Rare Branched Fatty Acids Characterize the Lipid Composition of the Intra-Aerobic Methane Oxidizer “Candidatus Methylomirabilis oxyfera”

Abstract

bThe recently described bacterium "Candidatus Methylomirabilis oxyfera" couples the oxidation of the important greenhouse gas methane to the reduction of nitrite. The ecological significance of "Ca. Methylomirabilis oxyfera" is still underexplored, as our ability to identify the presence of this bacterium is thus far limited to DNA-based techniques. Here, we investigated the lipid composition of "Ca. Methylomirabilis oxyfera" to identify new, gene-independent biomarkers for the environmental detection of this bacterium. Multiple "Ca. Methylomirabilis oxyfera" enrichment cultures were investigated. In all cultures, the lipid profile was dominated up to 46% by the fatty acid (FA) 10-methylhexadecanoic acid (10MeC 16:0 ). Furthermore, a unique FA was identified that has not been reported elsewhere: the monounsaturated 10-methylhexadecenoic acid with a double bond at the ⌬7 position (10MeC 16:1⌬7 ), which comprised up to 10% of the total FA profile. We propose that the typical branched fatty acids 10MeC 16:0 and 10MeC 16:1⌬7 are key and characteristic components of the lipid profile of "Ca. Methylomirabilis oxyfera." The successful detection of these fatty acids in a peatland from which one of the enrichment cultures originated supports the potential of these unique lipids as biomarkers for the process of nitrite-dependent methane oxidation in the environment.

High rates of anaerobic methane oxidation in freshwater wetlands reduce potential atmospheric methane emissions

“…10-Methylhexadecanoic acid, a characteristic lipid for 'Methylomirabilis oxyfera', a freshwater bacterium capable of oxidizing methane with nitrite 33 , was either not detected at these sites or found in rather low abundance (o1.5% of all fatty acids; data not shown). This proposed biomarker for n-damo 48 was not sufficiently depleted in 13 C to unambiguously support a relationship with AOM (Supplementary Table 3); a conclusive relationship of this biomarker to AOM is complicated by the evidence for chemoorganoautotrophy of M. oxyfera 49 , in analogy to some ANME archaea 47 , and additional sources of this biomarker such as sulphate-reducing 50 and anammox bacteria 51 . The other proposed diagnostic lipid for M. oxyfera, 10-methyl-hexadec-7-enoic acid 48 , was not detected in any sample.…”

Autotrophic Carbon Dioxide Fixation via the Calvin-Benson-Bassham Cycle by the Denitrifying Methanotroph “Candidatus Methylomirabilis oxyfera”

“…Isotope values were determined by elemental analysis, followed by isotope ratio mass spectrometry (with a Thermo Flash 2000 elemental analyzer coupled to a Thermo DeltaV information request management system). Lipid extraction of the subsample for compound-specific analysis was carried out as described previously (40). In short, after saponification the obtained extracts were methylated with boron trifluoride (BF 3 ) in methanol and subsequently separated into apolar and polar fractions by column chromatography over activated alumina (Al 2 O 3 ).…”

Rare bacteriohopanepolyols as markers for an autotrophic, intra-aerobic methanotroph

“…Both BHP-hexol IVa and the 3-methyl-BHP-hexol IVb could be detected in the field samples from Brunssummerheide, where genomic and fatty acid analyses had previously verified the presence of Methylomirabilis spp. (Kool et al, 2012;Zhu et al, 2012). This demonstrates the potential for these unusual BHPs as diagnostic biomarker for Methylomirabilis spp.…”

“…enrichment cultures. In this section of the soil profile a counter gradient of methane and nitrate was observed, coinciding with a peak in abundance of Methylomirabilis spp.-like bacteria as measured by quantitative polymerase chain reaction (qPCR) and an Methylomirabilis spp.-specific fatty acid (Kool et al, 2012;Zhu et al, 2012). All horizons of the soil profile contained C 30 , C 31 and C 32 regular hopanols (Ia, Ic, and Ie) after periodic acid treatment.…”

“…Biomass was obtained from four enrichment cultures capable of nitrite-dependent methane oxidation that had been enriched from three different environments in the Netherlands, as previously described in Kool et al (2012). Two enrichment cultures of M. oxyfera originated from sediment from a freshwater ditch in the 'Ooijpolder' (Ooij-1 and Ooij-2) (Ettwig et al, 2009(Ettwig et al, , 2010, the third enrichment culture of M. oxyfera was derived from sludge from a wastewater treatment plant in Lieshout (Luesken et al, 2011).…”