fermentation process techniques modes and types

fermentation details

1. Fermentation process

2. fermentation is  a metabolic process that produces that converts sugars to acids, gases and alcohol through the action of enzymes. it occurs in yeast, bacteria and oxygen starving muscle cells the science of fermentation is known as zymology in microorganisms, fermentation is the primary means of producing adenosine triphosphate (ATP) by the degradation of organic nutrients anaerobically. for example, yeast enzymes convert sugars and starches into alcohol, while proteins are converted to peptides/amino acids

3. hh Fig. Pilot scale fermenter

4. Introduction to inoculum development

5. Characteristics of proper inoculum 1. Healthy, active state thus minimizing the length of the lag phase in the subsequent fermentation. 2. Available in sufficiently large volumes to provide an inoculum of optimum size. 3. In a suitable morphological form. 4. Free of contamination. 5. Have product-forming capabilities ‘ Process adopted to produce an inoculum meeting these criteria is called inoculum development’

6. Growth media – have less nutrients Fermentation media –have more nutrients 1.Synthetic :level and conc of media can be controlled, exact composition is known and can be optimized as per req. (expensive due to pure ingredients) 2. Crude : unknown composition, gives high yield, contains high conc of nutrients, anti-foaming agents, growth factors etc.

7. Steps in inoculum preparation: small scale fermentation processes Preservation culture Generation culture on agar slants, this is then sub-cultured to: Working culture At this stage microbes starts growing This culture is then used as inoculum in small scale fermentation processes

8. Additional steps for large scale fermentation processes After working culture preparation, the sterile saline water or liquid nutrient medium containing glass beads is added to the agar slants and shaken so that microbial suspension is prepared. This suspension is transferred to flatbed bottle which contains sterile agar medium. The microbes are allowed to grow by incubating the bottle Now these microbial cells are then transferred to shaker flask containing sterile liquid nutrient medium and is placed on rotary shaker bed in an incubator Microbes grow rapidly due to aeration Now microbial cells from shaker flask can be used as seed culture which are then added to small fermenters and allowed to grow for 1-2 days This stimulates conditions that exists in large fermenters to be used further Finally microbial cells are transferred to large fermenters

9. Development of inocula for bacterial processes Objective: to produce an active inoculum which will give as short a lag phase as possible in subsequent culture Inoculum size -3 to 10% of the culture volume Inocula should be transferred in the logarithmic phase of growth, when the cells are still metabolically active Usually the inoculum at its log phase of growth is transferred aspetically to production fermentation vessel Composition of inoculum medium and production medium is usually kept identical to minimize lag period of inoculum culture in fermentation process

10. E.g. The inoculum development program for vitamin B12 production by Pseudomonas denitrificans

11. Development of inocula for mycelial /fungal processes Majority of industrially important fungi and streptomycetes are capable of asexual sporulation, hence spore suspension is used as seed during an inoculum development major advantage of a spore inoculum is that is contains far more ' propagules ' than a vegetative culture.

12. Techniques developed to produce a high concentration of spores for use as an inoculum: Sporulation on solidified media Sporulation on solid media Sporulation in submerged culture

13. Sporulation on solidified media Most fungi and streptomycetes will sporulate on suitable agar media but a large surface area must be employed to produce sufficient spores. Parker (1950) described the 'roll-bottle' technique for the production of spores of Penicillium chrysogenum

14. hockenhull (1980) described the production of 10 10 spores of penicillium chrysogenum on a 300 -cm2 agar layer in a roux bottle ei sayed (1992) quoted the use of spore suspensions derived from agar media containing between 10 7 and 10 8 cm- 3. butterworth (1984) described the use of a roux bottle for the production of a spore inoculum of streptomyces clavuligerus for the production of clavulanic acid. the spores produced from one bottle containing 200 -cm2 agar surface could be used to inoculate a 75 -dm3 seed fermenter which, in turn, was used to inoculate a 1500-dm3 fermenter.

15. Sporulation on solid media Many filamentous organisms will sporulate profusely on the surface of cereal grains from which the spores may be harvested Substrates used: barley, hard wheat bran, ground maize and rice The sporulation of a given fungus is particularly affected by the amount of water added to the cereal before sterilization and the relative humidity of the atmosphere (preferably high)

16. Singh et al. (1968) have described a system for the sporulation of Aspergillus ochraces in which a 2.8-dm3 Fernbach flask containing 200 grams of 'pot' barley or 100 grams of moistened wheat bran produced 5*10 10 conidia after six days at 28° and 98% relative humidity. This was 5 times the number obtained from a Roux bottle batched with Sabouraud agar and 50 times the number obtained from such a vessel batched with Difco Nutrient Agar, incubated for the same time period

17. Podojil et al. (1984) quoted the use of millet for the sporulation of Streptomyces aureofaciens in the development of inoculum for the chlortetracycline fermentation

18. Sporulation in submerged culture more convenient than the use of solid or solidified media because it is easier to operate aseptically and it may be applied on a large scale The technique was first adopted by Foster et al. (1945) who induced submerged sporulation in Penicillium notatum by including 2.5% calcium chloride in a defined nitrate-sucrose medium

19. Rhodes et al. (1957) described the conditions necessary for the submerged sporulation of the griseofulvin -producing fungus, Penicillium patulum The media for submerged process was formulated such that nitrogen level had to be limited to between 0.05 and 0.1 % w/v and that good aeration had to be maintained. Submerged sporulation was induced by inoculating 600 cm3 of the above medium, in a 2-dm3 shake flask, with spores from a well- sporulated Czapek-Dox agar culture and incubating at 25° for 7 days. The resulting suspension of spores was then used as a 10% inoculum for a vegetative seed stage in a stirred fermenter, the seed culture subsequently providing a 10% inoculum for the production fermentation

20. S C A L E UP OF FERMENTATION PROCESS

21. S C A L E UP STUDIES Scale up studies are studies carried out at the laboratory or even pilot plant scale fermenters to yield data that could be used to extrapolate and build the large scale industrial fermenters. We try to build industrial size fermenters capable or close of producing the fermentation products as efficient as produced in small scale fermenters.

22. RULES FOLLOWED WHILE DOING SCALE-UP There are a few rules that are followed when doing scale up studies such as: Similarity in the geometry and configuration of fermenters used in scaling up. A minimum of three or four stages of increment in the scaling up of the volume of fermentation studies. Each jump in scale should be by a magnitude or power increase and not an increase of a few litre capacity. Slight increase in the working volume would not yield significant data for scale up operation.

23. Studies carried out during scale up includes Inoculum development Sterilization establishing the correct sterilization cycle at larger loads Environment parameters including nutrient availability, pH, Temperature, dissolved oxygen and dissolved carbon dioxide. Shear conditions, foam production.

24. Steps in scale-up Define product economics based on projected market size and competitive selling and provide guidance for allowable manufacturing costs. Conduct laboratory studies and scale up planning at the same time. Conduct preliminary studies larger than laboratory studies with equipment to be used to aid in plant design. Design and construct a pilot plant including provisions for process and environment controls, cleaning and sanitization systems, packaging and waste handling system and meeting regulatory agency requirements. Evaluate pilot plant results (product and process) including product economics to make any corrections and a decision on whether or not to proceed with a full scale plant development.

25. SCALE DOWN FERMENTATION PROCESS

26. in scale up studies the main objective is to carry out studies on smaller bioreactors in order to gain data and confidence and predict the behaviour how things actually will behave in large production fermenter. scale down studies are also used during the operation of large industrial scale fermenters in trouble shooting or trying to optimize the industrial scale fermentation. this method is called the fermentation monitoring experiment the goal when scaling down is to create a small-scale or lab-scale system that mimics the performance of its large-scale (pilot or manufacturing) counterpart, when both the process parameters are varied within their operating ranges and also when a process parameter deviates outside its operating range.

27. the main type of studies in scale down such as: 1 medium design 2 medium sterilization 3 inoculation procedures 4 number of generations 5 mixing 6 oxygen transfer rate

28. Streptomycin production

29. CONTENTS  Introduction  Chemical composition  Medium The Hockenhul Medium  Fermentation process Phase 1 Phase2 Phase 3  Recovery & purification  Uses  References

30. INTR O DUCTION  Streptomycin is an bactericidal antibiotic drug belonging to class aminoglycosides.  Used against TB  Derived from actinobacterium Streptomyces griseus .  Used against gram negative bacteria .  Dihydrostrepomycin prepared by hydrogenation of streptomycin with platinum as catalyst & is commercially more successful.

31. CHEMICAL COMPOSITION  Chemically, it contains 3 sugars derived from glucose with C, N, O & H elements.  Chemical formula – C 21 H 39 N 7 O 12

32. MEDIUM  Medium is a nutritive substance in which cultures are grown for scientific purposes.  The culture medium for streptomycin consists of – Carbon source : starch, dextrin, glucose, glycerol & other economically available material. Nitrogen source : natural agricultural by-products, soybean meal, corn steep liquor, cotton seed flour, casein hydrolyte, or yeast & its extract. Inorganic N salts like ammonium sulphate & ammonium nitrates are also used. Animal oils, vegetable oils and mineral oils are also used.  INOCULUM – S.griseus spores maintained in soil stocks or lyophilized in carrier are inoculated into sporulation medium which builds up mycelial inoculum.

33. THE HOCKENHUL MEDIUM  GLUCOSE 2.5%  EXTRACTED SOYA MEAL 4%  DISTILLERS DRIED SOLUBLE 0.5%  SODIUM CHLORIDE 0.25%  Ph 7.3 – 7.5

34. FERMENTATION PROCESS  Spores of S.griseus are inoculated into a medium to establish a culture with high mycelial biomass for introduction into inoculum tank, using inoculum to initiate the fermentation process.  Yield in production vessel responds to high aeration & agitation conditions. Other conditions involve- Temperature range 25-30°C pH range 7-8 Time 5-7 days  The fermentation process for production of Streptomycin involves 3 phases.

35. PHASE 1  Initial fermentation phase and there is little production of streptomycin.  Rapid growth with production of mycelial biomass .  Proteolytic enzymatic activity of S.griseus releases NH 3 from soya meal, raising the pH to 7.5  Characterized by release of ammonia.  Carbon nutrients of soya meal are utilized for growth.  Glucose is slowly utilized with slight production of Streptomycin.

36. PHASE 2  Little production of mycelia .  Glucose added to the medium & the NH 3 released from soya meal are consumed.  pH remains fairly constant ranging between 7.6 to 8.

37. PHASE 3  Final phase of fermentation.  Depletion of carbohydrates from medium.  Streptomycin production ceases & bacterial cells begin to lyse.  Ammonia from lysed cells increase the pH.

38. RECOVERY & PURIFICATION  Mycelium is separated from broth by filteration & streptomycin is recovered.  Recovery process – broth is acidified, filtered & neutralized. Then its subjected to column containing cation exchange resin to adsorb Streptomycin from the broth & column is washed with water & streptomycin eluted with HCl before concentration in vacuum almost to dryness.

39. CONTINUED. .  The streptomycin is dissolved in methanol & filtered.  Acetone is used in filterate to precipitate the antibiotic.  Percipitate is washed with acetone & dried in vacuo.  Purification is done by dissolving in methanol to form pure S. chloride complex. Further by, adsorbing it onto activated charcoal & eluting with acid alcohol.

40. USES  Treatment of diseases Tuberculosis Plague Veterinary medicine against gram negative bacteria.  Pesticide & fungicide.  Cell culture.  Protein purification.

41. Industrial production of chemical acids: glutamic acid

42. Glutamic acid Glutamic acid is an α-amino acid that used in biosynthesis of proteins. It contains an α-amino group which is in the protonated − NH3+ . An α-carboxylic acid group which is in the deprotonated − COO . And a side chain carboxylic acid. Polar negatively charged (at physiological pH), aliphatic amino acid. It is non-essential in humans, meaning the body can synthesize it.

43. Glutamic Acid Food Production: As flavor enhancer, to improve flavor. As nutritional supplement. Beverage As flavor enhancer: in soft drink and wine. Cosmetics As Hair restorer: in treatment of Hair Loss. As Wrinkle: in preventing aging. Agriculture/Animal Feed As nutritional supplement: in feed additive to enhance nutrition. Other Industries As intermediate: in manufacturing of various organic chemicals.

44. Biosynthesis of Glutamic acid Reactants Products Enzymes Glutamine + H 2 O → Glu + NH 3 GLS, GLS2 NAcGlu + H 2 O → Glu + Acetate (unknown) α-ketoglutarate + NADPH + NH 4 + → Glu + NADP + + H 2 O GLUD1, GLUD2 α-ketoglutarate + α-amino acid → Glu + α-oxo acid transaminase 1-pyrroline-5-carboxylate + NAD + + H 2 O → Glu + NADH ALDH4A1 N-formimino-L-glutamate + FH 4 ⇌ Glu + 5-formimino-FH 4 FTCD An amino acid precursor is converted to the target amino acid using 1 or 2 enzymes. Allows the conversion to a specific amino acid without microbial growth, thus eliminating the long process from glucose. Raw materials for the enzymatic step are supplied by chemical synthesis. The enzyme itself is either in isolated or whole cell form which is prepared by microbial fermentation.

45. Industrial Production and use of Microorganisms Industrial microbiology Microorganisms, typically grown on a large scale, to produce products or carry out chemical transformations. The glutamic acid is produced through the fermentation process Major organism used is Corynebacterium glutamicum . Classic methods are used to select for high-yielding microbial variants. Corynebacterium glutamicum

46. The manufacturing process of glutamic acid by fermentation comprises :- fermentation, crude isolation, purification processes. There are 3 types of fermentation are used: (1) Batch Fermentation. (2) Fed-batch Fermentation. (3) Continuous Fermentation. Industrial production of glutamic acid

47. (1)Batch Fermentation Widely use d in the production of most of amino acids. Fermentation is a closed culture system which contains an initial, limited amount of nutrient. A short adaptation time is usually necessary (lag phase) before cells enter the logarithmic growth phase (exponential phase). Nutrients soon become limited and they enter the stationary phase in which growth has (almost) ceased. In glutamic acid fermentations, production of the acid normally starts in the early logarithmic phase and continues through the stationary phase.

48. For economical reasons the fermentation time should be as short as possible with a high yield of the amino acid at the end. A second reason not to continue the fermentation in the late stationary phase is the appearance of contaminant- products. The lag phase can be shortened by using a higher concentration of seed inoculum. The seed is produced by growing the production strain in flasks and smaller fermenters.

49. (2) Fed-batch fermentation Batch fermentations which are fed continuously, or intermittently, with medium without the removal of fluid. In this way the volume of the culture increases with time. The residual substrate concentration may be maintained at a very low level. This may result in a removal of catabolite repressive effects and avoidance of toxic effects of medium components. Oxygen balance. The feed rate of the carbon source (mostly glucose) can be used to regulate cell growth rate and oxygen limitation,especially when oxygen demand is high in the exponential growth phase.

50. (3) Continuous fermentation In continuous fermentation, an open system is set up. Sterile nutrient solution is added to the bioreactor continuously. And an equivalent amount of converted nutrient solution with microorganisms is simultaneously removed from the system.

51. Natural product such as sugar cane is used. Then, the sugar cane is squeezed to make molasses. The heat sterilize raw material and other nutrient are put in the tank of the fermenter. The microorganism ( Corynebacterium glutamicum ) producing glutamic acid is added to the fermentation broth. The microorganism reacts with sugar to produce glutamic acid. Then, the fermentation broth is acidified and the glutamic acid is crystallized. Industrial production of glutamic acid

52. Separation and purification After the fermentation process, specific method is require to separate and purify the amino acid produced from its contaminant products, which include: Centrifugation. Filtration. Crystallisation. Ion exchange. Electrodialysis. Solvent extraction. Decolorisation. Evaporation.

53. The glutamic acid crystal is added to the sodium hydroxide solution and converted into monosodium glutamate (MSG). MSG is more soluble in water, less likely absorb moisture and has strong umami taste. The MSG is cleaned by using active carbon, which has many micro holes on their surface. The clean MSG solution is concentrated by heating and the monosodium glutamate crystal is formed. The crystal produce are dried with a hot air in a closed system. Then, the crystal is packed in the packaging and ready to be sold . Separation and purification of Glutamic acid

54. Industrial Production of Amino Acid (L-Lysine)  l ys i n e is t h e a m ino a c id that co v e r s m o r e than 90% of total world amino acid production.  Synthesis of lysine 80% by Fermentation , 20% by chemical synthesis.

55. Lysine Production Fermentation Process Submerged Fermentation Aerobic Fermentation Mode of Operation Batch Process Fed-Batch Process Fermenter Type Stirred Tank Reactors Air Lift Bioreactors

56. Lysine Production Corynebactrium glutamicum (ATCC 13287) Gram positive Soil Bacterium Non-Motile Rod shaped Non – spore producing Non-pathogenic bacterium Electron micrograph of C. glutamicum 4. Microorganism

57. Lysine Production 5. Fermentation Media Carbon Source : Cane Molasses Nitrogen Source : Corn steep liquor / Soybean meal Minerals and Salts : KH2PO4/K2HPO4, CaCO3 Trace Elements : Corn steep liquor A n t i f o a m i n g A g e n t s : P E G - 20 0, S ili c o ne based oils

58. Lysine Production Process Parameters Optimum pH : 7.2 Optimum Temperature: 35-37 °C Time : 100 hours production cycle

59. Lysine Production Procedure The process can be divided into three main parts: Fermentation; Product Recovery; Product Concentration, Drying and Packaging.

60. Fermentation The culture media used in the batch and fed-batch phases of fermentation are prepared by mixing process water, glucose and nutrients. The fermentation step is performed in fed-batch mode and under aerobic conditions. In the batch phase, the microorganism seed is fed into the fermenters, which have been filled previously with the fermentation batch medium. After glucose exhaustion, the batch phase is finished and the fed-batch phase is started. .

61.  During the fed-batch phase, glucose and nutrients are continuously supplied until the desired L-lysine concentration is achieved.  At the end of the fermentation, the broth is sent to a buffer tank to provide a continuous flow to the downstream process steps.

62. Product Recovery  The fermentation broth is sent to an ultrafiltration system for the removal of cell debris and other suspended solids.  Subsequently, the liquor from ultrafiltration is fed to ion-exchange columns, where L- lysine is selectively adsorbed.  The adsorbed L-lysine is eluted from the ion- exchange resins by washing with an aqueous ammonia solution.

63. Product Concentration, Drying and Packaging  The L-lysine eluted from the ion-exchange columns is mixed with mother liquor from the product-filtration step and concentrated by evaporation.  The concentrated lysine solution is acidified with hydrochloric acid, and free L-lysine is converted to L-lysine HCl.  The L-lysine HCl solution is then sent to the crystallizer, and lysine salt is filtered. The mother liquor is recycled to the evaporator and the wet cake is conveyed to dryers.  Final dry L-lysine-HCl (98.5 wt.%) is obtained and sent to a packaging line before being stored in bags.

64. Lysine production plant  Lysine production plant of the BASF AG located in Gunsan, South Korea with an annual capacity of about 1lakh tons. C op y r i g ht BA S F A G- T h e chemical company (2003).

65. Applications and Uses of L-Lysine  Used as nutrition supplements in food, beverage, pharmaceutical, agriculture/animal feed, and various other industries.  Used as flavor enhancer in food production.  In Pharmaceutical L-Lysine is widely used as Nervous system drugs and Nutritional therapy in Pharmaceutical.  L-Lysine is widely used in poultry feed to improve growth and egg production and in fish feed to improve growth.

66. Ethanol production

67. CONTENTS: Ethanol Why do we need ethanol? Ethanol Fermentation Substrates for ethanol production. Microorganisms utilized for production of ethanol. Biochemistry of the reaction Immobilization of cells. Process of ethanol production Ethanol as a biofuel.

68. ETHANOL- GRAIN ALCOHOL/ ETHYL ALCOHOL Ethanol is a volatile , flammable , colorless liquid with a slight chemical odor. It is used as an antiseptic, a solvent, and a fuel.

69. WHY DO WE NEED ETHANOL? INGREDIENT Principle ingredient in alcoholic beverages like beer, wine, or brandy. EFFECTIVE SOLVENT Mixes easily with water & many organic compounds FOOD ADDITIVES Ethanol can help evenly distribute food colorings, as well as it enhances the flavor of food extracts. ASTRINGENT Acts as an astringent to help clean skin, In lotions as a PRESERVATIVE and used in other pharmaceutical products. DISTILLERS GRAINS By-product of ethanol production which can be fed to livestock either wet or dried. BIOFUEL It can be blended with gasoline and used in motor vehicles. E T H A N O L

70. ETHANOL FERMENTATION A biological process in which sugars such as glucose, fructose, and sucrose are converted into cellular energy and thereby produces ethanol and CO2 as metabolic waste products. It is an anaerobic process . Performed by microbes such as yeast and bacteria. The type of the organism chosen mostly depends on the nature of the substrate used. Among the yeast, Saccharomyces cerevisiae is the most commonly used, while among the bacteria, Zymomonas mobilis is the most frequently employed for ethanol production.

71. SUBSTRATES FOR ETHANOL PRODUCTION The substrates are chosen based on the regional availability and economical efficiency . Categories of substrates are : SUCROSE CO N T A I N I NG MATERIALS Sugarcane Sugar beet Sugar sorghum STARCHY MATERIALS Corn Other starchy materials( sweet potato, wheat etc.) LIGNOC E LLU L OSI C AND CELLULOSIC MATERIALS Maize silage Barley hull Paper sludge Wood etc

72. MICROORGANISMS INVOLVED Microorganisms such as yeasts and bacteria play an essential role in ethanol production by fermenting a wide range of sugars to ethanol. They are used in industrial plants due to valuable properties in ethanol yield (>90.0% theoretical yield), ethanol tolerance (>40.0 g/L), ethanol productivity (>1.0 g/L/h), growth in simple, inexpensive media and undiluted fermentation broth with resistance to inhibitors and retard contaminants from growth condition. These microorganisms provide the enzymes needed to catalyze the reaction.

73. Sa c charo m y c es cerevisiae zymomonas mobilis high specific growth rate (0.27). high ethanol tolerance up to 130g/l. a broad ph range for production ( ph 3.5-7.5). consumes glucose faster than s.cerevisiae , leading to higher ethanol productivity. anaerobic carbohydrate metabolism is carried out through the Entner - Doudoroff pathway, where only one mole of ATP is produced per mole of glucose used, thus reducing the amount of glucose that is converted to biomass rather than ethanol. has no amylases. specific growth rate of 0.13. can tolerate high concentrations of ethanol . ethanol production is coupled with yeast cell growth by- products like glycerol, organic acids, are also produced. uses the Embden-Meyerhof Pathway, generating 2 moles of ATPs under anaerobic conditions. SOME PROPERTIES OF MICROORGANISMS USED IN FERMENTATION

74. BIOCHEMISTRY OF THE REACTION

75. BIOCHEMISTRY CONTD… The overall reaction can be divided in two steps: 1. Glycolysis – where the yeast breaks do w n glucose to form 2 pyruvate molecules 2. Fermentation - where the 2 pyruvate molecules are converted into 2 CO2 molecules and 2 molecules of ethanol. before pyruvate can be converted to ethanol, it is first converted into an intermediary molecule- acetaldehyde , by decarboxylation of pyruvate and this releases CO2. Now acetaldehyde is converted to ethanol by reduction Enzyme responsible for decarboxylation is pyruvate decarboxylase while for reduction of acetaldehyde is alcohol dehydrogenase .

76. PROCESS OF ETHANOL PRO D UCTION The proces is carried out in a fermenter/ bioreactor. There are five basic steps for ethanol production. These are- 1.Pretreatment of raw materials depends on the chemical composition of the raw material/substrate, sugary raw materials require mild or no pretreatment, cellulosic and lignocellulosic materials need extensive pretreatment, involves Liquefaction and Saccharification . 2. Preparation of nutrient solution (media) media composition must contains specific nutrients, such as trace elements, vitamins, nitrogen, phosphorus, growth regulators etc. fermentation performance of the microbes highly depends on the composition of media. thermotolerant vitamins ( inositol, pantothenic acid, and biotin) are required to obtain rapid fermentation and high ethanol levels.

77. Process contd… 3 . Preparation Of Inoculum INOCULUM : a small amount of substance containing bacteria or any other micoorganism from a pure culture which is used to start a new culture. the desired organism is isolated and organisms are first cultured in flasks under aerobic condition to increase the size of the inoculum .

78. Process contd… 4. Fermentation process For the production of ethanol to occur properly, following conditions should be maintained- Fermentation conditions- Temperature : a moderate temperature of 25ᵒC to 50ᵒC [ if the temperature is too low, the yeast will be inactive and if it is too high, the enzymes in the yeast will be denatured and will stop working] pH : 4.0-4.5 Sugar concentration : higher glucose concentration rate does not enhance the production of ethanol due to substrate inhibition at a higher glucose concentration in the system. Intrinsic Factors- Culture medium Dissolved Oxygen : Aeration is initially required for good growth of the organisms. Later, anaerobic conditions are created by withdrawal of oxygen coupled with production of CO2. Immobilization Other micronutrients.

79. Process contd… production of biomass in aerobic conditions and production of ethanol in anaerobic conditions. As the fermentation is complete, the fermentation broth contains ethanol in the range of 6-9% by volume. This represents about 90-95% conversion of substrate to ethanol. Corn Fermenter Ethanol

80. Process contd… 5. Recovery of ethanol Energy demanding step. Fermentation by-products are mostly removed by distillation. Distillation is the most dominant and recognized industrial purification technique . It utilizes the differences of volatilities of components in a mixture. Principle : by heating a mixture, low boiling point components are concentrated in the vapor phase. By condensing this vapor, more concentrated less volatile compounds is obtained in liquid phase .

81. It involves two main problems : Separation of volatile compounds - impurities with similar boiling points to ethanol lodges in ethanol even after distillation. Cost We cannot completely separate ethanol from water since they are strongly bound to each other due to the presence of -OH group in both of them ( ethanol and water). An additional method must be utilized to remove all the water from ethanol ( since the ethanol fraction contains about 5% water and 95% ethanol) : DEHYDRATION . material used in this is called ZEOLITE . Zeolite absorbs the water into it, but the ethanol will not go into the zeolite ( because the pore sizes of zeolite are too small to allow the ethanol to enter.)

82. ETHANOL AS A BIOFUEL Ethanol fuel is cost effective compared to other biofuels. Ecologically effective. Minimizes global warming Easily accessible Minimizes dependence on fossil fuels Contributes to creation of employment to the country Opens up untapped agricultural sector Variety of sources of raw material Ethanol is classified as a renewable energy source

83. Production of Penicillin by Fermentation

84. • Alexander Fleming, 1952 Chains of conidia (spores) produced by hyphal branch from mycelium

85. Mechanism : Penicillium fungi blocks the growth of gram positive bacteria in culture

86. The strain Penicillium chrysogenum Penicillium notatum Mold conidiophores, fruiting structures, sporangia, conidia, and asexual spores of Penicillium notatum, also known as Penicillium chrysogenum. The mold is commonly found in homes, is used in the production of green- and blue-mold cheese, and is used to produce the antibiotic penicillin. Penicillin was the first antibiotic to be discovered by Alexander Fleming. Magnification of X600

87. Penicillin synthesis involves: Media Inocula preparation Process and control parameters (ph, Aeration , Agitation , Temp etc ) Downstream processing (Recovery and Purification)

88. M ed ia Component Concentration(gm/ltr) Cornsteep liquor 21.88 (NH 4 ) 2 SO 4 10.70 KH 2 PO 4 2.74 Sodium phenyl acetate 15.90 Glucose 81.90 MgSO 4 7H 2 O,CaCO 3, antifoam oil qs

89. • • Total Composition of Typical Media: Solids (40-60%), lactic acid (12-27%),total nitrogen(7.4-7.8%) , amino nitrogen(2.6-3.3%),reducing sugars as glucose (1.5- 14%) mag necium (18-20%). Carbon source: Lactose in concentration of 6% satisfactory. Cornsteep (greatly enhances the yeield of penicillin ). And/or one of the protein rich oil cakes like cotton seed and groundnu t. One or more sugars like lactose , sucrose and glucose and glucose along with a vegetable oil like soybean oil, groundnut oil Nitrogen source : Sodium nitrate,ammonium sulphate,ammonium acetate, ammonium lactate,cornsteep liquor etc.serve as. • • •

90. • Amino acids such as L-cystine,L-cysteine and valine are important in the synthesis of the b-lactum thiazolidine ring system of the penicillins. Mineral salts including sources of sulphate and phosphate Precursors are used to increase the yield of penicillin by the fermentation The requisite precursor, eg. phenylacetic acid,phenoxy acetic acid and phenylacetamide are commonly used as precursors. • •

91. Inoculation methods • Various media employed in the manufacture of penicillin can be inoculated by several methods, like .. s urface culture : surface of the medium is inoculated with dry spores. the spore material is applied in such a way as to cover the surface as uniformly as possible. submerged culture : production medium is inoculated with dry spores,by pellet inocula or by ungerminated spores can be prepared in sterile 0.1% soap solutions, in sterile water containing 100 ppm of sodium lauryl sulphonate.pellet inoculation saves time in the production stage. pellet inocula are prepared by growing mycelium from mold spores under submerged conditions.

92. F e r m en t e r

93. Fed-Batch Penicillin Production

94. Conditions of fermentation • • Optimum Temperature : 25 O C. Optimum ph Range: 5 to 7.5, lower ph range yield penicillin substantially lower Buffering agent : Calcium carbonate , however it is not suitable in surface culture production as it decreases the growth of the molds and the yield of penicillin.P.chrysogenum being strictly aerobic, Rate of Aeration: adequate aeration of the fermenter is essential, rate vary from around 0.5 volume of air/volume of liquid/minute., Effectiveness may be enhanced by increase in pressure of abt 20lb/sq inch. Aeration rate is also attained by the use of proper type of stirrer and at correct speed. Antifoam agents such as tributyl citrate, octadecanol, and lard oil, prevent excessive foam formation during the production of penicillin by submerged culture method. Prevention of contamination during the production of penicillin is essential because contamination usually causes rapid destruction of penicillin. Sterilization of facilities and media are easily achieved through steam. • • • • • •

95. Isolation and Purification The first step is the recovery process is the removal of mycelium or cells by filtration or centrifuging. Second step is to remove the antibiotic from the spent production medium by solvent extraction, adsorption or precipitation. Additional solvent extraction,distillation,sublimation, column chromatography or other methods accomplish purification. Semi-synthetic penicillins. Semi synthetic such as penicillin such as Ampicillin, Methicillin, Oxocillin, Propicllin are prepared by chemical acylation of 6-aminopenicillanic acid.

96. PRODUCTION OF PROTEASE ENZYME

97. Alkaline Protease One of the class of protease enzyme. An extracellular enzyme. Performs proteolysis , that is, protein catabolism by hydrolysis of the peptide bonds. Active at alkaline pH 8 to 12 and at temperature 30⁰-80⁰C. Molecular weight is about 20,000 to 45,000 Dalton. The structure is determined by X-ray crystallography. EC (Enzyme Commission) Number: 3.4.21–24.99 In 1971 , Japanese scientist Koki Horikoshi first reported the production of alkaline protease from bacteria.

98. Sources of Alkaline Protease Bacteria Fungi Bacillus subtilis Aspergillus flavus Bacillus pumilus Aspergillus fumigatus Bacillus licheniformis Aspergillus melleus Bacillus altitudinis Aspergillus niger Bacillus firmus Fusarium graminearum Bacillus amyloliquefaciens Penicillium griseofulvim Bacillus proteolyticus Penicillium lilacinus Thermomonospora fusca Scedosporium apiosermum Genetic Engineering of Microbes More than 50% of the industrially important enzymes are now produced from genetically engineered microorganisms . Methods Used: Conventional mutagenesis (UV or chemical exposure) or Recombinant DNA technology.

99. Production Process of Alkaline Protease Enzyme Isolation of Microorganisms Development and Preparation of Inoculum Preparation of Fermentation Media Optimization of Media Fermentation Process Enzyme Extraction and Assay Protein Assay Alkaline Protease Purification Ammonium sulphate precipitation Ultracentrifugation Flocculation Chromatography Electrophoresis Characterization of Purified Alkaline Protease Packaging

100. Alkaline Protease in Leather Industry Stages Enzymes involved Function of Enzymes Curing Enzymes are not directly involved To preserve hides and skins Soaking Alkaline Protease To remove non fibrillar proteins Dehairing Neutral and Alkaline Protease To remove hair Bating Trypsin and Alkaline Protease To make soft, supple and pliable Degreasing Lipase and Acid Protease To remove fats Tanning Enzymes are not directly involved To influence the quality of tanning Waste Processing Trypsin and Alkaline Protease Tanned waste processing

101. Advantages of Using Enzymes in Leather Industry Significant reduction of using chemicals. Simplification of processes. Creates of an ecologically conducive atmosphere for the workers. Leathers have shown better strength and quality. Saves time. Environment friendly. Leather wastes can be hydrolyzed by enzymes.

102. Mushroom Cultivation

103. What Are Mushrooms ? The mushroom is a fruiting body of microorganisms called fungi. To propagate, it forms a mycelium initates growth stage, generating spores in the gills for dispersal. A s m u sh r o o m s l a c k c h lo r o p h y ll, they do n ’ t  p h oto syn thesi z e ( p r ocess ene rgy f r om su n lig h t ) li k e green plants.  Mushrooms themselves are tasty, popular to eat and a beneficial source of nutrients for people.

104.  Much of Asia’s environment is suitable for cultivating many different types of mushrooms.  In addition, the low costs associated with growing mushrooms helps farmers get started and make relatively quick and good financial returns, positively contributing to the country’s economy.

105.  Mushrooms play a significant role in forest ecology, as they help decompose dead plants and animals, including dead trees, branches, leaves, fruits, seeds and animal droppings on the ground.

106. Mushrooms Varieties and their Values There are more than 30,000 identified types of mushrooms worldwide. 99% of these are safely edible and roughly 1% is poisonous. Yet there are still many undiscovered mushroom species and the effects of some mushrooms on human health remain unknown

107. A wide assortment of mushrooms is eaten around the world. Champignon and Field Mushrooms are popular in Europe. Shitake Mushrooms are consumed mostly in China and Japan, while Thai people prefer Yanagi Mushrooms or Straw Mushrooms.

108. Some mushrooms have medicinal qualities and their popularity is rising too. Nowadays, almost every country devotes more attention to research, experimentation, selection and development of mushrooms .

109. Nutritional Benefits Mushrooms are very popular in many countries and often considered to be as nutritious as meat. India, Taiwan, Japan, Korea and Thailand have the highest global export rates of mushrooms. Scientific research has shown that mushrooms contain many kinds of carbohydrates, proteins and fat, B-complex vitamins , important minerals ,

110. Types of Mushrooms

111. Continued………

112. Key environmental factors to consider for mushroom cultivation Temperature:- Straw Mushrooms grow well at 38-40 degree Celsius, which is the best temperature for producing spores. Fibers grow well at 35-38 degree Celsius while caps grow at 30 degree Celsius. If it is too hot, mushroom caps will be small and open faster than usual. Light – Even though light is necessary for the growth and assembly of fibers and in order to produce mushroom caps, it is not essential for the mushrooms’ growth. On the contrary, light darkens the mushrooms’ color, unlike growing them in the dark (which whitens them).

113. pH Levels – The pH level is important for the growth of mushrooms. Straw mushrooms are neutral or a little acidic. A suitable pH level for straw mushrooms and other mushrooms is between 5 and 8. Oxygen – In every stage of mushroom growth oxygen is needed, especially when the caps are coming out and after they have bloomed. If there is too much carbon dioxide in the mushroom bed, fibers will grow slower or stop growing, the mushrooms will grow abnormally and their skin will be affected.

114. Continued……..

115. Key steps in mushroom production The key generic steps in mushroom production – a cycle that takes between one to three months from start to finish depending on species – are:  identifying and cleaning a dedicated room or building in which temperature, moisture and sanitary conditions can be controlled to grow mushrooms in choosing a growing medium and storing the raw ingredients in a clean place under cover and protected from rain;

116. pasteurising or sterilizing the medium and bags in which, or tables on which, mushrooms will be grown (to exclude other fungi that would compete for the same space – once the selected fungi has colonized the substrate it can fight off the competition). seeding the beds with spawn (spores from mature mushrooms grown on sterile media); maintaining optimal temperature, moisture, hygiene and other conditions for mycelium growth and fruiting, which is the most challenging step; adding water to the substrate to raise the moisture content since it helps ensure efficient sterilization; harvesting and eating, or processing, packaging and selling the mushrooms; cleaning the facility and beginning again.

117. GANODERMA LUCIDUM THE KING OF HERBS  Ganoderma Lucidum (Red Reishi ) “Bright Shining skin”. Reishi began to be mass produced in the 70’s.

118. 100% CERTIFIED ORGANIC

119. Ganoderma Health Benefits •

120. Submerged Fermentation of Ganoderma lucidum The advantage of submerged fermentation over traditional basidiocarp cultivation is the reduction in the time spent to obtain the product of interest. The production of basidiocarp takes at least 3 to 5 months, while reasonable amounts of ganoderic acid and polysaccharides can be obtained by submerged fermentation after only 2 to 3 weeks.

121. Specific Effects Of Reishi Effect on Tumor Liver Protection & Detoxification Effect on Cardiovascular Effect on Hypertension Treatment of Diabetes

122. Effect on Hepatitis B it was also d i s cove red t ha t ex t r ac t o f g . lucidum could probably augment the rate of toxin transformation and subsequent bile excretion, thereby acting as a liver detoxicant and protectant.

123. Effect on Hypertension Effect on Hypertension G. lucidum is also i n l o w e ri n g h y pe r t en s iv e b l ood This is due t o t he p res enc e o f effective pressure. la n o s ta ne d e ri v ati v e s e s pec i a ll y gan o de r i c acids B, D, F, H, K, S and Y which exert their hypotensive activities.

124. Effect on Wound Healing  Patients with diabetic wounds were healed between 15 to 22 days. This might be due to the glucan from the cell walls of G. lucidum that could activate the fibroblast migration in order to achieve wound healing and tissue proliferation.

125. Effect on Tumor  Poor performance of Immune System is main cause of Tumor. Reishi can best regulate and activate the immune system and increase self defense capability against tumor.  It becomes one of the most effective medicines for anti-tumor, prevent cancer, and supplement to cancer treatment. Reishi possesses hardly any toxic to human body. This unique feature of enhancing immunity without toxigenicity is the definite advantage of Reishi over any other immune system intensifier.

126. Liver Protection & Detoxification R eishi p r ote c t the liver from damaged by various physiological and biological factors. It is also suitable for treating chronic hepatitis, effectively eliminating the related symptoms as dizzy, fatigue, and so on. It can be used to treat chronic toxicosis, the various kinds of chronic hepatitis, and other hepatic diseases.

127. Effect on Cardiovascular Effect on Cardiovascular Clinical studies and experiments with animals confirm that Reishi can effectively dilate coronary artery, increase coronary vessel blood flow, and improve circulation in cardiac muscle capillaries, thus increase the supply of oxygen and energy to cardiac muscle. Therefore Reishi helps to protect the heart from shortage of blood supply, and it is ideal for both curing and preventing heart diseases like nausea. Reishi can reduce the level of blood cholesterol, liporotein and triglycerides in hypertensive patient All these effects contribute to preventing various kinds of stroke.

128. Treatment of Diabetes The constitutes in Ganoderma lucidum that reduce blood glucose are Ganoderma B and C. The principle is by enhancing utilization of blood glucose by body tissues. Ganoderma lucidum serves as a substitute to insulin to inhibit release of fatty acids. It thus improves symptoms in high blood glucose and high urine glucose patients. Blood glucose will be reduced from 173 to 116, cholesterol from 233 to 179, beat-protein from 580 to 465.

129. Biotransformation

130. It is the specific modification of a definite compound to a distinct product with structural similarity, by the use of biological catalysts including microorganisms like fungi or bacteria. The biological catalyst can be described as an enzyme, or a whole, dead microorganism that contains an enzyme or several enzymes produced in it. Biotransformation is also known to comply with the green chemistry strategy today. Green chemistry is a term used for sustainable chemical industrial manufacturing processes towards achieving minimal waste production and energy consumption

131. year process 5000 BC Vinegar production 800 BC Casein hydrolysis with chymosin for cheese production 1670 Orlean process for the industrial bio-oxidation of ethanol to acetic acid 1934 Regioselective biooxidation of sorbite to sorbose for Reichstein Vitamin C synthesis 1950 Bioconversion of steroids 1970 Hydrolysis of penicillin to 6-aminopenicillanic acid 1974 Glucose to fructose isomerisation with immobilized glucose isomerase 1990 Hydrolysis by protease (trypsin) of porcine insuline to human insulin 1995 biotransformation of nicotinonitrile to nicotinamide