PTT 203 Biochemical Engineering

1. PTT 203 Biochemical Engineering Kinetics MICROBIAL GROWTH IN BATCH CULTURE WEEK 9-10 By: MADAM NOORULNAJWA DIYANA YAACOB

2. Lecture Outline • Introduction • Batch Growth Characteristics • Growth Stages, Effects of Environmental Conditions, Product Formation, Mathematical Models

3. Cell growth • Microbial growth is an autocatalytic reaction • The rate of growth is directly related to cell concentration • Characterized by the net specific growth rate:

4. Cell growth • The net specific growth rate is the difference between a gross specific growth rate (μg, h-1) and the rate of loss of cell mass due to cell death (kd, h-1): • Microbial growth can also be described in terms of cell number, N: where μR is the net specific replication rate (h-1)

5. Batch Growth –Determining Cell Number Density • Hemocytometer • Direct microscopic count • Counts all cells present (viable and non-viable) • Immediate result • Agar plates • Counts only living cells • Delayed result • Assumption: each viable cell will yield 1 colony • Results expressed in CFUs(colony-forming units) • Particle counters • Counts all cells present (viable and non-viable) • Suitable for discrete cells in a particulate-free medium • Can distinguish between cells of different sizes

6. Hemocytometer

7. The 4 outer squares, marked 1- 4, each cover a volume of 10 4mL. • The inner square, marked as 5, also covers a volume of 10 4mL, but is further subdivided into 25 smaller squares. • Each of the 25 smaller squares is further divided into 16 squares, which are the smallest gradations on the hemocytometer.

8. Viable Cell Count

10. Coulter Particle Counter

11. Determining Cell Mass Concentration –Direct Methods • Dry cell weight (DCW) • A sample of fermentation broth is centrifuged, washed, and dried at 80°C for 24hrs • Off-line measurement; wet cell weights (WCW) can performed in-process • Packed cell volume • Like wet cell weight, but measures cell pellet volume • Optical density (OD) • Turbidity –based on the absorption of light by suspended cells in culture media

12. Determining Cell Mass Concentration –Indirect Methods • In many fermentation processes, particularly with moulds, direct methods cannot be used • Indirect methods are therefore employed, based on the measurement of substrate consumption and/or product formation • Intracellular components of cells such as RNA, DNA and protein can be measured as indirect indicators of cell growth • Concentration of RNA/cell weight varies significantly during a batch growth cycle, while DNA and protein concentrations per cell weight remain fairly constant, and can therefore be used as reasonable measures of cell growth

13. Time-Dependent Changes in Cell Composition and Cell Size AzotobactervinelandiiGrowth in Batch Culture

14. Batch Growth

15. Batch Growth Curve Growth Phases Lag Exponential Deceleration Stationary Death/Decline

16. Lag Phase • Occurs immediately after inoculation and is a period of adaptation for the cells to their new environment • New enzymes are synthesized, synthesis of other enzymes is repressed • Intracellular machinery adapts to the new conditions • May be a slight increase in cell mass and volume, but no increase in cell number • The lag phase can be shortened by high inoculum volume, good inoculum condition (high % of living cells), age of inoculum, nutrient-rich medium

17. Influence of [Mg2+] on Lag Phase Duration in E. aerogenesCulture • E. aerogenes requires Mg2+ to activate the enzyme phosphatase, which is required for energy generation by the organism • The concentration of Mg2+ in the medium is indirectly proportional to the duration of the lag phase

18. Exponential Growth Phase • In this phase, the cells have adjusted to their new environment • At this point the cells multiply rapidly (exponentially) • Balanced growth –all components of a cell grow at the same rate • Growth rate is independent of nutrient concentration, as nutrients are in excess • The first order exponential growth rate expression is:

19. Exponential Growth Phase (cont’d) • An important parameter in the exponential phase is the doubling time (time required to double the microbial mass) • A graph of ln X versus t produces a straight line on a semi-logarithmic plot: • The doubling time based on cell number is expressed as:

20. Exponential Growth Phase (cont…) t

21. Deceleration Phase • Very short phase, during which growth decelerates due to either: • Depletion of one or more essential nutrients, or, • The accumulation of toxic by-products of growth (e.g. Ethanol in yeast fermentations) • Period of unbalanced growth: td=td’ • Cells undergo internal restructuring to increase their chances of survival • Followed quickly by the Stationary Phase

22. Stationary Phase • Starts at the end of the Deceleration Phase, when the net growth rate is zero (no cell division, or growth rate is equal to death rate) • Cells are still metabolically active, and can produce secondary metabolites • Primary metabolites are growth-related products, while secondary metabolites are non-growth-related • Many antibiotics and some hormones are produced as secondary metabolites • Secondary metabolites are produced as a result of metabolite deregulation

23. Stationary Phase (cont’d) • During this phase, one or more of the following phenomena may occur: • Total cell mass concentration may stay constant, but the number of viable cells may decrease • Cell lysis may occur, and viable cell mass may drop. A second growth phase may occur as cells grow on lysis products from the dead cells (cryptic growth) • Cells may not be growing, but may have active metabolism to produce secondary metabolites

24. Stationary Phase (cont’d) • During the stationary phase, the cell catabolizes cellular reserves for new building blocks and for energy-producing monomers • This is called endogenous metabolism • The cell must expend maintenance energy in order to stay alive • The equation that describes the conversion of cellular mass into energy, or the loss of cell mass due to lysis during the stationary phase is:

25. Death Phase • The death or decline phase is characterized by the expression: • Where Ns is the concentration of cells at the end of the stationary phase, and is the first-order death-rate constant • A plot of ln N versus t yields a line of slope –kd’

26. Death Phase • Cell lysis (spillage) may occur • Rate of cell decline is first-order where: –kd= 1st order death rate constant, Xs = conc. of cell at end of stationary phase • Growth can be re-established by transferring to fresh media

27. Yield Coefficients • Growth kinetics are generally further described by defining stoichiometrically related parameters • Yield coefficients are defined based on the amount of consumption of a given material • For example, the growth yield coefficient is: • For organisms growing aerobically on glucose, Yx/s is typically 0.4 to 0.6 g/g, for most yeast and bacteria; anaerobic growth is much less efficient

28. Aerobic and Anerobic Growth Yields of S. faecalison Glucose

29. Yield Coefficients • At the end of a batch growth period, there is an apparent or observed growth yield: • The apparent yield is not a true constant for compounds that can be used as both a carbon and energy source, but the true growth yield (YX/S) is constant ΔS

30. Yield Coefficients • Yield coefficients can also be defined for other substrates or for product formation: • YX/O2 is typically 0.9 to 1.4 g/g for most yeast and bacteria, but is much lower for highly reduced substrates (e.g. methane, CH4)

31. Summary of Yield Factors for Aerobic Growth

32. The Maintenance Coefficient • The maintenance coefficient is used to describe the specific rate of substrate uptake for cellular maintenance: • However, during the Stationary Phase, where little external substrate is available, endogenous metabolism of biomass components is used for maintenance energy • Maintenance energy is the energy required to repair damaged cellular components, to transfer nutrients and products in and out of cells, for motility, and to adjust the osmolarity of the cells’ interior volume

33. Microbial Products • Microbial products can be classified into three major categories • Growth-associated products • Non-growth-associated products • Mixed-growth-associated products • Growth-associated products • These products are produced simultaneously with microbial growth • Specific rate of product formation is proportional to the specific growth rate, μg • Note that μg is not equal to μnet, the net specific growth rate, when endogenous metabolism is occurring

34. Growth-Associated Products • The rate expression for product formation in growth-associated production is: • Where qp is the rate of product formation (h-1) • The production of a constitutive (continuously produced, as opposed to inducible) enzyme is an example of a growth-associated product

35. Non-Growth-Associated Products • Non-growth-associated product formation takes place during the Stationary Phase, when the growth rate is zero • Specific rate of product formation is constant: • Many secondary metabolites, such as most antibiotics (e.g. penicillin), are non-growth-associated products

36. Mixed-Growth-Associated Products • Mixed-growth-associated product formation takes place during the Deceleration (slow growth) and Stationary Phases • The specific rate of product formation is given by the Luedeking-Piret equation: • If α= 0, the product is completely non-growth associated; If β= 0, the product is completely growth-associated • Examples: lactic acid fermentation, production of xanthan gum, some secondary metabolites

37. Product Yield Coefficients (cont…) • Growth-associated product formation • Non-growth-associated product formation • Mixed-growth-associated product formation

38. Environmental Factors • Patterns of microbial growth and product formation are influenced by environmental factors such as temperature, pH and dissolved oxygen concentration (D.O.) • Microorganisms can be classified by their optimum growth temperatures, Topt • Psychrophiles: (Topt< 20°C) • Mesophiles: (20°C < Topt< 50°C) • Thermophiles: (Topt> 50o°C) • As the temperature increases towards Topt, the growth rate doubles every ~10°C

39. Optimum Growth Temperature

40. Optimum Growth Temperature

41. Effect of Temperature on Cell Growth • Above Topt the growth rate decreases and thermal death may occur • The net specific replication rate for temperatures above Topt is expressed by: • Both and vary with temperature according to the Arrhenius equation: • Where: • Ea =activation energy for growth ≈ 10-20 kcal/mol • Ed =activation energy for death ≈ 60-80 kcal/mol

42. Arrhenius Plot of Growth Rate of E. Coli Legend: (●) Growth on rich, complex medium (○) Growth on glucose-mineral salts medium

43. Effect of pH on Cell Growth • pH affects the activity of enzymes, and therefore the microbial growth rate • Acceptable pH’s for growth are typically within 1 or 2 pH units of the optimum pH • pH range varies by organism: • bacteria (most) pH = 3 to 8 • yeast pH = 3 to 6 • plants pH = 5 to 6 • animals pH = 6.5 to 7.5

44. Effect of pH on Cell Growth • The optimal pH for growth may be different from the optimal pH for product formation (e.g. Pichiapastoris) • Microorganism have the ability to control pH inside the cell, but this requires maintenance energy • pH can change due to: • Utilization of substrates; NH4+ releases H+, NO3- consumes H+ • Production of organic acids, amino acids, CO2, bases

45. Effect of pH on Cell Growth (cont…)

46. Effect of Dissolved O2 on Cell Growth • At high cell concentrations, the rate of oxygen consumption may exceed the rate of O2 supply • When oxygen is the rate-limiting factor, specific growth rate varies with [DO] according to saturation (Michaelis-Menten) kinetics • Below a critical concentration, growth approaches a first-order rate dependence on DO (oxygen is a limiting substrate) • Above a critical concentration, the growth rate becomes independent of DO (oxygen is non-limiting))

47. Effect of Dissolved O2 on Cell Growth (cont…) Facultative aerobic cells Obligate aerobic cells Saturation kinetics Saturation kinetics

48. Effect of Dissolved O2 on Cell Growth • The saturated DO concentration for water at 25°C and 1 atm is ~7 ppm • The presence of dissolved salts and organics can alter the saturation value • Increasing temperatures decrease the saturation value • The critical oxygen concentration is about 5%-10% of the saturated DO concentration for bacteria and yeast, and about 10%-50% of [DO]sat for moulds, since they grow as large spheres in suspended culture (diffusion issues)

49. Other Effects on Cell Growth • Dissolved CO2 can have a profound effect on the performance of microorganisms • Very high DCO2 concentrations can be toxic to some cells • On the other hand, cells require a certain minimum DCO2 level for proper metabolic function • Ionic strength (I); too high dissolved salts is inhibitory to membrane function (membrane transport of nutrients, osmotic pressure): where : Ci = molar concentration of ion i Zi = ion charge

50. Other Effects on Cell Growth • The redox potential is an important parameter that affects the rate and extent of many oxidative-reductive reactions • In fermentation media, the redox potential is a complex function of DO, pH, and other ion concentrations, such as reducing and oxidizing agents • Substrate concentrations significantly above stoichiometric requirements are inhibitory to cellular functions • Inhibitory levels of substrates vary depending on cell type and substrate • Typical maximum non-inhibitory concentrations of some nutrients are –glucose, 100 g/l; ethanol, 50 g/l for yeast, much less for other organisms; ammonium, 5 g/l; phosphate, 10 g/l; nitrate, 5 g/l