Contrasting Pathways for Anaerobic Methane Oxidation in Gulf of Mexico Cold Seep Sediments.

Gulf of Mexico sediments harbor numerous hydrocarbon seeps associated with high sedimentation rates and thermal maturation of organic matter. These ecosystems host abundant and diverse microbial communities that directly or indirectly metabolize components of the emitted fluid. To investigate microbial function and activities in these ecosystems, metabolic potential (metagenomic) and gene expression (metatranscriptomic) analyses of two cold seep areas of the Gulf of Mexico were carried out. Seeps emitting biogenic methane harbored microbial communities dominated by archaeal anaerobic methane oxidizers of phylogenetic group 1 (ANME-1), whereas seeps producing fluids containing a complex mixture of thermogenic hydrocarbons were dominated by ANME-2 lineages. Metatranscriptome measurements in both communities indicated high levels of expression of genes for methane metabolism despite their distinct microbial communities and hydrocarbon composition. In contrast, the transcription level of sulfur cycle genes was quite different. In the thermogenic seep community, high levels of transcripts indicative of syntrophic anaerobic oxidation of methane (AOM) coupled to sulfate reduction were detected. This syntrophic partnership between the dominant ANME-2 and sulfate reducers potentially involves direct electron transfer through multiheme cytochromes. In the biogenic methane seep, genes from an ANME-1 lineage that are potentially involved in polysulfide reduction were highly expressed, suggesting a novel bacterium-independent anaerobic methane oxidation pathway coupled to polysulfide reduction. The observed divergence in AOM activities provides a new model for bacterium-independent AOM and emphasizes the variation that exists in AOM pathways between different ANME lineages. IMPORTANCE Cold seep sediments are complex and widespread marine ecosystems emitting large amounts of methane, a potent greenhouse gas, and other hydrocarbons. Within these sediments, microbial communities play crucial roles in production and degradation of hydrocarbons, modulating oil and gas emissions to seawater. Despite this ecological importance, our understanding of microbial functions and methane oxidation pathways in cold seep ecosystems is poor. Based on gene expression profiling of environmental seep sediment samples, the present work showed that (i) the composition of the emitted fluids shapes the microbial community in general and the anaerobic methanotroph community specifically and (ii) AOM by ANME-2 in this seep may be coupled to sulfate reduction by Deltaproteobacteria by electron transfer through multiheme cytochromes, whereas AOM by ANME-1 lineages in this seep may involve a different, bacterium-independent pathway, coupling methane oxidation to elemental sulfur/polysulfide reduction.

Comparative metagenomics of hydrocarbon and methane seeps of the Gulf of Mexico.

Oil and gas percolate profusely through the sediments of the Gulf of Mexico, leading to numerous seeps at the seafloor, where complex microbial, and sometimes animal communities flourish. Sediments from three areas (two cold seeps with contrasting hydrocarbon composition and a site outside any area of active seepage) of the Gulf of Mexico were investigated and compared. Consistent with the existence of a seep microbiome, a distinct microbial community was observed in seep areas compared to sediment from outside areas of active seepage. The microbial community from sediments without any influence from hydrocarbon seepage was characterized by Planctomycetes and the metabolic potential was consistent with detrital marine snow degradation. By contrast, in seep samples with methane as the principal hydrocarbon, methane oxidation by abundant members of ANME-1 was likely the predominant process. Seep samples characterized by fluids containing both methane and complex hydrocarbons, were characterized by abundant Chloroflexi (Anaerolinaceae) and deltaproteobacterial lineages and exhibited potential for complex hydrocarbon degradation. These different metabolic capacities suggested that microorganisms in cold seeps can potentially rely on other processes beyond methane oxidation and that the hydrocarbon composition of the seep fluids may be a critical factor structuring the seafloor microbial community composition and function.

Characterization of enhanced-fluidity liquid hydrophilic interaction chromatography for the separation of nucleosides and nucleotides.

Hydrophilic interaction chromatography (HILIC) is a liquid chromatographic separation mechanism commonly used for polar biological molecules. The use of enhanced-fluidity liquid chromatography (EFLC) with mixtures of methanol/water/carbon dioxide is compared to acetonitrile/water mobile phases for the separation of nucleosides and nucleotides under HILIC conditions. Enhanced-fluidity liquid chromatography involves using common mobile phases with the addition of substantial proportions of a dissolved gas which provides greater mobile phase diffusivity and lower viscosity. The impact of varying several experimental parameters, including temperature, addition of base, salt, and CO2 was studied to provide optimized HILIC separations. Each of these parameters plays a key role in the retention of the analytes, which demonstrates the complexity of the retention mechanism in HILIC. The tailing of phosphorylated compounds was overcome with the use of phosphate salts and the addition of a strong base; efficiency and peak asymmetry were compared with the addition of either triethylamine (TEA), 1,4-diazabicyclo [2.2.2] octane (DABCO) or 1,5-diazabicyclo [4.3.0] non-5-ene (DBN). DBN and DABCO both led to increased efficiency and lower peak asymmetry; DBN provided the best results. Sodium chloride and carbon dioxide were added to enhance the selectivity between the analytes, giving a successful isocratic separation of nucleosides and nucleotides within 8 min. The retention mechanism involved in EFL-HILIC was explored by varying the temperature and the mole fraction of CO2. These studies showed that partitioning was the dominant mechanism. The thermodynamics study confirmed that the solvent strength is maintained in EFLC and that a change in entropy was mainly responsible for the improved selectivity. The selectivity using methanol/water/carbon dioxide varied greatly compared to that obtained with acetonitrile/water. Finally while this study highlights the optimization of EFL-HILIC for the separation of nucleosides and nucleotides under isocratic conditions, this is also an example of the broad range of polarities of compounds that EFL-HILIC can separate.

Enhanced fluidity liquid chromatography for hydrophilic interaction separation of nucleosides.

The application of enhanced fluidity liquid (EFL) mobile phases to improving isocratic chromatographic separation of nucleosides in hydrophilic interaction liquid chromatography (HILIC) mode is described. The EFL mobile phase was created by adding carbon dioxide to a methanol/buffer solution. Previous work has shown that EFL mobile phases typically increase the efficiency and the speed of the separation. Herein, an increase in resolution with the addition of carbon dioxide is also observed. This increase in resolution was achieved through increased selectivity and retention with minimal change in separation efficiency. The addition of CO2 to the mobile phase effectively decreases its polarity, thereby promoting retention in HILIC. Conventional organic solvents of similar nonpolar nature cannot be used to achieve similar results because they are not miscible with methanol and water. The separation of nucleosides with methanol/aqueous buffer/CO2 mobile phases was also compared to that using acetonitrile/buffer mobile phases. A marked decrease in the necessary separation time was noted for methanol/aqueous buffer/CO2 mobile phases compared to acetonitrile/buffer mobile phases. There was also an unusual reversal in the elution order of uridine and adenosine when CO2 was included in the mobile phase.