Methane-Oxidizing Communities in Lichen-Dominated Forested Tundra Are Composed Exclusively of High-Affinity USCα Methanotrophs

Upland soils of tundra function as a constant sink for atmospheric CH4 but the identity of methane oxidizers in these soils remains poorly understood. Methane uptake rates of −0.4 to −0.6 mg CH4-C m−2 day−1 were determined by the static chamber method in a mildly acidic upland soil of the lichen-dominated forested tundra, North Siberia, Russia. The maximal CH4 oxidation activity was localized in an organic surface soil layer underlying the lichen cover. Molecular identification of methanotrophic bacteria based on retrieval of the pmoA gene revealed Upland Soil Cluster Alpha (USCα) as the only detectable methanotroph group. Quantification of these pmoA gene fragments by means of specific qPCR assay detected ~107pmoA gene copies g−1 dry soil. The pmoA diversity was represented by seven closely related phylotypes; the most abundant phylotype displayed 97.5% identity to pmoA of Candidatus Methyloaffinis lahnbergensis. Further analysis of prokaryote diversity in this soil did not reveal 16S rRNA gene fragments from well-studied methanotrophs of the order Methylococcales and the family Methylocystaceae. The largest group of reads (~4% of all bacterial 16S rRNA gene fragments) that could potentially belong to methanotrophs was classified as uncultivated Beijerinckiaceae bacteria. These reads displayed 96–100 and 95–98% sequence similarity to 16S rRNA gene of Candidatus Methyloaffinis lahnbergensis and “Methylocapsa gorgona” MG08, respectively, and were represented by eight species-level operational taxonomic units (OTUs), two of which were highly abundant. These identification results characterize subarctic upland soils, which are exposed to atmospheric methane concentrations only, as a unique habitat colonized mostly by USCα methanotrophs.

Multifunctional Periphytic Biofilms: Polyethylene Degradation and Cd2+ and Pb2+ Bioremediation under High Methane Scenario

Priority pollutants such as polyethylene (PE) microplastic, lead (Pb2+), and cadmium (Cd2+) have attracted the interest of environmentalists due to their ubiquitous nature and toxicity to all forms of life. In this study, periphytic biofilms (epiphyton and epixylon) were used to bioremediate heavy metals (HMs) and to biodegrade PE under high (120,000 ppm) methane (CH4) doses. Both periphytic biofilms were actively involved in methane oxidation, HMs accumulation and PE degradation. Epiphyton and epixylon both completely removed Pb2+ and Cd2+ at concentrations of 2 mg L−1 and 50 mg L−1, respectively, but only partially removed these HMs at a relatively higher concentration (100 mg L−1). Treatment containing 12% 13CH4 proved to be most effective for biodegradation of PE. A synergistic effect of HMs and PE drastically changed microbial biota and methanotrophic communities. High-throughput 16S rRNA gene sequencing revealed that Cyanobacteria was the most abundant class, followed by Gammaproteobacteria and Alphaproteobacteria in all high-methane-dose treatments. DNA stable-isotope probing was used to label 13C in a methanotrophic community. A biomarker for methane-oxidizing bacteria, pmoA gene sequence of a 13C-labeled fraction, revealed that Methylobacter was most abundant in all high-methane-dose treatments compared to near atmospheric methane (NAM) treatment, followed by Methylococcus. Methylomonas, Methylocystis, Methylosinus, and Methylocella were also found to be increased by high doses of methane compared to NAM treatment. Overall, Cd+2 had a more determinantal effect on methanotrophic activity than Pb2+. Epiphyton proved to be more effective than epixylon in HMs removal and PE biodegradation. The findings proved that both epiphyton and epixylon can be used to bioremediate HMs and biodegrade PE as an efficient ecofriendly technique under high methane concentrations.

Characteristics of aerobic methane-oxidising bacterial community at the sea-floor surface of the Nankai Trough

Methane hydrate is one of the new energy sources, but methane leakage could cause environmental issues such as marine ecosystem changes and global warming. The methane-oxidising bacterial (MOB) community could reflect the methane concentration, thus it may be an indicator of methane leakage. We obtained two sea-floor surface samples from a methane seep area and 12 samples from other general sea-floor areas of the Nankai Trough for the detection and phylogenetic analysis of the particulate methane monooxygenase (pmoA) gene. Using quantitative polymerase chain reaction analysis, the methane seep samples were found to have 106 copies of the pmoA gene per gram of sediment, whereas the general sea-floor area samples of the Nankai Trough contained 103–104 copies of the gene per gram of sediment. Phylogenetic analysis of the pmoA gene sequences showed that the sequences detected in the general and methane seep area samples differed significantly. Specifically, the pmof1–pmor primer pair could detect pmoA genes for the methane seep area, whereas pmoA gene from the general seafloor samples could be detected only using the A189–mb661 primer pair. The results of this study may facilitate the detection of unintended leakage of methane at methane hydrate production sites by monitoring MOB communities using pmoA-targeted phylogenetic analysis and quantification.

Ammonia impacts methane oxidation and methanotrophic community in freshwater sediment

Lacustrine ecosystems are an important natural source of greenhouse gas methane. Aerobic methanotrophs are regarded as a major regulator controlling methane emission. Excess nutrient input can greatly influence carbon cycle in lacustrine ecosystems. Ammonium is believed to be a major influential factor, due to its competition with methane as the substrate for aerobic methanotrophs. To date, the impact of ammonia on aerobic methanotrophs remains unclear. In the present study, microcosms with freshwater lake sediment were constructed to investigate the influence of ammonia concentration on aerobic methanotrophs. Ammonia influence on the abundance of pmoA gene was only observed at a very high ammonia concentration, while the number of pmoA transcripts was increased by the addition of ammonium. pmoA gene and transcripts differed greatly in their abundance, diversity and community compositions. pmoA transcripts were more sensitive to ammonium amendment than pmoA gene. Methane oxidation potential and methanotrophic community could be impacted by ammonium amendment. This work could add some new sights towards the links between ammonia and methane oxidation in freshwater sediment.

Vertical and horizontal distribution of sediment nitrite-dependent methane-oxidizing organisms in a mesotrophic freshwater reservoir

In the present study, we investigated the spatial change of sediment nitrite-dependent anaerobic methane-oxidizing (n-damo) organisms in the mesotrophic freshwater Gaozhou Reservoir (6 different sampling locations and 2 sediment depths (0–5 cm, 5–10 cm)), one of the largest drinking water reservoirs in China. The abundance of sediment n-damo bacteria was quantified using quantitative polymerase chain reaction assay, while the richness, diversity, and composition of n-damo pmoA gene sequences were characterized using clone library analysis. Vertical and horizontal changes in sediment n-damo bacterial abundance occurred in Gaozhou Reservoir, with 1.37 × 105 to 8.24 × 105 n-damo 16S rRNA gene copies per gram of dry sediment. Considerable horizontal and vertical variations of n-damo pmoA gene diversity (Shannon index = 0.32–2.50) and composition also occurred in this reservoir. Various types of sediment n-damo pmoA genes existed in Gaozhou Reservoir. A small proportion of n-damo pmoA gene sequences (19.1%) were related to those recovered from “Candidatus Methylomirabilis oxyfera”. Our results suggested that sediment n-damo pmoA gene diversity might be regulated by nitrite, while n-damo pmoA gene richness might be governed by multiple environmental factors, including total organic carbon, total phosphorus, nitrite, and total nitrogen.

Isolation of a methane-oxydizing bacterium for the study on single cell protein production from methane

Single cell protein (SCP) can be produced from biomass of different types of microorganisms that have high protein content such as yeast, filamentous fungi, algae and bacteria. In comparison to animal and plant protein sources, this kind of protein has several advantages, namely high protein and nutrient contents, being produced in fermenters with the use of variety of organic wastes, independence in agriculture land or climate conditions. Methane oxidizing bacteria (MOB) are considered as good candidates for SCP production and have been intensively studied recently. In the present study, a MOB strain BG3 was isolated from wastewater of an anaerobic digester via enrichment and isolation procedures using methane as the only carbon and energy sources. Strain BG3 comprised of oval-shaped cells of 0,8-1´16 -1,8 μm in size, almost nonmotile. Based on comparative analyses of the 16S rDNA partial sequences, strain BG3 was identified as a member of the Methylomonas genus, the most closely related species was Methylomonas koyamae (97% homology). This was also confirmed by analyses of sequence of the pmoA gene, encoding for a-subunit in the methane-monooxygenase in the strain. Besides methane, strain BG3 also utilized methanol for the growth. It has been shown that methane-fed culture of strain BG3 could produce 68.69 g crude protein per 100 g CDW and the according methane to biomass conversion efficiency was 2,8 m3 methane×kg-1 dry biomassas. Owing the capability of utilization of methane, the second important greenhouse gas, for the production of protein source for animal feed, strain BG3 would have a great application potential in Vietnam. Strain BG3 was designated as Methylomonas sp. BG3 and its 16S rDNA and pmoA gene sequences were deposited at the GenBank with accession numbers of KJ081955 and KJ081956, respectively.