Temperature and Inoculum Origin Influence the Performance of Ex-Situ Biological Hydrogen Methanation.

The conversion of H2 into methane can be carried out by microorganisms in a process so-called biomethanation. In ex-situ biomethanation H2 and CO2 gas are exogenous to the system. One of the main limitations of the biomethanation process is the low gas-liquid transfer rate and solubility of H2 which are strongly influenced by the temperature. Hydrogenotrophic methanogens that are responsible for the biomethanation reaction are also very sensitive to temperature variations. The aim of this work was to evaluate the impact of temperature on batch biomethanation process in mixed culture. The performances of mesophilic and thermophilic inocula were assessed at 4 temperatures (24, 35, 55 and 65 °C). A negative impact of the low temperature (24 °C) was observed on microbial kinetics. Although methane production rate was higher at 55 and 65 °C (respectively 290 ± 55 and 309 ± 109 mL CH4/L.day for the mesophilic inoculum) than at 24 and 35 °C (respectively 156 ± 41 and 253 ± 51 mL CH4/L.day), the instability of the system substantially increased, likely because of a strong dominance of only Methanothermobacter species. Considering the maximal methane production rates and their stability all along the experiments, an optimal temperature range of 35 °C or 55 °C is recommended to operate ex-situ biomethanation process.

Functional Group Distribution of the Carrier Surface Influences Adhesion of Methanothermobacter thermautotrophicus.

Various support carriers are used for high-density retention of methanogenic archaea in anaerobic wastewater treatment systems. Although the physicochemical properties of carrier materials and microorganisms influence the adhesion of methanogenic archaea, details about the underlying mechanism remain poorly characterized. We applied seven types of chemical surface modifications to carbon felts to clarify the adhesion properties of Methanothermobacter thermautotrophicus, a representative thermophilic hydrogenotrophic methanogen. The relationship between carrier surface properties and methanogen adhesion was evaluated. M. thermautotrophicus adhesion was significantly increased up to 2.6 times in comparison with control on carbon felts treated with NaOH, HCl, H2SO4, or Na2HPO4. Treated carbon felts showed a lower water contact angle, but no correlation between the carrier surface contact angle and methanogen adhesion was observed. On the other hand, at the surface of the carrier that showed improved adhesion of methanogens, the ratio of -COOH : -OH was 1 : 0.65. Such a ratio was not observed with treated carriers for which methanogen adhesion was not improved. Therefore, in the adhesion of M. thermautotrophicus, the functional group abundance was important as well as physical surface properties such as the hydrophobicity. Hydrogenotrophic methanogens are involved in active methanation during the startup of anaerobic digestion. Additionally, these methanogenic archaea function as methanogenic cathode catalysts. Therefore, anaerobic digestion performance will greatly improve by controlling the adhesion of hydrogenotrophic methanogens such as M. thermautotrophicus.

A systematic approach re-analyzing the effects of temperature disturbance on the microbial community of mesophilic anaerobic digestion.

Microbial communities are key drivers of ecosystem processes, but their behavior in disturbed environments is difficult to measure. How microbial community composition and function respond disturbances is a common challenge in biomedical, environmental, agricultural, and bioenergy research. A novel way to solve this problem is to use a systems-level perspective and describe microbial communities as networks. Based on a mesophilic anaerobic digestion system of swine manure as a tool, we propose a simple framework to investigate changes in microbial communities via compositions, metabolic pathways, genomic properties and interspecies relationships in response to a long-term temperature disturbance. After temperature disturbance, microbial communities tend towards a competitive interaction network with higher GC content and larger genome size. Based on microbial interaction networks, communities responded to the disturbance by showing a transition from acetotrophic (Methanotrichaceae and Methanosarcinaceae) to methylotrophic methanogens (Methanomassiliicoccaceae and Methanobacteriaceae) and a fluctuation in rare biosphere taxa. To conclude, this study may be important for exploring the dynamic relationships between disturbance and microbial communities as a whole, as well as for providing researchers with a better understanding of how changes in microbial communities relate to ecological processes.

Culture- and metagenomics-enabled analyses of the Methanosphaera genus reveals their monophyletic origin and differentiation according to genome size.

The genus Methanosphaera is a well-recognized but poorly characterized member of the mammalian gut microbiome, and distinctive from Methanobrevibacter smithii for its ability to induce a pro-inflammatory response in humans. Here we have used a combination of culture- and metagenomics-based approaches to expand the representation and information for the genus, which has supported the examination of their phylogeny and physiological capacity. Novel isolates of the genus Methanosphaera were recovered from bovine rumen digesta and human stool, with the bovine isolate remarkable for its large genome size relative to other Methanosphaera isolates from monogastric hosts. To substantiate this observation, we then recovered seven high-quality Methanosphaera-affiliated population genomes from ruminant and human gut metagenomic datasets. Our analyses confirm a monophyletic origin of Methanosphaera spp. and that the colonization of monogastric and ruminant hosts favors representatives of the genus with different genome sizes, reflecting differences in the genome content needed to persist in these different habitats.

Methanogens in humans: potentially beneficial or harmful for health.

Methanogens are anaerobic prokaryotes from the domain archaea that utilize hydrogen to reduce carbon dioxide, acetate, and a variety of methyl compounds into methane. Earlier believed to inhabit only the extreme environments, these organisms are now reported to be found in various environments including mesophilic habitats and the human body. The biological significance of methanogens for humans has been re-evaluated in the last few decades. Their contribution towards pathogenicity has received much less attention than their bacterial counterparts. In humans, methanogens have been studied in the gastrointestinal tract, mouth, and vagina, and considerable focus has shifted towards elucidating their possible role in the progression of disease conditions in humans. Methanoarchaea are also part of the human skin microbiome and proposed to play a role in ammonia turnover. Compared to hundreds of different bacterial species, the human body harbors only a handful of methanogen species represented by Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, Methanomassiliicoccus luminyensis, Candidatus Methanomassiliicoccus intestinalis, and Candidatus Methanomethylophilus alvus. Their presence in the human gut suggests an indirect correlation with severe diseases of the colon. In this review, we examine the current knowledge about the methanoarchaea in the human body and possible beneficial or less favorable interactions.

Biogas production from food waste via co-digestion and digestion- effects on performance and microbial ecology.

In this work, performance and microbial structure of a digestion (food waste-only) and a co-digestion process (mixture of cow manure and food waste) were studied at mesophilic (37 °C) and thermophilic (55 °C) temperatures. The highest methane yield (480 mL/g VS) was observed in the mesophilic digester (MDi) fed with food waste alone. The mesophilic co-digestion of food waste and manure (McoDi) yielded 26% more methane than the sum of individual digestions of manure and food waste. The main volatile fatty acid (VFA) in the mesophilic systems was acetate, averaging 93 and 172 mg/L for McoDi and MDi, respectively. Acetate (2150 mg/L) and propionate (833 mg/L) were the main VFAs in the thermophilic digester (TDi), while propionate (163 mg/L) was the major VFA in the thermophilic co-digester (TcoDi). The dominant bacteria in MDi was Chloroflexi (54%), while Firmicutes was dominant in McoDi (60%). For the mesophilic reactors, the dominant archaea was Methanosaeta in MDi, while Methanobacterium and Methanosaeta had similar abundance in McoDi. In the thermophilic systems, the dominant bacteria were Thermotogae, Firmicutes and Synergistetes in both digesters, however, the relative abundance of these phyla were different. For archaea, the genus Methanothermobacter were entirely dominant in both TDi and TcoDi.

Low Pressure Tolerance by Methanogens in an Aqueous Environment: Implications for Subsurface Life on Mars.

The low pressure at the surface of Mars (average: 6 mbar) is one potentially biocidal factor that any extant life on the planet would need to endure. Near subsurface life, while shielded from ultraviolet radiation, would also be exposed to this low pressure environment, as the atmospheric gas-phase pressure increases very gradually with depth. Few studies have focused on low pressure as inhibitory to the growth or survival of organisms. However, recent work has uncovered a potential constraint to bacterial growth below 25 mbar. The study reported here tested the survivability of four methanogen species (Methanothermobacter wolfeii, Methanosarcina barkeri, Methanobacterium formicicum, Methanococcus maripaludis) under low pressure conditions approaching average martian surface pressure (6 mbar - 143 mbar) in an aqueous environment. Each of the four species survived exposure of varying length (3 days - 21 days) at pressures down to 6 mbar. This research is an important stepping-stone to determining if methanogens can actively metabolize/grow under these low pressures. Additionally, the recently discovered recurring slope lineae suggest that liquid water columns may connect the surface to deeper levels in the subsurface. If that is the case, any organism being transported in the water column would encounter the changing pressures during the transport.

Anaerobic digestion of food waste stabilized by lime mud from papermaking process.

The effects of lime mud from papermaking process (LMP) addition as buffer agent and inorganic nutrient on the anaerobic digestion stability of food waste (FW) were investigated under mesophilic conditions with the aim of avoiding volatile fatty acids accumulation, and inorganic elements deficiency. When LMP concentration ranged from 6.0 to 10g/L, the FW anaerobic digestion could maintain efficient and stable state. These advantages are attributed to the existence of Ca, Na, Mg, K, Fe, and alkaline substances that favor the methanogenic process. The highest CH4 yield of 272.8mL/g-VS was obtained at LMP and VS concentrations of 10.0 and 19.8g/L, respectively, with the corresponding lag-phase time of 3.84d and final pH of 8.4. The methanogens from residue digestates mainly consisted of Methanobrevibacter, coccus-type and sarcina-type methanogens with LMP addition compared to Methanobacteria in control. However, higher concentration of LMP inhibited methanogenic activities and methane production.

Physiological and genetic basis for self-aggregation of a thermophilic hydrogenotrophic methanogen, Methanothermobacter strain CaT2.

Several thermophilic hydrogenotrophic methanogens naturally aggregate in their habitats in association with hydrogen-producing bacteria for efficient transfer of the methane fermentation intermediates to produce methane. However, physiology of aggregation and the identity of aggregation-specific genes remain to be elucidated. Here, we isolated and characterized a hydrogen and formate-utilizing Methanothermobacter sp. CaT2 that is capable of self-aggregation and utilizing formate. CaT2 produced methane from propionate oxidation in association with a syntrophic propionate-oxidizing bacterium faster than other methanogens, including Methanothermobacter thermautotrophicus ΔH and Methanothermobacter thermautotrophicus Z-245. CaT2 also aggregated throughout the culture period and was coated with polysaccharides, which was not found on the ΔH and Z-245 cells. Sugar content (particularly of rhamnose and mannose) was also higher in the CaT2 cells than the ΔH and Z-245 cells. Comparative genomic analysis of CaT2 indicated that four candidate genes, all of which encode glycosyltransferase, were involved in aggregation of CaT2. Transcriptional analysis showed that one glycosyltransferase gene was expressed at relatively high levels under normal growth conditions. The polysaccharide layer on the CaT2 cell surface, which is probably assembled by these glycosyltransferases, may be involved in cell aggregation.

The intestinal archaea Methanosphaera stadtmanae and Methanobrevibacter smithii activate human dendritic cells.

The methanoarchaea Methanosphaera stadtmanae and Methanobrevibacter smithii are known to be part of the indigenous human gut microbiota. Although the immunomodulatory effects of bacterial gut commensals have been studied extensively in the last decade, the impact of methanoarchaea in human's health and disease was rarely examined. Consequently, we studied and report here on the effects of M. stadtmanae and M. smithii on human immune cells. Whereas exposure to M. stadtmanae leads to substantial release of proinflammatory cytokines in monocyte-derived dendritic cells (moDCs), only weak activation was detected after incubation with M. smithii. Phagocytosis of M. stadtmanae by moDCs was demonstrated by confocal microscopy as well as transmission electronic microscopy (TEM) and shown to be crucial for cellular activation by using specific inhibitors. Both strains, albeit to different extents, initiate a maturation program in moDCs as revealed by up-regulation of the cell-surface receptors CD86 and CD197 suggesting additional activation of adaptive immune responses. Furthermore, M. stadtmanae and M. smithii were capable to alter the gene expression of antimicrobial peptides in moDCs to different extents. Taken together, our findings strongly argue that the archaeal gut inhabitants M. stadtmanae and M. smithii are specifically recognized by the human innate immune system. Moreover, both strains are capable of inducing an inflammatory cytokine response to different extents arguing that they might have diverse immunomodulatory functions. In conclusion, we propose that the impact of intestinal methanoarchaea on pathological conditions involving the gut microbiota has been underestimated until now.

Long-term defaunation increases the abundance of cellulolytic ruminococci and methanogens but does not affect the bacterial and methanogen diversity in the rumen of sheep.

Protozoa are commensal eukaryotes in the rumen of herbivores. Protozoa are large producers of hydrogen, which is utilized by methanogenic archaea to produce methane, a greenhouse gas. The removal of protozoa from the rumen (defaunation) decreases methanogenesis, but also negatively affects fiber digestion, which is the main function of the rumen. The aim of this study was to examine the effect of long-term defaunation on the structure of the microbiota and particularly methanogenic archaea and fibrolytic bacteria to better understand the microbial mechanisms responsible for the decrease in methanogenesis and fibrolysis. The trial was conducted in 5 adult sheep subjected successively to long-term defaunation (2 yr), refaunation (12 wk), and short-term defaunation (10 wk). Methanogens were enumerated by quantitative PCR targeting the rrs (16S ribosomal RNA subunit) and mcrA (methyl coenzyme-M reductase) genes. The rrs gene was used to quantify the 3 major culturable rumen cellulolytic bacterial species (i.e., Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) and total bacteria. Bacterial and methanogen diversity was also examined by PCR-DGGE (PCR-denaturing gradient gel electrophoresis) analysis targeting the rrs and mcrA genes, respectively. Total rumen bacterial density estimated as rrs copies per gram of DM of rumen content increased in response to long- and short-term defaunation (+1 log, P < 0.001), but without noticeable shifts in diversity. Defaunation increased the rrs copies per gram of DM of rumen content of R. albus and R. flavefaciens (+2 log, P < 0 0.001), but did not affect that of F. succinogenes. Despite a 20% reduction in methane emission in the 2 defaunated periods, the mcrA and rrs copies of methanogens per gram of DM of rumen content increased (+1 log, P < 0.001) in the absence of protozoa, whereas the diversity of the dominant methanogenic community was not modified. This study shows no major difference between long- and short-term defaunation in abundance and diversity of bacteria and archaea. It also provides evidence that monitoring the abundance and diversity of methanogens is not sufficient to comprehend the microbial mechanisms leading to a reduction in methane emissions by ruminants. This study also reports for the first time in sheep a selective effect of defaunation on the abundance of cellulolytic bacterial species.

The methanogen-specific transcription factor MsvR regulates the fpaA-rlp-rub oxidative stress operon adjacent to msvR in Methanothermobacter thermautotrophicus.

Methanogens represent some of the most oxygen-sensitive organisms in laboratory culture. Recent studies indicate that they have developed mechanisms to deal with brief oxygen exposure. MsvR is a transcriptional regulator that has a domain architecture unique to a select group of methanogens. Here, runoff in vitro transcription assays were used to demonstrate that MsvR regulates transcription of the divergently transcribed fpaA-rlp-rub operon in Methanothermobacter thermautotrophicus in addition to transcription from its own promoter. The protein products of the fpaA-rlp-rub operon have previously been implicated in oxidative stress responses in M. thermautotrophicus. Additionally, electrophoretic mobility shift assays (EMSAs) and DNase I footprinting were used to confirm a binding site inferred by bioinformatic analysis. Sequence mutations within these binding sites did not significantly alter EMSA shifting patterns on longer templates but did on shorter 50-bp fragments encompassing only the region containing the binding sites. Footprinting confirmed that the regions protected for the longer mutant templates are at different positions within the intergenic region compared to those seen in the intact intergenic region. Oxidized and reduced preparations of MsvR demonstrated different EMSA binding patterns and regions of protection on the intergenic sequence, suggesting that MsvR may play a role in detecting the redox state of the cell.

Isolation and characterization of a new strain of Methanothermobacter marburgensis DX01 from hot springs in China.

Strain DX01, a thermophilic methanogen, was isolated from a hot spring in China. Strain DX01 grew only on H2/CO2. The DNA G+C content is 52 mol% and optimal growth temperature is 65 degrees C. The cell pellet is brick red. By analyzing 16S rRNA sequence, methyl-coenzyme M reductase I, gamma subunit protein sequences, we determined the DX01 strain to be closely related to the species of Methanothermobacter marburgensis. In addition, Methanothermobacter thermautotrophicus delta H(T) and strain DX01 had clear differences in their biochemical composition and protein expression profiles. Based on the above analysis, we propose that strain DX01 is a novel strain within thermoautotrophicus the species of M. marburgensis, namely M. marburgensis DX01. The isolation and characterization of the new M. marburgensis DX01 strain expands the known range of the Methanothermobacter genus.

Differential stability of TATA box binding proteins from archaea with different optimal growth temperatures.

The TATA box binding protein (TBP) is involved in promoter recognition, the first step of transcription initiation. TBP is universally conserved and essential in archaea and eukaryotes. In archaea, TBPs have to be stable and to function in species that cover an extremely wide range of optimal growth temperatures (OGTs), from below 0 degrees C to more than 100 degrees C. Thus, the archaeal TBP family is ideally suited to study the evolutionary adaptation of proteins to an extremely wide range of temperatures. We characterized the thermostability of one mesophilic and one thermophilic TBP by infrared spectroscopy. Transition temperatures (T(m)s) of thermal unfolding have been determined using TBPs from Methanosarcina mazei (OGT 37 degrees C) and from Methanothermobacter thermautotrophicus (OGT 65 degrees C). Furthermore, the influence of protein and salt concentration on thermostability has been characterized. Together with previous studies, our results reveal that the T(m)s of archaeal TBPs are closely correlated with the OGTs of the respective species. Noteworthy, this is also true for the TBP from M. mazei representing the first characterized TBP from a mesophilic archaeon. In contrast, the only characterized eukaryotic TBP of the mesophilic plant Arabidopsis thaliana has a T(m) more than 40 degrees C above the OGT.

Flagellum mediates symbiosis.

We report here molecular mechanisms underlying a bacteria-archaeon symbiosis. We found that a fermentative bacterium used its flagellum for interaction with a specific methanogenic archaeon. The archaeon perceived a bacterial flagellum protein and activated its metabolism (methanogenesis). Transcriptome analyses showed that a substantial number of genes in the archaeon, including those involved in the methanogenesis pathway, were up-regulated after the contact with the flagellum protein. These findings suggest that the bacterium communicates with the archaeon by using its flagellum.

MTH1745, a protein disulfide isomerase-like protein from thermophilic archaea, Methanothermobacter thermoautotrophicum involving in stress response.

MTH1745 is a putative protein disulfide isomerase characterized with 151 amino acid residues and a CPAC active-site from the anaerobic archaea Methanothermobacter thermoautotrophicum. The potential functions of MTH1745 are not clear. In the present study, we show a crucial role of MTH1745 in protecting cells against stress which may be related to its functions as a disulfide isomerase and its chaperone properties. Using real-time polymerase chain reaction analyses, the level of MTH1745 messenger RNA (mRNA) in the thermophilic archaea M. thermoautotrophicum was found to be stress-induced in that it was significantly higher under low (50 degrees C) and high (70 degrees C) growth temperatures than under the optimal growth temperature for the organism (65 degrees C). Additionally, the expression of MTH1745 mRNA was up-regulated by cold shock (4 degrees C). Furthermore, the survival of MTH1745 expressing Escherichia coli cells was markedly higher than that of control cells in response to heat shock (51.0 degrees C). These results indicated that MTH1745 plays an important role in the resistance of stress. By assay of enzyme activities in vitro, MTH1745 also exhibited a chaperone function by promoting the functional folding of citrate synthase after thermodenaturation. On the other hand, MTH1745 was also shown to function as a disulfide isomerase on the refolding of denatured and reduced ribonuclease A. On the basis of its single thioredoxin domain, function as a disulfide isomerase, and its chaperone activity, we suggest that MTH1745 may be an ancient protein disulfide isomerase. These studies may provide clues to the understanding of the function of protein disulfide isomerase in archaea.

Specific DNA binding of a potential transcriptional regulator, inosine 5'-monophosphate dehydrogenase-related protein VII, to the promoter region of a methyl coenzyme m reductase I-encoding operon retrieved from Methanothermobacter thermautotrophicus strain DeltaH.

Two methyl coenzyme M reductases (MCRs) encoded by the mcr and mrt operons of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus DeltaH are expressed in response to H(2) availability. In the present study, cis elements and trans-acting factors responsible for the gene expression of MCRs were investigated by using electrophoretic mobility shift assay (EMSA) and affinity particle purification. A survey of their operator regions by EMSA with protein extracts from mrt-expressing cultures restricted them to 46- and 41-bp-long mcr and mrt upstream regions, respectively. Affinity particle purification of DNA-binding proteins conjugated with putative operator regions resulted in the retrieval of a protein attributed to IMP dehydrogenase-related protein VII (IMPDH VII). IMPDH VII is predicted to have a winged helix-turn-helix DNA-binding motif and two cystathionine beta-synthase domains, and it has been suspected to be an energy-sensing module. EMSA with oligonucleotide probes with unusual sequences showed that the binding site of IMPDH VII mostly overlaps the factor B-responsible element-TATA box of the mcr operon. The results presented here suggest that IMPDH VII encoded by MTH126 is a plausible candidate for the transcriptional regulator of the mcr operon in this methanogen.

The Mth60 fimbriae of Methanothermobacter thermoautotrophicus are functional adhesins.

The ability to adhere onto surfaces is of very high importance for microorganisms, enabling them to stay in a favourable habitat for life. In the case of Bacteria cell surface organelles called fimbriae/pili have been shown to be used for adhesion; corresponding cell surface appendages of Archaea have not yet been defined. The first detailed characterization of archaeal fimbriae, namely those of Methanothermobacter thermoautotrophicus, allowed us to identify mth60 as the main structural fimbrin gene. Recombinant expression of mth60 in Escherichia coli was used to generate sufficient amounts of Mth60 to induce antibodies in rabbits. The antiserum reacted specifically with the 16 kDa fimbrial glycoprotein and could specifically detach adhering M. thermoautotrophicus cells from various surfaces. In addition we proved that cells adhering to solid surfaces - organic and inorganic ones - express many more fimbriae than cells growing in liquid cultures. The Mth60 fimbriae therefore are used by M. thermoautotrophicus as adhesins.