Methanotroph-derived bacteriohopanepolyol signatures as a function of temperature related growth, survival, cell death and preservation in the geological record.

Interpretation of bacteriohopanepolyol (BHP) biomarkers tracing microbiological processes in modern and ancient sediments relies on understanding environmental controls of production and preservation. BHPs from methanotrophs (35-aminoBHPs) were studied in methane-amended aerobic river-sediment incubations at different temperatures. It was found that: (i) With increasing temperature (4°C-40°C) a 10-fold increase in aminopentol (associated with Crenothrix and Methylobacter spp. growth) occurred with only marginal increases in aminotriol and aminotetrol; (ii) A further increase in temperature (50°C) saw selection for the thermophile Methylocaldum and mixtures of aminopentol and C-3 methylated aminopentol, again, with no increase in aminotriol and aminotetrol. (iii) At 30°C, more aminopentol and an aminopentol isomer and unsaturated aminopentol were produced after methanotroph growth and the onset of substrate starvation/oxygen depletion. (iv) At 50°C, aminopentol and C-3 methylated aminopentol, only accumulated during growth but were clearly resistant to remineralization despite cell death. These results have profound implications for the interpretation of aminoBHP distributions and abundances in modern and past environments. For instance, a temperature regulation of aminopentol production but not aminotetrol or aminotriol is consistent with and, corroborative of, observed aminopentol sensitivity to climate warming recorded in a stratigraphic sequence deposited during the Paleocene-Eocene thermal maximum (PETM).

Lipids as paleomarkers to constrain the marine nitrogen cycle.

Global climate is, in part, regulated by the effect of microbial processes on biogeochemical cycling. The nitrogen cycle, in particular, is driven by microorganisms responsible for the fixation and loss of nitrogen, and the reduction-oxidation transformations of bio-available nitrogen. Within marine systems, nitrogen availability is often the limiting factor in the growth of autotrophic organisms, intrinsically linking the nitrogen and carbon cycles. In order to elucidate the state of these cycles in the past, and help envisage present and future variability, it is essential to understand the specific microbial processes responsible for transforming bio-available nitrogen species. As most microorganisms are soft-bodied and seldom leave behind physical fossils in the sedimentary record, recalcitrant lipid biomarkers are used to unravel microbial processes in the geological past. This review emphasises the recent advances in marine nitrogen cycle lipid biomarkers, underlines the missing links still needed to fully elucidate past shifts in this biogeochemically-important cycle, and provides examples of biomarker applications in the geological past.

The Bacteriohopanepolyol Inventory of Novel Aerobic Methane Oxidising Bacteria Reveals New Biomarker Signatures of Aerobic Methanotrophy in Marine Systems.

Aerobic methane oxidation (AMO) is one of the primary biologic pathways regulating the amount of methane (CH4) released into the environment. AMO acts as a sink of CH4, converting it into carbon dioxide before it reaches the atmosphere. It is of interest for (paleo)climate and carbon cycling studies to identify lipid biomarkers that can be used to trace AMO events, especially at times when the role of methane in the carbon cycle was more pronounced than today. AMO bacteria are known to synthesise bacteriohopanepolyol (BHP) lipids. Preliminary evidence pointed towards 35-aminobacteriohopane-30,31,32,33,34-pentol (aminopentol) being a characteristic biomarker for Type I methanotrophs. Here, the BHP compositions were examined for species of the recently described novel Type I methanotroph bacterial genera Methylomarinum and Methylomarinovum, as well as for a novel species of a Type I Methylomicrobium. Aminopentol was the most abundant BHP only in Methylomarinovum caldicuralii, while Methylomicrobium did not produce aminopentol at all. In addition to the expected regular aminotriol and aminotetrol BHPs, novel structures tentatively identified as methylcarbamate lipids related to C-35 amino-BHPs (MC-BHPs) were found to be synthesised in significant amounts by some AMO cultures. Subsequently, sediments and authigenic carbonates from methane-influenced marine environments were analysed. Most samples also did not contain significant amounts of aminopentol, indicating that aminopentol is not a useful biomarker for marine aerobic methanotophic bacteria. However, the BHP composition of the marine samples do point toward the novel MC-BHPs components being potential new biomarkers for AMO.

Analysis of non-derivatised bacteriohopanepolyols by ultrahigh-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Traditional investigation of bacteriohopanepolyols (BHPs) has relied on derivatisation by acetylation prior to gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/MS (LC/MS) analysis. Here, modern chromatographic techniques (ultrahigh-performance liquid chromatography (UPLC)) and new column chemistries were tested to develop a method for BHP analysis without the need for derivatisation. METHODS: Bacterial culture and sedimentary lipid extracts were analysed using a Waters Acquity Xevo TQ-S triple quadrupole mass spectrometer in positive ion atmospheric pressure chemical ionisation (APCI) mode. Waters BEH C18 and ACE Excel C18 were the central columns evaluated using a binary solvent gradient with 0.1% formic acid in the polar solvent phase in order to optimise performance and selectivity. RESULTS: Non-amine BHPs and adenosylhopane showed similar performance on each C18 column; however, BHP-containing terminal amines were only identified eluting from the ultra-inert ACE Excel C18 column. APCI-MS/MS product ion scans revealed significant differences in fragmentation pathways from previous methods for acetylated compounds. The product ions used for targeted multiple reaction monitoring (MRM) are summarised. CONCLUSIONS: UPLC/MS/MS analysis using an ACE Excel C18 column produced superior separation for amine-containing BHPs and reduced run times from 60 to 9 min compared with previous methods. Unexpected variations in fragmentation pathways between structural subgroups must be taken into account when optimising MRM transitions for future quantitative studies. Copyright © 2016 John Wiley & Sons, Ltd.

Mimicking the oxygen minimum zones: stimulating interaction of aerobic archaeal and anaerobic bacterial ammonia oxidizers in a laboratory-scale model system.

In marine oxygen minimum zones (OMZs), ammonia-oxidizing archaea (AOA) rather than marine ammonia-oxidizing bacteria (AOB) may provide nitrite to anaerobic ammonium-oxidizing (anammox) bacteria. Here we demonstrate the cooperation between marine anammox bacteria and nitrifiers in a laboratory-scale model system under oxygen limitation. A bioreactor containing 'Candidatus Scalindua profunda' marine anammox bacteria was supplemented with AOA (Nitrosopumilus maritimus strain SCM1) cells and limited amounts of oxygen. In this way a stable mixed culture of AOA, and anammox bacteria was established within 200 days while also a substantial amount of endogenous AOB were enriched. 'Ca. Scalindua profunda' and putative AOB and AOA morphologies were visualized by transmission electron microscopy and a C18 anammox [3]-ladderane fatty acid was highly abundant in the oxygen-limited culture. The rapid oxygen consumption by AOA and AOB ensured that anammox activity was not affected. High expression of AOA, AOB and anammox genes encoding for ammonium transport proteins was observed, likely caused by the increased competition for ammonium. The competition between AOA and AOB was found to be strongly related to the residual ammonium concentration based on amoA gene copy numbers. The abundance of archaeal amoA copy numbers increased markedly when the ammonium concentration was below 30 µM finally resulting in almost equal abundance of AOA and AOB amoA copy numbers. Massive parallel sequencing of mRNA and activity analyses further corroborated equal abundance of AOA and AOB. PTIO addition, inhibiting AOA activity, was employed to determine the relative contribution of AOB versus AOA to ammonium oxidation. The present study provides the first direct evidence for cooperation of archaeal ammonia oxidation with anammox bacteria by provision of nitrite and consumption of oxygen.

New anaerobic, ammonium-oxidizing community enriched from peat soil.

Anaerobic ammonium-oxidizing (anammox) bacteria have been recognized as an important sink for fixed nitrogen and are detected in many natural environments. However, their presence in terrestrial ecosystems has long been overlooked, and their contribution to the nitrogen cycling in natural and agricultural soils is currently unknown. Here we describe the enrichment and characterization of anammox bacteria from a nitrogen-loaded peat soil. After 8 months of incubation with the natural surface water of the sampling site and increasing ammonium and nitrite concentrations, anammox cells constituted 40 to 50% of the enrichment culture. The two dominant anammox phylotypes were affiliated with "Candidatus Jettenia asiatica" and "Candidatus Brocadia fulgida." The enrichment culture converted NH(4)(+) and NO(2)(-) to N(2) with the previously reported stoichiometry (1:1.27) and had a maximum specific anaerobic ammonium oxidation rate of 0.94 mmol NH(4)(+)·g (dry weight)(-1)·h(-1) at pH 7.1 and 32°C. The diagnostic anammox-specific lipids were detected at a concentration of 650 ng·g (dry weight)(-1), and pentyl-[3]-ladderane was the most abundant ladderane lipid.