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

Abstract

Homoacetogenic bacteria are versatile microbes that use the acetyl coenzyme A (acetyl-CoA) pathway to synthesize acetate from CO 2 and hydrogen. Likewise, the acetyl-CoA pathway may be used to incorporate other 1-carbon substrates (e.g., methanol or formate) into acetate or to homoferment monosaccharides completely to acetate. In this study, we analyzed the fractionation of pure acetogenic cultures grown on different carbon substrates. While the fractionation of Sporomusa sphaeroides grown on C 1 compounds was strong ( C1 , ؊49‰ to ؊64‰), the fractionation of Moorella thermoacetica and Thermoanaerobacter kivui using glucose ( Glu ‫؍‬ ؊14.1‰) was roughly one-third as strong, suggesting a contribution of less-depleted acetate from fermentative processes. For M. thermoacetica, this could indeed be validated by the addition of nitrate, which inhibited the acetyl-CoA pathway, resulting in fractionation during fermentation ( ferm ‫؍‬ ؊0.4‰). In addition, we determined the fractionation into microbial biomass of T. kivui grown on H 2 /CO 2 ( anabol. ‫؍‬ ؊28.6‰) as well as on glucose ( anabol. ‫؍‬ ؉2.9‰).A cetogenic bacteria are a widespread microbial group unified by their ability to use the reductive acetyl coenzyme A (acetylCoA) pathway, which allows chemolithoautotrophic growth on hydrogen and carbon dioxide and is the only known pathway that combines carbon dioxide fixation with energy conservation (1-6). Most acetogens can use the reductive acetyl-CoA pathway for autotrophic growth, reducing 2 carbon dioxide molecules with 4 hydrogen molecules to liberate 1 acetate molecule. Under these conditions, CO 2 reduction with H 2 yields a ⌬G o = value of Ϫ95 kJ/mol. This is barely enough to generate 1 ATP molecule using either a sodium-or a proton-dependent electron motive force (7). On the other hand, the acetyl-CoA pathway can be accompanied by the fermentation of carbon substrates. Under these autotrophic conditions, the energy yield for, e.g., glucose fermentation to 3 acetate molecules is expressed by the equation ⌬G o = ϭ Ϫ310.9 kJ/mol. In contrast to fermentations of glucose by most other anaerobes, which yield only 1 to 3 ATP molecules via substratelevel phosphorylation during glycolysis, acetogens can redirect the reducing equivalents to the reductive acetyl-CoA pathway, allowing the generation of an additional ATP molecule as described above (3,8).One approach to evaluating the contributions of different bacterial pathways to certain compound pools in environmental systems is to use the natural abundance of stable carbon isotopes to track the metabolic activity of certain microorganisms in the environment (9, 10): In principle, each metabolic pathway is characterized by a specific preference for a certain carbon isotope (usually the lighter 12 C) during catalysis, resulting in a characteristic depletion of 13 C between the substrate and the end product. Mathematically, this depletion is characterized by the so-called apparent fractionation factor (␣) or enrichment factor (ε); ε is calculated as 10 3 (1 Ϫ ␣). For...

Illuminating microbial species‐specific effects on organic matter remineralization in marine sediments

“…Previous studies suggest that most of the acetate and/or other low MW organic acids in marine sediments appear to be generated through heterotrophic rather than autotrophic acetogenesis (Heuer et al ., ; Ijiri et al ., ; Lever et al ., ), consistent with the idea that autotrophic acetogens would be easily outcompeted by other microbes (e.g., methanogens) in marine sediments due to the very low energy yield of this metabolism (Lever, ). Thus, for the acetate and/or other fermentation products endmember (referred to generically as acetate), the δ 13 C value was set to −44 ± 1‰ for both on‐ and off‐axis sediment incubations, based on the expected fractionation in heterotrophic acetogenesis that would yield acetate depleted in 13 C by ~19.5‰ (Freude and Blaser, ), relative to the initial organic carbon pool. For the on‐axis sediment incubations, we constrained the ranges of possible Δ 14 C signatures and set the Δ 14 C value of acetate to −400 ± 50‰, which suggests hydrothermal‐derived petroleum compounds are the likely source of substrates for fermentation.…”

Spatial and temporal trends in Precambrian nitrogen cycling: A Mesoproterozoic offshore nitrate minimum

“…Diagenetic effects can be larger (on the order of ~20‰ depending on the substrate) if organic degradation is catalyzed by microbes using the Wood-Ljungdahl pathway, which includes methanogens, methanotrophs, and acetogens (Freude and Blaser, 2016). The range of δ 13 C org in the Bangemall samples is too large (~10‰) to be explained solely by diagenetic effects that do not involve the Wood-Ljungdahl pathway.…”

“…However, the fact that multiple basins through time show a similar trend suggests there may be (i) autotrophic carbon fixation under a gradient of nutrient availability, or (ii) photoautotrophic biomass mixed with varying proportions of secondary chemoautotrophic and/or heterotrophic biomass, as proposed for the contemporaneous Belt and Jixian basins (Guo et al, 2013;Stüeken, 2013). Such secondary metabolisms could include methanogenesis, acetogenesis, and methanotrophy that use the Wood-Ljungdahl pathway and are known to cause large fractionations in carbon isotopes (Freude and Blaser, 2016) We cannot rule out either possibility, and it is also not clear why the reverse gradient is seen in the Mesoproterozoic Belt basin (Stüeken, 2013). The samples from the Roper Group do not show a gradient in δ 13 C org values.…”

Illuminating microbial species-specific effects on organic matter remineralization in marine sediments

“…Thus, our mass balance model presumed three 342 major carbon sources: phytoplankton-derived compounds, microbially-produced acetate and/or 343 other fermentation products, and pre-aged OC. value was set to -44 ± 1‰ for both on-and off-axis sediment incubations, based on the expected 359 fractionation heterotrophic acetogenesis which would have resulted in acetate as being ~19.5‰ 360 more depleted relative to initial substrate (Freude and Blaser, 2016). Most of the acetate in 361 marine sediments appears to be generated through heterotrophic rather than autotrophic 362 acetogenesis (Heuer et al, 2009;Lever et al, 2010;Ijiri et al, 2010), consistent with the idea 363 that autotrophic acetogens would be easily outcompeted by other microbes (e.g., methanogens) 364 in marine sediments due to the very low energy yield of this metabolism (Lever, 2012).…”