Chapter 3

1. Chapter 3 Energetics Respiration Growth

2. Types of energy metabolism • Chemoheterotrophs • Energy source (electron donor):organic carbon • Carbon source: organic carbon An electron donor is an energy source An electron acceptor is a respiratory substrate

3. Chemoheterotrophs Electron donor Carbon source Organic C Carbon dioxide H2O Electron acceptor O2aerobic respiration NO3, Fe(III), Mn(IV), SO4, CO2anaerobic respiration

4. Types of energy metabolism • Chemoautotrophs • Energy source (electron donor): reduced form of an inorganic chemical • Carbon source: inorganic carbon-CO2

5. Chemoautotrophs NH4+, Fe(II), Mn(II), HS- Electron donor Carbon source Carbon dioxide Inorganic C Organic carbon Electron acceptor O2aerobic respiration H2O NO3, Fe(III), Mn(IV), SO4, CO2anaerobic respiration N2O, Fe(II), Mn(II), H2S, CH4

6. Types of energy metabolism • Photoautotrophs • Energy source: light • Electron donor: water • Carbon source: inorganic carbon

7. Photoautotrophs Energy source Electron donor Light (l) Water Carbon source, electron acceptor Carbon dioxide Inorganic C (CO2) Organic carbon Light (l) Electron donor O2 H2O

8. Types of energy metabolism • Photoheterotrophs • Energy source: light • Electron donor: water • Carbon source: organic carbon

9. Photoheterotrophs Energy source Light (l) Electron donor Water Carbon source Organic C CO2 Organic C Terminal electron acceptor Fe-S clusters in Photo System 1

10. Energy Yield In a chemical reaction, only part of the energy is used to do work. Energy available for work is called “free energy” or DG. The rest of the energy is lost to entrophy. DG = -RT log Keq where Keq = [C] [D] / [A] [B] from rxn: A + B C + D If logKeq is a negative value, this means the reaction can only proceed if energy is added (endothermic rxn). When logKeq is a negative value, DG is positive. If logKeq is a positive value, this means the reaction is favored and, in fact, gives off energy (exothermic rxn). When logKeq is a positive value, DG is negative.

11. Energy yield from different electron acceptors • 6O2 6H2Oaerobic respiration -686 • 24 NO3 12N2anaerobic respiration -36 • SO4 H2S anaerobic respiration -40 • CO2 CH2O photosynthesis +115 Terminal electron acceptor DG (kcal/mol) e- Which ones are exothermic and which ones are endothermic?

12. Reduction Potential Redox couples +0.85 O2/H2O +0.75 NO3/ N2 electron acceptor 1.28 1.22 oxidation state (volts) 0.00 -0.22 SO4/H2S 0.25 -0.47 CH2O/CO2 (CH2O CO2) electron donor

13. stopped Energy yield relationship between electron acceptor and electron donor Electron ReductionElectron Reduction Difference AcceptorPotential (V) Donor Potential (V)(V) O2 H2O +0.81 CH2O CO2 -0.47 -1.28 NO3 N2 +0.75 CH2O CO2 -0.47 -1.22 SO4 H2S -0.22 CH2O CO2 -0.47 -0.25 The sign and magnitude of the difference represents how much free energy is available to the cell to do work.

14. Bacterial Growth

15. Batch Culture-closed system stationary death N2 Cell Density Nutrient limitation N1 log lag t1 t2 Time N2 - N1 = #cells produced/unit time (growth rate) t2 – t1

16. Natural systems • Natural environments that behave as closed systems • Not many exist. There is always some energy coming in from outside the system • Natural environments that behave as open systems • Most inhabitable environments

17. Continuous Culture More closely mimics natural systems • Chemostat • Control flow rate and concentration of growth-limiting nutrient of liquid medium entering and exiting a growth chamber (bioreactor) • Control • pH • Temperature • Concentration of terminal electron acceptor (TEA) • Concentration of toxic by-products of metabolism Engineered wastewater treatment system operates like a chemostat

18. Chemostat:open system X=cell number S=limiting nutrient conc. D=dilution (flow) rate Energy source (electron donor) Inoculate vessel (Terminal electron acceptor)

19. By controlling flow rate or dilution rate, one can control growth rate (m) of the bacteria • dX/dt = mX –DX, where X is cell biomass in mass/volume, m is specific growth rate (1/t), and D is the dilution rate (1/t) • At steady state, when biomass in reactor remains constant, m = D • A chemostat reactor allows the maintenance of steady state conditions for extended periods of time

20. Processes occurring in a chemostat Stationary phase (X) m = D mmax

21. Limiting nutrient • In previous slide, the substrate that was plotted was the nutrient that limited the rate of growth • This could be any nutrient • Carbon source • Nitrogen source • Phosphorus source • Electron donor or electron acceptor • The chemistry of the limiting nutrient will influence how much cell biomass (cell yield) is produced at mmax

22. Cell Yield (Y) • Not all of the carbon added as the carbon source is converted to cell biomass • A fraction is respired as CO2 during the transformation of the carbon to energy (ATP) • Cell yield coefficient is defined as the amount of biomass produced per unit substrate consumed

24. Yield coefficient Carbon source 0.4 glucose Pentachlorophenol (PCP) 0.05 1.49 octadecane Cell Yield

25. Biochemical basis of cell yield • In case of PCP, it is a new chemical that microbes have only encountered since its initial production in 1936 • Consequently, microbes have not had time to evolve efficient enzyme reactions and metabolic pathways to convert it to biomass • A lot of energy is required to break the C-Cl bonds-energy not available for biomass production

26. Biochemical basis of cell yield • In case of octadecane, it is a component of crude oil that microbes have encountered for millions of years • Consequently, microbes have had time to evolve efficient enzyme reactions and metabolic pathways to convert it to biomass. • Octadecane is a highly reduced form of carbon (contains only C-H bonds) and can thus store more energy than compounds that are less reduced or have more oxygen atoms such as carbohydrates (CH2O)

27. Study calculations • Go over Example Calculation on page 50 of text • cells are growing on glucose • yield coefficient = 0.4 (0.4 g cell mass produced from 1 g of glucose consumed) • What percentage of glucose carbon is converted to cell mass and what percentage to CO2?

28. 1 mol of glucose is equivalent to 180g cell mass produced from 1 mol glucose = 180g x 0.4 = 72g cell mass is expressed as C5H7NO2 (mol. wt. = 113g/mol) moles of cell mass produced from 1 mol of glucose 72g cell mass/113 g/mol cell mass = 0.64 mol cell mass In terms of carbon: for cell mass, (0.64 mol cell mass)(5 mol C/mol cell mass)(12 g/mol C) = 38.4g C for substrate (glucose), (1 mol substrate)(6 mol C/mol glucose)(12 g/mol C) = 72 g C % of glucose carbon found in cell mass is (38.4 g C/72 g/C)x100 =53% by difference 47% glucose carbon is released as CO2

29. Substrate Glucose 53 Octadecane 93 Pentachlorophenol 10% % substrate consumed that ends up in cell mass

30. How do metabolic pathways evolve in bacteria • Lateral gene transfer • plasmids • bacteriophage • Naked DNA in the environment (tranformation)

31. Developing genetically-engineered microbes and plants to carry out specific remediation activities when indigenous organisms can’t do the job • Naphthalene-degrading plasmid (NAH) • Xylene-degrading plasmid (XYL) • Conjugation occurred between 2 cells that had the different plasmids containing genes that encode both degradative pathways. One recombinant cell can now degrade both compounds

32. Same genetic manipulations were carried out with octane-degrading plasmid (OCT) and camphor-degrading plasmid (CAM). • The plasmids fused into one plasmid when transferred into a bacterial strain to form a genetically-engineered bacterial strain that could degrade both octane and camphor. • The strain carrying the NAH & XYL plasmids were mated with the strain that carried the OCT/CAM plasmid to produce a new construct that did a good job growing on and degrading crude oil.

33. Summary • Microbes as a group are metabolically diverse • Amount of energy a cell can extract from a chemical is determined by intrinsic chemistry of the chemical • Abundance of the limiting nutrient in the environment controls the growth rate of the cell