Media for industrial fermentation

Media for industrial fermentation process, substrates used as carbon, nitrogen sources, growth factors, inducers

1. R. NITHYA, M. Sc., M. Phil ., (Ph. D) ASSISTANT PROFESSOR DEPARTMENT OF BIOTECHNOLOGY SRI ADI CHUNCHNAGIRI WOMENS COLLEGE, CUMBUM THENI DT, TAMIL NADU, INDIA MEDIA FOR INDUSTRIAL FERMENTATION

2. MEDIA FORMULATION Micro-organisms used for fermentation process grow on or in growth medium which satisfies the nutritional needs of microbes. Complete analysis is needed to be done to establish the most favourable medium for the growth of the microbe used for fermentation. Formulating medium at lab scale can be done by adding main ingredients like water, carbon source, nitrogen source, minerals and other supplements in pure form and in required quantities which supports the growth of the microbe whereas, the same may not support the satisfactory growth of the same organism at industrial level.

3. Following criteria need to be satisfied for the material to be treated as medium at industrial level. It should give maximum yield of product . It should give minimum yield of undesired product. It should be consistently available throughout the year. It should be cheap .

4. MEDIA FOR INDUSTRIALL FERMENTATION The media used for the growth of microorganisms in industrial fermentation must contain all the elements in a suitable form for the synthesis of cellular substances as well as the metabolic products. In the laboratory, pure defined chemicals may be used for culturing microorganisms. For industrial fermentations, undefined and complex substrates are frequently used for economic reasons. Cheaper substrates are advantageous since they minimize the production cost of the fermented products. Wastes from agriculture, and by products of other industries are generally preferred.

5. TYPES OF MEDIA The media used in fermentation processes may be Synthetic or Crude Synthetic media Media with all the requisite constituents in a pure form in the desired proportion represents synthetic media Crude media The non-synthetic media with naturally available source are better suited for fermentation. Crude media with an addition of requisite synthetic constituents is ideal for good product yield in fermentation.

6. The most frequently used substrates for industrial fermentation are Carbon source Nitrogen source Vitamins and Growth factors Minerals Water Chelators Inducers and Elicitors Inhibitors Antifoams

7. Medium component Defined Component Un-defined component Carbon source Glucose, Fructose, Glycerol, Xylose , Sucrose, Starch Molasses, Meat extract, Peptone, Plant extracts and Materials (Cellulosic, lignoncellulosic and hemicellulosic materials, Starch complex, etc…) Nitrogen source Ammonium and Nitrate Salts Yeast extract, Amino acid complex, Casein Phosphate Mono and di -phosphate salts In traces of complex C- and N sources CULTIVATION MEDIA

8. Medium component Defined Component Un-defined component Sulphur Ammonium and Magnesium sulphate In traces of complex C- and N sources Magnesium Mainly Magnesium sulphate In In traces of complex C- and N sources Mn , Mo, Fe, Zn, etc.. In form of Inorganic salts In traces of complex C- and N sources Vitamins and Growth factors Added in pure form of vitamin and growth factors preparation Yeast extract, and may found also as traces in some C- and N-sources CULTIVATION MEDIA

9. SUBSTRATES USED AS CARBON SOURCES Carbohydrates – most predominant source of energy in fermentation industry Refined & pure Carbohydrates – glucose or sucrose are rarely used for economic reasons. Substrates such as Molasses Malt extract Starch, dextrin and cellulose Whey Methanol and Ethanol used as carbon sourc e

10. MOLASSES - By product of sugar industry - cheapest sources of carbohydrates - Sugarcane molasses (sucrose 48%) - Sugar beet molasses (sucrose 33%) - Molasses also contains nitrogenous substances Vitamins Trace elements - Hydrol molasses – byproduct of glucose production from corn used as a fermentation substrate.

11. MALT EXTRACT Malt extract - an aqueous extract of malted barely contains 80% Carbohydrates (glucose, fructose, sucrose, maltose). 4.5% Nitrogen compounds (proteins, peptides, amino acids, purines , pyrimidines )

12. STARCH, DEXTRIN AND CELLULOSE - The polysaccharides – starch, dextrin and cellulose can be metabolized by microorganisms. - Frequently used for industrial production of alcohol. - low cost - Widely available

13. WHEY - Byproduct of dairy industry - Good source of carbon. - Used for the production of Alcohol Single cell protein Vitamin B12 Lactic acid and Gibberellic acid

14. METHANOL AND ETHANOL Some microbes utilize methanol or ethanol as carbon source . Methanol - cheapest source – utilized by only a few bacteria and yeasts. Used for the production of single cell protein Ethanol - Expensive Used for Acetic acid production

15. SUBSTRATES USED AS NITROGEN SOURCES Inorganic nitrogen sources Ammonium salts Free ammonia Organic nitrogen sources Urea Corn steep liquor Yeast extracts Soy meal Peptones

16. INORGANIC NITROGEN SOURCES Ammonium salts or free ammonia – cheap inorganic nitrogen sources All the microorganisms are not capable of utilizing them. Hence their use is limited .

17. ORGANIC NITROGEN SOURCES UREA Good source of nitrogen Cheapest source of organic forms of nitrogen

18. CORN STEEP LIQUOR - This is formed during starch Production from corn. - Rich in nitrogen(4%) - It is rich in several amino acids Alanine , valine , methionine , Arginine , threonine, glutamate .

19. YEAST EXTRACTS It contains 8% nitrogen and are rich in amino acids, peptides and vitamins. It is produced from baker’s Yeast through autolysis (at 50-55 C) or Through plasmolysis (high concentration of NaCl ). Very good sources for many industrially important microorganisms

20. SOY MEAL After extracting the soy bean oil from the soy bean seeds, the left out residue is soy meal. It is rich in proteins(50%) Carbohydrates (30%) It is used in antibiotic production

21. PEPTONES - Protein hydrolysates - Source of peptones include meat, soy meal, peanut seeds cotton seeds and sunflower seeds - Proteins include casein, gelatin and keratin can be hydrolyzed to yield peptones. - Peptone derived from animal sources - more nitrogen content than plant sources - Expensive, not widely used in industries

22. SOURCES OF GROWTH FACTORS Some of the microorganisms are not capable of synthesizing one or more growth factors such as vitamins. Vitamins are expensive in pure form, hence crude sources are preferred. Yeast extract is a rich source of almost all growth factors.

23. MINERALS All microorganisms require certain mineral elements for growth and metabolism. Major elements - magnesium, phosphorous, potassium, sulphur, calcium and chlorine Minor elements - cobalt, copper, iron, manganese, molybdenum and zinc are added. E.g. Corn steep liquor contains a wide range of minerals that will satisfy the minor and trace mineral needs.

24. WATER • All fermentation processes, except SSF, require vast quantities of water. • Important for ancillary services like heating, cooling, cleaning and rinsing . • Important factors to consider when assessing suitability of a water supply are: pH, dissolved salts and effluent contamination. • Water is hard , it is treated to remove salts such as calcium carbonate, iron and chlorine. • For plant and animal cell culture, the water must be highly purified. Reuse of water is important - It reduces water cost by 50%

25. CHELATORS Metal binders – It is a chemical compound that react with metal ions to form a stable water soluble complex. Metals are distributed in chelated form Some chelating agents are added to form complexes with metal ions which are gradually utilised by microorganisms. Examples : EDTA, Citric acid, phosphates . Phosphates are widely used as a chelating agent . Used as a required quantity otherwise it will inhibit the growth of microorganism.

26. INDUCERS AND ELICITORS If product formation is dependent upon the presence of a specific inducer compound or a structural analogue, it must be incorporated into the culture medium or added at a specific point during the fermentation. The majority of enzymes of industrial interest are inducible. Inducers are often substrates such as starches or dextrin's for amylase. In plant cell culture the production of secondary metabolites, such as flavanoids and terpenoids can be triggered by adding elicitors .

27. INDUCERS

28. OXYGEN • Supplied in the form of air containing about 21% (v/v) oxygen • Oxygen requirements may vary widely depending upon the carbon source. • The specific oxygen uptake rate of a microorganism increases with increase in the dissolved oxygen concentration up to a certain point referred to as the critical level. • Maximum biomass production is achieved by satisfying the organism’s maximum specific oxygen demand by maintaining the dissolved oxygen concentration greater than the critical level.

29. INHIBITORS Inhibitors are used to redirect metabolism towards the target product and reduce formation of other metabolic intermediates. Inducers are added at a certain point to prevent further metabolism of the target product. An example of an inhibitor specifically employed to redirect metabolism is sodium bisulphite

30. INHIBITORS

31. ANTIFOAMS • Antifoams are necessary to reduce foam formation during fermentation. • Foaming is largely due to media proteins that become attached to the air-broth interface where they denature to form a stable foam “skin” that is not easily disrupted. • If uncontrolled the foam may block air filters , resulting in the loss of aseptic conditions; the fermenter becomes contaminated and Microorganisms are released into the environment

32. ANTIFOAMS - Alcohols; stearyl and octyl decanol - Esters - Fatty acids and derivatives ( glycerides ) linseed oil, cotton seed oil, olive oil, castor oil, sunflower oil, rapeseed oil and cod liver oil. - Silicones - Sulphonates - Alkaterge C, oxazaline , poly-propylene glycol.

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