No abstract

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 ؉ ⅐ g (dry weight) ؊1 ⅐ h ؊1 at pH 7.1 and 32°C. The diagnostic anammox-specific lipids were detected at a concentration of 650 ng ⅐ g (dry weight)؊1 , and pentyl-[3]-ladderane was the most abundant ladderane lipid.Anaerobic ammonium-oxidizing (anammox) bacteria form a deep-branching, monophyletic group within the planctomycetes and anaerobically oxidize ammonium to dinitrogen gas with nitrite as an electron acceptor (14). They are active at redox transition zones, particularly in oceanic oxygen minimum zones (6, 10, 21, 24) and in other marine ecosystems (3,8,9,22). The discovery of anammox bacteria led to the realization that a substantial part of the nitrogen loss that is observed in the marine environment-up to 50% of the total nitrogen turnover-was due to the activity of these bacteria (2). There are also several studies reporting the presence of anammox bacteria in many freshwater and marine sediments (5,13,25,27,29). However, little is known to date about the distribution, diversity, and activity of anaerobic ammonium oxidation in terrestrial ecosystems. Only this year, anammox bacteria were detected in permafrost and agricultural soils by a 16S rRNA-based molecular approach (12). Nevertheless, anammox bacteria originating from terrestrial ecosystems have not yet been enriched in the laboratory under controlled conditions, and the physiology of these bacteria is still unknown.Since anammox bacteria depend on the concomitant presence of nitrate/nitrite (NO x ) and ammonium under oxygenlimited conditions, oxic-anoxic interfaces in terrestrial ecosystems should provide a suitable habitat for anammox bacteria.

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.

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.

The Long chain Diol Index (LDI) is a relatively new organic geochemical proxy for sea surface temperature (SST), based on the abundance of the C30 1,15-diol relative to the summed abundance of the C28 1,13-, C30 1,13-and C30 1,15-diols. Here we substantially extend and reevaluate the initial core top calibration by combining the original dataset with 172 data points derived from previously published studies and 262 newly generated data points. In total, we considered 595 globally distributed surface sediments with an enhanced geographical coverage compared to the original calibration. The relationship with SST is similar to that of the original calibration but with considerably increased scatter. The effects of freshwater input (e.g., river runoff) and long-chain diol contribution from Proboscia diatoms on the LDI were evaluated. Exclusion of core-tops deposited at a salinity < 32 ppt, as well as core-tops with high Probosciaderived C28 1,12-diol abundance, resulted in a substantial improvement of the relationship between LDI and annual mean SST. This implies that the LDI cannot be directly applied in regions with a strong freshwater influence or high C28 1,12-diol abundance, limiting the applicability of the LDI. The final LDI calibration (LDI=0.0.0325×SST+0.1082; R 2 = 0.88; n = 514) is not statistically different from the original calibration of Rampen et al. (2012)