Download T10 AD bioenergetics

Bioenergetics of Anaerobic Microbial
Reactions
Traditional and new processes
Methanogenesis
Archaebacteria
All methane producing bacteria (MPB) are archeabacteria
Archeabacteria are different from all other life forms
(3rd primary kingdom)
Eukaryotes
Plants
Animals
Fungi 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
Archeabacteria
Methanogens
Halobacterium etc.
Prokaryotes
Eubacteria
Cyanobacteria etc.
2. No murein in cell wall (→ penicillin resistant)
3. Different protein synthesis (→ streptomycin resistant)
4. More subunits in RNA polymerase
5. Unique coenzymes (e.g. F 420)
Special features in archaebacteria:
High temperature tolerance (115°C)
Acid resistance (pH 1, at 90°C)
Unique use of light ( no photosystem)
Pyrodiction
Sulfolobus
Halobacterium
Archaebacteria are likely to be the earliest life forms
(Symbiont hypothesis)
Methanogenesis
Pathway of CO2 Reduction
CO2
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H2
MF
MP MFCOH
H2
MF
MPCH2
H2
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Methylene - C
MPCH3
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Methyl - C
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Methane
CoM
MP
CoMCH3
CoM
ATP
CH4
Formyl - C
H2
Only the last step allows the generation of 1ATP
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
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
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• In the presence of hydrogen methanol is only used as
electron acceptor → methyl – respiration.
H2 + CH3OH → CH4 + H2O
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• 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
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Methyl - respiration is the only way of ATP generation in
methanogenesis
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
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.
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
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.
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Theory of Bryant on Interspecies Hydrogen Transfer
Hypothesis of Marvin P. Bryant: Interspecies Hydrogen Transfer,
CO2
CH4
Ethanol
H2
Acetate
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)
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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
CO2
CH4
Ethanol
H2
Acetate
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)?
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Bryant’s hypothesis was not readily accepted:
CO2
CH4
Ethanol
H2
Acetate
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Energy Sharing
DGo'=
+ 9.6 kJ/mol
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)
DGo'=
- 135.6 kJ/mol
CH4
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)
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Energy Sharing
DGo'=
+ 9.6 kJ/mol
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.
DGo'=
- 135.6 kJ/mol
CH4
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.
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Significance of Interspecies Hydrogen Transfer
Eventually Bryan’s hypothesis was not
only confirmed but found to be a general
feature of anaerobic systems (e.g. rumen,
anaerobic digesters, sediments)
DGo'=
+ 9.6 kJ/mol
DGo'=
- 135.6 kJ/mol
CH4
CO2
Ethanol
H2
Acetate
The interspecies hydrogn can be
intercepted by chemical and biological
means (more powerful H2 users than
methanogens.
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)
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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
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same reaction?
Considering pH for thermodynamic calculations
DG= 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 ?
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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.
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Visualising Effect of Substrate and Product
concentration on Reaction Energetics
Page 12:46 in study guide
The G calculations of the previous slides conclude that:
1.  [substrate]   energy released (more downhill)
2.  [product]   energy released (less downhill)
+100
+100
G 0
G 0
0.1
1
10
100
-100
Effect of substrate concentration
on energetics of the reaction
0.1
1
10
100
-100
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
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Visualising Effect of H2 on energetics of H2
consumption and Production
Page 12:46 in study guide
The G calculations of the previous slides conclude that:
1.  [H2]   energy released for H2 consumption
2.  [H2]   energy released for H2 production
+100
+100
G 0
G 0
0.1
1
10
-100
Effect of H2 on energetics
of H2 consumption
100
0.1
1
10
100
-100
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
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Visualising Effect of H2 on energetics of H2
consumption and Production
Page 12:46 in study guide
The G calculations of the previous slides conclude that:
1.  [H2]   energy released for H2 consumption
2.  [H2]   energy released for H2 production
There is only a narrow
concentration range at which
both reactions are energetically
favourable (G negative).
+100
G 0
0.1
1
10
100
-100
Effect of H2 on energetics
of H2 consumption and production
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).
Visualising Effect of H2 on energetics of H2
consumption and Production
Page 12:46 in study guide
1,000,000
100,000
In text books
[H2] is often plotted
against G.
10,000
[H2]
(ppm)
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G positive
endergonic
“uphill”
G=0
G negative
exergonic
“downhill”
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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:
Fermentative Bacteria
(e.g. Clostridia)
Hydrolysis and fermentation
Volatile fatty acids (VFA)
alcohols, amino acids
Acetogenesis via hydrogen
production
OHPA
H2
CO2
Methanogenesis of acetate and
H2
Acetate
MPB
CO2
CH4
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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
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What is the expected CH4 concentration in
biogas for different organics
Compound
Glucose
Ethanol
Lactate
Propionate
Butyrate
Butanol
Oxalate
Oxalate
Formate
Methanol
Methane
CO2
Electons
Carbons
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122
123
143
204
246
22
22
21
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E/C
OS
%CH4 content
4
6
4
4.7
5
6
1
1
2
6
8
0
0
-2
0
-0.7
-1
-2
3
3
2
-2
-4
+4
50
75
50
59
62.5
75
12.5
12.5
25
75
100
0
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