Abstract
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 when examined by 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 spp.’) anammox bacteria, using HPLC and gas chromatographiy (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 spp.’ synthesises 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 when examined by HPLC. As BHT-x is currently only known to be produced by ‘Ca. Scalindua spp.’, it may be a biomarker for marine anammox.
Highlights
In anoxic and low-oxygen marine systems, anaerobic ammonium oxidation removes bioavailable nitrogen by converting ammonium and nitrite into dinitrogen gas (Strous et al, 1999)
We investigated an unscreened anammox enrichment culture of ‘Ca. Brocadia sp.’ for the presence of lateeluting BHT isomers and re-evaluated a culture of ‘Ca. Kuenenia stuttgartiensis’ to determine if trace quantities of late-eluting BHT isomer were present in this non-marine anammox bacterium
Chromatographic separation and identification of late-eluting BHT isomers Analyses of acetylated Bligh & Dyer extraction (BDE) from M. palustris, A. pasteurianus, K. xylinus, Frankia spp., ‘Ca. Brocadia sp.’, ‘Ca. S. profunda’ and ‘Ca. S. brodeae’ revealed that all contained a BHT isomer that eluted after BHT-34S when measured using high performance liquid chromatography (HPLC) and gas chromatographic (GC), and non-acetylated BDE measured by ultra high performance liquid chromatography (UHPLC) (Figs. 2-4; Lengger et al, 2019; Peiseler and Rohmer, 1992; Rosa-Putra et al, 2001; Rush et al, 2014, 2019; van Winden et al, 2012)
Summary
In anoxic and low-oxygen marine systems, anaerobic ammonium oxidation (anammox) removes bioavailable nitrogen by converting ammonium and nitrite into dinitrogen gas (Strous et al, 1999). This limits the availability of a major nutrient for phytoplankton and may have pronounced effects on biogeochemical cycling in the ocean. Amongst the five currently known genera of anammox bacteria, four are primarily found in non-marine/freshwater environments: ‘Candidatus Brocadia’, ‘Ca. Jettenia’, ‘Ca. Kuenenia’ and ‘Ca. Anammoxoglobus’ (Kartal et al, 2007; Kuypers et al, 2003; Quan et al, 2008; Schmid et al, 2000; Strous et al, 1999). ‘Ca. Scalindua’ genus is typically reported only in marine systems (Schmid et al, 2007; Woebken et al, 2007; Villanueva et al, 2014), it was reported to be the dominant genus in a rice paddy (Wang and Gu, 2013)
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