esm 219

1. ESM 219 Lecture 5: Growth and Kinetics

3. Figure: 06-06a-b Caption: The rate of growth of a microbial culture. (a) Data for a population that doubles every 30 min. (b) Data plotted on an arithmetic (left ordinate) and a logarithmic (right ordinate) scale. Figure: 06-06a-b Caption: The rate of growth of a microbial culture. (a) Data for a population that doubles every 30 min. (b) Data plotted on an arithmetic (left ordinate) and a logarithmic (right ordinate) scale.

4. Figure: 06-07a-b Caption: Method of estimating the generation times (g) of exponentially growing populations with generation times of (a) 6 h and (b) 2 h from data plotted on semilogarithmic graphs. The slope of each line is equal to 0.301/g and n equals the number of generations that have occurred in the time, t. All numbers are expressed in scientific notation; that is, 10,000,000 is 1 x 107, 60,000,000 is 6 x 107, and so on. Figure: 06-07a-b Caption: Method of estimating the generation times (g) of exponentially growing populations with generation times of (a) 6 h and (b) 2 h from data plotted on semilogarithmic graphs. The slope of each line is equal to 0.301/g and n equals the number of generations that have occurred in the time, t. All numbers are expressed in scientific notation; that is, 10,000,000 is 1 x 107, 60,000,000 is 6 x 107, and so on.

5. Exponential Phase Growth

6. Monod Growth Kinetics

7. Monod Growth Kinetics First-order region, S << KS, the equation can be approximated as m = mmaxS/Ks Center region, Monod �mixed order� kinetics must be used Zero-order region, S >> KS, the equation can be approximated by m = mmax

8. Determining Monod parameters Double reciprocal plot (Lineweaver Burke) Commonly used Caution that data spread are often insufficient Other linearization (Eadie Hofstee) Less used, better data spread Non-linear curve fitting More computationally intensive Progress-curve analysis (for substrate depletion) Less lab work (1 curve), more uncertainty

9. Michaelis Menten Kinetics Used when microbe population is constant = non-growing (or short time spans) Derivable from first principles (enzyme-substrate binding rates and equilibria expressions) Parameter determination methods used for Monod calculations (i.e. Lineweaver Burke)

14. Monod vs. Michaelis-Menten:recap of differences Monod Growth Empirical Ks m, 1/t Michaelis Menten No growth; constant E Derived from theory Km v, mg/L-t

15. Substrate Depletion Kinetics The rate of biodegradation or biotransformation is a focus of environmental studies Substrate consumption rates have often been described using �Monod kinetics� S is the substrate concentration [mg/L] X is the biomass concentration [mg/ L] k is the maximum substrate utilization rate [sec-1] KS is the half-saturation coefficient [mg/L]

16. Substrate Depletion Kinetics Since And Then And

17. Modeling Substrate Depletion Three main methods for modeling Monod kinetics (mid range concentrations) First-order decay (low concentration of S, applicable to many natural systems) Zero-order decay (substrate saturated)

18. Modeling First-Order Decay dS/dt= kS where k is a pseudo first order constant Generally assumes nothing about limiting substrates or electron acceptors Degradation rate is proportional to the concentration Generally used as a fitting parameter, encompassing a number of uncertain parameters

19. Monod Kinetics First-order region, S << KS, the equation can be approximated by exponential decay (C = C0e�kt) Center region, Monod kinetics must be used Zero-order region, S >> KS, the equation can be approximated by linear decay (C = C0 � kt)

20. Microbial Kinetics in Modeling Fate of a Substrate Use mass balance framework for modeling fate of substance, S Choose appropriate �ideal� reactor analogy (usually batch or complete mix) Substitute appropriate reaction expression into the framework

21. Mass Balance: Batch example

22. Mass Balance: Batch example

23. Mass Balance: Batchexample of exponential decayS0 = 100 mg/L, k =-0.2/hr

24. Mass Balance: CFSTR

25. Mass Balance: CFSTR

26. Chemostat: CFSTR for Microbial Growth

27. Chemostat: CFSTR for Microbial Growth

28. Figure: 06-13 Caption: Schematic for a continuous culture device (chemostat). In such a device, the population density is controlled by the concentration of limiting nutrient in the reservoir, and the growth rate is controlled by the flow rate (see Figure 6.15). Both parameters can be set by the experimenter. Figure: 06-13 Caption: Schematic for a continuous culture device (chemostat). In such a device, the population density is controlled by the concentration of limiting nutrient in the reservoir, and the growth rate is controlled by the flow rate (see Figure 6.15). Both parameters can be set by the experimenter.

29. Figure: 06-15 Caption: Steady-state relationships in the chemostat. The dilution rate is determined from the flow rate and the volume of the culture vessel. Thus, with a vessel of 1000 ml and a flow rate through the vessel of 500 ml/h, the dilution rate would be 0.5 h-1. Note that at high dilution rates, growth cannot balance dilution, and the population washes out. Note also that although the population density remains constant during steady state, the growth rate (doubling time) can vary over a wide range. Thus, the experimenter can obtain populations with widely varying growth rates without affecting population density. Figure: 06-15 Caption: Steady-state relationships in the chemostat. The dilution rate is determined from the flow rate and the volume of the culture vessel. Thus, with a vessel of 1000 ml and a flow rate through the vessel of 500 ml/h, the dilution rate would be 0.5 h-1. Note that at high dilution rates, growth cannot balance dilution, and the population washes out. Note also that although the population density remains constant during steady state, the growth rate (doubling time) can vary over a wide range. Thus, the experimenter can obtain populations with widely varying growth rates without affecting population density.

30. Environmental Factors Temperature pH Salinity Oxygen Concentration

31. Environmental Factors Extremophiles can tolerate or perhaps require extreme conditions in any of the above. Cellular compensation outside of their optima can reduce growth rate and yield.

32. Figure: 06-16 Caption: Effect of temperature on growth rate and the molecular consequences for the cell. The three cardinal temperatures vary by organism. Figure: 06-16 Caption: Effect of temperature on growth rate and the molecular consequences for the cell. The three cardinal temperatures vary by organism.

33. Figure: 06-17 Caption: Relation of temperature to growth rates of a typical psychrophile, a typical mesophile, a typical thermophile, and two different hyperthermophiles. The temperature optima of the example organisms are shown on the graph. Figure: 06-17 Caption: Relation of temperature to growth rates of a typical psychrophile, a typical mesophile, a typical thermophile, and two different hyperthermophiles. The temperature optima of the example organisms are shown on the graph.

34. Figure: 06-22 Caption: The pH scale. Note that although some microorganisms can live at very low or very high pH, the cell�s internal pH remains near neutrality. Figure: 06-22 Caption: The pH scale. Note that although some microorganisms can live at very low or very high pH, the cell�s internal pH remains near neutrality.

35. Figure: 06-23 Caption: Effect of sodium ion concentration on growth of microorganisms of different salt tolerances or requirements. The optimum NaCl concentration for marine microorganisms such as V. fischeri is about 3%; for extreme halophiles, it is between 15 and 30%, depending on the organism. Figure: 06-23 Caption: Effect of sodium ion concentration on growth of microorganisms of different salt tolerances or requirements. The optimum NaCl concentration for marine microorganisms such as V. fischeri is about 3%; for extreme halophiles, it is between 15 and 30%, depending on the organism.

36. Figure: 06-24a Caption: Structures of some common compatible solutes in microorganisms. The structures of glutamate and proline, other common solutes, were shown in Figure 3.12. The formal name of ectoine is 1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylate. Note that all the compounds shown here are very polar (water soluble) molecules. Figure: 06-24a Caption: Structures of some common compatible solutes in microorganisms. The structures of glutamate and proline, other common solutes, were shown in Figure 3.12. The formal name of ectoine is 1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylate. Note that all the compounds shown here are very polar (water soluble) molecules.

37. Figure: 06-24b Caption: Structures of some common compatible solutes in microorganisms. The structures of glutamate and proline, other common solutes, were shown in Figure 3.12. Note that all the compounds shown here are very polar (water soluble) molecules. Figure: 06-24b Caption: Structures of some common compatible solutes in microorganisms. The structures of glutamate and proline, other common solutes, were shown in Figure 3.12. Note that all the compounds shown here are very polar (water soluble) molecules.

38. Figure: 06-25a-e Caption: Aerobic, anaerobic, facultative, microaerophilic, and aerotolerant anaerobe growth, as revealed by the position of microbial colonies (depicted here as black dots) within tubes of thioglycolate broth culture medium. A small amount of agar has been added to keep the liquid from becoming disturbed and the redox dye, resazurin, which is pink when oxidized and colorless when reduced, is added as a redox indicator. (a) Oxygen penetrates only a short distance into the tube, so obligate aerobes grow only at the surface. (b) Anaerobes, being sensitive to oxygen, grow only away from the surface. (c) Facultative aerobes are able to grow in either the presence or the absence of oxygen and thus grow throughout the tube. However, better growth occurs near the surface because these organisms can respire. (d) Microaerophiles grow away from the most oxic zone. (e) Aerotolerant anaerobes grow throughout the tube. However, growth is no better near the surface because these organisms can only ferment. Figure: 06-25a-e Caption: Aerobic, anaerobic, facultative, microaerophilic, and aerotolerant anaerobe growth, as revealed by the position of microbial colonies (depicted here as black dots) within tubes of thioglycolate broth culture medium. A small amount of agar has been added to keep the liquid from becoming disturbed and the redox dye, resazurin, which is pink when oxidized and colorless when reduced, is added as a redox indicator. (a) Oxygen penetrates only a short distance into the tube, so obligate aerobes grow only at the surface. (b) Anaerobes, being sensitive to oxygen, grow only away from the surface. (c) Facultative aerobes are able to grow in either the presence or the absence of oxygen and thus grow throughout the tube. However, better growth occurs near the surface because these organisms can respire. (d) Microaerophiles grow away from the most oxic zone. (e) Aerotolerant anaerobes grow throughout the tube. However, growth is no better near the surface because these organisms can only ferment.

39. Figure: 06-27 Caption: Four-electron reduction of O2 to water by stepwise addition of electrons. All the intermediates formed are reactive and toxic to cells except for water, of course. Figure: 06-27 Caption: Four-electron reduction of O2 to water by stepwise addition of electrons. All the intermediates formed are reactive and toxic to cells except for water, of course.

40. Figure: 06-29 Caption: Method for testing a microbial culture for the presence of catalase. A heavy loopful of cells from an agar culture was mixed on a slide with a drop of 30% hydrogen peroxide. The immediate appearance of bubbles is indicative of the presence of catalase. The bubbles are O2 produced by the reaction H2O2 + H2O2 ? 2 H2O + O2. Figure: 06-29 Caption: Method for testing a microbial culture for the presence of catalase. A heavy loopful of cells from an agar culture was mixed on a slide with a drop of 30% hydrogen peroxide. The immediate appearance of bubbles is indicative of the presence of catalase. The bubbles are O2 produced by the reaction H2O2 + H2O2 ? 2 H2O + O2.