Reductase Alpha Subunit Research Articles

Microbial mechanism underlying the effect of biochar supported nano zero-valent iron on the anaerobic digestion of food waste

Anaerobic digestion (AD) is one of the effective methods to treat the food waste (FW). In this study, biochar supported nano zero-valent iron (BC-nZVI) was prepared by carbon thermal reduction process. The effects of concentrations (1, 2 and 3 g/L) of BC-nZVI on methane production, microorganisms and metabolic mechanism during the AD of food waste were investigated. The results showed that 1 g/L of BC-nZVI was the best concentration for methane production. The cumulative methane production was enhanced by 29.54% at BC-nZVI 1 g/L. Microbial community analysis showed that BC-nZVI mainly increased the abundances of hydrolytic bacteria (including Bacteroidetes_vadinHA17, Proteiniphilum, Syntrophobacter) and acetoclast (Methanosaeta). Further exploration of the mechanism using PICRUSt2 version based on 16 S rRNA gene sequence analysis indicated that the addition of BC-nZVI promoted the abundances of enzymes related to methane metabolism such as the phosphate acetyltransferase and the methyl-coenzyme M reductase alpha subunit, which made the AD system stable.

Effects of PFOA on the physicochemical properties of anaerobic granular sludge: Performance evaluation, microbial community and metagenomic analysis

The impact of perfluorooctanoic acid (PFOA) on the anaerobic granular sludge was evaluated through a sequential batch experiment. Results showed that PFOA inhibited the chemical oxygen demand (COD) removal rate of the sludge and the dosage of 100 mg/L PFOA was more obvious. However, this negative effect would gradually weaken with the adaptation of microorganisms. For the 50 mg/L PFOA experimental group, the proteins content in the extracellular polymeric substances (EPS) of the anaerobic granular sludge increased from 1.53 mg/g to 3.65 mg/g. Meanwhile, PFOA inhibited the 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) activity of the anaerobic granular sludge. Furthermore, 100 mg/L PFOA reduced the relative abundance of Proteobacteria by 5.99% and Longilinea by 1.11%. 100 mg/L PFOA mainly restricted COD removal by affecting the glycolysis process, with the abundances of glucokinase and pyruvate kinase reduced by 8% and 28.1%, respectively. Compared with the control group, the relative abundance of the methyl-coenzyme M reductase alpha subunit increased by 84%, respectively, under 100 mg/L PFOA.

Preferential Use of the Perchlorate over the Nitrate in the Respiratory Processes Mediated by the Bacterium Azospira sp. OGA 24

Here we report the results obtained for a strain isolated from a polluted site and classified as Azospira sp. OGA 24. The capability of OGA 24 to utilize perchlorate and nitrate and the regulation of pathways were investigated by growth kinetic studies and analysis of messenger RNA (mRNA) expression of the genes of perchlorate reductase alpha subunit (pcrA), chlorite dismutase (cld), and periplasmic nitrate reductase large subunit (napA). In aerobic conditions and in a minimal medium containing 10 mM acetate as carbon source, 5.6 ± 0.34 mmol L−1 perchlorate or 9.7 ± 0.22 mmol L−1 nitrate were efficiently reduced during the growth with 10 mM of either perchlorate or nitrate. In anaerobiosis, napA was completely inhibited in the presence of perchlorate as the only electron acceptor, pcrA was barely detectable in nitrate-reducing conditions. The cell growth kinetics were in accordance with expression data, indicating a separation of nitrate and perchlorate respiration pathways. In the presence of both compounds, anaerobic nitrate consumption was reduced to 50% (4.9 ± 0.4 vs. 9.8 ± 0.15 mmol L−1 without perchlorate), while that of perchlorate was not affected (7.2 ± 0.5 vs. 6.9 ± 0.6 mmol L−1 without nitrate). Expression analysis confirmed the negative effect of perchlorate on nitrate respiration. Based on sequence analysis of the considered genes and 16S ribosomal gene (rDNA), the taxonomic position of Azospira sp. OGA 24 in the perchlorate respiring bacteria (PRB) group was further defined by classifying it in the oryzae species. The respiratory characteristics of OGA 24 strain make it very attractive in terms of potential applications in the bioremediation of environments exposed to perchlorate salts.

Palmitic acid accumulation limits methane production in anaerobic co-digestion of fats, oils and grease with municipal wastewater sludge

Anaerobic co-digestion of fats, oils and grease (FOG) with municipal wastewater sludge offers the opportunity to increase methane yields; yet the impact of FOG on overall process dynamics and the associated microbial communities is not well understood. This study employed lab-scale batch anaerobic co-digestion assays to advance the understanding of the co-digestion process through studying the dynamics of formation and consumption of intermediates along the anaerobic digestion pathway and correlating these to temporal analysis of methanogenic activity as well as end-point microbial community structure. Methane production was delayed during sludge co-digestion with FOG, but not during digestion of sludge alone. Palmitic acid, a long-chain fatty acid (LCFA), accumulated during co-digestion to concentrations above 16 mM, resulting in a lag in methane production. Acetate, hydrogen, and formate, carbon and/or energy sources for methanogens during methane production, did not accumulate during the lag-phase. Expression of the mcrA gene, which encodes the methyl coenzyme M reductase alpha subunit necessary for catalyzing the final step of methanogenesis, increased when co-digesters were spiked with acetate during the lag-phase, indicating no direct total inhibition from palmitic acid to methanogenic activity in assays with a 23% FOG loading (based on total g VS). End-point microbial community analyses revealed distinct community structure and composition differences between co-digestion assays and controls, and between biological replicates with variable responses. The hydrogenotrophic methanogen genus Methanoculleus dominated adapted co-digesters, but not inhibited co-digesters suggesting its importance for adapting to FOG loadings. Palmitic acid accumulation resulting from distinct microbial community composition and structure thus characterizes stalled or inhibited FOG co-digesters.

The influence of soil temperature, methanogens and methanotrophs on methane emissions from cold waterlogged paddy fields

Paddy fields are major sources of atmospheric methane (CH4). However, CH4 emissions from cold-waterlogged paddy fields, a major type of paddy soil in China, remain unclear. Here we investigated the CH4 emissions and associated influential factors in cold-waterlogged paddy fields at two sites (Yangxin County and Daye City) in Hubei Province, South China. Normal paddy fields matched with parental material and cropping system were used as the controls. The CH4 emissions from cold-waterlogged fields were significantly higher than those from normal fields with (3.0–4.4-fold) or without (3.5–8.6-fold) rice. Rice planting increased CH4 emissions by 59–78% in cold-waterlogged fields and by 85–247% in normal fields. CH4 instantaneous fluxes were positively correlated with soil temperature and methanogen mcrA (methyl coenzyme M reductase alpha subunit) and methanotroph pmoA (methane monooxygenase) copy numbers at the annual scale. Under rice planting, mcrA copy number was higher in cold-waterlogged fields than in normal fields at both sites, whereas pmoA copy number had the same trend at the Daye site only. Soil temperature and water content influenced mcrA and pmoA copy numbers in the normal paddy fields, whereas soil organic matter content was more influential in the cold-waterlogged paddy fields. These findings suggest that perennial waterlogging is a prerequisite for substantial CH4 emissions from cold-waterlogged paddy fields, and it promotes the proliferation of methanogens and methanotrophs under rice planting. Therefore, CH4 production-oxidation processes are more active in cold-waterlogged paddy fields than in normal paddy fields.

Long-term submergence of non-methanogenic oxic upland field soils helps to develop the methanogenic archaeal community as revealed by pot and field experiments

The community structure of methanogenic archaea is relatively stable, i.e., it is sustained at a high abundance with minimal changes in composition, in paddy field soils irrespective of submergence and drainage. In contrast, the abundance in non-methanogenic oxic soils is much lower than that in paddy field soils. This study aimed to describe methanogenic archaeal community development following the long-term submergence of non-methanogenic oxic upland field soils in pot and field experiments. In the pot experiment, a soil sample obtained from an upland field was incubated under submerged conditions for 275 d. Soil samples periodically collected were subjected to culture-dependent most probable number (MPN) enumeration, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of archaeal 16S rRNA gene, and quantitative PCR analysis of the methyl-coenzyme M reductase alpha subunit gene (mcrA) of methanogenic archaea. The abundance of methanogenic archaea increased from 102 to 103 cells g−1 dry soil and 104 to 107 copies of mcrA gene g−1 dry soil after submergence. Although no methanogenic archaeon was detected prior to incubation by the DGGE analysis, members from Methanocellales, Methanosarcinaceae, and Methanosaetaceae proliferated in the soils, and the community structure was relatively stable once established. In the field experiment, the number of viable methanogenic archaea in a rice paddy field converted from meadow (reclaimed paddy field) was monitored by MPN enumeration over five annual cycles of field operations. Viability was also determined simultaneously in a paddy field where the plow layer soil from a farmer's paddy field was dressed onto the meadow (dressed paddy field) and an upland crop field converted from the meadow (reclaimed upland field). The number of viable methanogenic archaea in the reclaimed paddy field was below the detection limit before the first cultivation of rice and in the reclaimed upland field. Then, the number gradually increased over five years and finally reached 103–104 cells g−1 dry soil, which was comparable to that in the dressed paddy field. These findings showed that the low abundance of autochthonous methanogenic archaea in the non-methanogenic oxic upland field soils steadily proliferated, and the community structure was developed following repeated and long-term submergence. These results suggest that habitats suitable for methanogenic archaea were established in soil following repeated and long-term submergence.

Effects of increased precipitation combined with nitrogen addition and increased temperature on methane fluxes in alpine meadows of the Tibetan Plateau

Climate change and anthropogenic activities have resulted in increased atmospheric methane (CH4) concentration. Increased nitrogen deposition and precipitation accompanies climate warming and can change soil carbon and nitrogen dynamics and microbial processes and alter CH4 fluxes. To quantify the sink of the vast alpine meadows of the Tibetan Plateau and to examine how precipitation addition (P), warming (W), and nitrogen addition (N) affect CH4 fluxes in alpine meadows, we conducted continuous 3-growing season experiments in an alpine meadow using the static chamber and gas chromatograph method. Soil CH4 samples were collected during the early, peak, and late stages of the growing season from 2015 to 2017. Our results suggested that neither P, W, nor N had an interaction effect on soil CH4 uptake. P significantly increased and decreased the copies number of particulate methane monooxygenase alpha subunit (pmoA) and methyl-coenzyme M reductase alpha subunit (mcrA), respectively. However, P significantly decreased CH4 uptake, particularly under the combined treatment of P and N. Compared with the control, CH4 uptake decreased under P, N, PW, and PN by 50.64%, 6.24%, 39.37%, and 75.06%, respectively, whereas under W and WN CH4 uptake increased by 16.19% and 7.56%, respectively. Soil CH4 uptake was positively correlated with soil temperature and pmoA and negatively correlated with soil moisture and NH4+-N content. CH4 uptake was significantly affected by the sampling period. CH4 uptake was significantly lower rates during peak growing season compared with those during the early and late stages of the growing season. Our results suggest that, (1) CH4 fluxes of alpine grassland ecosystems are more sensitive to P than W or N, and (2) precipitation controls CH4 flux response to increasing nitrogen deposition in alpine meadows on the Tibetan Plateau. Therefore, future research should focus on the response and feedback of CH4 uptake to changes in precipitation.

Methanogenic degradation of tetramethylammonium hydroxide by Methanomethylovorans and Methanosarcina

This study evaluated the methanogens responsible for methanogenic degradation of tetramethylammonium hydroxide (TMAH) in a continuous flow bioreactor. The enriched methanogens attained an estimated maximum specific TMAH degradation rate and half-saturation constant of 39.5 mg TMAH/gVSS/h and 820 mg/L, following the Monod-type kinetic expression for methanogenic TMAH degradation. Presence of sulfide more than 20 mg/L significantly extended lag period and slowed down specific TMAH degradation rates. The results of terminal restriction fragment length polymorphism (T-RFLP), cloning/sequencing, and quantitative real-time PCR analyses targeting on the methyl coenzyme M reductase alpha subunit (mcrA) genes retrieved from the bioreactor and batch experiments indicated that Methanomethylovorans species were the dominant methanogens responsible for methanogenic degradation of TMAH. The isolated TMAH-degrading methanogen from the bioreactor, however, was identified closely related to Methanosarcina mazei. It is likely that a very low TMAH environment in the bioreactor favored the growth of Methanomethylovorans hollandica, while the much higher TMAH in the isolation growth medium proliferated Methanosarcina mazei.

Contribution of Ruminal Fungi, Archaea, Protozoa, and Bacteria to the Methane Suppression Caused by Oilseed Supplemented Diets.

Dietary lipids can suppress methane emission from ruminants, but effects are variable. Especially the role of bacteria, archaea, fungi and protozoa in mediating the lipid effects is unclear. In the present in vitro study, archaea, fungi and protozoa were selectively inhibited by specific agents. This was fully or almost fully successful for fungi and protozoa as well as archaeal activity as determined by the methyl-coenzyme M reductase alpha subunit gene. Five different microbial treatments were generated: rumen fluid being intact (I), without archaea (–A), without fungi (–F), without protozoa (–P) and with bacteria only (–AFP). A forage-concentrate diet given alone or supplemented with crushed full-fat oilseeds of either safflower (Carthamus tinctorius) or poppy (Papaver somniferum) or camelina (Camelina sativa) at 70 g oil kg−1 diet dry matter was incubated. This added up to 20 treatments with six incubation runs per treatment. All oilseeds suppressed methane emission compared to the non-supplemented control. Compared to the non-supplemented control, –F decreased organic matter (OM) degradation, and short-chain fatty acid concentration was greater with camelina and safflower seeds. Methane suppression per OM digested in –F was greater with camelina seeds (−12 vs.−7% with I, P = 0.06), but smaller with poppy seeds (−4 vs. −8% with I, P = 0.03), and not affected with safflower seeds. With –P, camelina seeds decreased the acetate-to-propionate ratio and enhanced the methane suppression per gram dry matter (18 vs. 10% with I, P = 0.08). Hydrogen recovery was improved with –P in any oilseeds compared to non-supplemented control. No methane emission was detected with the –A and –AFP treatments. In conclusion, concerning methanogenesis, camelina seeds seem to exert effects only on archaea and bacteria. By contrast, with safflower and poppy seeds methane was obviously reduced mainly through the interaction with protozoa or archaea associated with protozoa. This demonstrated that the microbial groups differ in their contribution to the methane suppressing effect dependent on the source of lipid. These findings help to understand how lipid supplementation and microbial groups interact, and thus may assist in making this methane mitigation tool more efficient, but await confirmation in vivo.

Origin and fate of methane in the Eastern Tropical North Pacific oxygen minimum zone

Oxygen minimum zones (OMZs) contain the largest pools of oceanic methane but its origin and fate are poorly understood. High-resolution (<15 m) water column profiles revealed a 300 m thick layer of elevated methane (20–105 nM) in the anoxic core of the largest OMZ, the Eastern Tropical North Pacific. Sediment core incubations identified a clear benthic methane source where the OMZ meets the continental shelf, between 350 and 650 m, with the flux reflecting the concentration of methane in the overlying anoxic water. Further incubations characterised a methanogenic potential in the presence of both porewater sulphate and nitrate of up to 88 nmol g−1day−1 in the sediment surface layer. In these methane-producing sediments, the majority (85%) of methyl coenzyme M reductase alpha subunit (mcrA) gene sequences clustered with Methanosarcinaceae (⩾96% similarity to Methanococcoides sp.), a family capable of performing non-competitive methanogenesis. Incubations with 13C-CH4 showed potential for both aerobic and anaerobic methane oxidation in the waters within and above the OMZ. Both aerobic and anaerobic methane oxidation is corroborated by the presence of particulate methane monooxygenase (pmoA) gene sequences, related to type I methanotrophs and the lineage of Candidatus Methylomirabilis oxyfera, known to perform nitrite-dependent anaerobic methane oxidation (N-DAMO), respectively.

Analysis of methane-producing and metabolizing archaeal and bacterial communities in sediments of the northern South China Sea and coastal Mai Po Nature Reserve revealed by PCR amplification of mcrA and pmoA genes.

Communities of methanogens, anaerobic methanotrophic archaea and aerobic methanotrophic bacteria (MOB) were compared by profiling polymerase chain reaction (PCR)-amplified products of mcrA and pmoA genes encoded by methyl-coenzyme M reductase alpha subunit and particulate methane monooxygenase alpha subunit, respectively, in sediments of northern South China Sea (nSCS) and Mai Po mangrove wetland. Community structures representing by mcrA gene based on 12 clone libraries from nSCS showed separate clusters indicating niche specificity, while, Methanomicrobiales, Methanosarcinales clades 1,2, and Methanomassiliicoccus-like groups of methanogens were the most abundant groups in nSCS sediment samples. Novel clusters specific to the SCS were identified and the phylogeny of mcrA gene-harboring archaea was updated. Quantitative polymerase chain reaction was used to detect mcrA gene abundance in all samples: similar abundance of mcrA gene in the surface layers of mangrove (3.4∼3.9 × 106 copies per gram dry weight) and of intertidal mudflat (5.5∼5.8 × 106 copies per gram dry weight) was observed, but higher abundance (6.9 × 106 to 1.02 × 108 copies per gram dry weight) was found in subsurface samples of both sediment types. Aerobic MOB were more abundant in surface layers (6.7∼11.1 × 105 copies per gram dry weight) than the subsurface layers (1.2∼5.9 × 105 copies per gram dry weight) based on pmoA gene. Mangrove surface layers harbored more abundant pmoA gene than intertidal mudflat, but less pmoA genes in the subsurface layers. Meanwhile, it is also noted that in surface layers of all samples, more pmoA gene copies were detected than the subsurface layers. Reedbed rhizosphere exhibited the highest gene abundance of mcrA gene (8.51 × 108 copies per gram dry weight) and pmoA gene (1.56 × 107 copies per gram dry weight). This study investigated the prokaryotic communities responsible for methane cycling in both marine and coastal wetland ecosystems, showing the distribution characteristics of mcrA gene-harboring communities in nSCS and stratification of mcrA and pmoA gene diversity and abundance in the Mai Po Nature Reserve.

Phylogenetic Diversity of aprA Genes in Subseafloor Sediments on the Northwestern Pacific Margin off Japan.

Markedly diverse sequences of the adenosine-5′-phosphosulfate reductase alpha subunit gene (aprA), which encodes a key enzyme in microbial sulfate reduction and sulfur oxidation, were detected in subseafloor sediments on the northwestern Pacific off Japan. The aprA gene sequences were grouped into 135 operational taxonomic units (90% sequence identity), including genes related to putative sulfur-oxidizing bacteria predominantly detected in sulfate-depleted deep sediments. Our results suggest that microbial ecosystems in the subseafloor biosphere have phylogenetically diverse genetic potentials to mediate cryptic sulfur cycles in sediments, even where sulfate is rarely present.

Microbial community diversity in anaerobic reactors digesting turkey, chicken, and swine wastes.

The microbial community structures of two continuous stirred tank reactors digesting turkey manure with pine wood shavings as well as chicken and swine manure were investigated. The reactor fed with chicken/swine wastes displayed the highest organic acids concentration (up to 15.2 g/l) and ammonia concentration (up to 3.7 g/l ammonium nitrogen) and generated a higher biogas yield (up to 366 ml/g(VS)) compared with the reactor supplied with turkey wastes (1.5- 1.8 g/l of organic acids and 1.6-1.7 g/l of ammonium levels; biogas yield was up to 195 ml/g(VS)). The microbial community diversity was assessed using both sequencing and profiling terminal restriction fragment length polymorphisms of 16S rRNA genes. Additionally, methanogens were analyzed using methyl coenzyme M reductase alpha subunit (mcrA) genes. The bacterial community was dominated by members of unclassified Clostridiales with the prevalence of specific clostridial phylotypes in each reactor, indicating the effect of the substrate type on the community structure. Of the methanogenic archaea, methanogens of the genus Methanosarcina were found in high proportions in both reactors with specific methanosarcinas in each reactor, whereas the strict hydrogenotrophic methanogens of Methanoculleus sp. were found at significant levels only in the reactor fed with chicken/swine manure (based on the analyses of 16S rRNA gene). This suggests that among methanogenic archaea, Methanosarcina species which have different metabolic capabilities, including aceticlastic and hydrogenotrophic methanogenesis, were mainly involved in anaerobic digestion of turkey wastes.

Molecular analysis of methanogens involved in methanogenic degradation of tetramethylammonium hydroxide in full-scale bioreactors.

This study investigated methanogenic communities involved in degradation of tetramethylammonium hydroxide (TMAH) in three full-scale bioreactors treating TMAH-containing wastewater. Based on the results of terminal-restriction fragment-length polymorphism (T-RFLP) and quantitative PCR analyses targeting the methyl-coenzyme M reductase alpha subunit (mcrA) genes retrieved from three bioreactors, Methanomethylovorans and Methanosarcina were the dominant methanogens involved in the methanogenic degradation of TMAH in the bioreactors. Furthermore, batch experiments were conducted to evaluate mcrA messenger RNA (mRNA) expression during methanogenic TMAH degradation, and the results indicated that a higher level of TMAH favored mcrA mRNA expression by Methansarcina, while Methanomethylovorans could only express considerable amount of mcrA mRNA at a lower level of TMAH. These results suggest that Methansarcina is responsible for methanogenic TMAH degradation at higher TMAH concentrations, while Methanomethylovorans may be important at a lower TMAH condition.

Construction of aligned database of dsrA, a gene encoding dissimilatory sulfite reductase alpha subunit, for metagenomic studies of sulfate-reducing bacteria

A sulfite reductase gene sequence database, which could facilitate analysis of metagenomic data of a functional gene from sulfate-reducing bacteria, is described. The database contains 127 aligned nucleotide sequences of a gene (dsrA), encoding a dissimilatory sulfite reductase alpha subunit, retrieved from Reference Sequence of GenBank that provides only well-annotated genome sequences. The dsrA gene sequences were screened for length and aligned using four multiple sequencing alignment programs, Mafft, Muscle, Mothur, and Clustal Omega. In addition to the dsrA gene sequences, 16S ribosomal RNA (rRNA) gene sequences for the bacterial species appearing in the database were also retrieved from the GenBank and aligned by a public database of SILVA in Mothur to examine any potential relationship between taxonomy of the bacteria and distribution of a functional gene. The aligned dsrA and 16S rRNA gene sequences were used to construct neighbor-joining phylogenetic trees for comparison of the alignments for relative agreement among the aligned sequence databases. This method of aligned reference database construction using public database can help provide better insight into microbial communities by suggesting an alternative approach of studying metagenomic analysis of functional genes involved in important ecological processes.

Influences of the substrate feeding regime on methanogenic activity in biogas reactors approached by molecular and stable isotope methods

In order to better understand the effects of the substrate feeding regime on methanogenesis during anaerobic digestion in biogas reactors, four continuous stirred tank reactors operated under mesophilic conditions were investigated. In addition to standard physicochemical parameters, the stable isotopic signatures of CH4 and CO2 before and after daily feeding were analyzed. The activity of the methanogens was assessed by methyl coenzyme M reductase alpha-subunit (mcrA/mrtA) gene transcript analysis. Two different feeding regimes i.e. single vs. double consecutive feeding of the otherwise same daily maize silage load were investigated. During the first phase, a single feeding of the whole daily dose increased the biogas production within 70–80 min from around 0.5 to 2.0 L/h. This increase was associated with a transient increase of the acetic acid concentration and a corresponding decrease of the pH. Only moderate increase in biogas yield and VFA concentration (mainly acetate) was observed when the daily substrate was apportioned into two feedings. However, the overall daily gas production was similar in both cases.Regardless of the feeding regime, significantly depleted δ13CH4 and minor changes in the CO2 content of biogas were observed after feeding, which were followed by enrichment of δ13CH4. This period was associated with detectable changes in activity of methanogenic communities monitored by terminal restriction fragment length polymorphism analysis based on the transcripts of mcrA/mrtA genes. Methanoculleus and Methanobacterium spp. were the predominant methanogens in all reactors, while Methanosarcina spp. activity was only significant in two reactors. The activity of Methanoculleus and Methanosarcina spp. increased after the feeding in these reactors, which was followed by a depletion of δ13C in the produced gas. In both reactors, the less depleted isotopic values were detected before the second feeding, when Methanobacterium was the most active genus. Variations in reactor performance and methanogenic community characteristics were attributed to inoculum heterogeneity and stochastic factors during the reactor set up.

Identification of Methanogenic archaea in the Hyporheic Sediment of Sitka Stream

Methanogenic archaea produce methane as a metabolic product under anoxic conditions and they play a crucial role in the global methane cycle. In this study molecular diversity of methanogenic archaea in the hyporheic sediment of the lowland stream Sitka (Olomouc, Czech Republic) was analyzed by PCR amplification, cloning and sequencing analysis of the methyl coenzyme M reductase alpha subunit (mcrA) gene. Sequencing analysis of 60 clones revealed 24 different mcrA phylotypes from hyporheic sedimentary layers to a depth of 50 cm. Phylotypes were affiliated with Methanomicrobiales, Methanosarcinales and Methanobacteriales orders. Only one phylotype remains unclassified. The majority of the phylotypes showed higher affiliation with uncultured methanogens than with known methanogenic species. The presence of relatively rich assemblage of methanogenic archaea confirmed that methanogens may be an important component of hyporheic microbial communities and may affect CH4 cycling in rivers.

Methanomethylovorans uponensis sp. nov., a methylotrophic methanogen isolated from wetland sediment

A novel mesophilic, methylotrophic, methanogenic archaeon, designated strain EK1(T), was enriched and isolated from wetland sediment. Phylogenetic analysis showed that strain EK1(T) was affiliated with the genus Methanomethylovorans within the family Methanosarcinaceae, and shared the highest 16S rRNA and methyl-coenzyme M reductase alpha-subunit gene sequence similarity with the type strain of Methanomethylovorans hollandica (98.8 and 92.6 %, respectively). The cells of strain EK1(T) were observed to be Gram-negative, non-motile and irregular cocci that did not lyse in 0.1 % (w/v) sodium dodecyl sulfate. Methanol, mono-, di- and trimethylamine, dimethyl sulfide and methanethiol were found to be used as catabolic and methanogenic substrates, whereas H2/CO2, formate, 2-propanol and acetate were not. Growth was observed at 25-40 °C (optimum, 37 °C), at pH 5.5-7.5 (optimum, pH 6.0-6.5) and in the presence of 0-0.1 M NaCl (optimum, 0 M). Growth and methane production rates were stimulated in the presence of H2/CO2 although methane production and growth yields were not significantly affected; acetate, formate, 2-propanol and CO/CO2/N2 did not affect methane production. CoCl2 (0.6-2.0 μM) and FeCl2 (25 mg/l) stimulated growth, while yeast extract and peptone did not. The DNA-DNA hybridization experiment revealed a relatedness of <20 % between EK1(T) and the type strains of the genus Methanomethylovorans. The DNA G+C content of strain EK1(T) was determined to be 39.2 mol%. Based on the polyphasic taxonomic study, strain EK1(T) represents a novel species belonging to the genus Methanomethylovorans, for which the name Methanomethylovorans uponensis sp. nov. is proposed. The type strain is strain EK1(T)(=NBRC 109636(T) = KCTC 4119(T) = JCM 19217(T)).

Rapid extraction of total RNA from an anaerobic sludge biocoenosis

In order to assess the activity of metabolic pathways during anaerobic biogas production, it is necessary to isolate total RNA from the anaerobic sludge. mRNA activity profiling complements the quantification of excreted metabolites for a comprehensive anaerobic digestion model (ADM1). Four non-commercial total RNA extraction protocols were examined to extract total RNA from suspended solids of anaerobic sludge. The most suitable protocol was identified and optimised. In relation to total RNA extraction efficiency, total RNA purity and RNA integrity, the best homogenisation method was a combined method of nitrogen grinding and bead beating. When bead beating or nitrogen grinding was used alone for homogenisation, total RNA extraction efficiency was lower than when both homogenisation methods were applied. Depending on the homogenisation method, the whole RNA extraction procedure takes approximately 2 to 3 h, which is as fast as when using commercial available soil RNA extraction kits. The proposed method is rapid in extracting total RNA from a biocoenosis present in an anaerobic sludge environment. Furthermore, we could apply any of the extracted homogenization methods for reverse transcription and subsequent PCR amplification of the gene for the methyl coenzyme M reductase alpha subunit (mcrA/mrtA).

Characterization of the methanogen community in a household anaerobic digester fed with swine manure in China

Household anaerobic digesters have been installed across rural China for biogas production, but information on methanogen community structure in these small biogas units is sparsely available. By creating clone libraries for 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes, we investigated the methanogenic consortia in a household biogas digester treating swine manure. Operational taxonomic units (OTUs) were defined by comparative sequence analysis, seven OTUs were identified in the 16S rRNA gene library, and ten OTUs were identified in the mcrA gene library. Both libraries were dominated by clones highly related to the type strain Methanocorpusculum labreanum Z, 64.0% for 16S rRNA gene clones and 64.3% for mcrA gene clones. Additionally, gas chromatography assays showed that formic acid was 84.54% of the total volatile fatty acids and methane was 57.20% of the biogas composition. Our results may help further isolation and characterization of methanogenic starter strains for industrial biogas production.