Abstract
Clostridium acetobutylicum has traditionally been used for production of acetone, butanol, and ethanol (ABE). Butanol is a commodity chemical due in part to its suitability as a biofuel; however, the current yield of this product from biological systems is not economically feasible as an alternative fuel source. Understanding solvent phase physiology, solvent tolerance, and their genetic underpinning is key for future strain optimization of the bacterium. This study shows the importance of a [NiFe]-hydrogenase in solvent phase physiology. C. acetobutylicum genes ca_c0810 and ca_c0811, annotated as a HypF and HypD maturation factor, were found to be required for [NiFe]-hydrogenase activity. They were shown to be part of a polycistronic operon with other hyp genes. Hydrogenase activity assays of the ΔhypF/hypD mutant showed an almost complete inactivation of the [NiFe]-hydrogenase. Metabolic studies comparing ΔhypF/hypD and wild type (WT) strains in planktonic and sessile conditions indicated the hydrogenase was important for solvent phase metabolism. For the mutant, reabsorption of acetate and butyrate was inhibited during solventogenesis in planktonic cultures, and less ABE was produced. During sessile growth, the ΔhypF/hypD mutant had higher initial acetone: butanol ratios, which is consistent with the inability to obtain reduced cofactors via H2 uptake. In sessile conditions, the ΔhypF/hypD mutant was inhibited in early solventogenesis, but it appeared to remodel its metabolism and produced mainly butanol in late solventogenesis without the uptake of acids. Energy filtered transmission electron microscopy (EFTEM) mapped Pd(II) reduction via [NiFe]-hydrogenase induced H2 oxidation at the extracelluar side of the membrane on WT cells. A decrease of Pd(0) deposits on ΔhypF/hypD comparatively to WT indicates that the [NiFe]-hydrogenase contributed to the Pd(II) reduction. Calculations of reaction potentials during acidogenesis and solventogenesis predict the [NiFe]-hydrogenase can couple NAD+ reduction with membrane transport of electrons. Extracellular oxidation of H2 combined with the potential for electron transport across the membrane indicate that the [NiFe}-hydrogenase contributes to proton motive force maintenance via hydrogen cycling.
Highlights
Solventogenic Clostridia have been used to produce the commodity chemicals acetone, butanol, and ethanol from renewable feedstocks via the acetone, butanol, and ethanol (ABE) fermentation process [1,2]
Increased expression during solvent phase suggests the C. acetobutylicum [NiFe]-hydrogenase is important for solventogenic growth phase, but its exact role is not understood
Wild type and derivative strains were maintained as anaerobic spore suspensions at room temperature in potato glucose medium (PGM) containing: 150.0 g·L−1 potato, 10.0 g·L−1 glucose, 0.5 g·L−1 (NH4 )2 SO4, and 3.0 g·L−1 CaCO3 [15]
Summary
Solventogenic Clostridia have been used to produce the commodity chemicals acetone, butanol, and ethanol from renewable feedstocks via the acetone, butanol, and ethanol (ABE) fermentation process [1,2]. Internal production of H2 by the [FeFe]-hydrogenase increases intracellular pH (via proton reduction) and it provides oxidized ferredoxin for central metabolism [8,10]. An siRNA knock down of the [NiFe]-hydrogenase in C. saccharoperbutylacetonicum N1, to our knowledge the only published study of Clostridial [NiFe]-hydrogenase activity, showed a marked decrease in butanol production, thereby indicating physiological evidence of hydrogen uptake [13]. Increased expression during solvent phase suggests the C. acetobutylicum [NiFe]-hydrogenase is important for solventogenic growth phase, but its exact role is not understood. The hypF/hypD mutant displayed altered metabolism, as consistent with a defect in hydrogen uptake, resulting in decreased ABE output and altered product ratios in early solventogenesis. Together with energetic calculations and sequence analysis, the results indicate the [NiFe]-hydrogenase likely couples H2 oxidation with electron transport and intracellular NAD+. Reduction, thereby acting as a crude proton pump that conserves energy via hydrogen cycling
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