Metabolic controls on the carbon isotope fractionations of bacterial fermentation

Abstract Microbial fermentation facilitates the initial breakdown of organic matter into small molecules, and is thought to be the rate-limiting step for mineralization under anoxic conditions. Fermentation is understudied in modern and ancient biogeochemistry due to a lack of environmental biomarkers that would constrain its activity. It has long been assumed that fermentation, like respiration, does not express significant carbon isotope fractionations, precluding isotopic signals as a means of studying it in nature. Here, we tested this idea by growing pure cultures of four fermenting bacteria on glucose and measuring the carbon isotope compositions of the organic acids and alcohols produced. We found that fermentation exhibits a strong carbon isotope fractionation, ranging from -6‰ to +16‰, depending on the fermentation product. This range can even be observed within a single organism. Using bioisotopic models that track site-specific isotope enrichments through metabolic networks, we constrained the enzymes responsible for these fractionations. Our models reproduced in vivo organic acid δ13C values in all four organisms. These findings demonstrate that acetate 13C-enrichment is likely a consistent signature of fermentation. Furthermore, our study suggests that fermentation imposes an anaerobic trophic carbon isotope fractionation as organic carbon is passed from fermenters to secondary degraders like sulfate reducers. Looking to the geologic past, this trophic fractionation could have imprinted isotopic signals on the three billion year record of sedimentary organic carbon, specifically the inverse δ13C pattern of Precambrian acyclic isoprenoid and n-alkane biomarkers. Pervasive evidence of fermentation in the rock record suggests its under-appreciated role in biogeochemical cycles throughout Earth history. Significance Statement Microorganisms drive the global cycling of elements like carbon and regulate the Earth’s climate on both human and geologic timescales. Of particular importance is the microbial breakdown of organic matter, which generates greenhouse gases like methane. Fermenting bacteria play a crucial role in anaerobic carbon degradation. However, they remain largely invisible to our analyses. We explored the natural abundance of carbon stable isotopes in the molecular products of fermentation as a tool to study this metabolism in modern ecosystems and the ancient biosphere. We identified significant isotopic signals in molecules produced by fermenting bacteria and compared them to previously observed isotopic signals in molecular fossils in sedimentary rocks, which may provide evidence of microbial fermentation on the Precambrian Earth. Competing Interest Statement The authors have declared no competing interest.