It does not always take two to tango: "Syntrophy" via hydrogen cycling in one bacterial cell

Abstract

Interspecies hydrogen transfer in anoxic ecosystems is essential for the complete microbial breakdown of organic matter to methane. Acetogenic bacteria are key players in anaerobic food webs and have been considered as prime candidates for hydrogen cycling. We have tested this hypothesis by mutational analysis of the hydrogenase in the model acetogen Acetobacterium woodii. Hydrogenase-deletion mutants no longer grew on H₂ + CO₂ or organic substrates such as fructose, lactate, or ethanol. Heterotrophic growth could be restored by addition of molecular hydrogen to the culture, indicating that hydrogen is an intermediate in heterotrophic growth. Indeed, hydrogen production from fructose was detected in a stirred-tank reactor. The mutant grew well on organic substrates plus caffeate, an alternative electron acceptor that does not require molecular hydrogen but NADH as reductant. These data are consistent with the notion that molecular hydrogen is produced from organic substrates and then used as reductant for CO₂ reduction. Surprisingly, hydrogen cycling in A. woodii is different from the known modes of interspecies or intraspecies hydrogen cycling. Our data are consistent with a novel type of hydrogen cycling that connects an oxidative and reductive metabolic module in one bacterial cell, "intracellular syntrophy."

Fig. 1. The modularity of acetogenesis in A. woodii.

Shown are the oxidation of fructose to acetate in the oxidative branch (left) and the reduction of CO₂ to acetate (right) in the reductive branch (WLP). Redox balancing is achieved by a third module, in which the Rnf complex and the electron-bifurcating hydrogenase produce the reductants required for the the WLP. Fd, ferredoxin; Fd²⁻, reduced ferredoxin; THF, tetrahydrofolate; HDCR, hydrogen-dependent CO₂ reductase; CODH/ACS, carbon monoxide dehydrogenase/acetyl-CoA synthetase; Co-FeS-P, corronoid iron-sulfur protein.

Fig. 2. Verfication of the hydBA deletion in A. woodii ΔpyrEvia PCR and Western Blot analysis.

Agarose gel showing PCR products of the wild type (WT), pyrE (ΔpyrE) and hydBA mutant (ΔhydBA) using primers which anneal outside the hydBA region (a). Detection of HydB and HydA in crude extract of the wild type and hydBA mutant blotted on a nitrocellulose membrane and by using antibodies against HydB and HydA (b). M, Marker; NC, negative control; WT, A. woodii wild type.

Fig. 3. Growth and metabolite concentrations in A. woodii wild type and hydBA mutant in bicarbonate-supplied medium supplemented with 20 mM fructose + hydrogen.

Displayed are the optical densities of the wild type (■) and the hydBA mutant (▲) as well as the hydrogen concentration in the headspace of the wild type (×) and the hydBA mutant (+) cultures (a). The fructose consumption of the wild type (◇) and hydBA mutant (▽) together with the concomitant production of acetate in the wild type (◆) and hydBA (▼) mutant was monitored over time (b). (n = 2).

Fig. 4. Off-line and off-gas data for stirred-tank reactor fermentations of A. woodii wild type.

The wild type was grown in complex medium with 20 mM fructose as carbon and energy source and the cultivation broth was sparged with 10 ml min⁻¹ 20 % CO₂ in nitrogen. The upper half shows the average off-line values for OD₆₀₀ (◆), concentration of fructose (■) and acetic acid (▲) of four cultivations whilst the bottom half shows off-gas flow rates for hydrogen (solid line) and carbon dioxide (dashed line). The grey areas in the bottom half depict the variation of the off-gas values.

Fig. 5. Schematic overview of fructose metabolism of A. woodii using formate instead of CO₂ as electron acceptor in the reductive branch.

Fd, ferredoxin; Fd²⁻, reduced ferredoxin.

Fig. 6. Schematic overview of the fructose metabolism of A. woodii using caffeate as final electron acceptor and growth of A. woodii wild type and the hydBA mutant on different carbon sources with caffeate as electron acceptor.

Fd, ferredoxin; Fd²⁻, reduced ferredoxin (a). Cells were grown in complex medium supplemented with either 20 mM fructose + 6 mM caffeate (b), 80 mM lactate + 4 mM caffeate (c) or 50 mM ethanol + 4 mM caffeate (d). Growth of the wild type + caffeate (■), hydBA mutant + caffeate (▲), wild type without caffeate (□), hydBA mutant without caffeate (△) were measured over time at 600 nm. Caffeate was added when concentrations dropped below 1 mM and its utilisation by the wild type (×) and hydBA mutant  (+) was monitored over time (n = 2).