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Hydrogen uptake thresholds and kinetics by methanogens in pure culture and defined mixed cultures of syntrophic hydrogen-producing and hydrogen-consuming bacteria
Abstract
The purpose of this dissertation was to improve the understanding of interspecies hydrogen transfer in methanogenic environments by studying the kinetics and thermodynamics of hydrogen uptake by methanogenic bacteria. It was shown that the half-saturation constant for hydrogen uptake (K$\sb{\rm m})$ by methanogenic bacteria was lower by an order of magnitude than previous studies (K$\sb{\rm m}$ = 386 nM for Methanobacterium formicicum). These values for the K$\sb{\rm m}$ were determined using the infinite dilution method, which avoided mass and phase transfer limitations by coculturing hydrogen-producing bacteria with methanogenic bacteria. The kinetic parameters for two methanogenic bacteria, M. formicicum and strain SR5M were compared. M. formicicum was enriched and isolated under 80% hydrogen and 20% carbon dioxide in the headspace, while SR5M was isolated from a butyrate-degrading enrichment that was serially transferred at least six times. Hydrogen uptake kinetics were fit to a modified form of the Michaelis-Menton equation with an added term for the hydrogen uptake threshold. It was found that SR5M had a lower maximum hydrogen uptake velocity (V$\sb{\rm max})$ and K$\sb{\rm m}$ (V$\sb{\rm max}$ = 7.1 $\mu$mol hydrogen per mg protein per min, K$\sb{\rm m}$ = 205 nM hydrogen) than M. formicicum (V$\sb{\rm max}$ = 11.0 $\mu$mol hydrogen per mg protein per min, K$\sb{\rm m}$ = 386 nM hydrogen). The hydrogen uptake thresholds of methanogenic bacteria were shown to be dependent on the thermodynamic properties of methanogenesis. As the parameters of the Nernst equation were varied, the hydrogen uptake threshold changed so as to maintain a constant available free energy $\rm(\Delta G\sb{r})$ from methanogenesis. The hydrogen uptake threshold for methanogenesis is 11-15 nM (15-20 ppm) and represents a $\rm\Delta G\sb{r}$ of $-$15 to $-$20 kJ/mol methane. This suggested that methanogenesis was coupled to an energy conservation step which had a minimum available free energy requirement, which is the translocation of protons by a membrane-bound ATPase to synthesize ATP. In order to test this hypothesis, hydrogen uptake was uncoupled from proton motive force generation (PMF) by the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). The PMF acts as the intermediate between methanogenesis and ATP synthesis. When CCCP was added to a culture of M. formicicum, the hydrogen uptake threshold fell to 1-2 ppm in the headspace, at which the $\rm\Delta G\sb{r}$ of methanogenesis is zero (thermodynamic equilibrium). The magnitude of the $\rm\Delta G\sb{r}$ from methanogenesis at the hydrogen uptake threshold was twice the electrochemical potential of protons $(\tilde{\mu}\sb{\rm H+}$ or PMF $\times$ F). This strongly suggests that two moles of protons are pumped from inside the cell to the outside per mole of methane produced during methanogenesis.
Subject Area
Microbiology|Ecology
Recommended Citation
Mobarry, Bruce Kevin, "Hydrogen uptake thresholds and kinetics by methanogens in pure culture and defined mixed cultures of syntrophic hydrogen-producing and hydrogen-consuming bacteria" (1993). Doctoral Dissertations Available from Proquest. AAI9408316.
https://scholarworks.umass.edu/dissertations/AAI9408316