Deepak Kumaresan

Life without light: microbial diversity and evidence of sulfur- and ammonium-based chemolithotrophy in Movile Cave

Microbial diversity in Movile Cave (Romania) was studied using bacterial and archaeal 16S rRNA gene sequence and functional gene analyses, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), soxB (sulfate thioesterase/thiohydrolase) and amoA (ammonia monooxygenase). Sulfur oxidizers from both Gammaproteobacteria and Betaproteobacteria were detected in 16S rRNA, soxB and RuBisCO gene libraries. DNA-based stable-isotope probing analyses using 13C-bicarbonate showed that Thiobacillus spp. were most active in assimilating CO2 and also implied that ammonia and nitrite oxidizers were active during incubations. Nitrosomonas spp. were detected in both 16S rRNA and amoA gene libraries from the ‘heavy’ DNA and sequences related to nitrite-oxidizing bacteria Nitrospira and Candidatus ‘Nitrotoga’ were also detected in the ‘heavy’ DNA, which suggests that ammonia/nitrite oxidation may be another major primary production process in this unique ecosystem. A significant number of sequences associated with known methylotrophs from the Betaproteobacteria were obtained, including Methylotenera, Methylophilus and Methylovorus, supporting the view that cycling of one-carbon compounds may be an important process within Movile Cave. Other sequences detected in the bacterial 16S rRNA clone library included Verrucomicrobia, Firmicutes, Bacteroidetes, alphaproteobacterial Rhodobacterales and gammaproteobacterial Xanthomonadales. Archaeal 16S rRNA sequences retrieved were restricted within two groups, namely the Deep-sea Hydrothermal Vent Euryarchaeota group and the Miscellaneous Crenarchaeotic group. No sequences related to known sulfur-oxidizing archaea, ammonia-oxidizing archaea, methanogens or anaerobic methane-oxidizing archaea were detected in this clone library. The results provided molecular biological evidence to support the hypothesis that Movile Cave is driven by chemolithoautotrophy, mainly through sulfur oxidation by sulfur-oxidizing bacteria and reveal that ammonia- and nitrite-oxidizing bacteria may also be major primary producers in Movile Cave.

Effect of earthworms on the community structure of active methanotrophic bacteria in a landfill cover soil

In the United Kingdom, landfills are the primary anthropogenic source of methane emissions. Methanotrophic bacteria present in landfill biocovers can significantly reduce methane emissions via their capacity to oxidize up to 100% of the methane produced. Several biotic and abiotic parameters regulate methane oxidation in soil, such as oxygen, moisture, methane concentration and temperature. Earthworm-mediated bioturbation has been linked to an increase in methanotrophy in a landfill biocover soil (AC Singer et al., unpublished), but the mechanism of this trophic interaction remains unclear. The aims of this study were to determine the composition of the active methanotroph community and to investigate the interactions between earthworms and bacteria in this landfill biocover soil where the methane oxidation activity was significantly increased by the earthworms. Soil microcosms were incubated with 13C-CH4 and with or without earthworms. DNA and RNA were extracted to characterize the soil bacterial communities, with a particular emphasis on methanotroph populations, using phylogenetic (16S ribosomal RNA) and functional methane monooxygenase (pmoA and mmoX) gene probes, coupled with denaturing gradient-gel electrophoresis, clone libraries and pmoA microarray analyses. Stable isotope probing (SIP) using 13C-CH4 substrate allowed us to link microbial function with identity of bacteria via selective recovery of ‘heavy’ 13C-labelled DNA or RNA and to assess the effect of earthworms on the active methanotroph populations. Both types I and II methanotrophs actively oxidized methane in the landfill soil studied. Results suggested that the earthworm-mediated increase in methane oxidation rate in the landfill soil was more likely to be due to the stimulation of bacterial growth or activity than to substantial shifts in the methanotroph community structure. A Bacteroidetes-related bacterium was identified only in the active bacterial community of earthworm-incubated soil but its capacity to actually oxidize methane has to be proven.

Microbiology of Lonar Lake and other soda lakes

Soda lakes are saline and alkaline ecosystems that are believed to have existed throughout the geological record of Earth. They are widely distributed across the globe, but are highly abundant in terrestrial biomes such as deserts and steppes and in geologically interesting regions such as the East African Rift valley. The unusual geochemistry of these lakes supports the growth of an impressive array of microorganisms that are of ecological and economic importance. Haloalkaliphilic Bacteria and Archaea belonging to all major trophic groups have been described from many soda lakes, including lakes with exceptionally high levels of heavy metals. Lonar Lake is a soda lake that is centered at an unusual meteorite impact structure in the Deccan basalts in India and its key physicochemical and microbiological characteristics are highlighted in this article. The occurrence of diverse functional groups of microbes, such as methanogens, methanotrophs, phototrophs, denitrifiers, sulfur oxidizers, sulfate reducers and syntrophs in soda lakes, suggests that these habitats harbor complex microbial food webs that (a) interconnect various biological cycles via redox coupling and (b) impact on the production and consumption of greenhouse gases. Soda lake microorganisms harbor several biotechnologically relevant enzymes and biomolecules (for example, cellulases, amylases, ectoine) and there is the need to augment bioprospecting efforts in soda lake environments with new integrated approaches. Importantly, some saline and alkaline lake ecosystems around the world need to be protected from anthropogenic pressures that threaten their long-term existence.

Active methylotrophs in the sediments of Lonar Lake, a saline and alkaline ecosystem formed by meteor impact

Lonar Lake is a unique saline and alkaline ecosystem formed by meteor impact in the Deccan basalts in India around 52 000 years ago. To investigate the role of methylotrophy in the cycling of carbon in this unusual environment, stable-isotope probing (SIP) was carried out using the one-carbon compounds methane, methanol and methylamine. Denaturing gradient gel electrophoresis fingerprinting analyses performed with heavy 13C-labelled DNA retrieved from sediment microcosms confirmed the enrichment and labelling of active methylotrophic communities. Clone libraries were constructed using PCR primers targeting 16S rRNA genes and functional genes. Methylomicrobium, Methylophaga and Bacillus spp. were identified as the predominant active methylotrophs in methane, methanol and methylamine SIP microcosms, respectively. Absence of mauA gene amplification in the methylamine SIP heavy fraction also indicated that methylamine metabolism in Lonar Lake sediments may not be mediated by the methylamine dehydrogenase enzyme pathway. Many gene sequences retrieved in this study were not affiliated with extant methanotrophs or methylotrophs. These sequences may represent hitherto uncharacterized novel methylotrophs or heterotrophic organisms that may have been cross-feeding on methylotrophic metabolites or biomass. This study represents an essential first step towards understanding the relevance of methylotrophy in the soda lake sediments of an unusual impact crater structure.

Spatial and temporal diversity of methanotrophs in a landfill cover soil are differentially related to soil abiotic factors

Methanotrophs present in landfill cover soil can limit methane emissions from landfill sites by oxidizing methane produced in landfill. Understanding the spatial and temporal distribution of populations of methanotrophs and the factors influencing their activity and diversity in landfill cover soil is critical to devise better landfill cover soil management strategies. pmoA-based microarray analyses of methanotroph community structure revealed a temporal shift in methanotroph populations across different seasons. Type II methanotrophs (particularly Methylocystis sp.) were found to be present across all seasons. Minor shifts in type I methanotroph populations were observed. In the case of spatial distribution, only minor differences in methanotroph community structure were observed with no recognizable patterns (both vertical and horizontal) at a 5 m scale. Correlation analysis between soil abiotic parameters (total C, N, NH4 (+) , NO3 (-) and water content) and distribution of methanotrophs revealed a lack of conclusive evidence for any distinct correlation pattern between measured abiotic parameters and methanotroph community structure, suggesting that complex interactions of several physico-chemical parameters shape methanotroph diversity and activity in landfill cover soils.

A novel pathway producing dimethylsulphide in bacteria is widespread in soil environments.

The volatile compound dimethylsulphide (DMS) is important in climate regulation, the sulphur cycle and signalling to higher organisms. Microbial catabolism of the marine osmolyte dimethylsulphoniopropionate (DMSP) is thought to be the major biological process generating DMS. Here we report the discovery and characterization of the first gene for DMSP-independent DMS production in any bacterium. This gene, mddA, encodes a methyltransferase that methylates methanethiol and generates DMS. MddA functions in many taxonomically diverse bacteria including sediment-dwelling pseudomonads, nitrogen-fixing bradyrhizobia and cyanobacteria, and mycobacteria including the pathogen Mycobacterium tuberculosis. The mddA gene is present in metagenomes from varied environments, being particularly abundant in soil environments, where it is predicted to occur in up to 76% of bacteria. This novel pathway may significantly contribute to global DMS emissions, especially in terrestrial environments and could represent a shift from the notion that DMSP is the only significant precursor of DMS.

Microbiology of Movile Cave—A Chemolithoautotrophic Ecosystem

Discovered in 1986, Movile Cave is an unusual cave ecosystem sustained by in situ chemoautotrophic primary production. The cave is completely isolated from the surface and the primary energy sources are hydrogen sulfide and methane released from hydrothermal fluids. Both condensation and acid corrosion processes contribute to the formation of Movile Cave. Invertebrates, many of which are endemic to Movile Cave, are isotopically lighter in both carbon and nitrogen than surface organisms, indicating that they derive nutrition from chemoautotrophic primary producers within the cave. Here we review work on the microbiology of the Movile Cave ecosystem, with particular emphasis on the functional diversity of microbes involved in sulfur, carbon and nitrogen cycling, and discuss their role in chemosynthetic primary production.

Bacterial metabolism of methylated amines and identification of novel methylotrophs in Movile Cave.

Movile Cave, Romania, is an unusual underground ecosystem that has been sealed off from the outside world for several million years and is sustained by non-phototrophic carbon fixation. Methane and sulfur-oxidising bacteria are the main primary producers, supporting a complex food web that includes bacteria, fungi and cave-adapted invertebrates. A range of methylotrophic bacteria in Movile Cave grow on one-carbon compounds including methylated amines, which are produced via decomposition of organic-rich microbial mats. The role of methylated amines as a carbon and nitrogen source for bacteria in Movile Cave was investigated using a combination of cultivation studies and DNA stable isotope probing (DNA-SIP) using 13C-monomethylamine (MMA). Two newly developed primer sets targeting the gene for gamma-glutamylmethylamide synthetase (gmaS), the first enzyme of the recently-discovered indirect MMA-oxidation pathway, were applied in functional gene probing. SIP experiments revealed that the obligate methylotroph Methylotenera mobilis is one of the dominant MMA utilisers in the cave. DNA-SIP experiments also showed that a new facultative methylotroph isolated in this study, Catellibacterium sp. LW-1 is probably one of the most active MMA utilisers in Movile Cave. Methylated amines were also used as a nitrogen source by a wide range of non-methylotrophic bacteria in Movile Cave. PCR-based screening of bacterial isolates suggested that the indirect MMA-oxidation pathway involving GMA and N-methylglutamate is widespread among both methylotrophic and non-methylotrophic MMA utilisers from the cave.

Alpha- and Gammaproteobacterial Methanotrophs Codominate the Active Methane-Oxidizing Communities in an Acidic Boreal Peat Bog

ABSTRACT The objective of this study was to characterize metabolically active, aerobic methanotrophs in an ombrotrophic peatland in the Marcell Experimental Forest, in Minnesota. Methanotrophs were investigated in the field and in laboratory incubations using DNA-stable isotope probing (SIP), expression studies on particulate methane monooxygenase (pmoA) genes, and amplicon sequencing of 16S rRNA genes. Potential rates of oxidation ranged from 14 to 17 μmol of CH4 g dry weight soil−1 day−1. Within DNA-SIP incubations, the relative abundance of methanotrophs increased from 4% in situ to 25 to 36% after 8 to 14 days. Phylogenetic analysis of the 13C-enriched DNA fractions revealed that the active methanotrophs were dominated by the genera Methylocystis (type II; Alphaproteobacteria), Methylomonas, and Methylovulum (both, type I; Gammaproteobacteria). In field samples, a transcript-to-gene ratio of 1 to 2 was observed for pmoA in surface peat layers, which attenuated rapidly with depth, indicating that the highest methane consumption was associated with a depth of 0 to 10 cm. Metagenomes and sequencing of cDNA pmoA amplicons from field samples confirmed that the dominant active methanotrophs were Methylocystis and Methylomonas. Although type II methanotrophs have long been shown to mediate methane consumption in peatlands, our results indicate that members of the genera Methylomonas and Methylovulum (type I) can significantly contribute to aerobic methane oxidation in these ecosystems.

Earthworm activity in a simulated landfill cover soil shifts the community composition of active methanotrophs.

Landfills represent a major source of methane in the atmosphere. In a previous study, we demonstrated that earthworm activity in landfill cover soil can increase soil methane oxidation capacity. In this study, a simulated landfill cover soil mesocosm (1 m × 0.15 m) was used to observe the influence of earthworms (Eisenia veneta) on the active methanotroph community composition, by analyzing the expression of the pmoA gene, which is responsible for methane oxidation. mRNA-based pmoA microarray analysis revealed that earthworm activity in landfill cover soil stimulated activity of type I methanotrophs (Methylobacter, Methylomonas, Methylosarcina spp.) compared to type II methanotrophs (particularly Methylocystis spp.). These results, along with previous studies of methanotrophs in landfill cover soil, can now be used to plan in situ field studies to integrate earthworm-induced methanotrophy with other landfill management practises in order to maximize soil methane oxidation and reduce methane emissions from landfills.