Carbon Isotope Fractionation of 11 Acetogenic Strains Grown on H ₂ and CO ₂

Abstract

Acetogenic bacteria are able to grow autotrophically on hydrogen and carbon dioxide by using the acetyl coenzyme A (acetyl-CoA) pathway. Acetate is the end product of this reaction. In contrast to the fermentative route of acetate production, which shows almost no fractionation of carbon isotopes, the acetyl-CoA pathway has been reported to exhibit a preference for light carbon. In Acetobacterium woodii the isotope fractionation factor () for 13 C and 12 C has previously been reported to be ‫؍‬ ؊58.6‰. To investigate whether such a strong fractionation is a general feature of acetogenic bacteria, we measured the stable carbon isotope fractionation factor of 10 acetogenic strains grown on H 2 and CO 2 . The average fractionation factor was TIC ‫؍‬ ؊57.2‰ for utilization of total inorganic carbon and acetate ‫؍‬ ؊54.6‰ for the production of acetate. The strongest fractionation was found for Sporomusa sphaeroides ( TIC ‫؍‬ ؊68.3‰), the lowest fractionation for Morella thermoacetica ( TIC ‫؍‬ ؊38.2‰). To investigate the reproducibility of our measurements, we determined the fractionation factor of 21 biological replicates of Thermoanaerobacter kivui. In general, our study confirmed the strong fractionation of stable carbon during chemolithotrophic acetate formation in acetogenic bacteria. However, the specific characteristics of the bacterial strain, as well as the cultural conditions, may have a moderate influence on the overall fractionation.A cetate is a central intermediate in the anaerobic degradation of organic matter and originates either from fermentation of organic compounds or from reduction of CO 2 with H 2 via the acetyl coenzyme A (acetyl-CoA) pathway (acetogenesis). The produced acetate serves as an important substrate for other microbes, e.g., for acetoclastic methanogens. The pathways involved in acetate turnover can be differentiated if the fractionation factors (ε) of the individual processes (e.g., fermentation, acetogenesis, and methanogenesis) are known (1). The fractionation factor can be quantified from the natural abundance of 13 C in acetate, organic carbon, inorganic carbon, and methane, which can all be measured in environmental samples.In anoxic environments and pure cultures of anaerobic microorganisms mainly the acetate degradation processes (methanogenesis and sulfate or sulfur reduction) have been investigated for the isotopic fractionation factors (2-10). The production of acetate, however, is less well characterized. In general it has been reported that fermentation exhibits only a weak (Ͻ5‰) fractionation (11-13). On the other hand, acetate production via chemolithotrophic acetogenesis fractionates stronger. However, it has thus far only been studied in Acetobacterium woodii (2). There a mean fractionation factor of ε ϭ Ϫ58.6‰ Ϯ 0.7‰ was reported, with both branches of the acetyl-CoA pathway contributing equally. Thus, the fractionation factor for total acetate (ε acetate total ϭ Ϫ57.3‰ Ϯ 2.3‰) was similar to that of the methyl group of acetate (ε methyl ϭ Ϫ58.2‰ Ϯ 3.1‰). These dat...

Carbon Isotope Fractionation during Catabolism and Anabolism in Acetogenic Bacteria Growing on Different Substrates

“…Furthermore, it has been shown that there is no intramolecular fractionation during acetate formation: the isotope values of the methyl group were similar to the ␦ 13 C values of the overall acetate signal (35). The range of fractionations for different C 1 compounds reported in this study is well within the range reported previously (Ϫ38‰ to Ϫ68‰) for acetogenic cultures grown on H 2 /CO 2 (34,35). And now our data on the conversion of different C 1 compounds to acetate suggest similar discrimination against 13 C irrespective of the individual C 1 compound.…”

“…The data presented in this report confirmed the overall strong fractionation of acetogens using the acetyl-CoA pathway when H 2 /CO 2 was the substrate (34,35); in addition, it could be shown that the fractionation of cells grown on different C 1 compounds likewise resulted in very strong fractionation and was largely independent of substrate usage (different C 1 compounds). This is in contrast to the findings of several published reports where the rate-limiting step (usually the initial step of a reaction cascade) primarily determined the overall fractionation (20-23, 25, 27, 55, 58).…”

“…The acetyl-CoA pathway imparts strong isotopic enrichment of Ϫ55‰ to Ϫ60‰ during acetate formation with H 2 /CO 2 (80/ 20) as the substrate (34)(35)(36). In contrast, the fractionation associated with acetate fermentation by microbial glycolytic fermentation is weak (Ͻ5‰) (37)(38)(39)(40).…”

“…Therefore, we expect that two-thirds of the acetate released originates from the oxidative decarboxylation of pyruvate and the remaining one-third from the acetylCoA pathway. This suggests that the ␦ 13 C value is affected by both pathways and is a combination of the weak fractionation (Ͻ5‰) during glycolysis and oxidative decarboxylation (37)(38)(39)(40) and the strong fractionation (Ϫ38‰ to Ϫ68‰) caused by the acetyl-CoA pathway (34)(35)(36). To further test the contribution of each reaction, we added nitrate as an alternative electron acceptor, since nitrate can be used by some acetogens instead of the acetyl-CoA pathway to deplete the reducing equivalents produced by glycolysis (44)(45)(46).…”

Carbon flow from volcanic CO2 into soil microbial communities of a wetland mofette

“…Assuming an average isotopic fractionation (i.e., e acetate/CO2 ) of -57% (Gelwicks et al, 1989;Heuer et al, 2010;Blaser et al, 2013), we would expect acetogenesis to produce acetate with d 13 C-values around -59% in the mofette soil, where d 13 C CO2 is on average -1.95±0.06% (Bräuer et al, 2004). However, the observed d 13 C of pore-water acetate ranged from -33.6% to -13.9%.…”