A Bacterial Electron-bifurcating Hydrogenase

The Wood-Ljungdahl pathway of anaerobic CO(2) fixation with hydrogen as reductant is considered a candidate for the first life-sustaining pathway on earth because it combines carbon dioxide fixation with the synthesis of ATP via a chemiosmotic mechanism. The acetogenic bacterium Acetobacterium woodii uses an ancient version of the pathway that has only one site to generate the electrochemical ion potential used to drive ATP synthesis, the ferredoxin-fueled, sodium-motive Rnf complex. However, hydrogen-based ferredoxin reduction is endergonic, and how the steep energy barrier is overcome has been an enigma for a long time. We have purified a multimeric [FeFe]-hydrogenase from A. woodii containing four subunits (HydABCD) which is predicted to have one [H]-cluster, three [2Fe2S]-, and six [4Fe4S]-clusters consistent with the experimental determination of 32 mol of Fe and 30 mol of acid-labile sulfur. The enzyme indeed catalyzed hydrogen-based ferredoxin reduction, but required NAD(+) for this reaction. NAD(+) was also reduced but only in the presence of ferredoxin. NAD(+) and ferredoxin reduction both required flavin. Spectroscopic analyses revealed that NAD(+) and ferredoxin reduction are strictly coupled and that they are reduced in a 1:1 stoichiometry. Apparently, the multimeric hydrogenase of A. woodii is a soluble energy-converting hydrogenase that uses electron bifurcation to drive the endergonic ferredoxin reduction by coupling it to the exergonic NAD(+) reduction.     

Distribution of culturable microorganisms in Fennoscandian Shield groundwater

Microbial populations in 16 groundwater samples from six Fennoscandian Shield sites in Finland and Sweden were investigated. The average total cell number was 3.7×10⁵ cells ml⁻¹, and there was no change in the mean of the total cell numbers to a depth of 1390 m. Culture media were designed based on the chemical composition of each groundwater sample and used successfully to culture anaerobic microorganisms from all samples between 65 and 1350 m depth. Between 0.0084 and 14.8% of total cells were cultured from groundwater samples. Sulfate-reducing bacteria, iron-reducing bacteria and heterotrophic acetogenic bacteria were cultured from groundwater sampled at 65–686 m depth in geographically distant sites. Different microbial populations were cultured from deeper, older and more saline groundwater from 863 to 1350 m depth. Principal component analysis of groundwater chemistry data showed that sulfate- and iron-reducing bacteria were not detected in the most saline groundwater. Iron-reducing bacteria and acetogens were cultured from deep groundwater that contained 0.35–3.5 mM sulfate, while methanogens and acetogens were cultured from deep sulfate-depleted groundwater. In one borehole from which autotrophic methanogens were cultured, dissolved inorganic carbon was enriched in ¹³C compared to other Fennoscandian Shield groundwater samples, suggesting that autotrophs were active. It can be concluded that a diverse microbial community is present from the surface to over 1300 m depth in the Fennoscandian Shield.     

Dynamics of sulfidogenesis associated to methanogenesis in horizontal-flow anaerobic immobilized biomass reactor

Two bench-scale horizontal anaerobic fixed bed reactors were tested to remove both sulfate and organic matter from wastewater. First, the reactors (R1 and R2) were supplied with synthetic wastewater containing sulfate and a solution of ethanol and volatile fatty acids. Subsequently, R1 and R2 were fed with only ethanol or acetate, respectively. The substitution to ethanol in R1 increased the sulfate reduction efficiency from 83% to nearly 100% for a chemical oxygen demand to sulfate (COD/sulfate) ratio of 3.0. In contrast, in R2, the switch in carbon source to acetate strongly decreased sulfidogenesis and the maximum sulfate reduction achieved was 47%. Process stability in long-term experiments and high removal efficiencies of both organic matter and sulfate were achieved with ethanol as the sole carbon source. The results allow concluding that syntrophism instead of competition between the sulfate reducing bacteria and acetoclastic methanogenic archaeal populations prevailed in the reactor.     

Glycolate as a metabolic substrate for the acetogen Moorella thermoacetica

Glycolate was growth supportive for Moorella thermoacetica ATCC 39073. Growth occurred in undefined and basal culture media containing 20 mM glycolate and was proportional to the concentration of glycolate (10–40 mM). Nitrate inhibited glycolate-dependent growth in the basal medium. Acetate and cell biomass were the major end products recovered from glycolate. The molar ratio (moles of glycolate required to synthesize a mole of acetate) averaged 1.4 and was in close agreement with the theoretical ratio of 1.3 for glycolate-derived acetogenesis. Glycolate-dependent growth yielded approximately 3-fold less biomass per pair of reducing equivalents utilized than did oxalate- or glyoxylate-dependent growth.     

Characterization of the psychrotolerant acetogen strain SyrA5 and the emended description of the species Acetobacterium carbinolicum

A psychrotolerant, obligate anaerobic, acetogenic bacterium designated strain SyrA5 was isolated from black anoxic sediment of a brackish fjord. Cells were Gram-positive, non-sporeforming rods. The isolate utilized H(2)/CO(2), CO, fructose, glucose, ethanol, ethylene glycol, glycerol, pyruvate, lactate, betaine and the methyl-groups of several methoxylated benzoic derivatives such as syringate, trimethoxybenzoate and vallinate. The optimum temperature for growth was 29 degrees C, whilst slow growth occurred at 2 degrees C. The strain grew optimally with NaCl concentrations below 2.7% (w/v), but growth occurred up to 4.3% (w/v) NaCl. Growth was observed in the range from pH 5.9 to 8.5, optimum at pH 8. The G+C content was 44.1 mol%. Based upon 16S rRNA gene sequence analysis and DNA-DNA reassociation studies, the organism was classified in the genus Acetobacterium. Strain SyrA5 shared a 16S rRNA sequence similarity with A. carbinolicum of 100%, a fthfs gene (which codes for the N5,N10 tetrahydrofolate synthetase) sequence identity of 98.5-98.7% (amino acid sequence similarities were 99.4-100%) and a RNA-DNA hybridization homology of 64-68%. Despite a number of phenotypic differences between strain SyrA5 and A. carbinolicum we propose including strain SyrA5 as a subspecies of A. carbinolicum for which we propose the name Acetobacterium carbinolicum subspecies kysingense. The type strain is SyrA5 (=DSM 16427(T), ATCC BAA-990).     

Characterization of two strains of Pelobacter carbinolicus isolated from anaerobic digesters

In anaerobic industrial digesters treating wastewaters from food industry, the sludges showed high capacities to degrade ethanol. The main syntrophic ethanoldegrading organisms were characterized as Pelobacter carbinolicus. During acetoin degradation, butanediol isomers were shown to be transiently produced. In coculture with Methanobrevibacter arboriphilus, the strains oxidized not only primary monoalcohols but also 1,2- and 1,3-diols. Ecological importance as well as metabolic pathways of diol degradation are discussed.