1. Effects of Environmental Conditions on Growth Kinetics

2. Effects of environmental conditions on growth kinetics How environment conditions affect growth kinetics? Environmental conditions: temperature, pH and dissolved-oxygen concentration Temperature affects the performance of cells. Organisms can be classified in 3 groups: 1)Psychrophiles (T opt < 20°C) 2)Mesophiles (T opt = 20 to 50 °C) 3)Thermophiles (T opt > 50°C) As the temperature is increased toward optimal growth temperature, the growth rate approximately doubles for every 10 °C increase in temperature.

3. Above the optimal temperature range, the growth rate decreases and thermal death may occur. The net specific replication rate can be expressed by Eq. 9.19 (for temperature above optimal level): At high temperatures, the thermal death rate exceeds the growth rate, which causes a net decreases of viable cells. Both u R and k d vary with temperature according to the Arrhenius equation: Where Ea and Ed are activation energies for growth and thermal death. Ea (10-20 kcal/mol) Ed (60-80 kcal/mol) Thermal death is more sensitive to temperature changes than the microbial growth. Microbial growth

4. Product formation Temperature also affects product formation. Temperature optimum for product formation and growth is different. The yield coefficient (Yx/s) is also affected by temperature. Example: a single-cell protein production. When temperature above the optimum temperature, the maintenance requirements of cells increase, resulting a decrease in the yield coefficient. Temperature also affects product formation. Temperature optimum for product formation and growth is different. The yield coefficient (Yx/s) is also affected by temperature. Example: a single-cell protein production. When temperature above the optimum temperature, the maintenance requirements of cells increase, resulting a decrease in the yield coefficient. Rate-limiting step in fermentation At high temperatures, the rate of bioreaction might become higher than the diffusion rate, and the diffusion then would be the rate- limiting step. Example:An immobilized cell system. The activation energy of molecular diffusion =6 kcal/mol. The activation energy of most bioreaction is 10 kcal/mol. So diffusion limitation must be carefully considered in high temperatures At high temperatures, the rate of bioreaction might become higher than the diffusion rate, and the diffusion then would be the rate- limiting step. Example:An immobilized cell system. The activation energy of molecular diffusion =6 kcal/mol. The activation energy of most bioreaction is 10 kcal/mol. So diffusion limitation must be carefully considered in high temperatures

5. Hydrogen-ion concentration (pH) Affect the activity of enzymes and therefore the microbial growth rate. The optimal pH for growth may be different from that for product formation. Different organisms have different pH optima: 1.Bacteria (pH 3 to 8) 2.Yeast (pH 3 to 6) 3.Molds (pH 3 to 7) 4.Plant cells (pH 5 to 6) 5.Animal cells (pH 6.5 to 7.5)  Many organism have mechanism to maintain intracellular pH at relatively constant level in the presence of fluctuations in environmental pH  When pH differs from the optimal value, the maintenance-energy requirement increase  One consequence of different pH optima is that the pH of the medium can be used to select one organism over another.  Many organism have mechanism to maintain intracellular pH at relatively constant level in the presence of fluctuations in environmental pH  When pH differs from the optimal value, the maintenance-energy requirement increase  One consequence of different pH optima is that the pH of the medium can be used to select one organism over another.

6. AMMONIA (NH3)  If ammonium is the sole nitrogen source, hidrogen ions are released into the medium as a result of the microbial utilization of ammonia  Resulting in a decrease in pH NITRATE  If nitrate is the sole nitrogen source, hydrogen ions are removed from the medium to reduce nitrate to ammonia  Resulting in an increase in pH ORGANIC ACID PRODUCTION / BASES pH can change because of organic acid or bases production In most fermentation, pH can change or vary substantially by: SUPPLY OF CO2 Seawater or animal cell Variation of specific growth rate with pH is depicted by Figure 6.8

8. Dissolved Oxygen  Important substrate in aerobic fermentations and may be a limiting substrate  At high cell concentrations, the rate of oxygen consumption may exceed the rate of oxygen supply, leading to oxygen limitation  When oxygen is the rate-limiting factor, specific growth rate varies with dissolved-oxygen concentration; below a critical concentration, growth or respiration approaches a first-order rate dependence on the dissolved-oxygen concentration. Above a critical oxygen concentration, the growth rate becomes independent of the dissolved-oxygen concentration. Figure 6.9 depicts the variation of specific growth rate with dissolved oxygen concentration.

10. O2 is a growth-rate limiting factor when the DO level is below the critical DO concentration. In this case, another medium component (glucose, ammonium) becomes growth- extent limiting. EXAMPLE: Azotobacter vinelandii at a DO = 0.05 mg/l, the growth rate is about 50% of maximum, even if a large amount of glucose is present. However, the maximum amount of cells formed is not determined by the DO, as the O2 is continually resupplied. If glucose were totally consumed, growth would cease even if DO = 0.05mg/l. Thus the extent of growth (mass of cells formed) would depend on glucose, while the growth rate for most of the culture period would depend on the value of DO. O2 is a growth-rate limiting factor when the DO level is below the critical DO concentration. In this case, another medium component (glucose, ammonium) becomes growth- extent limiting. EXAMPLE: Azotobacter vinelandii at a DO = 0.05 mg/l, the growth rate is about 50% of maximum, even if a large amount of glucose is present. However, the maximum amount of cells formed is not determined by the DO, as the O2 is continually resupplied. If glucose were totally consumed, growth would cease even if DO = 0.05mg/l. Thus the extent of growth (mass of cells formed) would depend on glucose, while the growth rate for most of the culture period would depend on the value of DO.

11. C crit = 5 to 10% of the saturated DO concentration (bacteria and yeast) C crit = 10 to 50% of the saturated DO concentration (mold cultures) Saturated DO concentration in water at 25°C and 1 atm pressure = 7ppm Therefore the presence of dissolved salts and organics can alter the saturation value, while increasingly high temperatures decrease the saturation value Saturated DO concentration in water at 25°C and 1 atm pressure = 7ppm Therefore the presence of dissolved salts and organics can alter the saturation value, while increasingly high temperatures decrease the saturation value

12. O 2 transfer from gas bubbles to cells is usually limited by oxygen transfer through the liquid film surrounding the gas bubbles. The rate of oxygen transfer from the gas to liquid phase is given by: k L = oxygen transfer coefficient (cm/h) a = the gas-liquid interfacial area (cm 2 /cm 3 ) k L a = the volumetric oxygen transfer coefficient (h -1 ) C* = saturated DO concentration (mg/L) C L = the actual DO concentration in broth (mg/L) N O2 = rate of oxygen transfer (mg O 2 /l.h) OTR = Oxygen Transfer Rate Oxygen Transfer Rate (OTR)

13. Oxygen Uptake Rate (OUR) q O2 = specific rate of oxygen consumption (mg O2/g dw cells-h) Yx/o = yield coefficient on oxygen (g dw cells / g O2) X = cell concentration (g dw cells / l) q O2 = specific rate of oxygen consumption (mg O2/g dw cells-h) Yx/o = yield coefficient on oxygen (g dw cells / g O2) X = cell concentration (g dw cells / l) When oxygen is the rate –limiting step, the rate of O2 consumption is equal to the rate of oxygen transfer. or

14. Redox potential Parameter that affects the rate and extent of many oxidative-reductive reactions In a fermentation medium, the redox potential is a complex function of DO, pH, and other ion concentrations (reducing and oxidizing agents). The electrochemical potential of fermentation medium can be express by the following equation:  To reduce the redox potential of a fermentation media – (N 2, cystein HCL, Na 2 S  To increase the redox potential – O 2, oxidizing agents

15. Dissolved Carbon Dioxide (DCO 2 ) DCO2 concentration in medium fermentation may have effect on performance of organism. Very high DCO2 concentration may be toxic to some cells Cells require a certain DCO2 level for proper metabolic functions. DCO2 in medium can be controlled by: 1.Changing the DCO2 content of the air supply 2.Agitation speed DCO2 concentration in medium fermentation may have effect on performance of organism. Very high DCO2 concentration may be toxic to some cells Cells require a certain DCO2 level for proper metabolic functions. DCO2 in medium can be controlled by: 1.Changing the DCO2 content of the air supply 2.Agitation speed

16. Ionic Strength of Fermentation Media Affects 1.The transport of certain nutrients in and out of cells 2.The metabolic functions of cells 3.The solubility of certain nutrients (DO) The ionic strength is given by the following equation: C = concentration of an ion Zi = charge of ion I = ionic strength of the medium

17. High substrate concentrations that are significantly above stoichiometric requirements are inhibitory to cellular functions and depending on the type of cells and substrate. SubstrateConcentration (g/L) 1. Inhibitory concentration Glucose (yeast fermentation) 200 2.NaCl40 3.Refractory compunds (phenol, toluene, methanol) 1 4. Noninhibitory concentration: Nutrients: Ammonium Phosphate Nitrate 5 10 5

18. Heat generation by microbial growth About 40-50% of the energy stored in a carbon and energy source is converted to biological energy (ATP) during aerobic metabolism and the rest of energy is released as heat. For actively growing cells, the maintanance requirement is low and heat evolution is directly related to growth. The heat generated during microbial growth can be calculated using the heat of combustion of the substrate and of cellular material.

19. A schematic of an entalphy balance for microbial utilization of substrate The heat of combustion of the substrate is equal to the sum of the metabolic heat and the heat of combustion of the cellular material Hs = Heat of combustion of substrate Hc = Heat of combustion of cells 1/Yc = Metabolic heat evolved per gram of cells

20. Traditional Batch Fermentation Yogurt Soy Sauce @ Kicap Traditional Fermented Food @ Tapai Tempeh

21. Introduction : Soy Sauce @ Kicap Soya sauce @ kicap Salty soy sauce @ kicap asin Sweetened soy sauce @ kicap manis Fermentation Soya beans “a liquid food condiment which is used to add flavour and colour to the Oriental diet (Yong and Wood, 1974) History  invented in China as condiment for ~2500 years  widely used in East & Southeast Asia & in some Western dishes  2 methods : traditional brewing method (fermentation) & non – brewed method (chemical-hydrolyzation)

22. FLOW CHART FOR SOY SAUCE PRODUCTION Soy Beans Pretreatment of soy beans (cleaning, soaking, cooking and draining) Fungus culture + wheat flour Mix Incubated in koji room (48 hours) Koji Brine solution Incubate Moromi (2 months)

23. ExtractionResidue Filtrate Additives Packaging Pasteurization Soy sauce @ Kicap Chinese soy sauce Indonesian soy sauce Japanese soy sauce @ Shoyu

25. Yogurt Yogurt (yoghurt) is a healthy source of protein, calcium, magnesium, and other essential vitamins, whose active bacterial cultures aid in digestion. Make Your Own… It tastes better It's better for you : no preservatives no sugar no chemicals added It's less expensive There's no packaging waste

26. Make your own yogurt… Add milk Heat 80°C (10-20 min) Cool rapidly to about 45°C Pitch your yogurt Incubate ~ 4 - 18 hours (custardlike) Stir well Chill overnight Stir & ENJOY !!! Stir, cover, warm

27. Traditional Fermented Food @ Tapai Origin  Found throughout much of East- and Southeast Asia  It is a sweet or sour alcoholic paste  Tapai can be made from a variety of carbohydrate sources  Tapai is also used to make alcoholic beverages

30. Tempeh Origin  Natural culturing and controlled fermentation process  It originated from Indonesia, invented by the Javanese  Made from soybeans  Tempeh fermentation process  higher content of protein, dietary fiber and vitamins  Tempeh is used worldwide in vegetarian cuisine

31. Source: Tempeh Wizard’s Guild

32. Summary Batch Culture a large-scale closed system culture in which cells are grown in a fixed volume of nutrient culture medium under specific environmental conditions (e.g. nutrient type, temperature, pressure, aeration, etc.) up to a certain density in a tank or fermentor, harvested and processed