Environmental controls on bacteriohopanepolyol signatures in estuarine sediments

PhD ThesisTo date, research on the fate of methane in marine settings has mainly focused on anaerobic microbial processes. An alternative fate for methane is aerobic methane oxidation (AMO) by methanotrophic bacteria which takes place in aerobic surface sediments and the overlying water column. Tracing methanotroph activity in past environments can be achieved via analysis of a distinctive suite of biomarkers called bacteriohopanepolyols (BHPs). BHPs are membrane lipids produced by many prokaryotes comprising a pentacyclic triterpenoid structure with an extended polyfunctionalised side chain. Although, there is much debate about the role of BHPs, studies suggest they regulate cell membrane fluidity, however, the factors controlling their expression are poorly constrained. They have a wide range of structural variation which varies between bacterial phyla and species. The major BHPs produced by methanotrophs are collectively known as the 35-aminoBHPs, most commonly including 35-aminobacteriohopane-32,33,34-triol (aminotriol), 35-aminobacteriohopane-31,32,33,34-tetrol (aminotetrol) and 35-aminobacteriohopane-30,31,32,33,34-pentol (aminopentol), with aminopentol seen as a diagnostic marker for Type I methanotrophs from the phylum Gammaproteobacteria. The changes in methanotroph community composition in estuarine sediments under a range of environmental perturbations and the effect this had on BHP composition, namely the 35-aminoBHPs, was assessed. Aerobic microcosms inoculated with River Tyne (UK) estuarine sediment with a 5% methane amended headspace (unless otherwise stated), were subjected to a range of environmental perturbations; methane concentration (0.1-5%), temperature (4-60°C), pH (4-9) and salinity (1-150 g/L NaCl). Methane oxidation rates were monitored and methanotroph diversity was determined by targeting the particulate methane monooxygenase gene (pmoA). Methane oxidation was observed between 4 and 50°C, at all tested pH values and up to salinities of 70 g/L; however, methanotroph community composition varied with temperature, pH and salinity and these changes were reflected in the 35-aminoBHP signatures quantified by LC-MS analysis. For example, aminopentol was not enriched at pH 9 when the unusual Type I Methylomicrobium spp. were dominant, whilst the maximal production of C-3 methylated aminopentol was witnessed at 50°C when a Methylocaldum sp. was enriched. The hpnR gene, required for the methylation of BHPs at the C-3 position, was also identified in sediments at the aforementioned temperature. Novel iv compounds, identified after the analysis of six previously untested Type I marine methanotrophs within this study, were also found in microcosm sediments in varying abundances. The effect that of growth stage on 35-aminoBHP abundance was determined by analysing aerobic microcosms inoculated with River Tyne estuarine sediment over a 28 day period at times before and after methane oxidation. It revealed the continued production of aminopentol at mesophilic temperatures after methane oxidation was complete. This may have implications for the interpretation of the sedimentary record where aminopentol witnessed in marine settings may not represent periods of significant methane oxidation but rather a response to methane limiting conditions. Anaerobic producers of BHPs were investigated and the preservation and/or degradation of individual compounds was assessed in long-term studies. Microcosms inoculated with anoxic River Tyne estuarine sediment were subjected to sulphate-reducing and methanogenic conditions over a period of 706 and 665 days respectively. Changes in BHP composition over time were quantified by LC-MS with compounds including bacteriohopane-32,33,34,35-tetrol (BHT) and adenosylhopane found to be more resistant to degradation over the course of the study compared to bacteriohopane-32,33,34,35-tetrol cyclitol ether (BHT cyclitol ether). This indicates that some compounds are more resistant to degradation over time compared with others