1. Microbial Fermentation (Enzymology, Metabolic pathways and Fermentation aspects) OSAMA O. IBRAHIM, PH.D. CONSULTANT BIOTECHNOLOGY GURNEE IL. 60031 BIOINNOVATION04@YAHOO.COM

2. Agenda  Introduction.  Fermentation media.  Industrial microorganisms.  Types of fermentation.  Batch fermentation.  Fed-Batch fermentation.  Growth rate.  Continuous fermentation.  Effect of flow rate on substrate concentration.  Important factors for continuous fermentation.  Classification of fermentation. 2

3. Agenda (Cont.)  Enzymes.  Enzymes equilibrium state.  Factors effects enzymes catalytic activity.  Natural mechanisms for regulating enzyme activity.  Enzymes activators.  Classification of enzymes.  Fermentor systems.  Fermentation process.  Microbial cell breakage systems. 3

4. Introduction  The fermentation industry is composed of five major bio-ingredient categories.  They are: - Proteins & amino acids. - Organic acids. - Antibiotics. - Enzymes. - Vitamins & hormones. 4

5. Introduction (cont.)  Fermentation industry is driven by: - The cost and availability of feed-stocks. - The efficiency of industrial microorganism. - Fermentation condition and optimization. - Down stream process and end-product recovery efficiency. - Fermentation by-product utilization. - Utility consumption and labor cost. 5

6. Fermentation media  Optimum balance of the media is mandatory for cells propagation and for the maximum production of target metabolite (end-product).  Media compositions: - Carbon source. - Nitrogen source. - Minerals. - Growth factors. - Precursors (mutants). 6

7. Industrial microorganisms  Microbial screening. - Wild strains.  Microbial yield improvement - Mutation. - Recombinant DNA. - Genetically engineered.  Microbial selection.  Industrial microorganism 7 93 C 43 C 21 C 4 C

8. Types of fermentation  Solid State fermentation (SSF).  Liquid State fermentation (LSF) Surface culture & submerged culture 8

9. Solid State Fermentation (SSF)  SSF process can be defined as microbial growth on particles without presence of free water.  Particles are a solid culture substrate such as rice or wheat bran saturated with water and inoculated with (mold, yeast, bacteria) in controlled room temperature.  It is 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). 9

10. SSF process and applications Applications :  Potentially many high value products such as extra-cellular enzymes, primary metabolites, and antibiotics could be produced in SSF.  It is estimated that nearly a third of industrial enzyme produced in Japan is made by SSF process.  Production of organic and ethanol from starchy substrates.  Digestibility of fibers and lignocelluloses materials for both human and animal consumption. 10

11. Liquid State fermentation (LSF) [Submerged culture] - Submerged culture is performed in tanks which can reach in size for over 100,000 gallons. - It is ideal for the growing unicellular organisms such as bacteria and yeast. LSF methods: - Batch fermentation. - Fed-batch fermentation. - Continuous fermentation. - Semi-continuous fermentation. 11

12.  Considered to be a closed system.  The sterilized media in the fermenter is inoculated with the microorganism.  Incubation is allowed under the optimum conditions (aeration, agitation, temperature).  During entire fermentation nothing is added except air, antifoam and acid/base. 12 Batch fermentation

13. Fed-Batch fermentation  It is enhancement of batch fermentation.  Continue adding the nutrients (feeding) in a small doses during the fermentation.  The method in controlling nutrients feeding process is by measuring methods.  The main advantage of fed-batch fermentation is the elimination of catabolite repression (feed-back inhibition). 13

14. Microbial growth rate 14 Primary metabolites Secondary metabolites

15. Batch fermenter system 15

16. Continuous fermentation  It is an open system.  Continuously sterile nutrient is added and the converted nutrient is taken out from the fermentor.  In continuous process cell loss as a result of outflow must be balanced by growth of the microorganism. 16

17. Effect of flow rate on substrate concentration 17 The relationship between biomass (X), the concentration of limiting nutrients (C),and the dilution rate (D) are important factors in continuous

18. Continuous fermenter system 18

19. Important factors for continuous fermentation  The system must be stable for at least 500 hours.  Maintaining sterile conditions for all period of fermentation time.  The composition of nutrients must be constant all the time.  Maintaining the strain stability for constant high production yield (concerning about reverse mutation). 19

20. Semi-continuous fermentation  Semi-continuous fermentations, in which a fraction of a fermentation is replaced with fresh media at regular intervals. 20

21. Classification of fermentation  Classification is according to product formation: - TYPE I: Substrate P product Substrate A B C D P product - Type II: Substrate A B C D P-Primary Metabolites E F G P-Secondary Metabolites 21 A E 1 E2E2 E3E3 E4E4 ExEx E1E1 E2E2 E3E3 E4E4 Es 1 Es 2 Es 3 Es 4

22. Enzymes  Enzymes are active biological molecules responsible for thousands of metabolic process that sustain life.  Most enzymes are proteins, although some catalytic enzymes are RNA molecules.  In enzyme reactions, the molecules at the beginning of the process, called substrates and converted into different molecules that called end-products.  Enzymes are very specific as which reaction they catalyze and the substrate that involved in the reaction.  Depends on enzyme activity, the bioconversion to end- products can be faster and reached the equilibrium state rapidly. 22

23. Enzymes equilibrium state 23 E + S ES E + P Substrate binding E= enzyme S= Substrate Catalytic step E=enzyme P= product E + S E + P

24. Enzyme/substrate interaction 24

25. Factors Effects Enzymes Catalytic Activity  Temperature: The optimum is generally 40-60 0 C. Some enzymes exhibit an optimum at almost 100 0 C.  Value of PH: The optimum generally in the range from 5-7. Extreme values of 1.5-10.5 have been found.  Activation: Many chemical activates the catalytic enzymes activity, Such as inorganic ions.  Inhibitors: Many chemical inhibits the catalytic enzymes activity  There are two types of enzymes inhibition: Irreversible inhibitors (competitive inhibition) and reversible inhibitors (uncompetitive inhibition).  Substrate inhibition: High concentration of substrate may inhibit the catalytic activity of an enzyme.  End-product inhibition : In the case of multi enzyme system (catalytic inhibition). 25

26. Factors effecting enzymes activity 26

27. Activators (Cofactors and Coenzymes)  Some enzymes do not need any additional components to show full activity.  Cofactors can be either inorganic (metals) or organic compounds (flavin and heme).  Coenzymes include NAD +, NADP + and ATP.  These coenzymes transfer chemical group between enzymes.  The chemical groups carried by the hydride ion (H + ) carried by NADH or NADPH. NAD + + 2H + + 2e - NADH / NADP + + 2O - 2 + 2H + NADPH + 2O 2  Or phosphate groups carried by ATP. ATP +H 2 o ADP+ P 1 (-7.3kcal/ mole ) / ATP +H 2 o AMP + PP 1 (-14kcal/mole) 27

28. Enzymes inhibitors and activators mechanism 28

29. Feedback inhibition and precursor activation 29

30. Natural mechanisms for regulating enzyme activity 30

31. Classification of enzymes  The International of Biochemistry and Molecular Biology developed a nomenclature for enzymes (EC number).  Each enzyme is classified by sequence of four numbers preceded by EC. (E.C. 5.3.1.18 Glucose isomerase)  The top-level classification is: - EC1 Oxidoreductases (catalyze oxidation/reduction reactions). - EC2 Tranferases (transferee a functional group). - EC3 Hydrolases (catalyze the hydrolysis of various bonds). - EC4 Lyases (cleave bonds by mean of hydrolysis /oxidation). - EC5 Isomerases (isomerization within same molecule). - EC6 Ligase ( join two molecules with covalent bonds). 31

32. Enzymes production Inoculums Flask Seed tank Fermentor Enzyme recovery 32 Constitutive enzymes: The microorganism produce the enzyme in minimal fermentation media. Inducible enzymes: The microorganism require adding inducible agents in the media to produce the target enzyme. Extracellular enzymes: The microorganism secrete the enzyme in the fermentation media. Intracellular enzymes: The microorganism produce the enzyme inside the cell.

33. Mi crobial cell breakage systems 33

34. Enzymes (Conclusion)  Enzymes are usually sold based on the activity (u/ml or u/gm).  If the efficiency of enzymes are considered, their cost, is based on active enzyme protein u/mg protein (specific activity).  The commercial exploitation of enzymes range from high-volume but low cost (industrial enzymes) to low volume, but high cost (enzymes for medical, scientific and analytical use).  Workers handling industrial enzymes should use protective clothing and eye protection.  Food enzymes if foods processing have bees shows to be safe through many years of manufacturing practice. 34

35. Thank You for your attention 35