Bioremediation

Its a short presentation on bioremediation for easy understanding

1. Bioremediation Presented by, Amrutha .S. Joy II Msc biotechnology

2. Introduction Bioremediation is the use of living organisms, primarily microorganisms, for the degradation of hazardous chemicals in soil sediments, water, or other contaminated materials into less toxic forms microorganisms metabolize the chemicals to produce carbon dioxide or methane, water and biomass enzymatically transformed to metabolites that are less toxic or innocuous in some instances, the metabolites formed are more toxic than the parent compound. For example, perchloroethylene and trichloroethylene may degrade to vinyl chloride .

3. Bioremediation organisms genus pseudomonas are the most predominant microorganisms that degrade xenobiotics Xenobiotics ----hydrocarbons , phenols, organo phosphates, polycyclic aromatics and naphthalein Other microorganisms are Mycrobacterium , Mycococcus , Nitrosomonas , Nocardia , Penicillium , Phanerochaete etc

4. Microorganisms (Pure cultures) helpful in bioremediation Pollutant Microorganism(s ) Atrazine   Acinetobacter species Pseudomonas sp Carbon tetrachloride   Pseudomonas stutzeri Chlorpyrifos   Aspergillus niger Trichoderma viride 2,4,6-Trichlorophenol   Alcaligenes eutrophus   2,4-Dichlorophenoxyacetic acid   Ralstonia eutropha Mono and dichloro aromatic compounds Psedomonas putida Alkanes Psedomonas olevorans

5. Mechanism of Bioremediation microorganisms use the organic contaminants ( nitrogen, phosphorus, and minor nutrients such as sulfur and trace elements) for their growths metabolism modes are broadly classified as aerobic and anaerobic . Aerobic transformations occur in the presence of molecular oxygen, with molecular oxygen serving as the electron acceptor. This form of metabolism is known as aerobic respiration . Anaerobic reactions occur only in the absence of molecular oxygen and the reactions are subdivided into ------anaerobic respiration ------fermentation , and -------methane fermentation .

6. Fermentation organic compounds serve as both electron donors and electron acceptors. can proceed only under strictly anaerobic conditions. end products depend on the type of microorganisms but usually include a number of acids, alcohols, ketones , and gases such as CO 2 and CH 4 .

7. Reductant electron donor Oxidant electron donor End products Aerobic respiration Organic substrates (benzene, toluene, phenol) NH 4 Fe 2+ S 2–       O 2 O 2 O 2 O 2   CO 2 , H 2 O NO 2 – , NO 3 – , H 2 O Fe 3+ SO­ -- Anaerobic respiration Organic substrates (benzene, toluene, phenol, trichloroethylene) Organic substrates (benzene, trichloroethylene) H 2 H 2       NO 3 – SO 4 2 – SO 4 2 – CO 2       N 2 , CO 2 , H 2 O, Cl – S 2- , H 2 O, CO 2 , Cl – S 2- ,H 2 O CH 4 , H 2 O Fermentation Organic substrates   Organic compounds   CO 2 , CH 4 Table;2 :Summary of Metabolism Modes

8. different modes of Microbial transformations of organic compounds Degradation - ----initial substrate no longer exists Mineralization ------- complete conversion of the organic structure to inorganic forms such as CO 2 , H 2 O, and Cl – . Detoxification ------transformation of the compound to some intermediate form that is nontoxic or less toxic. activation ---- The process of forming toxic end products or intermediate products

9. Microorganisms are capable of catalyzing a variety of reaction Hydrolysis —frequently conducted outside the microbial cell by exoenzymes . Hydrolysis is simply a cleavage of an organic molecule with the addition of water . Cleavage —cleaving of a carbon–carbon bond ------- An organic compound is split or a terminal carbon is cleaved off an organic chain . Oxidation —breakdown of organic compounds using an electrophilic form of oxygen . Reduction —breakdown of organic compounds by a nucleophilic form of hydrogen or by direct electron delivery . Dechlorination —the chlorinated compound becomes an electron acceptor; in this process, a chlorine atom is removed and is replaced with a hydrogen atom . Dehydrogenation —an oxidation–reduction reaction that results in the loss of two electrons and two protons, resulting in the loss of two hydrogen atoms . Dehydrohalogenatio n —results in the loss of a hydrogen and chlorine atom from the organic compound . Substitution —these reactions involve replacing one atom with another.  

10. Factors affecting bioremediation Moisture : influences the rate of contaminant metabolism because it influences the kind and amount of soluble materials that are available as well as the osmotic pressure and pH of terrestrial and aquatic systems. A range of 50-80% is optimal for biodegradation.   Nutrients : nitrogen , phosphorous, potassium, sulfur , copper, and trace element etc are required If nutrients are not available in sufficient amounts, microbial activity will become limited . Nitrogen and phosphorous are deficient in the contaminated environment. These are usually added to the bioremediation system in a useable form (e.g., as ammonium for nitrogen and as phosphate for phosphorous). Oxygen level ; increasing the concentration of electron acceptors and nutrients in ground water and surface water . Oxygen is the main electron acceptor for aerobic bioremediation. pH : Soil pH may affect the availability of nutrients. pH of 6.5 to 8.5 is generally optimal for biodegradation in most aquatic and terrestrial systems and values ranging

11.   Temperature Temperature directly influences the rate of biodegradation by controlling the rates of enzyme catalysed reactions . Temperature of 15-65 C is optimal for biodegradation. Chemical nature of pollutant In general, aliphatic compound are more easily degraded than aromatic ones . Presence of cyclic ring structure and length chains or branches decrease the efficiency of biodegradation . Water soluble compounds are more easily degraded . The presence of halogen inhibit biodegradation.

12. Methods of bioremediation On the basis of removal and transportation of the wastes for the treatment, basically there are two methods: in-situ bioremediation and ex-situ bioremediation.

13. In situ bioremediation In situ bioremediation involves a direct approach for the microbial degradation of pollution (soil, ground water). Biostimulation ------Addition of adequate quantities of nutrients at the site promote microbial growth is done. When microorganisms are imported to a contaminated site to enhance degradation, the process is called as “ Bio-augmentation applied for clean-up of oil spillages, beaches etc. There are two types of in situ bioremediation – # intrinsic # engineered .

14. Intrinsic bioremediation Conversion of environmental pollutants into the harmless forms through the innate capabilities of naturally occurring microbial population The intrinsic that is inherent capacity of microorganisms to metabolize the contaminants should be tested at the laboratory and field levels before use for intrinsic bioremediation. Through site monitoring programmes progress of intrinsic bioremediation should be recorded time to time. The conditions of site that favour intrinsic bioremediation are ground water flow throughout the year, carbonate minerals to buffer acidity produced during biodegradation, supply of electron acceptors and nutrients for microbial growth and absence of toxic compounds . Bioremediation of waste mixture containing metals such as Hg, Pb , and cyanide at toxic concentration can create problem The environmental factors such as pH, concentration, temperature and nutrient availability determine whether or not biotransformation takes place.

15. Engineered in situ bioremediation Intrinsic bioremediation is satisfactory at some places, but it is slow process due to the poorly adapted microorganisms, limited ability of electron acceptor and nutrients, cold temperature and high concentration of contaminants . When site conditions are not suitable, bioremediation requires construction of engineered system to supply materials that stimulate microorganisms. Engineered in situ bioremediation accelerates the desired biodegradation reactions by encouraging growth of more microorganisms via optimizing physico - chemical conditions. Oxygen and electron acceptors (e. g NO 3 - , SO 4 2- ) and nutrients (e. g nitrogen and phosphorous) promote microbial growth in surface. When contamination is deeper, amended water is injected through wells. But in systems both extraction and injection wells are used in combination to control the flow of contaminated ground water combined with above ground bioreactor treatment and subsequent reinjection of nutrients spiked effluent are done.

16. In situ bioremediation Techniques Bioventing Biosparging Bioslurping Phytoremediation

17. Bioventing It is a promising technology that stimulates the natural in situ biodegradation of any aerobically degradable compounds in soil by providing oxygen to existing soil microorganisms. It typically uses low air flow rates to provide only enough oxygen to sustain microbial activity . Oxygen is most commonly supplied through direct air injection into residual contamination in soil . In addition to degradation of adsorbed fuel residuals, volatile compounds are biodegraded as vapors move slowly through biologically active soil . Bioventing techniques have been successfully used to remediate soils contaminated by petroleum hydrocarbons, no chlorinated solvents, some pesticides, wood preservatives, and other organic chemicals. This technique shows considerable promise of stabilizing or removing inorganics from soil as it can induce changes in the valence state of inorganics and cause adsorption, uptake, accumulation, and concentration of inorganics in micro or macro organisms . However , several factors may limit the applicability and effectiveness of the process for example highly saturated soils, extremely low moisture content or low permeability soils negatively affect the bioventing performance.   Fig 1: Bioventing system

18. Biosparging It involves the injection of a gas (usually air or oxygen) and occasionally gas-phase nutrients, under pressure, into the saturated zone to promote aerobic biodegradation. In air sparging , volatile contaminants also can be removed from the saturated zone by desorption and volatilization into the air stream. Typically , biosparging is achieved by injecting air into a contaminated subsurface formation through a specially designed series of injection wells. The air creates an inverted cone of partially aerated soils surrounding the injection point Fig 2: Biosparging system

19. The air displaces pore water, volatilizes contaminants, and exits the saturated zone into the unsaturated zone . While in contact with ground water, oxygen dissolution from the air into the ground water is facilitated and supports aerobic biodegradation. A number of contaminants have been successfully addressed with biosparging technology, including gasoline components such as benzene, toluene, ethyl benzene, and xylenes . Biosparging is most often recommended at sites impacted with mid-weight petroleum hydrocarbon contaminants, such as diesel and jet fuels. Lighter contaminants, such as gasoline, tend to be easily mobilized into the unsaturated zone and physically removed. Heavier contaminants, such as oils, require longer remedial intervals because of reduced microbial bioavailability with increasing carbon chain length .

20. Bioslurping also known as multi-phase extraction is effective in removing free product that is floating on the water table. Bioslurping combines the two remedial approaches of bioventing and vacuum-enhanced free-product recovery . Bioventing stimulates aerobic bioremediation of contaminated soils in in situ, while vacuum-enhanced free-product recovery extracts -- light , nonaqueous -phase liquids (LNAPLs ) from the capillary fringe and the water table Bioslurping is limited to 25 feet below ground surface as contaminants cannot be lifted more than 25 feet by this method.

21. A bioslurping tube with adjustable height is lowered into a ground water well and installed within a screened portion at the water table. A vacuum is applied to the bioslurping tube and free product is “slurped” up the tube into a trap or oil water separator for further treatment . Removal of the LNAPL results in a decline in the LNAPL elevation, which in turn promotes LNAPL flow from outlying areas toward the bioslurping well. As the fluid level in the bioslurping well declines in response to vacuum extraction of LNAPL, the bioslurping tube also begins to extract vapours from the unsaturated zone. This vapour extraction promotes soil gas movement, which in turn increases aeration and enhances aerobic biodegradation. Fig 3: Bioslurping system

22. Phytoremediation Phytoremediation is an in situ technique that uses plants to remediate contaminated soils . Phytoremediation is most suited for sites where other remediation options are not costs effective, low-level contaminated sites, or in conjunction with other remediation techniques. Deep rooted trees, grasses, legumes, and aquatic plants all have application in the phytoremediation field. Phytoremediation has been used to remove PAH, 2,4,6-trinitrotoluene (TNT), hexahyro-1,3,5-trinitro-1,3,5 triazine etc. Plants are able to remove pollutants from the groundwater and store, metabolize, or volatilize them . Also, roots also help support a wide variety of microorganisms in the subsurface. These microorganisms can then degrade the contaminants . The roots also provide organic carbon sources to promote cometabolism in the rizosphere .

23. Sl.no Aquatic/semi aquatic plants Terrestrial plants 1 2 3 Water hyacinth ( Eichhornia crassipes ) Duckweed ( Lemna minor ) Water velvet ( Azolla pinnata ) Indian mustard (Brassica juncea ) Sun flower ( Helianthus annus ) Alyssum Table 3: List of some plants used in Phytoremediation

24. Advantages of in situ bioremediation Cost effective, with minimal exposure to public or site personnel Sites of bioremediation remain minimally disrupted

25. Disadvantages of in situ bioremediation Very time consuming process Sites are directly exposed to environmental factors such as temperature, oxygen supply etc . Microbial degrading ability varies seasonally

26. Ex situ bioremediation Ex situ bioremediation involves removal of waste materials and their collection at the place to facilitate microbial degradation. On the basis of phases of contaminated materials ex situ bioremediation is classified in to two : # Solid phase system # Slurry phase system

27. Solid phase system Solid waste system includes organic wastes (e. g leaves, animal manure and agriculture wastes) and problematic wastes ( e. g. domestic and industrial wastes, sewage sludge and municipal solid wastes bioremediation techniques: Composting Land farming

28. Composting Composting is aerobic, thermophilic treatment process in which contaminated material is mixed with bioremediation microorganisms. This is a controlled biological process by which organic contaminants (e.g., PAHs) are converted by microorganisms to safe, stabilized by products. Typically , thermophilic conditions -----54 to 65°C pH ------6-9. In composting, soils are excavated and mixed with bulking agents and organic amendments, such as wood chips and vegetative wastes, to enhance the porosity of the mixture to be decomposed. Degradation of the bulking agent heats up the compost, creating thermophilic conditions. Oxygen content usually is maintained by frequent mixing, such as daily or weekly turning off windrows. Surface irrigation often is used to maintain moisture content. Temperatures are controlled, to a degree, by mixing, irrigation, and air flow, but are also dependent on the degradability of the bulk material and ambient conditions.

29. Organisms involved in composting: actinomycetes (a filamentous type of bacteria) Fungi ( molds , yeasts) and protozoa , earth worms ,insects, mites and ants. Mechanism of composting The bacteria bring out the decomposition of macromolecules namely proteins and lipids, besides generating energy. Fungi and actinomycetes degrade cellulose and other complex organic compounds. Composting may be divided into 3 stages with refernce to changes in temperature i . Mesophilic stage : The fungi ( Aspergillus , Mucor . Penicillium ) and acid producing bacteria ( Pseudomonas, Bacillus ) are active in this stage, and the temperature increases to 40 C. ii.Thermophilic stage : As the composting proceeds, the temperature rises from 40 C to70 C. Thermophilic bacteria ( Thermus , Bacillus ) , Thermophilic fungi ( Absidia ) and actinomycetes ( Sterpyomyce s, Micropolyspora ) are active at this stage. Thermophilic stage is associated with high rate and maximum degradation of organic materials. iii.Cooling stage : The microbial degradative activity slows down and the thermophilic organisms are replaced by mesophil bacteria and fungi. Cooling stage is associated with formation of water,pH stabilization and completion of humeic acid formation

30. D esigns commonly applied for composting : Aerated static piles —Compost is formed into piles and aerated with blowers or vacuum pumps. Mechanically agitated in-vessel composting —Compost is placed in a reactor vessel, in which it is mixed and aerated. Windrow composting —Compost is placed in long, low, narrow piles (i.e., windrows) and periodically mixed with mobile equipment.

31. Windrow composting is the least expensive method, but has the potential to emit larger quantities of VOCs . In-vessel composting is generally the most expensive type, but provides for the best control of VOCs. Aerated static piles , especially when a vacuum is applied, offer some control of VOCs and are typically in an intermediate cost range, but will require off gas treatment . Berms may also be needed to control runoff during composting operations. Runoff may be managed by retention ponds, provision of a roof. Composting has been successfully applied to soils and biosolids contaminated with petroleum hydrocarbons (e.g., fuels, oil, grease), solvents, chlorophenols , pesticides, herbicides, PAHs, and nitro-aromatic explosives. For TNT, complete mineralization has been difficult to demonstrate via composting. TNT may bind to soil, resulting in low microbial bioavailability and apparent disappearance . Composting is not likely to be successful for highly chlorinated substances, such as PCBs, or for substances that are difficult to degrade biologically.

32. Land farming also called Land treatment useful in treating aerobically degradable contaminants . This process is suitable for non-volatile contaminants at sites where large areas for treatment cells are available . Land treatment of site-contaminated soil usually entails the tilling of an 8-12 inch layer of the soil to promote aerobic biodegradation of organic contaminants. The soils are periodically tilled to aerate the soil, and moisture is added when needed . In some cases, amendments may be added to improve the tilth of the soil, supply nutrients, moderate pH, or facilitate bioremediation . Figure 4:landfarming technique.

33. Typically , full-scale land treatment would be conducted in a prepared-bed land treatment unit—an open, shallow reactor with an impermeable lining on the bottom and sides to contain leachate, control runoff, and minimize erosion and with a leachate collection system under the soil layer . In some cases, hazardous wastes (such as highly contaminated soils) or process wastes ( such as distillate residues) may be treated in land treatment units. In these cases, the waste may be applied to a base soil layer.

34. Slurry phase treatment The contaminated solid materials ( soil, degraded sediments etc.), microorganisms and water formulated into slurry are brought within a bioreactor a triphasic system involving three major components: water, suspended particulate matter and air. water serves as suspending medium where nutrients, trace elemnts,pH adjustment chemicals and desorbed contaminants are dissolved. Suspended particulate matter includes a biologically inert substratum consisting of contaminants (soil particles) and biomass attached to soil matrix or free in suspended medium. Air provides oxygen for bacterial growth . Biologically thre are two types of slurry phase reactors : Aerated lagoons Low shear air lift reactors.

35. Aerated lagoons commonly used for treatment of municipal waste water. Nutrients and aeration are pumped to the reactor. Mixers are fitted to mix different components and form slurry, whereas surface aerators provide air required for microbial growth. The process may used as single stage or multistage operation . If the waste contains volatiles ,this reactor is not appropriate. Fig 5: Aerated lagoon system

36. Low shear air lift reactors: useful when the waste contains volatile components : These are cylindrical tanks which is made up of stainless steel . In this bioreactor pH, temperature, nutrient addition, mixing and oxygen can be controlled as desired . Shaft is equipped with impellers. It is driven by motor set up at the top. The rake arms are connected with blades which is used for resuspension of coarse materials and tend to settle on the bottom of the bioreactor . Air diffusers are placed radially along the rake arm. Airlift provides to bottom circulation of contents in reactor . Baffles make the hydrodynamic behaviour of slurry-phase bioreactors. Pre treatment process includes size fractionation of solids , soil washing ,milling to reduce particle size slurry preparation . Certain surfactants such as anthracene , pyrene etc are added to enhance the rate of biodegradation. These act as cosubstrate and utilize as carbon source. Co substrates also induce the production of beneficial enzymes. Fig 6: Low shear air lift reactors

37. Factors affecting slurry phase biodegradation pH(optimum 5.5-8.5 ) moisture content temperature( 20-30 C ) Mixing Nutrients Microbial population(naturally occurring microorganisms are satisfactory,genetically engineered microorganisms for layer compound may be added ) Reactor operation (batch and continouse )

38. Advantages of ex situ bioremediation Better controlled and more efficient process . Process can be improved by enrichment with desired microorganisms . Time required in short.

39. Disadvantages of ex situ bioremediation Very coastly process . Sites of pollution are highly disturbed . There may be disposal problem after the process is complete.  

40. Conclusion: In situ and ex situ biodegradation technologies are increasingly selected to remediate contaminated sites, either alone or in combination with other source control measures Bioremediation technologies have proven effective in remediating fuels and VOCs and are often able to address diverse organic contaminants including, PAHs, pesticides and herbicides, and nitro-aromatic compounds (such as explosives), potentially at lower cost than other remediation options. Some bioremediation techniques are also able to address heavy metal contamination. Bioremediation continues to be an active area of research, developent , and demonstration for its applications to diverse contaminated environments . A unique feature of bioremediation is the diversity of its application to solids, liquids, and liquid–solid mixtures, involving both in situ and ex situ environments . Amendments may be necessary to support or enhance the biodegradation processes to improve the timeframe involved to achieve clean-up goals. Site characterization and long-term monitoring are necessary to support system design and sizing as well as to verify continued performance. There are also regulatory requirements to be addressed regarding system design, implementation, operation, and performance, including the disposition of liquid effluents and other wastes resulting from the treatment process

41. Reference R.C Dubey BIOTECHNOLOGY;2 ND edition; Page no.571-576 . United states environmental protection agency; In Situ and Ex Situ Biodegradation Technologies for Remediation of Contaminated Sites;PDF file . Anushree Malik ; Enviornmental microbiology;Pdf file . Jera Williams; Bioremediation of Contaminated Soils: A Comparison of In Situ and Ex Situ Techniques;Pdf file . FUNDAMENTAL PRINCIPLES OF BIOREMEDIATION(An Aid to the Development of Bioremediation Proposals) ;Pdf file. U . satyanarayana and U Chakrapani ; BIOTECHNOLOGY;3 rd edition; page no. 718-721.    

42. Thank you