A unique bacteriohopanetetrol stereoisomer of marine anammox

Anaerobic ammonium oxidation (anammox) is a major process of bioavailable nitrogen removal from marine systems. Previously, a bacteriohopanetetrol (BHT) isomer, with unknown stereochemistry, eluting later than BHT using high performance liquid chromatography (HPLC), was detected in ‘Ca. Scalindua profunda’ and proposed as a biomarker for anammox in marine paleo-environments. However, the utility of this BHT isomer as an anammox biomarker is hindered by the fact that four other, non-anammox bacteria are also known to produce a late-eluting BHT stereoisomer. The stereochemistry in Acetobacter pasteurianus, Komagataeibacter xylinus and Frankia sp. was known to be 17β, 21β(H), 22R, 32R, 33R, 34R (BHT-34R). The stereochemistry of the late-eluting BHT in Methylocella palustris was unknown. To determine if marine anammox bacteria produce a unique BHT isomer, we studied the BHT distributions and stereochemistry of known BHT isomer producers and of previously unscreened marine (‘Ca. Scalindua brodeae’) and freshwater (‘Ca. Brocadia sp.’) anammox bacteria using HPLC and gas chromatographic (GC) analysis of acetylated BHTs and ultra high performance liquid chromatography (UHPLC)-high resolution mass spectrometry (HRMS) analysis of non-acetylated BHTs. The 34R stereochemistry was confirmed for the BHT isomers in Ca. Brocadia sp. and Methylocella palustris. However, ‘Ca. Scalindua sp.’ synthesise a stereochemically distinct BHT isomer, with still unconfirmed stereochemistry (BHT-x). Only GC analysis of acetylated BHT and UHPLC analysis of non-acetylated BHT distinguished between late-eluting BHT isomers. Acetylated BHT-x and BHT-34R co-elute by HPLC. As BHT-x is currently only known to be produced by ‘Ca. Scalindua spp.’, it may be a biomarker for marine anammox.