Bacterial physiology highlighted by the δ13C fractionation of bacteriohopanetetrol isomers

Lipid biomarkers, such as the various bacteriohopanetetrol (BHT) isomers studied here, are useful tools in tracing bacterially mediated nitrogen and carbon cycle processes affecting greenhouse gas emissions, including the anaerobic oxidation of ammonia. Three BHT isomers occur commonly in the environment. By gas chromatography, BHT-34S elutes first; it is produced by numerous bacteria. The two later eluting isomers are more constrained in their origin. The marine anammox bacteria ‘Ca. Scalindua’ is the only known producer of a BHT isomer of unknown stereochemistry (BHT-x), making BHT-x a diagnostic biomarker in anoxic marine settings. The BHT-34R isomer is produced by three freshwater aerobic heterotrophic producers (Frankia spp., Acetobacter pasteurianus, and Komagataeibacter xylinus), a freshwater serine-cycle (Type II) methanotroph (Methylocella palustris), and the freshwater anammox ‘Ca. Brocadia’, which makes the detection of freshwater anammox using BHT-34R more complicated. We investigated whether the source of BHT-34R in freshwater environments could be ascertained via its δ ¹³C value. We used conventional on-column gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) (as opposed to high temperature GC-C-IRMS) to determine the δ ¹³C composition of acetylated BHT isomers in cultured bacteria and bacterial enrichments. We combined these with bulk biomass and substrate δ ¹³C compositions to establish carbon isotopic fractionation factors. The two anammox genera had large fractionation factors from dissolved inorganic carbon (DIC) to biomass (Δ ¹³C _{biomass – DIC} = –43.8 to –26.4 ‰) and to BHTs (Δ ¹³C _{BHT – DIC} = –53.8 to –38.2 ‰), which clearly distinguished them from the freshwater aerobic heterotrophic producers (Δ ¹³C _{biomass – substrate} = –2.3 to –0.1 ‰; Δ ¹³C _{BHT – substrate} = –12.8 to 5.2 ‰). Methylocella assimilated mainly carbon from DIC, rather than from methane, into its biomass and BHT, and previous work suggested this assimilation comes with relatively small fractionation. Thus, in peatlands, the BHT δ ¹³C values of Methylocella would not reflect the low δ ¹³C values of biogenic methane. Consequently, the presence of BHT-34R with low δ ¹³C values relates to ‘Ca. Brocadia’ and presents a novel tool to trace anammox in freshwater environments.