Bioenergetics of Anaerobic Microbial Reactions

1. Bioenergetics of Anaerobic Microbial Reactions Traditional and new processes

2. Methanogenesis Archaebacteria All methane producing bacteria (MPB) are archeabacteria Archeabacteria are different from all other life forms (3rd primary kingdom) Eukaryotes Archeabacteria Prokaryotes Eubacteria Cyanobacteria etc. Plants Animals Fungi etc. Methanogens Halobacterium etc. This new taxonomic division of lifeforms into three kingdoms is based on phylogenetically conservative features: 1. Ether linked membrane lipids rather than ester links 2. No murein in cell wall (→ penicillin resistant) 3. Different protein synthesis (→ streptomycin resistant)

3. 4. More subunits in RNA polymerase 5. Unique coenzymes (e.g. F 420) Special features in archaebacteria: High temperature tolerance (115°C) Pyrodiction Acid resistance (pH 1, at 90°C) Sulfolobus Unique use of light ( no photosystem) Halobacterium Archaebacteria are likely to be the earliest life forms (Symbiont hypothesis)

4. Methanogenesis 0 8 6 4 2 1 1 1 1 1 Pathway of CO2 Reduction CO2 H2 MF MFCOH Formyl - C MP H2 MF MPCH2 Methylene - C H2 Methyl - C MPCH3 CoM MP CoMCH3 H2 ATP CoM CH4 Methane Only the last step allows the generation of 1ATP

5. Methanogenesis Electron transport chain CH3 - CoM + F420 2 H+ CH4 + F420 + CoM ADP + Pi 2 H+ ATP Membrane ATP - ase Principle mechanism of ATP generation: Methyl - Respiration

6. 0 6 8 6 8 2 1 1 0 1 1 1 Methanogenesis Acetate and Methanol Metabolism • During methanol conversion to methane only 1 ATP is generated via methyl – respiration. 4 CH3OH → 3 CH4 + CO2 + 2 H2O • In the presence of hydrogen methanol is only used as electron acceptor → methyl – respiration. H2 + CH3OH → CH4 + H2O

7. 6 8 2 0 8 1 1 1 1 2 • Acetoclastic methanogens split acetate into a methyl group and a carboxy group. The two electrons of the carboxy group are transferred to the methyl group → methyl – respiration. CH3 - COOH → CH4 + CO2 Methyl - respiration is the only way of ATP generation in methanogenesis

8. Overall the energy conserving reaction in methanogenesis is from proton translocation via electron transfer to the methyl group generated during metabolism. Methanogenesis = anaerobic methyl respiration

9. Revision: Gibbs Free Energy Change The Gibbs Free Energy (G) of substrates and products can be calculated. For exergonic (=spontaneous = downhill) reactions the G of the substrates is higher than that of the products. Hence the change in G (=ΔG) for exergonic reactions is negative. This ΔG is the driving force of the reaction. As the reaction proceeds, the diminishing substrate concentration and increasing product concentration cause the difference in G to become smaller and smaller until an equilibrium is reached at which ΔG = zero.

10. Revision: Gibbs Free Energy Change Handbooks provide values of the Gibbs Free Energy Change as: ΔGo assumes that all reactants and products are a unity (concentrations are 1 M and gas partial pressures are 100 kPa) ΔGo’ assumes that all reactants and products are at unity, however the proton concentration is not 1 M (pH 0) but 10-7 M ( pH 7). This value makes more sense for most biological systems ΔG is the actual Free Energy Change under experimental conditions and changes any moment as the reaction proceeds

11. Methanobacillus omelianskii, observed features 1. degrades ethanol to acetate and methane gas: CH3-CH2OH + CO2 --> CH3-COOH + CH4 2. Also grows with H2 as e-donor and CO2 as e- acceptor: 4H2 + CO2 --> CH4 + 2H2O Suspicions about the purity of culture were raised because: 1. H2 inhibited ethanol degradation but not CH4 production (Preference for H2?) 2. No growth on ethanol after prolonged cultivation on H2 + CO2 (Mutagenic loss of ethanol dehydrogenease?) 3. Bubbling inert N2 gas through ethanol degrading culture --> CH4 production stopped but ethanol oxidation to acetate continued.

12. CO2 CH4 Ethanol H2 Acetate Theory of Bryant on Interspecies Hydrogen Transfer Hypothesis of Marvin P. Bryant: Interspecies Hydrogen Transfer, Claims: 1. Methanobacillus consists of an association of two microbes: 2. Pelobacter depends on H2 removal by MPB or N2 flushing. 3. MPB needs needs Pelobacter to provide H2 as e-donor. 4. Both depend on each other (syntrophy)

13. CO2 CH4 Ethanol H2 Acetate Bryant’s hypothesis was not readily accepted: Lack of driving force of reaction 1: CH3-CH2OH + H2O --> CH3-COO- + H+ + 2 H2 DGo'= + 9.6 kJ/mol Bryant's argument: reaction 2 has excess driving force 4 H2 + HCO3- + H+ --> CH4 + 3 H2O DGo'= - 135.6 kJ/mol Total reaction: DGo'= - 116.4 kJ/mol Question: Energy sharing possible (pulley analogy)?

14. CO2 CH4 Ethanol H2 Acetate Bryant’s hypothesis was not readily accepted:

15. Energy Sharing DGo'= DGo'= + 9.6 kJ/mol - 135.6 kJ/mol CH4 How can two different microbes share the common energetic potential? The actual free energy change of the reaction DG depends on the product to substrate ratio (P/S) and can be calculated from the standard DG= DGo + 5.69 kJ * log (P/S) CO2 Ethanol H2 Acetate For high substrate concentrations (P/S less than 1) the DG will be more favourable (negative). Interspecies hydrogen transfer was postulated to operate at extremely low H2 partial pressures o 1- 10 Pa (compared to 100,000 Pa for standard conditions)

16. Energy Sharing DGo'= DGo'= + 9.6 kJ/mol - 135.6 kJ/mol CH4 Interspecies hydrogen transfer was postulated to operate at extremely low H2 partial pressures o 1- 10 Pa (compared to 100,000 Pa for standard conditions. Low H2 increases the driving force of reaction 1 while it decreases the driving force of reaction 2. CO2 Ethanol H2 Acetate Reaction1 Reaction 2 A sharing in driving force is possible by lowering H2 until reaction 1 becomes feasible (exergonic) but not so low that reaction 2 becomes endergonic.

17. Significance of Interspecies Hydrogen Transfer DGo'= DGo'= + 9.6 kJ/mol - 135.6 kJ/mol Eventually Bryan’s hypothesis was not only confirmed but found to be a general feature of anaerobic systems (e.g. rumen, anaerobic digesters, sediments) The interspecies hydrogn can be intercepted by chemical and biological means (more powerful H2 users than methanogens. CH4 CO2 Ethanol H2 Acetate Reaction1 Reaction 2 Other substrates found to degrade via H2 transfer include all fatty acids, alcohols, many amino acids 30 % of electron flow in anaerobic digesters passes via H2. Use of energetic calculations critical to understand (exploit, control, optimise) many biological reactions)

18. How can the energetic values be determined ?: The example of Methanobacillus omelianskii and interspecies hydrogen transfer has shown the importance of DG calculations: Example problem: Establish the standard DGo for the anaerobic conversion of ethanol (12 e-) to acetate (8 e-) by Pelobacter (syntrophic partner in M. omlianskii) 1. Establish proper equation: (4 electrons transferred) CH3-CH2OH + H2O <--> CH3-COO- + H+ + 2 H2 (-181.75) + (-237.18) <--> (-369.4) + 0 + 0 2. Look up the standard Gibbs free energy of formation (Gfo) 3. Subtract the Gfo of substrates from Gfo of products --> DGo= + 49.53 kJ/mol 4. This gives the energetic situation (negative = spontaneous = exergonic = downhill) for standard conditions: Room temperature, partial pressure of all gases = 100 kPa, all concentrations 1 mol/L. Why is the established value not the one calculated by Bryant for the same reaction?

19. Considering pH for thermodynamic calculationsDG= DGo + 5.69 kJ * log (P/S) Standard conditions imply that also protons are at a concentration of 1 mol/L (pH 0 !). How to convert reaction energetics to consider pH 7 rather than 0: 1. Use formula : DG= DGo + 5.69 kJ * log (P/S) DG= 49.53 kJ + 5.69 kJ * log (P/S) 2. Replace P by the concentration desired: DG= 49.53 kJ + 5.69 kJ * log (0.0000001 /S) = 49.53 kJ - 39.83 kJ = +9.7 kJ 3. Conclusion: Reaction requires less energy to be run but is still not spontaneous as DG is positive. What about the other products and substrates ?

20. How to calculate actual reaction energetics by considering all P and S concentrations CH3-CH2OH + H2O --> CH3-COO- + H+ + 2 H2 DG= DGo + 5.69 kJ * log ((P1*P2*P3 ) / (S1*S2*S3)) Example : Does the reaction become favourable given that P1= aceate =1 mM, = 0.001 of std. cond. P2= protons = 10-7 M P3=H2 = 10 Pa = 0.0001 of std. cond. S1= ethanol = 10 mM = 0.01 of std. cond. 1. Use formula : DG= 49.53 kJ + 5.69 kJ * log (P1*P2*P3/(S1*S2*S3)) 2. Replace P and S by the concentrations desired: DG= 49.53 kJ + 5.69 kJ * log (0.001*10-7*(0.0001)2/(0.01*1))= DG= 49.53 kJ - 5.69 kJ * log 10-16= 49.53 kJ - 5.69 kJ * 16 = -41.51 kJ 3. Conclusion: Reaction is now thermodynamically possible and can support growth.

21. Visualising Effect of Substrate andProduct concentration on Reaction Energetics • Page 12:46 in study guide • The G calculations of the previous slides conclude that: •  [substrate]   energy released (more downhill) •  [product]   energy released (less downhill) +100 G 0 -100 +100 G 0 -100 0.1 1 10 100 0.1 1 10 100 Effect of substrate concentration on energetics of the reaction Effect of product concentration on energetics of reaction Remember: energy released means the change in energy is negative.  energy released means G becomes more negative

22. Visualising Effect of H2 on energetics of H2 consumption andProduction • Page 12:46 in study guide • The G calculations of the previous slides conclude that: •  [H2]   energy released for H2 consumption •  [H2]   energy released for H2 production +100 G 0 -100 +100 G 0 -100 0.1 1 10 100 0.1 1 10 100 Effect of H2 on energetics of H2 consumption Effect of H2 on energetics of H2 production Remember: energy released means the change in energy is negative.  energy released means G becomes more negative

23. Visualising Effect of H2 on energetics of H2 consumption andProduction • Page 12:46 in study guide • The G calculations of the previous slides conclude that: •  [H2]   energy released for H2 consumption •  [H2]   energy released for H2 production • There is only a narrow • concentration range at which • both reactions are energetically • favourable (G negative). • Both reactions can co-exist. • Both bacterial groups can • obtain energy. • Lowering H2 by one microbe • improves the energetic situation • of the other (energy sharing). +100 G 0 -100 0.1 1 10 100 Effect of H2 on energetics of H2 consumption and production

24. Visualising Effect of H2 on energetics of H2 consumption andProduction Page 12:46 in study guide 1,000,000 100,000 In text books [H2] is often plotted against G. 10,000 [H2] (ppm) 10 G positive endergonic “uphill” G=0 G negative exergonic “downhill”

25. Anaerobic digestion, a 3 stage process Complex organic matter (starch, cellulose, fats, protein) In contrast to aerobic degradation, AD requires different groups of specialised bacteria: Hydrolysis and fermentation Acetogenesis via hydrogen production Methanogenesis of acetate and H2 Fermentative Bacteria (e.g. Clostridia) Volatile fatty acids (VFA) alcohols, amino acids OHPA H2 CO2 Acetate MPB CO2 CH4

26. Anaerobic digestion, role of H2 concentration • 1/3 of the electron flow proceeds via H2 • H2 concentration is very low (1/ 40,000 of atmosphere = 40 ppm) but flux is high • If H2 increases higher than 100 ppm to 1000 ppm the OHPA can not operate any more (DG is positive) • Extremely low H2 level is critical • Digester overloading with easily fermetable organics (sugars) • fast H2 production • H2 production > H2 consumption • H2 accumulation • OHPA don’t convert organic acids • fermenting bacteria produce more VFA than H2 acetate • drop in pH • killing MPB • Overloading of anaerobic digesters with excess substrate results in failure due to H2 buildup • Bottleneck similar to Crabtree effect , acidification • Methanogenesis of acetate and H2

27. What is the expected CH4 concentration in biogas for different organics Compound Electons E/C OS %CH4 content Carbons Glucose 246 4 0 50 Ethanol 122 6 -2 75 Lactate 123 4 0 50 Propionate 143 4.7 -0.7 59 Butyrate 204 5 -1 62.5 Butanol 246 6 -2 75 Oxalate 22 1 3 12.5 Oxalate 22 1 3 12.5 Formate 21 2 2 25 Methanol 61 6 -2 75 Methane 81 8 -4 100 CO2 01 0 +4 0