MIC 303 INDUSTRIAL AND ENVIRONMENTAL MICROBIOLOGY

1. MIC 303INDUSTRIAL AND ENVIRONMENTAL MICROBIOLOGY MICROBIAL GROWTH KINETICS Mdm Aslizah Mohd Aris

2. TYPE OF MICROBIAL GROWTH SYSTEM • 3 major type: • Batch culture • Continuous or Chemostat Culture • Fed-batch Culture

3. BATCH CULTURE

4. BATCH CULTURE • Definition: A closed system with limited amount of nutrient (no additional of medium added) • Cells grows through several phases: i) Lag phase ii) Log phase iii) Deceleration phase iv) Stationary phase v) Death phase

5. i) Lag phase • Time of adaptation of cell to the environment or medium (reorganization of micro molecular contituents). • Length of lag phase may vary (depend on specific circumstances). • Shorter lag time is recommended for industry, can be achieved using suitable inoculum (active or not) and environmental condition. • Synthesis or inhibition of the enzyme or cell structure components may occur. • Typical effects: • Low cell number / cell concentration. • No changes in substrate pH. • No changes in substrate concentration. • No product formation.

6. ii) Log phase • Illustrate by a linear line of the plot of log cell mass vs time. • During this phase, a growth at steady state where specific growth rate, µ are fixed. • Cell growth at maximum attainable rate. • Typical effects in log phase: • Rapid increase in cell concentration. • Rapid changes in substrate pH. • Substrate concentration decreased. • Product formation starts.

7. iii) Deceleration phase • Growth rate slowly decreases due to: • Consumption of nutrient or essential nutrient become depleted (substrate limitation). • Accumulation of toxic product.

8. iv) Stationary phase • Start when the growth rate begins to decrease • Nutrients have been used (finished) • Accumulation of product that inhibit the growth • Growth rate become zero – cell stop dividing • At equilibrium growth rate = death rate • Typical effects: • Microbial deaths balance production of new cells, cell mass may be constant. • The number of viable cell decreased, lysis cell may occur so the biomass will decreased .

9. iv) Death phase • Typical effects: • Substrate depletion • Rapid decline in cell numbers / biomass concentration. • Substrate concentration became until zero

10. GROWTH VS MULTIPLICATION CURVE

11. COMPARISON BETWEEN GROWTH AND MULTIPLICATION CURVE

12. GROWTH CURVE • X = Biomass concentration • Individual phases: 1: Lag phase 2: Accelaration phase 3: Balanced growth 4: Deceleration phase 5: Stationary phase 6: Death phase

13. MULTIPLICATION CURVE • N = No of cell • Individual phases: 1: Lag phase 2: Accelaration phase 3: Balanced growth 4: Deceleration phase 5: Stationary phase 6: Death phase

14. PRODUCTION KINETICS

15. To determine the metabolic parameters • Need data on: • substrate uptake with time • with and without product formation • product generation with time • with and without cell growth • cell growth with time

16. Specific growth rate: • Where: • dx= Change in biomass concentration. • dt= Change in incubation time. • x = biomass concentration. • Specific growth rate, µ expressed in reciprocal time unit (h-1).

17. During batch cultivation, specific growth rate changes • continuosly from zero to the max value µmax. • µmax depends on microorganisms, physical, chemical • conditions. • Typical values of µmax:

18. By plotting the growth curve of the microorganisms, then determine the instavenous µ value at each sampling time by ascertaining the tangent at the point of contact on the growth curve. • The highest value obtained (from 24-72h) is the µmax.

19. The Yield Coefficient (Y) • A measure of the overall efficiency of the conversion of substrate to cell mass or specific product: • Y is not constant, will vary depending on organism, pH, temperature and substrate

20. Substrate Utilization and Product Formation (Yp/s) g/l product produced g/g carbon sources utilized g/l product produced g/L biomass formed YP/S = YP/X = = g/g = g/g Economic yield (Yp/x)

21. Batch Productivity Productivity – a measure of product ( or biomass) produced per unit time (g/L/h). Product formation of growth – link product is closely related with growth rate. Productivity in batch culture will be a greatest when growth rate max (µmax).

22. X max - Xo Productivity (R batch) = T final – T initial Where; X max = maximum cell concentration at stationary phase Xo = initial cell during inoculation T final = time during which organism growing at µmax T initial = time which organism not growing at µmax, including lag phase, deceleration phase period of batching, sterilizing and so on.

24. CONTINUOUS CULTURE

25. CONTINUOUS CULTURE Fresh fermentation media is continuosly added to the reactor while fermenter broth containing biomass, products and unused nutrient are continuosly removed. Exponential growth in batch culture may be prolonged by the addition of fresh medium to the vessel. Growth can be maintained for long duration Continuous feeding to a culture at a suitable rate formation of new biomass by the culture is balanced by the loss of cell from the vessel STEADY STATE.

26. Application of Continuous Culture Biomass production Growth associated product or primary metabolite – e.g: ethanol, citric acid Not suitable for non-growth associated or secondary metabolite – e.g: antibiotic

27. Important • When referring to continuous culture systems, the terms used in batch culture (lag, exponential, stationary, death phase) have no meaning because the system is operating continuously and growth cannot segregated into phases

29. FED-BATCH CULTURE

30. FED BATCH CULTURE Extending the batch culture by feeding continuously or periodically with medium with no removal of culture from the vessel. Somewhere between batch and continuous culture. A volume of medium is inoculated with the organism and allowed to grow for a batch period of time. Subsequently, a feed is initiated into the fermenter when a “quasi steady state” is obtained. Quasi steady state: when the growth limiting substrate has depleted.

31. PRODUCT FORMATION

32. Production kinetics • Classified based on the relationship between product synthesis and energy generation in the cell • Growth associated. • Non-growth associated. • Mixed-growth associated.

33. Products • Growth-associated. • produced at the same time as cell growth. • constitutive enzymes (ones that are normally present). • glucose isomerase. • metabolic intermediates. • pyruvate, citrate, acetate. • Non-growth-associated. • takes place during the stationary phase (m=0) • secondary metabolites. • Antibiotics. • Mixed - growth associated / Partially growth associated. • takes place during growth and stationary phases. • metabolic by-products. • lactate, ethanol. • secondary metabolites.

34. Garden’s Law of Product Formation. Growth-associated Mixed-growth associated Non-growth associated

35. Primary Metabolite Released as a result of metabolic processes which are essential for the life of the micro-organism e.g. ethanol from Saccharomyces cerevisiae. Thus, primary metabolites are produced throughout the growth of the micro-organism, especially through the exponential phase.

36. Secondary Metabolite A substance which is not essential for the life of the micro-organisms and which is not produced as a result of the growth process e.g. penicillin. Secondary metabolites are produced after the exponential growth phase has stopped. This is important because it means that secondary metabolites such as penicillin cannot be produced in continuous fermenters – which deliberately maintain the micro-organism in the exponential growth stage.

37. Production of secondary metabolite starts when exponential growth stops and growth of cells starts to slow. • Adding a lot of extra nutrients at time T (see graph) will simply increase the growth of the micro-organism but not formation of the product. • However, by adding a small amount of extra nutrients at this time, the amount of product formed can be increased.