Isolation and characterization of a thermophilic, obligately anaerobic and heterotrophic marine Chloroflexi bacterium from a Chloroflexi dominated microbial community associated with a Japanese shallow hydrothermal system, and proposal for Thermomarinilinea lacunofontalis gen. nov., sp. nov.

A novel marine thermophilic and heterotrophic Anaerolineae bacterium in the phylum Chloroflexi, strain SW7T, was isolated from an in situ colonization system deployed in the main hydrothermal vent of the Taketomi submarine hot spring field located on the southern part of Yaeyama Archipelago, Japan. The microbial community associated with the hydrothermal vent was predominated by thermophilic heterotrophs such as Thermococcaceae and Anaerolineae, and the next dominant population was thermophilic sulfur oxidizers. Both aerobic and anaerobic hydrogenotrophs including methanogens were detected as minor populations. During the culture-dependent viable count analysis in this study, an Anaerolineae strain SW7T was isolated from an enrichment culture at a high dilution rate. Strain SW7T was an obligately anaerobic heterotroph grew with fermentation, and non-motile thin rods 3.5-16.5 μm in length and 0.2 μm in width constituting multicellular filament. Growth was observed between 37-65 ℃ (optimum 60℃), pH 5.5-7.3 (optimum pH 6.0), 0.5-3.5% (w/v) NaCl concentration (optimum 1.0%). Based on physiological and phylogenetic features of a new isolate, we propose a new species representing a novel genus Thermomarinilinea: the type strain of Thermomarinilinea lacunofontalis sp. nov., is SW7T (= JCM15506T = KCTC5908T)

Overcoming organic and nitrogen overload in thermophilic anaerobic digestion of pig slurry by coupling a microbial electrolysis cell

The combination of the anaerobic digestion (AD) process with a microbial electrolysis cell (MEC) coupled to an ammonia stripping unit as a post-treatment was assessed both in series operation, to improve the quality of the effluent, and in loop configuration recirculating the effluent, to increase the AD robustness. The MEC allowed maintaining the chemical oxygen demand removal of the whole system of 46 ± 5% despite the AD destabilization after doubling the organic and nitrogen loads, while recovering 40 ± 3% of ammonia. The AD-MEC system, in loop configuration, helped to recover the AD (55% increase in methane productivity) and attained a more stable and robust operation. The microbial population assessment revealed an enhancement of AD methanogenic archaea numbers and a shift in eubacterial population. The AD-MEC combined system is a promising strategy for stabilizing AD against organic and nitrogen overloads, while improving the quality of the effluent and recovering nutrients for their reutilization.Postprint (author's final draft

ThermoPhyl : a software tool for selecting phylogenetically optimized conventional and quantitative-PCR taxon-targeted assays for use with complex samples

The ability to specifically and sensitively target genotypes of interest is critical for the success of many PCR-based analyses of environmental or clinical samples that contain multiple templates.Next-generation sequence data clearly show that such samples can harbour hundreds to thousands of operational taxonomic units; a richness which precludes the manual evaluation of candidate assay specificity and sensitivity using multiple sequence alignments. To solve this problem we have developed and validated a free software tool which automates the identification of PCR assays targeting specific genotypes in complex samples. ThermoPhyl uses user-defined target and non-target sequence databases to assess the phylogenetic sensitivity and specificity of thermodynamically optimised candidate assays derived from primer design software packages. ThermoPhyl takes its name from its central premise of testing Thermodynamically optimal assays for Phylogenetic specificity and sensitivity and can be used for two primer (traditional PCR) or two primers with an internal probe (e.g. TaqMan® qPCR) applications and potentially for oligonucleotide probes.Here we describe the use of ThermoPhyl for traditional PCR and qPCR assays. PCR assays selected using ThermoPhyl were validated using 454 pyrosequencing of a traditional specific PCR assay and with a set of four genotype-specific qPCR assays applied to estuarine sediment samples

Methyl Coenzyme M Reductase (mcrA) Gene Abundance Correlates with Activity Measurements of Methanogenic H2/CO2-Enriched Anaerobic Biomass

Biologically produced methane (CH4) from anaerobic digesters is a renewable alternative to fossil fuels, but digester failure can be a serious problem. Monitoring the microbial community within the digester could provide valuable information about process stability because this technology is dependent upon the metabolic processes of microorganisms. A healthy methanogenic community is critical for digester function and CH4 production. Methanogens can be surveyed and monitored using genes and transcripts of mcrA, which encodes the α subunit of methyl coenzyme M reductase – the enzyme that catalyses the final step in methanogenesis. Using clone libraries and quantitative polymerase chain reaction, we compared the diversity and abundance of mcrA genes and transcripts in four different methanogenic hydrogen/CO2 enrichment cultures to function, as measured by specific methanogenic activity (SMA) assays using H2/CO2. The mcrA gene copy number significantly correlated with CH4 production rates using H2/CO2, while correlations between mcrA transcript number and SMA were not significant. The DNA and cDNA clone libraries from all enrichments were distinctive but community diversity also did not correlate with SMA. Although hydrogenotrophic methanogens dominated these enrichments, the results indicate that this methodology should be applicable to monitoring other methanogenic communities in anaerobic digesters. Ultimately, this could lead to the engineering of digester microbial communities to produce more CH4 for use as renewable fuel

Relating Methanogen Community Structure and Anaerobic Digester Function

Much remains unknown about the relationships between microbial community structure and anaerobic digester function. However, knowledge of links between community structure and function, such as specific methanogenic activity (SMA) and COD removal rate, are valuable to improve anaerobic bioprocesses. In this work, quantitative structure–activity relationships (QSARs) were developed using multiple linear regression (MLR) to predict SMA using methanogen community structure descriptors for 49 cultures. Community descriptors were DGGE demeaned standardized band intensities for amplicons of a methanogen functional gene (mcrA). First, predictive accuracy of MLR QSARs was assessed using cross validation with training (n = 30) and test sets (n = 19) for glucose and propionate SMA data. MLR equations correlating band intensities and SMA demonstrated good predictability for glucose (q2 = 0.54) and propionate (q2 = 0.53). Subsequently, data from all 49 cultures were used to develop QSARs to predict SMA values. Higher intensities of two bands were correlated with higher SMA values; high abundance of methanogens associated with these two bands should be encouraged to attain high SMA values. QSARs are helpful tools to identify key microorganisms or to study and improve many bioprocesses. Development of new, more robust QSARs is encouraged for anaerobic digestion or other bioprocesses, including nitrification, nitritation, denitrification, anaerobic ammonium oxidation, and enhanced biological phosphorus removal

Relating Anaerobic Digestion Microbial Community and Process Function

Anaerobic digestion (AD) involves a consortium of microorganisms that convert substrates into biogas containing methane for renewable energy. The technology has suffered from the perception of being periodically unstable due to limited understanding of the relationship between microbial community structure and function. The emphasis of this review is to describe microbial communities in digesters and quantitative and qualitative relationships between community structure and digester function. Progress has been made in the past few decades to identify key microorganisms influencing AD. Yet, more work is required to realize robust, quantitative relationships between microbial community structure and functions such as methane production rate and resilience after perturbations. Other promising areas of research for improved AD may include methods to increase/control (1) hydrolysis rate, (2) direct interspecies electron transfer to methanogens, (3) community structure–function relationships of methanogens, (4) methanogenesis via acetate oxidation, and (5) bioaugmentation to study community–activity relationships or improve engineered bioprocesses

Insights into the Ecological Roles and Evolution of Methyl-Coenzyme M Reductase-Containing Hot Spring Archaea

Several recent studies have shown the presence of genes for the key enzyme associated with archaeal methane/alkane metabolism, methyl-coenzyme M reductase (Mcr), in metagenome-assembled genomes (MAGs) divergent to existing archaeal lineages. Here, we study the mcr-containing archaeal MAGs from several hot springs, which reveal further expansion in the diversity of archaeal organisms performing methane/alkane metabolism. Significantly, an MAG basal to organisms from the phylum Thaumarchaeota that contains mcr genes, but not those for ammonia oxidation or aerobic metabolism, is identified. Together, our phylogenetic analyses and ancestral state reconstructions suggest a mostly vertical evolution of mcrABG genes among methanogens and methanotrophs, along with frequent horizontal gene transfer of mcr genes between alkanotrophs. Analysis of all mcr-containing archaeal MAGs/genomes suggests a hydrothermal origin for these microorganisms based on optimal growth temperature predictions. These results also suggest methane/alkane oxidation or methanogenesis at high temperature likely existed in a common archaeal ancestor

Plane of nutrition affects the phylogenetic diversity and relative abundance of transcriptionally active methanogens in the bovine rumen

peer-reviewedMethane generated during enteric fermentation in ruminant livestock species is a major contributor to global anthropogenic greenhouse gas emissions. A period of moderate feed restriction followed by ad libitum access to feed is widely applied in cattle management to exploit the animal’s compensatory growth potential and reduce feed costs. In the present study, we utilised microbial RNA from rumen digesta samples to assess the phylogenetic diversity of transcriptionally active methanogens from feed-restricted and non-restricted animals. To determine the contribution of different rumen methanogens to methanogenesis during dietary restriction of cattle, we conducted high-throughput mcrA cDNA amplicon sequencing on an Illumina MiSeq and analysed both the abundance and phylogenetic origin of different mcrA cDNA sequences. When compared to their unrestricted contemporaries, in feed-restricted animals, the methanogenic activity, based on mcrA transcript abundance, of Methanobrevibacter gottschalkii clade increased while the methanogenic activity of the Methanobrevibacter ruminantium clade and members of the Methanomassiliicoccaceae family decreased. This study shows that the quantity of feed consumed can evoke large effects on the composition of methanogenically active species in the rumen of cattle. These data potentially have major implications for targeted CH4 mitigation approaches such as anti-methanogen vaccines and/or tailored dietary management

Elevated CO2 and Warming Altered Grassland Microbial Communities in Soil Top-Layers.

As two central issues of global climate change, the continuous increase of both atmospheric CO2 concentrations and global temperature has profound effects on various terrestrial ecosystems. Microbial communities play pivotal roles in these ecosystems by responding to environmental changes through regulation of soil biogeochemical processes. However, little is known about the effect of elevated CO2 (eCO2) and global warming on soil microbial communities, especially in semiarid zones. We used a functional gene array (GeoChip 3.0) to measure the functional gene composition, structure, and metabolic potential of soil microbial communities under warming, eCO2, and eCO2 + warming conditions in a semiarid grassland. The results showed that the composition and structure of microbial communities was dramatically altered by multiple climate factors, including elevated CO2 and increased temperature. Key functional genes, those involved in carbon (C) degradation and fixation, methane metabolism, nitrogen (N) fixation, denitrification and N mineralization, were all stimulated under eCO2, while those genes involved in denitrification and ammonification were inhibited under warming alone. The interaction effects of eCO2 and warming on soil functional processes were similar to eCO2 alone, whereas some genes involved in recalcitrant C degradation showed no significant changes. In addition, canonical correspondence analysis and Mantel test results suggested that NO3-N and moisture significantly correlated with variations in microbial functional genes. Overall, this study revealed the possible feedback of soil microbial communities to multiple climate change factors by the suppression of N cycling under warming, and enhancement of C and N cycling processes under either eCO2 alone or in interaction with warming. These findings may enhance our understanding of semiarid grassland ecosystem responses to integrated factors of global climate change