Towards stable chemical fossils for anaerobic ammonium-oxidizing bacteria in palaeoenvironmental studies: novel cyclic aliphatic hydrocarbons as potential dia- and catagenetic products of ladderane lipids

Anaerobic ammonium oxidation (anammox) is an important process in modern global nitrogen cycling. However, the contribution of anammox to ancient nitrogen biogeochemical cycles cannot currently be accounted for because the most characteristic lipid biomarkers of anammox bacteria, ladderane lipids containing concatenated series of cyclobutane moieties, do not preserve as such into the catagenetic window in the sedimentary record. Alternative, more stable, lipid biomarkers potentially thermal products of ladderanes, are needed to be able to investigate the presence anammox in thermally more mature sediments and inform on past marine oxygen depletion, nitrogen cycling, and the formation of organic matter-rich sediments such as black shale deposits. Here, we describe novel cyclic hydrocarbons produced by the simulated thermal maturation of anammox bacterial biomass. Alkyl branched tricyclic hydrocarbons were present in pyrolysates generated by hydrous pyrolysis experiments at temperatures >200 °C, in the window of oil generation. Two-dimensional gas chromatography-mass spectrometry revealed that these artificial maturation experiments generated multiple series of cyclic hydrocarbons. A representative product was isolated by preparative gas chromatography, but was shown to be composed of two co-eluting components. Their structures were partially identified, by way of two-dimensional nuclear magnetic resonance (NMR) spectroscopy, to be 4-octyl-tricyclo[7.3.0.0^2,8]dodecanes with the three concatenated rings comprising of 7, 4, and 5 carbon atoms. This ring assemblage was further confirmed through NMR spectroscopy and mass spectral comparisons with four synthesized model compounds. The isolated components had a stable carbon isotopic composition similarly depleted in¹³C as the ladderane fatty acids of the original anammox biomass, consistent with a ladderane lipid source. The C₂₀ hydrocarbons could have formed from thermal ether cleavage and ring-opening of the C₂₀-[5]- or [3]-ladderane glycerol ether, common membrane lipids of anammox bacteria. Molecular mechanic calculations were used to generate the relative structural stability of stereoisomers and the possible conversion steps to the formation of these components. These indicated that the 7-4-5 components were the result of the breakdown and rearrangement of bonds in the cyclobutane rings from biosynthetically-derived 4-4-4-4-4 ladderane lipids. These components were not detected in thermally mature sediment and rocks screened using conventional GC–MS analysis, however, by using more advanced chromatography methods these lipids may be still be helpful biomarkers for assessing past anammox in thermally more mature sediments and crude oils.