Methane oxidation and methylotroph population dynamics in groundwater mesocosms.

Extraction of natural gas from unconventional hydrocarbon reservoirs by hydraulic fracturing raises concerns about methane migration into groundwater. Microbial methane oxidation can be a significant methane sink. Here, we inoculated replicated, sand-packed, continuous mesocosms with groundwater from a field methane release experiment. The mesocosms experienced thirty-five weeks of dynamic methane, oxygen and nitrate concentrations. We determined concentrations and stable isotope signatures of methane, carbon dioxide and nitrate and monitored microbial community composition of suspended and attached biomass. Methane oxidation was strictly dependent on oxygen availability and led to enrichment of 13 C in residual methane. Nitrate did not enhance methane oxidation under oxygen limitation. Methylotrophs persisted for weeks in the absence of methane, making them a powerful marker for active as well as past methane leaks. Thirty-nine distinct populations of methylotrophic bacteria were observed. Methylotrophs mainly occurred attached to sediment particles. Abundances of methanotrophs and other methylotrophs were roughly similar across all samples, pointing at transfer of metabolites from the former to the latter. Two populations of Gracilibacteria (Candidate Phyla Radiation) displayed successive blooms, potentially triggered by a period of methane famine. This study will guide interpretation of future field studies and provides increased understanding of methylotroph ecophysiology.

An oligotrophic deep-subsurface community dependent on syntrophy is dominated by sulfur-driven autotrophic denitrifiers.

Subsurface lithoautotrophic microbial ecosystems (SLiMEs) under oligotrophic conditions are typically supported by H2 Methanogens and sulfate reducers, and the respective energy processes, are thought to be the dominant players and have been the research foci. Recent investigations showed that, in some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa, methanogens contribute <5% of the total DNA and appear to produce sufficient CH4 to support the rest of the diverse community. This paradoxical situation reflects our lack of knowledge about the in situ metabolic diversity and the overall ecological trophic structure of SLiMEs. Here, we show the active metabolic processes and interactions in one of these communities by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Dominating the active community are four autotrophic ß-proteobacterial genera that are capable of oxidizing sulfur by denitrification, a process that was previously unnoticed in the deep subsurface. They co-occur with sulfate reducers, anaerobic methane oxidizers, and methanogens, which each comprise <5% of the total community. Syntrophic interactions between these microbial groups remove thermodynamic bottlenecks and enable diverse metabolic reactions to occur under the oligotrophic conditions that dominate in the subsurface. The dominance of sulfur oxidizers is explained by the availability of electron donors and acceptors to these microorganisms and the ability of sulfur-oxidizing denitrifiers to gain energy through concomitant S and H2 oxidation. We demonstrate that SLiMEs support taxonomically and metabolically diverse microorganisms, which, through developing syntrophic partnerships, overcome thermodynamic barriers imposed by the environmental conditions in the deep subsurface.

Opuntia ficus-indica cladodes as feedstock for ethanol production by Kluyveromyces marxianus and Saccharomyces cerevisiae.

The feasibility of ethanol production using an enzymatic hydrolysate of pretreated cladodes of Opuntia ficus-indica (prickly pear cactus) as carbohydrate feedstock was investigated, including a comprehensive chemical analysis of the cladode biomass and the effects of limited aeration on the fermentation profiles and sugar utilization. The low xylose and negligible mannose content of the cladode biomass used in this study suggested that the hemicellulose structure of the O. ficus-indica cladode was atypical of hardwood or softwood hemicelluloses. Separate hydrolysis and fermentation and simultaneous saccharification and fermentation procedures using Kluyveromyces marxianus and Saccharomyces cerevisiae at 40 and 35 °C, respectively, gave similar ethanol yields under non-aerated conditions. In oxygen-limited cultures K. marxianus exhibited almost double the ethanol productivity compared to non-aerated cultures, although after sugar depletion utilization of the produced ethanol was evident. Ethanol concentrations of up to 19.5 and 20.6 g l(-1) were obtained with K. marxianus and S. cerevisiae, respectively, representing 66 and 70 % of the theoretical yield on total sugars in the hydrolysate. Because of the low xylan content of the cladode biomass, a yeast capable of xylose fermentation might not be a prerequisite for ethanol production. K. marxianus, therefore, has potential as an alternative to S. cerevisiae for bioethanol production. However, the relatively low concentration of fermentable sugars in the O. ficus-indica cladode hydrolysate presents a technical constraint for commercial exploitation.

Hydrogen and dark oxygen drive microbial productivity in diverse groundwater ecosystems.

Around 50% of humankind relies on groundwater as a source of drinking water. Here we investigate the age, geochemistry, and microbiology of 138 groundwater samples from 95 monitoring wells (<250 m depth) located in 14 aquifers in Canada. The geochemistry and microbiology show consistent trends suggesting large-scale aerobic and anaerobic hydrogen, methane, nitrogen, and sulfur cycling carried out by diverse microbial communities. Older groundwaters, especially in aquifers with organic carbon-rich strata, contain on average more cells (up to 1.4 × 107 mL-1) than younger groundwaters, challenging current estimates of subsurface cell abundances. We observe substantial concentrations of dissolved oxygen (0.52 ± 0.12 mg L-1 [mean ± SE]; n = 57) in older groundwaters that seem to support aerobic metabolisms in subsurface ecosystems at an unprecedented scale. Metagenomics, oxygen isotope analyses and mixing models indicate that dark oxygen is produced in situ via microbial dismutation. We show that ancient groundwaters sustain productive communities and highlight an overlooked oxygen source in present and past subsurface ecosystems of Earth.

Metagenomic analysis of ethylene glycol contamination in anaerobic digestion.

Anaerobic digestion is an established method for the biological conversion of waste feedstocks to biogas and biomethane. While anaerobic digestion is an excellent waste management technique, it can be susceptible to toxins and pollutants from contaminated feedstocks, which may have a detrimental impact on a digester's efficiency and productivity. Ethylene glycol (EG) is readily used in the heat-transfer loops of anaerobic digestion facilities to maintain reactor temperature. Failure of the structural integrity of these heat transfer loops can cause EG to leak into the digester, potentially causing a decrease in the resultant gas yields. Batch fermentations were incubated with 0, 10, 100 and 500 ppm (parts per million) of EG, and analysis showed that the EG was completely metabolised by the digester microbiome. The concentrations of EG tested showed significant increases in gas yields, however there were no significant changes to the digester microbiome.

A metagenomic window into carbon metabolism at 3 km depth in Precambrian continental crust.

Subsurface microbial communities comprise a significant fraction of the global prokaryotic biomass; however, the carbon metabolisms that support the deep biosphere have been relatively unexplored. In order to determine the predominant carbon metabolisms within a 3-km deep fracture fluid system accessed via the Tau Tona gold mine (Witwatersrand Basin, South Africa), metagenomic and thermodynamic analyses were combined. Within our system of study, the energy-conserving reductive acetyl-CoA (Wood-Ljungdahl) pathway was found to be the most abundant carbon fixation pathway identified in the metagenome. Carbon monoxide dehydrogenase genes that have the potential to participate in (1) both autotrophic and heterotrophic metabolisms through the reversible oxidization of CO and subsequent transfer of electrons for sulfate reduction, (2) direct utilization of H2 and (3) methanogenesis were identified. The most abundant members of the metagenome belonged to Euryarchaeota (22%) and Firmicutes (57%)-by far, the highest relative abundance of Euryarchaeota yet reported from deep fracture fluids in South Africa and one of only five Firmicutes-dominated deep fracture fluids identified in the region. Importantly, by combining the metagenomics data and thermodynamic modeling of this study with previously published isotopic and community composition data from the South African subsurface, we are able to demonstrate that Firmicutes-dominated communities are associated with a particular hydrogeologic environment, specifically the older, more saline and more reducing waters.

Deep subsurface mine stalactites trap endemic fissure fluid Archaea, Bacteria, and Nematoda possibly originating from ancient seas.

Stalactites (CaCO3 and salt) from water seeps are frequently encountered in ceilings of mine tunnels whenever they intersect water-bearing faults or fractures. To determine whether stalactites could be mineralized traps for indigenous fracture water microorganisms, we analyzed stalactites collected from three different mines ranging in depth from 1.3 to 3.1 km. During sampling in Beatrix gold mine (1.4 km beneath the surface), central South Africa, CaCO3 stalactites growing on the mine tunnel ceiling were collected and observed, in two cases, to contain a living obligate brackish water/marine nematode species, Monhystrella parvella. After sterilization of the outer surface, mineral layers were physically removed from the outside to the interior, and DNA extracted. Based upon 16S and 18S rRNA gene sequencing, Archaea, Bacteria, and Eukarya in different combinations were detected for each layer. Using CT scan and electron microscopy the inner structure of CaCO3 and salt stalactites were analyzed. CaCO3 stalactites show a complex pattern of lamellae carrying bacterially precipitated mineral structures. Nematoda were clearly identified between these layers confirming that bacteria and nematodes live inside the stalactites and not only in the central straw. Salt stalactites exhibit a more uniform internal structure. Surprisingly, several Bacteria showing highest sequence identities to marine species were identified. This, together with the observation that the nematode M. parvella recovered from Beatrix gold mine stalactite can only survive in a salty environment makes the origin of the deep subsurface colonization enigmatic. The possibility of a Permian origin of fracture fluids is discussed. Our results indicate stalactites are suitable for biodiversity recovery and act as natural traps for microorganisms in the fissure water long after the water that formed the stalactite stopped flowing.

Comparisons of the composition and biogeographic distribution of the bacterial communities occupying South African thermal springs with those inhabiting deep subsurface fracture water.

South Africa has numerous thermal springs that represent topographically driven meteoric water migrating along major fracture zones. The temperature (40-70°C) and pH (8-9) of the thermal springs in the Limpopo Province are very similar to those of the low salinity fracture water encountered in the South African mines at depths ranging from 1.0 to 3.1 km. The major cation and anion composition of these thermal springs are very similar to that of the deep fracture water with the exception of the dissolved inorganic carbon and dissolved O2, both of which are typically higher in the springs than in the deep fracture water. The in situ biological relatedness of such thermal springs and the subsurface fracture fluids that feed them has not previously been evaluated. In this study, we evaluated the microbial diversity of six thermal spring and six subsurface sites in South Africa using high-throughput sequencing of 16S rRNA gene hypervariable regions. Proteobacteria were identified as the dominant phylum within both subsurface and thermal spring environments, but only one genera, Rheinheimera, was identified among all samples. Using Morisita similarity indices as a metric for pairwise comparisons between sites, we found that the communities of thermal springs are highly distinct from subsurface datasets. Although the Limpopo thermal springs do not appear to provide a new window for viewing subsurface bacterial communities, we report that the taxonomic compositions of the subsurface sites studied are more similar than previous results would indicate and provide evidence that the microbial communities sampled at depth are more correlated to subsurface conditions than geographical distance.

Biting of anthropophilic Culicoides fulvithorax (Diptera: Ceratopogonidae), a vector of Mansonella perstans in Nigeria.

Anthropophilic Culicoides were investigated in a rural community endemic for Mansonella perstans in Ijebu North area of western Nigeria between December 2003 and October 2004. Three hundred and fifty-nine adults of Culicoides fulvithorax collected by human bait in the morning were dissected for Mansonella perstans larvae, and 1.95% of infection rate was found. Seasonal abundance of C. fulvithorax was investigated by monthly biting rates, and showed that higher prevalence was observed in rainy season, with peak in September. Culicoides prevalence was positively correlated with rainfall and relative humidity, but not temperature. Human perceptions on the behavior of these biting midges were determined by interviewing 854 self-selected villagers, of which 86.5% of the interviewees confessed having experienced Culicoides bites. Between 76.5 and 99.1% of the various age groups complained body reactions to Culicoides bites. Itching was the most frequent body reaction. No interviewees associated Culicoides with transmission of any parasitic infections. The results showed need to adequately control Culicoides in the community.