Rachel Schwartz-Narbonne, Philippe Schaeffer, Ellen C. Hopmans, Margot Schenesse, E. Alex Charlton, Martin Jones, Jaap Damste, Muhammad Farhan Ul Haque, Mike Jetten, Sabine Lengger

Anaerobic ammonium oxidation (anammox) is a significant process for bioavailable nitrogen removal from marine systems. 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. Four non-marine, non-anammox genera also produce late-eluting BHT stereoisomers. Of these, the stereochemistry in Acetobacter pasteurianus, Komagataeibacter xylinus and Frankia sp. was shown to be 17β, 21β(H), 22R, 32R, 33R, 34R (BHT-34R), while the stereochemistry of the late-eluting BHT in Methylocella palustris was unknown. We studied the BHT distributions and stereochemistry of these known BHT isomer producers and of previously unscreened marine (‘Ca. Scalindua brodeae’) and freshwater (‘Ca. Brocadia sp.’) anammox species and genera 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 a still unconfirmed stereochemistry (BHT-x). ‘Ca. Kuenenia sp.’ was confirmed not to produce any late-eluting BHT stereoisomers. 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 so far only known to be produced by ‘Ca. Scalindua spp.’, it may be applied as a biomarker for marine anammox. As ‘Ca. Brocadia sp.’ produces BHT-34R, this BHT isomer is a potential, albeit less specific, biomarker for anammox in non-marine settings.