Methanogen Abundance Thresholds Capable of Differentiating In Vitro Methane Production in Human Stool Samples.

BACKGROUND: Intestinal methane (CH4) gas production has been associated with a number of clinical conditions and may have important metabolic and physiological effects. AIMS: In this study, taxonomic and functional gene analyses and in vitro CH4 gas measurements were used to determine if molecular markers can potentially serve as clinical tests for colonic CH4 production. METHODS: We performed a cross-sectional study involving full stool samples collected from 33 healthy individuals. In vitro CH4 gas measurements were obtained after 2-h incubation of stool samples and used to characterize samples as CH4 positive (CH4+) and CH4 negative (CH4-; n = 10 and 23, respectively). Next, we characterized the fecal microbiota through high-throughput DNA sequencing with a particular emphasis on archaeal phylum Euryarchaeota. Finally, qPCR analyses, targeting the mcrA gene, were done to determine the ability to differentiate CH4+ versus CH4- samples and to delineate major methanogen species associated with CH4 production. RESULTS: Methanobrevibacter was found to be the most abundant methane producer and its relative abundance provides a clear distinction between CH4+ versus CH4- samples. Its sequencing-based relative abundance detection threshold for CH4 production was calculated to be 0.097%. The qPCR-based detection threshold separating CH4+ versus CH4- samples, based on mcrA gene copies, was 5.2 × 105 copies/g. CONCLUSION: Given the decreased time-burden placed on patients, a qPCR-based test on a fecal sample can become a valuable tool in clinical assessment of CH4 producing status.

Mechanical and structural properties of archaeal hypernucleosomes.

Many archaea express histones, which organize the genome and play a key role in gene regulation. The structure and function of archaeal histone-DNA complexes remain however largely unclear. Recent studies show formation of hypernucleosomes consisting of DNA wrapped around an 'endless' histone-protein core. However, if and how such a hypernucleosome structure assembles on a long DNA substrate and which interactions provide for its stability, remains unclear. Here, we describe micromanipulation studies of complexes of the histones HMfA and HMfB with DNA. Our experiments show hypernucleosome assembly which results from cooperative binding of histones to DNA, facilitated by weak stacking interactions between neighboring histone dimers. Furthermore, rotational force spectroscopy demonstrates that the HMfB-DNA complex has a left-handed chirality, but that torque can drive it in a right-handed conformation. The structure of the hypernucleosome thus depends on stacking interactions, torque, and force. In vivo, such modulation of the archaeal hypernucleosome structure may play an important role in transcription regulation in response to environmental changes.

Links between the rumen microbiota, methane emissions and feed efficiency of finishing steers offered dietary lipid and nitrate supplementation.

Ruminant methane production is a significant energy loss to the animal and major contributor to global greenhouse gas emissions. However, it also seems necessary for effective rumen function, so studies of anti-methanogenic treatments must also consider implications for feed efficiency. Between-animal variation in feed efficiency represents an alternative approach to reducing overall methane emissions intensity. Here we assess the effects of dietary additives designed to reduce methane emissions on the rumen microbiota, and explore relationships with feed efficiency within dietary treatment groups. Seventy-nine finishing steers were offered one of four diets (a forage/concentrate mixture supplemented with nitrate (NIT), lipid (MDDG) or a combination (COMB) compared to the control (CTL)). Rumen fluid samples were collected at the end of a 56 d feed efficiency measurement period. DNA was extracted, multiplexed 16s rRNA libraries sequenced (Illumina MiSeq) and taxonomic profiles were generated. The effect of dietary treatments and feed efficiency (within treatment groups) was conducted both overall (using non-metric multidimensional scaling (NMDS) and diversity indexes) and for individual taxa. Diet affected overall microbial populations but no overall difference in beta-diversity was observed. The relative abundance of Methanobacteriales (Methanobrevibacter and Methanosphaera) increased in MDDG relative to CTL, whilst VadinCA11 (Methanomassiliicoccales) was decreased. Trimethylamine precursors from rapeseed meal (only present in CTL) probably explain the differences in relative abundance of Methanomassiliicoccales. There were no differences in Shannon indexes between nominal low or high feed efficiency groups (expressed as feed conversion ratio or residual feed intake) within treatment groups. Relationships between the relative abundance of individual taxa and feed efficiency measures were observed, but were not consistent across dietary treatments.

Chromatinization of Escherichia coli with archaeal histones.

Nucleosomes restrict DNA accessibility throughout eukaryotic genomes, with repercussions for replication, transcription, and other DNA-templated processes. How this globally restrictive organization emerged during evolution remains poorly understood. Here, to better understand the challenges associated with establishing globally restrictive chromatin, we express histones in a naive system that has not evolved to deal with nucleosomal structures: Escherichia coli. We find that histone proteins from the archaeon Methanothermus fervidus assemble on the E. coli chromosome in vivo and protect DNA from micrococcal nuclease digestion, allowing us to map binding footprints genome-wide. We show that higher nucleosome occupancy at promoters is associated with lower transcript levels, consistent with local repressive effects. Surprisingly, however, this sudden enforced chromatinization has only mild repercussions for growth unless cells experience topological stress. Our results suggest that histones can become established as ubiquitous chromatin proteins without interfering critically with key DNA-templated processes.

High-Level Abundances of Methanobacteriales and Syntrophobacterales May Help To Prevent Corrosion of Metal Sheet Piles.

Iron sheet piles are widely used in flood protection, dike construction, and river bank reinforcement. Their corrosion leads to gradual deterioration and often makes replacement necessary. Natural deposit layers on these sheet piles can prevent degradation and significantly increase their life span. However, little is known about the mechanisms of natural protective layer formation. Here, we studied the microbially diverse populations of corrosion-protective deposit layers on iron sheet piles at the Gouderak pumping station in Zuid-Holland, the Netherlands. Deposit layers, surrounding sediment and top sediment samples were analyzed for soil physicochemical parameters, microbially diverse populations, and metabolic potential. Methanogens appeared to be enriched 18-fold in the deposit layers. After sequencing, metagenome assembly and binning, we obtained four nearly complete draft genomes of microorganisms (Methanobacteriales, two Coriobacteriales, and Syntrophobacterales) that were highly enriched in the deposit layers, strongly indicating a potential role in corrosion protection. Coriobacteriales and Syntrophobacterales could be part of a microbial food web degrading organic matter to supply methanogenic substrates. Methane-producing Methanobacteriales could metabolize iron, which may initially lead to mild corrosion but potentially stimulates the formation of a carbonate-rich protective deposit layer in the long term. In addition, Methanobacteriales and Coriobacteriales have the potential to interact with metal surfaces via direct interspecies or extracellular electron transfer. In conclusion, our study provides valuable insights into microbial populations involved in iron corrosion protection and potentially enables the development of novel strategies for in situ screening of iron sheet piles in order to reduce risks and develop more sustainable replacement practices.IMPORTANCE Iron sheet piles are widely used to reinforce dikes and river banks. Damage due to iron corrosion poses a significant safety risk and has significant economic impact. Different groups of microorganisms are known to either stimulate or inhibit the corrosion process. Recently, natural corrosion-protective deposit layers were found on sheet piles. Analyses of the microbial composition indicated a potential role for methane-producing archaea. However, the full metabolic potential of the microbial communities within these protective layers has not been determined. The significance of this work lies in the reconstruction of the microbial food web of natural corrosion-protective layers isolated from noncorroding metal sheet piles. With this work, we provide insights into the microbiological mechanisms that potentially promote corrosion protection in freshwater ecosystems. Our findings could support the development of screening protocols to assess the integrity of iron sheet piles to decide whether replacement is required.

Methanotrophy - Environmental, Industrial and Medical Applications.

Aerobic methanotrophs are an intriguing group of microbes with the singular ability to consume methane as their sole source of carbon and energy. As such, methanotrophs are receiving increased attention to control methane emissions to limit future climate change. Methanotrophs have a wide range of other applications, including pollutant remediation and methane valorization (e.g. conversion of methane to protein, bioplastics, and biodiesel amongst other products). Methanotrophs also produce a novel copper-binding compound, methanobactin, that has significant potential for the treatment of copper-related human pathologies. Here we provide an overview of aerobic methanotrophy, describe current and future applications of these unique microbes, as well as discuss various strategies one can consider to better realize the opportunities these microbes present.

Diversity of Methane Cycling Microorganisms in Soils and Their Relation to Oxygen.

Microorganisms are important players in the global methane cycle. Anaerobic methanogenic archaea are largely responsible for methane production, while aerobic methanotrophic bacteria, as well as anaerobic methanotrophic bacteria and archaea, are involved in methane oxidation. In anoxic wetland soils, methanogens produce methane, while methanotrophs act as a filter and reduce methane emissions. In the predominantly oxic upland soils, aerobic methanotrophs oxidize atmospheric methane. This review gives an overview of the diversity of methanogenic and methanotrophic microorganisms, highlights recent discoveries and provides information concerning their occurrence in soils. Recent findings indicate that the methanogenic and methanotrophic lifestyles are more widespread in microorganisms than previously thought, and that the metabolic versatility of some methane-cycling organisms is broader than known from well characterized cultivated organisms. It also turned out that the control of methanogenic and methanotrophic bacteria by oxygen is more complex than previously thought. The implications this finding may have for the life of these microorganisms in soils and on soil methane fluxes is discussed.

Metagenomic Approaches Unearth Methanotroph Phylogenetic and Metabolic Diversity.

Methanotrophic microorganisms utilize methane as an electron donor and a carbon source. To date, the capacity to oxidize methane is restricted to microorganisms from three bacterial and one archaeal phyla. Most of our knowledge of methanotrophic metabolism has been obtained using highly enriched or pure cultures grown in the laboratory. However, many methanotrophs currently evade cultivation, thus metagenomics provides a complementary approach for gaining insight into currently unisolated microorganisms. Here we synthesize the studies using metagenomics to glean information about methanotrophs. We complement this summary with an analysis of methanotroph marker genes from 235 publically available metagenomic datasets. We analyze the phylogenetic and environmental distribution of methanotrophs sampled by metagenomics. We also highlight metabolic insights that methanotroph genomes assembled from metagenomes are illuminating. In summary, metagenomics has increased methanotrophic foliage within the tree of life, as well as provided new insights into methanotroph metabolism, which collectively can guide new cultivation efforts. Lastly, given the importance of methanotrophs for biotechnological applications and their capacity to filter greenhouse gases from a variety of ecosystems, metagenomics will continue to be an important component in the arsenal of tools needed for understanding methanotroph diversity and metabolism in both engineered and natural systems.

CH4 flux and methanogen community dynamics from five common emergent vegetations in a full-scale constructed wetland.

To investigate the effects of emergent plants on CH4 efflux and elucidate the key factors responsible for these effects, annual monitoring of CH4 emissions and methanogen community dynamics in a full-scale constructed wetland (CW) was conducted. Five emergent plants (Typha orientalis, Cyperus alternifolius, Arundo domax, Iris pseudacorus, and Thalia dealbata) commonly used in CWs were selected for investigation. The greatest CH4 flux (annual mean 19.4 mg m-2 h-1) was observed from I. pseudacorus, while the lowest CH4 flux (7.1 mg m-2 h-1) was observed from Thalia dealbata. The CH4 flux from five emergent plants showed marked seasonal variation. Total nitrogen (TN) and total phosphorous (TP) were weakly correlated with CH4 emissions, whereas total carbon (TC) and root biomass of plants were positively correlated with CH4 emissions. Quantitative real-time PCR (q-PCR) analysis indicated that the gene abundance of eubacterial 16S rRNA, particulate methane monooxygenase (pmoA) and methyl coenzyme M reductase (mcrA) significantly differed among plant species. Differences in TC, root biomass, and dissolved oxygen (DO) caused by plant species were potential factors responsible for differences in methanogens, methanotrophs, and CH4 emissions. Methanobacteriaceae, Methanoregulaceae, Methanomicrobiaceae, and Methanosarcinaceae were the dominant families of methanogens. The pathways of methanogenesis from the five emergent plants differed, with the main pathway being hydrogenotrophic, while both hydrogenotrophic and acetotrophic methanogens were involved in A. domax. Redundancy analysis (RDA) further indicated that emergent plant types had a profound influence on the methanogenic communities. Taken together, these results suggest emergent plant species can significantly influence CH4 fluxes in CW through microbial communities, biochemical pathways for methanogenesis, TC, and DO. Furthermore, plant species in CWs should be considered an important factor in evaluating greenhouse gases emission. Finally, it is necessary to effectively manage CWs vegetation to maximize their environmental benefits. Graphical abstract ᅟ.

In Vitro Response of Rumen Microbiota to the Antimethanogenic Red Macroalga Asparagopsis taxiformis.

The red macroalga Asparagopsis taxiformis has been shown to significantly decrease methane production by rumen microbial communities. This has been attributed to the bioaccumulation of halogenated methane analogues produced as algal secondary metabolites. The objective of this study was to evaluate the impact of A. taxiformis supplementation on the relative abundance of methanogens and microbial community structure during in vitro batch fermentation. Addition of A. taxiformis (2% organic matter) or the halogenated methane analogue bromoform (5 µM) reduced methane production by over 99% compared to a basal substrate-only control. Quantitative PCR confirmed that the decrease in methane production was correlated with a decrease in the relative abundance of methanogens. High-throughput 16S ribosomal RNA gene amplicon sequencing showed that both treatments reduced the abundance of the three main orders of methanogens present in ruminants (Methanobacteriales, Methanomassiliicoccales and Methanomicrobiales). Shifts in bacterial community structure due to the addition of A. taxiformis and 5 µM bromoform were similar and concomitant with increases in hydrogen concentration in the headspace of the fermenters. With high potency and broad-spectrum activity against rumen methanogens, A. taxiformis represents a promising natural strategy for reducing enteric methane emissions from ruminant livestock.

Community structure of the metabolically active rumen bacterial and archaeal communities of dairy cows over the transition period.

Dairy cows experience dramatic changes in host physiology from gestation to lactation period and dietary switch from high-forage prepartum diet to high-concentrate postpartum diet over the transition period (parturition +/- three weeks). Understanding the community structure and activity of the rumen microbiota and its associative patterns over the transition period may provide insight for e.g. improving animal health and production. In the present study, rumen samples from ten primiparous Holstein dairy cows were collected over seven weeks spanning the transition period. Total RNA was extracted from the rumen samples and cDNA thereof was subsequently used for characterizing the metabolically active bacterial (16S rRNA transcript amplicon sequencing) and archaeal (qPCR, T-RFLP and mcrA and 16S rRNA transcript amplicon sequencing) communities. The metabolically active bacterial community was dominated by three phyla, showing significant changes in relative abundance range over the transition period: Firmicutes (from prepartum 57% to postpartum 35%), Bacteroidetes (from prepartum 22% to postpartum 18%) and Proteobacteria (from prepartum 7% to postpartum 32%). For the archaea, qPCR analysis of 16S rRNA transcript number, revealed a significant prepartum to postpartum increase in Methanobacteriales, in accordance with an observed increase (from prepartum 80% to postpartum 89%) in relative abundance of 16S rRNA transcript amplicons allocated to this order. On the other hand, a significant prepartum to postpartum decrease (from 15% to 2%) was observed in relative abundance of Methanomassiliicoccales 16S rRNA transcripts. In contrast to qPCR analysis of the 16S rRNA transcripts, quantification of mcrA transcripts revealed no change in total abundance of metabolically active methanogens over the transition period. According to T-RFLP analysis of the mcrA transcripts, two Methanobacteriales genera, Methanobrevibacter and Methanosphaera (represented by the T-RFs 39 and 267 bp), represented more than 70% of the metabolically active methanogens, showing no significant changes over the transition period; minor T-RFs, likely to represent members of the order Methanomassiliicoccales and with a relative abundance below 5% in total, decreased significantly over the transition period. In accordance with the T-RFLP analysis, the mcrA transcript amplicon sequencing revealed Methanobacteriales to cover 99% of the total reads, dominated by the genera Methanobrevibacter (75%) and Methanosphaera (24%), whereas the Methanomassiliicoccales order covered only 0.2% of the total reads. In conclusion, the present study showed that the structure of the metabolically active bacterial and archaeal rumen communities changed over the transition period, likely in response to the dramatic changes in physiology and nutritional factors like dry matter intake and feed composition. It should be noted however that for the methanogens, the observed community changes were influenced by the analyzed gene (mcrA or 16S rRNA).

Identification of an Intestinal Microbiota Signature Associated With Severity of Irritable Bowel Syndrome.

BACKGROUND & AIMS: We have limited knowledge about the association between the composition of the intestinal microbiota and clinical features of irritable bowel syndrome (IBS). We collected information on the fecal and mucosa-associated microbiota of patients with IBS and evaluated whether these were associated with symptoms. METHODS: We collected fecal and mucosal samples from adult patients who met the Rome III criteria for IBS at a secondary/tertiary care outpatient clinics in Sweden, as well as from healthy subjects. The exploratory set comprised 149 subjects (110 with IBS and 39 healthy subjects); 232 fecal samples and 59 mucosal biopsy samples were collected and analyzed by 16S ribosomal RNA targeted pyrosequencing. The validation set comprised 46 subjects (29 with IBS and 17 healthy subjects); 46 fecal samples, but no mucosal samples, were collected and analyzed. For each subject, we measured exhaled H2 and CH4, oro-anal transit time, and the severity of psychological and gastrointestinal symptoms. Fecal methanogens were measured by quantitative polymerase chain reaction. Numerical ecology analyses and a machine learning procedure were used to analyze the data. RESULTS: Fecal microbiota showed covariation with mucosal adherent microbiota. By using classic approaches, we found no differences in fecal microbiota abundance or composition between patients with IBS vs healthy patients. A machine learning procedure, a computational statistical technique, allowed us to reduce the 16S ribosomal RNA data complexity into a microbial signature for severe IBS, consisting of 90 bacterial operational taxonomic units. We confirmed the robustness of the intestinal microbial signature for severe IBS in the validation set. The signature was able to discriminate between patients with severe symptoms, patients with mild/moderate symptoms, and healthy subjects. By using this intestinal microbiota signature, we found IBS symptom severity to be associated negatively with microbial richness, exhaled CH4, presence of methanogens, and enterotypes enriched with Clostridiales or Prevotella species. This microbiota signature could not be explained by differences in diet or use of medications. CONCLUSIONS: In analyzing fecal and mucosal microbiota from patients with IBS and healthy individuals, we identified an intestinal microbiota profile that is associated with the severity of IBS symptoms. TRIAL REGISTRATION NUMBER: NCT01252550.

Archaeal communities of Arctic methane-containing permafrost.

In the present study, we used culture-independent methods to investigate the diversity of methanogenic archaea and their distribution in five permafrost samples collected from a borehole in the Kolyma River Lowland (north-east of Russia). Total DNA was extracted from methane-containing permafrost samples of different age and amplified by PCR. The resulting DNA fragments were cloned. Phylogenetic analysis of the sequences showed the presence of archaea in all studied samples; 60%-95% of sequences belonged to the Euryarchaeota. Methanogenic archaea were novel representatives of Methanosarcinales, Methanomicrobiales, Methanobacteriales and Methanocellales orders. Bathyarchaeota (Miscellaneous Crenarchaeota Group) representatives were found among nonmethanogenic archaea in all the samples studied. The Thaumarchaeota representatives were not found in the upper sample, whereas Woesearchaeota (formerly DHVEG-6) were found in the three deepest samples. Unexpectedly, the greatest diversity of archaea was observed at a depth of 22.3 m, probably due to the availability of the labile organic carbon and/or due to the migration of the microbial cells during the freezing front towards the bottom.

Changes in microbial communities associated with gas hydrates in subseafloor sediments from the Nankai Trough.

Little is known about the microbial distribution patterns in subseafloor sediments. This study examines microbial diversity and activities in sediments of the Nankai Trough, where biogenic gas hydrates are deposited. Illumina sequencing of 16S rRNA genes revealed that the prokaryotic community structure is correlated with hydrate occurrence and depth but not with the sedimentary facies. The bacterial phyla 'Atribacteria' lineage JS1 and Chloroflexi dominated in all samples, whereas lower taxonomic units of Chloroflexi accounted for community variation related to hydrate saturation. In archaeal communities, 'Bathyarchaeota' was significantly abundant in the hydrate-containing samples, whereas Marine Benthic Group-B dominated in the upper sediments without hydrates. mcrA gene sequences assigned to deeply branching groups and ANME-1 were detected only in hydrate-containing samples. A predominance of hydrogenotrophic methanogens, Methanomicrobiales and Methanobacteriales, over acetoclastic methanogens was found throughout the depth. Incubation tests on hydrate-containing samples with a stable isotope tracer showed anaerobic methane oxidation activities under both low- and seawater-like salinity conditions. These results indicate that the distribution patterns of microorganisms involved in carbon cycling changed with gas hydrate occurrence, possibly because of the previous hydrate dissociation followed by pore water salinity decrease in situ, as previously proposed by a geochemical study at the study site.

Changes in microbial community structures due to varying operational conditions in the anaerobic digestion of oxytetracycline-medicated cow manure.

Management of manure containing veterinary antibiotics is a major concern in anaerobic treatment systems because of their possible adverse effects on microbial communities. Therefore, the aim of study was to investigate how oxytetracycline (OTC) influences bacteria and acetoclastic and hydrogenotrophic methanogens under varying operational conditions in OTC-medicated and non-medicated anaerobic cow manure digesters. Concentrations of OTC and its metabolites throughout the anaerobic digestion were determined using ultraviolet-high-performance liquid chromatography (UV-HPLC) and tandem liquid chromatography-mass spectrometry (LC/MS/MS), respectively. Fluorescent in situ hybridization, denaturing gradient gel electrophoresis, cloning, and sequencing analyses were used to monitor changes in microbial community structures. According to the results of analytical and molecular approaches, operating conditions highly influence active microbial community dynamics and associate with biogas production and elimination of OTC and its metabolites during anaerobic digestion of cow manure in the presence of an average initial concentration of 2.2 mg OTC/L. The impact of operating conditions has a drastic effect on acetoclastic methanogens than hydrogenotrophic methanogens and bacteria.

Methane production improvement by modulation of solid phase immersion in dry batch anaerobic digestion process: Dynamic of methanogen populations.

Several 60L dry batch anaerobic digestion (AD) reactors were implemented with or without liquid reserve on cattle manure. The immersed part modulation of cattle manure increased the methane flow of about 13%. The quantitative real time PCR and the optimized DNA extraction were implemented and validated to characterize and quantify the methanogen dynamic in dry batch AD process. Final quantities of methanogens converged toward the same level in several inocula at the end of AD. Methanogen dynamic was shown by dominance of Methanosarcinaceae for acetotrophic methanogens and Methanobacteriales for the hydrogenotrophic methanogens. Overall, methanogens populations were stabilized in liquid phase, except Methanosaetaceae. Solid phase was colonized by Methanomicrobiales and Methanosarcinaceae populations giving a support to biofilm development. The methane increase could be explained by a raise of Methanosarcinaceae population in presence of a total contact between solid and liquid phases. Methanosarcinaceae was a bio-indicator of the methane production.

Abundance and potential metabolic activity of methanogens in well-aerated forest and grassland soils of an alpine region.

Although methanogens were recently discovered to occur in aerated soils, alpine regions have not been extensively studied for their presence so far. Here, the abundance of archaea and the methanogenic guilds Methanosarcinales, Methanococcales, Methanobacteriales, Methanomicrobiales and Methanocella spp. was studied at 16 coniferous forest and 14 grassland sites located at the montane and subalpine belts of the Northern Limestone Alps (calcareous) and the Austrian Central Alps (siliceous) using quantitative real-time PCR. Abundance of archaea, methanogens and the methanogenic potentials were significantly higher in grasslands than in forests. Furthermore, methanogenic potentials of calcareous soils were higher due to pH. Methanococcales, Methanomicrobiales and Methanocella spp. were detected in all collected samples, which indicates that they are autochthonous, while Methanobacteriales were absent from 4 out of 16 forest soils. Methanosarcinales were absent from 10 out of 16 forest soils and 2 out of 14 grassland soils. Nevertheless, together with Methanococcales they represented the majority of the 16S rRNA gene copies quantified from the grassland soils. Contrarily, forest soils were clearly dominated by Methanococcales. Our results indicate a higher diversity of methanogens in well-aerated soils than previously believed and that pH mainly influences their abundances and activities.

Effects of freezing storage on the DNA extraction and microbial evaluation from anaerobic digested sludges.

BACKGROUND: The anaerobic digestion is one of the most spread renewable energy technology. The input biomasses included various environmental problematic wastes such as sludge coming from wastewater treatment plant (WWTP) and organic fraction of municipal solid waste (OFMSW). As biomolecular procedures have become important tools for the microbial characterisation of anaerobic samples coming from the reactors, it is crucial sampling and extracting properly DNA in order to employ such types of techniques. The current study is aimed to evaluate how freezing temperature and length of storage at -20 °C influence both the extracted DNA yield and microbial community quantifications from digested sludge samples collected at full-scale plants. RESULTS: From WWTP sludge samples, we observed a reduction of DNA concentration comparing fresh and stored samples for 10 days at -20 °C (ANOVA test p < 0.0001), with an estimated DNA loss of approximately 65% for such types of samples, however the methanogen communities can be assessed respecting the fresh conditions. From OFMSW sludge samples, we observed a reduction in extracted DNA (-90%), after 120 frozen days, while microbial communities are determined respecting the fresh conditions within 2 months of frozen storage. CONCLUSIONS: The remarkable effect of frozen storage on sludge samples suggests as the better procedure to perform the DNA extraction from fresh sample. On the other hand it is not generally possible, so approximately 2 months of storage at -20 °C appears to be suitable time at which DNA concentrations remain sufficient to perform coherent microbial characterization through quantitative qRT-PCR.

Improved Monitoring of Semi-Continuous Anaerobic Digestion of Sugarcane Waste: Effects of Increasing Organic Loading Rate on Methanogenic Community Dynamics.

The anaerobic digestion of filter cake and its co-digestion with bagasse, and the effect of gradual increase of the organic loading rate (OLR) from start-up to overload were investigated. Understanding the influence of environmental and technical parameters on the development of particular methanogenic pathway in the biogas process was an important aim for the prediction and prevention of process failure. The rapid accumulation of volatile organic acids at high OLR of 3.0 to 4.0 gvs·L⁻¹·day⁻¹ indicated strong process inhibition. Methanogenic community dynamics of the reactors was monitored by stable isotope composition of biogas and molecular biological analysis. A potential shift toward the aceticlastic methanogenesis was observed along with the OLR increase under stable reactor operating conditions. Reactor overloading and process failure were indicated by the tendency to return to a predominance of hydrogenotrophic methanogenesis with rising abundances of the orders Methanobacteriales and Methanomicrobiales and drop of the genus Methanosarcina abundance.

Methanol induces low temperature resilient methanogens and improves methane generation from domestic wastewater at low to moderate temperatures.

Low temperature (<20 °C) limits bio-methanation of sewage. Literature shows that hydrogenotrophic methanogens can adapt themselves to low temperature and methanol is a preferred substrate by methanogens in cold habitats. The study hypothesizes that methanol can induce the growth of low-temperature resilient, methanol utilizing, hydrogenotrophs in UASB reactor. The hypothesis was tested in field conditions to evaluate the impact of seasonal temperature variations on methane yield in the presence and absence of methanol. Results show that 0.04% (v/v) methanol increased methane up to 15 times and its effect was more pronounced at lower temperatures. The qPCR analysis showed the presence of Methanobacteriales along with Methanosetaceae in large numbers. This indicates methanol induced the growth of both the hydrogenotrophic and acetoclastic groups through direct and indirect routes, respectively. This study thus demonstrated that methanol can impart resistance in methanogenic biomass to low temperature and can improve performance of UASB reactor.