solids settle to the bottom of
the pond where they undergo anaerobic decomposition.
Anaerobic microbial activity results in the production of gases such as methane,
hydrogen sulfide, carbon dioxide, and nitrogen. Hydrogen sulfide production by sulfate-
reducing bacteria may encourage the growth of photo synthetic bacteria, namely the
purple sulfur bacteria (Chromatium, Thiocapsa, Thiopedia) (Edeline, 1988; Holm and
Vennes, 1970; Houghton and Mara, 1992).
Blooms of Rhodospirillaceae (non-sulfur purple bacteria) in sewage lagoons have also
been documented (Jones, 1956). Photosynthetic bacte ria are found below the algal
layers in facultative ponds. They help protect pond algae from the deleterious effect of
H2S. Although some of the carbon is lost to the atmosphere as CO
2 or CH4, most of it is
converted to microbial biomass.
Unless the microbial cells are removed by sedimentation or some other solids removal
process (e.g., intermittent sand filtration), little carbon reduction is obtained in

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oxidation ponds. Furthermore, the microbial cells in pond effluents may exert an
oxygen demand in receiving waters (U.S. EPA, 1977).
Zooplankton Activity
Zooplankton (rotifera, cladocera, and copepoda) prey on algal and bacterial cells and
can play a significant role in controlling these populations. Their activity is thus
important to the operation of the pond. Cladocera (e.g., Daphnia) are filter feeders that
feed mostly on bacterial cells and detrital particles, but less on filamentous algae. They
are therefore helpful in reducing the turbidity of pond effluents. Zooplankton biomass
can, however, be adversely affected in stratified ponds. This is due to the high
concentrations of unionized ammonia resulting from high pHs associated with algal
photosynthesis (Arauzo, 2003).
Effect of Temperature on Pond Operation
Temperature plays an important role with regard to the activity of phototrophs and
heterotrophs in wastewater ponds. It also significa ntly affects anaerobic waste
degradation in the pond sediments. No methanogenic activity and subsequent reduction
of sludge volume occurs at temperatures below 15°C. Loading of BOD varies from 2.2
g/m
2
.day in cold climates to 5.6 g/m
2
.day in warmer climates (Hammer, 1986). During
the colder months, the ponds become anaerobic becau se of the lack of solar radiation
and, hence, photosynthesis.
One of the empirical equations developed for expressing the loading rate of a pond is the
Gloyna equation, which gives the pond volume as a f unction of the prevailing
temperature (Gloyna, 1971):

where
V = pond volume (m
3
);
N = number of people contributing the waste;
q = per capita waste contribution (L d
-1
);
La = BOD (mg L
-1
);
and Tm = average water temperature of coldest month (°C).
In warm climates, the effluent has a BOD of <30 mg/L. However, the suspended solid
concentration may be high due to the presence of algal cells in the pond effluents.
Removal of Suspended Solids, Nitrogen, and Phosphor us by Ponds

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Oxidation pond effluents often have a high level of suspended solids composed mostly of
algal cells and wastewater solids (Reed et al., 1988). The algae often exert an oxygen
demand in the receiving stream. Thus, the lagoon ef fluents need to be treated by
intermittent sand filters, microstrainers, or constructed wetlands (Steinmann et al.,
2003). To obtain a high-quality effluent with low turbidity (about 1 NTU), the pond
effluent can be treated by chemical coagulation, clarification, and passage through a
continuous backwash sand filter (e.g., Dynasand filter). This sand filter can handle high
algae concentrations because of its countercurrent flow pattern and continuous
backwashing of the sand particles. The clean sand is deposited afterwards on top of the
filter (Fraser and Pan, 1998).
Nitrogen is removed by ponds by a number of mechani sms, including nitrification/
denitrification, volatilization as ammonia, and alg al uptake. Ponds remove
approximately 40–80 percent of nitrogen. Phosphorus removal by ponds is low. Only 26
percent removal was obtained in two experimental lagoons in series under the arid
climate of Marrakech, Morocco (Mandi et al., 1994). Phosphorus removal can be
increased by in-pond treatment with iron and aluminum salts or with lime.
Other types of ponds
Aerobic ponds are shallow ponds (0.3–0.5 m deep) and are generally mixed to allow the
penetration of light necessary for algal growth and subsequent oxygen generation. The
detention time of wastewater is generally 3–5 days. Aerated lagoons are 2–6 m deep with
a detention time of <10 days. They are used to treat high-strength domestic wastewater.
They are mechanically aerated with air diffusers or mechanical aerators. Treatment (i.e.,
BOD removal) depends on aeration time, temperature, and type of wastewater. At 20°C,
there is an 85 percent BOD removal with an aeration period of 5 days. Faulty operation
of the aerated lagoon may result in foul odors.Anaerobic ponds (Hammer, 1986) have a
depth of 2.5–9 m and a relatively long detention time of 20–50 days (Metcalf and Eddy,
1991; Reed et al., 1988). These ponds serve as a pr etreatment step for high-BOD
organic wastes with high protein and fat content (e.g., meat wastes) and with high
concentration of suspended solids. Organic matter is biodegraded under anaerobic
conditions to CH
4, CO2, and other gases such as H2S.
These ponds do not require expensive mechanical aeration and generate small amounts
of sludge. Some problems associated with these ponds are the production of odorous
compounds (e.g., H
2S), sensitivity to toxicants, and the requirement of relatively high

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temperatures. Anaerobic digestion of wastewater is virtually halted at <10°C. These
lagoons are not suitable for domestic wastewaters, which have a characteristic low
BOD.
Maturation or tertiary ponds are 1–2 m deep and ser ve as tertiary treatment for
wastewater effluents from activated sludge or trickling filters. The detention time is
approximately 20 days. Oxygen provided by surface reaeration and algal photosynthesis
is used for nitrification. Their role is to further reduce BOD, suspended solids, and
nutrients (nitrogen and phosphorus), and to further inactivate pathogens.
Pathogen removal by oxidation ponds
Removal or inactivation of pathogens in oxidation ponds is controlled by a variety of
factors, among which are temperature, sunlight, pH, lytic action of bacteriophages,
predation by macroorganisms, and attachment to settleable solids.
ANAEROBIC PROCESSES
Anaerobic digestion consists of a series of microbiological processes that convert
organic compounds to methane and carbon dioxide, and reduce the volatile solids by 35
percent to 60 percent, depending on the operating conditions (U.S. EPA, 1992a). The
microbiological nature of methanogenesis was discovered more than a century ago
(Koster, 1988). While several types of microorganis ms are implicated in aerobic
processes, anaerobic processes are driven mostly by bacteria and methanogens.
Anaerobic digestion has long been used for the stabilization of wastewater sludges.
Later on, however, it was successfully used for the treatment of industrial and domestic
wastewaters. This was made possible through a bette r understanding of the
microbiology of this process and through improved reactor designs.
Anaerobic digestion has several advantages over aerobic processes (Lettinga, 1995;
Lettinga et al., 1997; Sahm, 1984; Speece, 1983; Switzenbaum, 1983):
Anaerobic digestion uses readily available CO2 as an electron acceptor. It requires no
oxygen, the supply of which adds substantially to the cost of wastewater treatment.
Anaerobic digestion produces lower amounts of stabilized sludge (3–20 times less than
aerobic processes) since the energy yields of anaerobic microorganisms are relatively
low. Most of the energy derived from substrate breakdown is found in the final product,
CH
4. With regard to cell yields, 50 percent of organic carbon is converted to biomass
under aerobic conditions, whereas only 5 percent is converted into biomass under
anaerobic conditions. The net amount of cells produ ced per metric ton of COD

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destroyed is 20–150 kg, as compared with 400–600 kg for aerobic digestion. However,
the amount of biomass produced can be theoretically reduced by uncoupling
metabolism in aerobic activated sludge. Under laboratory conditions, the addition of
para-nitrophenol, a known uncoupler of oxidative phosphorylation, led to a 30 percent
decrease in biomass production in activated sludge (Low et al., 2000). Heavy metals,
such as Cu, Zn, and Cd, can also act as uncouplers of microbial metabolism.
Anaerobic digestion produces a useful gas, methane. This biogas contains about 90
percent of the energy, has a calorific value of approximately 9000 kcal/m
3
, and can be
burned on site to provide heat for digesters or to generate electricity. Little energy
(3–5 percent) is wasted as heat. The biogas has been exploited for centuries as an
inexpensive source of energy. Today, millions of small-scale plants operate worldwide
and produce heat and light (Cowan and Burton, 2002) . The biogas yield can be
improved by lysing biosolids microorganisms. This i s achieved by re-treating the
biosolids by mechanical (high pressure, sonication), thermal (biosolids exposure for
short periods to high temperatures ranging from 60 to 225°C), or chemical (alkaline
treatment with NaOH or lime) means (Sanders et al., 2002). Methane production
contributes to the BOD reduction in digested sludge.
There is a reduction of energy required for wastewater treatment.
Anaerobic digestion is suitable for high-strength industrial wastes.
There is a possibility of applying high loading rates to the digester.
There is preservation of the activity of anaerobic microorganisms, even if the digester
has not been fed for long periods of time.
Anaerobic systems can biodegrade xenobiotic compounds such as chlorinated aliphatic
hydrocarbons (e.g., trichloroethylene, trihalomethanes) and recalcitrant natural
compounds such as lignin (see Chapter 19).
Some investigators predict that anaerobic processes will be used increasingly in the
future.
Some disadvantages of anaerobic digestion are:
It is a slower process than aerobic digestion.
It is more sensitive to upsets by toxicants.
Start-up of the process requires long periods, although the use of high-quality seed
material (e.g., granular sludge) can speed up the process.

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As regards biodegradation of xenobiotic compounds v ia co-metabolism, anaerobic
processes require relatively high concentrations of primary substrates (Rittmann et al.,
1988).
Single-Stage Digestion
Anaerobic digesters are large fermentation tanks provided with mechanical mixing,
heating, gas collection, sludge addition and withdrawal, and supernatant outlets
(Metcalf and Eddy, 1991) (Fig.).
Sludge digestion and settling occur simultaneously in the tank. Sludge stratifies and
forms several layers from the bottom to the top of the tank: digested sludge, actively
digesting sludge, supernatant, a scum layer, and gas.
Higher sludge loading rates are achieved in the high-rate version where sludge is
continuously mixed and heated.


Two-Stage Digestion
This process consists of two digesters (Metcalf and Eddy, 1991) (Fig. 13.3); one tank is
continuously mixed and heated for sludge stabilization and the other one for thickening
and storage prior to withdrawal and ultimate disposal. Although conventional high-rate
anaerobic digestion and two-stage anaerobic digestion achieve comparable methane
yield and COD stabilization efficiency, the latter process allows operation at much
higher loading rates and shorter hydraulic retention times (Ghosh et al., 1985).

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Process microbiology
Consortia of microorganisms, mostly bacteria and me thanogens, are involved in the
transformation of complex high-molecular-weight org anic compounds to methane.
Furthermore, synergistic interactions between the various groups of microorganisms
are implicated in anaerobic digestion of wastes. The overall reaction is shown in Eq.
(Polprasert, 1989):

Although some fungi and protozoa (anaerobic protozoa were found in landfill material;
Finlay and Fenchel, 1991) may be found in anaerobic digesters, bacteria and
methanogens are undoubtedly the dominant microorgan isms. Large numbers of strict
and facultative anaerobic bacteria (e.g., Bacteroides, Bifidobacterium, Clostridium,
Lactobacillus, and Streptococcus) are implicated in the hydrolysis and fermentation of
organic compounds.
ANAEROBIC DIGESTION
Four categories of microorganisms are involved in t he transformation of complex
materials into simple molecules such as methane and carbon dioxide. These microbial
groups operate in a synergistic relationship (Archer and Kirsop, 1991; Barnes and
Fitzgerald, 1987; Koster, 1988; Sahm, 1984; Sterritt and Lester, 1988; Zeikus, 1980):
(Fig.).

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Group 1: Hydrolytic Bacteria
Consortia of anaerobic bacteria break down complex organic molecules (e.g., proteins,
cellulose, lignin, lipids) into soluble monomer molecules such as amino acids, glucose,
fatty acids, and glycerol. The monomers are directly available to the next group of
bacteria. Hydrolysis of the complex molecules is catalyzed by extracellular enzymes
such as cellulases, proteases, and lipases. However, the hydrolytic phase is relatively
slow and can be limiting in anaerobic digestion of wastes such as raw cellulolytic
wastes that contain lignin (Polprasert, 1989; Speece, 1983).
Group 2: Fermentative Acidogenic Bacteria
Acidogenic (i.e., acid-forming) bacteria (e.g., Clostridium) convert sugars, amino acids,
and fatty acids to organic acids (e.g., acetic, propionic, formic, lactic, butyric, or
succinic acids), alcohols and ketones (e.g., ethanol, methanol, glycerol, acetone),
acetate, CO
2, and H2. Acetate is the main product of carbohydrate fermentation. The
products formed vary with the bacterial type as wel l as with culture conditions
(temperature, pH, redox potential).
Group 3: Acetogenic Bacteria

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Acetogenic bacteria (acetate and H2-producing bacteria) such as Syntrobacter wolinii
and Syntrophomonas wolfei (McInernay et al., 1981) convert fatty acids (e.g., propionic
acid, butyric acid) and alcohols into acetate, hydrogen, and carbon dioxide, which are
used by the methanogens. This group requires low hy drogen tensions for fatty acid
conversion, necessitating a close monitoring of hydrogen concentration. Under relatively
high H
2 partial pressure, acetate formation is reduced and the substrate is converted to
propionic acid, butyric acid, and ethanol rather than methane. There is a symbiotic
relationship between acetogenic bacteria and methanogens. Methanogens help achieve
the low hydrogen tension required by acetogenic bac teria. Online methods for
measuring the concentration of total VFAs (volatile fatty acids) and dissolved hydrogen
are available and can serve as good monitoring tools for the functioning of anaerobic
digestion processes (Bjo¨rnsson et al., 2001).
Ethanol, propionic acid, and butyric acid are converted to acetic acid by acetogenic
bacteria according to the following reactions:

Acetogenic bacteria grow much faster than methanogens. The former group has ammax
of approximately 1 h
-1
, whereas the mmax of the latter is around 0.04 h
-1
(Hammer,
1986).
Group 4: Methanogens
As mentioned before, anaerobic digestion of organic matter in the environment releases
approximately 500 million tons of methane/year into the atmosphere, representing
about 0.5 percent of the organic matter derived from photosynthesis (Kirsop, 1984;
Sahm, 1984). The fastidious methanogens occur naturally in deep sediments or in the
rumen of herbivores. Methanogenic microorganisms gr ow slowly in wastewater and
their generation times range from 3 days at 35°C to as high as 50 days at 10°C.
Methanogens use a limited number of substrates that include acetate, H
2, CO2, formate,
methanol, and methylamines. All of these substrates are reduced to methylCoM (CH
3–
S–CoM), which is converted to CH
4 by methylCoM reductase (Ritchie et al., 1997).
Methanogens are subdivided into two subcategories:

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Hydrogenotrophic methanogens (i.e., hydrogen-using chemolithotrophs) convert
hydrogen and carbon dioxide into methane:

Most of the methanococcales and methanobacteriales use H2 and CO2 (Ritchie et al.,
1997)
Acetotrophic methanogens, also called acetoclastic or acetate-splitting methanogens,
convert acetate into methane and CO
2:

This group comprises two main genera: Methanosarcina (Smith and Mah, 1978) and
Methanothrix (Huser et al., 1982) and Methanosaeta (Ritchie et al., 1997). During
thermophilic (58°C) digestion of lignocellulosic waste, Methanosarcina was the
dominant acetotrophic methanogen encountered in the bioreactor. After 4 months,
Methanosarcina (µ
max = 0.3 day
-1
; Ks = 200 mg/L) was displaced by Methanothrix (µmax
= 0.1 day
-1
; Ks = 30 mg/L). It was postulated that the competi tion in favor of
Methanothrix was due to the lower acetate Ks value of this organism (Gujer and
Zehnder, 1983; Koster, 1988; Zinder et al., 1984).
About two-thirds of methane is derived from acetate conversion by acetotrophic
methanogens. The other third is the result of carbon dioxide reduction by hydrogen
(Mackie and Bryant, 1981).
Methanogens belong to a separate domain, the archaea, and differ from bacteria in the
following characteristics (Sahm, 1984):
They differ in cell wall composition; for example, the cell wall of methanogens lacks
peptidoglycan.
They also differ in the composition of the cell membranes, which are made of branched
hydrocarbon chains attached to glycerol by ether linkages.
Methanogens have a specific coenzyme F420, a 5-deazaflavin analog, which acts as an
electron carrier in metabolism. Its oxidized form absorbs light at 420 nm (Cheeseman et
al., 1972). This blue-green fluorescent coenzyme has been proposed for quantifying
methanogens in mixed cultures (van Beelen et al., 1983). F
420 determination in cell
extracts is carried out by extraction followed by fluorescence measurement or by high-
performance liquid chromatography (HPLC) with fluor imetric detection (Peck, 1989).
Methanogenic colonies can be distinguished from non methanogenic ones by using

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fluorescence microscopy (Edwards and methanogenic c onsortia can be misleading as
regards the determination of acetoclastic methanogenic activity (Dolfing and Mulder,
1985). Another nickelcontaining coenzyme, F
430, is also unique to methanogens.
Methanogens belong to the kingdom of euryarchaeota within the archaea domain. They
are strictly anaerobic and thrive in oxygen-free environments such as freshwater and
marine sediments, swamps, landfills, the rumen of cows, or in anaerobic digesters. A
key coenzyme involved in methane production is methyl coenzyme M.
Methanogens have ribosomal RNA sequences that diffe r from those of bacteria and
eukaryotes.
Methanogens were grouped into four orders: Methanob acteriales (e.g.,
Methanobacterium, Methanobrevibacter, Methanothermu s), Methanomicrobiales (e.g.,
Methanomicrobium, Methanogenium, Methanospirillum, Methanococcoides),
Methanococcales (e.g., Methanococcus), and Methanosarcinales. At least 49 species of
methanogens have been described (Vogels et al., 1988) and more are being discovered
Factors controlling anaerobic digestion
Efficient AD requires the development and maintenan ce of an optimised internal
environment to facilitate biological activity. This is of particular importance in the
commercial setting and a number of both physical and chemical factors must be taken
into account to achieve it, of which the most important are:
Temperature
As mentioned previously, in commercial systems, digesters are operated at around 35
◦C (mesophilic) or 55 ◦C (thermophilic). Irrespective of which approach is adopted for
any particular application, a relatively constant temperature is essential for the process
to run at its greatest efficiency.
Retention period
Although the amount of biowaste degraded depends on its character, the availability of
bacteria and the time allowed for processing, temperature governs both the rate of
breakdown itself and the particular bacterial species present in the digester. Hence,
there is a direct relationship between temperature and the retention period. Some AD
technologies have attempted to shorten the retention period by separating the stages of
the process within the digester. The separation of the acidogenic and methanogenic
stages permits each to be optimised and this has been well demonstrated at laboratory
scale using a completely mixed digester, with phase-isolation being achieved by pH

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manipulation. Despite the greater efficiency, higher biogas yield and enhanced process
stability claimed, it has seen little large-scale use, probably as a result of the higher
cost implications of such a system.
Agitation
The agitation of the digester contents has a number of benefits, one of the most obvious
being that it helps to mix up material, evening out any localised concentrations, thus
also helping to stop the formation of ‘dead zones’ or scum.
In addition, it increases the waste’s availability to the bacteria, helps remove and
disperse metabolic products and also acts to ensure a more uniform temperature within
the digester. There have been some suggestions that efficient mixing enhances methane
production, but the evidence is inconclusive, so it seems likely that this may only be of
noticeable benefit for some systems or operational regimes.
Wetness
Anaerobic digestion is a wet process and any biowaste which is too dry in its natural
state will require the addition of a suitable liquid, typically water, recycled
AD process liquor or slurries, either sewage or agricultural, before processing can begin.
In order to minimise digester size, so-called ‘dry’ systems have tended to dominate the
commercial world, but the relatively thicker contents inevitably demand more energy to
mix effectively, off-setting much of the advantage. Comparisons of ‘wet’ or ‘dry’
approaches, like those of mesophilic or thermophilic processes, generally yield no clear
winner. Each system has particular advantages and a pplications for certain kinds of
biowaste, and selecting the right one for any given use is almost always best done on
this basis.
Feedstock
As with composting, the particle size and nature of the material to be treated play an
important role. The ease of breakdown is largely defined by the characteristics of the
biowaste material to be treated, but generally finer particles allow for better processing
and a homogeneous slurry or suspension is the ideal feedstock for AD. It must be
stressed, however, that some biowaste types, particularly the likes of lignin-rich, woody
material, are relatively resistant to this process.
Loading rate
Loading depends on the characteristics of the waste, its degree of wetness, digester
volume, the expected retention period and similar system design parameters. It is

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typically expressed as the chemical oxygen demand per cubic metre of digester void-
space (COD/m3) or, for continuous or semi-continuou s process, per unit time
(COD/day, COD/hr).
pH and volatile fatty acids concentration
These are interdependent factors which need to be c onsidered together. Adequate
process control and digester optimisation requires suitable pH monitoring, since many
of the bacteria involved are pH sensitive. In particular, acidogens, having better
tolerance to acidity, may produce acids faster than the increasingly inhibited
methanogens can use it, in conditions of low pH, leading to spiralling acidity and the
potential for process collapse. A number of acid–base reactions exist within the typical
AD process, which lead to a measure of natural, inbuilt resistance to major pH swings.
However, under certain circumstances, the need for external interference may arise and
the amount of such intervention necessary to maintain proper equilibrium will depend
on the nature of the material. For some wastes, pH control may only be necessary
during start-up or in overload conditions; for others where acidity is habitually shown
to be a problem, continuous control may be necessary.
Volatile fatty acid concentration is one of the most important process indicators.
Elevated VFAs are characteristic of AD instability and thus they may be the first
indication of a developing problem, though the actual cause may be less immediately
obvious. Inadequate mixing, excessive loading, poor temperature control or bacterial
inhibition can all lead to an increase in VFAs and a decrease in pH. Considering the
inconvenience and cost of being forced to empty a s ick reactor, commercial AD
operations rely greatly on routine monitoring of this kind.
ANAEROBIC FILTERS
Anaerobic filters were first introduced at the beginning of the last century and further
developed in 1969 by Young and McCarty.
These filters are the anaerobic equivalent of trickling filters. They contain support media
(rock, gravel, plastic) with a void space of approximately 50 percent or more (Frostell,
1981; Jewell, 1987). The bulk of anaerobic microorganisms grow attached to the filter
medium, but some form flocs that become trapped inside the filter medium. The upflow
of wastewater through the reactor helps retain suspended solids in the column. This
process is particularly efficient for wastewaters rich in carbohydrates (Sahm, 1984). The
loading rate varies with the type of waste and with the type of support medium. It

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generally falls within the range of 5–20 kg COD m
-3
d
-1
(Barnes and Fitzgerald, 1987).
This system achieves modest BOD removal but higher removal of solids. Approximately
20 percent of the BOD is converted to methane.

Another version of the anaerobic filter is the thin film reactor developed by van den
Berg and collaborators (1981). This reactor contains several clay tubes 5–10 cm in
diameter. Incoming wastewater flows downward and is treated by the 1 to 3 mm thick
anaerobic biofilm that develops on the surfaces of the clay tubes (Fig.).


ANAEROBIC SLUDGE
The upflow anaerobic sludge blanket (UASB) uses imm obilized biomass to allow the
retention of the sludge in the treatment system. It was introduced at the beginning of
the century and, after numerous modifications, it was put into commercial use in the
Netherlands for the treatment of industrial wastewater generated by the food industry
(e.g., beet-sugar, corn, and potato starch).
The UASB-type digester consists of a bottom layer of packed sludge, a sludge blanket
and an upper liquid layer (Lettinga, 1995) (Fig.).

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Wastewater flows upward through a sludge bed, which is covered with a floating
blanket of active bacterial flocs. Settler screens separate the sludge flocs from the
treated water and gas is collected at the top of the reactor (Schink, 1988). This process
results in the formation of a compact granular sludge, which settles well and which
withstands the shear force caused by the upflow of wastewater. The sludge is
immobilized by the formation of highly settleable microbial aggregates that grow into
distinct granules (<1–5 mm) that have a high VSS co ntent and specific activity.
Immunological techniques, scanning electron microsc opic (SEM) examination, and
energy-dispersive X-ray analysis of granular sludge have shown that the granules are
composed of methanogens such as Methanothrix soehng enii (Brummeler et al., 1985;
Hulshoff Pol et al., 1982; 1983), Methanobacterium, Methanobrevibacter,
Methanosarcina, as well as Ca precipitates (Vissier et al., 1991; Wu et al., 1987).
Scanning electron microscopy and transmission electron microscopy (TEM) have shown
that the granules are three-layered structures (MacLeod et al., 1990).



The inner layer consists of Methanothrix-like cells, which may act as nucleation centers
necessary for the initiation of granule development. The middle layer consists of
bacterial rods that include both H
2-producing acetogens and H2-consuming organisms.
The outermost layer consists of a mixture of rods, cocci, and filamentous

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microorganisms. This layer compiles a mixture of fe rmentative and H 2-producing
organisms. Thus, a granule appears to harbor the necessary physiological groups to
convert organic compounds to methane. The layered s tructure of anaerobic sludge
granules was confirmed using 16S rRNA-targeted probes (Harmsen et al., 1996; Liu et
al., 2002a; Sekiguchi et al., 1999). Both mesophili c and thermophilic granules
displayed layered structures, with the outer layer containing mostly bacterial cells while
the inner layer contained mostly archaeal cells (Methanosaeta-, Methanobacterium-,
Methanospirillum-, and
Methanosarcina-like cells) (Sekiguchi et al., 1999).
Micro-electrodes, in combination with genetic probes, are useful in showing pH,
methane, and sulfide profiles within the granules. This approach provides useful
information on the interaction between methanogens and sulfate-reducing bacteria
(SRB) within the granules. For example, in granules fed glucose as a substrate, it was
shown that pH increased from the outer to the inner portion of the granules (i.e.,
conversion of acids to methane in the inner portion). This technology also proved that
sulfate reduction occurred at the outer portion of the granule while FeS precipitation
occurred in the inner portion (Yamaguchi et al., 2001). In methanogenic–sulfidogenic
bioreactors, the SRB were located in the surface layer down to a depth of 100 mm,
while the methanogens were located in the core of the aggregates (Santegoeds et al.,
1999).
The microbiological composition of granules depends on the type of growth substrate
(Grotenhuis et al., 1991). Factors affecting the rate of granulation include wastewater
characteristics (higher rate when wastewater is composed of soluble carbohydrates),
conditions of operation (e.g., sludge loading rate), temperature, pH, and availability of
essential nutrients (Hulshoff Pol et al., 1983; Wu et al., 1987). Problems associated with
granulation include deterioration of sludge granules, attachment of fast-growing
bacteria, flotation, and calcium carbonate scaling (Lettinga et al., 1997). The treatment
of distillery wastewater by the UASB process resulted in 92 percent BOD removal (Pipyn
et al., 1979).
Novel versions of UASB reactors include, among others, the expanded granular sludge
bed (EGSB) reactor, which functions at high flow velocities and allows the treatment of
very low-strength wastewaters even at temperatures lower than 108C (Kato et al., 1994;
Rebac et al., 1995).

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MEMBRANE BIOREACTORS
Anaerobic Attached-Film Expanded-Bed and Fluidized- Bed Reactors
These reactors were introduced during the 1970s. So me features distinguish the
expanded beds from the fluidized ones (Fig.).
Bed expansion caused by the upflow of wastewater is much greater in expanded than in
fluidized beds. Wastewater flows upward through a sand bed (diameter <1 mm), which
provides a surface area for biofilm growth. The flow rate is high enough to obtain an
expanded or fluidized bed.

This in turn necessitates the recirculation of the wastewater through the bed. This
process is effective for the treatment of low-strength organic substrates (COD less than
600 mg/L) at short hydraulic retention times (several hours) and allowing high solid
retention times (SRT) (Speece, 1983; Switzenbaum, 1983). This process offers several
advantages:
Good contact is achieved between wastewater and microorganisms.
Clogging and channeling are avoided.
High biomass concentrations can be achieved, and th is is associated with reduced
reactor volume.
Biofilm thickness can be controlled.
Successful treatment of low-strength wastewater (≤600 mg/L COD) is attained at low
temperature and at relatively short hydraulic retention times (<6 h).
Some investigators have argued that fluidized-bed reactors can be applicable to aerobic
treatment. An advantage of such an application would be lower detention times, and

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thus, higher loading rates. Furthermore, nitrification would be favored as a result of the
retention of the slow-growing nitrifiers in the biofilm (Rittmann, 1987).
Some of the disadvantages of this process are the high energy necessary for sufficient
upflow velocity for bed expansion and the relatively large volume (30–40 percent)
occupied by the support material. Some have proposed an anaerobic expanded micro-
carrier bed (MCB) to solve these problems. Powdered zeolite is used as support material
in the MCB process, which promotes the formation of granular sludge as in the UASB
process (Yoda et al., 1989).
Anaerobic Rotating Biological Contactor
An anaerobic rotating biological contactor is similar to its aerobic counterpart except
that the reactor is sealed to create anaerobic conditions (Laquidara et al., 1986) (Fig.).
This process allows greater disk submergence becaus e oxygen transfer is not
considered. Development of the attached anaerobic b iofilm is a function of applied
organic loading and time.

Approximately 85 percent COD removal is achievable even at loading rates as high as
90 g/m
2
/day COD (Laquidara et al., 1986).
At this organic loading, methane is produced at a rate of 20 L/m
2
/day. Some of the
advantages of this fixed-film anaerobic system are (Laquidara et al., 1986).
Potential for higher organic loadings;
Cell residence time independent of hydraulic detention time;
Low production of waste solids;
Ability to withstand toxic shock loads;
Methane production.
REVERSE OSMOSIS
Osmosis is the process where a solvent (e.g., water ) moves from an area of low
concentration to high across a semipermeable membra ne which does not allow the

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dissolved solids to pass. In reverse osmosis, a pressure greater than the osmotic
pressure is applied so the flow is reversed. Pure water will then flow through the
membrane from the concentrated solution.
Over the last 30 years, a number of membrane processes have evolved, which make use
of a pressure driving force and a semi-permeable me mbrane in order to effect a
separation of components in a solution or colloidal dispersion. The separation is based
mainly on molecular size, but to a lesser extent on shape and charge. The three main
processes are reverse osmosis (hyperfiltration), ultrafiltration and microfiltration. The
dimensions of the components involved in these separations are given in Fig. and are
typically in the range of less than 1 nm to over 1000 nm.

A brief summary of the main differences between them, in terms of the components
which are rejected by the membranes, is also illustrated. More recently the process term
‘nanofiltration’ has been introduced, which is somewhere between reverse osmosis (RO)
and ultrafiltration, bringing about a separation of low molecular weight components
such as monovalent ions and salts from organic comp ounds such as sugars. These
pressure-activated processes can also be regarded a s a continuous spectrum of
processes, with no obvious distinct boundaries between them. However, it should be
noted that the sizes of the components being separated range over several orders of
magnitude, so it is highly likely that the separation mechanisms and hence the
operating strategies may change as we move through the spectrum.
Terminology
The feed material is applied to one side of a membrane. The feed is usually a low
viscosity fluid, which may sometimes contain suspended matter and which is subjected

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to a pressure. In most cases the feed flows in a direction parallel to the membrane
surface and the term cross-flow filtration is used to describe such applications. Dead-
end systems are used, but mainly for laboratory scale separations. The stream which
passes through the membrane under the influence of this pressure is termed the
permeate (filtrate). After removal of the required amount of permeates, the remaining
material is termed the concentrate or retentate. The extent of the concentration is
characterised by the concentration factor df), which is the ratio of the feed volume to the
final concentrate volume.
The process can be illustrated simply in Fig.(a).

From a single membrane processing stage, two fracti ons are produced, named the
concentrate and permeate. The required extent of concentration may not be achieved in
one stage, so the concentrate may be returned to th e same module for further
concentration or taken to other modules in a cascad e, or multistage process. The
permeate may also be further treated in a separate process.
In terms of size considerations alone, one extreme is a membrane with very small pore
diameters (tight pores). In this case the permeate will be pure water because even small
molecular weight solutes will be rejected by the membrane; high-pressure driving forces
are required to overcome frictional resistance and osmotic pressure gradients. If the
permeate is predominantly water, then the process is known as reverse osmosis or
hyperfiltration; it is similar in its effects to evaporation or freeze-concentration. A
concentrate will be produced, in which there is virtually no alteration in the proportion
of the solid constituents. In some applications it is the permeate which is the required
material; for example the production of ‘drinking water’ from sea-water or ‘pure water’
from brackish water. The best processes are those where both the concentrate and the
permeate are fully utilised. There have been severa l comparisons made between

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evaporation and reverse osmosis, in terms of capital costs, energy costs and product
quality (Renner, 1991). In general terms RO is less energy intensive and can improve
product quality. Some limitations are the high capital costs, membrane replacement
costs and extent of concentration, which is not as high as that obtainable by
evaporation.
If a fluid, for example milk, is separated from water by a semi-permeable membrane
(Fig.b), there will be a flow of water from the water to the milk, in order to equalise the
chemical potential of the two fluids; this is termed osmosis. This flow of water can be
stopped by applying a pressure to the milk. This pressure that stops the flow is termed
the osmotic pressure. If a pressure greater than the osmotic pressure is applied, the
water will flow from the milk to the water, thereby reversing the natural process of
osmosis and achieving a concentration of the milk. Therefore in reverse osmosis, the
pressure applied needs to be in excess of the osmotic pressure. Osmotic pressure (T) is
a colligative property, the pressure being dependent upon the number of particles and
their molecular weight.
Osmotic pressures are highest for low molecular weight solutes, so the highest osmotic
pressures arise for salt and sugar solutions. Concentration of such solutions results in
a large increase in their osmotic pressure. On the other hand, proteins and other
macromolecules do not produce high osmotic pressure s. There will only be small
increases during their concentration as well as small differences in osmotic pressure
between the feed and permeate in ultrafiltration.

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Values for osmotic pressures are not easy to find in the literature and a selection of
values is given in Table.
A further complication with foods and other biological systems is their complexity, with
not just one but many components. In reverse osmosis the applied pressure must
exceed the osmotic pressure, and the driving-force term in reverse osmosis is normally the
difference between the applied pressure and the osm otic pressure. It could be that
osmotic pressure is one of the factors that limits the extent of concentration. One
suggested experimental method for measuring osmotic pressure is to determine the
pressure that would give zero flux, by extrapolatio n. In ultrafiltration and
microfiltration, there is little osmotic pressure difference over the membrane as the low
molecular weight components are almost freely permeating.
As the membrane pore size increases, the membrane b ecomes permeable to low
molecular weight solutes in the feed; even the transport mechanisms are likely to
change. Lower pressure driving forces are required as osmotic pressure differences
between the feed and permeate are reduced. However, molecules of a larger molecular
weight are still rejected by the membrane. Therefore some separation of the solids
present in the feed takes place; the permeate conta ins low molecular weight
components at approximately the same concentration as they are in the feed, and the
concentrate contains large molecular weight components at an increased concentration,
compared to the feed.
Note that some of the low molecular weight componen ts will be retained in the
concentrate. It is this fractionation and concentra tion process that makes the
ultrafiltration process more interesting than reverse osmosis, although, as mentioned
earlier, there is no sharp demarcation between the processes. More porous membranes
still allow not only sugars and salts, but also macromolecules, to pass through, but
retain particular matter and fat globules, i.e. greater than 100 nm; this is termed
microfiltration.
Concentration factor and rejection
Two important processing parameters for all pressur e activated processes are the
conceirtration factor cf) and the membrane rejection characteristics. The concentration
factor is defined as follows:

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where VF = feed volume and Vc = final concentrate volume.
The term volume reduction factor (VRF) is sometimes used:

Thus a process with a concentration factor of 10 would have a volume reduction factor
of 90%.
The permeate volume (Vp) equals the feed volume minus the concentrate volume
(assuming no losses)

As soon as the concentration factor exceeds 1, the volume of permeate will exceed that
of the concentrate. Concentration factors may range from as low as 1.5 for some viscous
materials, to up to 50 for dilute protein solutions, e.g. chhana whey (Jindal and
Grandison, 1992). Generally higher concentration factors are used for ultrafiltration
than for reverse osmosis, e.g. up to 25-30 for UF of cheese-whey, compared to 5 for RO
of cheese-whey.
A mass balance for the process can be applied and i s useful for estimating the
distribution of components between the permeate and concentrate, or for estimating the
losses that are incurred in practical situations.
The rejection or retention factor (R) of any component is defined as

where CF is the concentration of component in the feed and cp is the concentration in
the permeate.
It can be determined experimentally for each and every component in the feed, by
sampling the feed and permeate at the same time and analysing the component in
question. It is a very important property of a membrane, as it will influence the extent
(quality) of the separation that can be achieved.
Rejection values normally range between 0 and 1; sometimes they are expressed as
percentages (0-1 00%).
when cp = 0; R = 1; all the component is retained in the feed
when cp = CF R = 0; the component is freely permeating.
An ideal RO membrane would give a rejection value for all components of 1, whilst an
ideal UF membrane, being used to concentrate a high molecular weight component or

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remove a low molecular weight component would give respective rejection values of 1
and 0. If the concentration factor and rejection value are known, the yield of any
component, which is defined as the fraction of that component present in the feed,
which is recovered in the concentrate, can be estimated. Obviously for reverse osmosis,
the yield for an ideal membrane is 1.0.
Membrane characteristics
The main terms used to describe membranes are microporous or asymmetric.
Microporous membranes have a uniform porous structu re throughout, although the
pore size may not be uniform across the thickness of the membrane. They are usually
characterized by a nominal pore size and no particle larger than this will pass through
the membrane. In contrast to this, most membranes u sed for ultrafiltration are of
asymmetric type, having a dense active layer or skin of 0.5-1 pm in thickness, and a
further support layer which is much more porous and of greater thickness (Fig.).

Overall the porosity of these membranes is high, although the surface porosity may be
low, with quoted values in the range 0.3-15% (Fane and Fell, 1987). Often the porous
path may be quite tortuous, the distance covered by the solvent or solute being much
greater than the thickness of the membrane; the term tortuosity has been used as a
measure of this property.
Transport phenomena and concentration polarisation
A very important consideration for pressure-driven membrane processes is that the
separation takes place not in the bulk of solution, but in a very small region close to the
membrane, known as the boundary layer, as well as o ver the membrane itself. This
gives rise to the phenomenon of concentration polarisation over the boundary layer.
(Note that in streamline flow the whole of the fluid will behave as a boundary layer.) It is
manifested by a quick and significant reduction (2-10 fold) in flux when water is
replaced by the feed solution, for example in a dynamic start.

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Concentration polarisation occurs whenever a component is rejected by the membrane.
As a result, there is an increase in the concentrat ion of that component at the
membrane surface, together with a concentration gra dient over the boundary layer.
Eventually a dynamic equilibrium is established, wh ere the convective flow of the
component to the membrane surface equals the flow of material away from the surface,
either in the permeate or back into the bulk of the solution by diffusion, due to the
concentration gradient established. This increase in concentration, especially of large
molecular weight components, offers a very significant additional resistance. It may also
give rise to the formation of a gelled or fouling layer on the surface of the membrane.
Membrane processes can be operated under batch or c ontinuous conditions. The
simplest system is a batch process. The feed is usu ally recycled, as sufficient
concentration is rarely achieved in one pass. Flux rates are initially high but decrease
with time.
Energy costs are high because the pressure is released each time. Residence times are
long. Batch operations are usually restricted to sm all-scale operations. Batch
processing with top-up is used in situations when the entire feed volume will not fit into
the feed tank. Batch processing times can be estimated (predicted) if the relationship
between flux rate and product concentration is known. In many cases there is a linear
relationship between the flux and the log of the Concentration.

Continuous processes may be single-stage (feed and bleed) or multistage processes,
depending upon the processing capacity required. The simplest continuous system is a
single-stage process with recycle. Once the retenta te has reached its final
concentration, a feed and bleed system is operated. Steady state conditions are
achieved and product is withdrawn and replaced by fresh feed, at a rate which keeps its
composition constant. One drawback is that the conc entration in the module is the
same as the final product concentration, so the process is operating at its highest total

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solids content. Therefore, flux rates are low and solids yields may be reduced. The
instrumentation and ancillaries for a batch process and a single-stage continuous
process are shown in Fig.

Some of these problems can be alleviated by multist age processes. In multistage
processes, the feed may pass once through each stag e (single pass), or be recycled
within the stage. Single-pass operations are used in situations where a high degree of
concentration is achieved in one pass through the s tage. Within each stage, the
modules may be arranged in parallel or series. The stages are then arranged in series,
with a pump or bleed system between the stages. There may be up to six such stages
arranged in series in some larger plants.

Fig(c) illustrates a three-stage process. Each stage contains 24 modules, four banks in
parallel, each containing six modules in series.
ULTRA FILTRATION
Introduction
Ultrafiltration offers the opportunity to concentrate large molecular weight components
without the application of heat or a change of phase. Such components are rejected by

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the membrane, whereas the permeate produced will co ntain the low molecular weight
components present in the food, at a concentration similar to that in the feed. This
results in an increase in their concentration both on a wet weight and dry weight basis
in the solution. It is a pressure-activated process, with pressures in the range of 1-15
bar; these pressures are considerably lower than those used in reverse osmosis. For
many heat labile macromolecules, e.g. proteins and starches, concentration by UF at
ambient temperature will minimise heat-induced reac tions which may adversely
influence their functional behaviour in foods. Some important functional properties are
solubility, foaming capacity, gelation, emulsification capacity, fat and water binding
properties.
In the case of enzymes or pharmaceutical agents, their biological activity needs to be
conserved. It also affords the opportunity to separ ate small molecular weight
components from complex mixtures, containing compon ents with a wide range of
molecular weights. There have also been investigations into using UF for protein
fractionation, but this is not straight forward due to the diffuse nature of the
membranes and their selectivity. UF is also very us eful for recovering valuable
components from food processing waste streams and fermentation broths. Probably the
greatest impetus has come from the dairy industry and dairying applications. However,
in all applications, flux decline due to concentration polarisation and fouling are
probably the two most important practical aspects.
There are various factors which will influence the outcome of the process, such as the
concentration factor and rejection.
The extent of the concentration is defined by the concentration factor cf), defined as
Vf/Vc. Usually the permeate is the biggest fraction by volume. Milk for cheese making is
concentrated by UF fivefold, whereas cheese whey is concentrated twentyfold for the
production of protein concentrates. Sometimes the resulting permeates are further
concentrated by reverse osmosis.
Rejection or retention factors
The rejection or retention factor (R) of any component is defined as

where cF is the concentration of component in the feed and cp is the concentration in
the permeate.

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The rejection is determined experimentally for each component in the feed, by sampling
the feed and permeate at the same time and analysin g that component. It is very
important and will influence the extent (quality) of the separation achievable.
Rejection values normally range between 0 and 1; sometimes they are expressed as
percentages (0 to 100%).
when cp =O; R = 1; all the component is retained in the feed
when cp = cF R = 0; the component is freely permeating
In ultrafiltration experiments, some workers have measured negative rejection, i.e.
cp > cF, particularly for minerals. It is not immediately obvious why this should have
occurred. Possible explanations for this are higher concentrations at the membrane
surface than in the bulk, due to concentration polarisation. However, this is unlikely to
be the case for freely permeating species. Another explanation is the basis on which
concentration is measured (Glover, 1985). This may arise when there is substantial fat
in the feed which is rejected by the material. It is suggested that concentrations be
expressed in the aqueous portion. A third explanation lies in the Donnan effect; Donnan
predicted and later demonstrated that concentration of electrolyte in the solutions on
either side of a dialysis membrane were unequal whe n the colloid on one side was
electrically charged.
For example, at low pH values, where proteins are likely to be positively charged, this
could lead to higher concentrations of cations in the permeate.
Rejection characteristics can readily be determined for different substances using
different membranes. This is one practical way of s electing the most appropriate
membrane for a particular application. Rejection values may also be influenced by
operating conditions.
An ‘ideal’ ultrafiltration membrane would have a re jection value of 1.0 for high
molecular weight components and zero for low molecular weight components. However,
typical values observed for real membranes are between 0.9 and 1.0 for high molecular
weights and between 0 and 0.1 for low molecular wei ght components. Values for
minerals often are usually in the region of 0.1, but may be as high as 0.5, if the mineral
binds to macromolecules. It is important to appreciate that any component wi th a
rejection value greater than 0 will increase in concentration during the course of
an ultrafiltration process. Rejection values can be used to check the integrity and

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performance of a membrane. Some values for componen ts in dairy processing are given
in Table.

Note the relatively high values for minerals, which suggest some binding to the proteins,
particularly for calcium and magnesium. Membrane ma nufacturer’s sometimes present
performance data in terms of rejection values of a range of components of different
molecular weights (see Table).

This will give some guidelines in terms of selection. However, very rarely are those
components selected that one is interested in. An alternative form of representation
widely used is the molecular weight cut-off value.
Yield
Ultrafiltration is now being used to concentrate an d recover some very valuable
compounds. The yield or recovery of a component is a very important variable, as it will
strongly influence the economics of the process. The yield of a component is defined as
the fraction of that component, originally present in the feed, which is retained in the
concentrate. For recovery of components it is important to have a high yield. However,
when washing out components, such as toxins, the yield should be low.
Pekformance of ultrafiltration systems
Permeate flux

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In UF process applications, the two most important parameters are the membrane
rejection and the flow rate of permeate or permeate flux, hereafter abbreviated to 'flux'.
The flux will probably be measured in gallons/min o r litres/hour, but it is usually
presented in terms of volume per unit time per unit area (1 m
-2
h
-l
).
Expressed this way it allows a ready comparison of the performance of different
membrane configurations with different surface areas. Flux values may be as low as 5
or as high as 450 1 m
-2
h
-1
. The flux is one of the major factors influencing the viability
of many processes.
UF processes have been subject to a number of modelling processes, in an attempt to
predict flux rates and rejection values from the physical properties of the solution, the
membrane characteristics and the hydrodynamics of t he flow situation, in order to
optimize the performance of the process.
Transport phenomena and concentration polarisation
Ultrafiltration is usually regarded as a sieving process and in this sense the
mechanisms are simpler than for RO. However, it is important to remember that for
pressure-driven membrane processes, the separation takes place not in the bulk of
solution, but in a very small region close to the membrane, known as the boundary
layer, as well as over the membrane itself. This gives rise to the phenomenon of
concentration polarisation over the boundary layer. (Note that in streamline flow the
whole of the fluid will behave as a boundary layer.)Concentration polarisation occurs
whenever a component is rejected by the membrane.
As a result, there is an increase in the concentration of that component at the
membrane surface, and a concentration gradient over the boundary layer. This increase
in concentration offers a very significant additional resistance, and for macromolecules
may also give rise to the formation of a gelled or fouling layer on the surface of the
membrane (see Fig.).

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It is interesting to note that the boundary layer does not establish itself immediately at
the point where the fluid first contacts the membrane. Rather it takes some distance for
it to be fully established. This distance taken for it to be fully established has been
defined as the entry length, and the process of establishment is illustrated for a tubular
membrane.
Howell et al. (1990) have analysed flux conditions over the entry length and have
concluded that the flux and wall concentrations change quite considerably over the
developing boundary layer, although changes were less marked for a fouled membrane.
There would also be less likelihood of operating in the pressure-independent region in
the entry length, so there could be an improvement to the flux by use of higher
pressures. They reported that the entrance length may be greater than 1 m, but that
most of the benefits to flux which could occur using higher pressures would be over the
first 20 cm.
One much used model considers a number of resistanc es in series. Therefore, during
the transfer of components from the bulk of the solution to the permeate, the main
resistances are due to the membrane (Rm), the fouling layer (Rf) and the polarisation
layer (Rp).
Therefore the flux can be expressed as

where µ is the viscosity of the solvent. The pressure term may be modified to (∆ρ –∆π),
to account for differences in osmotic pressure, but in most UF applications, the osmotic
pressure differences (∆π ) are negligible
Fouling
In most practical applications, fouling of the membrane takes place and this is a major
operating problem in ultrafiltration. Fouling material collects on the surface of the
membrane (and perhaps internally) and gives rise to a steady decline in the permeate
flux. This could be particularly important for continuous processes operating at steady
state, where a long-term decline in the flux would be extremely detrimental to the
process. It could also give rise to a reduced life for the membrane, due to more stringent
cleaning regimes being needed to remove the fouling.
Fouling is almost impossible to avoid. Removal of colloidal and particulate matter is of
paramount importance prior to processing and should always be carried out. However,

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both Fane and Fell (1987) and McGregor (1986) have described situations where fouling
was apparent, even with ‘pure’ water. McGregor also reported that prolonged exposure
to 200 ppm of sodium hypochlorite caused considerab le flux decline. When more
complex materials are involved, such as proteins, interactions can occur between the
proteins and the membrane material; for example proteins may bind to the membrane
by hydrophobic effects, charge transfer such as hydrogen bonding and electrostatic
interactions, or through combinations of these. Conditions that minimise the amount of
binding to the membrane should be useful in reducing fouling. The two characteristics
which appear to strongly influence fouling are the physicochemical properties of the
membrane and porosity and morphology of the surface. It is also not easy to assess the
individual contributions made by concentration polarisation and fouling toward flux
decline. Wu et al.
(1991) suggest that concentration polarisation is responsible for the rapid initial flux
decline, which is followed after about ten minutes by a long and gradual flux decline,
caused by fouling. Fane and Fell (1987) report that the flux decline due to concentration
polarisation is largely reversible and is therefore different in nature to that caused by
fouling. Work on measuring the membrane resistance (R,) during these early stages of
concentration polarisation showed an increase in its value, suggesting that some fouling
was taking place within the pores, as well as on the surface.
Other suggestions are that there are three phases i n flux decline: (1) due to
concentration polarisation, taking place very quickly (seconds); (2) due to initial
adsorption of protein onto the surface; and (3) due to further adsorption and an
increase in deposit thickness.
Diafiltration
An extension of ultrafiltration employs the addition of water at some stage during the
concentration process. It should be remembered that during ultrafiltration, the
concentration of any component in the retentate can never decrease (unless there is a
true negative rejection). At best, for zero rejection, it will remain constant. However the
amount (yield) of a component of low rejection value is significantly reduced, as is the
dry weight composition, compared to a substance with a much higher rejection.
To effect a reduction in concentration, the retentate needs to be diluted with water;
such a process is known as diafiltration. The net effect of diafiltration is to wash out

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more of the lower molecular weight components. The two main modes of operation are
discontinuous dinfiltration (DDF) and continuous diafiltration (CDF).
Ultrafiltration applications
Some applications of iiltrafiltration will now be discussed, which take advantage of the
opportunity to concentrate macromolecules or to rem ove small molecular weight
components, at ambient temperature, without changing pH or ionic environment.
A wide range of membrane materials is available, the most common being cellulose
acetate, polyamides, polysulphones and polyethersulphones; each with different flux
characteristics, rejection values, and other physicochemical characteristics, such as
charge and extent of hydrophobicity.
Molecular weight cut-offs range from 2000 to 300 000, with operating temperatures up
to 80°C, over the pH range 1 to 14.
Ultrafiltration is also very useful for recovering valuable components from
foodprocessing waste stream and fermentation broths. Probably the greatest impetus
has come from the dairy industry and dairying appli cations. However, in all
applications, flux decline due to concentration polarisation and fouling are probably the
two most important practical aspects.
DAIRY APPLICATIONS
Milk is chemically complex, containing components o f a wide range of molecular
weights, such as protein, fat, lactose, minerals an d vitamins. It also contains
microorganisms, enzymes and perhaps antibiotics and other contaminants. An idea of
the complexity of milk is given by Walstra and Jenness (1984), who list well over 50
components. Whole milk contains about 30-35% protein and about the same amount of
fat (dry weight basis). Therefore it is an ideal fluid for membrane separation processes,
in order to manipulate its composition, thereby providing a variety of products or
improving the stability of a colloidal system. The same applies to skim-milk,
standardised milk and some of its by-products such as cheese whey. Skim milk can be
concentrated up to seven times and full-cream milk up to about five times (Kosikowski,
1986). Milk can be derived from a number of different species, cows' milk being the
most common, with milk from buffalo, goats and shee p being drunk in substantial
quantities throughout the world. Milk is either consumed in its liquid form or converted
to a wide variety of products. Surplus milk is usually preserved as skim milk powder
and butter or anhydrous butterfat. At one time the most valuable component was the

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fat, with cream products and butter fetching high returns. Skim milk was therefore a
by-product of cream and butter manufacture, along with lesser quantities of buttermilk.
Most of the skim milk was dried, and in some situations fed to animals. However, a
further important trend over the last ten years has been the move to a more health-
conscious diet, and in this sense skim milk is more widely used as the starting material
for yoghurts, low-fat cheeses and other desserts.Ultrafiltered milk also forms the
starting material for some of these types of product (de Boer and Koenraads, 1991).
Cheese is a very important product derived from milk. In cheese production, most of the
fat and the casein fractions are incorporated into the curd. However the by-product of
cheese manufacture is whey, which incorporates the whey proteins (about 20% of milk
protein).

The compositions of whey and skim milk are given in Table.
An IDF publication (International Dairy Federation, 1979) gives a summary of the
rejection values obtained during the ultrafiltration of sweet whey, acid whey, skim milk
and whole milk, using a series of industrial membranes.
It should be noted that protein rejections are based on Kjeldahl nitrogen x 6.38. Where
this is the case, it is also measuring non-protein nitrogen (NPN). Rejection values
obtained may not be a true reflection of the behaviour of the protein. Such rejection
values could be lower than expected for materials containing substantial amounts of
NPN. One example is chhana whey, which is produced from a heat coagulated cheese
and contains substantial NPN (Jindal and Grandison, 1992).
However, low rejection values could also be indicative of excessive protein leakage, and
may well warrant investigation if not expected. Rejection values based on true protein
can be determined by Kjeldahl or by using electrophoresis or high performance liquid

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chromatography (HPLC) for analysis and will give a clearer picture of the behaviour of
the proteins.
Rejection values for ash are interesting and are higher than would be expected from
their molecular weight. This would suggest that binding of minerals to the protein is
occurring. During ultrafiltration of whey and buttermilk, Hiddink et al. (1978) observed
that at pH 6.6, anions such as C1- and NO, were preferentially removed. However, at
pH 3.2, cations such as Na', K', and Ca2+ were pref erentially removed. This is an
example of the Donnan effect. Maximum ash removal w as obtained by ultrafiltration at
pH 6.6, followed by diafiltration at pH 3 to 3.5.
Bastian et 111. (1991) compared the rejection values during ultrafi ltration and
diafiltration of whole milk. They found that the rejection of lactose, riboflavin, calcium,
sodium and phosphorus was higher during diafiltrati on than ultrafiltration.
Diafiltration of acidified milk gave rise to lower rejections of calcium, phosphorus and
sodium. Premaratne and Cousin (1991) have performed a detailed study on the
rejection of vitamins and minerals during ultrafiltration of skimmed milk. During a five-
fold concentration the following minerals were concentrated by the following factors: Zn
(4.9), Fe (4.9), Cu (4.7), Ca (4.3), Mg (4.0) and Mn (3.0), indicating high rejection values.
On the other hand, most of the B vitamins examined were almost freely permeating.
The use of nanofiltration or partial demineralisation has been discussed by Kelly et al.
(1991) and its effects on lactose crystallisation, which was improved by about 8% at a
concentration factor of 3, by Guu and Zall (1992).
Both cheese whey and skim milk contain substantial protein and other nutrients. A
great deal of attention has been paid to ultrafiltration of these products to increase their
functionality and hence profitability.
Cheese whey contains only about 10-12% protein on a dry weight basis. However the
proteins are soluble and have excellent functional properties. The main thrust has been
toward using ultrafiltration to increase the protein content, in order to produce
concentrates, which could then be dried to produce high protein powders (concentrates
and isolates) with useful functional properties. Some typical concentration factors (f)
used are as follows:
f'= 5 protein content (dwb) about 35% (similar to skimmed milk)
f'= 20 protein content about 65%
f'= 20; plus diafiltration protein content about 80%

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The product starts to become very viscous at a conc entration factor of about 20.
Therefore if a protein concentrate with higher total solids is required, diafiltration is
required. Skim milk has been investigated also. However because of its original higher
protein content, concentration factors of about 7 are the maximum achievable. Protein
concentrates based on skim milk have not received the same amount of commercial
interest as those based on whey proteins. However, it has been suggested that the
concentrates can be further modified to produce an interesting range of products with
good whipping and foaming characteristics. Rajagopalan and Cheryan (1991) reported
that it was not possible to produce a pure protein isolate by ultrafiltration and
diafiltration of skim milk, due to a high mineral rejection. An isolate containing about
90% protein and 8% ash was obtained.
Yoghurt and other fermented products have been made from skim milk and whole milk
concentrated by ultrafiltration (Renner and El-Sala m, 199 1). Whey protein
concentrates have also been incorporated (de Boer and Koenraads, 1991). Production of
labneh, which is a strained or concentrated yoghurt at about 21% total solids, has been
described by Tamime et al. (1991), by preconcentrating milk to 21% TS. Inorgan ic
membranes have also been used for skim milk, and Da ufin et al. (1992) have
investigated the cleanability of these membranes us ing different detergents and
sequestering agents.
As well as exploiting the functional properties of whey proteins, full cream milk has
been concentrated by UF, prior to cheesemaking. The UF concentrate has been
incorporated directly into the cheese vats. Some advantages of this process include:
increased yield, particularly of whey protein, lower rennet and starter utilisation,
smaller vats, or even complete elimination of vats, little or no whey drainage and better
control of cheese weights. Lawrence (International Dairy Federation, 1989), suggests
that concentration below a factor of 2 gives protein standardisation, reduced rennet and
vat space, but no increased yield. At concentration factors greater than 2, an increased
yield is found.
Some problems result from considerable differences in the way the cheese matures and
hence its final texture and flavour. The types of cheese that can be made in this way
include: Camembert type cheese, mozzarella, feta and many soft cheeses. Those which
are difficult include the hard cheeses such as Cheddar and also cottage cheese; the
problems are mainly concerned with poor texture. Some debate about compositional

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standards for cheeses produced by such technology exists. More discussion is given by
Kosikowski (1986).
Further reviews on the technological problems arisi ng during the conversion of
retentate into cheeses are discussed by Lelievre and Lawrence (1988). Spangler etal.
(1990a) have investigated how the quality of Gouda cheese was affected by
concentration factor (3.6-5), rennet concentration and coagulation temperature. The
best cheeses were made from the five-fold concentration and a rennet concentration of
0.14%. Higher rennet concentrations lead to the development of very bitter flavoured
cheese. Spangler et al. (1990b), for example, found that Gouda production f rom
ultrafiltered milk could be improved by attention to detail; such as preacidification of
milk prior to ultrafiltration; the amount of rennet was also very important. Sachdeva
and Reuter (1991) produced acceptable chhana by ultrafiltration; it had lower moisture
content, giving a yield improvement of 31.4% (product basis) and 16.4% on a dry matter
basis. Quarg is also produced from ultrafiltered milk.
Whole milk has been concentrated five times by ultrafiltration and the retentate heated
at 120°C for 5 min before being recombined with the permeate prior to pasteurization
(Kosikowski and Mistry, 1990). This procedure is claimed to produce an extended shelf
life product with a superior flavour to a conventionally pasteurised product. Sweetsuir
and Muir (1985) have investigated the production of sterile concentrates, produced by
the ultrafiltration of skim milk, with and without the addition of fat. Such concentrates
were able to withstand sterilisation at 120°C for 7 min. The organoleptic qualities were
improved by the presence of fat and the heat stability was improved by procedures
which reduced the levels of salts in the concentrates.Protein hydrolysates produced
from milk proteins have been discussed by Donnelly (1991). Ultrafiltration is an
extremely valuable method of concentrating and reco vering many of the minor
components, particularly enzymes from raw milk, many of which would be inactivated
by pasteurisation.
TREATMENT OF INDUSTRIAL EFFLUENTS (DAIRY, DISTILLER Y, TANNERY,
TEXTILE, PAPER AND SUGAR INDUSTRIES)
The mechanisms by which pollution or waste may be reduced at source are varied. They
may involve changes in technology or processes, alteration in the raw materials used or
a complete restructuring of procedures. Generally s peaking, biotechnological

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interventions are principally limited to the former aspects, though they may also prove
instrumental in permitting procedural change.
The main areas in which biological means may be rel evant fall into three broad
categories:
process changes;
biological control;
bio-substitutions.
In the following discussions of these three groups, it is not suggested that the examples
cited are either comprehensive or exhaustive; they are simply intended to illustrate the
wide potential scope of applications open to biotechnology in clean manufacturing. For
precisely the reasons mentioned in respect of the economic aspects of this particular
area of industrial activity, the field is a fast evolving one and many more types of
biotechnological interventions are likely in the future, especially where commercial
pressures derive a competitive advantage.
Process Changes
Replacement of existing chemical methods of production with those based on microbial
or enzyme action is an important potential area of primary pollution prevention and is
one role in which the use of genetically modified organisms could give rise to significant
environmental benefit. Biological synthesis, either by whole organisms or by isolated
enzymes, tends to operate at lower temperatures or, as a result of high enzymatic
specificity, gives a much purer yield with fewer byproducts, thus saving the additional
cost of further purification. There are many examples of this kind of industrial usage of
biotechnology. In the cosmetics sector, there is a high demand for isopropyl myristate
which is used in moisturising creams. The conventional method for its manufacture has
a large energy requirement, since the process runs at high temperature and pressure to
give a product which needs further refinement befor e it is suitable for use. An
alternative approach, using enzyme-based esterification offers a way to reduce the
overall environmental impact by deriving a cleaner, odour-free product, and at higher
yields, with lower energy requirements and less waste for disposal.
TANNERY OR LEATHER INDUSTRY
The leather industry has a lengthy history of using enzymes. In the bating process,
residual hair and epidermis, together with nonstructural proteins and carbohydrates,
are removed from the skins, leaving the hide clean, smooth and soft.

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Traditionally, pancreatic enzymes were employed. Moreover, something in the region of
60% of the input raw materials in leather manufactu ring ultimately ends up being
discarded and enzyme additions have long been used to help manage this waste. Recent
advances in biotechnology have seen the upsurge in the use of microbially-derived
biological catalysts, which are cheaper and easier to produce, for the former
applications, and the possibility of converting was te products into saleable
commodities, in the latter.
As well as these improvements on existing uses of biotechnology, new areas of clean
application are emerging for tanners. Chemical methods for unhairing hides dissolve
the hairs, making for efficient removal, but adding to the treatment cost, and the
environmental implications, of the effluents produced, which are of high levels of COD
and suspended solids. Combining chemical agents and biological catalysts significantly
lessens the process time while reducing the quantities of water and chemicals used. The
enzymes also help make intact hair recovery a possibility, opening up the prospect of
additional income from a current waste. It has been estimated that, in the UK, for a
yearly throughput of 400 000 hides, enzymatic unhairing offers a reduction of around
2% of the total annual running costs (BioWise 2001) . While this may not seem an
enormous contribution, two extra factors must be borne in mind. Firstly, the leather
industry is very competitive and, secondly, as effluent treatment becomes increasingly
more regulated and expensive; the use of clean manu facturing biotechnology will
inevitably make that margin greater.
Degreasing procedures are another area where biotechnological advances can benefit
both production and the environment, since conventi onal treatments produce both
airborne volatile organic compounds (VOCs) and surfactants. The use of enzymes in
this role not only gives better results, with a more consistent quality, better final colour
and superior dye uptake, but also considerably reduces VOC and surfactant levels. The
leather industry is also one of the places where biosensors may have a role to play. With
the ability to give almost instantaneous detection of specific contaminants, they may
prove of value in giving early warning of potential pollution problems by monitoring
production processes as they occur.
TEXTILE INDUSTRY
There is a long tradition of the use of biological treatment methods in the clothing and
textile industry, dating back to the first use of amylase enzymes from malt extract, at

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the end of the nineteenth century, to degrade starch-based sizes for cheap and effective
reduction of fabric stiffness and improvement to its drape. Currently, novel enyzmatic
methods provide a fast and inexpensive alternative to traditional flax extraction by
breaking down the woody material in flax straw, reducing the process time from seven
to ten days, down to a matter of hours.
The enzyme-based retting processes available for use on hemp and flax produce finer,
cleaner fibres, and, consequently, novel processing techniques are being developed to
take advantage of this. Interest is growing in the production of new, biodegradable
polymeric fibres which can be synthesised using mod ified soil bacteria, avoiding the
current persistence of these materials in landfills, long after garments made from them
are worn out.
In natural fibre production enzymes are useful to remove the lubricants which are
introduced to prevent snagging and reduce thread breakage during spinning, and to
clean the natural sticky secretions present on silk. The process of bioscouring for wool
and cotton uses enzymes to remove dirt rather than traditional chemical treatments
and bio-bleaching uses them to fade materials, avoiding both the use of caustic agents
and the concomitant effluent treatment problems suc h conventional methods entail.
Biological catalysts have also proved effective in shrink-proofing wool, improving quality
while ameliorating the wastewater produced, and red ucing its treatment costs,
compared with chemical means.
A process which has come to be called biopolishing involves enzymes in shearing off
cotton microfibres to improve the material’s softness and the drape and resistance to
pilling of the eventual garments produced.
Biostoning has been widely adopted to produce ‘stone-washed’ denim, with enzymes
being used to fade the fabric rather than the original pumice stone method, which had
higher water consumption and caused abrasion to the denim.
However, perhaps the most fitting example of environmental biotechnology in the textile
industry, though not really in a ‘clean technology’ role, is the incorporation of adsorbers
and microbes within a geotextile produced for use i n land management around
railways. Soaking up and subsequently biodegrading diesel and grease, the textile
directly reduces ground pollution, while also providing safer working conditions for
track maintenance gangs and reducing the risk of fire.

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PAPER INDUSTRY
The pulp and paper industry is quite old. In India, more than 150 paper and board
mills with an installed capacity of nearly 3 million tones year–1 are in operation of
which 36 are the large mills with a production capacity >55 tones day–1, and the rest
are small mills with production capacity <30 tones day–1. The large pulp-paper mills
equipped with soda recovery discharge about 270 to 450 l effluent kg–1 of paper
containing 40 to 50 g lignin kg–1 bleached paper produced. Contrary to that, the small
paper mills without soda recovery discharge nearly 300 to 400 l of black liquor effluent
containing 200 to 250 g lignin kg–1 of paper manufactured.
More than 150×10
6
tons of pulp is produced annually and about 50×10
6
tons of lignin
together with the chemicals used is released from the P&P industry indicating that a lot
of efforts have to be undertaken to handle the enor mous amounts of hazardous
potential.
One of the major problems of effluent discharge from the pulp and paper industry is its
brown/black color, generally known as black liquor. The color of these wastewaters is
primarily due to lignin and its derivatives, which are discharged in such effluents
mainly from the pulping, bleaching, and chemical recovery stages of the plant. High
molecular-weight chlorinated lignins are generally not removed from the effluents.
These products include chlorolignins, chlorophenols, and chloroaliphatics. Besides,
these paper mill effluents are highly alkaline and alter the pH of the soil and water
bodies into which they are discharged.
SUGAR INDUSTRY
Wastewaters containing molasses are generated by distilleries, fermentation industries,
sugar mills, Pharmaceutical companies and other molasses-based industries. Molasses
from sugarcane industry is the common raw material used in ethanol production due to
its easy availability and low cost.
India is the second largest producer of ethanol in Asia. There are 319 distilleries in
India with an installed capacity of 3.25 billion litres of alcohol. The Central Pollution
Control Board (CPCB) categorizes distillery industry among 17 top polluting industries
in India. For every one litre of alcohol produced, 10–15 l of spent-wash are generated
and thereby a typical distillery producing ethanol from cane molasses generates nearly
half million liters of spent-wash daily.

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Approximately, 40 billion litres of spent-wash is generated annually in India alone for
the production of 2.3 billion litres of alcohol. Distillery is one of the most highly
polluting and growth-oriented industries in India with reference to the extent of water
pollution and the quantity of wastewater generated. The Population equivalent of
distillery waste based on BOD has been reported to be as high as 6.2 billion, which
means that the contribution of distillery waste in India to organic pollution is
approximately seven times more than the contribution by the entire population.
These contain mostly dark brown colored recalcitrant compounds collectively termed as
melanoidin polymers which are the product of Maillard reaction between the amino
acids and carbonyl groups present in molasses.
With their high biochemical and chemical oxygen dem and, these effluents are
environmental hazards. When released in water bodies they cause oxygen depletion and
associated problems, and/or if released in soil they reduce the soil alkalinity and
manganese availability, inhibit seed germination and affect vegetation. Besides causing
unaesthetic discoloration of water and soil, melanoidin pigments are also toxic to
microorganisms present in soil and water.
Dark brown color of these effluents is highly resistant to microbial degradation and
other biological treatments. Melanoidins have recal citrant compounds; thus the
conventional treatment methods are not effective for complete color removal from this
stream and color can even be increased during anaer obic treatments, due to re-
polymerization of compounds. Anaerobic digestion of effluents produces dark brown
sludge which is used as fertilizer and the colored waters are discharged after diluting
them several folds with water. Thus ultimately fresh water resource which is a precious
commodity in most parts of the world is wasted. The spentwash is highly colored with
an extremely high Chemical Oxygen Demand (COD) load and contains high percentage
of dissolved organic and inorganic matter. The Biochemical Oxygen Demand (BOD) and
COD, the index of its polluting character, typically range between 35,000–50,000 mg L-
1 and 80,000–1,00,000 mg L-1 respectively.
Apart from high organic content, distillery wastewater also contains nutrients in the
form of nitrogen, phosphorus and potassium that can lead to eutrophication of water
bodies. Spent-wash disposal even after conventional treatment is hazardous and has a
high pollution potential due to the accumulation of non-biodegradable recalcitrant
compounds, which are mostly colored and in a highly complex state. Melanoidins have

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anti-oxidant properties causing toxicity to many microorganisms involved in wastewater
treatment processes. Lowering of pH value of the streams, increasing organic load and
obnoxious smell are some of the major problems due to distillery wastewater. The
distillery wastewater poses a serious threat to water quality in several regions of the
country. Disposal on land is equally detrimental causing a reduction in soil alkalinity
and inhibition of seed germination. In addition to pollution, increasingly stringent
environmental regulations are forcing distilleries to improve existing treatment and also
explore alternative methods for effluent management.

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UNIT: 3 BIOMINING AND BIODIESEL
Bioleaching of ores to retrieve scarce metals, Bio – mining; Biodiesel production
from Jatropa, Pongamia and Castor

BIOLEACHING OF ORES TO RETRIEVE SCARCE METALS
Bioleaching is a process based on the ability of microorganism (bacteria and fungi) to
transform solid compounds into soluble and extracta ble elements, which can be
recovered. It represents a 'clean technology' in the mining industries with low cost and
capital inputs required as compared to conventional methods that are very expensive
for the recovery of metal ions from low and lean grade ores (Devasia and Natarajan,
2004).
The bioleaching allows the cycling of metals by a process close to natural biochemical
cycles reducing the demand for resources such as or es, energy or landfill space
(Devasia and Natarajan, 2004; Xu and Ting, 2009).
Copper was first utilized for making tools in about 6000 BC. All industries for their
equipments are dependent on metals mined from ores. Besides, their significant role in
technology progression, their natural abundance in the environment sometimes inflicts
danger to the environment. For instance, cadmium causes lungs cancer whereas lead
causes cancer in kidney etc (LaGrega, 1994). Furthermore, mining wastes disposals as
well as industrial effluents release huge amounts of toxic metals into the atmosphere
are sources of many health as well as environmental hazards. Ever increasing demand
for metals from limited sources, the sustainable development approach seems a feasible
solution. In order to appreciate sustainable develo pment framework for metals
production, a brief background of metal resources i s necessary generally metal
resources can be classify into two main groups:
a) Naturally occurring resources, providing majority of metals for industries
b) Second hand resources e.g. industrial wastes and used materials
An ore contain metals in the form of minerals as well as naturally occurring inorganic
substance, or an aggregate of minerals, gangue minerals where gangue is undesired
minerals which associated with ore and are mostly nonmetallic in nature.
Types of ore
Ores are divided into:
High grade ores, the ones in which metals concentration is relatively high.

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Low grade ores, the one with little concentration of metals like shale and schist etc.
Techniques for extraction of metals
Extraction metallurgy
In a broader sense, extractive metallurgy or mining is the extraction of metals from raw
materials by physical and chemical processes involving their properties in bulk as well
as at the atomic level. Generally, the first step is the separation of undesired minerals;
gangue from ore followed by "ore dressing" which involves disintegration to small sizes
makes separation of different kinds of minerals. Separation of valuable fragments based
on some physical properties such as density, magnetic properties as well as surface
energy leads to assistance of chemical process to be performed with higher efficiency.
Finally, in the extraction process, the metal is produced. Different techniques can be
utilized during extraction process are given below:
Pyrometallurgy techniques follow high temperature heating process. These processes
are mainly applied to oxide ores. In this process the ore is heated in the presence of
heating agent in furnace resulting in the production of molten metal and slag along with
impurities which is separated from metal at working temperature.
Pyrometallurgical techniques involve following steps
Preliminary treatment such as roasting and calcinations which changes the physical
and chemical properties of the ore to make it suitable for extraction purposes. These
pretreatments followed by the
Liquidification and smelting in which metals become melted and separated in to
number of liquid layers containing metals (Alcock, 1976).
Hydrometallurgy techniques involve aqueous solutions as well as inorganic solvents
to achieve the desired product. This technique is utilized when high purity of metallic
product is required with less environmental hazards. This technique is performed in
liquid environment, involving different steps like leaching of desired metal contents in
aqueous solvent followed by the removal of impurities from metal surface. At the end,
metals recovered either as precipitates (Arsdale, 1953).
Electrometallurgical techniques involve various electrical operations for metals
processing. These electrical energy operations increase the efficiency of the process but
also increase the cost of the process. These include electrical furnace processing,
electrowinning and electrorefining (Alcock, 1976).

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Electrowinning involves the extraction of metals in purified form from their ore using
appropriate electrolytes, among various types of electrolytes, sulphuric acid is most
preferred (Felsher et al., 2000).
Electrolysis. In which an electric current splits a liquid (molten salts) in to its chemical
parts.
Biohydrometallurgy techniques involve microbes in metals extraction e.g. bioleaching,
biosorption, biobenefication etc. Biohydrometallurgy has originated about 2000 years
prior to detection of microorganisms (Rossi, 1990). Hallberg and Rickard, (1973)
reported the microbial recovery of copper from the copper deposit of the Falun Mine in
central Sweden since 1687. Rossi (1990) cited in his book “Biohydrometallurgy “that
copper was microbially recovered from copper deposits in Rio Tinto in Spain in 1752 on
commercial scale (GerbelIa, 1940). Rudolfs and Helbronner (1922) reported the
extraction of zinc sulphide from low-grade zinc sulphide ores economically. Bacterial
leaching of metals from acid mine drainage appeared in 1950's (Colmer et al., 1950;
Temple and Colmer, 1951; Leathen et al., 1953). Kennecott Copper Corporation in
1950s patented copper bioleaching for the first time (Zimmerley et al, 1958).
Today, biohydrometallurgy is an interdisciplinary p romising technology used for
obtaining valuable metal compounds from low grade o res and for detoxification of
industrial waste products (Rossi, 1990; Brombacher et al., 1997). Moreover, it has
provided information about companies applying biole aching techniques for metals
recovery. Many reports exhibited recovery of precious metals from industrial wastes
(Francis and Dodge, 1990; Strasser et al., 1994; Bousshard et al., 1996), sewage sludge
(Blais et al., 1993; Benmussa et al., 1994; Strasser et al., 1995), soi1 (Bartlett, 1993),
coal (Dugan, 1986) as well as fossil fuels (Andrews et al., 1993).
Microbial weathering/ bioleaching
Microbial leaching is the mining of metals from their ores using microbes.
Microbial technology offers an economic substitute for the mining industry, at a time
when high grade mineral resources are being exhausted (Lawrence, 1994).
In general, bioleaching is a process described as “Dissolution of metals from their
mineral sources by certain naturally inhabiting microbes” or the use of microorganisms
to transform elements so that the elements can be e xtracted from a material when
water is filtered through it (Lundgren and Malouf, 1983).
Importance of bioleaching:

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Worldwide assets of high grade ores are going to an end as a result of rapidly increasing
metals demand. Up till now, low and discarded ore deposits are the substitute of these
exhausted reserves of high grade ores but problems exist in the extraction of metals
from these discarded ores, using conformist techniques; high consumption of energy
and less output. Another most important crisis is environmental outlay due to the
elevated level of contamination from these technolo gies hardens environmental
standards (Devasia and Natarajan 2004). There is growing concern that the heavy metal
contents of soils are increasing as the result of industrial, mining, agricultural and
domestic activities, all through the world. Not like many other pollutants, heavy metals
are difficult to remove from the environment. These metals cannot be chemically or
biologically degraded and are ultimately indestructible (Anjum et al., 2010).
The poisonous effects of heavy metals result mainly from the interaction of metals with
proteins in form of enzymes and inhibition of metabolic processes. When heavy metals
accumulated in soils, such as copper, cadmium, lead, iron, manganese, zinc, nickel,
mercury and chromium can be present in toxic concen tration for plants, animals, and
human and also to aquatic life, therefore reliable remediation techniques are required to
clean up the site. In contrast to organic pollutants, metals are not mineralized by
microorganisms but can be oxidized or reduced i.e. transformed to different redox
stages or complexed by organic metabolites (Ren et al., 2009).
Biohydrometallurgy including biotechnology have bee n introduced in different
industries in well-mechanized and engineered systems for the sake of energy production
during mining operation in different parts of the world for the last 4 decades.
Biohydrometallurgy encompass different disciplines on the basis of interaction between
metals and microbes like bioremediation, biosorption, bioaccumulation and bioleaching
(Brombacher, 1997; Devasia and Natarajan 2004). All these processes are usually slow
but more environment friendly, require less energy consumption than physicochemical
processes which require more energy and release harmful gases like sulphur dioxide
and produce environmental hazards (Mishra et al., 2004). This process has the
potential to offer a much needed step-change in the technology for processing low grade
ores (Tzeferis and Agatzinin-Leonardou, 1994; Valix et al., 2001a).
Forms of microbial leaching:
There are two types of microbial leaching on the basis of microbes which are known:
Chemolithoautotrophic leaching and Heterotrophic leaching

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Chemolithoautotrophic (Autotrophic) leaching.
Chemolithoautotrophic is a term, which encompasses the autotrophic creatures that
oxidize sulphur as a source of energy, mostly Acidithiobacillus type, leaching processes,
which have been surveyed intensively. It is a suitable type of leaching for minerals rich
in sulphur or sulphides (Anjum et al., 2009a).
Heterotrophic leaching.
Heterotrophic is a different form of microbial leaching in which the organisms that need
organic carbon sources to survive the leaching processes, in this type of leaching,
metabolites excreted by microorganisms are the results of chemical reactions, occur
between ore and microorganisms. This type of leaching is suitable for minerals low in
sulphur and sulphides, under low pH conditions due to organic acid production by
microorganisms; at pH values of 6-9 when some leach ing by-products or non-acidic
complexing agents are produced as metabolites; or where pH is high due to ammonia
production from catabolism of proteins (Sukla et al., 1995).
Heterotrophic microorganisms with leaching activity reported previously are mostly
filamentous fungi and bacteria. Metal leaching by h eterotrophic microorganisms
generally involves an indirect as well as direct process with microbial production of
organic acids, amino acids and other metabolites. These metabolites dissolve metals
from minerals by displacement of metal ions from the ore matrix by hydrogen ions or by
the formation of soluble metal complexes and chelates. The most important species of
fungi are Aspergillus and Penicillium Sp. because of their ability to excrete abundant
concentration of organic acids (Rezza et al., 2001; Anjum et al., 2010).
Mechanism of bioleaching
Bacterial leaching
A generalized reaction can be used to articulate the biological oxidation of a mineral
sulphide involved in leaching:

Where M is the bivalent metal. Until now, the biole aching/biobeneficiation of ores
includes the following two types of mechanism, invo lved in microbial metal
solubilization of sulfide minerals.
Direct microbial leaching: In this mechanism, microorganisms have direct conta ct
with mineral surface. Exact mechanism is still not completely understood but specified

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for autotrophs, can attach to the specific surface not to the whole mineral surface thus,
metals solubilization (Ehrlich, 1996) is due to electrochemical interaction. In this case,
cells attach to the mineral surface directly by establishing close physical contact
between the bacterial cell and the mineral sulfide surface and the oxidation to sulfate
takes place via several enzymatically catalyzed steps. The adsorption of cells to
suspended mineral particles takes place within some minutes or hours (Lizama and
Suzuki, 1991).
In this way, microorganism development and the heav y metal leaching take place
together. This technique is easy to perform, but microbial metabolism and growth can
negatively affect by dissolved metal ions thus, lim iting the bioleaching process
efficiency. In this process pyrite is oxidized to sulfate according to following reactions:

The bacterial oxidation of pyrite is summarized by the following reaction:

(Holmstrom, 2000; Evangelou, 2001).
Non-iron metal sulfides like cuvellite (CuS), chalcocite (Cu
2S), sphalerite (ZnS), galena
(PbS), molybdenite (MoS
2), stibnite (Sb2S2), cobaltite (CoS), millerite (NiS) can be
oxidized by acidophiles by direct interaction.

(Devasia and Natarajan, 2004; Brocht et al., 2004; Conner, 2005; Leahy et al., 2005;
McGuire et al., 2001; Sadowski, 2005).
Indirect microbial leaching: Another mechanism, called ‘indirect’ mechanism, involves
the generation of organic and inorganic acids by fungal and bacterial strains (Sadowski,
2005). Bioleaching carried out in two stages (Camselle et al., 1998). In the first one, the
microorganisms are allowed to grow in an adequate medium under appropriate cultural
conditions to produce active metabolites for the leaching process. Afterwards, the spent
culture medium under aggressive leaching conditions (low pH, high temperature, etc.) is
used, in the second stage as a leaching agent for the mineral in the absence of growing
microorganisms. Therefore, increase the metals remo val. In many cases direct
mechanism is favored over indirect mechanism due to the direct physical contact of
microbes with the minerals surface (Gehrke et al., 1995)

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Fungal leaching: Fungal leaching of metals from minerals are mainly based on three
principle mechanisms reported by Berthlin, (1993) such as
Acidolysis: Protonation of oxygen atoms occurred around the s urface of metallic
compounds. The protons and oxygen associated with water displace the metal from the
surface.
Complexolysis: This mechanism is somewhat slower than acidolysis . Metal complex
formation results in the solubilization of the metal ion e.g. complex of oxalic acid and
iron, magnesium as well as citric acid and calcium and magnesium (Berthlin et al.,
1993). Besides, this process often reduces the toxicity of heavy metals towards fungi.
Redoxolysis: Reduction of metal ions in an acidic environment, such as reduction of
ferric iron and manganese under the influence of oxalic acid (Brombacher et
al., 1997; Berthelin, 1993). A series of organic acids are formed by fungal metabolism
resulting in organic acidolysis, complex and chelat e formation (Berthelin, 1993;
Burgstaller et al., 1992). The metabolic process of fungi is parallel to those of higher
plants with the exception of carbohydrate synthesis. Organic acids are produced from
glucose in glycolytic pathway (Nalini and Sharma, 2004). Bioleaching processes are
mediated by complex formation between extracted org anic acids and ores thus,
increasing metal dissolution by lowering the pH and increasing the load of soluble
metals by complexation/chelating into soluble organic-metallic complexes (Vasan et al.,
2001).
Most active leaching fungi are from the genera Penicellium and Aspergillus
(Burgstaller and Schinner, 1993). Aspergillus niger exhibited good potential in
generating organic acids like oxalic and citric, malic and tartaric acids, effective for
metal solubilization (Mulligan et al. 2004; Anjum et al., 2009). Best recovery of metals
has been achieved by citric and oxalic acids as reported earlier (Tarosova, 1995).
BIOMINING
Biomining is a general term used to describe the use of microorganisms to facilitate the
extraction of metals from sulfide or iron-containing ores or concentrate. The metal-
solubilization process is due to a combination of chemistry and microbiology: chemistry,
because the solubilization of the metal is considered to be mainly a result of the action
of ferric iron and/or acid on the mineral, and microbiology, because microorganisms
are responsible for producing the ferric iron and acid. Since the metal is extracted into
water, the process is also known as bioleaching and sometimes as biooxidation (used in

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the case of gold recovery where the metal remains in the mineral, and, therefore, the
term bioleaching is inappropriate)
Microbial diversity in bioleaching
Autotrophic microorganisms
Several microbes can play an important role in acti ve removal of sulphurous
compounds as well as metals from minerals and fossil fuels originated from ores, shales
and schist etc. These include bacteria, tremendously acidophilic (flourishing at pH
below 3) called chemolithoautotrophs, (Brierley, 1995; Ehrlich, 1996) utilize either
reduced inorganic sulfur or ferrous (II) iron as energy source and grow autotrophically
by fixing CO
2 from atmosphere and converted to organic carbon by Carbon Benson
cycle (Sand et al., 1995).
Bioleaching of metals by autotrophs
Some recent research demonstrated the microbial rec overy of rare metal ions like
vanadium, uranium and radionuclide, molybdenum and aluminum, etc by
chemolithotrophic bacteria in acidic environment ( acidithiobacillus ferrooxidans,
acidithiobacillus thiooxidans, sulfolobus caldarius) (Konopka et al., 1993; Tasa, 1998;
Anjum et al., 2009a). These bacteria utilized sulphide minerals as a source of energy
present in shale as impurities.
These bacteria remove pyritic sulfur from coals, schist and shales (Groudev and
Groudeva, 2001). These practices are now used for the solubilization of number of metal
ions from their sulfide minerals such as sulfidic copper and refractory gold ores (Nemati
et al., 1998; Pina et al., 2005) as well as manganese and iron ores (Abdelouas et al.,
2000). Acidithiobacillus thiooxidans isolated by Waksman and Joffe, (1922) oxidize
elemental sulfur rapidly (Rossi, 1990) whereas Acidithiobacillus ferrooxidans removed
>95 % of the pyrite contents from kerogen (organic compound) rich shale within four
weeks of contact time and assessed oxidative dissolution of pyrite from shale residue
(Mishra et al., 1984).
The enhanced metal leaching from shales by Fe- and S-oxidizing bacteria has been
reported for Polish and Russian black shales (Iskra et al., 1980; Konopka et al., 1993).
When Thiobacillus ferrooxidans was added to a mixture of sulfide minerals during
flotation, the cells adhered preferentially to pyrite and suppressed its floatability
(Nagaoka et al., 1999) thus, by using the microbes, pyrite could be estranged from
chalcocite, molybdenite, millerite, and Galena (Hackl, 1997). Menon and Dave (1993)

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studied the leaching of metals from ore sample obtained from Ambamata mines using
shake flask, tank and column leaching techniques and recovered 63, 38 and 17% of Zn,
Pb and Cu respectively.
Willscher and Bosecker (2003) investigated that bacterial strains leached up to
38% of the manganese and up to 46% of the magnesium and were more selective than
chemical processes. Scanning electron microscopy (SEM) analysis of the solid material
after leaching showed considerably more surface erosion of the biologically leached
material than observed in the control experiments.
Soil samples contaminated with crude oil under go bioremediation by using bacterial
strains. Methyl isocyanate tests (MIC) were performed to determine the tolerance of
bacteria to vanadium salt as investigated by Jenifer et al. (2004).
Mishra et al. (2004) found bacterial leaching technique, an economical alternative in the
mining industries and recovered metals like zinc, copper, aluminum, nickel, lead,
manganese and iron from different mine drainage, wa ste sludge, river
sediments,electronic scrap.
Uryga et al. (2004) extracted cobalt, copper and arsenic from two flotation byproducts
of mine whereas microbes were isolated from water that were well adopted to metals
leaching and shrinking core model was applied to bioleaching of metals from various
particle size fractions.
Pradhan et al. (2006) reported effective recovery of 39% of alumina at 10% ore density
just after six days by Bacillus circulans in situ bioleaching and found bacterial leaching
more efficient in alumina recovery than fungal bioleaching.
Orquidea et al. (2008) assumed Cuban serpentines as one of the richest deposits of Ni
and Co in the world. In direct leaching experiments, Acidithiobacillus thiooxidans were
used to produce inorganic acids as metabolites using elemental sulphur as energy
source in batch experiments. Higher percentage of metals solubilization like Co (100%)
and Ni (80%) achieved from laterite tailings just after 15 days of incubation. In indirect
bioleaching experiments using sulphuric acid metabo lites, significant recovery of Ni
(79%) and Co (55%) was also detected.
Junya et al. (2009) utilized the sulphate reducing bacteria for the retrieval of valuable
metals from a bioleaching solution. Batch and continuous experiments were carried
out. Hydrogen sulphide was obtained in the first reactor followed by subsequent metal

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precipitation in the second reactor, resulted in the faster precipitation of valuable
metals in the form of sulphide.
Heterotrophic microorganisms
Heterotrophic microbes like fungi require organic supplements to enhance their growth
and contribute to metal extraction as a result of excretion of organic and inorganic acid
metabolites e.g., lactic, oxalic, citric, gluconic, malic, phytic as well as sulphuric acid
(Burgstaller and Schinner, 1993; Bosecker, 2001). Aspergillus sp. and penicillium sp. are
widely used species for bioleaching of metals from ores.
Generally, fungi can work well between pH ranges of 2-8, a temperature range of
20-40
o
C. Fungi have relatively high tolerability to heavy metals (Burgstaller and
Schinner, 1993) therefore have been utilized in the past for the leaching of
carbonaceous low grade ores, mining wastes. Degradation of persistent carbon sources,
such as charcoal and black shale, is accelerated by fungal activity which results in the
liberation of inorganic minerals and metals (Wengel et al., 2006).
Bioleaching of metals by heterotrophs
Several species of fungi can be used for bioleaching process. Enormous literature has
been published concerning the ability of fungi to e xtract metals from different
resources. Wenberg et al. (1971) extracted metals from carbonaceous low grade ores
using fungal strains whereas Silverman and Munoz (l971) detected titanium from rock
by fungal activities. Golab and Orlowska (1988) reported tartaric acid and citric acid
production by Aspergillus Niger to leach out zinc oxide up to 90%. Groudev (1990)
carried out bioleaching of gold from ores and gold dust using Penecillium spp.
Penicillium spp. can produce some organic acids such as citric acid, tartaric acid, lactic
acid and malic acid etc, so it can be used for bioleaching of different ores. The use of
microorganisms in ore leaching to extract metals has been commercialized. Earlier
investigations have revealed that bacteria and fungi could be effectively used to extract
iron and silica from clays, sands and low grade ores (Ogurtsova et al., 1990).
Ehrlich and Rossi (1990) accounted that not only ba cterial strains but also fungal
strains can solubilize metals like Ca, Mg, and Zn from silicates might be due to the fact
that these strains produce organic acids (oxalic and citric), have affinity towards metals
and extraction of silicon from aluminum silicate ha s resulted from complexation
reaction of metals instead of direct attack of acids.
Burgstaller et al. (1992) reported 30g/L zinc oxide recovery from filter dust

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correspond to the maximum amount of H+ availability of organic acid metabolites (citric
acid) produced by penicillium sp. upto 9 days of leaching period.
Tzeferis (1994a; 1994b; 1994c) has conducted research work on nickel recovery of up to
72% from non-sulphide ore using fungal strain. He h as studied variety of fungal
screening, adaptation and leaching techniques in order to obtain optimum conditions
for nickel recovery. In his study, molasses has been detected as suitable substrate for
fungi while Mulier et al (1995) studied the leaching of zinc from an industrial filter dust
using Penicillium sp. And two bacterial strains and compared the effect of amino acid
and citric acid in leaching process. Citric acid showed better results as compared to
amino acid. Cameselle et al. (1998, 2003) investigated better recovery of iron from
kaolin with oxalic acid instead of citric acid produced by Aspergillus Niger. Bousshard et
al., (1996) conducted leaching experiments in shake flasks for the recovery of different
metals like Al, Cd, Cr, Cu, Zn and Mn from fly ash using Aspergillus Niger. Agatzini and
Tzeferis (1997) leached up to 60% and 50% of Ni and Co, respectively, from nonsulfidic
nickel ores using Aspergillus and Penicillium spp. They showed the presence of citric,
oxalic and other organic acids in the leach liquors , indicating their role in the
bioleaching process.
Bosecker (1997) extracted copper, uranium and gold from low grade ore as a result of
production of organic acids and chelating and complexing compounds excreted
into the environment by microbial activities of fungi and bacteria whereas Shanableh
and Ginige (1999) reported the fungal recovery of Cr, Ni, Zn, Cu, Cd and Pb up to 50%,
79%, 45%, 24%, 30% and 82% respectively from ore.
Bioleaching of important elements like Al, Fe, Co, Cu, Zn, Sn, Pb and Ni from their ores
using fungal species like Penicilli as well as Aspergilli has been examined earlier (Sukla
et al., 1993; Castro, 2000; Valix et al., 2000; Valix et al., 2001; Sayer and Gadd, 2001;
Brandl et al., 2001; Rezza, 2001; Tang, 2004)
Different experiments performed by Mulligan et al. (2004) have shown that two fungal
strains (Aspergillus niger, Penicillium simplicissimum) were able to mobilize Cu and Sn
by 65%, and Al, Ni, Pb, and Zn by more than 95% due to the production of some
organic acids such as citric acid, tartaric acid, lactic acid and malic acid etc.
Rashid et al. (2001) studied the behavior of different fungal strains towards metals
bioleaching. Aspergillus niger showed better production of organic acid metabolites at
5% ore density used for the recovery of aluminum (4g/L) from ore.

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Venkateshwara et al. (2002) examined 20 isolates of fungal strains of genera
Aspergillus, Penicillium and Rhizopus for copper extraction from low grade chalcopyrite
ore.
Experiments were conducted with 1 % ore density in shaking flasks for 20 days. Out of
20, four strains showed better solubilization of copper. Maximum recovery (78 mg/L)
was detected in case of Aspergillus flavus as well as Aspergillus niger strain whereas
Valix et al. (2000a) found Penicillium and Aspergillus spp. most efficient
microoorganisms not only in organic acids production but also in leaching of nickel up
to 36 %, cobalt 54 % and iron 76 % from laterite in direct leaching process that were
comparable with chemical leaching recovery of up to 79.5 Ni, 71 Co and 50 wt % Fe
whereas nickel ions have been extracted as function of time on laterite gangue as
reported by Valix et al. (2000b).
LEACHING TECHNIQUES
Leaching techniques can be divided into three main areas depending upon the working
volume.
Laboratory-scale (0-10dm
3
): Laboratory-scale leaching techniques can be categorized
into two main groups; the first type involves a qualitative or semiquantitative analysis of
an ore using microbes. This class includes manometric and stationary flask techniques.
The second type, on the other hand, engrosses the assessment of quantitative measures
in an analytical approach including air-lift percolator and shake flask, tank, and
pressure bioleaching (Rossi, 1990; Bosecker, 1994; Devasia and Natarajan, 2004).
Manometric technique: This technique, involves the assessment of evolved gases at
constant temperature (Rossi, 1990).
Stationary flask technique: This technique the simplest of all methods has co st
effective equipments and experimental simplicity. In this procedure, sample kept at
constant temperature. The opening is plugged by absorbent cotton to allow only filtered
air to enter. The sample is sterilized by ultraviolet light at ambient temperature. The
culture medium, substrate and inoculum are then introduced into the flask. The area of
air-liquid contact should be maximized to favor the entrance of air into the liquid
medium. This technique is helpful to determine the metals microbes’ interaction, the
physiology of microorganisms; however, it is limited to use (Rossi, 1990).
Shake flask technique: This technique is more advantageous than stationary flask
technique. Erlenmeyer flasks containing growth medium, are fixed and incubated in

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rotary shaker to assure the mixing and homogenizati on of medium thereafter,
enhancing the dissolution of atmospheric gases essential for microorganisms such as
oxygen and carbon dioxide. Experimental conditions should be kept constant for
accurate measurement of kinetic parameters. This te chnique has been utilized in
number of applications (Rossi, 1990; Farbiszewska-Kiczma et al., 2004; Torma, 1988;
Beyer et al., 1986; Jong-Un et al., 2005).
Pilot-plant (< 10m
3
): For developing reliable models for commercial-scale plants,
laboratory scale experiments are not considered rel iable though providing useful
information in a relatively short time. Commercial scale tests are carried out under
different conditions. Particle size is a critical factor in metals dissolution; large particle
size, less exposed surface area leads to decreases accessibility of solution and
microorganisms to valuable metals thus results in decrease metals solubilization (Rossi,
1990). Moreover, the controlled environmental condition in laboratory-scale tests may
not be applicable to commercial-scale plants. More reliable commercial scale plants can
be modeled under controlled environmental condition s by following pilot-plant
techniques of different types, such as column leaching and agitated tanks and reactors
(Chae, and Wadsworth, 1979; Lizama, 2004; Lizama et al., 2005; Szubert et al., 2006).
Commercial-scale (> 10m
3
): Commercial scale bioleaching techniques has major
applications in the mining industry, first patented by the copper industry in the early
1950's and subsequently followed by mining operation of gold (Olson, 1994), uranium
(Khalid et al. ,1993) and zinc (Agate, 1996) from low grade ores on commercial level.
Commercial scale leaching has various forms of application:
In situ leaching: It is the recovery of metals from ore without removing ore body from
rocks.
Dump leaching: This technique is more suitable for the extraction of metals from low
and lean grade ores by stripping of open-cast mines form the dump (Rossi, 1990;
Ehrlich, 1996; Devasia and Natarajan, 2004).
Heap leaching: This technique apply for metals recovery from runoff mine ore on
proper site after pretreatments such as crushing, screening or even partial roasting
(Chae and Wadsworth, 1979; Bosecker, 1997; Devasia and Natarajan, 2004; Lizama,
2004; Lizama et al., 2005).
Vat leaching: In vat leaching, ore after pretreatment dumped on concrete vat

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lined with acid proof material and vat fed through bottom opening and leaches solution
percolates upwards through the ore mass and overflo w is pumped into the next vat
(Rossi, 1990; Devasia and Natarajan, 2004).
Reactor leaching: This method is more expensive due to the elevated installation and
operating costs thus, limited to the products of conventional concentration processes.
This process occur in reactors consisting of sufficiently stirred tanks in which the ore to
be leached resides for a time (Rossi, 1990).
Approximately 10 % of the world's copper was obtained by microbiological leaching in
1980 as reported by Ingledew, (1990) whereas recently, it is estimated 15-30 % of the
world production of copper (Brombacher et al., 1997). Furthermore, Brierley (1995)
reported 1992, 2.1 kt gold, of which about 20 % was obtained by microbial activities.
BIODIESEL PRODUCTION FROM JATROPA
Introduction
Biodiesel is an alternative fuel made from renewabl e biological sources such as
vegetable oils both (edible and non edible oil) and animal fats. Vegetable oils are usually
esters of glycol with different chain length and degree of saturation. It may be seen that
vegetable contains a substantial amount of oxygen in their molecules.
Practically the high viscosity of vegetable oils (30- 200 Centistokes) as compared to that
to Diesel (5.8- 6.4 Centistokes) leads to unfavorable pumping, inefficient mixing of fuel
with air contributes to incomplete combustion, high flash point result in increased
carbon deposit formation and inferior coking. Due to these problems, vegetable oil
needs to be modified to bring the combustion related properties closer to those of Diesel
oil. The fuel modification is mainly aimed at reducing the viscosity and increasing the
volatility.
One of the most promising processes to convert vegetable oil into methyl ester is the
transesterification, in which alcohol reacts with triglycerides of fatty acids (vegetable oil)
in the presence of catalyst. Jatropha vegetable oil is one of the prime non edible sources
available in India. The vegetable oil used for biodiesel production might contain free
fatty acids which will enhance saponification reaction as side reaction during the
transesterification process.
All countries are at present heavily dependent on petroleum fuels for transportation and
agricultural machinery. The fact that a few nations together produce the bulk of
petroleum has led to high price fluctuation and unc ertainties in supply for the

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consuming nations. This in turn has led them to look for alternative fuels that they
themselves can produce. Among the alternatives bein g considered are methanol,
ethanol, biogas and vegetable oils.
Vegetable oils have certain features that make them attractive as substitute for Diesel
fuels.
The various uses of J. curcas components:


Source of jatropha Oil:
The plant that is generally cultivated for the purpose of extracting jatropha oil is
Jatropha curcas. The seeds are the primary source from which the oil is extracted.

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Owing to the toxicity of jatropha seeds, they are not used by humans. The major goal of
jatropha cultivation, therefore, is performed for the sake of extracting jatropha oil.
Analysis of jatropha curcus seed shows the following chemical compositions.
Moisture: 6.20%
Protein: 18.00%
Fat: 38.00%
Carbohydrates: 17.00%
Fiber: 15.50%
Ash: 5.30%
The oil content is 25-30% in the seed. The oil contains 21% saturated fatty acids and
79% unsaturated fatty acids. These are some of the chemical elements in the seed,
cursin, which is poisonous and render the oil not appropriate for human consumption.
Oil has very high saponification value and being extensively used for making soap in
some countries. Also oil is used as an illuminant in lamps as it burns without emitting
smoke. It is also used as fuel in place of, or along with kerosene stoves.
PROCESSING TECHNIQUES
Natural vegetable oils and animal fats are pressed to obtain crude oil which contains
free fatty acids, phospholipids, sterols, water, od orants and other impurities
(Openshaw, 2000). Because of these compounds, high viscosity, low volatility and the
polyunsaturated character of the vegetable oils, they cannot be used as fuel directly in
compression engines (Banapurmath et al., 2008; Srivastava and Prasad, 2000). The
specifications of the seed oil of J. curcas are outlined in Table

The fatty acid composition of the seed oil of J. curcas is compared with other vegetable
oils in Table.

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Jatropha seed oil has about 72% unsaturated fatty acids with oleic acid predominatly
followed by lenoleic acid. The viscosity of Jatropha oil is considerably lower than those
reported for some common and tested oils at 30°C such as soybean (31cSt), cottonseed
(36cSt), and sunflower (43cSt) and pointing to its suitability for use as diesel fuel
(Akintayo, 2004; Kamman and Phillip, 1985).
To overcome the problems highlighted above of using the vegetable oils directly, the oils
require chemical modification so that they can match the properties of fossil diesel. The
processing techniques that are mainly used to convert vegetable oils including Jatropha
oil into fuel form are direct use and blending, pyrolysis, microemulsification and
transesterification (Demirbas, 2000; Ma and Hanna, 1999; Nwafor, 2003). Although
production of biodiesel is a mature technology, there is still a lot ongoing research to
improve the quality and yield of the biodiesel from vegetable oils.
Micro-emulsion
The problem of the high viscosity of vegetable oils was solved by micro-emulsions with
solvents such as methanol, ethanol, and 1-butanol (Agarwal, 2007). A microemulsion is
defined as a colloidal equilibrium dispersion of optically isotropic fluid microstructures
with dimensions generally in the 1 - 150 nm range f ormed spontaneously from two
normally immiscible liquids and one or more ionic or non-ionic amphiphiles (Ma and
Hanna, 1999). The components of a biodiesel micro- emulsion include diesel fuel,
vegetable oil, alcohol, and surfactant and cetane improver in suitable proportions.
Alcohols such as methanol and ethanol are used as viscosity lowering additives, higher
alcohols are used as surfactants and alkyl nitrates are used as cetane improvers.
Microemulsions can improve spray properties by expl osive vaporisation of the low

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boiling constituents in the micelles. Micro-emulsion results in reduction in viscosity
increase in cetane number and good spray characters in the biodiesel. According to
Srivastava and Prasad (2000), short term performance of micro-emulsions of aqueous
ethanol in soybean oil was nearly as good as that of No. 2 diesel, despite the lower
cetane number and energy content.
However, continuous use of micro-emulsified diesel in engines causes problems like
injector needle sticking, carbon deposit formation and incomplete combustion.
Pyrolysis (thermal cracking)
Pyrolysis can be defined as the conversion of one substance into another by means of
heat in the absence of air (or oxygen) or by heat in the presence of a catalyst which
result in cleavage of bonds and formation of a variety of small molecules. The pyrolysis
of vegetable oil to produce biofuels has been studied and found to produce alkanes,
alkenes, alkadienes, aromatics and carboxylic acids in various proportions (Ma and
Hanna, 1999; Alencar et al., 1983; Peterson, 1986). The equipment for thermal cracking
and pyrolysis is expensive for modest biodiesel production particularly in developing
countries. Furthermore, the removal of oxygen durin g the thermal processing also
removes any environmental benefits of using an oxygenated fuel (Ma and Hanna, 1999).
Another disadvantage of pyrolysis is the need for separate distillation equipment for
separation of the various fractions. Also the product obtained was similar to gasoline
containing sulphur which makes it less ecofriendly (Ranganathan et al., 2007).
Transesterification (alcoholysis)
Transesterification of vegetable oils is the most popular method of producing biodiesel.
Transesterification (alternatively alcoholysis) is the reaction of a fat or oil (trigylceride)
with an alcohol to form fatty acid alkyl esters (valuable intermediates in oleo chemistry),
methyl and ethyl esters (which are excellent substitutes for biodiesel) and glycerol as
shown:

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Transesterification as an industrial process is usually carried out by heating an excess
of the alcohol with vegetable oils under different reaction conditions in the presence of
an inorganic catalyst. The reaction is reversible and therefore excess alcohol is used to
shift the equilibrium to the products side. The alcohols that can be used in the
transesterification process are methanol, ethanol, propanol, butanol and amyl alcohol,
with methanol and alcohol being frequently used. The reactions are often catalysed by
an acid, a base or enzyme to improve the reaction r ate and yield. Alkali-catalysed
transesterification is much faster than acid-catalysed transesterification and is most
often used commercially (Ma and Hanna, 1999; Rangan athan et al., 2008; Agarwal and
Agarwal, 2007). The alkalis which are used include sodium hydroxide, potassium
hydroxide, and carbonates. Sulphuric acid, sulfonic acids, and hydrochloric acids are
the usual acid catalysts. After transesterification of trigylcerides, the products are a
mixture of esters, glycerol, alcohol, catalyst and tri-, di- and monogylcerides which are
then separated in the downstream (Ma and Hanna, 199 9; Freedman et al., 1986;
Demirbas, 2005).
The process of transesterification brings about drastic change in viscosity of the
vegetable oil. The high viscosity component, glycerol, is removed and hence the product
has low viscosity like the fossil fuels. The biodiesel produced is totally miscible with
mineral diesel in any proportion. Flash point of th e biodiesel is lowered after
transesterification and the cetane number is improved. The yield of biodiesel in the
process of transesterification is affected by several process parameters which include;
presence of moisture and free fatty acids (FFA), reaction time, reaction temperature,
catalyst and molar ratio of alcohol and oil.
The effect of moisture and free fatty acids
The gylceride should have an acid value less than 1 and all materials should be
substantially anhydrous. An acid value greater than 1 requires that the process uses
more sodium hydroxide to neutralise the free fatty acids. Transesterification yields are
significantly reduced if the reactants do not meet this requirement (Freedman et al.,
1986; Goodrum, 2002; Dorado et al., 2002; Ma et al. , 1998). The presence of water
causes the transesterification reaction to partially change to saponification, which
produces soap and thus lowering the yield of esters. Saponification also renders the
separation of ester and glycerol difficult since it increases the viscosity and form gels
(Berchmans and Hirata, 2008).

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Most of the biodiesel is currently made from edible oils by using methanol and alkaline
catalyst. However, there are large amounts of low cost oils and fats that cannot be
converted to biodiesel using methanol and alkaline catalyst because they contain high
amounts of free fatty acids and water. In some instances, crude J. curcas oil quality
gradually deteriorates due to improper handling and inappropriate storage conditions
which cause various chemical reactions such as hydr olysis, polymerization and
oxidation to occur. Improper handling and prolonged exposure of crude J. curcas oil will
result in an increase in the concentration of free fatty acids and water. The presence of
high concentration of free fatty acids can significantly reduce the yield of methyl esters.
Two-step process, acid-catalysed esterification process and followed by base-catalysed
transesterification process have been developed for these oils in which initially the free
fatty acids are converted to fatty acid methyl esters by an acid catalysed pretreatment
and then transesterified using alkaline catalyst in the second step (Berchmans and
Hirata, 2008; Ghadge and Raheman, 2005; Velkovic et al., 2006). A two-stage
transesterification process for crude J. curcas L. seed oil with high content of free fatty
acids was studied by Berchmans and Hirata (2008). T he first stage was acid
pretreatment process which reduced the free fatty level to less than 1%. The second
stage, alkali base catalysed transesterification process gave 90% methyl ester yield.
The effect of reaction time
The conversion rate increases with reaction time and therefore is important in the
transesterification process. Freedman et al. (1986) studied the transesterification of
peanut, cotton-seed, sunflower and soybean oils under methanol to oil ratio of 6:1,
0.5% sodium methoxide catalyst and 60°C. About 80% yield was observed after 1
minute for soybean and sunflower oils. After an hour, yields (93 - 98%) were almost the
same for the four oils. Similar results were reported by Ma et al. (1998). No similar
studies have been reported for J. curcas oil.
The effect of reaction temperature
The transesterification process can occur at different temperature depending on the oil
used. Generally the reaction is carried out close to the boiling point of methanol (60 -
70°C) at atmospheric pressure at molar ratio (alcohol to oil) of 6:1 (Srivastava and
Prasad, 2000; Pramanik, 2003; Huaping et al., 2006). Freedman et al. (1984) observed
that temperature clearly influenced the reaction rate and yield of esters when they
investigated transesterification of soybean oil with methanol (6:1) at 32, 45 and 60°C.

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The effect of molar ratio
The stoichiometric ratio for transesterification requires 3 mole of alcohol per mole of
triglyceride to yield 3 mole fatty esters and 1 mole of glycerol. The transesterification
reaction is shifted to the right by using excess alcohol or removing one of the products
from the reaction mixture continuously. A molar rat io of 6:1 (with alkali as the
catalysts) is normally used in industrial processes to obtain yields of methyl esters
higher than 98% by weight. Ratios greater than 6:1 do not increase the yield but rather
interfere with separation of glycerol because there is an increase in glycerol solubility.
When glycerine remains in solution, it helps drive the equilibrium back to the left,
lowering the yield of esters (Tomasevic and Marinkovic, 2003). When using acid catalyst
the desirable product is obtained with 1 mol% of sulphuric acid with molar ratio of 30:1
at 65°C and conversion of 99% is achieved in 50 h.
The effect of catalysts
To make the transesterification process possible a catalyst in the form of an alkali, acid
or lipase enzyme is required.
Alkali catalyst
Alkali-catalysed transesterification is much faster than acid-catalysed
transesterification and is less corrosive to industrial equipment and therefore is the
most often used commercially (Ma and Hanna, 1999; R anganathan et al., 2008;
Agarwal, 2007; Marchetti et al., 2007). Sodium hydroxide or potassium hydroxide is
used as basic catalyst with methanol or ethanol as well as the vegetable oil. Sodium
hydroxide is cheaper and is the widely used in large scale-processing. The alkaline
catalyst concentration in the range of 0.5 - 1% by weight yield 94 - 99% conversion of
most vegetable oils into esters. There are several disadvantages in using an alkaline
catalysis process although it gives high conversion levels of triglycerides to their
corresponding methyl esters in short reaction times. The process is energy intensive,
recovery of glycerol is difficult, the alkaline catalyst has to be removed from the product,
alkaline wastewater generated requires treatment and the level of free fatty acids and
water greatly interfere with the reaction. The risk of free acid or water contamination
results in soap formation that makes the separation process difficult (Fukuda et al.,
2001; Barnwal and Sharma, 2005).

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Acid catalyst
The second conversional way of making the biodiesel is to use the triglycerides with
alcohol and an acid. Sulphuric acid, sulfonic acids, and hydrochloric acids are the
usual acid catalysts but the most commonly used is sulphuric acid. Acid catalysts are
used if the triglyceride has a higher free fatty acid content and more water. Although
the yields could be high, the corrosiveness of acids may cause damage to the equipment
and the reaction rate can be low, sometimes taking more than day to finish (Freedman
et al., 1984). According to some authors, the reactions are also slow, requiring typically
temperature above 100°C and more than 3 h to comple te the conversion (Meher et al.,
2006). For example, Freedman et al. (1986) studied the transesterification of soybean oil
in the presence of 1% sulphuric acid with alcohol/oil molar ratio 30:1 at 65°C and the
conversion was completed in 20 h.
Lipase catalyst
Recently, enzymatic transesterification has attracted much attention for biodiesel
production as it produces high purity product (esters) and enables easy separation from
the by-product, glycerol (Devanesan et al., 2007; Mamoru et al., 2001; Oznur and
Melek, 2002; Ranganathan et al., 2008). The enzyme that was found to be capable of
catalysing transesterification is lipase. Lipase can be obtained from microorganisms like
Mucor miehei, Rhizopus oryzae, Candida antarctica, Pseudomonas fluorescens and
Pseudomonas cepacia. Enzymatic biodiesel production is possible using both
intracellular and extracellular lipases. Biocompat ibility, biodegradability and
environmental acceptability of the biotechnological procedure when using lipase as a
catalyst are the desired properties in this alternative biodiesel production method
(Marchetti et al., 2007; Devanesan et al., 2007). However, the use of extracellular lipase
as a catalyst requires complicated recovery, purification and immobilisation processes
for industrial application (Bank et al., 2001). Consequently, the direct use of whole cell
biocatalyst of intracellular lipases has received considerable research efforts (Devanesan
et al., 2007; Kaieda et al., 1999; Matsumoto et al. , 2001). For the industrial
transesterification of fats and oils, Pseudomonas species immobilised with sodium
alginate gel can be used directly as a whole cell b io-catalyst (Foidl et al., 1996;
Devanesan et al., 2007; Mohamed and Uwe, 2003; Yong and Siyi, 2007).
Devanesan et al. (2007) reported maximum yield (72% ) of biodiesel from
transesterification of Jatropha oil and short chain alcohol (methanol on hexane) using

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immobilized P. fluorescens at the optimum conditions of 40°C, pH 7.0, molar ratio of
1:4, amount of beads of 3 g and reaction time of 48 h.
In all the work in literature on lipases, the enzymes or whole cells are immobilised and
used for catalysis. The advantage of immobilisation is that the enzyme can be reused
without separation. Also the operating temperature of the process is low (50°C)
compared to other techniques which operate at harsh conditions. However, the cost of
enzymes remains a barrier for its industrial implem entation (Neslon et al., 1996;
Shimada et al., 2002). In order to increase the cost effectiveness of the enzymatic
process, the enzyme (both intracellular and extracellular) is reused by immobilising in a
suitable biomass support particle and that has resulted in considerable increase in
efficiency (Ranganathan et al., 2008, Neslon et al, 1996; Jackson and King, 1996). The
advantages and disadvantages of using lipases are summarised in Table.
Various alcohols are being investigated for the transesterification process using lipase
including methanol, ethanol, iso-propanol and butanol. Jackson and King (1996) used
immobilised lipases as biocatalysts for transesterification of corn oil in flowing
supercritical carbon dioxide and reported an ester conversion of more than 98%. But
the activity of immobilized enzyme is inhibited by methanol and glycerol present in the
mixture. The use of tert-butanol as a solvent, continuous removal of glycerol, stepwise
addition of methanol are found to reduce the inhibitory effects, thereby increasing the
cost effectiveness of the process (Li et al., 2006; Samukawa et al., 2000; Royon et al.,
2007).
Effective methanolysis using extracellular lipase has been reported to improve by
stepwise addition of methanol through which 90 - 95% conversion can be achieved even
after 50 and 100 cycles of repeated operation (Shimada et al., 2002; Samukawa et al.,
2000; Watanabe et al., 2000). The efficiency of transesterifcation process using lipase
can be significantly increased by using intracellular lipase (whole cell immobilisation)
instead of extracellular lipase which demands compl ex purification stages before
immobilisation. This can clearly reduce cost of the transesterification production as
reported by Matsumoto et al. (2001), Ban et al. (2001) and Hama et al. (2004), when R.
oryzae for the transesterification process of vegetable oils was used. Tamalampudi et al.
(2008) recently reported that whole cell R. oryzae immobilised onto bio-mass support
particles which catalysed the methanolysis of Jatropha oil more efficiently than
Novozym 435.

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The production of biodiesel using a biocatalyst eliminates the disadvantages of the
alkali process by producing product of very high purity with less or no downstream
operations (Meher et al., 2006; Fukuda et al., 2001; Modi et al., 2007). The process of
producing biodiesel using immobilised lipase has no t yet been implemented at
industrial scale.
On the other hand, in general the production cost of a lipase catalyst is significantly
greater than that of an alkaline one. There are no comparative studies in the literature
on the most appropriate transesterification technique which can be used to produce
biodiesel from J. curcas L. Theadvantages and disadvantages of the methods o f
transesterification are summarised in Table
FUEL PROPERTIES OF JATROPHA BIODIESEL
The fuel properties of Jatropha biodiesel are summarized in Table.

Jatropha biodiesel has comparable properties with those of f ossil biodiesel and
conforms to the latest standards for biodiesel. Standardisation is a prerequisite for
successful market introduction and penetration by b iodiesel, and many countries
including Austria, Germany (DIN), Italy, France, United States (AST D) have defined
standards for biodiesel.
Advantages of biodiesel
Provides a domestic, renewable energy supply.
Biodiesel is carbon neutral because the balance between the amount of CO
2 emissions
and the amount of CO2 absorbed by the plants producing vegetable oil is equal.
Biodiesel can be used directly in compression ignition engines with no substantial
modifications of the engine.
Blending of biodiesel with diesel fuel increases engine efficiency.
The higher flash point of biodiesel makes its storage safer.
Biodiesel is non-toxic.

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Biodiesel degrades four times faster than diesel.
CO, CO
2 and UBHC, PAH, soot and aromatics emissions are re duced in biodiesel and
its blends than in fossil diesel because biodiesel is oxygen in structure and it burns
clearly all the fuels.
It is biodegradable.
Disadvantages of biodiesel
More expensive due to less production of vegetable oil.
Blends of biodiesel above 20% can cause engine main tenance problems and even
sometimes damage the engine in the long term.
BIODIESEL PRODUCTION FROM PONGAMIA The potential of P. pinnata oil as a source of fuel for the biodiesel industry is well
recognized. Moreover, the use of vegetable oils from plants such as P. pinnata has the
potential to provide an environmentally acceptable fuel, the production of which is
greenhouse gas neutral, with reductions in current diesel engine emissions.
Importantly, the successful adoption of biofuels is reliant on the supply of feedstock
from non-food crops with the capacity to grow on marginal land not destined to be used
for the cultivation of food crops. In this regard P. pinnata is a strong candidate to
contribute significant amounts of fuel feedstock, meeting both of these criteria. Existing
feedstocks such as palm oil and canola are costly, making the production of biodiesel
economically marginal. Sources such as tallow and waste oil from food outlets are seen
as variable in availability and/or of low quality. Pongamia pinnata belongs to the sub
family Fabeacae (Papilionaceae). It is also called Derris indica and Pongamia glabra. It is
a medium sized evergreen tree with a spreading crown and a short bole. The tree is
planted for shade and is grown as ornamental tree. It is one of the few nitrogen fixing
trees producing seeds containing 30-40% oil. The natural distribution is along coasts
and river banks in lands and native to the Asian subcontinent. It is also cultivated
along road sides, canal banks and open farm lands.
Material and Methods
Transesterification Reaction:
Transesterification or alcoholysis is the displacement of alcohol from an ester by
another in a process similar to hydrolysis, except an alcohol is used instead of water15
this process has been widely used to reduce the high viscosity of triglycerides. The
transesterification reaction is represented by the general equation as below

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Some feedstock must be pretreated before they can go through the transesterification
process. Feedstock with less than 5 % free fatty acid, do not require pretreatment.
When an alkali catalyst is added to the feedstock’s (with FFA > 5 %), the free fatty acid
react with the catalyst to form soap and water as shown in the reaction below:

If methane is used in this process it is called methanolysis.
Methanolysis of glyceride is represented. Transesterification is one of the reversible
reactions. However, the presence of a catalyst (a strong acid or base) accelerates the
conversion. In the present work the reaction is conducted in the presence of base
catalyst. The mechanism of alkali-catalyzed transesterification is described below. The
first step involves the attack of the alkoxide ion to the carbonyl carbon of the
triglyceride molecule, which results in the formation of tetrahedral intermediate. The
reaction of this intermediate with an alcohol produces the alkoxide ion in the second
step. In the last step the rearrangement of the tetrahedral intermediate gives rise to an
ester and a diglyceride. The same mechanism is appl ied to diglyceride and
monoglyceride.
Experimental set up
The experimental set up is shown in figure.
A 2000 ml three necked round –bottom flask was used as a reactor. The flask was
placed in heating mantle whose temperature could be controlled within +2 0C. One of
the two side necks was equipped with a condenser and the other was used as a thermo
well. A thermometer was placed in the thermo well c ontaining little glycerol for
temperature measurement inside the reactor. A blade stirrer was passed through the
central neck, which was connected to a motor along with speed regulator for adjusting
and controlling the stirrer speed.

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Materials:
Feedstock: karanja crude oil,
Acid catalyst: H
2SO4,
Base Catalyst: NaOH of 1% w/w of oil
Reactant: Methanol to oil ratio -molar ratio is 13%,
Reactor vessel
Electricity and timer.
Pretreatment:
In this method, the karanja oil is first filtered to remove solid material then it is
preheated at 110
o
C for 30 min to remove moisture (presence of moisture responsible for
saponification in the reaction). After this demoisturisation of oil we removed available
wax, carbon residue, unsaponificable matter and fiber. These are present in a very
small quantity and carried out some important tests of oil that are given in table.

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Process flowchart


Esterification:
Karanja oil contains 6%- 20% (wt) free fatty acids. The methyl ester is produced by
chemically reacting karanja oil with an alcohol (methyl), in the presence of catalyst
(KOH). A two stage process is used for the transesterification of karanja oil. The first
stage (acid catalyzed) of the process is to reduce the free fatty acids (FFA) content in

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karanja oil by esterification with methanol (99% pure) and acid catalyst sulfuric acid
(98% pure) in one hour time at 57
o
C in a closed reactor vessel.
The karanja crude oil is first heated to 50oC and 0.5% (by wt) sulfuric acid is to be
added to oil then methyl alcohol about 13% (by wt) added. Methyl alcohol is added in
excess amount to speed up the reaction. This reaction was proceed with stirring at 700
rpm and temperature was controlled at 55-57
o
C for 90 min with regular analysis of FFA
every after 25-30 min. When the FFA is reduced upto 1%, the reaction is stopped. The
major obstacle to acid catalyzed esterification for FFA is the water formation. Water can
prevent the conversion reaction of FFA to esters from going to completion. After
dewatering the esterified oil was fed to the transesterification process
Transesterification- Base catalyzed reaction:
Mixing of alcohol and catalyst
The catalyst used is typically sodium hydroxide (NaOH) with 1% of total quantity of oil
mass. It is dissolved in the 13% of distilled methanol (CH
3OH) using a standard agitator
at 700 rpm speed for 20 minutes.The alcohol - catalyst solution was prepared freshly in
order to maintain the catalytic activity and prevent the moisture absorbance. After
completion it is slowly charged into preheated esterified oil.
Transesterification Reaction:
When the methoxide was added to oil, the system was closed to prevent the loss of
alcohol as well as to prevent the moisture. The tem perature of reaction mix was
maintained at 60 to 65
o
C (that is near to the boiling point of methyl alcohol) to speed up
the reaction. The recommended reaction time is 70 m in. The stirring speed is
maintained at 560- 700rpm. Excess alcohol is normally used to ensure total conversion
of the fat or oil to its esters. The reaction mixture was taken each after 20 min. for
analysis of FFA. After the confirmation of completion of methyl ester formation, the
heating was stopped and the products were cooled and transferred to separating funnel.
Settling and Separation:
Once the reaction is complete, it is allowed for settling for 8-10 hours in separating
funnel. At this stage two major products obtained that are glycerin and biodiesel. Each
has a substantial amount of the excess methanol that was used in the reaction. The
glycerin phase is much denser than biodiesel phase and is is settled down while
biodiesel floted up. The two can be gravity separated with glycerin simply drawn off the

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bottom of the settling vessel. The amount of transesterified karanja biodiesel (KOME)
and glycerine by this process are given in table

Alcohol Removal:
Once the glycerin and biodiesel phases were been separated, the excess alcohol in each
phase was removed by distillation. In either case, the alcohol is recovered using
distillation equipment and is re-used. Care must be taken to ensure no water
accumulates in the recovered alcohol stream.
Methyl Ester Wash:
Once separated from the glycerin and alcohol removal, the crude biodiesel was purified
by washing gently with warm water to remove residual catalyst or soaps.
The biodiesel was washed by air bubbling method up to the clear water was drained
out. This shows the impurities present in biodiesel was removed completely.
Dring of Biodiesel:
This is normally the end of the production process to remove water present in the
biodiesel which results in a clear amber-yellow liquid with a viscosity similar to petro
diesel. In some systems the biodiesel is distilled in an additional step to remove small
amounts of color bodies to produce a colorless biodiesel.
Product quality
Prior to use a commercial fuel, the finished biodie sel must be analyzed using
sophisticated analytical equipment to ensure it meets ASTM specifications. The most
important aspects of biodiesel production to ensure trouble free operation in diesel
engines are: complete reaction, removal of glycerin, removal of catalyst, removal of
alcohol, absence of free fatty acids.
BIODIESEL PRODUCTION FROM CASTOR
The major problem associated with the use of pure vege- table oils as fuels for diesel
engines is caused by high fuel viscosity in compression ignition. The vegetable oils are
all highly viscous, with viscosities ranging 10-20 times those of No. 2 Diesel fuel.
Amongst vegetable oils in the context of viscosity, castor oil is in a class by itself, with a

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viscosity more than 100 times that of No. 2 Diesel fuel (MSDS of No. 2 Diesel Fuel −
PetroCard). Due to their high viscosity and low volatility, they do not burn completely
and form deposits in the fuel injector of die- sel engines. Furthermore, acrolein (a highly
toxic sub- stance) is formed through thermal decomposition of glycerol.
Dilution, micro-emulsification, pyrolysis and transesterification are the four techniques
applied to solve the problems encountered with the high fuel viscosity. Amongst the
four techniques, chemical conversion of the oil to its corresponding fatty ester is the
most promising solution to the high viscosity probl em. This process - chemical
conversion of the oil to its corresponding fatty ester, and thus biodiesel − is called
transesterification.
Transesterification Process
The transesterification process will be adopted for the preparation of ethyl ester or
methyl ester of Castor (Ricinus communis) oil. In the preparation of ethyl ester
(biodiesel), five distinct stages will be involved,
Heating of oil.
Preparation of alkaline mixture.
Adding of alkaline alcohol to oil and stirring the mixture.
Settling of separation of glycerol.
Washing of ethyl ester with water.
The biodiesel can be obtained by transesterification of castor oil using either ethanol or
methanol as the Trans- esterification agent. The extraction of biodiesel from castor oil,
in the presence of the catalysts faster with methanol as the transesterification agent
compared with ethanol. The maximum yield of esters depends on the reaction time and
that is 1 hour with methanol or of 5 hours with ethanol. However, while similar yields of
fatty acid es-ters may be obtained following ethanolysis or metha-nolysis, the reaction
times required to attain them are very different, with methanolysis being much more
rapid. The transesterification of castor oil via ethanolysis or methanolysis can be
improved through the develop-ment of more efficient catalytic systems and processes, to
maintain kinetic control of the reaction, and by optimiza-tion of purification procedures.

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UNIT: 4 BIOREMEDIATION
Concept and principles, Bioremediation using microb es, In situ and ex situ
bioremediation, biosorption and bioaccumulation of heavy metals;
Phytoremediation, bioremediation of xenobiotics ( h eavy metals, pesticides, oil
slicks, plastic). Bioremediation of soil and water contaminated with hydrocarbons
and surfactants, biofilms.

CONCEPT AND PRINCIPLES
Bioremediation is the use of microorganisms for the degradation of hazardous
chemicals in soil, sediments, water, or other conta minated materials. Often the
microorganisms metabolize the chemicals to produce carbon dioxide or methane, water
and biomass. Alternatively, the contaminants may be enzymatically transformed to
metabolites that are less toxic or innocuous.
It should be noted that in some instances, the metabolites formed are more toxic than
the parent compound. For example, perchloroethylene and trichloroethylene may
degrade to vinyl chloride.
There are at least five critical factors that should be considered when evaluating the use
of bioremediation for site clean up. These factors are:
Magnitude, toxicity, and mobility of contaminants.
It is imperative that the site be properly investigated and characterized to determine the
(a) horizontal and vertical extent of contamination; (b) the kinds and concentrations of
contaminants at the site; (c) the likely mobility of contaminants in the future, which
depends in part on the geological characteristics of the site.
Proximity of human and environmental receptors.
Whether bioremediation is the appropriate cleanup remedy for a site is dependent on
whether the rate and extent of contaminant degradation is sufficient to maintain low
risks to human or environmental receptors.
Degradability of contaminants.
The biodegradability of a compound is generally high if the compound occurs naturally
in the environment (e.g., petroleum hydrocarbons). Often, compounds with a high
molecular weight, particularly those with complex r ing structures and halogen
substituents, degrade more slowly than simpler straight chain hydrocarbons or low

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molecular weight compounds. Whether synthetic compo unds are metabolized by
microorganisms is largely determined by whether the compound has structural features
similar to naturally occurring compounds. The rate and extent to which the compound
is metabolized in the environment is often determined by the availability of electron
acceptors and other nutrients.
Planned site use.
A critical factor in deciding whether bioremediation is the appropriate cleanup remedy
for a site is whether the rate and extent of contaminant degradation is sufficient to
reduce risks to acceptable levels.
Ability to properly monitor.
There are inherent uncertainties in the use of bioremediation for contaminated soils
and aquifers due to physical, chemical and biologic al heterogeneities of the
contaminated matrix. It is important to recognize that biological processes are dynamic
and, given current knowledge, often lack the predic tability of more conventional
remediation technologies. Thus, it is important to insure that unacceptable risks do not
develop in the future. These risks may include migration of contaminants to previously
uncontaminated media and the failure of bioremediation
to achieve acceptable contaminant concentrations.
The remainder of this document will focus on the factors that influence the rate and
extent of contaminant degradation by microorganisms. These can be broadly grouped
into two classes of factors: (a) biological factors and (b) environmental factors. The
biological factors are primarily concerned with the numbers of specific kinds of
microorganisms present and the expression and activity of metabolic enzymes, in other
words, the amount of “catalyst” present.
The environmental factors include chemical and physical characteristics that influence
the bioavailability of contaminants, the availability of other nutrients, the activity of
biological processes (temperature and pH, for examp le), characteristics of the
contaminants with respect to how they interact with the site’s geochemical and
geological characteristics.
Potential Advantages and Disadvantages of Bioremedi ation Technologies
The use of intrinsic or engineered bioremediation processes offers several potential
advantages that are attractive to site owners, regulatory agencies, and the public. These
include:

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Lower cost than conventional technologies.
Contaminants usually converted to innocuous products.
Contaminants are destroyed, not simply transferred to different environmental media.
Nonintrusive, potentially allowing for continued site use.
Relative ease of implementation.
However, there are potential disadvantages to bioremediation as well, these include:
May be difficult to control.
Amendments introduced into the environment to enhan ce bioremediation may cause
other contamination problems.
May not reduce concentration of contaminants to required levels.
Requires more time.
May require more extensive monitoring.
Lack of (hydraulic) control.
Dynamic process, difficult to predict future effectiveness.
BIOREMEDIATION USING MICROBES - IN SITU AND EX SITU BIOREMEDIATION
Bioremediation strategies can be categorized based on the location of contaminant
biodegradation and the aggressiveness of the remedi ation (Madsen, 1997). In situ
bioremediation is performed with the contaminated m aterial left in its natural or
original position. Ex situ bioremediation involves the removal of contaminated material
from its original position and its treatment in a bioreactor system. Both in situ and ex
situ technologies can be applied to solid-, slurry-, and vapor-phase systems. The
aggressiveness of in situ and ex situ bioremediation approaches ranges widely, but they
can be divided into two broad classes (NRC, 1993; Madsen, 1997). At the passive end of
the spectrum is intrinsic or natural bioremediation. This approach relies solely on the
innate capabilities of naturally occurring microorganisms to degrade the contaminants
in situ. In more aggressive bioremediation applications, actions are taken to modify the
site or contaminated material to promote and enhance the biodegradative activities of
microorganisms (e.g., via biostimulation or bioaugmentation). These technologies are
typically referred to as engineered or enhanced bioremediation approaches.
IN SITU TECHNIQUES
The fundamental basis of in situ engineered bioremediation involves introducing oxygen
and nutrients to the contaminated area by various m ethods, all of which ultimately
work by modifying conditions within the soil or groundwater. There are three major

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techniques commonly employed, namely biosparging, bioventing and injection recovery.
In many respects, these systems represent extreme versions of a fundamentally unified
technology, perhaps best viewed as individual applications of a treatment spectrum as
will, hopefully, become clearer from the descriptions of each which follow. As set out
previously, the major benefits of in situ methods are their low intrusion, which enables
existing buildings and site features to remain undisturbed, their relative speed of
commencement and the reduced risk of contamination spread.
Biosparging
Biosparging is a technique used to remediate contam ination at, or below, the water
table boundary, a generalised diagram of which appears in Fig.

In effect, the process involves superaeration of the groundwater, thereby stimulating
accelerated contaminant biodegradation. Though the primary focus of the operation is
the saturated zone, the permeability of the overlying soil has a bearing on the process,
since increased oxygenation of this stratum inevitably benefits the overall efficiency of
remediation.
Air is introduced via pipes sunk down into the contaminated area and forms bubbles in
the groundwater. The extra oxygen made available in this way dissolves into the water,
also increasing the aeration of the overlying soil, stimulating the activity of resident
microbes, which leads to a speeding up of their nat ural ability to metabolise the
polluting substances. The method of delivery can range from relatively simple to the
more complicated, dependent on individual requirements.
One of the major advantages of this is that the required equipment is fairly standard
and readily available, which tends to keep installation costs down. Typically the sparger
control system consists of a pressure gauge and relief valve to vent excess air pressure,
with associated flow meters and filter systems to clean particulates from the input.
More sophisticated versions can also include data loggers, telemetry equipment and
remote control systems, to allow for more precise process management. It should be

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obvious that extensive and comprehensive site inves tigation, concentrating on site
geology and hydrogeology in particular, is absolutely essential before any work starts.
Bioventing
Bioventing is a technique used to remediate contami nation above the water table
boundary, and again a generalised diagram appears in Fig.

This process also involves superaeration, again with the intention of stimulating
accelerated breakdown of the pollutants present, though this time it is taking place
within the soil itself, instead of the groundwater. Bioventing is not generally suitable for
remediating sites with a water table within one metre of the surface, or for heavy or
waterlogged soils, since air is flow compromised under these conditions.
Air is introduced from a compressor pump, via a central pipe, or set of pipes, dependent
on the size of the area to be treated, down into the region of contamination. The extra
oxygen availability thus achieved, as in the previous approach described, stimulates the
resident microbes, which then treat the polluting substances. The air flow through the
soil is further driven by vacuum extractors’ peripheral to the treatment zone, which
increases the dissolved oxygen levels of the soil water and thus facilitates uptake by the
native microorganisms.
Volatile compounds, which are either present as part of the original contamination, or
generated as byproducts of the biological treatment , are often mobilised during
processing and thus more easily extracted. However, in many practical applications, the
air extraction rate is adjusted to maximise decomposition underground, thus reducing a
separate requirement for surface treatment of volatile compounds.
As with the biosparger, control devices typically regulate the pressure, filters clean
particles from the intake and the flow rate is monitored in operation, with data loggers
and telemetry systems again featuring in the more complex applications.

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Unsurprisingly, bioventing also requires extensive and comprehensive site investigation
before commencement, not least because the proper positioning of the necessary system
of pipework is essential to the proper functioning of this technique.
Injection recovery
The injection and recovery method, for which a generalised diagram appears in Fig.,
makes use of the movement of groundwater through the zone of contamination to assist
the remediation process. Although, as mentioned in the introductory comments, this
approach shares many functional similarities with the preceding technologies, it is
essentially more sophisticated and refined, with th e biological treatment being
effectively divided into two complementary stages. Thus, what may be considered a
‘virtual’ bioreactor is established within the soil matrix, with the actual clean-up activity
taking place both within the groundwater and also externally to it.

The major characteristic of this technique is the two-well system sunk into the ground,
the ‘injection well’ and the ‘recovery well’, the former being located ‘upstream’ of the
latter. Nutrients and air are forced down the injection well, and as they flow through the
contamination, they stimulate the growth and activi ty of the indigenous micro-
organisms, which begin the process of remediation. Groundwater, now rich in
contaminant, microbes, microbial metabolites and contaminant breakdown products is
extracted via the ‘recovery well’ from beyond the contaminated zone. It then undergoes
additional biological treatment above ground in an associated bioreactor vessel,
frequently where it is subjected to highly aerobic conditions, before being reinjected,
having been further replenished with air and nutrients. This cycle may be repeated
many times in the course of treatment. Process control is achieved by having separated

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out the aeration, nutrient addition and biotreatment phases into isolated near-episodic
events and the facility for direct analysis of the abstracted water enables treatment
progress to be monitored with much greater certainty. As a consequence, the injection
recovery method neatly overcomes many of the traditional criticisms of in situ treatment
techniques, particularly in respect of difficulties in ensuring true optimization of
conditions and determining the end-point.
Of course, this technique does not avoid the necessity for thorough site investigation
and geological surveys, since it is clearly imperative that the particulars of the
subterranean water flow, soil depth and underlying geology are known in considerable
detail.
Site monitoring for biotechnological applications
Environmental monitoring is well established as a separate science in its own right and
many notable books have been written to describe th e various approaches and
techniques relevant to its many practical applications. It is then, clearly, beyond the
scope of this work to reiterate these discussions and the reader is recommended to
examine such publications at first hand should detailed information be required.
However, it is worth noting that for some sites it may be necessary to continue
monitoring into the future. Under these circumstances, a comprehensive environmental
management and audit scheme can be put in place to monitor environmental effects of
such operations and Fig. shows a suitable illustrative outline. The results would then,
of course, feed back into the decision-making process and ultimately help to shape the
ongoing environmental management regime of the site.

EX SITU TECHNIQUES
Again, there are three principal approaches in common use, namely land farming, soil
banking and soil slurry bioreactors. Though inevitably there are distinct similarities

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between all applications of bioremediation, for obvious reasons of fundamental biology,
these techniques are generally more distinct and separate.
The major benefits of ex situ methods are the greater ease of process optimization and
control, relatively shorter treatment time and the increased potential for the safe
introduction of specialised organisms, if and as required. However, the increased
transport costs, additional land requirement and higher levels of engineering often
combine to make these technologies more costly options.
Land farming
This technique is effectively accelerated natural attenuation, taking place offsite, within
constructed earthwork banking to provide what is essentially a low-tech bioreactor. The
pretreatment stage involves the soil being excavated from site, screened for rocks,
rubble and any other oversize inclusions before typically being stored prior to the
commencement of actual remediation, either at the original location or on arrival at the
treatment site.
The processing itself takes place in lined earthworks isolated from the surroundings by
an impermeable clay or high density polyethylene (H DPE) liner, as shown
diagrammatically in Fig., and typically relies on the activities of indigenous micro-
organisms to bring about the remediation, though specialist bacteria or fungi can be
added if required. The soil to be treated is laid on a sand layer, which itself stands on a
gravel bed, through which a series of drainage pipes have been laid. An impermeable
clay or polymer lining isolates the whole system from direct contact with the underlying
ground. Water and nutrients are added to stimulate biological activity and soil aeration
is maintained by means of turning or ploughing.

The inherent simplicity of the process, however, ma kes its effectiveness highly
dependent on soil characteristics and climatic conditions. For example, heavy clay soils,
make attaining adequate oxygenation difficult and u niform nutrient distribution is

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almost impossible to achieve. In colder climes, it may be necessary to cover the soil to
overcome the worst effects of the weather. Throughout the treatment itself, a process of
sampling and monitoring helps to assess progress an d compliance with required
standards and, at completion, the treated soil can be removed either for return to
original site or use elsewhere.
Soil banking
In some respects, soil banking is an inverted version of the previous system, ranging
from a long row of soil at its simplest, to a more engineered approach, with aeration
pipes, a drainage layer, impermeable liner and a reservoir to collect leachate.
Just as with the previous approach, soil is excavated and screened, often also being
stored prior to treatment. As the name suggests, the soil to be processed is formed into
banks, sometimes with the addition of filler material like chaff, wood chips or shredded
organic matter, if the character of the contaminated soil requires it to improve the
overall texture, ease of aeration, water-holding capacity or organic matter content. This
technique is sometimes termed ‘soil composting’ because of the similarity it has with
the windrow method of treatment for biowaste material.
It is not a true example of the compost process, though there are many functional
parallels between these procedures and the same windrow turning equipment may be
used in either. Often these rows are covered, either with straw or synthetic blanketing
materials, to conserve heat and reduce wash-out. Accordingly, this method is generally
better suited to colder and wetter climates and is typically faster than land farming.
Indigenous micro-organisms are again the principal agents of remediation, though
specialized bacterial or fungal cultures can be introduced as required, and nutrients
added to optimise and enhance their activities.
To further boost the speed and efficiency of this treatment approach, particularly when
space is limited, a more sophisticated version, often termed ‘engineered biopiling’, is
sometimes used to ensure greater process control. Leachate is collected in a reservoir
and recirculated through the pile to keep the soil moist and return the microbes it
contains and a series of pipes within the pile or the underlying drainage layer forces air
through the biopile itself. The increased air flow also permits VOCs to be managed more
efficiently and having the whole system above an impermeable geotextile liner prevents
leachate migration to the underlying ground.

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In both versions of soil banking, a regime of sampling and monitoring is established
which again aids process assessment and control. After treatment is concluded, the soil
may be returned to the original site for use, or taken elsewhere.
Both land farming and soil banking are relatively u nsophisticated approaches,
effectively utilising the mechanisms of natural attenuation to bring about the necessary
clean-up, though enhancing and accelerating the pro cess, having first isolated,
concentrated and contained the material to be treated. The final commonly encountered
technology to be described in this section is a more engineered approach, which works
by increasing the levels of water, nutrients and dissolved oxygen available to the
microbes.
Soil slurry reactor
In most respects, this system shares essentially similar operating principles to the
activated sludge system described in the next chapt er, which is used in treating
effluents. Fig. shows a schematic representation of this method.

After excavation, the soil is introduced into a mixing tank, where slurry is produced by
combining it with water. Nutrients are then added to stimulate microbial growth. The
suspension formed is transferred to a linked series of wellaerated slurry reactors, and
micro-organisms within them progressively treat the contaminants. Clarifiers and
presses thicken the treated slurry and dewater it, the recovered liquid component being
recirculated to the mixing tank to act as the wetting agent for the next incoming batch
of soil, while the separated solids are removed for further drying followed by reuse or
disposal.
Process selection and integration

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However, when complex mixtures of compounds are required to be treated, combining a
series of different individual process stages within a series of interlinked bioreactors,
may often be a more appropriate and effective response.
Dependent on the specific type of contaminants, this may necessitate a sequence of
both aerobic and anaerobic procedures, or even one which combines biological and
chemical steps to achieve the optimum remediation system. Under such circumstances,
clearly each bioreactor features conditions designed to optimize specific biological
processes and degrade particular contaminants.
It should be clear from the preceding discussions t hat the actual process of
bioremediation employed will depend on a number of factors, amongst others relating to
the site itself, the local area, economic instruments, reasons for remediation and the
benefits and limitations of the actual technologies. Hence, it should not be difficult to
see that for any given contamination event, there m ay be more than one possible
individual approach and, indeed, as described earlier, the potential will often exist for
using integrated combinations of technologies to maximise the effectiveness of the
overall response. In this way, though dependent on many external variables, a mix and
match assemblage of techniques may represent the in dividual best practicable
environmental option (BEPO). The merging of an ex situ treatment, like, for example,
soil washing via a slurry reactor, to offer an intensive and immediate lessening of
pollution effect, with a slower in situ process to polish the site to a final level, has much
to recommend it, both environmentally and commercia lly. Accordingly, it seems
reasonable to conclude that the prevalence and relative importance of such approaches
will be likely to grow over the coming years.
BIOSORPTION AND BIOACCUMULATION OF HEAVY METALS
Biosorption is a physiochemical process that occurs naturally in certain
biomass which allows it to passively concentrate an d bind contaminants onto its
cellular structure. Though using biomass in environ mental cleanup has been in
practice for a while, scientists and engineers are hoping this phenomenon will provide
an economical alternative for removing toxic heavy metals from industrial wastewater
and aid in environmental remediation.
Pollution interacts naturally with biological systems. It is currently uncontrolled,
seeping into any biological entity within the range of exposure. The most problematic
contaminants include heavy metals, pesticides and other organic compounds which can

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be toxic to wildlife and humans in small concentration. There are existing methods for
remediation, but they are expensive or ineffective.
However, an extensive body of research has found th at a wide variety of commonly
discarded waste including eggshells, bones, peat, fungi, seaweed, yeast and carrot peels
can efficiently remove toxic heavy metal ions from contaminated water. Ions from
metals like mercury can react in the environment to form harmful compounds
like methylmercury, a compound known to be toxic in humans. In addition, adsorbing
biomass, or biosorbents, can also remove other harm ful metals
like: arsenic, lead, cadmium, cobalt, chromium anduranium.
Biosorption may be used as an environmentally friendly filtering technique. There is no
doubt that the world could benefit from more rigorous filtering of harmful pollutants
created by industrial processes and all-around human activity.
The idea of using biomass as a tool in environmental cleanup has been around since
the early 1900s when Arden and Lockett discovered c ertain types of living bacteria
cultures were capable of recovering nitrogen and phosphorus from raw sewage when it
was mixed in an aeration tank.
This discovery became known as the activated sludge process which is structured
around the concept of bioaccumulation and is still widely used in wastewater treatment
plants today. It wasn't until the late 1970s when scientists noticed the sequestering
characteristic in dead biomass which resulted in a shift in research from
bioaccumulation to biosorption.
Though bioaccumulation and biosorption are used syn onymously, they are very
different in how they sequester contaminants:
Biosorption:
Biosorption is a metabolically passive process, meaning it does not require energy, and
the amount of contaminants a sorbent can remove is dependent on kinetic equilibrium
and the composition of the sorbents cellular surface. Contaminants are adsorbed onto
the cellular structure. Bioaccumulation is an active metabolic process driven by energy
from a living organism and requires respiration.
Bioaccumulation:
Bioaccumulation occurs by absorbing contaminants wh ich are transferred onto and
within the cellular surface. Both bioaccumulation and biosorption occur naturally in all
living organisms however, in a controlled experiment conducted on living and dead

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strains of bacillus sphaericus it was found that the biosorption of chromium ions was
13–20% higher in dead cells than living cells.
In terms of environmental remediation, biosorption is preferable to bioaccumulation
because it occurs at a faster rate and can produce higher concentrations. Since metals
are bound onto the cellular surface, biosorption is a reversible process whereas
bioaccumulation is only partially reversible
Uses:
Even though the term biosorption may be relatively new, it has been put to use in many
applications for a long time. One very widely known use of biosorption is seen
in activated carbon filters. They can filter air and water by allowing contaminants to
bind to their incredibly porous and high surface area structure. The structure of the
activated carbon is generated as the result of charcoal being treated with oxygen.
Another type of carbon, sequestered carbon, can be used as a filtration media. It is
made by carbon sequestration, which uses the opposi te technique as for creating
activated carbon. It is made by heating biomass in the absence of oxygen. The two
filters allow for biosorption of different types of contaminants due to their chemical
compositions — one with infused oxygen and the other without
PHYTOREMEDIATION
Bioremediation as a biotechnological intervention for cleaning up the residual effects of
previous human activities on a site, typically relies on the inherent abilities and
characteristics of indigenous bacteria, fungi or plant species.
The use of plants, including bioaccumulation, phytoextraction, phytostabilisation and
rhizofiltration, all of which are sometimes collectively known as phytoremediation.
Thus, the biological mechanisms underlying the relevant processes are biosorption,
demethylation, methylation, metal-organic complexat ion or chelation, ligand
degradation or oxidation. Microbes capable of utilising a variety of carbon sources and
degrading a number of typical contaminants, to a gr eater or lesser extent, are
commonly found in soils. By enhancing and optimising conditions for them, they can be
encouraged to do what they do naturally, but more swiftly and/or efficiently. This is the
basis of the majority of bioremediation and proceeds by means of one of the three
following general routes.
Mineralisation, in which the contaminant is taken up by microbe species, used as a food
source and metabolised, thereby being removed and destroyed. Incomplete, or staged,

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decomposition is also possible, resulting in the generation and possible accumulation of
intermediate byproducts, which may themselves be fu rther treated by other micro-
organisms.
Cometabolism, in which the contaminant is again taken up by microbes but this time, is
not used as food, being metabolised alongside the organism’s food into a less hazardous
chemical. Subsequently, this may in turn be mineralised by other microbial species.
Immobilisation, which refers to the removal of contaminants, typically metals, by means
of adsorption or bioaccumulation by various micro-organism or plant species.
Terrestrial Phyto-Systems (TPS)
The importance of pollution, contaminated land and the increasing relevance of
bioremediation have been discussed in previous chapters. Phytoremediation methods
offer significant potential for certain applications and, additionally, permit much larger
sites to be restored than would generally be possible using more traditional remediation
technologies. The processes of photosynthesis described earlier in this chapter are
fundamental in driving what is effectively a solar- energy driven, passive and
unengineered system and hence may be said to contribute directly to the low cost of the
approach.
A large range of species from different plant group s can be used, ranging from
pteridophyte ferns, to angiosperms like sunflowers, and poplar trees, which employ a
number of mechanisms to remove pollutants. There ar e over 400 different species
considered suitable for use as phytoremediators. Am ongst these, some
hyperaccumulate contaminants within the plant biomass itself, which can subsequently
be harvested, others act as pumps or siphons, removing contaminants from the soil
before venting them into the atmosphere, while others enable the biodegradation of
relatively large organic molecules, like hydrocarbons derived from crude oil. However,
the technology is relatively new and so still in the development phase. The first steps
toward practical bioremediation using various plant-based methods really began with
research in the early 1990s and a number of the resulting techniques have been used
in the field with reasonable success.
In effect, phytoremediation may be defined as the direct in situ use of living green plants
for treatment of contaminated soil, sludges or grou ndwater, by the removal,
degradation, or containment of the pollutants present. Such techniques are generally
best suited to sites on which low to moderate levels of contamination are present fairly

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close to the surface and in a relatively shallow band. Within these general constraints,
phytoremediation can be used in the remediation of land contaminated with a variety of
substances including certain metals, pesticides, so lvents and various organic
chemicals.
Metal Phytoremediation
The remediation of sites contaminated with metals typically makes use of the natural
abilities of certain plant species to remove or stabilise these chemicals by means of
bioaccumulation, phytoextraction, rhizofiltration or phytostabilisation.
Phytoextraction
The process of phytoextraction involves the uptake of metal contaminants from within
the soil by the roots and their translocation into the above-ground regions of the plants
involved. Certain species, termed hyperaccumulators, have an innate ability to absorb
exceptionally large amounts of metals compared to most ordinary plants, typically 50–
100 times as much (Chaney et al. 1997, Brooks et al. 1998) and occasionally
considerably more. The original wild forms are often found in naturally metal-rich
regions of the globe where their unusual ability is an evolutionary selective advantage.
Currently, the best candidates for removal by phytoextraction are copper, nickel and
zinc, since these are the metals most readily taken up by the majority of the varieties of
hyperaccumulator plants. In order to extend the potential applicability of this method of
phytoremediation, plants which can absorb unusually high amounts of chromium and
lead are also being trialled and there have been some recent early successes in attempts
to find suitable phytoextractors for cadmium, nickel and even arsenic. The removal of
the latter is a big challenge, since arsenic behaves quite differently from other metal
pollutants, typically being found dissolved in the groundwater in the form of arsenite or
arsenate, and does not readily precipitate. There have been some advances like the
application of bipolar electrolysis to oxidise arsenite into arsenate, which reacts with
ferric ions from an introduced iron anode, but gene rally conventional remediation
techniques aim to produce insoluble forms of the me tal’s salts, which, though still
problematic, are easier to remove. Clearly, then, a specific arsenic-tolerant plant
selectively pulling the metal from the soil would be a great breakthrough. One attempt
to achieve this which has shown some promise involves the Chinese ladder brake fern,
Pteris vittata, which has been found to accumulate arsenic in concentrations of 5 grams

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per kilogramme of dry biomass. Growing very rapidly and amassing the metal in its root
and stem tissue, it is easy to harvest for contaminant removal.
Hyperaccumulation
Hyperaccumulation itself is a curious phenomenon an d raises a number of
fundamental questions. While the previously mention ed pteridophyte, Pteris vittata,
tolerates tissue levels of 0.5% arsenic, certain strains of naturally occurring alpine
pennycress (Thlaspi caerulescens) can bioaccumulate around 1.5% cadmium, on the
same dry weight basis. This is a wholly exceptional concentration. Quite how the
uptake and the subsequent accumulation is achieved are interesting enough issues in
their own right. However, more intriguing is why so much should be taken up in the
first place. The hyperaccumulation of copper or zinc, for which there is an underlying
certain metabolic requirement can, to some extent, be viewed as the outcome of an
over-efficient natural mechanism. The biological basis of the uptake of a completely
nonessential metal, however, particularly in such a mounts, remains open to
speculation at this point. Nevertheless, with plants like Thlaspi showing a zinc removal
rate in excess of 40 kg per hectare per year, their enormous potential value in
bioremediation is very clear. In a practical application, appropriate plants are chosen
based on the type of contaminant present, the regional climate and other relevant site
conditions. This may involve one or a selection of these hyperaccumulator species,
dependent on circumstances. After the plants have b een permitted to grow for a
suitable length of time, they are harvested and the metal accumulated is permanently
removed from the original site of contamination. If required, the process may be
repeated with new plants until the required level of remediation has been achieved. One
of the criticisms commonly levelled at many forms of environmental biotechnology is
that all it does is shift a problem from one place to another. The fate of harvested
hyperaccumulators serves to illustrate the point, since the biomass thus collected,
which has bioaccumulated significant levels of contaminant metals, needs to be treated
or disposed of itself, in some environmentally sensible fashion.
Typically the options are either composting or incineration. The former must rely on co-
composting additional material to dilute the effect of the metal-laden hyperaccumulator
biomass if the final compost is to meet permissible levels; the latter requires the ash
produced to be disposed of in a hazardous waste landfill. While this course of action
may seem a little unenvironmental in its approach, it must be remembered that the

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void space required by the ash is only around a tenth of that which would have been
needed to landfill the untreated soil. An alternative that has sometimes been suggested
is the possibility of recycling metals taken up in this way. There are few reasons, at
least in theory, as to why this should not be possible, but much of the practical reality
depends on the value of the metal in question. Dried plant biomass could be taken to
processing works for recycling and for metals like gold, even very modest plant content
could make this economically viable. By contrast, low value materials, like lead for
example, would not be a feasible prospect. At the moment, nickel is probably the best
studied and understood in this respect. There has been considerable interest in the
potential for biomining the metal out of sites which have been subject to diffuse
contamination, or former mines where further tradit ional methods are no longer
practical. The manner proposed for this is essentia lly phytoextraction and early
research seems to support the economic case for drying the harvested biomass and
then recovering the nickel. Even where the actual p ost-mining residue has little
immediate worth, the application of phytotechnological measures can still be of benefit
as a straightforward clean-up. In the light of recent advances in Australia, using the
ability of eucalyptus trees and certain native grasses to absorb metals from the soil, the
approach is to be tested operationally for the decontamination of disused gold mines
(Murphy and Butler 2002). These sites also often contain significant levels of arsenic
and cyanide compounds. Managing the country’s minin g waste is a major expense,
costing in excess of Aus$30 million per year; success in this trial could prove of great
economic advantage to the industry.
The case for metals with intermediate market values is also interesting. Though
applying a similar approach to zinc, for instance, might not result in a huge commercial
contribution to the smelter, it would be a benefit to the metal production and at the
same time, deal rationally with an otherwise unresolved disposal issue. Clearly, the
metallurgists would have to be assured that it was a worthwhile exercise. The recycling
question is a long way from being a workable solution, but potentially it could offer a
highly preferable option to the currently prevalent landfill route.
Rhizofiltration
Rhizofiltration is the absorption into, or the adsorption or precipitation onto, plant roots
of contaminants present in the soil water. The principal difference between this and the
previous approach is that rhizofiltration is typically used to deal with contamination in

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the groundwater, rather than within the soil itself, though the distinction is not always
an easy one to draw. The plants destined to be used in this way are normally brought
on hydroponically and gradually acclimatised to the specific character of the water
which requires to be treated. Once this process has been completed, they are planted
on the site, where they begin taking up the solution of pollutants. Harvesting takes
place once the plants have become saturated with co ntaminants and, as with the
phytoextraction, the collected biomass requires some form of final treatment. The
system is less widely appreciated than the previous technology, but it does have some
very important potential applications. Sunflowers were reported as being successfully
used in a test at Chernobyl in the Ukraine, to remo ve radioactive uranium
contamination from water in the wake of the nuclear power station accident.
Phytostabilisation
In many respects, phytostabilisation has close similarities with both phytoextraction
and rhizofiltration in that it too makes use of the uptake and accumulation by,
adsorption onto, or precipitation around, the roots of plants. On first inspection, the
difference between these approaches is difficult to see, since in effect, phytostabilisation
does employ both extractive and filtrative techniques. However, what distinguishes this
particular phytoremediation strategy is that, unlike the preceding regimes, harvesting
the grown plants is not a feature of the process. In this sense, it does not remove the
pollutants, but immobilises them, deliberately concentrating and containing them
within a living system, where they subsequently remain. The idea behind this is to
accumulate soil or groundwater contaminants, lockin g them up within the plant
biomass or within the rhizosphere, thus reducing their bio-availability and preventing
their migration off site. Metals do not ultimately degrade, so it can be argued that
holding them in place in this way is the best practicable environmental option for sites
where the contamination is low, or for large areas of pollution, for which large-scale
remediation by other means would simply not be possible.
A second benefit of this method is that on sites where elevated concentrations of metals
in the soil inhibits natural plant growth, the use of species which have a high tolerance
to the contaminants present enables a cover of vegetation to be re-established. This can
be of particular importance for exposed sites, minimizing the effects of wind erosion,
wash off or soil leaching, which otherwise can sign ificantly hasten the spread of
pollutants around and beyond the affected land itself.

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BIOREMEDIATION OF XENOBIOTICS
(HEAVY METALS, PESTICIDES, OIL SLICKS, PLASTIC)
HEAVY METALS
In addition to metals such as iron and manganese, w hich are largely nontoxic to
microorganisms and animals, there are a series of metals that have varied toxic effects
on microorganisms and homeothermic animals. Microor ganisms play important roles in
modifying the toxicity of these metals (table).

The “metals” can be considered in broad categories. The “noble metals” tend not to
cross the vertebrate blood-brain barrier but can ha ve distinct effects on
microorganisms. Microorganisms also can reduce ionic forms of noble metals to their
elemental forms.
The second group includes metals or metalloids that microorganisms can methylate to
form more mobile products called organometals. Some organometals can cross the
blood-brain barrier and affect the central nervous system of vertebrates.
Organometals contain carbon-metal bonds. These bond s are their unique identifying
characteristics. The mercury cycle is of particular interest and illustrates many
characteristics of those metals that can be methylated. Mercury compounds were widely
used in industrial processes over the centuries. One has only to think of Lewis Carroll’s
allusion to this problem when he wrote of the Mad Hatter in Alice in Wonderland. At
that time mercury was used in the shaping of felt hats. Microorganisms methylated
some of the mercury, thus rendering it more toxic to the hatmakers. A devastating
situation developed in southwestern Japan when larg e-scale mercury poisoning
occurred in the Minamata Bay region because of industrial mercury released into the

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marine environment. Inorganic mercury that accumula ted in bottom muds of the bay
was methylated by anaerobic bacteria of the genus Desulfovibrio. Such methylated
mercury forms are volatile and lipid soluble and the mercury concentrations increased
in the food chain (by the process of biomagnification).
The mercury was ultimately ingested by the human po pulation, the “top consumers,”
through their primary food source—fish—leading to severe neurological disorders.
A similar situation has occurred in many of the freshwater lakes in the north-central
United States and in Canada, where mercury compound s were used to control
microbial growth in pulp mills. Even decades later the fish in lakes downstream from
these pulp mills cannot yet be used for food, and fishing is only for recreation.
The third group of metals occurs in ionic forms directly toxic to microorganisms. The
metals in this group also can affect more complex organisms. However, plasma proteins
react with the ionic forms of these metals and aid in their excretion unless excessive
long-term contact and ingestion occur. Relatively high doses of these metals are
required to cause lethal effects. At lower concentrations many of these metals serve as
required trace elements. The differing sensitivity of more complex organisms and
microorganisms to metals forms the basis of many antiseptic procedures developed over
the last 150 years.
The noble metals, although microorganisms tend to d evelop resistance to them,
continue to be used in preference to antibiotics in some medical applications. Examples
include the treatment of burns with silver-containing antimicrobial compounds and the
use of silver-plated catheters.
The Mercury Cycle
Interactions between the atmosphere, aerobic water, and anaerobic sediment are
critical. Microorganisms in anaerobic sediments, primarily Desulfovibrio, can transform
mercury to methylated forms that can be transported to water and the atmosphere.
These methylated forms also undergo biomagnificatio n. The production of volatile
elemental mercury (Hg0) releases this metal to waters and the atmosphere. Sulfide, if
present in the anaerobic sediment, can react with ionic mercury to produce less soluble
HgS.

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Metabolic Activity and Metal Biotransformations
The metabolic activity of microorganisms may result in the solubilization, precipitation,
chelation, biomethylation, or volatilization of heavy metals (Bremer and Geesey, 1991;
Iverson and Brinckman, 1978). Microbial activity may result in the following results:
Production of strong acids such as H2SO4 by chemoautotrophic bacteria (e.g.,
Acidibacterium, (formerly Thiobacillus), which dissolve minerals.
Production of organic acids (e.g., citric acid), that dissolve but that also chelate metals
to form metallorganic molecules.
Production of ammonia or organic bases, which precipitate heavy metals as hydroxides.
Extracellular metal precipitation: Sulfate-reducing bacteria produce H
2S, which
precipitates heavy metals as insoluble sulfides. Another example is Klebsiella planticola
Strain Cd-1, which can precipitate Cd in the presence of thiosulfate (Sharma et al.,
2000).
Production of extracellular polysaccharides, which can chelate heavy metals and thus
reduce their toxicity (Bitton and Freihofer, 1978).
Ability of certain bacteria (e.g., sheathed filamentous bacteria) to fix Fe and Mn on their
surface in the form of hydroxides or some other insoluble metal salts.
Biotransformation by bacteria that have the ability to biomethlylate or volatilize (e.g.,
Hg), oxidize (e.g., As), or reduce (e.g., Cr) heavy metals Cadmium (Cd2þ) can be

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accumulated by bacteria (e.g., E. coli, B. cereus) and fungi (e.g., Aspergillus niger). This
metal can also be volatilized in the presence of vitamin B12 . As for mercury, lead can
be methylated by bacteria (e.g., Pseudomonas, Alcaligenes,
Flavobacterium) to tetramethyl lead (CH
3)4Pb.
Fungi (e.g., Aspergillus, Fusarium) are capable of transforming arsenic to trimethyl
arsine, a volatile form with a garlic-like odor. Methanogens convert inorganic arsenic to
dimethylarsine under anaerobic conditions.
Selenium is methylated to volatile alkylselenides through the metabolic activity of
bacteria (Aeromonas, Flavobacterium) and fungi (Penicillium, Aspergillus). As seen for
mercury, methylcobalamin is the methyl donor.
Chromium-rich wastewaters are generated by industrial processes such as leather
tanning, metal plating, and cleaning. An Enterobacter cloacae strain, isolated from
municipal wastewater, was able to reduce hexavalent chromium (Cr6þ) to trivalent
chromium (Cr3þ), which precipitates as a metal hydroxide at neutral pH, reducing the
bioavailability and toxicity of this metal (Ohtake and Hardoyo, 1992).
PESTICIDES
According to the definition by the International Union of Pure and Applied Chemistry,
the term biodegradation is “Breakdown of a substance catalyzed by enzymes in vitro or
in vivo. This may be characterized for the purpose of hazard assessment such as:
Primary
Alteration of the chemical structure of a substance resulting in loss of a specific
property of that substance.
Environmentally acceptable
Biodegradation to such an extent as to remove undesirable properties of the compound.
This often corresponds to primary biodegradation but it depends on the circumstances
under which the products are discharged into the environment.
Ultimate
Complete breakdown of a compound to either fully ox idized or reduced simple
molecules (such as carbon dioxide/methane, nitrate/ammonium and water).
It should be noted that the biodegradation products can be more harmful than the
substance degraded.”
Microbial degradation of chemical compounds in the environment is an important route
for the removal of these compounds. The biodegradat ion of these compounds, i.e.,

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pesticides, is often complex and involves a series of biochemical reactions. Although
many enzymes efficiently catalyze the biodegradatio n of pesticides, the full
understanding of the biodegradation pathway often requires new investigations. Several
pesticide biodegradation studies have shown only the total of degraded pesticide, but
have not investigated in depth the new biotransformed products and their fate in the
environment.
Organochorine pesticides


Figure shows some structures of organochlorine pesticides.
The organochlorine pesticides are known to be highly persistant in the environment.
This class of pesticides includes the chlorinated d erivatives of diphenyl ethane
(dichlorodiphenyltrichloroethane - DDT, its metabol ites
dichlorodiphenyldichloroethylene - DDE, dichlorodiphenyldichloroethane - DDD, and
methoxychlor), hexachlorobenzene (HCB), the group of hexachlorocyclohexane (ϒ-HCH,
ϒ-HCH, ϒ-HCH, ϒ-HCH, or lindane), the group of cyclodiene (aldrin, dieldrin, endrin,

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chlordane, nonachlor, heptachlor and heptachlor-epo xide), and chlorinated
hydrocarbons (dodecachlorine, toxaphene, and chlordecone)
Microbial degradation of organochloride pesticides
The fate of pesticides in the environment is determined by both biotic and abiotic
factors. The rate at which different pesticides are biodegraded varies widely. Some
pesticides such as DDT and dieldrin have proven to be recalcitrant. Consequently, they
remain in the environment for a long time and accumulate into food chains for decades
after their application to the soil.
Most of the studies involving the biodegradation of organochlorine pesticides are done
in pure cultures. The culture is usually isolated f rom a soil sample, generally
contaminated with organochlorine pesticides. The strains are characterized and tested
with different concentrations of the pesticide studied. DDT-metabolising microbes have
been isolated from a range of habitats, including animal feces, soil, sewage, activated
sludge, and marine and freshwater sediments.
The degradation of organochlorine pesticides by pure cultures has been proven to occur
in situ.
Biodegradation of DDT residues largely involves co-metabolism, that is, it requires the
presence of an alternative carbon source, in which microorganisms growing at the
expense of a substrate are able to transform DDT re sidues without deriving any
nutrient or energy for growth from the process
Under reducing conditions, reductive dechlorination is the major mechanism for the
microbial conversion of both the o,p'-DDT and p,p'-DDT isomers of DDT to DDD.
The reaction involves the substitution of an aliphatic chlorine for a hydrogen atom.
Using metabolic inhibitors together with changes in pH and temperature, Wedemeyer
(1967) found that discrete enzymes were involved in the metabolism of DDT by
Aerobacter aerogenes. The suggested pathway for the anaerobic transformation of DDT
by bacteria is shown in Figure.

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Degradation proceeds by successive reductive dechlorination reactions of DDT to yield
2,2-bis(p-chlorophenyl)ethylene (DDNU), which is then oxidise d to 2,2- bis(p-
chlorophenyl)ethanol (DDOH). Further oxidation of D DOH yields bis(pchlorophenyl)
acetic acid (DDA) which is decarboxylated to bis(p-chlorophenyl)methane (DDM). DDM
is metabolized to 4,4’dichlorobenzophenone (DBP) or , alternatively, may undergo
cleavage of one of the aromatic rings to form p-chlorophenylacetic acid (PCPA).
Under anaerobic conditions DBP was not further metabolized.
Table 1 presents some of the microorganisms that were able to degrade organochlorine
pesticides. Among microorganisms, bacteria comprise the major group involved in
organochlorine degradation, especially soil habitants belonging to genera Bacillus,
Pseudomonas, Arthrobacter and Micrococcus.
Organophosphate pesticides
The organophosphorus pesticides (OP) are all esters of phosphoric acid and are also
called organophosphates, which include aliphatic, phenyl and heterocyclic derivatives.

Owing to large-scale use of OP compounds, contamina tions of soil and water systems
have been reported from all parts of the world. In light of this, bioremediation provides a

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suitable way to remove contaminants from the enviro nment as, in most cases, OP
compounds are totally mineralized by the microorganisms. Most OP compounds are
degraded by microorganisms in the environment as a source of phosphorus and /or
carbon.
Classification of Pesticides
Thus, the OP pesticides can be hydrolyzed and detox ified by carboxylesterase and
phosphotriesterase enzymes. The organophosphorates possess an efficient insecticide
activity, due to its characteristic of irreversibly inhibiting the enzyme
acetylcholinesterase in the nervous system, which acts in both insects and in mammal.
In man, the organophosphates are absorbed through a ll routes, reaching high
concentrations in fatty tissues, liver, kidneys, salivary glands, thyroid, pancreas, lungs,
stomach, intestines and, at smaller proportions, in the central nervous system (SNC)
and muscles. However, the organophosphates do not a ccumulate in the human
organism, as it is readily biotransformed in the liver. The excretion of these compounds
and of their metabolites is quite fast, taking place mostly in the urine and, at small
proportions, in the feces, usually within 48 h.
Microbial degradation of organophosphate pesticides
Methyl parathion (O,O-dimethyl-O-(p-nitro-phenylphosphorothioate) is one of the most
used organophosphorus pesticides. This product is widely used throughout the world
and its residues are regularly detected in a range of fruits and vegetables. Investigation
of microbial degradation is useful for developing insecticide degradation strategies using
microorganisms. Bacteria with the ability to degrade methyl parathion have been
isolated worldwide.
Two bacteria identified as Pseudomonas putida and Acinetobacter rhizosphaerae, able to
rapidly degrade the organophosphate fenamiphos, were isolated. Denaturating gradient
gel electrophoresis analysis revealed that the two isolates were dominant members of
the enrichment culture. Clone libraries further showed that bacteria belonging to α, β,
γ, Proteobacteria and Bacteroidetes were also present in the final enrichment, but were
not isolated. Both strains hydrolyzed FEN to fenamiphos phenol and ethyl hydrogen
isopropylphosphoramidate (IPEPAA), which was further transformed, only by P. putida.
The two strains were using FEN as C and N source. C ross-feeding studies with other
pesticides showed that P. putida degraded OPs with a P–O–C linkage.

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Thus, both bacteria were able to hydrolyze FEN, without prior formation of FSO or
FSO2, to FEN-OH which was further transformed only by P. putida (Figure), suggesting
elimination of environmentally relevant metabolites.

In addition, P. putida was the first wild-type bacterial isolate able to degrade OPs. All
the above characteristics of P. putida and its demonstrated ability to remove aged
residues of FEN highlight its high bioremediation potential.
Herein, it was shown that the construction of genetically engineered microorganism
(GEM) and the dual-species consortium has the potential to be used in the degradations
of different kinds of pesticides. These studies show the benefits of bioremediation in
multiple pesticidecontaminated environments and mineralization of toxic intermediates
in the environment, which can lead to complete bioremediation of contaminated sites
that have an adverse effect.
OIL SLICKS
Several methods exist for containing and cleaning up oil spills in aquatic environments.
Chapter two describes how mechanical equipment, suc h as booms and skimmers, is
used to block the spread of oil, concentrate it into one area, and remove it from the
water. Chemical and biological treatment of oil can be used in place of mechanical
methods, especially in areas where untreated oil may reach shorelines and sensitive
habitats where a cleanup becomes difficult and environmentally damaging. Alternative
treatment typically involves adding chemical or biological agents to spilled oil and also
includes in-situ burning.
Types of substances used

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Two types of substances commonly used in responding to an oil spill are (1) dispersing
agents and (2) biological agents.
Dispersing Agents
Dispersing agents, also called dispersants, are chemicals that contain surfactants, or
compounds that act to break liquid substances such as oil into small droplets. In an oil
spill, these droplets disperse into the water column, where they are subjected to natural
processes—such as wind, waves, and currents—that he lp to break them down further.
This helps to clear oil from the water surface, making it less likely that the oil slick will
reach the shoreline. The effectiveness of a dispersant is determined by the composition
of the oil it is being used to treat and the method and rate at which the dispersant is
applied. Heavy crude oils do not disperse as well a s light- to mediumweight oils.
Dispersants are most effective when applied immediately following a spill, before the
lightest components in the oil have evaporated.
Environmental factors, including water salinity and temperature, and conditions at sea
influence the effectiveness of dispersants. Studies have shown that many dispersants
work best at salinity levels close to that of normal seawater. While dispersants can work
in cold water, they work best in warm water.
Some countries rely almost exclusively on dispersants to combat oil spills because
frequently rough or choppy conditions at sea make m echanical containment and
cleanup difficult. However, dispersants have not been used extensively in the United
States because of difficulties with application, disagreement among scientists about
their effectiveness, and concerns about the toxicit y of the dispersed mixtures.
Dispersants used today are much less toxic than those used in the past, but few long-
term environmental effects tests have been conducted after a dispersant application.
The EPA encourages the monitoring of areas that may see increased dispersant use.
These problems are being overcome, however. New tec hnologies that improve the
application of dispersants are being designed. The effectiveness of dispersants is being
tested in laboratories and in actual spill situations, and the information collected is
being used to help design more effective dispersants. In addition, the EPA maintains an
authorized list of chemical and biological agents for use on oil spills.

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Biological Agents
Biological agents are nutrients, enzymes, or microorganisms that increase the rate at
which natural biodegradation occur. Biodegradation is a process by which
microorganisms such as bacteria, fungi, and yeasts break down complex compounds
into simpler products to obtain energy and nutrients.
Biodegradation of oil is a natural process that slowly— over the course of weeks,
months, or years—removes oil from the environment. However, rapid removal of spilled
oil from shorelines and wetlands may be necessary in order to minimize potential
environmental damage to these sensitive habitats.
Bioremediation technologies can help biodegradation processes work faster.
Bioremediation refers to the act of adding materials to the environment, such as
fertilizers or microorganisms, that will increase the rate at which natural biodegradation
occurs. Furthermore, bioremediation is often used after all mechanical oil recovery
methods have been used. Two bioremediation approaches have been used in the United
States for oil spill cleanups—biostimulation and bioaugmentation.
Biostimulation is the method of adding nutrients such as phosphorus and nitrogen to
a contaminated environment to stimulate the growth of the microorganisms that break
down oil. Limited supplies of these necessary nutrients usually control the growth of
native microorganism populations. When nutrients ar e added, the native
microorganism population can grow rapidly, potentia lly increasing the rate of
biodegradation.
Bioaugmentation is the addition of microorganisms to the existing native oil-degrading
population. Sometimes species of bacteria that do not naturally exist in an area will be
added to the native population. As with nutrient addition, the purpose of seeding is to
increase the population of microorganisms that can biodegrade the spilled oil. This
process is seldom needed, however, because hydrocarbon-degrading bacterial exist
almost everywhere and non-indigenous species are of ten unable to compete
successfully with native microorganisms.
During the Exxon Valdez oil spill cleanup and restoration activities, the Alaska
Regional Response Team authorized the use of biorem ediation products, including

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biostimulation and bioaugmentation. Nutrient additi on use was approved for
approximately 100 miles of the Prince William Sound shoreline. Data collected through
a monitoring protocol required by the State of Alaska indicated that nutrient addition
accelerated the natural degradation of oil with no observed eutrophication or toxicity.
Proof of the effectiveness of bioremediation as an oil spill cleanup technology was
developed on the shoreline of Delaware Bay in 1994. This EPA-funded study, which
involved an intentional release of light crude oil onto small plots, demonstrated a
several-fold increase in biodegradation rate due to the addition of fertilizer compared to
the unfertilized control plots. Bioaugmentation or seeding with native microorganisms
did not result in faster biodegradation.
IN-SITU BURNING of oil involves the ignition and controlled combustion of oil. It can be
used when oil is spilled on a water body or on land. The National Oil and Hazardous
Substances Contingency Plan authorizes in-situ burn ing as a cleanup method but
requires approval from the regional response team (RRT) before it can be used. RRT can
provide approval through pre-authorization plans and agreements among the federal
and state agencies. Insitu burning is typically used in conjunction with mechanical
recovery on open water. Fire resistant booms are often used to collect and concentrate
the oil into a slick that is thick enough to burn.
Many factors influence the decision to use in-situ burning on inland or coastal waters.
Elements affecting the use of burning include water temperature, wind direction and
speed, wave amplitude, slick thickness, oil type, and the amount of oil weathering and
emulsification that have occurred. Weathering is a measure of the amount of oil already
having escaped to the atmosphere through evaporation. Emulsification is the process of
oil mixing with water. Oil layer thickness, weathering, and emulsification are usually
dependent upon the time period between the actual s pill and the start of burn
operations. For many spills, there is only a short “window of opportunity” during which
in-situ burning is a viable option.
General guidelines for the use of in-situ burning on a water body are as follows:
• Wind speeds of less than 23 mph,
• Waves less than 3 feet in height,
• Minimum slick thickness of 2-3 mm, depending upon oil type,

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• Less than 30 percent evaporative loss, and
• Emulsification of less than 25 percent water content.
The major issues for in-situ burning of inland spil ls are proximity to human
populations (burning must take place at least three miles away from population at
risk), soil type, water level, erosion potential, vegetation species and condition, and
wildlife species presence. Burning may actually allow oil to penetrate further into some
soils and shoreline sediments.
Because it releases pollutants into the air, in-situ burning requires careful air quality
monitoring. Devices are pre-deployed near populations to measure particulate levels. If
air quality standards are exceeded, the burn will be terminated.
Because in-situ burning uses intense heat sources, it poses additional danger to
response personnel. Igniting an oil slick requires a device that can deliver an intense
heat source to the oil.
Vessel-deployed ignition devices are soaked with a volatile compound, lit, and allowed to
drift into an oil slick. During the Exxon Valdez cleanup effort, plastic bags filled with
gelled gasoline were ignited and placed in the path of oil being towed in a containment
fire-boom. Hand-held ignition systems can be thrown into oil slicks but require
personnel to be in close proximity to the burning oil. A recently developed ignition
device called the “Helitorch,” delivers a falling stream of burning fuel from a helicopter,
allowing personnel to maintain a safer distance from the burning slick and distribute
ignition sources over a wider area.
Although it can be effective in some situations, in-situ burning is rarely used on marine
spills because of widespread concern over atmospher ic emissions and uncertainty
about its impacts on human and environmental health . However, burning of inland
spills is frequently used in a number of states. All burns produce significant amounts of
particulate matter, dependent on the type of oil being burned. Burning oil delivers
polycyclic aromatic hydrocarbons, volatile organic compounds, carbon dioxide, and
carbon monoxide into the air in addition to other c ompounds at lower levels. In
addition, when circumstances make it more difficult to ignite the oil, an accelerant such
as gasoline may need to be added, possibly increasing the toxicity of the volatilizing

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particles. Lack of data regarding the environmental and human health effects of
burning has also discouraged its use.
In-situ burning will be used more often as federal response agencies learn from its
behavior and effects. As in the case of the New Carissa, a Japanese freighter that ran
aground at the entrance to Coos Bay in Oregon on Fe bruary 4, 1999, the conditions
were favorable for burning. The ship was carrying approximately 360,000 gallons of
bunker fuel. Early assessment of the vessel revealed that it was leaking fuel. In order to
reduce the potential for oil to spill from the vessel during impending storms, responders
ignited the grounded ship with incendiary devices in an attempt to burn the fuel in the
cargo holds.
Despite its drawbacks, in-situ burning may be an ef ficient cleanup method under
certain conditions where there are few negative effects on humans or the environment.
These conditions include remote areas, areas with herbaceous or dormant vegetation,
and water or land covered with snow or ice. In thes e circumstances, burning can
quickly prevent the movement of oil to additional areas, eliminate the generation of oily
wastes, provide a cleanup means for affected areas with limited access for mechanical
or physical removal methods, or provide an additional level of cleanup when other
methods become ineffective. When oil is spilled into water containing a layer or chunks
of ice, burning can often remove much more oil than conventional means. Burning can
also help to eliminate some volatile compounds that might otherwise evaporate off a
slick.
Although limited, research and development for in-situ burning in the areas of training,
fire-resistant booms, and ignition systems have increased in recent years. Investigation
into inland environments and vegetative species that are more tolerant of burns is also
yielding results which can aid responders. As data regarding the effects of burning oil
on the environment and human population increase, c onsideration and use of in-situ
burning may become more frequent when spills occur.
PLASTICS
Plastics are man-made long chain polymeric molecule s. They are widely used,
economical materials characterized by excellent all-round properties, easy molding and
manufacturing. Traditionally plastics are very stable and not readily degraded in the

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ambient environment. As a result, environmental pollution from synthetic plastics has
been recognized as a large problem.
Most of this plastic waste has been accumulating in landfills. Therefore, in order to save
capacity for plastic waste disposal, there is a growing interest both in the development
of newer, readily biodegradable plastics and in the biodegradation of conventional
plastic waste.
Types of plastics and usage
Plastics are synthetic polymers. There are two main processes in the manufacture of
synthetic polymers. The first involves breaking the double bond in the original olefin by
additional polymerization to form new carbon-carbon bonds, the carbon-chain
polymers. For example, the fabrication of polyolefins, such as polyethylene and
polypropylene, is based on this general reaction. The second process is the elimination
of water (or condensation) between a carboxylic acid and an alcohol or amine to form
polyester or polyamide. Polyurethane is also made by this general reaction.
Plastics are divided into two groups: thermoplastic s and thermoset plastics.
Thermoplastics are the products of the first kind of general reaction mentioned above.
Thermoplastics can be repeatedly softened and hardened by heating and cooling. In
thermoplastics, the atoms and molecules are joined end-to-end into a series of long,
sole carbon chains. These long carbon chains are in dependent of the others. This
structure in which the backbone is solely built of carbon atoms makes thermoplastics
resistant to degradation or hydrolytic cleavage of chemical bonds. Consequently,
thermoplastics are considered non-biodegradable pla stics. Thermoset plastics are
synthesized from the second kind of general reaction stated above. They are solidified
after being melted by heating. The process of changing from the liquid state to the solid
state is irreversible.
Distinguished from the linear structure of thermoplastics, thermoset plastics have a
highly cross-linked structure. Since the main chain of thermoset plastics is made of
heteroatoms, it is possible that they are potentially susceptible to be degraded by the
hydrolytic cleavage of chemical bonds such as ester bonds or amide bonds.
Main plastics and their applications

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Biodegradable plastics

Degradation of thermoplastic polyolefins
Generally speaking, synthetic polyolefins are inert materials whose backbones consist
of only long carbon chains. This characteristic structure makes polyolefins non-
susceptible to degradation by microorganisms. Howev er, a comprehensive study of
polyolefin biodegradation has shown that some microorganisms could utilize polyolefins
with low molecular weight. This biodegradation always follows photo-degradation and
chemical degradation. Although traditional polyolefins are non-biodegradable, their
biodegradability is enhanced when blending with sta rch or other polyesters. The
degradation of blends of low density polyethylene (PE) or isotactic polypropylene (PP)
with glycerol plasticized starch (GS). Glycerol mono-ethers, fatty alcohols or epoxidized
rubber were required as compatibilizers. The results showed that the blends were
subject to different kinds of degradation. However, the degree of degradation was a
function of the type of polymer and the blend composition. The biodegradation of the

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polyolefin chain was clearly observed. Therefore, with the development of starch-plastic
as well as the discovery of other additives added to synthetic plastics, biodegradable
polyolefins provide an attractive option for reducing plastic waste in the environment.
Biodegradable Polyolefins
Traditionally, polyolefins are considered to be nonbiodegradable for three reasons. First,
the hydrophobic character of polyolefins makes this material resistant to hydrolysis.
Secondly, the use of anti-oxidants and stabilizers during manufacture keeps polyolefins
from oxidation and biodegradation. Thirdly, polyolefins have high molecular weights36
of 4000 to 28,000. Therefore, to make polyolefins biodegradable, these factors have to
be considered. The molecular weight of biodegradable polyolefins must be less than
500. Accordingly, the principle of making biodegradable polyolefins involves adding
special additives to the synthetic polyolefins so that the modified structures are
susceptible to photo-degradation and chemical degradation.
As a result, the long carbon chains are broken to shorter segments and their molecular
weights are reduced below 500. Microorganisms can t hen assimilate the polyolefins
monomeric and oligomeric breakdown products previou sly derived from photo and
chemical degradations.
As commercial products, synthetic polyolefins resist oxidation and biodegradation
because of the presence of anti-oxidants and stabil izers. The use of pro-oxidant
additives makes polyolefins oxo-bio-degradable. First, pro-oxidant activities can change
the polyolefins’ surface from hydrophobic character to hydrophilic. Secondly, pro-
oxidants can catalyze the breakdown the long chain of polyolefins and produce lower
molecular weight products either during photolysis or thermolysis. The thermo-
oxidative degradation of polyethylene films during composting conditions and in the
presence of pro-oxidant additives. The metal combinations were the most active pro-
oxidants. To be active as catalysts, it is necessary that two metal ions of similar stability
be involved in the metal combinations, and also when the two metal ions are oxidized
by oxidants, the oxidation number of the metal ion must be only one unit different from
the one before oxidation.
For example, Mn (manganese) is a suitable metal participating in metal combination for
pro-oxidant activity. As an oxidation-reduction catalyst, two Mn2+ ions with similar
stability can form and would be oxidized to Mn3+ an d then later reduced to Mn2+.
Thus, when polyolefins are exposed to the environme nt, a free radical chain in the

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material can react with oxygen from the atmosphere and produce hydro-peroxides that
can, in turn, be hydrolyzed and photolyzed. Also the pro-oxidant catalyzes the reaction
of chain scission in the polymer, producing low molecular mass oxidation products,
such as carboxylic acids, alcohols and ketones. Furthermore, peroxidation modifies the
material surface character from hydrophobic to hydr ophilic. Consequently,
microorganisms can access the material surface, bio-assimilate the low molecular mass,
hydrophilic oxidant products and facilitate the biodegradation process.
Starch can also be blended into the polymers for producing biodegradable polyolefins.
However, as mentioned earlier, without the addition of a suitable pro-oxidant system,
biodegradation will simply cause the removal of starch and leave behind shorter chains
of unmodified polyolefin. The amount of starch requ ired to be added to synthetic
polyolefins needs to be optimized. If the amount of starch is too high, the mechanical
properties of the material may be adversely affected. On the contrary, if the amount of
starch is too low, the material may not biodegrade.
Biodegradation of Polyethylene
Since polyethylene (PE) is widely used as packaging material, considerable research not
only on biodegradable polyethylene but also on biodegradation of polyethylene has been
recently conducted. Biodegradation of polyethylene is known to occur by two
mechanisms: hydro-biodegradation and oxobiodegradat ion. These two mechanisms
agree with the modifications due to the two additives, starch and pro-oxidant, used in
the synthesis of biodegradable polyethylene. Starch blend polyethylene has a
continuous starch phase that makes the material hydrophilic and, therefore, catalyzed
by amylase enzymes.
Microorganisms can easily access, attack and remove this part. Thus the hydrophilic
polyethylene with the matrix continues to be hydro-biodegraded. For the biodegradable
polyethylene synthesized by adding pro-oxidant addi tive, biodegradation occurs
following photo-degradation and chemical degradation. The pro-oxidant is a metal
combination. After transition metal catalyzed thermal peroxidation, biodegradation of
the low molecular weight oxidation products occurs sequentially.
Degradation of Polyesters
There are two kinds of polyesters: aliphatic and aromatic. Their biodegradability is
completely different. Pure aromatic polyesters are quite insensitive to any hydrolytic
degradation. It was observed that direct microbial or enzymatic attack of pure aromatic

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polyester was not significant. However, other research has recently claimed that
aromatic polyester could be disintegrated by microb ial strains of Trichosporum
andArthrobacter in a time scale of weeks. Some growth of Aspergillus Niger was found
on the surface of aromatic polyesters.
Degradation of Polyurethane
Polyurethane (PUR) is commonly utilized as a constituent material in many products
including furniture, coating, construction materials, fibers, and paints.
Structurally, PUR is the condensation product of polyisocyanate and polyol having
intramolecular urethane bonds (carbonate ester bond, -NHCOO-). The urethane bond in
PUR has been reported to be susceptible to microbial attack. The hydrolysis of ester
bonds in PUR is postulated to be the mechanism of P UR biodegradation. The
breakdown products of the biodegradation are derived from polyester segment in PUR
when ester bonds are hydrolyzed and cleaved. Three types of PUR degradation have
been identified in literature: fungal biodegradation, bacterial biodegradation and
degradation by polyurethanase enzymes. For example, four species of fungi, Curvularia
senegalensis, Fusarium solani, Aureobasidium pullulans and Cladosporium sp, were
obtained from soil and found to degrade ester-based polyurethane.
Degradation of Polyhydroxyalkanoates
Bacteria produce polyhydroalkanoates as energy storage materials. A good example of
this is polyhydroxybutyrate (PHB), which is made by numerous microorganisms.26
PHAs are easily metabolized. The enzymes responsibl e for the biodegradation, PHA
depolymerases, have wide substrate specificity. PHAs and PHBs are recently finding
commercial interest. They may also find applications as blends and additives similar to
starch based plastics.
BIOREMEDIATION OF SOIL AND WATER CONTAMINATED WITH HYDROCARBONS
AND SURFACTANTS
Hydrocarbons—compounds consisting of carbon and hyd rogen alone—are common
groundwater contaminants and often amenable to bioremediation. Releases of gasoline
and other refined petroleum products represent a ma jor source of hydrocarbon
contamination.

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The BTEX compounds are of particular concern and re gulatory importance because
they constitute a significant fraction of gasoline and are relatively soluble and highly
toxic. Benzene is a known human carcinogen.
PAHs containing from two to five fused aromatic rings are also of significant concern
because of the mutagenicity and carcinogenicity of several of these compounds and
their tendency to bioaccumulate. PAHs are generated from the incomplete combustion
of organic matter. Extensive PAH contamination is associated with coal gasification
sites (manufactured-gas plants), as well as the production and use of the coal tar
creosote, a wood preservative. Methyl tert-butyl ether (MTBE) is a fuel additive that was
used originally as a replacement for tetraethyllead to increase the octane rating of
gasoline and prevent engine knocking when leaded ga soline was phased out in the
1970s and 1980s. The 1990 Clean Air Act Amendments led to the addition of increased
amounts of MTBE to reformulated gasoline; however, MTBE is a problematic
groundwater pollutant and its use as a fuel additive is being phased out, due to
concerns about its potential carcinogenicity, taste and odor problems at very low MTBE

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concentrations, and its tendency to migrate through the subsurface more rapidly than
do hydrocarbon co-contaminants such as the BTEX com pounds.
BTEX Bacteria that can aerobically biodegrade BTEX compo unds are indigenous at
nearly all contaminated sites. Aerobic biodegradation of a BTEX compound is initiated
by one of many different monooxygenases and/or diox ygenases that can react with
these compounds. Dioxygenase-mediated attack of the aromatic nucleus of benzene
yields catechol (1,2-dihydroxybenzene)
Biodegradation of benzene, toluene, ethylbenzene, and xylene (BTEX) via initial
Oxygenase-catalyzed reactions that lead to the form ation of catechols, which
undergo ring cleavage and are ultimately converted to central metabolic pathway
intermediates. Note that individual arrows frequent ly encompass multiple
reactions.
The aliphatic side chains and aromatic rings of toluene, ethylbenzene, or the xylene
isomers also serve as sites of initial attack by oxygenases, which generally lead to
formation of substituted catechols. The presence of hydroxyl groups on adjacent
carbons prepares the aromatic ring for further reaction with ring cleavage dioxygenases
through the insertion of two additional oxygen atom s. Ring cleavage serves two
important functions:
Regeneration of the NAD(P)H that was invested in ac tivating the ring for cleavage,
andgeneration of metabolic intermediates that are u sed in synthesis and energy
generation.
The aromatic ring may be opened between the hydroxy l groups, via the ortho cleavage
(β-ketoadipate) pathway, or adjacent to the hydroxyl groups, via the meta cleavage (TOL)
pathway. The products of both ring fission reactions are further degraded to form key
intermediates in central metabolic pathways.
Field and laboratory studies have also demonstrated that biodegradation of BTEX
compounds can occur under different anaerobic terminal electron-accepting processes
(TEAPs), including Fe(III), nitrate, and sulfate reduction and methanogenesis. Toluene is
often the most readily degraded BTEX compound under anaerobic conditions. The
anaerobic biodegradation of the other BTEX compound s has been observed at some
contaminated sites, but not others. In particular, benzene is frequently recalcitrant or
biodegraded only after lengthy lag periods. Thus, while growth of bacterial strains on

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benzene under denitrifying conditions has been obse rved. Anaerobic benzene
degradation appears to be a highly site-specific process.
Anaerobic bacteria use several different strategies to initiate the biodegradation of
BTEX compounds; however, they all appear to direct the contaminants to formation of
benzoyl-CoA as a central biodegradation intermediate, analogous to the formation of
catecholic compounds during aerobic BTEX biodegradation.
The CoA (coenzyme A) substituent is analogous to the dihydroxy groups in catecholic
compounds in that it prepares the aromatic nucleus for subsequent (reduction)
reactions that lead to destabilization and cleavage of the ring under anaerobic
conditions.
The initial attack on toluene involves the formation of a new carbon–carbon bond
through the reaction of the methyl group with fumarate. The fumarate addition reaction
initiates toluene biodegradation under a broad rang e of anaerobic TEAPs, and
analogous reactions have been observed for other methylated aromatics (including m-
xylene) and methylene groups in aliphatic compounds . Anaerobic ethylbenzene
biodegradation is initiated by a dehydrogenation reaction, an oxidation that results in
the hydroxylation of the aliphatic substituent but, unlike reactions mediated by
oxygenases, uses water rather than molecular oxygen as the coreactant.
PAHs
Under aerobic conditions, biodegradation of PAHs with two or three aromatic rings such
as naphthalene, anthracene, and phenanthrene often occurs readily via reactions that
are analogous to the biodegradation of the monoarom atic BTEX compounds.
Monooxygenase- or dioxygenase-catalyzed reactions lead to the formation of catechols
or o-phthalate (1,2-benzenedicarboxylate) intermediates that can be attacked by
dioxygenases leading to eventual ring cleavage.
Similar reactions may also contribute to the biodegradation of four- and five-ring PAHs;
however, the solubilities of these higher-molecular-weight PAHs are extremely low and
limit the biodegradation of these compounds. Bacter ia use different trategies for
increasing the limited bioavailability of PAHs, including the formation of biofilms on
PAH crystals and production of biosurfactants that enhance their dissolution.
Evaluation of bioavailability and treatment strategies are discussed below. Utilization of
naphthalene as the sole carbon and energy source ha s been demonstrated under
denitrifying and sulfate-reducing conditions, and biodegradation of several other PAHs

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under these TEAPs has been observed. Under sulfate-reducing conditions, naphthalene
is converted to 2-methylnaphthalene, which is further transformed through the addition
of fumarate (as observed for toluene and m-xylene).
MTBE
The MTBE molecule contains both a stable ether bond and bulky methyl branching,
structural features that are often resistant to biodegradation. Under aerobic conditions,
cometabolism of MTBE can be mediated by organisms growing on short
(C3–C5) normal and branched alkanes or other organic compounds that may be present
as co-contaminants due to their presence in gasoline.
Aerobic growth on MTBE has also been observed. Meta bolic and co-metabolic
biodegradation of MTBE under aerobic conditions is initiated by a monooxygenase-
mediated attack on the methoxy group and leads to the formation of tert-butyl alcohol
(TBA) plus either formaldehyde or formic acid, depending on the degradation pathway.
In some cases, TBA persists; however, TBA can putat ively be funneled into central
metabolic pathways via several routes and can be us ed by some aerobic
microorganisms as the sole source of carbon and energy. Nevertheless, MTBE and TBA
frequently persist at contaminated sites.
This suggests that microorganisms that can biodegra de MTBE and/or TBA are not
abundant in the environment. Another possibility is that the concentration of MTBE
present at contaminated sites is below the minimum substrate level or threshold
needed to sustain growth. There is evidence suggesting that substantial amounts of
MTBE can be biodegraded anaerobically at a number o f gasoline-contaminated sites,
often after a lengthy lag period. However, in some cases, anaerobic biodegradation of
MTBE does not proceed past TBA, which is an unaccep table bioremediation end
product, due to its toxicity.
Chlorinated Aliphatic Hydrocarbons
Chlorinated aliphatic hydrocarbons (CAHs), including chlorinated methanes, ethanes,
and ethenes, have a number of industrial uses. In particular, their widespread use as
solvents (e.g., in the removal of grease from metal, clothing, and other materials) has led
to frequent contamination of soil and groundwater t hrough spills and improper
disposal, particularly at military and industrial sites and dry-cleaning facilities.
Contamination with CAHs is of concern because of th eir toxicity to humans and, in
many cases, known or likely carcinogenicity.

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As discussed above, polar carbon–halogen bonds may serve as the site of initial attack
for each of the three major biodegradation mechanisms—hydrolysis, oxidation involving
electrophilic oxygen, and reduction—although individual CAHs vary with respect to
their susceptibility to transformation via a given mechanism. Hydrolysis is most
commonly observed for CAHs with two or fewer chlorine substituents on a given carbon.
Hydrolytic attack of dichloromethane and 1,2-dichloroethane by some organisms allows
them to use the CAHs as a source of carbon and ener gy, and longer halogenated
alkanes are also subject to hydrolysis. The selection of an oxidative or reductive
biodegradation-based cleanup approach depends on the predominant redox conditions
in the contaminated groundwater, the relative susceptibility of the contaminant(s) to
oxidation and reduction reactions, the physiological capabilities of the indigenous
microorganisms, and the availability of co-substrates, including electron donors and/or
oxygen. For example, there are many challenges associated with the implementation of
bioremediation strategies based on co-metabolic rea ctions. As discussed above,
biodegradation of co-metabolic substrates occurs re latively slowly compared to
metabolic processes due to competition with growth substrates for key enzymes,
diversion of coreactants in metabolic reactions, and/or cellular damage caused by
transformation product toxicity. Nevertheless, if the contaminated groundwater is
aerobic and a potential source of carbon and energy (e.g., toluene or methane) is
present, it may be reasonable to select a bioremediation strategy based on aerobic co-
metabolism. Co-metabolic oxidation has also been used successfully to bioremediate
TCE-contaminated groundwater through the careful addition of both oxygen and either
phenol or toluene as the source of carbon and energy.
Only the most highly chlorinated aliphatic hydrocarbons (e.g., carbon tetrachloride,
TeCA, and PCE) appear to be resistant to aerobic co-metabolic transformations.
On the other hand, all chlorinated methanes, ethane s, and ethenes can undergo
reductive dechlorination reactions, although reductive dechlorination of more highly
chlorinated CAHs (e.g., PCE and TCE) tends to occur at faster rates than does
transformation of the less chlorinated analogs (e.g., DCE and VC). Thus, efforts to
bioremediate anaerobic groundwater systems contamin ated with highly chlorinated
aliphatic hydrocarbons typically focus on promoting reductive dechlorination processes.
Often, this involves the addition of electron donors, a practice known as biostimulation,
due to the limited availability of suitable electron donors at most CAH-contaminated

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sites. In particular, biostimulation of reductive dechlorination reactions is an effective
bioremediation practice for CAHs that can serve as terminal electron acceptors in
dehalorespiration.
CAHs can serve as terminal electron acceptors, and their susceptibility to co-metabolic
biotransformations and utilization as an electron donor under aerobic and anaerobic
conditions. However, the biodegradation of CAHs is an active area of research, and new
information on the metabolic roles that these compo unds can fulfill is constantly
emerging. For example, utilization of chloromethane and cis-1,2-DCE as electron
donors under anaerobic and aerobic conditions, resp ectively, has been reported.
Further, several chlorinated ethanes, including 1,1 ,1-trichloroethane, 1,1,2-
trichloroethane, 1,1-dichloroethane, and 1,2-dichloroethane, are now known to serve as
terminal electron acceptors for certain dehalorespirers.
Halogenated Aromatic Hydrocarbons
Several categories of halogenated aromatic hydrocar bons are of environmental
significance, including polychlorinated biphenyls (PCBs) and polybrominated diphenyl
ethers (PBDEs). PCBs were widely used in electrical capacitors and transformers and
dielectric and hydraulic fluids until the 1970s, when PCB use was banned in the United
States. Spills and improper disposal practices led to widespread and persistent
contamination of the environment with PCBs. The concern over PCBs is due primarily
to their tendency to partition into organic matter and thus bioaccumulate in ecological
food chains. Unlike PCBs, PBDEs break down with heat.
In the process, they release bromine radicals that help quench combustion processes.
These properties have led to their extensive use as flame retardants in plastics and
textiles and their broad dissemination in the environment. PBDEs levels are also
increasing rapidly in animal tissues and human breast milk, which is of concern due to
the endocrine-disrupting action of some PBDEs.
PCBs
Up to 209 distinct PCB molecules (or congeners) that differ with respect to the numbers
and positions of chlorine substituents on the biphenyl backbone can be found.

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Structure and numbering of polychlorinated biphenyl s (PCBs) and polybrominated
diphenyl ethers (PBDEs).
However, PCBs were typically produced and sold comm ercially as mixtures of 60 to 90
congeners under the U.S. trade name Aroclor. The Aroclor product numbers indicate
the overall degree of chlorination in the mixture. For example, Aroclor
1242 contains 12 carbon atoms and 42% chlorine by w eight, which corresponds to an
average of approximately three chlorines per biphenyl molecule. Microorganisms utilize
strategies similar to those involved in the biodegradation of CAHs and the BTEX
compounds to biodegrade PCBs. However, individual c ongeners vary with respect to
their susceptibility to various biotransformation mechanisms, which can complicate
efforts to detoxify Aroclors under a given set of conditions. Under aerobic conditions,
lightly chlorinated PCBs (generally those with three or fewer chlorines) are converted to
dihydroxylated intermediates by dioxygenases, reactions that are analogous to the ring
activation mechanisms in BTEX biodegradation. The dioxygenases typically attack the 2
and 3 positions (Figure 8.5) on the more lightly chlorinated ring and are hindered by
chlorine substituents in these positions. The activated ring undergoes meta cleavage, as
in toluene biodegradation, and leads to the formation of a chlorobenzoic acid, which is
not transformed by most PCB-degrading bacteria but generally can be mineralized by
chlorobenzoate-degrading populations. Other bacteria can dihydroxylate certain PCBs
in the 3 and 4 positions via a similar biodegradation pathway. Bacteria that can
aerobically degrade lightly chlorinated PCBs (either metabolically or cometabolically
while growing on biphenyl or another primary substrate) appear to be fairly common in
contaminated soils. Thus, under aerobic conditions, the composition of an Aroclor
mixture is expected to shift toward more highly chlorinated congeners, which tend to

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persist under aerobic conditions. Conversely, the m ore highly chlorinated PCB
congeners are better suited than the lightly chlorinated congeners for reductive
dechlorination. Several microbial reductive dechlorination processes that target
chlorines in different positions have been identified, although dechlorinations in the
para and meta positions (relative to the biphenyl linkage) generally are dominant.
Recently, a Dehalococcoides population and a related strain that can respire PCBs as
terminal electron acceptors have been identified.
PBDEs
The nomenclature and number of PBDE congeners are t he same as for the PCBs.
Studies of the biodegradability of PBDEs have largely focused on the potential for
dechlorination of the fully brominated decabromodip henyl ether and other highly
brominated congeners, due to their potential for biodegradation to less brominated
congeners, which are more toxic and bioavailable.
Microbial reductive dechlorination of highly brominated PBDEs has been observed in
both complex cultures and in pure dehalorespiring cultures maintained on chlorinated
electron acceptors. Up to five bromines were removed by the dehalorespiring strains. At
least two aspects of anaerobic PBDE and PCB biodegr adation are similar. First,
dechlorination of the more highly halogenated congeners occurred more slowly than did
PBDEs containing fewer bromine substituents, presum ably due to the reduced
bioavailability of the more hydrophobic, highly brominated congeners. Second, removal
of bromines occurred predominantly in the para and Meta positions (relative to the
ether linkage).
BIOFILMS.
Biofilms consist of microorganisms immobilized at a substratum surface and typically
embedded in an organic polymer matrix of microbial origin. They develop on virtually all
surfaces immersed in natural aqueous environments, including both biological (aquatic
plants and animals) and abiological (concrete, metal, plastics, stones). Biofilms form
particularly rapidly in flowing aqueous systems where a regular nutrient supply is
provided to the microorganisms. Extensive microbial growth, accompanied by excretion
of copious amounts of extracellular organic polymers, thus leads to the formation of
visible slimy layers (biofilms) on solid surfaces.
Most of the human gastrointestinal tract is coloniz ed by specific groups of
microorganisms (the normal indigenous microbiota that give rise to natural biofilms. At

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times, these natural biofilms provide protection for pathogenic species, allowing them to
colonize the host. Insertion of a prosthetic device into the human body often leads to
the formation of biofilms on the surface of the device. The microorganisms primarily
involved are Staphylococcus epidermidis, other coagulase-negative staphylococci, and
gram-negative bacteria. These normal skin inhabitan ts possess the ability to
tenaciously adhere to the surfaces of inanimate prosthetic devices. Within the biofilms
they are protected from the body’s normal defense m echanisms and also from
antibiotics; thus the biofilm also provides a source of infection for other parts of the
body as bacteria detach during biofilm sloughing.
Some examples of biofilms of medical importance include:
Cystic fibrosis patients harboring great numbers of Pseudomonas aeruginosa that
produce large amounts of alginate polymers, which inhibit the diffusion of antibiotics
Teeth, where biofilm forms plaque that leads to tooth decay.
Contact lenses, where bacteria may produce severe eye irritation, inflammation, and
infection
Air-conditioning and other water retention systems where potentially pathogenic
bacteria, such as Legionella species, may be protected from the effects of chlorination by
biofilms.
Microorganisms tend to create their own microenvironments and niches, even without
having a structured physical environment available, by creating biofilms. These are
organized microbial systems consisting of layers of microbial cells associated with
surfaces. Such biofilms are an important factor in almost all areas of microbiology, as
shown in figurea.
Simple biofilms develop when microorganisms attach and form a monolayer of cells.
Depending on the particular microbial growth environment (light, nutrients present and
diffusion rates), these biofilms can become more complex with layers of organisms of
different types (figure b).
A typical example would involve photosynthetic organisms on the surface, facultative
chemoorganotrophs in the middle, and possibly sulfate-reducing microorganisms on the
bottom. More complex biofilms can develop to form a four-dimensional structure (X,Y,
Z, and time) with cell aggregates, interstitial pores, and conduit channels (figure c).

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This developmental process involves the growth of attached microorganisms, resulting
in accumulation of additional cells on the surface, together with the continuous
trapping and immobilization of freefloating microor ganisms that move over the
expanding biofilm. This structure allows nutrients to reach the biomass, and the
channels are shaped by protozoa that graze on bacteria. These more complex biofilms,
in which microorganisms create unique environments, can be observed by the use of
confocal scanning laser microscopy (CSLM).
The diversity of nonliving and living surfaces that can be exploited by biofilm-forming
microorganisms is illustrated in figure.

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These include surfaces in catheters and dialysis units, which have intimate contact
with human body fluids. Control of such microorganisms and their establishment in
these sensitive medical devices is an important part of modern hospital care.
Microorganisms that form biofilms on living organisms such as plants or animals have
additional advantages. In these cases the surfaces themselves often release nutrients,
in the form of sloughed cells, soluble materials, and gases. These biofilms also can play
major roles in disease because they can protect pathogens from disinfectants; create a
focus for later occurrence of disease, or release microorganisms and microbial products
that may affect the immunological system of a susceptible host.
Biofilms are critical in ocular diseases because Chlamydia, Staphylococcus, and other
pathogens survive in ocular devices such as contact lenses and in cleaning solutions.
Depending on environmental conditions, biofilms can become so large that they are
visible and have macroscopic dimensions. Bands of microorganisms of different colors
are evident as shown in figure.

These thick biofilms, called microbial mats, are found in many freshwater and marine
environments. These mats are complex layered microbial communities that can form at

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the surface of rocks or sediments in hypersaline and freshwater lakes, lagoons, hot
springs, and beach areas. They consist of microbial filaments, including cyanobacteria.
A major characteristic of mats is the extreme gradients that are present.
Light only penetrates approximately 1 mm into these communities, and below this
photosynthetic zone, anaerobic conditions occur and sulfate-reducing bacteria play a
major role. The sulfide that these organisms produce diffuses to the anaerobic lighted
region, allowing sulfur-dependent photosynthetic microorganisms to grow. Some believe
that microbial mats could have allowed the formation of terrestrial ecosystems prior to
the development of vascular plants, and fossil microbial mats, called stromatolites, have
been dated at over 3.5 billion years old.

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UNIT: 5 BIOWASTE TREATMENT
Microorganisms involved in the degradation of plant fibre, cell wall, lignin, fungal
de – lignifications and pulping of wood. Pitch prob lems in pulp and paper
processes and solving by enzymes or fungi. Hemicell ulases in pulp bleaching.
Solving slime problem in the pulp and paper industr y. Reduction of
organochlorine compounds in bleach plant effluents.
Solid wastes: Sources and management, waste as a so urce of energy. Production of
oils and fuels from solid waste, composting, vermic ulture, Biogas production,
methanol production from organic wastes, byproducts of sugar industries.

MICROORGANISMS INVOLVED IN THE DEGRADATION
OF PLANT FIBRE, CELL WALL
Plant cell wall polysaccharides are the most abundant organic compounds found in
nature. They make up 90% of the plant cell wall and can be divided into three groups:
cellulose, hemicellulose, and pectin. Cellulose represents the major constituent of cell
wall polysaccharides and consists of a linear polymer of β-1,4-linkedD-glucose residues.
The cellulose polymers are present as ordered struc tures (fibers), and their main
function is to ensure the rigidity of the plant cell wall.
Hemicelluloses are more heterogeneous polysaccharid es and are the second most
abundant organic structure in the plant cell wall. The major hemicellulose polymer in
cereals and hardwood is xylan. Xylan consists of a β-1,4-linked D-xylose backbone and
can be substituted by different side groups such as L-arabinose,D-galactose, acetyl,
feruloyl, p-coumaroyl, and glucuronic acid residues. A second hemicellulose structure
commonly found in soft- and hardwoods is (galacto) glucomannan, which consists of a
backbone of β-1, 4-linkedD-mannose and D-glucose residues withD-galactose side
groups. Softwoods contain mainly galactoglucomannan , whereas in hardwoods
glucomannan is the most common form. Xyloglucans ar e present in the cell walls of
dicotyledonae and some monocotylodonae (e.g., onion). Xyloglucans consist of a β-1,4-
linked D-glucose backbone substituted by D-xylose. L-Arabinose andD-galactose
residues can be attached to the xylose residues, an d L-fucose has been detected
attached to galactose residues in xyloglucan. Xylog lucans interact with cellulose

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microfibrils by the formation of hydrogen bonds, thus contributing to the structural
integrity of the cellulose network.
Pectins form another group of heteropolysaccharides and consist of a backbone of α-
1,4-linked D-galacturonic acid residues. In specific “hairy” regions the galacturonic acid
backbone is interrupted by α-1,2-linked L-rhamnose residues. Long side chains
consisting mainly of L-arabinose andD-galactose residues can be attached to these
rhamnose residues. In pectins of certain origins (e.g., sugar beet and apple), ferulic acid
can be present as terminal residues attached to O-5 of the arabinose residues or O-2 of
the galactose residues.
The hemicellulose and pectin polysaccharides, as well as the aromatic polymer lignin,
interact with the cellulose fibrils, creating a rigid structure strengthening the plant cell
wall. They also form covalent cross-links, which are thought to be involved in limiting
cell growth and reducing cell wall biodegradability.
Two types of covalent cross-links have been identif ied between plant cell wall
polysaccharides and lignin. The cross-link formed by diferulic acid bridges is studied in
most detail. Diferulic acid bridges between polysaccharides and lignin have been
identified in many plants. They have been shown to occur between arabinoxylans from
bamboo shoot cell walls, between pectin polymers in sugar beet, and between lignin and
xylan in wheat. A second type of cross-link is the ester linkage between lignin and
glucuronic acid attached to xylan, which was identi fied in beech wood. Recently,
indications of a third type of cross-linking have been reported involving a protein- and
pH-dependent binding of pectin and glucuronoarabinoxylan to xyloglucan. This not yet
fully characterized binding is dependent on the presence of fucose on the xyloglucan.

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Schematic presentation of the hairy region of pectin

Schematic presentation of the two galactoglucomanna n structures.

Schematic presentation of galactomannan.

Schematic presentation of xylan.

Schematic presentation of the repeating units of the two major xyloglucan
structures.
Microbiological degradation of wood

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The woods under uninjured bark of healthy trees are generally free from
microorganisms. Microbial degradation of wood can c ause undesirable changes in
colour, lustre, texture, odor, grains and structural integrity of wood thus causing huge
loss by destroying wood in forest and wood used in buildings, houses etc. The disease
causing microbes alone are responsible for losses amounting more than 65% of the
wood volume in forest. Microbes that degrade wood produce extracellular enzymes that
breakdown woody cell wall. Different kinds of micro organisms are involved in
degradation of wood. Growth characteristics of microorganisms in the wood and type of
degradative system results in different decay patte rns. During the process of
degradation, substrate changes continuously and results in successive change in the
microbial population.
Microorganisms found to colonize and degrade wood i nclude (a) Basidiomycetes (b)
Ascomycetes (c) Phycomycetes (d) Deuteromycetes and (e) Bacteria. The chemical and
structural effects of the attack on wood and resulting decay patterns can be correlated
with these groups of microorganisms. The greatest loss of wood, however, are due to the
basidiomycetous members of families Polysporaceae, Thelophoraceae and Agaricaceae.
Many a times interection between insect and microorganism plays a crucial role in wood
decay. Mechanical destruction by insects renders wood exposed for action of microbes.
Some insects are ectosymbionts with fungi and some termites prefer to colonize on
wood attacked by fungi.
Many wood degrading fungi feed exclusively on intra cellular contents whilst others
continue to decompose components of cell wall as well. This is mainly dependent upon
the hydrolytic efficiency based on enzyme secretion. Wood decaying microorganisms can
therefore be grouped broadly into:
Microorganisms utilizing cell contents- (but not degrading lignified cell walls)
a. Moulds b. Blue stain fungi.
Microorganisms that breakdown lignified cell walls:
(i) With limited degradation ability
(c) Bacteria (d) Soft rot fungi
Microorganisms with high degradation ability:

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(e) Brown rot fungi (f) White rot fungi.
Wood decaying fungi
Moulds belonging to Ascomycetes and Deuteromycetes mainly feed on dead cell
contents and their hyphae accumulate in ray parenchyma cells or in cell lumina after
penetrating pit-tori. The infestation resembles incipient soft rot. Many members of these
classes cause discoloration of wood due to their pigmented hyphae (Blue stain fungi).
These are common in softwoods but hardwoods are no exception. Initially their hyphae
grow in ray parenchyma cells occurring only rarely in trachieds. In hardwoods the
hyphae are also found in fibres and trachieds around the rays. Alternaria, Bispora,
Chloridium and Bhialophora spp. are some common bluing fungi. Important wood
deteriorating fungi and their effects on wood is given in Table.

Soft rot
Fungal attack on the lignified cell walls characterized by a soft decayed surface of wood
in contact with excessive moisture is called as soft rot. This type of degradation is

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caused by fungi belonging to Ascomycetes and Deuteromycetes that can cause limited
enzymatic degradation of wood.
These fungi principally attack carbohydrates mainly cellulose while lignin is modified or
degraded to lesser extent. Characteristically, the hyphae penetrate into the cell wall and
develop within S
2
layer causing regular and rhomboidal or long cylindrical cavities with
conically tapered ends. In early stages, soft rot fungi primarily penetrates through pits
and often cause exhaustion of storage materials in the cells, borehole formation begins
both on radial or tangential walls. In hardwood, the fungus may also attack the cell
walls of the lumen, causing corrosion and subsequen t lysis of S
3
and S
2
layers. In
softwood S
3
layer being resistant, the principal location of soft rot cavities is the S
2
layer.
Before invasion of the tracheid cell walls, the longitudinal hyphae in cell lumina branch
laterally and produce fine, hyaline, perforation hyphae which grow horizontally through
the S
3
layer into S
2
layer. Later, the hyphae branch into T-shape giving two branches
parallel to microfibrils, which grow at the same rate in opposite directions and continue
to follow spiral fribrillar structure.

Cavity formation is closely related to hyphal growth due to proximity of hydrolases.
Apart from fungal species, the pattern of cavities is also influenced by physiological
factors e.g. temperature, water content etc. Finally, the entire secondary wall becomes
perforated by confluent cavities leaving a collapsible middle lamella, thus causing
severe loss in strength. About 70 species of the genera Chaetomium, Sordaria, Peziza,
Conithyrium, Cytospora, Phoma, Pestalotia, Chephalosporium, Monosporium, Penicillium,
Alternaria, Bispora, Chloridium, Phialophora, Stemphylium, Torula, Graphium, Stilbella,
Doratomyces and Fusarium are capable of causing soft rot in different kinds of wood.
The attack of exclusive soft-rot fungi is evidenced under extreme moisture conditions.
These being the pioneers on newly exposed wood are followed by other group of fungi. A

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slow attack advancing inward after destruction of o uter wood layers, exclusive
degradation of polysaccharides (lignin remains intact), formation of chains of cavities in
the S
2
layers of tracheids and fibres are identifying features of soft-rot.
LIGNIN, FUNGAL DE – LIGNIFICATIONS,
The structural complexity of lignin, its high molecular weight and its insolubility make
its degradation very difficult. Extracellular, oxidative, and unspecific enzymes that can
liberate highly unstable products which further und ergo many different oxidative
reactions catalyze the initial steps of lignin depolymerization. This non-specific
oxidation of lignin has been referred to as ‘‘enzymatic combustion’’. White-rot fungi are
the microorganisms that most efficiently degrade li gnin from wood. Of these,
Phanerochaete chrysosporium is the most extensively.
Two major families of enzymes are involved in ligni nolysis by white-rot fungi:
peroxidases and laccases. Apparently, these enzymes act using low-molecular weight
mediators to carry out lignin degradation. Several classifications of fungi have been
proposed based on their ligninolytic enzymes. Some of them produce all of the major
enzymes, others only two of them, or even only one. In addition, reductive enzymes
including cellobiose oxidizing enzymes, arylal coho l oxidases, and aryl alcohol
dehydrogenases seem to play major roles in ligninolysis.
Two groups of peroxidases, lignin peroxidases (LiPs ) and manganese-dependent
peroxidases (MnPs), have been well-characterized. LiP has been isolated from several
white-rot fungi. The catalytic, oxidative cycle of LiP has been well-established and is
similar to those of other peroxidases. In most fungi, LiP is present as a series of
isoenzymes encoded by different genes. LiP is a glycoprotein with a heme group in its
active center. Its molecular mass ranges from 38 to 43 kDa and its pI from 3.3 to 4.7.
So far, it is the most effective peroxidase and can oxidize phenolic and non-phenolic
compounds, amines, aromatic ethers, and polycyclic aromatics with appropriate
ionization potential. Since LiP is too large to enter the plant cell, it degradation is
carried out only in exposed regions of lumen. This kind of degradation is found in
simultaneous wood decays. However, microscopic stud ies of selective lignin
biodegradation reveal that white-rot fungi remove the polymer from inside the cell wall.
An indirect oxidation by LiP of lowmolecular- weight diffusible compounds capable of
penetrating the cell wall and oxidizing the polymer has been suggested. However, this

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theory lacks evidence since low-molecular-weight in termediates such as veratryl
alcoholcation radical are too short-lived to act as mediators. MnPs are molecularly very
similar to LiPs and are also glycosylated proteins, but they have slightly higher
molecular masses, ranging from 45 to 60 kDa.
MnPs oxidize Mn(II) to Mn(III). They have a conventional peroxidase catalytic cycle, but
with Mn(II) as substrate. This Mn(II) must be chelated by organic acid chelators, which
stabilize the product Mn(III). Mn(III) is a strong oxidant that can leave the active center
and oxidize phenolic compounds, but it cannot attack non-phenolic units of lignin. MnP
generates phenoxy-radicals which in turn undergo a variety of reactions, resulting in
depolymerization. In addition, MnP oxidizes non-phenolic lignin model compounds in
the presence of Mn(II) via peroxidation of unsatura ted lipids. A novel versatile
peroxidase (VP), which has both manganese peroxidase and lignin peroxidase activities
and which is involved in the natural degradation of lignin has been described.
VP can oxidize hydroquinone in the absence of exogenous H
2O2 when Mn(II) is present
in the reaction. It has been suggested that chemica loxid ation of hydroquinones
promoted by Mn(II) could be important during the initial steps of wood biodegradations
because ligninolytic enzymes are too large to penetrate into non-modified wood cell
walls. Laccases are blue-copper phenoloxidases that catalyze the one-electron oxidation
mainly of phenolic compounds and non-phenolics in t he presence of mediators. The
phenolic nucleus is oxidized by removal of one electron, generating phenoxy-free-radical
products, which can lead to polymer cleavage. Wood- rotting fungi are the main
producers of laccases but this oxidase has been isolated from many fungi including
Aspergillus and the thermophilic fungi Myceliophora thermophila and Chaemotium
thermophilium. Recently, bacterial laccases-like proteins have been found. These
enzymes polymerized a low-molecular-weight, water-s oluble organic matter fraction
isolated from compost into high-molecular-weight products, suggesting the involvement
of laccase in humification during composting.
The potential biotechnological applications of white rot fungi or their ligninolytic
enzymes are many. The most promising applications m ay be biopulping and bleaching
of chemical pulps. White-rot fungi can degrade/mine ralize a wide variety of toxic
xenobiotics including polycyclic aromatic hydrocarbons, chlorophenols, nitrotoluenes,
dyes, and polychlorinated and biphenyls. An obvious application is in situ
bioremediation of contaminated soils. Other fields under research are the use of these

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fungi for biocatalysis in the production of fine chemicals and natural flavors (e.g.
vanillin), and the biotreatment of several waste waters such as bleach plant effluents or
other waste water containing lignin-like polymers, as is the cases of dye-industry
effluents and olive-oil-mill waste-waters.
Degradation of lignin and lignin-degrading enzymes has also been reported for
actinobacteria from the Streptomyces genus. Even th ough lignin biodegradation is
accepted as an aerobic process, some authors have r eported that anaerobic
microorganisms in the rumen may alter, if not partially degrade, portions of lignified
plant cells.
PULPING OF WOOD
Biopulping is defined as the appropriate treatment of wood chips with lignin-degrading
fungi prior to pulping. This biotreatment not only reduces energy consumption in the
process of pulping, but also improves paper strength and removes wood extractives,
leading to additional benefits such as fewer pitch problems and lower effluent
toxicity.The pulp and paper industry uses mechanicalor chemical pulping processes or
a combination of both to produce pulps. Mechanical pulping involves mechanical force
to separate wood fibers. With this method, the yield is high, and the paper produced is
of good quality. In chemical pulping processes the yield is low, but the pulp produced
has a higher strength.
Pretreatments of wood chips for mechanicaland chemicalpul ping with severalwhite-rot
fungi have been developed in several laboratories. Biomechanical pulping using this
microorganism has proven to be feasible from both the engineering and the economic
point of view. Global concerns for the preservation of forests and the elimination of
environmental pollution from pulp and paper industr ies have focused research on
alternative fibrous resources for papermaking, such as non-woody plants or
agricultural residues. Biopulping of non-woody plants with C. subvermispora, Pleurotus
and other basidiomycetes reduces the amount of electricalpower used for the refining
stage by as much as 30%and improves paper properties.
Fungal pretreatment removes some and/or modifies; t hese changes make it easier to
remove macromolecules, probably because they improv e chemical penetration and so
minimize the use of chemicals. Biopulping also benefits other pulping techniques such
as sulfite, organosolv and dissolving pulp production. Fungal treatment prior to kraft
pulping has received little attention. However, severalstud ies indicate that biokraft

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pulping with white-rot fungi increases pulp yield and strength and reduces the cooking
time during kraft pulping.
Bleaching of pulps using enzymes or ligninolytic fungi is known as biobleaching. The
use of ligninolytic enzymes and hemicellulases aids in pulp bleaching and decreases the
amount of chemicalbl each required to obtain a desirable brightness of pulps.
Vikarii et al.were the first to show that treating kraft pulps with fungal hemicellulases
reduces subsequent chlorine bleaching requirements. Other authors have confirmed
these studies. Xylanase treatments decrease chlorine demand for kraft pulp by 6–15%.
In recent years at least 15 patents dealing with enzymatic treatments to enhance kraft
pulps have been released. There are different hypotheses to explain the mechanism of
hemicelluloses prebleaching. One of them suggests that the hydrolysis of relocated and
reprecipitated xylan on the surface of pulp fibers renders the pulp more permeable,
thereby enhancing residuall ignin extraction. The second model suggests that lignin or
chromophores generated during kraft cooking react with carbohydrates moieties.
Hemicellulases liberate lignin by releasing xylan-chromophore fragments and lignin
extraction increases.
Mannanases interact synergistically with xylanases and improve biobleaching,
especially in softwood. Although promising results were obtained initially, mannanases
seem to be less effective than xylanases. Several x ylanases from different
microorganisms have proven to be effective for biobleaching of various kinds of pulps.
PITCH PROBLEMS IN PULP AND PAPER PROCESSES AND SOLV ING BY ENZYMES
OR FUNGI
Industrial Use of Fungi to Solve Pitch Problems
Several studies have shown that wood extractive com ponents such as triglycerides,
resin acids, and steryl esters are major components of paper machine pitch deposits. In
addition, pitch outbreaks are more common when resinous wood species are used and
during seasons when wood resin content is particularly high.
There is a living fungus, marketed to the pulp and paper industry, which metabolizes
and thus removes pitch. This fungus is a colorless strain of O. piliferum, an Ascomycete
of the same species of that often dominates in naturally seasoned piles. Marketed as
Cartapip, with different numbers denoting different strains such as 97 and 58, it is
commercialized as a powder inoculum. Moreover, Cartapip use results in a biocontrol
effect, i.e., the presence of Cartapip reduce growth of other, undesired organisms. One

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kilogram of the powder can treat about 1200 tons of wood chips. Industrial use involves
dispersing the powder in mill water and spraying it onto chips as they are conveyed to a
chip pile.
Selective breeding was used to obtain this isolate, which rapidly colonizes nonsterile
wood chips, rapidly degrades extractives, and is colorless and nonstaining. Most O.
piliferum strains are a bluish-black color. Growth of pigmented fungi on wood chips
reduces chip brightness and increases bleach usage when these chips are used to
produce TMP or sulfite pulp. Because Cartapip outco mpetes indigenous
microorganisms and maintains chip brightness, use o f this product reduces bleach
chemical usage during TMP production, in addition to reducing the extractive content of
chips and pulp, and alleviating pitch problems.
Use of treated chips has also been shown to increas e paper strength. Moreover,
treatment of wood chips with Cartapip also results in improved chemical pulping
efficiency. Reductions in kappa number were observe d during laboratory-scale kraft
and sulfite pulping. Wall et al. hypothesize that the improved pulping efficiency
observed experimentally is caused by more rapid and more uniform penetration of
steam and cooking chemicals in the fungally treated chips.
Two commercially available strains of O. piliferum, Cartapip 28 and Cartapip 58, have
been shown to degrade the extractives of both hardw oods and softwoods including
aspen, southern yellow pine, red pine, and spruce, Both fungi in two weeks reduced the
diethyl ether extractive content of fresh nonsterile southern yellow pine wood chips by
40%, or 22% if the chips were aged but not inoculated. In addition, the white strain,
Cartapip 58, maintained chip brightness. This study also showed that Cartapip 28
decreased the DCM extractive content of sterile southern yellow pine chips by 30% for a
2 week treatment. O. piliferum has also been shown to reduce more DCM extractives of
nonsterile red pine in 31 days (48% reduction) than in 21 days (32% reduction). O.
piliferum, strain Cartapip 97, also reduces the DCM extractives of spruce by 25% in a 2
week treatment, and the DCM extractive content of sterile loblolly pine chips by up to
35% in a 4 week treatment. Cartapip 97 reduced the acetone extractive content of fresh
nonsterile aspen chips by 36% after a 3 week incubation, or 13% for uninoculated aged
chips. Cartapip 28 and Cartapip 58 significantly decreased the fatty acid content and
the unidentified compound content, which includes w axes, alcohols, and sterols, of
southern yellow pine extractives l-6]. In particular, Cartapip 58 decreased esterified

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fatty acids by 60%. Both esterified fatty acids and nonsaponifiable compounds such as
waxes and steryl esters have been shown to be major components of industrial pitch
deposits. Both fungal treatment and natural microbial activity increased the free fatty
acid content of the extractives.
The increase in free fatty acid content results from initial hydrolysis of esterified fatty
acids to free fatty acids. The free fatty acid content of the Cartapip-treated chips is
lower than that of naturally aged chips, indicating further metabolism and removal of
these components by the fungus. In addition, analys is of the individual fatty acids
showed that fungal treatment significantly decreased the content of the three fatty acids
found in highest concentration in the untreated southern yellow pine chips oleic acid by
44%, linoleic acid by 64%, and palmitic acid by 45%.
The results of Cartapip treatment such as pitch rem oval and maintence of chip
brightness and improved paper machine runnability have been documented by use in
mills. In a TMP mill using southern yellow pine, a trial was performed comparing a two
week period using the Cartapip product on their woo d chips to a two week period
without product use, and the results are shown in Table.

Reductions in the DCM extractive content of secondary refiner pulp caused expected
reductions in alum, a pitch control chemical. Because Cartapip 97 is a colorless strain
that outcompetes indigenous microorganisms, its use results in brighter chips. This
effect was observed as a 36.9% reduction in bleach usage along with increased paper
brightness of 0.9%. In addition, strength properties were increased, probably due to the
lower extractive content of the paper. Brandal and Lindheim have shown an inverse
relationship between paper strength and pitch content.
A two month Cartapip 97 trial at a US TMP mill usin g southern yellow pine showed
significant reductions in the DCM extractives of wood chips and an increase in burst
index. A one week trial was performed at a mill in Northwestern USA using a blend of
60% lodgepole pine and spruce and 40% fir and hemlock. Only the pine/spruce mixture

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was treated because this mixture caused the most serious pitch problems. Cartapip 97
treatment reduced the averaged DCM extractive content of the reclaim chips by 25%.
Enzymatic Pitch Control in the Papermaking Process
Identification of Compound Causing Pitch Trouble
A new method using an adsorption resin was establis hed, instead of the previous
solvent fractionation method, in order to fractionate red pine pitch and to determine
what the components that were sticky were and causi ng pitch troubles. Pitch
compounds in red pine as well as deposited pitch were fractionated using the method
and analyzed by gas chromatography. The changes in pitch compounds during the
seasoning period and the contents of pitch in fresh wood were also investigated in great
detail to understand the seasoning mechanism. These investigations produced the
following results:
Pitch compounds could be fractionated into polar and nonpolar fractions.
Fresh wood contained more nonpolar compounds, espec ially in winter. The main
component consisted of triglycerides (TG).
96% of the fatty acids that composed TG was oleic and linoleic acids.
TG was rapidly decomposed and reduced during seasoning.
Deposited pitch in the papermaking process always contained much TG.
Based on these results, TG was estimated to be the key to pitch troubles. In general,
nonpolar compounds such as TG may easily adhere to hydrophobic surfaces, such as
rolls, by Van der Waals forces and build to become pitch deposits. It was hypothesized
by Hata and his coworkers that if TG in pulp slurry could be converted to less adhesive
components, pitch deposits would decrease. The conversion of TG would enable the use
of flesh wood with less probability of pitch trouble.
Application of Lipase
Lipase specifically hydrolyzes TG, and thus was not expected to affect the environment
or the paper quality. Three kinds of lipase, each produced by a different microorganism,
were used in the original work by Hata and colleagues.
Effect of Lipase Treatment on Prevention of Pitch Deposition
Resinuous materials extracted from red pine wood an d groundwood pulp (GP) were
treated with lipase, and their adhesiveness to the hydrophobic surface was determined,
the pitch deposits increased when the ratio of nonpolar compounds to polar compounds
increased. Thus, evidently the nonpolar compounds of the pitch materials had higher

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adhesiveness to hydrophobic material and seemed to play an important role in pitch
deposition. TG was shown to be a key material in pi tch deposition because the
enzymatic hydrolysis of TG reduced pitch deposition significantly.
TG was hydrolyzed to glycerol and fatty acids with the lipase, and the resulting glycerol
dissolved into water. Fatty acids existed in the form of aluminum salt in the presence of
alum, and were dispersed into the pulp slurry and fixed on the surface of fibers.

Application to Papermaking Process
Since the effect of lipase on reducing pitch deposits was confirmed, the technology was
applied to the actual papermaking process. To select optimum conditions for the lipase
treatment in mills, the following factors were investigated: the effects of enzyme
concentration, reaction temperature, reaction time, and agitating mode on the
hydrolysis of TG. The following results were obtained from the investigation:
It was necessary to have a strong mixing system to keep contact between enzyme and
TG for the effective reaction by enzyme.
Under sufficient mixing conditions, lipase 5 000 U/kgGP (300 ppm Lipase B) could
hydrolyze more than 80% of TG in the surface pitch (n-hexane extract from GP slurry)
within two hours.
No effect of the lipase treatment on the brightness and strength of pulp was observed.
HEMICELLULASES IN PULP BLEACHING
Hemicellulases, especially xylanases, have been applied in processes such as pulp
bleaching, baking, and clarification of juices, extraction of coffee, plant oils and starch
and as a feed supplement to improve digestion in animals. Other potential applications
include the conversion of xylan in wastes from agricultural and food industry into
xylose, and the production of fuel and chemical feedstocks.
In pulp bleaching, xylanases selectively degrade the accessible hemicellulose fraction of
woods and have been found to enhance the extractabi lity of lignin. Xylanases from
several T. lanuginosus strains have exhibited promising results when applied as a
bleaching agent to kraft and sul ￠te pulp produced from sugar cane bagasse,

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Eucalyptus and beech. Signi￠cant reduction of the use of bleaching chemicals required
to attain the desired kappa number was found while increased brightness and viscosity
was achieved.
Commercial xylanases are typically produced by mesophilic ￠lamentous fungi such as
Trichoderma reesei and A. Niger, which are excellent protein secretors. However, these
xylanases may not be sufficiently thermostable for processes where enzymes active at
higher temperatures have a competitive advantage. Therefore the thermostable xylanase
of T. lanuginosus may be suitable for such high temperature processes. Deinking of
laser-printed paper was achieved when treated individually, and with combinations of a
xylanase from strain DSM 5826, a mannanase from S. rolfsii and purified
endoglucanases from Gloeophyllum sepiarium and Gloe ophyllum trabeum. The enzyme
treatment in these studies was conducted at a temperature more appropriate for the
optimum temperature of the enzymes from the mesophi lic fungi than the xylanase from
the thermophilic fungus T. lanuginosus. However, the combination of enzymes resulted
in 50% more mannan and 11% more xylan being solubil ised than did the individual
enzymes and illustrated the synergistic effects of using enzymes together for treatment
of pulp.
Addition of xylanase at the correct dosage to cerealbased foods such as bread, pasta
and noodles yields more flexible, easy-to-handle dough thereby improving the final
baked product. A baking process based on the use of xylanolytic enzymes from T.
lanuginosus strains has been patented and was repor ted to improve the baking
properties of dough. Hemicellulases, in addition to cellulases, also play an important
role in the digestion of grass and hay by ruminants and a xylanase from T. lanuginosus
was shown to improve in vitro rumen degradation of wheat straw when used as a
supplement.
SOLVING SLIME PROBLEM IN THE PULP AND PAPER INDUSTR Y
REDUCTION OF ORGANOCHLORINE COMPOUNDS IN BLEACH PLA NT EFFLUENTS
In the last few years environmental protection agencies have become more restrictive
regarding air and water discharges from chemical pulp and paper mills. The reductions
of contaminants affect all stages of the paper industry, including pulping, bleaching,
and papermaking.

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Pulping processes release colored compounds such as residual lignins, carbohydrate
degradation products, and extractives (lipophilic compounds) into the effluent streams.
The removalof this residual lignin can significantly reduce the volume of the chemicals
used for bleaching, thus lowering the amount of haz ardous compounds formed in
bleach-plant waste waters. Residual lignins in pulps are the cause of their typicaldark
brownish color, which must be removed by multistage bleaching.
The use of elemental chlorine for bleaching leads to environmental problems including
release of toxic and recalcitrant chlorinated aromatics such as dioxins.
The number of paper industries using chlorine dioxide in elemental chlorine-free (ECF)
bleaching is decreasing, especially in Europe. Totally chlorine-free (TCF) output is
estimated as 15% of total production. TCF pulp prod uction is growing slowly.
Alternative chemicals for bleaching have been used, e.g. oxygen, hydrogen peroxide, and
ozone. These compounds have some disadvantages, and the paper loses quality during
the bleaching. Alternatively, environmentally friendly technologies, based on the
capacity of microorganisms to degrade cellulose, hemicellulose, or lignin, have been
developed.
Present-day treatments of pulp and paper-mill effluents were reviewed by Ali and
Sreekrishnan and by Thompson et al. All pulp and paper mills have facilities to assure
compliance with the regulations established by environmental agencies in their own
coun try. Even so, the residualwaters impose coloration and toxicity problems on the
receiving waters, causing environmental hazards. Currently, most paper industries use
the classical primary and secondary biological treatments. Almost half the paper
industries use the aerobic activated-sludge method for the secondary treatment. Some
industries apply physical and chemical tertiary treatments, especially to remove the
residual dark color; but these treatments are expensive and ineffective. The dark color
of these effluents is mainly due to lignin and its derivatives, released during the
different stages of pulp and paper-making processes. Lignin is converted into thio- and
alkali-lignin during the kraft process, and to lignosulfonates in the sulfite process.
Chlorolignins are the main byproducts from chemical wood-pulp bleaching. These
compounds of high molecular weight are not degraded by any of the above described
biological treatments, and their final fates are unknown. The discharge of these colored
wastes is not only a problem of aesthetics, but it has also been proven that
chlorolignins are toxic and mutagenic.

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Physicalan d chemical processes to remove the dark color are expensive and do not
solve the problem because the lignins persist, although in different form. An alternative
treatment is the use of ligninolytic microorganisms. Severalspecies of bacteria have
been studied for their decolorization abilities. While some of them are able to decolorize,
only a few strains metabolize lignin derivatives. Color removal was efficiently achieved
by employing the FPL/NCSU Mycor method, which uses Phanerochaete chrysosporium
in rotating biological contactors a later modification of this method, termed MYCOPOR,
was developed by Messner et al. Biological decolorization of paper mill wastes using
either fungal mycelia, pellets, or immobilized cells was achieved with different strains.
Also, soluble or immobilized ligninolytic enzymes c an be employed for effluent
decolorization. In addition to white-rot fungi, oth er strains evaluated for the
decolorization and decontamination of kraft effluen ts include ascomycetes and
Thermoascus aurantiacus.
SOLID WASTES: SOURCES AND MANAGEMENT
Source Typical waste generators Types of solid wastes
Residential Single and multifamily
dwellings
Food wastes, paper,
cardboard, plastics, textiles,
leather, yard wastes, wood,
glass, metals, ashes, special
wastes (e.g., bulky items,
consumer electronics, white
goods, batteries, oil, tires),
and household hazardous
wastes.).
Industrial Light and heavy
manufacturing, fabrication,
construction sites, power
and chemical plants.
Housekeeping wastes,
packaging, food wastes,
construction and demolition
materials, hazardous
wastes, ashes, special
wastes.

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Commercial Stores, hotels, restaurants,
markets, office buildings,
etc.
Paper, cardboard, plastics,
wood, food wastes, glass,
metals, special wastes,
hazardous wastes.
Institutional Schools, hospitals, prisons,
government centers.
Same as commercial.
Construction and
demolition
New construction sites,
road repair, renovation
sites, demolition of
buildings
Wood, steel, concrete, dirt,
etc.
Municipal services Street cleaning,
landscaping, parks,
beaches, other recreational
areas, water and
wastewater treatment
plants.
Street sweepings; landscape
and tree trimmings; general
wastes from parks, beaches,
and other recreational areas;
sludge.
Process
(manufacturing,
etc.)
Heavy and light
manufacturing, refineries,
chemical plants, power
plants, mineral extraction
and processing.
Industrial process wastes,
scrap materials, off-
specification products, slay,
tailings.
Agriculture Crops, orchards, vineyards,
dairies, feedlots, farms.
Spoiled food wastes,
agricultural wastes,
hazardous wastes (e.g.,
pesticides).

Management:
Introduction to solid waste management
Solid waste is the unwanted or useless solid materi als generated from combined
residential, industrial and commercial activities in a given area. It may be categorized

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according to its origin (domestic, industrial, commercial, construction or institutional);
according to its contents (organic material, glass, metal, plastic paper etc); or according
to hazard potential (toxic, non-toxin, flammable, radioactive, infectious etc).
Management of solid waste reduces or eliminates adverse impacts on the environment
and human health and supports economic development and improved quality of life. A
number of processes are involved in effectively managing waste for a municipality.
These include monitoring, collection, transport, processing, recycling and disposal.
Reduce, Reuse, Recycle
Methods of waste reduction, waste reuse and recycling are the preferred options when
managing waste. There are many environmental benefi ts that can be derived from the
use of these methods. They reduce or prevent green house gas emissions, reduce the
release of pollutants, conserve resources, save energy and reduce the demand for waste
treatment technology and landfill space. Therefore it is advisable that these methods be
adopted and incorporated as part of the waste management plan.
Waste reduction and reuse
Waste reduction and reuse of products are both meth ods of waste prevention. They
eliminate the production of waste at the source of usual generation and reduce the
demands for large scale treatment and disposal facilities. Methods of waste reduction
include manufacturing products with less packaging, encouraging customers to bring
their own reusable bags for packaging, encouraging the public to choose reusable
products such as cloth napkins and reusable plastic and glass containers, backyard
composting and sharing and donating any unwanted it ems rather than discarding
them.
All of the methods of waste prevention mentioned require public participation. In order
to get the public onboard, training and educational programmes need to be undertaken
to educate the public about their role in the process. Also the government may need to
regulate the types and amount of packaging used by manufacturers and make the
reuse of shopping bags mandatory.
Waste Collection
Waste from our homes is generally collected by our local authorities through regular
waste collection, or by special collections for recycling. Within hot climates such as that
of the Caribbean the waste should be collected at least twice a week to control fly
breeding, and the harbouring of other pests in the community. Other factors to consider

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when deciding on frequency of collection are the odours caused by decomposition and
the accumulated quantities.
Treatment & Disposal
Waste treatment techniques seek to transform the wa ste into a form that is more
manageable, reduce the volume or reduce the toxicity of the waste thus making the
waste easier to dispose of. Treatment methods are selected based on the composition,
quantity, and form of the waste material. Some waste treatment methods being used
today include subjecting the waste to extremely high temperatures, dumping on land or
land filling and use of biological processes to treat the waste. It should be noted that
treatment and disposal options are chosen as a last resort to the previously mentioned
management strategies reducing, reusing and recycling of waste (figure).

Thermal treatment
This refers to processes that involve the use of heat to treat waste. Listed below are
descriptions of some commonly utilized thermal treatment processes.
Incineration
Incineration is the most common thermal treatment process. This is the combustion of
waste in the presence of oxygen. After incineration, the wastes are converted to carbon
dioxide, water vapour and ash. This method may be u sed as a means of recovering
energy to be used in heating or the supply of electricity. In addition to supplying energy
incineration technologies have the advantage of reducing the volume of the waste,
rendering it harmless, reducing transportation costs and reducing the production of the
green house gas methane
Pyrolysis and Gasification

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Pyrolysis and gasification are similar processes they both decompose organic waste by
exposing it to high temperatures and low amounts of oxygen. Gasification uses a low
oxygen environment while pyrolysis allows no oxygen. These techniques use heat and
an oxygen starved environment to convert biomass in to other forms. A mixture of
combustible and non-combustible gases as well as pyroligenous liquid is produced by
these processes. All of these products have a high heat value and can be utilised.
Gasification is advantageous since it allows for the incineration of waste with energy
recovery and without the air pollution that is characteristic of other incineration
methods.
Open burning
Open burning is the burning of unwanted materials in a manner that causes smoke
and other emissions to be released directly into the air without passing through a
chimney or stack. This includes the burning of outdoor piles, burning in a burn barrel
and the use of incinerators which have no pollution control devices and as such release
the gaseous by products directly into the atmosphere. Openburning has been practiced
by a number of urban centres because it reduces the volume of refuse received at the
dump and therefore extends the life of their dumpsite. Garbage may be burnt because
of the ease and convenience of the method or because of the cheapness of the method.
In countries where house holders are required to pay for garbage disposal, burning of
waste in the backyard allows the householder to avoid paying the costs associated with
collecting, hauling and dumping the waste.
Open burning has many negative effects on both huma n health and the environment.
This uncontrolled burning of garbage releases many pollutants into the atmosphere.
These include dioxins, particulate matter, polycyclic aromatic compounds, volatile
organic compounds, carbon monoxide, hexachlorobenze ne and ash. All of these
chemicals pose serious risks to human health. The Dioxins are capable of producing a
multitude of health problems; they can have adverse effects on reproduction,
development, disrupt the hormonal systems or even c ause cancer. The polycyclic
aromatic compounds and the hexachlorobenzene are considered to be carcinogenic. The
particulate matter can be harmful to persons with respiratory problems such as asthma
or bronchitis and carbon monoxide can cause neurological symptoms.
The harmful effects of open burning are also felt by the environment. This process
releases acidic gases such as the halo-hydrides; it also may release the oxides of

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nitrogen and carbon. Nitrogen oxides contribute to acid rain, ozone depletion, smog and
global warming. In addition to being a green house gas carbon monoxide reacts with
sunlight to produce ozone which can be harmful. The particulate matter creates smoke
and haze which contribute to air pollution.
Dumps and Landfills
Sanitary landfills
Sanitary Landfills are designed to greatly reduce or eliminate the risks that waste
disposal may pose to the public health and environm ental quality. They are usually
placed in areas where land features act as natural buffers between the landfill and the
environment. For example the area may be comprised of clay soil which is fairly
impermeable due to its tightly packed particles, or the area may be characterised by a
low water table and an absence of surface water bodies thus preventing the threat of
water contamination. In addition to the strategic p lacement of the landfill other
protective measures are incorporated into its design. The bottom and sides of landfills
are lined with layers of clay or plastic to keep the liquid waste, known as leachate, from
escaping into the soil. The leachate is collected and pumped to the surface for
treatment. Boreholes or monitoring wells are dug in the vicinity of the landfill to monitor
groundwater quality. A landfill is divided into a series of individual cells and only a few
cells of the site are filled with trash at any one time. This minimizes exposure to wind
and rain. The daily waste is spread and compacted to reduce the volume, a cover is
then applied to reduce odours and keep out pests. When the landfill has reached its
capacity it is capped with an impermeable seal which is typically composed of clay soil.
Some sanitary landfills are used to recover energy. The natural anaerobic
decomposition of the waste in the landfill produces landfill gases which include Carbon
Dioxide, methane and traces of other gases. Methane can be used as an energy source
to produce heat or electricity. Thus some landfills are fitted with landfill gas collection
(LFG) systems to capitalise on the methane being produced. The process of generating
gas is very slow, for the energy recovery system to be successful there needs to be large
volumes of wastes.
These landfills present the least environmental and health risk and the records kept
can be a good source of information for future use in waste management, however, the
cost of establishing these sanitary landfills are high when compared to the other land
disposal methods.

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Controlled dumps
Controlled dumps are disposal sites which comply with most of the requirements for a
sanitary landfill but usually have one deficiency. They may have a planned capacity but
no cell planning, there may be partial leachate man agement, partial or no gas
management, regular cover, compaction in some cases, basic record keeping and they
are fenced or enclosed. These dumps have a reduced risk of environmental
contamination, the initial costs are low and the operational costs are moderate. While
there is controlled access and use, they are still accessible by scavengers and so there
is some recovery of materials through this practice.
Bioreactor Landfills
Recent technological advances have lead to the introduction of the Bioreactor Landfill.
The Bioreactor landfills use enhanced microbiological processes to accelerate the
decomposition of waste. The main controlling factor is the constant addition of liquid to
maintain optimum moisture for microbial digestion. This liquid is usually added by re
circulating the landfill leachate. In cases where leachate in not enough, water or other
liquid waste such as sewage sludge can be used. The landfill may use either anaerobic
or aerobic microbial digestion or it may be designe d to combine the two. These
enhanced microbial processes have the advantage of rapidly reducing the volume of the
waste creating more space for additional waste, they also maximise the production and
capture of methane for energy recovery systems and they reduce the costs associated
with leachate management. For Bioreactor landfills to be successful the waste should
be comprised predominantly of organic matter and sh ould be produced in large
volumes.
Biological waste treatment

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Composting
Composting is the controlled aerobic decomposition of organic matter by the action of
micro organisms and small invertebrates. There are a number of composting techniques
being used today. These include: in vessel composti ng, windrow composting,
vermicomposting and static pile composting. The process is controlled by making the
environmental conditions optimum for the waste deco mposers to thrive. The rate of
compost formation is controlled by the composition and constituents of the materials
i.e. their Carbon/Nitrogen (C/N) ratio, the temperature, the moisture content and the
amount of air.
The C/N ratio is very important for the process to be efficient. The micro organisms
require carbon as an energy source and nitrogen for the synthesis of some proteins. If
the correct C/N ration is not achieved, then application of the compost with either a
high or low C/N ratio can have adverse effects on both the soil and the plants. A high
C/N ratio can be corrected by dehydrated mud and a low ratio corrected by adding
cellulose. Moisture content greatly influences the composting process. The microbes
need the moisture to perform their metabolic functions. If the waste becomes too dry
the composting is not favoured. If however there is too much moisture then it is
possible that it may displace the air in the compost heap depriving the organisms of
oxygen and drowning them.
A high temperature is desirable for the elimination of pathogenic organisms. However, if
temperatures are too high, above 75
o
C then the organisms necessary to complete the
composting process are destroyed. Optimum temperatu res for the process are in the
range of 50-60
o
C with the ideal being 60
o
C.
Aeration is a very important and the quantity of air needs to be properly controlled
when composting. If there is insufficient oxygen the aerobes will begin to die and will be
replaced by anaerobes. The anaerobes are undesirable since they will slow the process,
produce odours and also produce the highly flammabl e methane gas. Air can be
incorporated by churning the compost.
Anaerobic Digestion
Anaerobic digestion like composting uses biological processes to decompose organic
waste. However, where composting can use a variety of microbes and must have air,
anaerobic digestion uses bacteria and an oxygen free environment to decompose the
waste. Aerobic respiration, typical of composting, results in the formation of Carbon

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dioxide and water. While the anaerobic respiration results in the formation of Carbon
Dioxide and methane. In addition to generating the humus which is used as a soil
enhancer, Anaerobic Digestion is also used as a method of producing biogas which can
be used to generate electricity.
Optimal conditions for the process require nutrients such as nitrogen, phosphorous
and potassium, it requires that the pH be maintained around 7 and the alkalinity be
appropriate to buffer pH changes, temperature should also be controlled.
Integrated Solid Waste Management
Integrated Solid Waste Management (ISWM) takes an o verall approach to creating
sustainable systems that are economically affordabl e, socially acceptable and
environmentally effective. An integrated solid waste management system involves the
use of a range of different treatment methods, and key to the functioning of such a
system is the collection and sorting of the waste. It is important to note that no one
single treatment method can manage all the waste ma terials in an environmentally
effective way. Thus all of the available treatment and disposal options must be
evaluated equally and the best combination of the a vailable options suited to the
particular community chosen. Effective management schemes therefore need to operate
in ways which best meet current social, economic, and environmental conditions of the
municipality.

WASTE AS A SOURCE OF ENERGY
Waste-to-energy conversions
Energy from waste is not a new concept, but it is a field which requires a serious
attention. There are various energy conversion technologies available to get energy from
solid waste, but the selection is based on the physicochemical properties of the waste,
the type and quantity of waste feedstock, and the desired form of energy.

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Conversion of solid waste to energy is undertaken u sing three main process
technologies: thermochemical, biochemical, and mechanical extraction.
Biochemical conversion:
Biochemical conversion processes make use of the en zymes of bacteria and other
microorganisms to breakdown biomass. Biochemical conversion is one of the few which
provide environment friendly direction for obtaining energy fuel from MSW.
In most of the cases, microorganisms are used to pe rform the conversion process:
anaerobic digestion and fermentation. Anaerobic digestion is the conversion of organic
material directly to a gas, termed biogas, which has a calorific value of around 20 to 25
MJ/Nm
3
with methane content varying between 45% and 75% a nd the remainder of
CO
2 (biomass conversion) with small quantities of other gasses such as hydrogen.
Fermentation is used commercially on a large scale in various countries to produce
ethanol from sugar crops. This produces diluted alcohols which then are needed to be
distilled and, thus, suffers from a lower overall process performance and high plant
cost.
Thermochemical conversion:
Thermal conversion is the component of a number of the integrated waste management
solutions proposed in the various strategies. Four main conversion technologies have
emerged for treating dry and solid waste: combustio n (to immediately release its
thermal energy), gasification, pyrolysis, and liquefaction (to produce an intermediate
liquid or gaseous energy carrier).
Combustion is the burning of biomass in air. It is used over a wide range of commercial
and industrial combustion plant outputs to convert the chemical energy stored in the
solid waste into heat or electricity using various items of process equipment, such as
boilers and turbines. It is possible to burn any type of biomass, but in practice,
combustion is feasible only for biomass with a moisture content <50%, unless the
biomass is pre-dried.
Gasification process means treating a carbon-based material with oxygen or steam to
produce a gaseous fuel. Gas produced can be cleaned and burned in a gas engine or
transformed chemically into methanol that can be used as a synthetic compound.
Pyrolysis is the heating of biomass in the absence of oxygen and results to liquid
(termed bio-oil or biocrude), solid, and gaseous fractions in varying yields depending on

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a range of parameters such as heating rate, tempera ture level, particle size, and
retention time.
Liquefaction is the low-temperature cracking of biomass molecules due to high pressure
and results in a liquid-diluted fuel. The advantage of this process, employing only low
temperatures of around 200°C to 400°C, has to compe te with comparably low yields
and extensive equipment prerequisites to provide the pressure levels needed (50 to 200
bars).
Mechanical extraction:
It can be used to produce oil from the seeds of sol id waste. Rapeseed oil can be
processed further by reacting it with alcohol using a process termed esterification to
obtain biodiesel, for example.
PRODUCTION OF OILS AND FUELS FROM SOLID WASTE
COMPOSTING
Composting is the natural process of 'rotting' or decomposition of organic matter by
microorganisms under controlled conditions. Raw organic materials such as crop
residues, animal wastes, food garbage, some municipal wastes and suitable industrial
wastes, enhance their suitability for application to the soil as a fertilizing resource, after
having undergone composting.
Compost is a rich source of organic matter. Soil organic matter plays an important role
in sustaining soil fertility, and hence in sustainable agricultural production. In addition
to being a source of plant nutrient, it improves the physico-chemical and biological
properties of the soil. As a result of these improvements, the soil: (i) becomes more
resistant to stresses such as drought, diseases and toxicity; (ii) helps the crop in
improved uptake of plant nutrients; and (iii) possesses an active nutrient cycling
capacity because of vigorous microbial activity. These advantages manifest themselves
in reduced cropping risks, higher yields and lower outlays on inorganic fertilizers for
farmers.
Types of composting
Composting may be divided into two categories by the nature of the decomposition
process. In anaerobic composting, decomposition occurs where oxygen (O) is absent or
in limited supply. Under this method, anaerobic micro-organisms dominate and develop
intermediate compounds including methane, organic acids, hydrogen sulphide and
other substances. In the absence of O, these compounds accumulate and are not

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metabolized further. Many of these compounds have strong odours and some present
phytotoxicity. As anaerobic composting is a low-temperature process, it leaves weed
seeds and pathogens intact. Moreover, the process usually takes longer than aerobic
composting. These drawbacks often offset the merits of this process, viz. little work
involved and fewer nutrients lost during the process.
Aerobic composting takes place in the presence of ample O. In this process, aerobic
microorganisms break down organic matter and produce carbon dioxide (CO
2),
ammonia, water, heat and humus, the relatively stable organic end product. Although
aerobic composting may produce intermediate compoun ds such as organic acids,
aerobic micro-organisms decompose them further. The resultant compost, with its
relatively unstable form of organic matter, has little risk of phytotoxicity. The heat
generated accelerates the breakdown of proteins, fats and complex carbohydrates such
as cellulose and hemi-cellulose. Hence, the processing time is shorter. Moreover, this
process destroys many micro-organisms that are human or plant pathogens, as well as
weed seeds, provided it undergoes sufficiently high temperature. Although more
nutrients are lost from the materials by aerobic composting, it is considered more
efficient and useful than anaerobic composting for agricultural production. Most of this
publication focuses on aerobic composting.
Composting objectives may also be achieved through the enzymatic degradation of
organic materials as they pass through the digestive system of earthworms. This
process is termed vermicomposting.
The aerobic composting process
The aerobic composting process starts with the formation of the pile. In many cases, the
temperature rises rapidly to 70-80 °C within the first couple of days. First, mesophilic
organisms (optimum growth temperature range = 20-45 °C) multiply rapidly on the
readily available sugars and amino acids (Figure 1). They generate heat by their own
metabolism and raise the temperature to a point where their own activities become
suppressed. Then a few thermophilic fungi and several thermophilic bacteria (optimum
growth temperature range = 50-70 °C or more) continue the process, raising the
temperature of the material to 65 °C or higher. This peak heating phase is important for
the quality of the compost as the heat kills pathogens and weed seeds.
The active composting stage is followed by a curing stage, and the pile temperature
decreases gradually. The start of this phase is identified when turning no longer reheats

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the pile. At this stage, another group of thermophilic fungi starts to grow. These fungi
bring about a major phase of decomposition of plant cell-wall materials such as
cellulose and hemi-cellulose. Curing of the compost provides a safety net against the
risks of using immature compost such as nitrogen (N) hunger, O deficiency, and toxic
effects of organic acids on plants.
Eventually, the temperature declines to ambient temperature. By the time composting
is completed, the pile becomes more uniform and less active biologically although
mesophilic organisms recolonize the compost. The material becomes dark brown to
black in colour. The particles reduce in size and become consistent and soil-like in
texture. In the process, the amount of humus increases, the ratio of carbon to nitrogen
(C:N) decreases, pH neutralizes, and the exchange capacity of the material increases.
Temperature changes and fungi populations in wheat straw compost

Note:
Solid line = temperature; broken line = mesophilic fungi population; dotted line =
thermophilic fungi population; left y-axis = fungal populations (logarithm of colony
forming units (cfu) per gram of compost plated onto agar); right y-axis = temperature in
centre of compost. a, b, c and d = heating phases.
Factors affecting aerobic composting
Aeration
Aerobic composting requires large amounts of O, particularly at the initial stage.
Aeration is the source of O, and, thus, indispensable for aerobic composting. Where the
supply of O is not sufficient, the growth of aerobic micro-organisms is limited, resulting

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in slower decomposition. Moreover, aeration removes excessive heat, water vapour and
other gases trapped in the pile. Heat removal is particularly important in warm climates
as the risk of overheating and fire is higher. Therefore, good aeration is indispensable
for efficient composting. It may be achieved by controlling the physical quality of the
materials (particle size and moisture content), pile size and ventilation and by ensuring
adequate frequency of turning.
Moisture
Moisture is necessary to support the metabolic activity of the micro-organisms.
Composting materials should maintain a moisture content of 40-65 percent. Where the
pile is too dry, composting occurs more slowly, while a moisture content in excess of 65
percent develops anaerobic conditions. In practice, it is advisable to start the pile with a
moisture content of 50-60 percent, finishing at about 30 percent.
Nutrients
Micro-organisms require C, N, phosphorus (P) and potassium (K) as the primary
nutrients. Of particular importance is the C:N ratio of raw materials. The optimal C:N
ratio of raw materials is between 25:1 and 30:1 although ratios between 20:1 and 40:1
are also acceptable. Where the ratio is higher than 40:1, the growth of micro-organisms
is limited, resulting in a longer composting time. A C:N ratio of less than 20:1 leads to
underutilization of N and the excess may be lost to the atmosphere as ammonia or
nitrous oxide, and odour can be a problem. The C:N ratio of the final product should be
between about 10:1 and 15:1.
Temperature
The process of composting involves two temperature ranges: mesophilic and
thermophilic. While the ideal temperature for the initial composting stage is 20-45 °C,
at subsequent stages with the thermophilic organisms taking over, a temperature range
of 50-70 °C may be ideal. High temperatures characterize the aerobic composting
process and serve as signs of vigorous microbial activities. Pathogens are normally
destroyed at 55 °C and above, while the critical point for elimination of weed seeds is 62
°C. Turnings and aeration can be used to regulate temperature.
Lignin content
Lignin is one of the main constituents of plant cell walls, and its complex chemical
structure makes it highly resistant to microbial degradation (Richard, 1996). This
nature of lignin has two implications. One is that lignin reduces the bioavailability of

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the other cell-wall constituents, making the actual C:N ratio (viz. ratio of biodegradable
C to N) lower than the one normally cited. The other is that lignin serves as a porosity
enhancer, which creates favourable conditions for aerobic composting. Therefore, while
the addition of lignin-decomposing fungi may in some cases increase available C,
accelerate composting and reduce N loss, in other cases it may result in a higher actual
C:N ratio and poor porosity, both of which prolong composting time.
Polyphenols
Polyphenols include hydrolysable and condensed tannins (Schorth, 2003). Insoluble
condensed tannins bind the cell walls and proteins and make them physically or
chemically less accessible to decomposers. Soluble condensed and hydrolysable tannins
react with proteins and reduce their microbial degradation and thus N release.
Polyphenols and lignin are attracting more attention as inhibiting factors. Palm et al.
(2001) suggest that the contents of these two substances be used to classify organic
materials for more efficient on-farm natural resource utilization, including composting.
pH value
Although the natural buffering effect of the composting process lends itself to accepting
material with a wide range of pH, the pH level should not exceed eight. At higher pH
levels, more ammonia gas is generated and may be lost to the atmosphere.
Techniques for effective aerobic composting
Simple replication of composting practices does not always give the right answer to
potential composters. This is because composting takes place at various locations and
under diverse climates, using different materials with dissimilar physical, chemical and
biological properties. An understanding of the principles and technical options and their
appropriate application may be helpful in providing the optimal environment to the
compost pile.
Improved aeration
In order to obtain the end product of uniform quality, the whole of the pile should
receive a sufficient amount of O so that aerobic micro-organisms flourish uniformly.
The methodologies deliberated in this publication made use of the techniques as
presented below.
Pile size and porosity of the material
The size of the pile is of great significance and finds mention in the sections on passive
composting of manure piles (Chapter 2) and turned wind-rows (Chapter 3). Where the

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pile or wind-row is too large, anaerobic zones occur near its centre, which slows the
process in these zones. On the other hand, piles or wind-rows that are too small lose
heat quickly and may not achieve a temperature high enough to evaporate moisture
and kill pathogens and weed seeds. The optimal size of the piles and wind-rows should
also consider such parameters as the physical property (porosity) of the materials and
the way of forming the pile. While more porous materials allow bigger piles, heavy
weights should not be put on top and materials should be kept as loose as possible.
Climate is also a factor. With a view to minimizing heat loss, larger piles are suitable for
cold weather. However, in a warmer climate, the same piles may overheat and in some
extreme cases (75 °C and above) catch fire.
Ventilation
Provision of ventilation complements efforts to optimize pile size. Ventilation methods
are varied. The simplest method is to punch holes in the pile at several points. The high
temperature compost method of Chinese rural composting involves inserting a number
of bamboo poles deep into the pile and withdrawing them a day later, leaving the pile
with ventilation holes.
Aeration is improved by supplying more air to the base of the pile where O deficiency
occurs most often. In addition to the above-mentioned vertical poles, Ecuador on-farm
composting uses a lattice of old branches at the base to allow more pile surface to come
into contact with the air, and the composting period is reduced to two to three months
in warm seasons. This technique is also practised in the rapid composting method
developed by the Institute of Biological Sciences (IBS) in the Philippines, where the
platform should be 30 cm above the ground. The passively aerated wind-rows method
uses a more sophisticated technique. It entails embedding perforated pipes throughout
the pile. As the pipe ends are open, air flow is induced and O is supplied to the pile
continuously. The aerated static pile method takes this aeration system a step further;
a blower generates air flow to create negative pressure (suction) in the pile and fresh air
is supplied from outside.
Turning
Once the pile is formed and decomposition starts, the only technique for improving
aeration is turning. As Table 1 shows, frequency of turning is crucial for composting
time. While the Indian Bangalore method requires six to eight months to mature, the
Indian Coimbatore method (turning once) reduces the time to four months, and the

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Chinese rural composting pit method (turning three times) reduces the time to three
months. An extreme example is the Berkley rapid composting method, which employs
daily turning to complete the process in two weeks. In some cases, turning not only
distributes air throughout the pile, it also prevents overheating as it kills all the
microbes in the pile and terminates decomposition. However, turning too frequently
might result in a lower temperature.
Inoculation
While some composters find improved aeration enough for enhanced microbial
activities, others may need inoculation of micro-organisms. Inoculum organisms
utilized for composting are mainly fungi such as Trichodermasp. (IBS rapid composting
and composting weeds and Pleurotus sp. (composting Coir Pith and composting weeds).
This publication also features 'effective micro-organisms' (EMs) (EM-based quick
compost production process. The inoculums are an affordable choice for those with
access to the market and also for resource-poor farmers. The production cost could be
reduced by using inoculums taken from compost pits (pit method of the Indian Indore
method), by purchasing the commercial product and multiplying it on the farm (EM-
based quick compost production process), and by utilizing native inoculums derived
from soils or plant leaves.
Supplemental nutrition
The techniques mentioned above often need to be complemented by the provision of
nutrients. One of the most common practices is to add inorganic fertilizers, particularly
N, in order to modify a high C:N ratio. Similarly, P is sometimes applied as the C:P ratio
of the material mix is also considered important (the ratio should be between 75:1 and
150:1). When micro-organisms are inoculated, they require sugar and amino acids in
order to boost their initial activities; molasses is often added for this purpose.
Table 1 Salient features of selected small-scale aerobic composting techniques
Method
Salient features
Duration
Substrate
size
reduction
Turnings
at
intervals
of (days)
Added aeration
provision
Microbial
inoculation
Supporting
microbial
nutrition
Indore pit +15,
+30, +60
Inoculum from
old pit
4
months

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Indore heap Shredded +42, +84 4
months
Chinese pit +30,
+60, +75
Superphosphate 3
months
Chinese
high
temperature
compost
Shredded +15 Aeration holes in
heap through
bamboo
poles/maize
stalks
Superphosphate 2
months
Ecuador
on-farm
composting
+21 Lattice of old
branches/poles at
heap base
2-3
months
in
summer;
5-6
months
in winter
Berkley
rapid
composting
Shredded
to small
size
Daily or
alternate
day
turning
2 weeks
with
daily
turning
& 3
weeks
with
alternate
day
turning
North
Dakota
State
University
hot
composting
Shredded +3 or +4 4-5 holes
punched in centre
of pile
0.12 kg N per
90 cm dry
matter
4-6
weeks
EM-based
quick
composting
+14, +21 EM Molasses 4-5
weeks
IBS rapid
composting
Shredded +7, +14,
then
Raised platform
ground/perforated
Trichodermasp. 3-7
weeks

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every 2
weeks
bamboo trunks
Shredding
Downsizing, or chopping up the materials, is a sound and widely-practised technique. It
increases the surface area available for microbial action and provides better aeration.
This technique is particularly effective and necessary for harder materials such as
wood.
Other measures
An example of other measures mentioned in this publication is the practice of adding
lime. Lime is thought to weaken the lignin structure of the plant materials and enhance
the microbial population. However, in some cases, liming is not recommended as the
pile may become too alkaline, resulting in significant N loss.
VERMICULTURE
Vermiculture is basically the science of breeding and raising earthworms. It defines the
thrilling potential for waste reduction, fertilizer production, as well as an assortment of
possible uses for the future. Vermicomposting is the process of producing organic
fertilizer or the vermicompost from bio-degradable materials with earthworms.
Composting with worms avoids the needless disposal of vegetative food wastes and
enjoys the benefits of high quality compost. The earthworm is one of nature’s pinnacle
“soil scientists.” Earthworms are liberated and cost effective farm relief. The worms are
accountable for a variety of elements including turning common soil into superior
quality. They break down organic matter and when they eat, they leave behind castings
that are an exceptionally valuable type of fertilizer.
Methodology
Vermiculture is the science of worm composting. Worms can eat their body weight each
day in fruit and vegetable scraps, leaving castings as the byproduct. Worm castings are
called worm compost.
Clean-up and Preparation of Vermi Beds
There are two vermi beds, 1 x 2 in size and made with hollow blocks. We have cleaned
each vermi beds and started to gather substrates.
Substrate Application
After some days of gathering, add the substrates to both vermi beds and put a mixture
of loam soil, carabao manure and partially decomposed leaves in the first vermi bed

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while in the second bed; Put a mixture of carabao manure, partially decomposed rice
straw and rice hull and shredded moist newspapers. The succeeding application made
used of mixed and different substrates.
Before putting the substrate, we made sure that the materials are cut or break into
smaller pieces. Finer materials could easily decompose (partial decomposition). We also
mixed the different media together well for the worms to easily digest these. We have
moistened the materials and cover the vermi beds wi th roof and tarpaulin cover to
initiate anaerobic decomposition. The substrates were kept in the beds for ten days
before we put the vermi worms. It took 10 to 15 day s to complete anaerobic
decomposition and only then that they are ready for worm consumption.
Introducing the Vermi Worms, Red wriggler (Eisenia foetida)
After 10 days upon putting the substrates into the vermi beds, introduce the vermi
worms into the substrate. Here the Red wriggler ( Eisenia foetida) is used in our
vermicompost. Aerobic decomposition lasts for 7 – 14 days depending on the materials
used and the ratio of the worms to the substrate.Within the period, moistened (not
soggy) the substrate regularly to provide the right moisture (60 - 80%) for the vermi
worms to grow and multiply.
Feeding the Vermi Worms
After introducing the red wrigglers, fed the worms by placing vegetable wastes and also
saluyot (Corchorus capsularis) leaves and malunggay (Moringa oleifera) leaves. The
vegetable wastes are placed in a different place each time for the worms to easily feed
into it. After two weeks, the red wrigglers have eaten the food waste leaving behind
worm casting or compost.
Harvesting of Vermicast
Harvesting will commence 10 to 14 days or 2 weeks a fter stocking of worms. Prior to
harvest, refrained from watering the substrate for the last three days to ease the
separation of castings from worms and likewise prev enting the castings to become
compact.
Re-Applying Substrates.
After the harvest of the vermi cast, substrates are applied in the vermi beds anew. In
the first bed, we put pure carabao manure without a ny loam soil like what we put
before. And in the second bed, we applied a mixture of carabao manure and partially
decomposed rice straw.

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Re-introducition of the Vermi Worms, Red wriggler (Eisenia foetida).
The application of new substrates into the vermi beds require the re-introduction of the
vermi worms or the red wrigglers (Eisenia foetida) for the continuity of the worm’s
culture and for their production of the vermi cast which are very good organic fertilizer .
After introducing the worms into the substrates, sprinkle water to keep the moisture
on which worms can easily digest these substrates. And these steps will go over and
over again until such time that the red wrigglers are cultured into a big number and
vermicast are produced well that it can be sold to gardening companies.
Advantages of Vermiculture and Vermicomposting
Vermiculture and vermicomposting is one of the most valuable ecological endeavors we
have engaged in as it caters not only environmental protection but also helped us
acquire knowledge on its proper methodology. Vermic ulture is environment friendly
since earthworms feed on anything that is biodegrad able, vermicomposting then
partially aids in the garbage disposal problems. No imported inputs required, worms are
now locally available and the materials for feeding are abundant in the locality as
market wastes, grasses, used papers and farm wastes. It is also highly profitable, both
the worms and castings are saleable.
Vermicompost does not have any adverse effect on so il, plant and environment. It
improves soil aeration and texture thereby reducing soil compaction. It improves water
retention capacity of soil because of its high organic matter content. It also promotes
better root growth and nutrient absorption and improves nutrient status of soil, both
macro-nutrients and micro-nutrients.
BIOGAS PRODUCTION
Anaerobic digestion is a natural process in which bacteria convert organic materials
into biogas. It occurs in marshes and wetlands, and in the digestive tract of ruminants.
The bacteria are also active in landfills where they are the principal process degrading
landfilled food wastes and other biomass. Biogas can be collected and used as a
potential energy resource. The process occurs in an anaerobic (oxygen-free)
environment through the activities of acid- and methane-forming bacteria that break
down the organic material and produce methane (CH
4
) and carbon dioxide (CO
2
) in a
gaseous form known as biogas.

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Dairy manure waste consists of feed and water that has already passed through the
anaerobic digestion process in the stomach of a cow, mixed with some waste feed and,
possibly, flush water. The environmental advantages of using anaerobic digestion for
dairy farm wastes include the reduction of odors, flies, and pathogens as well as
decreasing greenhouse gas (GHG) and other undesirable air emissions. It also stabilizes
the manure and reduces BOD. As large dairies become more common, the pollution
potential of these operations, if not properly managed, also increases. The potential for
the leaching of nitrates into groundwater, the pote ntial release of nitrates and
pathogens into surface waters, and the emission of odors from storage lagoons is
significantly reduced with the use of anaerobic digestion. There may also be a reduction
in the level of VOC emissions.
Elements of Anaerobic Digestion Systems
Anaerobic digester systems have been used for decades at municipal wastewater
facilities, and more recently, have been used to process industrial and agricultural
wastes. These systems are designed to optimize the growth of the methane-forming
(methanogenic) bacteria that generate CH
4
. Typically, using organic wastes as the major
input, the systems produce biogas that contains 55% to 70% CH
4
and 30% to 45% CO
2
.
On dairy farms, the overall process includes the following:
Manure collection and handling.
Key considerations in the system design include the amount of water and inorganic
solids that mix with manure during collection and handling
Pretreatment.
Collected manure may undergo pretreatment prior to introduction in an anaerobic
digester. Pretreatment—which may include screening, grit removal, mixing, and/or flow
equalization—is used to adjust the manure or slurry water content to meet process
requirements of the selected digestion technology. A concrete or metal collection/mix
tank may be used to accumulate manure, process water and/or flush water. Proper
design of a mix tank prior to the digester can limit the introduction of sand and rocks
into the anaerobic digester itself. If the digestion processes requires a thick manure
slurry, a mix tank serves a control point where water can be added to dry manure or
dry manure can be added to dilute manure. If the digester is designed to handle
manures mixed with flush and process water, the contents of the collection/mix tank

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can be pumped directly to a solids separator. A variety of solids separators, including
static and shaking screens are available and currently used on farms.
Anaerobic digestion
An anaerobic digester is an engineered containment vessel designed to exclude air and
promote the growth of methane bacteria. The digester may be a tank, a covered lagoon
(Figure), or a more complex design, such as a tank provided with internal baffles or with
surfaces for attached bacterial growth. It may be designed to heat or mix the organic
material. Manure characteristics and collection technique determine the type of
anaerobic digestion technology used. Some technologies may include the removal of
impurities such as hydrogen sulfide (H
2
S), which is highly corrosive.

By-product recovery and effluent use
It is possible to recover digested fiber from the effluent of some dairy manure digesters.
This material can then be used for cattle bedding or sold as a soil amendment. Most of
the ruminant and hog manure solids that pass through a separator will digest in a
covered lagoon, leaving no valuable recoverable by-product.
Biogas recovery
Biogas formed in the anaerobic digester bubbles to the surface and may accumulate
beneath a fixed rigid top, a flexible inflatable top, or a floating cover, depending on the
type of digester. The collection system, typically plastic piping, then directs the biogas
to gas handling subsystems.
Biogas handling.

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Biogas is usually pumped or compressed to the operating pressure required by specific
applications and then metered to the gas use equipment. Prior to this, biogas may be
processed to remove moisture, H
2
S, and CO
2
, the main contaminants in dairy biogas, in
which case the biogas becomes biomethane. (Partial removal of contaminants,
particularly H
2
S, will yield an intermediate product that we refer to in this report as
partially upgraded biogas). Depending on applications, biogas may be stored either
before or after processing, at low or high pressures (see Chapter 4).
Biogas use
Recovered biogas can be used directly as fuel for heating or it can be combusted in an
engine to generate electricity or flared. If the biogas is upgraded to biomethane,
additional uses may be possible
METHANOL PRODUCTION FROM ORGANIC WASTES
Methanol can be produced from concentrated carbon sources, such as natural gas,
coal, biomass, by-product streams or even carbon dioxide (CO
2) from fuel gases. A
simplified overview of the steps involved in methanol production is given in Figure.

In general, the plant confi gurations used for bio-methanol production show strong
similarities to coal-based methanol production via gasifi cation, with two notable
exceptions: bio-methanol from bio-gas (which is similar to methanol production from
natural gas) and bio-methanol from CO
2. The main processes in a conventional
methanol plant are: gasifi cation, gas cleaning, reforming of high hydrocarbons, water-
gas shift, hydrogen addition and/or CO
2 removal, and methanol synthesis and purifi
cation (Hamelinck & Faaij, 2002).

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If the feedstock consists of primary biomass, a pre-treatment of the raw material may be
required (e.g. chipping and drying of woody biomass or purification of liquid feedstock).
The feedstock is then gasifi ed into synthesis gas (syngas), a mixture of mainly carbon
monoxide (CO) and hydrogen (H
2), as well as carbon dioxide, water (H2O) and other
hydrocarbons. Using a limited amount of oxygen during feedstock heating (i.e. above
700°C) will improve the formation of CO and H
2 and reduce the amount of unwanted
CO
2 and H2O. However, if air is used as a source of oxygen, inert gases (e.g. nitrogen)
increase the gas fl ow through the gasifi er and downstream equipment (Mignard &
Pritchard, 2008), thus resulting in higher equipment (investment) costs (Hamelinck &
Faaij, 2006).
On the other hand, using pure oxygen is rather expensive. Therefore, an economic
optimum is to be found between oxygen purity and production costs based on electricity
prices and equipment costs. After gasifi cation, impurities and contaminants (e.g. tars,
dust and inorganic substances) are removed before the gas is passed through several
conditioning steps that optimise its composition for methanol synthesis.The aim of the
syngas conditioning step is to produce syngas that has at least twice as many H
2
molecules as CO molecules (Specht & Bandi, 1999). The optimal ratio of H
2 molecules
to CO molecules depends on the initial syngas composition, as well as the availability of
H
2.
The initial syngas composition depends on the carbon source and gasifi cation method
(Galindo Cifre & Badr, 2007). The concentrations of CO and H
2 can be altered in several
ways.
First, unprocessed syngas can contain small amounts of methane and other light
hydrocarbons with high energy content. These are reformed to CO and H
2 (Hamelinck &
Faaij, 2006) by high temperature catalytic steam reforming or by auto-thermal
reforming (ATR). These reform processes can lead to the formation of carbonaceous
residues that reduce the performance of catalysts, and there is currently no consensus
on which option is more cost-eff ective (Hamelinck & Faaij, 2006).
Second, the initial hydrogen concentration in the syngas is usually too low for optimal
methanol synthesis. To reduce the share of CO and increase the share of H
2, a water
gas-shift reaction (WGSR) can be used to convert CO and H
2O into CO2 and H2. CO2 can
also be removed directly using chemical absorption by amines. Other CO
2 removal
technologies (e.g. adsorption onto liquids, cryogenic separation and permeation through

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membranes) are being developed, but more time is needed for practical applications
(Olah et al., 2009).
Third, hydrogen can be produced separately and added to the syngas. Industrial
hydrogen is produced either by steam reforming of methane or electrolysis of water.
While electrolysis is usually expensive, it can offer important synergies if the oxygen
produced during electrolysis is used for partial oxidation in the gasifi cation step, thus
eliminating?? The need for air or oxygen production from air separation. However, from
an environmental point of view, it is estimated that electrolysis only makes sense if
renewable electricity is available (Specht et al., 1999; Clausen et al., 2010)
Fourth In addition, if electrolysis provides precisely enough oxygen for the gasifi cation,
the associated hydrogen production is not enough to meet the optimal stoichiometry in
the syngas. Therefore, CO
2 removal might be needed anyway to obtain an optimised
syngas (Specht & Bandi, 1999).
After conditioning, the syngas is converted into methanol by a catalytic process based
on copper oxide, zinc oxide or chromium oxide catalysts (Hamelinck & Faaij, 2006).
Distillation is used to remove the water generated during methanol synthesis. An
overview of major methanol production processes from various carbon sources is
presented in Figure, with the most important inputs and outputs, and the possible
addition of electrolysis
The technologies used in the production of methanol from biomass are relatively well-
known since they are similar to the coal gasification technology, which has been applied
for a long time. However, making biomass gasification cost-competitive has proven
difficult.
BYPRODUCTS OF SUGAR INDUSTRIES.
A special attention in the diversification strategy must be given to the efficient use of
the energy potential of sugar cane, which may be transformed into approximately 1
tonne of oil equivalent for every tonne of sugar produced. A typical (traditional) sugar
cane factory uses inefficiently all the available energy in its bagasse but it has been
shown that with a properly designed process, oriented to energy saving, it is possible to
operate with only 50 percent of that energy. Sugar cane processed using chemical and
biotechnologies, can produce a high number of produ cts, and is surpassed only by
those obtained from petrochemistry. Practically all products and by-products obtained
from sugar cane may become substrates for liquid or solid-state fermentation

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processes; using available second and third generation biotechnology a significant
number of production processes could be developed. The agro industrial character of
sugar cane processing allows for an industrial development in which all available
wastes and resources could be managed profitably wh ile avoiding pollution of the
environment. Sugar cane producing countries, have a significant advantage in
possessing a renewable raw material, which can be used in human and animal feeding,
and in the production of basic chemicals, with a yield not equalled by any other plant.
Furthermore, sugar cane has energy delivering capacity equivalent to five times that
used by the crop. All these factors taken together and the possibilities offered by further
genetic improvement, turns sugar cane into the ideal crop for the next century.
Bagasse fibre as a raw material
Bagasse obtained as a by-product of sugar cane processing, is composed of fibre, pith,
non-soluble solids and water; fibre represents about half of all components, and
includes cellulose, hemicellulose and lignin of low molecular weight. Its morphological
structure is not strong in comparison with other fi bres like those of wood; its
advantages are shown during chemical and mechanical treatments, since it does not
have to be submitted to severe processes. Another important advantage is that it is
directly obtained and concentrated in the sugar factory as a process by-product, thus
simplifying handling and transport operations. Every year, more than 200 million
tonnes of bagasse are obtained together with sugar, in all cane producing countries, 95
percent of which is used as fuel in the mills, which represent a saving of about 40
million tonnes of oil.
Pulp and paper, boards, furfural and animal feeds a re among the main products
obtained from bagasse not used as fuel. The most widespread use of bagasse around
the world is in the production of pulp and paper. There are about 90 factories in all, of
which three-quarters are established in Asia, Oceania and the Middle East, delivering
up to 2.5 percent of the total world production. Intense exploitation of wood has caused
depletion of forest reserves. This has triggered a trend to decrease this exploitation, to
implement reforestation policies and renew interest in tropical woods and annual plants
to be used in combination with traditional wood fibres. It becomes evident that it will be
necessary to rely on other fibre sources to fulfil the forecasted demands for the next
century. Among alternative fibres, it is accepted that bagasse is one of the best due to
its quality, cost and renewable character.

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Bagasse fibre can be used at present as an importan t component in printing and
cultural paper, but its use for industrial paper is still limited. One hectare of sugar cane
can produce, annually, about five tonnes of fibre for pulp and paper production, twice
as much as that produced by one hectare of wood wit h the same management. The
renewal period is fifteen times shorter for sugar cane. New developments in pulping are
expected to include a first step consisting of a microbiological treatment for fibre
preservation and its partial or total delignification in the unavoidable storage stage.
Consequently the rest of the process will have lower energy and chemicals demands.
Bleaching processes will turn to gas phase, aiming for better yields and the elimination
of pollutants in current processes.
Mechanical and thermo-mechanical pulping will becom e more popular in paper
production with a decreasing share of traditional chemical pulps. More chemicals will
be used in industrial paper production in order to obtain the desired specific properties.
The paper industry will maintain its trend of increasing the waste paper recycling in
order to reduce fresh fibre addition in the feed and as a contribution to the rational use
of natural resources. New technologies will decrease the present demand of water to one
third, reducing in the same proportion the pollution load of the wastes.
Fibres are also used for the production of bagasse boards, a production which is
expected to increase for the same reason because of the need to preserve forest
reserves, promoting the search for new alternatives for both types, particle and fibre
boards and new types as the MDF boards. Moulded ele ments, produced by similar
technologies, as those of boards will fulfil the quality requirements of a great deal of
industry and home products Boards and moulded eleme nts will show specific properties
for different purposes: furniture, liners, walls or construction forms. These products will
be designed and produced in such a way that they will be able to substitute wood in up
to 90 percent of its uses. They will be finished with radiationpolymerised resins, which
will result in a much better quality than that reached today and an improved surface
resistance. These polymers used for bonding and finishing may be produced from other
products and by-products such as furfural or sucrose; the lignin present in bagasse can
also be used as the bonding agent. Properties of lignin will be used for fulfilling
demands of moulded elements for domestic products, furniture, the automobile
industry, and packing mat erials.
Chemical and biotechnological development of cane s ugar by-products

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Most chemical firms have based their production mai nly on petro-chemistry, while
other alternatives like those offered by sugar cane, have received much less attention.
The economical causes for this situation are expect ed to change in the future.
Considering all of these sources, different possible alternatives for the production of
organic and speciality chemicals and related produc ts could be mentioned.
Biotechnologies of second and third generation have made available conventional and
new technologies for the production of: amino acids, vitamins, organic acids, solvents,
microbial polymers, proteins for human and animal c onsumption, enzymes for
industrial use, alcohol and co-products industry and such microbiological processes as:
silage, biologic treatments of lignocelulosic residues, biopulping and biobleaching
processes, anaerobic digestion of waste streams, and other alternatives. In addition,
more sophisticated technologies are available for their application in the pharmaceutical
industry. It may be considered that all sugar cane by-products could be used as
substrates for liquid or solid-state fermentation opening a wide range of possibilities for
production based on biochemical processes.
The development of a chemical industry from ethanol or alco-chemistry, as an
alternative or complement to petro-chemistry could allow the production of basic
products in all industry branches. The two most dev eloped alternatives are those
following the ethylene and acetaldehyde paths. The first one produces a great variety of
plastic materials in great demand and the second one develops acetate compounds,
which are the basis of the paint and varnish industries. Even when high efficiency and
relative low cost in petro-chemical production are perfectly known, the complexity of
plants, their economies of scale and high investment costs set true limits in those
countries with insufficient financial resources, limited markets or technological
development, and little or inexistent oil resources. Alco-chemistry technologies are
available and operating at industrial scale in some countries. A waste product from
alcohol production: fusel oil, a mixture of isomers of amylic and propylic alcohols, could
be recovered. Isoamylic alcohol, amylic alcohol, n-butanol and other compounds may be
separated through distillation showing some economical advantages due to low costs.
Furfural is an aldehyde produced by the acid hydrolysis of the pentosans present in
different crops.Cane bagasse is one of the best raw materials for this production. Low
toxicity of fufural makes it a valuable product for the chemical industry from which the
following could be derived: resins and plastics, pharmaceuticals, fufurylalcohol and its

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resins and monochloroacetic acid, herbicides, tetrahydrofufurylic alcohol and maleic
anhydride, tetrahydrofuran.
Based on sugar cane by-products some alternatives for herbicides production could be
mentioned: the first from furfural, and some others based on acetic or propionic acids
possibly derived from furfural or alcohol production wastes. The option of new herbicide
production starting with furfural has been studied for many years. The synthesis of
different products, which passed the screening tests with positive results, has been
successfully achieved. The results however can only be considered in the medium or
long term because of the high costs of development. Acetic acid obtained from furfural
distillation, and propionic acid from alcohol distilleries wastes may become, in the short
term, raw materials for conventional products such as 3-chloroacetic acid,
monochloroacetic acid or chloropropionic acid. Cellulose in bagasse could be used for
the development of a cellulose based chemical industry. Cellulose fibres obtained from
bagasse, showing high modules of wet resistance, could substitute up to 60 percent of
the fibres derived from cotton cellulose used today in commercial textiles.
Fibrana production processes use directly wet pulps and accelerated ageing of alkali
cellulose. Fibres are treated in processes, which allow them to reach high modules of
wet resistance, similar to those of cotton. Based on chemical pulps, other high demand
by-products of cellulose can be produced such as: cellophane, carboximethylcellulose,
cellulose acetate, cellulose nitrate, cellulosamide, chromatographic bases and others.
Most of these products would be manufactured in a combined way through the integral
use of the installations.
For the production of chemicals through thermo chem istry there are three main
options: gasification, pyrolysis and liquefaction. Studies up to the present show that the
most interesting alternative is that of gasification for production of synthesis gas as a
way which can be converted to methanol, ammonia and ethylene. At medium and long
terms, some of these products may be feasible but these alternatives are still in an
experimental stage.
Lignin is the second most abundant substance in nature, and at the same time one of
the least exploited up to the present. A great potential exists for its use in the chemical
industry. It is technologically feasible to obtain phenols for production of adhesives,
resins, fungicides, veterinary products, insecticides, and carbon. Lignosulphonates are
present in black liquors of sulfite processing. It is a very important product in the

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artificial board industry as a filler and / or additive in the ureaformaldehyde resin, and
for reducing costs and improving some board properties
Sucrochemistry as an alternative use of sugar
During the nineteen forties, technological processes using sucrose as raw material for
chemical production started to be referred to as sucrochemistry. In the last five decades,
about 10 000 technically feasible products have bee n developed from sucrose at
laboratory and pilot plant scale. Only about one hundred have also demonstrated their
economic feasibility and are being produced on a commercial scale.
Due to its high production level as a commercial, crystalline product, sucrose is one of
the most interesting substrate for the development of new chemical and microbiological
technologies. The presence of 8 hydroxyl groups allows broad possibilities of chemical
bonding becoming a.potential source for the production of chemicals with quite different
properties, through degradation, synthetic and micr obiological reactions. Through
controlled degradation reactions, it is possible to obtain an important number of
chemicals. However only through catalytic hydrogena tion it has been possible to
produce such important commercial products as: fruc tose, sorbitol and mannitol.
Through chemical synthesis and based on the high solubility of sucrose in water and
the presence of the high number of hydroxyl groups, it has been possible to produce:
ethers, esters from fatty acids, polymers and resins, among the most important, some of
which have reached commercial production. These rou tes show the drawback that
reaction products are not stable in those solvents in which sucrose itself is, turning
separation and purification stages very complex and expensive.
Sucrose ethers are obtained by the substitution of hydrogen ions by alkyl or aryl groups.
The properties of resulting compounds are not attractive from a commercial point of
view, limiting the development of this alternative; only urethanes have found an
industrial use and a market. Used in rigid foams (p olyurethane) they offer good
potentiality based on their prices and quality.
Fatty acid esters require most intense research work and a significan t number of
different processes. Some American, Japanese, Engli sh and French firms deliver
different products to the market, turning this alternative as the most important, with
the highest commercial potential.
Polyesters Of Fatty Acids are receiving special attention in the last years because of
their use in hypo caloric diets, and as blood cholesterol depressors. Some sucrose

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esters have shown their properties, when used in low concentration, as antimicrobial
agents, inhibiting bacterial growth. Cosmetic formulation with sucrose esters has had
good acceptance, due to their dermatological properties and their innocuity. In the same
way, they have been used in biodegradable detergents, for widespread use extracting
spilt oil from the sea and other aqueous deposits. The alternative of microbial
transformations of sucrose molecules allows the production of different biopolymer
such as dextran and xhantan; both are products used in foods, pharmaceuticals and in
oil extraction. The biopolymer polyhydroxibutirate showing similar properties to those of
polypropylene has not been able to compete with the latter due to high production
costs.
Based on sucrose, some other sweetening agents showing special properties have been
obtained becoming potential alternatives for sucrose. Among the most important the
following could be mentioned.
Chlored chemicals obtained through selective substitution of hydroxyls with chlorine
atoms are often products sweeter than sucrose. But there are others, which are
extremely bitter, showing specific properties. The use of these compounds awaits
approval by medical authorities.
Leucrose is a sucrose isomer with less sweetening power, innocuous, acceptable by
diabetics, approved as a human food. It is also used as basis for production of special
purpose tensoactives.
Sucralose was developed two decades ago through a modificatio n of the molecular
structure of sucrose.
It is 600 times sweeter and shows similar flavour characteristics. From the point of view
of human health it does not stimulate insulin formation, and it is eliminated from the
human body without showing chemical changes, thus being a non-caloric food.
L. sugars or "lefties" as they are also called, are obtained through a change in hydroxyl
groups. Their taste is close to that of sucrose while showing no calorific value, as they
are not metabolised, and proceed through the intestines without chemical changes.
Their production costs are high, and they have yet to complete tests for medical
approval. Another product reached as sugar modification is isomalt, showing functional
properties close to those ones of sucrose, with little commercialisation in Europe.
It can be generally stated that sucrose alternative molecules, to be used as raw material
for chemical production maintain the interest of researchers and producers due to the

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variety of applications that the products may have. Out of the three ways of reactions
that sucrose could be subjected to degradation, chemical and microbiological, the latter
appears to be the most interesting for the development of sucrochemistry.
Sources of animal feeding
The food, energy and environment problems are among the priority issues for the next
century. Sugar cane production, followed by a rational diversification strategy, might
have a significant place in this context because of its characteristic as a highly efficient
system for solar energy fixation and transformation into green matter. This biomass is
usable for energy and feeding purposes, while saving non-renewable sources of energy;
it has therefore a positive C02 fixation balance. This statement is relevant to countries
with tropical and subtropical climates where sugar cane can be grown and where socio-
economical, technical and agricultural considerations will probably determine the
optimal use of cereals mainly for direct human cons umption. The indiscriminate
extrapolation of feeding systems of moderate climate conditions to tropical countries
will most probably result in a sub-optimal solution in the latter.
Under tropical conditions, feeding systems might rely on sugar cane as a source for 60 -
80 percent of ruminant and swine feeding requiremen ts; this is true only if they are
adequately supplemented with other products, which must be imported or made
available locally. Sugar cane has a high percent of fibre with a low digestibility, which
becomes a limitation for its use in monogastric animals. To overcome this deficiency
and make an optimal use of the whole cane, alternatives have been developed for the
latter's separation into three basic components: the crop wastes that may be used in its
natural form or treated in a simple way for feeding bovines; the soluble sugars present
in the juice to be used for poultry and swine, and lastly the fibre, after being subjected
to simple treatments to raise its digestibility, to be given to ruminants.

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UNIT: 6 GLOBAL ENVIRONMENTAL PROBLEMS
Global warming, ozone depletion, UV – B, green hous e effect and acid rain, their
impact and management. Biodiversity and its conserv ation, status of biodiversity,
hotspots, Red data book.

GLOBAL WARMING
Global warming first emerged as a cause of global concern in the 1980's, a result of the
apparent rise in global temperatures, believed to be due to human activities, such as
burning of fossil fuels and destruction of forests.
Impact
on:
Effect: Consequence (-): Consequence (+):
Health
Malaria and cholera
increase, due to
temperature increase.
More money needed to
fight disease, strain on
medical services, rise in
death rate.

Vegetation
Shifting flora and fauna
to different areas.
Extinction of some
species.
Spread of pests and
disease, alteration in
crop yields, may
increase food shortages.
Canadian Prairies could
become major wheat
growing belt. Areas able to
grow different crops, for
example, citrus fruits in the
UK.
Weather
More extreme climates
in inland locations.
More frequent and
devastating hurricanes.
Unknown at present. Unknown at present.
Ocean
Sea temperatures
increase, sea levels
rise, shift in ocean
currents.
Changes in number of
fish stocks and their
location will impact the
fish industry.
Increase in fish stocks in
certain areas.
Landscape
Reduced snow cover in
some areas. Glaciers
Rise in sea levels.
Extended summer season
in some landscapes due to

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melt in Antarctica. higher temperatures,
increasing revenue.
Hydrology
Reduction of wetland
areas, as precipitation
is reduced. In some
places river flooding
may increase.
Great pressure on water
supplies. Problems for
HEP schemes and
irrigation.
Increased awareness of
water conservation
measures, less water
wastage.
Population
Reduction of areas
suitable for human
habitation, for example.
Lowland Bangladesh.
Increased population
densities increase
possibility of disease
and malnutrition.
Forced movement of
population from densely
populated coastal areas, to
interior locations.
Climate
Location of jet stream
may alter. Depressions
may shift south,
causing them to be
more intense.
Better forecasting
needed to warn people
of approaching storms.
Insurance premiums
will increase.
More accurate weather
forecasting developed.

OZONE DEPLETION
Ozone depletion occurs when the natural balance bet ween the production and
destruction of stratospheric ozone is tipped in favour of destruction. Although natural
phenomena can cause temporary ozone loss, chlorine and bromine released from man-
made compounds such as CFCs are now accepted as the main cause of this depletion.
It was first suggested by Drs. M. Molina and S. Rowland in 1974 that a man-made
group of compounds known as the chlorofluorocarbons (CFCs) were likely to be the
main source of ozone depletion. However, this idea was not taken seriously until the
discovery of the ozone hole over Antarctica in 1985 by the Survey. Chlorofluorocarbons
are not "washed" back to Earth by rain or destroyed in reactions with other chemicals.
They simply do not break down in the lower atmosphe re and they can remain in the
atmosphere from 20 to 120 years or more. As a consequence of their relative stability,
CFCs are instead transported into the stratosphere where they are eventually broken
down by ultraviolet (UV) rays from the Sun, releasi ng free chlorine. The chlorine
becomes actively involved in the process of destruction of ozone. The net result is that

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two molecules of ozone are replaced by three of molecular oxygen, leaving the chlorine
free to repeat the process:
Cl + O
3 = ClO + O2
ClO + O = Cl + O
2
Ozone is converted to oxygen, leaving the chlorine atom free to repeat the process up to
100,000 times, resulting in a reduced level of ozone. Bromine compounds, or halons,
can also destroy stratospheric ozone. Compounds con taining chlorine and bromine
from man-made compounds are known as industrial hal ocarbons. Emissions of CFCs
have accounted for roughly 80% of total stratospheric ozone depletion.
Naturally occurring chlorine has the same effect on the ozone layer, but has a shorter
life span in the atmosphere.
Chlorofluorocarbons
Chlorofluorocarbons or CFCs (also known as Freon) are non-toxic, non-flammable and
non-carcinogenic. They contain fluorine atoms, carbon atoms and chlorine atoms.The 5
main CFCs include CFC-11 (trichlorofluoromethane - CFCl3), CFC-12 (dichloro-
difluoromethane - CF2Cl2), CFC-113 (trichloro-trifluoroethane - C2F3Cl3), CFC-114
(dichloro-tetrfluoroethane - C2F4Cl2), and CFC-115 (chloropentafluoroethane -
C2F5Cl).CFCs are widely used as coolants in refrigeration and air conditioners, as
solvents in cleaners, particularly for electronic circuit boards, as a blowing agents in the
production of foam (for example fire extinguishers), and as propellants in aerosols.
Man-made CFCs however, are the main cause of strato spheric ozone depletion. CFCs
have a lifetime in the atmosphere of about 20 to 100 years, and consequently one free
chlorine atom from a CFC molecule can do a lot of damage, destroying ozone molecules
for a long time. Although emissions of CFCs around the developed world have largely
ceased due to international control agreements, the damage to the stratospheric ozone
layer will continue well into the 21
st
century.
Rocket Launches
The global market for rocket launches may require more stringent regulation in order to
prevent significant damage to
Earth’s stratospheric ozone layer in the decades to come, according to a new study by
researchers in California and

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Colorado. Future ozone losses from unregulated rocket launches will eventually exceed
ozone losses due to chlorofluorocarbons, or CFCs, which stimulated the 1987 Montreal
Protocol banning ozone-depleting chemicals, said
Martin Ross, chief study author from The Aerospace Corporation in Los Angeles. The
study, which includes the
University of Colorado at Boulder and Embry-Riddle Aeronautical University provides a
market analysis for estimating future ozone layer depletion based on the expected
growth of the space industry and known impacts of r ocket launches.” As the rocket
launch market grows, so will ozone-destroying rocket emissions,” said Professor Darin
Toohey of CU-Boulder’s atmospheric and oceanic sciences department.
UV – B
The depletion of the ozone layer leads, on the average, to an increase in ground-level
ultraviolet radiation, because ozone is an effective absorber of ultra-violet radiation. The
Sun emits radiation over a wide range of energies, with about 2% in the form of high-
energy, ultraviolet (UV) radiation. Some of this UV radiation (UV-B) is especially effective
in causing damage to living beings, the largest decreases in ozone during the past 15
years have been observed over Antarctica, especially during each September and
October when the ozone hole forms. During the last several years, simultaneous
measurements of UV radiation and total ozone have b een made at several Antarctic
stations. In the late spring, the biologically damaging ultraviolet radiation in parts of the
Antarctic continent can exceed that in San Diego, California, where the Sun is much
higher above the horizon. In areas where smaller ozone depletion has been observed,
UV-B increases are more difficult to detect. In particular, detection of trends in UV-B
radiation associated with ozone decreases can be further complicated by changes in
cloudiness, by local pollution, and by difficulties in keeping the detection instrument in
precisely the same condition over many years. Prior to the late 1980s, instruments with
the necessary accuracy and stability for measuremen t of small long-term trends in
ground-level UV-B were not available. Therefore, the data from urban locations with
older, less-specialized instruments provide much less reliable information, especially
since simultaneous measurements of changes in cloud iness or local pollution are not
available. When high-quality measurements have been made in other areas far from
major cities and their associated air pollution, decreases in ozone have regularly been
accompanied by increases in UV-B. This is shown in the figure below, where clear-sky

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measurements performed at six different stations demonstrate that ozone decreases
lead to increased UV-B radiation at the surface in amounts that are in good agreement
with that expected from calculations (the "model" curve).
GREEN HOUSE EFFECT
Many greenhouses gases exist naturally, and it is human actions that are increasing
their concentrations within the lower atmosphere. It is believed that as the amount of
greenhouse gases in the atmosphere increases, the a mount of long-wave (infra-
red) radiation in the lower atmosphere also increases, thus raising temperatures.
The table below outlines the major sources of greenhouses gases:
Gas Source Use
Way it increases global
warming
Water
vapour
Oceans, lakes, rivers, reservoirs.
Humans have little impact upon
levels.
Absorbs limited
outgoing
radiation.
Water vapour and clouds
are responsible for
nearly 98% of the
natural greenhouse
effect.
Carbon
dioxide
Burning of fossil fuels, and
forests, breathing animals, less
produced by southern hemisphere
(less land).
Absorption of
long wave
radiation.
Approximately 50%.
Methane
(CH4)
Much from break down of organic
matter by bacteria (rice paddy
fields) cows, swamps marshes.
As above. Approximately 18%.
Ozone
Naturally from some oxygen
atoms. Ozone in the troposphere
is due to chemical reactions
between sunlight and agents of
pollution.
Filters short
wave UV
radiation.
Difficult to estimate.
CFCs Fridges and aerosols.
25%, but increasing due
to ability to survive
within the atmosphere
for 100 years.

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Nitrous
oxide
Nitrate fertilisers, transport and
power stations (combustion).
Absorption of
long wave
radiation.
Approximately 6%.


ACID RAIN
Acid rain is rain consisting of water droplets that are unusually acidic because of
atmospheric pollution - most notably the excessive amounts of sulfur and nitrogen
released by cars and industrial processes. Acid rain is also called acid deposition
because this term includes other forms of acidic precipitation such as snow.
Acidic deposition occurs in two ways: wet and dry. Wet deposition is any form of
precipitation that removes acids from the atmosphere and deposits them on the Earth’s
surface. Dry deposition polluting particles and gases stick to the ground via dust and
smoke in the absence of precipitation. This form of deposition is dangerous however
because precipitation can eventually wash pollutants into streams, lakes, and rivers.
There are several important impacts of acid deposition on both natural and man-made
environments. Aquatic settings are the most clearly impacted by acid deposition though
because acidic precipitation falls directly into them. Both dry and wet deposition also
runs off of forests, fields, and roads and flows into lakes, rivers, and streams.
As this acidic liquid flows into larger bodies of water, it is diluted but over time, acids
can accrue and lower the overall pH of the body. Acid deposition also causes clay soils
to release aluminum and magnesium further lowering the pH in some areas. If the pH
of a lake drops below 4.8, its plants and animals risk death and it is estimated that
around 50,000 lakes in the United States and Canada have a pH below normal (about
5.3 for water). Several hundred of these have a pH too low to support any aquatic life.
Aside from aquatic bodies, acid deposition can significantly impact forests. As acid rain
falls on trees, it can make them lose their leaves, damage their bark, and stunt their
growth. By damaging these parts of the tree, it makes them vulnerable to disease,
extreme weather, and insects. Acid falling on a forest’s soil is also harmful because it
disrupts soil nutrients, kills microorganisms in the soil, and can sometimes cause a
calcium deficiency. Trees at high altitudes are also susceptible to problems induced by
acidic cloud cover as the moisture in the clouds blankets them.

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Damage to forests by acid rain is seen all over the world, but the most advanced cases
are in Eastern Europe. It’s estimated that in Germany and Poland, half of the forests
are damaged, while 30% in Switzerland have been affected.
Finally, acid deposition also has an impact on architecture and art because of its ability
to corrode certain materials. As acid lands on buildings (especially those constructed
with limestone) it reacts with minerals in the ston es sometimes causing it to
disintegrate and wash away. Acid deposition can also corrode modern buildings, cars,
railroad tracks, airplanes, steel bridges, and pipes above and below ground.
THEIR IMPACTS
Effects on Human and Animal Health
Increased penetration of solar UV-B radiation is likely to have profound impact on
human health with potential risks of eye diseases, skin cancer and infectious diseases.
UV radiation is known to damage the cornea and lens of the eye. Chronic exposure to
UV-B could lead to cataract of the cortical and posterior subcapsular forms. UV-B
radiation can adversely affect the immune system ca using a number of infectious
diseases. In light skinned human populations, it is likely to develop nonmelanoma skin
cancer (NMSC). Experiments on animals show that UV exposure decreases the immune
response to skin cancers, infectious agents and other antigens
Effects on Terrestrial Plants
It is a known fact that the physiological and developmental processes of plants are
affected by UV-B radiation. Scientists believe that an increase in UV-B levels would
necessitate using more UV-B tolerant cultivar and b reeding new tolerant ones in
agriculture. In forests and grasslands increased UV-B radiation is likely to result in
changes in species composition (mutation) thus altering the bio-diversity in different
ecosystems. UV-B could also affect the plant community indirectly resulting in changes
in plant form, secondary metabolism, etc. These cha nges can have important
implications for plant competitive balance, plant pathogens and bio-geochemical cycles.
Effects on Aquatic Ecosystems
While more than 30 percent of the world’s animal pr otein for human consumption
comes from the sea alone, it is feared that increased levels of UV exposure can have
adverse impacts on the productivity of aquatic systems. High levels of exposure in
tropics and subtropics may affect the distribution of phytoplanktons which form the
foundation of aquatic food webs. Reportedly a recent study has indicated 6-12 percent

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reduction in phytoplankton production in the marginal ice zone due to increases in UV-
B. UV-B can also cause damage to early development stages of fish, shrimp, crab,
amphibians and other animals, the most severe effects being decreased reproductive
capacity and impaired larval development.
Effects on Bio-geo-chemical Cycles
Increased solar UV radiation could affect terrestrial and aquatic bio-geo-chemical cycles
thus altering both sources and sinks of greenhouse and important trace gases, e.g.
carbon dioxide (CO2), carbon monoxide (CO), carbony l sulphide (COS), etc. These
changes would contribute to biosphere-atmosphere fe edbacks responsible for the
atmosphere build-up of these gases. Other effects of increased UV-B radiation include:
changes in the production and decomposition of plan t matter; reduction of primary
production changes in the uptake and release of imp ortant atmospheric gases;
reduction of bacterioplankton growth in the upper ocean; increased degradation of
aquatic dissolved organic matter (DOM), etc. Aquatic nitrogen cycling can be affected by
enhanced UV-B through inhibition of nitrifying bacteria and photo decomposition of
simple inorganic species such as nitrate. The marine sulphur cycle may also be affected
resulting in possible changes in the sea-to-air emissions of COS and dimethylsulfied
(DMS), two gases that are degraded to sulphate aero sols in the stratosphere and
troposphere, respectively.
Effects on Air Quality
Reduction of stratospheric ozone and increased penetration of UV-B radiation result in
higher photo dissociation rates of key trace gases that control the chemical reactivity of
the troposphere. This can increase both production and destruction of ozone and
related oxidants such as hydrogen peroxide which are known to have adverse effects on
human health, terrestrial plants and outdoor materials. Changes in the atmospheric
concentrations of the hydroxyl radical (OH) may change the atmospheric lifetimes of
important gases such as methane and substitutes of chlorofluoro carbons (CFCs).
Increased troposphere reactivity could also lead to increased production of particulates
such as cloud condensation nuclei from the oxidation and subsequent nucleation of
sulphur of both anthropogenic and natural origin (e.g. COS and DMS).
Effects on Materials
An increased level of solar UV radiation is known to have adverse effects on synthetic
polymers, naturally occurring biopolymers and some other materials of commercial

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interest. UV-B radiation accelerates the photo degradation rates of these materials thus
limiting their lifetimes. Typical damages range from discoloration to loss of mechanical
integrity. Such a situation would eventually demand substitution of the affected
materials by more photo stable plastics and other materials in future. In 1974, two
United States (US) scientists Mario Molina and F. Sherwood Rowland at the University
of California were struck by the observation of Lovelock that the CFCs were present in
the atmosphere all over the world more or less even ly distributed by appreciable
concentrations. They suggested that these stable CFC molecules could drift slowly up to
the stratosphere where they may breakdown into chlorine atoms by energetic UV-B and
UB-C rays of the sun. The chlorine radicals thus pr oduced can undergo complex
chemical reaction producing chlorine monoxide which can attack an ozone molecule
converting it into oxygen and in the process regenerating the chlorine atom again. Thus
the ozone destroying effect is catalytic and a small amount of CFC would be destroying
large number of ozone molecules. Their basic theory was then put to test by the
National Aeronautic Space Authority (NASA) scientists and found to be valid, ringing
alarm bells in many countries and laying the foundation for international action
Effects on Climate Change
Ozone depletion and climate change are linked in a number of ways, but ozone
depletion is not a major cause of climate change. Atmospheric ozone has two effects on
the temperature balance of the Earth. It absorbs solar ultraviolet radiation, which heats
the stratosphere. It also absorbs infrared radiation emitted by the Earth's surface,
effectively trapping heat in the troposphere. Therefore, the climate impact of changes in
ozone concentrations varies with the altitude at which these ozone changes occur. The
major ozone losses that have been observed in the l ower stratosphere due to the
human- produced chlorine- and bromine-containing ga ses have a cooling effect on the
Earth's surface. On the other hand, the ozone increases that are estimated to have
occurred in the troposphere because of surface-pollution gases have a warming effect
on the Earth's surface, thereby contributing to the "greenhouse" effect. In comparison to
the effects of changes in other atmospheric gases, the effects of both of these ozone
changes are difficult to calculate accurately. In the figure below, the upper ranges of
possible effects for the ozone changes are indicated by the open bars, and the lower
ranges are indicated by the solid bars.

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MANAGEMENT
International actions
The first international action to focus attention on the dangers of ozone depletion in the
stratosphere and its dangerous consequences in the long run on life on earth was
focused in 1977 when in a meeting of 32 countries in Washington D.C. a World plan on
action on Ozone layer with UNEP as the coordinator was adopted. As experts began
their investigation, data piled up and in 1985 in an article published in the prestigious
science journal, “Nature” by Dr.
Farman pointed out that although there is overall depletion of the ozone layer all over
the world, the most severe depletion had taken place over the Antarctica. This is what is
famously called as "the Antarctica Ozone hole". His findings were confirmed by Satellite
observations and offered the first proof of severe ozone depletion and stirred the
scientific community to take urgent remedial actions in an international convention
held in Vienna on March 22, 1985. This resulted in an international agreement in 1987
on specific measures to be taken in the form of an international treaty known as the
Montreal Protocol on Substances That Deplete the Ozone Layer. Under this Protocol the
first concrete step to save the Ozone layer was taken by immediately agreeing to
completely phase out chlorofluorocarbons (CFC), Halons,
Carbon tetrachloride (CTC) and Methyl chloroform (MCF) as per a given schedule
Montreal Protocol
In 1985 the Vienna Convention established mechanism s for international co-operation
in research into the ozone layer and the effects of ozone depleting chemicals (ODCs).
1985 also marked the first discovery of the Antarctic ozone hole.
On the basis of the Vienna Convention, the Montreal Protocol on Substances that Deplete
the Ozone Layer was negotiated and signed by 24 countries and by th e European
Economic Community in September 1987. The Protocol called for the Parties to phase
down the use of CFCs, halons and other man-made ODC s. The Montreal Protocol
represented a landmark in the international environmentalist movement. For the first
time whole countries were legally bound to reducing and eventually phasing out
altogether the use of CFCs and other ODCs. Failure to comply was accompanied by stiff
penalties. The original Protocol aimed to decrease the use of chemical compounds
destructive to ozone in the stratosphere by 50% by the year 1999. The Protocol was
supplemented by agreements made in London in 1990 a nd in Copenhagen in 1992,

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where the same countries promised to stop using CFCs and most of the other chemical
compounds destructive to ozone by the end of 1995.
Fortunately, it has been fairly easy to develop and introduce compounds and methods
to replace CFC compounds. In order to deal with the special difficulties experienced by
developing countries it was agreed that they would be given an extended period of
grace, so long as their use of CFCs did not grow significantly. China and India, for
example, are strongly increasing the use of air conditioning and cooling devices. Using
CFC compounds in these devices would be cheaper than using replacement compounds
harmless to ozone. An international fund was theref ore established to help these
countries introduce new and more environmentally friendly technologies and chemicals.
The depletion of the ozone layer is a worldwide problem which does not respect the
frontiers between different countries. It can only be affected through determined
international co-operation.
Australian Chlorofluorocarbon Management Strategy
It provides a framework for the responsible management and use of CFCs in Australia.
The strategy recognizes some continuing need for these chemicals in pharmaceutical
and laboratory uses, but commits to their gradual phasing out.
Environmental Protection (Ozone Protection) Policy2 000
This WA policy aims to minimize the discharge of ozone-depleting substances into the
environment, and has been extended to cover use of alternative refrigerants (where
relevant). This has been done to prevent current stocks of ozone-depleting substances
from being released to the atmosphere by trade’s people that are not accredited, or with
inadequate training and/or equipment working on sys tems that contain these
substances.
United Nations Environment Programme
Has published several assessments of the environmen tal effects of ozone depletion
(United Nations Environment Programme, 1998; World Meteorological Organization,
2002).
Ozone Protection and Synthetic Greenhouse Gas Mana gement Act 1989 (and
associated regulations and amendments) Was implemented by the Commonwealth
Government to meet its commitments under the Montreal Protocol.
Ultraviolet index forecast

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The Bureau of Meteorology has developed a model to predict the amount of ultraviolet
exposure and the times of day at which it will occur for 45 WA locations. It is designed
to help people minimize their exposure to dangerous levels of ultraviolet radiation.
BIODIVERSITY AND ITS CONSERVATION
Biodiversity" is most commonly used to replace the more clearly defined and long
established terms, species diversity and species richness. Biologists most often define
biodiversity as the "totality of genes, species, and ecosystems of a region". An advantage
of this definition is that it seems to describe most circumstances and presents a unified
view of the traditional three levels at which biological variety has been identified:
Species diversity
Ecosystem diversity
Genetic diversity
Biodiversity is not evenly distributed; rather it varies greatly across the globe as well as
within regions. Among other factors, the diversity of all living things (biota) depends on
temperature, precipitation, altitude, soils, geography and the presence of other species.
The study of the spatial distribution of organisms, species, and ecosystems, is the
science of biogeography.
STATUS OF BIODIVERSITY
The International Union for the Conservation of Nature (IUCN) has organized a global
assortment of scientists and research stations acro ss the planet to monitor the
changing state of nature in an effort to tackle the extinction crisis. The IUCN provides
annual updates on the status of species conservation through its Red List. The IUCN
Red List serves as an international conservation tool to identify those species most in
need of conservation attention and by providing a g lobal index on the status of
biodiversity. More than the dramatic rates of species loss, however, conservation
scientists note that the sixth mass extinction is a biodiversity crisis requiring far more
action than a priority focus on rare, endemic orendangered species. Concerns for
biodiversity loss covers a broader conservation man date that looks at ecological
processes, such as migration, and a holistic examination of biodiversity at levels beyond
the species, including genetic, population and ecos ystem diversity. Extensive,
systematic, and rapid rates of biodiversity loss threatens the sustained well-being of
humanity by limiting supply of ecosystem services that are otherwise regenerated by the
complex and evolving holistic network of genetic an d ecosystem diversity. While

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the conservation status of species is employed exte nsively in conservation
management, some scientists highlight that it is the common species that are the
primary source of exploitation and habitat alteration by humanity. Moreover, common
species are often undervalued despite their role as the primary source of ecosystem
services.
HOTSPOTS - RED DATA BOOK
While most in the community of conservation science "stress the importance"
of sustaining biodiversity, there is debate on how to prioritize genes, species, or
ecosystems, which are all components of biodiversity (e.g. Bowen, 1999). While the
predominant approach to date has been to focus effo rts on endangered species by
conserving biodiversity hotspots, some scientists (e.g) and conservation organizations,
such as the Nature Conservancy, argue that it is mo re cost effective, logical, and
socially relevant to invest in biodiversity coldspots. The costs of discovering, naming,
and mapping out the distribution every species, the y argue, is an ill advised
conservation venture. They reason it is better to understand the significance of the
ecological roles of species.
Biodiversity hotspots and coldspots are a way of re cognizing that the spatial
concentration of genes, species, and ecosystems is not uniformly distributed on the
Earth's surface. For example, "[...] 44% of all species of vascular plants and 35% of all
species in four vertebrate groups are confined to 25 hotspots comprising only 1.4% of
the land surface of the Earth."
Those arguing in favor of setting priorities for coldspots point out that there are other
measures to consider beyond biodiversity. They point out that emphasizing hotspots
downplays the importance of the social and ecological connections to vast areas of the
Earth's ecosystems where biomass, not biodiversity, reigns supreme. It is estimated
that 36% of the Earth's surface, encompassing 38.9% of the world’s vertebrates, lacks
the endemic species to qualify as biodiversity hotspot. Moreover, measures show that
maximizing protections for biodiversity does not capture ecosystem services any better
than targeting randomly chosen regions. Population level biodiversity (i.e. coldspots) are
disappearing at a rate that is ten times that at the species level. The level of importance
in addressing biomass versus endemism as a concern for conservation biology is
highlighted in literature measuring the level of threat to global ecosystem carbon stocks
that do not necessarily reside in areas of endemism. A hotspot priority approach would

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not invest so heavily in places such as steppes, the Serengeti, the Arctic, ortaiga. These
areas contribute a great abundance of population (n ot species) level
biodiversity and ecosystem services, including cultural value and planetary nutrient
cycling.

Those in favor of the hotspot approach point out th at species are irreplaceable
components of the global ecosystem, they are concen trated in places that are most
threatened, and should therefore receive maximal strategic protections.

ALL THE BESTALL THE BESTALL THE BESTALL THE BEST