Metabolic controls on the carbon isotope fractionations of bacterial fermentation

Microbial fermentation facilitates the initial breakdown of organic matter into small molecules and is thought to be the rate-limiting step of anoxic organic mineralization. However, fermentation is understudied in modern and ancient biogeochemistry due to a lack of environmental biomarkers. 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. With bioisotopic models that track site-specific isotope enrichments through metabolism, 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 widespread signature of fermentation. They also challenge traditional notions of controls on the isotope composition of lipids. Finally, our study suggests that fermentation imposes a 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 would suggest its underappreciated role in biogeochemical cycles throughout Earth history.

We gratefully acknowledge Professor Gérald Remaud for intramolecular isotopic analysis of glucose stocks. We thank Professor James B. McKinlay and Professor Ned Ruby for offering strains of Z. mobilis and V. fischeri, respectively. We thank Victoria Orphan for use of her laboratory facilities and Ludmilla Aristilde and Roger Summons for helpful discussions in the interpretation of the data. We are also grateful for the contributions of Jenny Wendt from Center for Marine Environmental Sciences (MARUM) and Daniel Felsmann from Thermo Fisher Scientific for their competent and dedicated support in operating the isotope ratio monitoring liquid chromatography/mass spectrometry system. Portions of the paper were developed from the doctoral thesis of E.P.M. Funding for this work came from an NSF Graduate Research Fellowship DGE-1745301 (to E.P.M.), a European Association of Organic Geochemists Research Award (to E.P.M.) and the NASA Astrobiology Institute grant # 80NSSC18M0094 (to A.L.S.). This work was also supported by the Deutsche Forschungsgemeinschaft through the Cluster of Excellence “The Ocean Floor–Earth’s Uncharted Interface” (project 390741603) (to V.B.H. and K.-U.H.).