Introduction to fermentation and range of fermentation processes

Introduction to fermentation and Range of fermentation processes

1. Fermentation
Compiled By
Dr. Uzma Hameed, AP
Biotechnology, GCU Lahore

2. •Fermentation
•Latin verb fervere
• to boil
•appearance of the action of yeast on extracts of
fruit or malted grain.
•Reason

Biochemical meaning:
“ The generation of energy by the catabolism of
organic compounds.”
•Industrial microbiological meaning:
“Any process for the production of product by the
mass culture of a microorganism.”
11/5/2023 Prof. Dr. Ikram-ul-Haq (SI) 2

3. •Fermentation
•Latin verb fervere
• to boil
•appearance of the action of yeast on extracts of
fruit or malted grain.
•Reason

11/5/2023 Prof. Dr. Ikram-ul-Haq (SI) 3

4. •Fermentation:
•Physical state of bioiling
•Zymology/ Zymurgy
•metaolicprocess
•organic substrate –Energy+Product
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5. History
•Since Neolithic era
•Food stuff especially berverages
•7000-6600 BCE (China
•5000 BCE (india)
•3150 BCE ( Egypt)
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6. •Zymology
•Science of fermentation
•Anaerobic catabolism
•Energy production process for microbes
•Schwann and his collegeusexperiment
•1837
•Boiling grape juice →no fermentation
•Heat killing
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7. •Louis Pasteur
•1857→lactic acid fermentation is caused by living
organisms.
•In 1860,→demonstrated milk souring by bacteria
•formerly thought to be merely a chemical change.
•identifying the role of microorganisms in food
spoilage led to the process of pasterizaiton
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8. •EduarBuechner
•German chemist
•1897 when the
•Extract/juice of yeast Fermentation without cell
•In 1907 –Nobel Prize in chemistry
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9. 11/5/2023 Prof. Dr. Ikram-ul-Haq (SI) 9

10. •Biochemical meaning:
“ The generation of energy by the catabolism of
organic compounds.”
•Industrial microbiological meaning:
“Any process for the production of product by the
mass culture of a microorganism.”
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11. •Strict biochemical meaning:
“ The fermentation is energy-generation process
in which organic compounds act as both electron
donors and electron acceptors.”
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12. •In biotechnological meaning:
“ Use of the biological system for the sake of
development/improvement /facilitation in the process
by mass culture of cells.”
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13. •Fermentation
•The term fermentation is derived from the Latin verb
fervere, to boil, thus describing the appearance of the
action of yeast on extracts of fruit or malted grain.
However, fermentation has come to have
different meanings to biochemists and to industrial
microbiologists.
•Biochemical meaning:
“ The generation of energy by the catabolism of
organic compounds.”
•Industrial microbiological meaning:
“Any process for the production of product by the
mass culture of a microorganism.”
11/5/2023 Prof. Dr. Ikram-ul-Haq (SI) 13

14. An introduction to fermentation processes
•The term “fermentation-Latin verb fervere, to boil
•describing the appearance of the action of yeast on the extracts of fruit or malted
grain.
•biochemical meaning relates to
•the generation of energy by the catabolism of organic compounds
•catabolism of sugar -oxidative process
•reduced pyridine nucleotides-must be reoxidizedfor the process to continue.
•Under aerobic conditions
•reoxidationof reduced pyridine nucleotide –ET via the cytochrome system
•oxygen as the terminal electron acceptor
•Under anaerobic conditions
•reoxidationof reduced pyridine nucleotide -coupled with the reduction of an
organic compound
•often a subsequent product of the catabolic pathway
•E.g. reduction of pyruvic acid to ethanol

15. •Different microbial taxa are capable of
reducing pyruvate to a wide range of end
products
•A-Lactic acid bacteria (Streptococcus,
Lactobacillus);
•B, Clostridium propionicum;
•C, Yeast, Acetobacter, Zymomonas, Sarcina
ventriculi, Erwiniaamylovora; D,
Enterobacteriaceae(coli-aerogenes); E,
Clostridia;
•F, Klebsiella;
•G, Yeast;
•H, Clostridia (butyric, butylic
•organisms);
•I, Propionic acid bacteria.

16. •Termfermentation→biochemicalsense
•anenergy-generationprocessinwhichorganiccompoundsactasbothelectron
donorsandterminalelectronacceptors.
•Industrial microbiology →much broader*
“any process for the production of product by the mass culture of a
microorganism”
•First large scale “industrial” process for microbial metabolite production
•Ethanol production /yeast / malt or fruit extracts
•Brewing and the production of organic solvents
•Biochemical→An aerobic process
•microbiological →product

17. RANGE OF FERMENTATION PROCESSES
•five major groups of commercially important fermentations:
1. Microbial cells (or biomass)
2. microbial enzymes
3. microbial metabolites
4. recombinant products
5. transformation (biotransformation)

18. MICROBIAL BIOMASS
•For commercial production of microbial biomass →two major processes:
•production of yeast
•baking industry
•production of microbial cells
•human/animal feed
•SCP
•Term by M.I.T. professor Carol Wilson
•better image than "microbial protein”
•dead, dry cells of micro-organisms such as yeast, bacteria, fungi and algae which grow
on different carbon sources
•Fermentation
•variety of microbial products
•proteinaceous ingredients

19. MICROBIAL BIOMASS
•Bakers’ yeast has been produced on a large scale since early 1900s
•yeast was produced as human food in Germany during the First World War
•However, it was not until the 1960s that the production of microbial biomass
as a source of food protein was explored to any great depth.
•a few large-scale continuous processes for animal feed production were
established in the 1970s.
•were based on hydrocarbon feedstocks
•could not compete against other high protein animal feeds,
•resulting in their closure in the late 1980s

20. MICROBIAL BIOMASS
•ICI & Rank HovisMcDougal
•Establishment of a process for the production of fungal biomass for human food
•Mycoprotein
•Cleared for human consumption (except edible mushrooms)
•Fusarium graminearum
•molasse, or glucose
•NH
3
•pH control
•Heat treatment
•RNA reduction
•Marketing
•In UK →pies
•a success →less fat than meat
•Fusarium venenatum
•Quornin the spotlight over allergy alerts

21. MICROBIAL BIOMASS
•Fusarium venenatum
•In 1985 by Marlow Foods
•a joint venture between
•RHM) and ICI
•Since 1985 (38 years ago)
•Quorn in the spotlight over allergy alerts

22. •PRUTEEN
•ICI
•Methylophilusmethylotropha
•Methanol & ammonia
•72% crude protein
•Marketing
•energy, vitamins, minerals & a highly balanced protein source
•methionine and lysine content of Pruteen
•compared very favorably with white fish meal
•Production plant
•a single largest fermentorin the world (2 x 10,000,000 L)
•a 60,000 tn/year

23. •TORUTEIN
•Food grade yeast: "Torula"
•Ethanol -expensive as a substrate
•Amoco Company, US
•government clearances →Toruteinin Canada and Sweden
•Torutein→a flavor enhancer of high nutritional value,
•a replacement for meat, milk & egg protein.
•not very successful in the United States
•since soya which is plentiful and cheap can serve
•an alternative or substitute to meat and egg diets.
•yeast is about 52% protein

24. 2. MICROBIAL ENZYMES
•Commercial enzymes sources:- plant, animal, and microbes
•microbial -enormous advantage
•can be produced in large quantities by fermentation techniques
•easier to improve the productivity of a microbial system compared with
•Controlled production processes
•may be exploited/modified to improve productivity
•E.g.
•induction by including inducers in the medium
•repression control
•removed by mutation & recombination techniques
•Advent of recombinant DNA technology
•Production of enzymes of animal/plant origin by microorganisms
•number of gene copies coding for the enzyme can be increased

29. MICROBIAL METABOLITES
•Microbial culture growth stages
•lag, log, stationary & death
•Behavior of a culture may also be
described according to the
products

30. Primary metabolites
•During the log phase of growth, the products produced
are either
•anabolites(products of biosynthesis)
•essential to the growth of the organism
•aminoacids, proteins,
•nucleotides, nucleicacids,
•lipids, carbohydrates, etc.
•catabolites(products of catabolism)
•suethanoland lactic acid
•primary products of metabolism

31. Primary metabolites
•Trophophase
•primary products are produced
•Many primary metabolism are of considerable economic
importance

32. Primary metabolites
•Primary metabolism
•economic importance
•synthesis by wild-type microbes
•sufficient to meet the requirements of the organism
•Industrial production of p. metabolies
•Improvement by cultural conditions
•control of the process environment of the producing
organism
•Modification of wild-type organism
•induced mutations
•recombinant DNA technology

33. •Example: Production of amino acids
•Increase in productivity by several orders of magnitude
•Previously -microbial processes were only successful and
competelablewith chemical industry
•for the production of relatively complex and high value
compounds.
•In recent years-advancement in metabolic engineering
(genomics, proteomics, and metabolomics)
•provided new powerful techniques to
•understand the physiology of “over-production”
•reengineer microorganisms to “over-produce” end products and
intermediates of primary metabolites

35. Secondary Metabolite
•idiophase-(equivalent to the stationary phase)
•During the deceleration and stationary phases,
•compounds which are not produced during the
trophophase
•do not appear to have any obvious function in cell
metabolism
•secondary metabolites
•secondary metabolism may occur in continuous
cultures at low

36. •growth rates
•property of slow-growing, as well as non-
•growing cells
•Growth of microbes in natural environment??
•idiophasestate that prevails in nature rather
•trophophas-a property of microbes in culture
•secondary product
•synthesized by only a relatively few different
microbial species

38. •Not all microorganisms undergo secondary metabolism
•common in microorganisms that differentiate such as
•filamentous bacteria and fungi and the sporingbacteria
•not found, in the Enterobacteriaceae.
•taxonomic distribution of secondary metabolism is quite
different from that of primary metabolism

39. •not all microorganisms undergo secondary metabolism
•common amongst microorganisms that
•differentiate such as
•filamentous bacteria and fungi and the sporingbacteria
•not found others
•for example, in the Enterobacteriaceae
•taxonomic distribution of secondary metabolism
•quite different from that of primary metabolism

40. secondary metabolites tend to be
elaborated from the intermediates and
products of primary metabolism
•primary biosynthetic routes are
common to the vast majority of
microorganisms,
•each secondary product would be
synthesized by only a relatively few
different microbial species.

41. •classification of microbial products into primary and secondary
metabolites
•is convenient
•artificial system
•Bushell (1988) –the classification
•“should not be allowed to act as a conceptual
Straitjacket
to consider
all products as
either primary or secondary metabolites.”

42. •It is sometimes difficult to categorize a product as primary or secondary
•kinetics of synthesis of certain compounds
•change depending on the cultural condition
•sec. metabolites importance in fermentation
•due to the effects they have on other organisms
•antimicrobial activity
•specific enzyme inhibitors
•growth promoters
•many with pharmacological

43. •Production in only low concentrations of secondary metabolites by wild-type
microorganisms
•Synthesis being controlled by
•Induction
•quorum sensing
•growth rate
•feedback systems
•& catabolite repression
•modulated by a range of effector molecules
•Many improvement methods

44. RECOMBINANT PRODUCTS
•advent of recombinant DNA technology
•extended range of potential fermentation products
•Genes cloning
•Gene from higher organisms →microbial cell
•synthesizing “foreign” proteins
•“heterologous
•derived from a different organism.”
•A wide range of microbial cells -as hosts
•Escherichia coli
•Saccharomyces cerevisiae
•filamentous fungi

46. RECOMBINANT PRODUCTS
•Animal cells culture
•fermentation systems
•widely used →production of heterologous proteins
•a number of novel problems had to be solve
•considered extremely fragile
•Very less achievable cell density
•Complex media
•Low productivity
•Prone to contamination

49. RECOMBINANT PRODUCTS
•Important factors in the design of these processes include
•secretion of the product,
•minimization of the degradation of the product,
•control of the onset of synthesis during the fermentation
•maximizing the expression of the foreign gene.
•Recombinant products
•interferon, insulin, human serum albumin, factors VIII and IX, epidermal growth
factor, calf chymosin, and bovine somatostatin

51. TRANSFORMATION PROCESSES
•Conversation to a structurally related and financially more valuable
compound.
•biotransformation
•Microbial cells
•microorganisms -as chiral catalysts with high positional specificity and
stereospecificity
•microbial processes
•more specific than purely chemical ones &enable
•addition, removal, or modification of functional groups at specific sites on a complex
molecule
•without the use of chemical protection

52. TRANSFORMATION PROCESSES
•Reactions
•Dehydrogenation
•Oxidation
•Hydroxylation
•Dehydration and condensation
•Decarboxylation
•Animation, Deamination
•Isomerization

53. TRANSFORMATION PROCESSES
•Microbial processes
•additional advantage over chemical reagents
•operating at relatively low temperatures and pressures
•no requirement for potentially polluting heavy-metal catalysts
•vinegar production -oldest established microbial transformation process
•(conversion of ethanol to acetic acid),
•the majority of these processes involve the production of high-value
compounds including
•steroids, antibiotics, and prostaglandins
•conversion of acetonitrile to acrylamideby Rhodococcusrhodochrous
•being used in the manufacturing of a bulk chemical
•20,000 metric tons being produced annually

55. •Novel application of microbial transformation
•use of microorganisms to mimic mammalian
metabolism
•Humans and animals
•drugs metabolism→(e.g) removal from the body
•metabolites →biologically active
•desirable effect or
•damage

56. •Imp. In drug development studies
•activity of administered drug + its metabolites
•significant amount of the metabolites
•Source
•Tissues, blood & urine, or faecesof the experimental animal
•concentration →often very low
•time-consuming
•expensive, & far from pleasant
•Biotransformationsby metabolic ability of microorganisms
•drug metabolites production in small-scale fermentation
•investigation of their biological activity and/or toxicity

57. •Problemof transformation fermentation process
•large biomass
•catalyze a single reaction
solution
•Immobilization on an inert support
•whole cells or isolated enzymes
•catalyze the reactions,
•Reusability

59. COMPONENT PARTS OF A FERMENTATION PROCESS
•six basic component parts:
1.Medium formulation
2. Sterilization
3. Inoculum preparation/ Produciton
4. Growth of the organism in the production fermenter
5. Product extraction and purification
6. Disposal of effluents

65. Types of fermentation
•Fermentation
•production of a wide variety of substances
•highly beneficial to individuals and industry
•gained immense importance due to their economic and Environmental advantages.
•Ancient techniques
•modified and refined to maximize productivity.
•This has also involved the development of new machinery and processes.
•Types
1. Solid State fermentation (SSF).
2. Liquid State fermentation (LSF)
Surface culture &
submerged culture

66. •SSF process can be defined as microbial growth on particles without
presence of free water.
•Particles are a solid culture substrate
•rice or wheat bran
•saturated with water
•inoculated with (mold, yeast, bacteria)
•controlled room temperature
•ideal for growing filamentous fungi.
•It has been used in Asia and developing nations.
•It is more cost effective
•smaller vessels lower water consumption,
•reduced waste water treatment costs,
•lower energy consumption, and less contamination problems.

68. •SSF employs natural raw materials as carbon source such as
•Cassava
•barley
•wheat bran
•sugarcane bagasse
•various oil cakeslike
•palm kernel cake, soybean cake,
ground nut oil cake,
•fruit pulps (e.g. apple pomace),
•saw dust, seeds (e.g. tamarind, jack fruit),
•coffee husk and coffee pulp,
•tea waste
•spent brewing

70. •This method is alternative to the production of enzyme in liquid by
subermergedfermentation.
•High volumetric productivity
•Relatively high concentration of product
•Less effluent generate and
•Simple fermentation equipment
•Factors Involved in SSF Process
•Selection of Micro-organisms
•Substrate
•Process Optimization
•Product Isolation & Purification

71. Selection of Micro-organism
•key factor
•Product yield
•Bacteria, Yeast and Filamentous Fungi
•Filamentous Fungi
•solid substrate fermentation
Substrate
•important role
•Improved growth of micro-organisms
•increased product yield
•Substrate choice
•physical support
•nutrients to growing culture

72. Process Optimization
•Optimization
•physico-chemical and biochemical Parameters
•Size, initial moisture, pH and pre-treatment of the substrate
•Relative humidity, temperature of incubation, agitation and aeration, age and
size of the inoculum
•Nutrient Supplementation such as N, P and trace elements, supplementation
of additional carbon source and inducers
•Extraction of product and its purification
•Extraction of product and its purification

73. Problems in SSF
•Heat Transfer:
•main difficulty
•to control the temperature during fermentation process
•Heat →metabolic activities of microorganisms
•substrate used →low thermal conductivity →heat removal →
slow.
•If heat generated →beyond certain level→
•product denaturation
•effect on growth of microbe
•ultimately ending up in reduction in yield and quality of product

74. Applications
•Production of Industrial Enzymes
•Production of Bio pesticides
•Production of Renewable Energies
•Bioleaching
•Bioremediation

75. Submerged fermentation
•isubstrate
•liquid state
•nutrients needed for growth
•Submerged Fermentation (SmF)/Liquid Fermentation (LF)
•free flowing liquid substrates
•such as molasses & broths

76. Submerged fermentation
•bioactive compounds
•secretion into the fermentation broth
•substrates utilization
•rapid
•constant replacement/supplementation with nutrients
•best suited for
•microorganisms --> high moisture requirement
•additional advantage
•Easier purification of products
•primarily used in the extraction of secondary metabolites
•liquid form

77. •Advantages
•Easiness of measuring process parameters
•Even distribution of nutrients and microorganisms
•Ability to control growth conditions
•Availability of high water content for the growth of microbes
•Disadvantages
•Use of expensive media and expensive equipment
•Complex and expensive downstream process and difficulty
in the waste disposal
•High power consumption

79. •Compared to solid state fermentation
•High energy and labour
•Utilizes free-flowing liquid substrates, such as molasses and broths
•Substrates are utilized quite rapidly, and need to be replaced
constantly, especially in continuous culturing
•Best suited for bacteria that require high moisture content
•Inoculum ratio is usually small
•Requires constant agitation

80. •Surface culture fermentation
•organism grow →surface of a liquid medium
•without agitation
•microbial films can be developed on surface of a sufficient packing medium
•can be in the form of a fixed bed andstones or plastic sheet
•incubation →culture filtrate separation
•Filteration/cell mass →productrecovery

81. •Surface culture fermentation
•This fermentation process is not much expensive.
•Easy to control process
•Less energy
•Important for the high acetic acid concentration.
•Surface culture fermentation is important for the large scale industrial
product formation
•Sometimes biomass may be reused
•generally time consuming method
•needs large, area or space
•Examples:
•alcohol production,
•beer production
•citric acid production

82. Application of surface culture fermentation
•production of citric acid from Aspergillus niger
•production of nicotinic acid from Aspergillus terrus
•Not much expensive fermentation
•Easy to control
•Important for high acetic acid concentration
•lower space is required
•important for the large scale industrial product formation

83. Important Criteria in the Choice of Organism
1. Nutritional characteristics
2. Optimum temperature of the organism
3. Suitibilitythe process & reaction of the organism with the
equipment
4. Stability & amenability to genetic manipulation
5. Productivity
6. Ease of product recovery from culture
83

84. •TYPES OF MICROBIAL CULTURE
•three models of fermentation used in industrial applications:
•batch
•continuous and
•fed batch fermentations

85. Microbial growth kinetics
BATCH CULTURE/Fermentation
Batch culture is a closed culture system that contains an initial,
limited amount of nutrient.
•Fermentation process
•In a single vessel (from start to end)
•No addition/removal of, a major substrate or product stream

87. •At t= 0
•Medium →All the nutrients + microorgansims
•Suitable temperature
•Gaseous exchange (oxygen exception
→aerobic microorganism)
•Antifoaming agent
•Acid or base (for pH control)
Change
culture medium
number of microorganisms
amount of the product produced

88. •At t= 0
•Medium →All the nutrients + microorgansims
•Suitable temperature
•Gaseous exchange (oxygen exception →aerobic microorganism)
•Antifoaming agent
•Acid or base (for pH control)
•Change
•culture medium
•number of microorganisms
•amount of the product produced

89. •Batch fermentation →six phases of microbial growth
(a) Lag phase:
(b)Acceleration phase:
(c)Log phase:
(d)Deceleration phase:
(e)Stationary phase:
(f) Death phase:
89

90. Log/exponential phase:
•Constant & maximum growth rate increase
•Dependson the inoculumsize
Eq. 3
•Where
•x=concentrationofmicrobialbiomass(gdm−3)
•t=time(h)
•d=asmallchange
Eq. 2
μ =specific growth rate =biomass produced /unit of biomass /unit of time (hours)

91. •On integration of Eq. 2
•x0 =original biomass concentration
•xt= biomass concentration after the time interval, t hours
•e = base of the natural logarithm
•On taking natural log

92. •Plot of natural log of biomass
•Straight line
•Slope = μ

93. •During the exponential phase
•nutrients are in excess
•Organism growth→maximum
•maximum specific growth rate=μ
max
•μ
max values
•under the prevailing conditions experiment
•affected by
•medium composition
•pH,
•temperature

95. Feb-batch fermentation
•Introduced by Yoshida, Yamane and Nakamoto (1973)
•Batch culture continuously/sequentially fed with medium
without the removal of culture fluids.
•Addition of freshly prepared culture media at regular intervals
•increases the volume of fermentation culture
•Example:
•Production of proteins from recombinant microorganisms

97. 3-Continuous fermentation
•In this type of fermentation the products are removed
continuously along with the cells and the same is
replenished with the cell girth and addition of fresh
culture media.
•This results in a steady or constant volume of the
contents of the fermentor.
•This type of fermentation is used for the production
of single cell protein (S.S.P), antibiotics and organic
solvents.
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99. •After inoculation microbial culture will pass through a number of phases (Growth
curve). Growth is a result ofconsumptionofnutrients.
•In batch system, at time t=0,
•the sterilized nutrient solution in the fermenter is inoculated with microorganisms
•incubation is allowed to proceed at a suitable temperature
•gaseous environment for a suitable period of time.
•In the course of the entire fermentation nothing is added, except
•oxygen (in case of aerobic microorganisms),
•an antifoam agent, acid or base to control pH (in case of fermenter).
•The composition of the medium, the biomass concentration and the metabolite
concentration generally change constantly as a result of metabolism of the cells
•After the inoculation of a sterile nutrient solution with microorganisms and
cultivation under physiological conditions, typical phases of growthare observed

106. •Introduction
•Range of fermentation process
•Outline of fermentation process
•Types of fermentation
•Microbial growth (batch culture, fed batch and continuous) & their
requirements during fermentation process
•Brief overview of pilot scale fermentation and bioreactor/fermentor
•types of fermenter,
•Brief overviewof downstream process