Upstream and down stream process

Fermentation process

1. UPSTREAM AND DOWNSTREAM PROCESS M.Phil Microbiology - NEETHU ASOKAN

2. FERMENTOR Closed vessels Used for fermentation Production of large scale process Fermenter is a closed vessels now it is called as bioreactor Fermenter is a old process used for cultivate the microorganisms Fermenter used for cultivate Prokaryotic cells (fungi bacteria) Bioreactors used for cultivate eukaryotic cells (mammalian ,,insects) INTRODUCTION M.Phil Microbiology - NEETHU ASOKAN

3. convert Occur by yeast and bacteria M.Phil Microbiology - NEETHU ASOKAN

4. HISTORICAL PERSPECTIVE It is known and practiced by humankind prehistoric times since. The production of beer, bread, wine etc., are established in ancient Egypt. In 1857 Pasteur developed the fermentation process. 1897 Eduard Buchner discovered that sucrose could be fermented to alcohol by yeast. In 1984 first constructed bioreactor by Stanberry and Whitaker M.Phil Microbiology - NEETHU ASOKAN

5. STRUCTURE OF FERMENTER M.Phil Microbiology - NEETHU ASOKAN

6. Components' of Fermenter Parts Working Water jacket Circulating the cooling water and remove the heat and generate the metabolic activity Steam inlet Sterilizes the fermenter between fermentation Heating plate Raise the temperature Nutrient inlet Where nutrients enter the fermenter Temperature and ph probe Maintain pH and temperature in neutral level Stirring paddle Stirs the reaction microorganisms are mixed with the nutrients, which stops them from sinking to the bottom and keeps the temperature well Harvesting outlet Where the products' are release Steam outlet Where steam comes out let Stainless steel Some microorganisms produce acid as waste which might corrode other metals Electric motor Connected centre shaft to rotate M.Phil Microbiology - NEETHU ASOKAN

7. TYPES OF FERMENTOR Continuous stirred tank bioreactor Bubble Colum bioreactor Air lift flurized bioreactor Internal loop airlift bioreactor External loop airlift bioreactor Tower bioreactor Photo bioreactor Cyclone Colum bioreactor Holl fibre bioreactor Cylinder conical bioreactor etc., M.Phil Microbiology - NEETHU ASOKAN

8. M.Phil Microbiology - NEETHU ASOKAN

9. The pre-fermentation stage Isolation Improvement Producing of microorganisms Screening method; isolate microbes to produce dicered products Two methods; primary screening checking the quality of microbes done in agar plate. Secondary screening checking the quntative of microbes done in liquid media UPSTREAM PROCESS M.Phil Microbiology - NEETHU ASOKAN

10. Microbes isolated from natural sources thus is improved to get productive strains by using Recombination Mutations Cell fusion Gene cloning Media formulation : growth medium must have essential nutrients for microbial growth for successful fermentation process Two kind media : Inoculum media : enrich the culture Production media : contain carbon and nitrogen Raw materials : corn molasses, cellulose, corn, streep liquor soybean, sugar, beet molasses, malt extract etc., M.Phil Microbiology - NEETHU ASOKAN

11. Upstream processing includes formulation of the fermentation medium, sterilization of air, fermentation medium and the Fermenter, inoculum preparation and inoculation of the medium. The fermentation medium should contain an energy source, a carbon source, a nitrogen source and micronutrients required for the growth of the microorganism along with water and oxygen, if necessary. A medium which is used for a large scale fermentation, in order to ensure the sustainability of the operation, should have the following characteristics; 1. It should be cheap and easily available 2. It should maximize the growth of the microorganism, productivity and the rate of formation of the desired product 3. It should minimize the formation of undesired products M.Phil Microbiology - NEETHU ASOKAN

12. Usually, waste products from other industrial processes, such as molasses, lignocelluloses wastes, cheese whey and corn steep liquor, after modifying with the incorporation of additional nutrients, are used as the substrate for many industrial fermentations. Sterilisation is essential for preventing the contamination with any undesired microorganisms. Air is sterilised by memb rane filtration while the medium is usually heat sterilised . Any nutrient component which is heat labile is filter-sterilised and later added to the sterilised medium. The fermenter may be sterilised together with the medium or separately.   M.Phil Microbiology - NEETHU ASOKAN

13. Inoculum build up is the preparation of the seed culture in amounts sufficient to be used in the large Fermenter vessel. This involves growing the microorganisms obtained from the pure stock culture in several consecutive Fermenter. This process cuts down the time required for the growth of microorganisms in the Fermenter, thereby increasing the rate of productivity. Then the seed culture obtained through this process is used to inoculate the fermentation medium. M.Phil Microbiology - NEETHU ASOKAN

14. Inoculum preparation procedure Test tube Pure culture under good condition Test tube Contain seed culture In Suitable conditions Conical flask Containing culture for large scale fermentation Fermenter contain medium and culture for mass culture contain suitable conditions Fermenter M.Phil Microbiology - NEETHU ASOKAN

15. The degradation of carbon compounds by cells or organisms under anaerobic conditions. Carbon compounds degraded Microorganisms Complex into simple one by FEREMENTATION PROCESS M.Phil Microbiology - NEETHU ASOKAN

16. Process Three cultural fermentation system used in bioreactors Batch culture fermentation Continuous culture fermentation Feed batch culture fermentation Batch culture fermentation It is closed system filled fresh medium and inoculated It is refilled for the next process of fermentation M.Phil Microbiology - NEETHU ASOKAN

17. Continuous reaction Fresh medium added continuously Shut down less frequently then batch system. M.Phil Microbiology - NEETHU ASOKAN

18. Feed batch Feed continuously added until maximum liquid fermenter volume reached. Then fermenter may be allowed to continue or completed partially or completely depending on the process M.Phil Microbiology - NEETHU ASOKAN

19. Define different modes of fermentation and known their limitation. Develop a suitable medium and perform a material balance. Deter-mind fermentation productivity and yields. Fermentation take place in absence of oxygen. The science of fermentation is know as zymology FERMENTATION PRINCIPLE M.Phil Microbiology - NEETHU ASOKAN

20. when fermentation is over, the desired product is recovered from the growth medium. Then the product is Extraction Purification and Packed of a biotechnological product from fermentation is refered to as DSP or product recovery or downstream processing. The end products include Antibiotics, Amino acid, Vitamins, Organic acid, Industrial enzyme, vaccines etc. It is complex and important as fermentation process. DOWNSTREAM PROCESS M.Phil Microbiology - NEETHU ASOKAN

21. DOWNSTREAM PROCESS M.Phil Microbiology - NEETHU ASOKAN

22. DESIRED PRODUCT IN CULTURE BROTH INTRACELLULAR PRODUC EXTRACELLULAR PRODUCT OR DISINTEGRATION PHYSICAL, CHEMICAL ENZYMATIC METHODS BROTH WITH SOLIDS AND LIQUID SOLID-LIQUID SEPARATION FLOTATION, FLOCCULATION, FILTRATION , CENTRIFUGATION M.Phil Microbiology - NEETHU ASOKAN

23. CONCENTRATION EVAPORATION, DIALYSIS, EXTRACTION, MEMBRANE FILTRATION, PRECIPITATION, ADSORPTION, DISTILLATION PURIFICATION ADSORPTION CHROMATOGRAPHY, ION- EXHANGE CHROMATOGRAPHY, GEL CHROMATOGRAPHY, etc. FORMULATION CHAMBER DRYER, TUMBLER DRYER DRUM DRYER, BELT DRER, THIN-LAYER DRYER M.Phil Microbiology - NEETHU ASOKAN

24. CELL -DISRUPTION OR DISINTEGRATION Mechanical methods: Shear forces in solid matter and solution. Non mechanical methods: Lysis Physical – freezing and thawing : now high osmotic pressure, shock Chemical – surface active agents, solvents, antibiotics etc., Enzymatic – lysozyme Drying Freeze-drying, Air drying, Pressure release, Drying with solvents M.Phil Microbiology - NEETHU ASOKAN

25. Primary operation Separation of whole cells Removal of cell debris Collection of protein precipitate Collection of inclusion bodies etc., SOLID-LIQUID SEPARATION OR CLARIFICATION M.Phil Microbiology - NEETHU ASOKAN

26. Solid-liquid separation (clarification) Coagulation Colloids into small flocs using simple electrolytes Flocculation Agglomeration of these small flocs into larger settle-able particles using polyelectrolytes. Flotation Enrichment of microorganisms Filtration The separation of suspended particles from liquid Centrifugation Gravitational force used for separate the particles M.Phil Microbiology - NEETHU ASOKAN

27. CONCENTRATION METHODS The purity or concentration of metabolite Evaporation –steam as heat source Extraction – the cell mass and more or less clear solution obtained Adsorption – special polymer resins (chemical) used for the isolation of hydrophilic metabolites that cannot be extract with organic solvents. Filtration – separation of biomolecules and particles [pore size] Precipitation – removal of product from the solvent Dialysis – semi permeable membrane M.Phil Microbiology - NEETHU ASOKAN

28. PURIFICATION Purify relatively low concentration of metabolic products The chromatography technique are used Purification is a main process in fermentation Desired product purification is important Many techniques are used for the purification M.Phil Microbiology - NEETHU ASOKAN

29. FORMULATION Products is depended on the maintenance of its activity and stability during distribution and storage. Concentrated solutions after removing most of the water. Drying : Withdrawal of water from the products Crystallization : Established method used for concentration of the bio-products. Widely used for final purification M.Phil Microbiology - NEETHU ASOKAN

30. MONITORING OF DSP T o keep control over the presence and concentration of target molecule Monitored by using sensor Mostly used in pharmaceuticals Monitor the UV-absorbance, conductivity, pH, molecular size, protein, bio-specific binding reactions etc., M.Phil Microbiology - NEETHU ASOKAN

31. MARKETING The formulated product is packed and sent to the market for the consumers. The purified product is modified before marketing. Product may be in aqueous form or in solid form. The nature of the product have 98-100% in pure for seal in market. The purity of the final product depends upon the successful operation of all these downstream processes. M.Phil Microbiology - NEETHU ASOKAN

32. Reference Text book of fermentation technology by H.A. Modi Text book of biotechnology by R.C Duby Text book of biotechnology by Dr. Prakash S Lohar Net references M.Phil Microbiology - NEETHU ASOKAN