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. ABSTRACT 42 Anaerobic ammonium oxidation (anammox) is a major process of bioavailable nitrogen 43 removal from marine systems. Previously, a bacteriohopanetetrol (BHT) isomer, with 44 unknown stereochemistry, eluting later than BHT using liquid 45 chromatography (HPLC), was detected in ‘ Ca . Scalindua profunda’ and proposed as a 46 biomarker for anammox in marine paleo-environments. However, the utility of this BHT 47 isomer as an anammox biomarker is hindered by the fact that four other, non-anammox 48 bacteria are also known to produce a late-eluting BHT stereoisomer. The stereochemistry in 49 Acetobacter pasteurianus , Komagataeibacter xylinus and Frankia sp . was known to be 17β, 50 21β(H), 22 R , 32 R , 33 R , 34 R (BHT-34 R ). The stereochemistry of the late-eluting BHT in 51 Methylocella palustris was unknown. To determine if marine anammox bacteria produce a 52 unique BHT isomer, we studied the BHT distributions and stereochemistry of known BHT 53 isomer producers and of previously unscreened marine (‘ Ca . Scalindua brodeae’) and 54 freshwater (‘ Ca . Brocadia sp.’) anammox bacteria using HPLC and gas chromatographic 55 (GC) analysis of acetylated BHTs and ultra high performance liquid chromatography 56 (UHPLC)-high resolution mass spectrometry (HRMS) analysis of non-acetylated BHTs. The 57 34 R stereochemistry was confirmed for the BHT isomers in Ca . Brocadia sp. and 58 Methylocella palustris. However, ‘ Ca . Scalindua sp.’ synthesise a stereochemically distinct 59 BHT isomer, with still unconfirmed stereochemistry (BHT- x ). Only GC analysis of 60 acetylated BHT and UHPLC analysis of non-acetylated BHT distinguished between late- 61 eluting BHT isomers. mass spectrometry (HPLC-MS) analyses BHTs were performed using an auto-injector - equipped HP 1100 series HPLC interfaced to a Bruker ion trap mass spectrometer and chromatography manager software the Strasbourg-CNRS. Separation was achieved on a Zorbax ODS column (4.6 mm x 250 mm, 5 µm) maintained at 30 °C. The injection volume was 10  L. eluted isocratically using a mixture of methanol/isopropanol 95:5 v/v with a flow rate of 1.0 mL min Detection achieved a APCI-MS pressure temperature 420 drying gas (N ) flow 6 L 350 °C, capillary voltage -4 kV, corona 4 µA, scanning range m/z 300-800.

isomer as an anammox biomarker is hindered by the fact that four other, non-anammox 48 bacteria are also known to produce a late-eluting BHT stereoisomer. The stereochemistry in 49 Acetobacter pasteurianus, Komagataeibacter xylinus and Frankia sp. was known to be 17β, In anoxic and low-oxygen marine systems, anaerobic ammonium oxidation (anammox) 68 removes bioavailable nitrogen by converting ammonium and nitrite into dinitrogen gas 69 (Strous et al., 1999). This limits the availability of a major nutrient for phytoplankton and 70 thus may have pronounced effects on biogeochemical cycling in the ocean. Marine anammox 71 is suggested to account for ca. 30% of the loss of bioavailable nitrogen from the global 72 oceans today (Ward, 2013). Reconstructing the presence of anammox in paleo-environments 73 is therefore particularly important for understanding the changes in the nitrogen cycle. Anammox is performed by bacteria belonging to the Planctomycetes. Anammox was first 76 recognized in an anaerobic waste water treatment system (Strous et al., 1999) and 77 subsequently in the environment (Kuypers et al., 2003). Amongst the five currently known      Here, we provided further insight into these biomarkers. We examined the stereochemistry    Anoxic conditions and pH were maintained as described above.

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All anammox cultures were harvested from the reactor and centrifuged (4000 x g, 20 min, 173 4°C) to obtain cell pellets, which were subsequently freeze-dried prior to analysis. tube was extracted using a monophasic mixture of water/methanol/chloroform (4 mL/10 199 mL/5 mL), sonicated for 15 min at 40 °C, followed by centrifugation at 4000 rpm for 5 min.

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The supernatant was transferred to a second centrifuge tube. The cellular residue pellet was 201 re-extracted twice using the same methods to third and fourth centrifuge tubes. Chloroform (5 202 mL) and water (5 mL) were added to centrifuge tubes 2-4 to obtain a biphasic mixture. These  pressure was 40 arbitrary units and the auxiliary gas (N 2 ) pressure was 10 arbitrary units. The  The MS 2 spectra of BHT and late-eluting BHT isomers were too similar to discriminate 341 between the different stereochemistries (Fig. 5).   authentic standard (Fig. 3). Co-injections of the authentic acetylated BHT-34R standard with 370 acetylated extracts from Frankia sp. strain Ea1-12 (Fig. 3c), M. palustris (Fig. 3d), and the 371 anammox bacterium 'Ca. Brocadia sp.' (Fig. 4e) confirmed the BHT-34R stereochemistry of 372 the late-eluting BHT isomer of these cultures. The late-eluting BHT isomer (BHT-x) from 373 'Ca. S. brodeae' did not co-elute with BHT-34R (Fig. 4f). To verify that other species  S. profunda' TLE was also co-injected with authentic BHT standards (Fig. 4g). Both 'Ca. 376 Scalindua' species were found to produce the BHT-x isomer. Of the known bacterial 377 producers of BHT isomers, BHT-x is only synthesized by marine anammox bacteria, 378 suggesting that it can be applied as a biomarker for paleo-marine anammox studies.

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The isomer elution order of non-acetylated BHTs when measured by UHPLC-HRMS was  We also acknowledge support through The Leverhulme Trust (Grant RPG2016-050) to JCM.

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The authors declare no competing financial or non-financial interests. We thank the