CEE 629 – Fall 2003 Environmental Microbial Biotechnology
Class notes – 9/15/05 Microbial Metabolic Diversity
Microbial Metabolic Diversity Broadly speaking, we will be considering the following questions: How do microbes make a living?
What chemical transformations are involved?
Which microbes are doing what?
Ultimately, we would like to address more advanced questions: Can we predict the microbial community based on the resident geochemistry?
Can we predict the geochemistry from the resident microbiology?
How can we control or harness microbe-mediated chemical transformations?
I. How do microbes make a living? The study of microbial metabolism is the study of how microbes make a living. Recall that “metabolism” encompasses both catabolism and anabolism. Catabolism = biochemical breakdown/transformation of compounds to produce energy Anabolism = biochemical synthesis/transformation of compounds to produce cell constituents Recall from general chemistry that thermodynamically unfavorable reactions (generally anabolic) can be driven by favorable reactions (generally catabolic). Thus, metabolism is a description of how microbes couple energy-generating reactions to energy-requiring reactions. Microbes are often broadly classified based on their modes of metabolism*: Examples: Chemoorganotrophs = energy from organic chemicals Chemolithotrophs = energy from inorganic chemicals Phototrophs = energy from light Heterotroph = carbon from organic compounds Autotroph = carbon from CO2 * Note that it is more appropriate to think in terms of classifying metabolisms (e.g. chemoorganotrophy) since some microbes can carry out multiple types of metabolism, depending on the environment they are living in.
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CEE 629 – Fall 2003 Environmental Microbial Biotechnology
Class notes – 9/15/05 Microbial Metabolic Diversity
In this class we are concerned with 1) harnessing the metabolic diversity of microbes to carry out biochemical transformations to benefit the environment; and 2) predicting and modeling the behavior of microbes and their communities that impact the environment. It is essential that we understand the physical and chemical limitations to metabolic diversity. To do this, we will consider further the concept of “bioenergetics”.
II. Bioenergetics In order to discuss how microbes use a series of individual chemical transformations to generate energy, we must introduce some principles of thermodynamics. A. Change in free energy Free energy = the amount of energy released during a reaction that is available to do useful work Definition of G For a (bio)chemical reaction, we introduce the concept of change in free energy
G o =
G of (products) - G of (reactants)
Where Gfo refers to the free energy of formation for a compound. Usually, Gfo values can be found in a reference table (see handout from Thauer et al. 1977). Some definitions: Go = standard free-energy change at 1 atm pressure and 1 M concentrations Go’ = free-energy change at standard conditions but pH = 7 G = free-energy change at conditions specified G’ = free-energy change at conditions specified but pH = 7 EXERGONIC reactions: If G 0, the reaction can take place spontaneously ENDERGONIC reactions: If G > 0, the reaction cannot take place spontaneously, but may be driven by an input of energy 3
CEE 629 – Fall 2003 Environmental Microbial Biotechnology
Class notes – 9/15/05 Microbial Metabolic Diversity
Example: What is the Go for conversion of acetate (CH3COO-) to HCO3- by the sulfate-reducing bacterium Desulfobacter postgatei? Calculation of Go for CH3COO- + SO42- 2 HCO3- + HSObtain Gfo from tables: HCO3- (aq)
Gfo =
-586.85 kJ
HS- (aq)
Gfo =
+12.05 kJ
CH3COO- (aq)
Gfo =
-369.41 kJ
SO42- (aq)
Gfo =
-744.63 kJ
Calculate Go Go = [2x(-586.85) + 12.05] – [-369.41 - 744.63] =
-47.61 kJ
(exergonic)
Note this example dealt with standard conditions, with all reactants and products at 1 mole per liter. However, standard conditions do not prevail in natural habitat The free energy of a reaction depends on the concentrations or partial pressures (if gas) of the reactants and products and is calculated from Go as follows: For the reaction aA + bB cC + dD
we calculate G using : G = G o + RT ln
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[C]c [D]d [A]a [B]b
CEE 629 – Fall 2003 Environmental Microbial Biotechnology
Class notes – 9/15/05 Microbial Metabolic Diversity
where [A] = actual concentration of A in moles per liter or in atm if a gas With R = 8.314 J K-1 mol-1 and ln(x) = 2.203 log(x), we obtain
G = G o + 5.71 log
[C]c [D]d [A]a [B]b
at 25 o C
Although the concentrations of substrates and products in the microbes’ environment are almost always lower than 1 M, it is common to calculate free energies for standard conditions. However, the actual concentration of H+ ions (i.e. the pH) is often taken into consideration. By convention, Go’ is the G of a reaction under standard conditions except for the H+ concentration which is 10-7 M. Example: What is the G for hydrogenotrophic methanogenesis under typical conditions found in lake sediment? HCO3- + 4H2 + H+ CH4 + 3H2O a) First calculate Go (under standard conditions) Obtain Gfo from tables: CH4 (g)
Gfo =
-50.45 kJ
H2O (aq)
Gfo =
-237.18 kJ
HCO3- (aq)
Gfo =
-586.85 kJ
4H2 (g)
Gfo =
0 kJ
H+ (aq)
Gfo =
0 kJ
Calculate Go Go = [-50.45 + 3x(-237.18)] – [-586.85 + 4x(0) + 0] = -175.14 kJ (exergonic)
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CEE 629 – Fall 2003 Environmental Microbial Biotechnology
Class notes – 9/15/05 Microbial Metabolic Diversity
b) Calculate G under typical conditions We can first look at Go’
G o ' = G o + 5.71 log
1 [H + ]
G o ' = 175.14 + 5.71 log
1 = -175.14 + 39.97 = -135.17 kJ/mol 10 -7
H2 in the headspace above methanogenic lake sediment is typically only a few pascals (1 Pa = 9.9 x 10-6 atm). How does this affect the thermodynamics of methane formation?
G' = G o '+5.71 log
PCH 4 [HCO-3 ]
- 5.71 log PH2 4
Assume [HCO3- ]= 10 mM and PCH4 = 0.5 atm. G’ = -135.17 + 5.71 log (50) – 22.8 log PH2 50 25
endergonic
0 exergonic
-25
G ' -50 (kJ / rxn) -75 -100 -125
HCO3- + 4 H2 + H+ --> CH4 + 3 H2O
-150 -7
-6
-5
-4
-3
-2
-1
0
Hydrogen partial pressure (log PH2)
G’ = -125 – 22.8 log PH2 = dependence of G’ on H2 partial pressure
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CEE 629 – Fall 2003 Environmental Microbial Biotechnology
Class notes – 9/15/05 Microbial Metabolic Diversity
We can derive a plot for any reaction that has hydrogen as either a reactant or a product. (For you to think about: how would the graph look for a reaction in which hydrogen is a product?) For a sulfate reducer 4 H2 + SO42- + H+ 4 H2O + HS-,
Go’ = -152 kJ/rxn
For an acetogen 4 H2 + 2HCO3- + H+ acetate + 4 H2O,
Go’ = -105 kJ/rxn
See handout (graph from Zinder 1993)
Conclusion: G’ for the reaction in situ is very dependent on the local PH2, which can be 10-4 to 10-5 atm. This has important implications for who can make a living in this type of environment.
III. Capturing energy: ATP synthesis Regardless of the way in which microbes generate energy, they must all have a mechanism for harnessing this energy in order to generate ATP. ATP = the universal molecular carrier of biological energy Energy-productivity = ATP production • When thinking about the potential productivity of microbial energyproducing chemical reactions, we must consider in terms of ATP production. • Knowing the Go’ (or even G’) of a particular energy producing chemical system is not helpful outside of the context of ATP production. • Must know if the energy yield is sufficient to support the production of ATP (i.e. is it worth it?) We need to know the amount of energy required to drive ATP production under conditions found within microbial cells (that is, on-site).
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CEE 629 – Fall 2003 Environmental Microbial Biotechnology
Class notes – 9/15/05 Microbial Metabolic Diversity
Convention is to treat the reaction in the direction of ATP hydrolysis ATP + H2O ADP + Pi
Go’ = -30.5 kJ/rxn (controversial?)
To calculate G’, we need to know intracellular levels of ATP, ADP, Pi Typical(?): [ATP] = 2 mM; [ADP] = 1 mM; [Pi] = 10 mM
G' = G o '+5.71 log
G' = 30.5 + 5.71 log
[ADP][Pi ] [ATP]
(0.001)(0.01) = - 30.5 -13.1 = - 43.6 kJ/mol (0.002)
Thus, the minimal free energy required for the synthesis of 1 mol ATP is approximately 40 to 50 kJ However, neither energy conservation during catabolism nor energy utilization during anabolism occurs with 100% efficiency. Part of the energy is always lost as heat or through increased entropy. Therefore, we can assume that synthesis of 1 mole of ATP requires > the minimal free energy calculated above, perhaps as high as 70 kJ/mole. Thus, if an energy generating reaction yields less than 50 kJ/mole, the reaction will have to turn of > 1 mole of substrate for the synthesis of 1 mole ATP.
IV. Redox equilibrium The energy-generating reactions described in part II above are all oxidationreduction reactions. That is, one reactant is oxidized and another is reduced. Microbes are remarkably versatile when it comes to pairing different chemicals as oxidants and reductants, in order to generate energy. To more fully understand the extent of this versatility, it is useful to split the reactions into their component oxidation and reduction half reactions for examination. Some terminology:
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CEE 629 – Fall 2003 Environmental Microbial Biotechnology
Class notes – 9/15/05 Microbial Metabolic Diversity
Reductant = compound that reacts by releasing electrons (donor) Oxidant = compound that reacts to take up electrons (acceptor) During a redox reaction, the reductant is oxidized and the oxidant is reduced. Express overall reaction as: A(ox) + B(red) A(red) + B(ox) Comprised of two half reactions: 1. A(ox) + n e- A(red) 2. B(red) n e- + B(ox) In this example, A is the electron acceptor and B is the electron donor. For a given pair of chemicals, how do we know which one will serve as the donor, and which will serve as the acceptor?? Each chemical has a particular tendency to give up or accept electrons. This tendency is expressed as the reduction potential (Eo, at standard conditions). Each half reaction has an associated Eo. The Eo is always referenced to the reaction written in the following form: A(ox) + n e- A(red). For most biochemically relevant compounds, reduction potentials can be looked up in a table. For a given redox couple (ie pair of half reactions), the one with a more positive Eo will always be written in the forward direction (the oxidized form will become reduced). This will become more obvious soon. By convention, reduction potentials are almost always given for pH 7 (Eo’). See handout on electron tower. Example: Nitrate reduction coupled to hydrogen oxidation Nitrate: NO3- + 2 e- + 2 H+ NO2- + H2O
Eo’ = +0.42 V
Hydrogen:
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CEE 629 – Fall 2003 Environmental Microbial Biotechnology
2 H+ + 2 e- H2
Class notes – 9/15/05 Microbial Metabolic Diversity
Eo’ = -0.42 V
In this example, since the Eo’ for nitrate is more positive than the Eo’ for hydrogen, the nitrate will become reduced. Net reaction: NO3- + H2+ NO2- + H2O How can we relate reduction potentials to free energy calculations? We know that redox reactions lead to energy generation. It turns out that the change in free energy associated with a reaction is proportional to the difference in reduction potential between the oxidant and reductant.
Redox potential and G G = - n F E where G = change in free energy associated with the redox reaction n = number of electrons transferred F = Faraday constant (98.48 kJ/V) E = difference in the redox potential of the two half reactions similarly, for standard conditions
Go’ = - n F Eo’ Note that, in order to be exergonic, a reaction must have a POSITIVE E. For a redox reaction A(ox) + B(red) A(red) + B(ox) G at any concentration of reactants and products becomes
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CEE 629 – Fall 2003 Environmental Microbial Biotechnology
G = G o + RT ln
[A(red)][B(ox)] [A(ox)][B(red)]
- nFE = - nFE o + RT ln
E = E o +
Class notes – 9/15/05 Microbial Metabolic Diversity
[A(red)][B(ox)] [A(ox)][B(red)]
RT [A(ox)][B(red)] ln nF [A(red)][B(ox)]
Thus, we can determine E for any conditions. Note we usually find E’ in tables, so this may need to be taken into consideration. For the two half reactions below, E = EA – EB A(ox) + n e- A(red) EA B(ox) + n e B(red) EB In order for reaction to proceed, E must be positive (so that G is negative). So, if EA is more positive than EB, the following reaction will occur: A(ox) + B(red) A(red) + B(ox) We can then look individually at the two reduction potentials:
RT [A(ox)] RT [B(ox)] ln ln nF [A(red)] nF [B(red)] This is the difference, (a) – (b), in the two reactions E = E A E B = E A o - E B o +
(a)
EA = EA o +
RT [A(ox)] ln nF [A(red)]
and (b)
EB = EBo +
RT [B(ox)] ln nF [B(red)]
These are the potentials of the half reactions.
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CEE 629 – Fall 2003 Environmental Microbial Biotechnology
Class notes – 9/15/05 Microbial Metabolic Diversity
The redox potential of a half reaction is given by the Nernst
E = Eo -
equation:
RT [reduced form] ln nF [oxidized form]
With T = 25o C and the constants:
E = Eo -
0.059 [reduced form] ln Eh n [oxidized form]
The symbol Eh is used instead of E for the redox potential of a half reaction at any given concentration. Can we use what we know about redox potentials to predict feasible microbial processes, and begin to address our more advanced questions (page 1)?
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