Anaerobic methanotroph 'Candidatus Methanoperedens nitroreducens' has a pleomorphic life cycle.

'Candidatus Methanoperedens' are anaerobic methanotrophic (ANME) archaea with global importance to methane cycling. Here meta-omics and fluorescence in situ hybridization (FISH) were applied to characterize a bioreactor dominated by 'Candidatus Methanoperedens nitroreducens' performing anaerobic methane oxidation coupled to nitrate reduction. Unexpectedly, FISH revealed the stable co-existence of two 'Ca. M. nitroreducens' morphotypes: the archetypal coccobacilli microcolonies and previously unreported planktonic rods. Metagenomic analysis showed that the 'Ca. M. nitroreducens' morphotypes were genomically identical but had distinct gene expression profiles for proteins associated with carbon metabolism, motility and cell division. In addition, a third distinct phenotype was observed, with some coccobacilli 'Ca. M. nitroreducens' storing carbon as polyhydroxyalkanoates. The phenotypic variation of 'Ca. M. nitroreducens' probably aids their survival and dispersal in the face of sub-optimal environmental conditions. These findings further demonstrate the remarkable ability of members of the 'Ca. Methanoperedens' to adapt to their environment.

Borgs are giant genetic elements with potential to expand metabolic capacity.

Anaerobic methane oxidation exerts a key control on greenhouse gas emissions1, yet factors that modulate the activity of microorganisms performing this function remain poorly understood. Here we discovered extraordinarily large, diverse DNA sequences that primarily encode hypothetical proteins through studying groundwater, sediments and wetland soil where methane production and oxidation occur. Four curated, complete genomes are linear, up to approximately 1 Mb in length and share genome organization, including replichore structure, long inverted terminal repeats and genome-wide unique perfect tandem direct repeats that are intergenic or generate amino acid repeats. We infer that these are highly divergent archaeal extrachromosomal elements with a distinct evolutionary origin. Gene sequence similarity, phylogeny and local divergence of sequence composition indicate that many of their genes were assimilated from methane-oxidizing Methanoperedens archaea. We refer to these elements as 'Borgs'. We identified at least 19 different Borg types coexisting with Methanoperedens spp. in four distinct ecosystems. Borgs provide methane-oxidizing Methanoperedens archaea access to genes encoding proteins involved in redox reactions and energy conservation (for example, clusters of multihaem cytochromes and methyl coenzyme M reductase). These data suggest that Borgs might have previously unrecognized roles in the metabolism of this group of archaea, which are known to modulate greenhouse gas emissions, but further studies are now needed to establish their functional relevance.

Spatial variations of activity and community structure of nitrite-dependent anaerobic methanotrophs in river sediment.

Rivers are an important site for methane emissions and reactive nitrogen removal. The process of nitrite-dependent anaerobic methane oxidation (n-damo) links the global carbon cycle and the nitrogen cycle, but its role in methane mitigation and nitrogen removal in rivers is poorly known. In the present study, we investigated the activity, abundance, and community composition of n-damo bacteria in sediment of the upper, middle, and lower reaches of Wuxijiang River (Zhejiang Province, China). The 13CH4 stable isotope experiments showed that the methane oxidation activity of n-damo was 0.11-1.88 nmol CO2 g-1 (dry sediment) d-1, and the activity measured from the middle reaches was significantly higher than that from the remaining regions. It was estimated that 3.27 g CH4 m-2 year-1 and 8.72 g N m-2 year-1 could be consumed via n-damo. Quantitative PCR confirmed the presence of n-damo bacteria, and their 16S rRNA gene abundance varied between 5.45 × 105 and 5.86 × 106 copies g-1 dry sediment. Similarly, the abundance of n-damo bacteria was significantly higher in the middle reaches. High-throughput sequencing showed a high n-damo bacterial diversity, with totally 152 operational taxonomic units being detected at 97 % sequence similarity cut-off. In addition, the n-damo bacterial community composition also varied spatially. The inorganic nitrogen (NH4+, NO2-, NO3-) level was found to be the key environmental factor controlling the n-damo activity and bacterial community composition. Overall, our results showed the spatial variations and environmental regulation of the activity and community structure of n-damo bacteria in river sediment, which expanded our understanding of the quantitative importance of n-damo in both methane oxidation and reactive nitrogen removal in riverine systems.

Response of potential activity, abundance and community composition of nitrite-dependent anaerobic methanotrophs to long-term fertilization in paddy soils.

The process of nitrite-dependent anaerobic methane oxidation (n-damo) catalysed by Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria is a novel pathway in regulating methane (CH4 ) emissions from paddy fields. Nitrogen fertilization is essential to improve rice yields and soil fertility; however, its effect on the n-damo process is largely unknown. Here, the potential n-damo activity, abundance and community composition of M. oxyfera-like bacteria were investigated in paddy fields under three long-term (32 years) fertilization treatments, i.e. unfertilized control (CK), chemical fertilization (NPK) and straw incorporation with chemical fertilization (SNPK). Relative to the CK, both NPK and SNPK treatments significantly (p < 0.05) increased the potential n-damo activity (88%-110%) and the abundance (52%-105%) of M. oxyfera-like bacteria. The variation of soil organic carbon (OrgC) content and inorganic nitrogen content caused by the input of chemical fertilizers and straw returning were identified as the key factors affecting the potential n-damo activity and the abundance of M. oxyfera-like bacteria. However, the community composition and diversity of M. oxyfera-like bacteria did not change significantly by the input of fertilizers. Overall, our results provide the first evidence that long-term fertilization greatly stimulates the n-damo process, indicating its active role in controlling CH4 emissions from paddy fields.

Active anaerobic methane oxidation and sulfur disproportionation in the deep terrestrial subsurface.

Microbial life is widespread in the terrestrial subsurface and present down to several kilometers depth, but the energy sources that fuel metabolism in deep oligotrophic and anoxic environments remain unclear. In the deep crystalline bedrock of the Fennoscandian Shield at Olkiluoto, Finland, opposing gradients of abiotic methane and ancient seawater-derived sulfate create a terrestrial sulfate-methane transition zone (SMTZ). We used chemical and isotopic data coupled to genome-resolved metaproteogenomics to demonstrate active life and, for the first time, provide direct evidence of active anaerobic oxidation of methane (AOM) in a deep terrestrial bedrock. Proteins from Methanoperedens (formerly ANME-2d) are readily identifiable despite the low abundance (≤1%) of this genus and confirm the occurrence of AOM. This finding is supported by 13C-depleted dissolved inorganic carbon. Proteins from Desulfocapsaceae and Desulfurivibrionaceae, in addition to 34S-enriched sulfate, suggest that these organisms use inorganic sulfur compounds as both electron donor and acceptor. Zerovalent sulfur in the groundwater may derive from abiotic rock interactions, or from a non-obligate syntrophy with Methanoperedens, potentially linking methane and sulfur cycles in Olkiluoto groundwater. Finally, putative episymbionts from the candidate phyla radiation (CPR) and DPANN archaea represented a significant diversity in the groundwater (26/84 genomes) with roles in sulfur and carbon cycling. Our results highlight AOM and sulfur disproportionation as active metabolisms and show that methane and sulfur fuel microbial activity in the deep terrestrial subsurface.

Optimized Cultivation and Syntrophic Relationship of Anaerobic Benzene-Degrading Enrichment Cultures under Methanogenic Conditions.

Current challenges in the anaerobic bioremediation of benzene are the lack of capable cultures and limited knowledge on the biodegradation pathway. Under methanogenic conditions, benzene may be mineralized by syntrophic interactions between microorganisms, which are poorly understood. The present study developed an optimized formula for anoxic medium to successfully promote the growth of the putative benzene degrader Deltaproteobacterium Hasda-A and enhance the benzene degradation activity of methanogenic enrichment cultures. Within 70| |d of incubation, the benzene degradation activity and relative abundance of Hasda-A in cultures in the new defined medium increased from 0.5 to >3| |mg L-1 d-1 and from 2.5% to >17%, respectively. Together with Hasda-A, we found a strong positive relationship between the abundances of superphylum OD1 bacteria, three methanogens (Methanoregula, Methanolinea, and Methanosaeta) and benzene degradation activity. The syntrophic relationship between these microbial taxa and Hasda-A was then demonstrated in a correlation analysis of longitudinal data. The involvement of methanogenesis in anaerobic benzene mineralization was confirmed by inhibition experiments. The high benzene degradation activity and growth of Hasda-A were quickly recovered in successive dilutions of enrichment cultures, proving the feasibility of using the medium developed in the present study to produce highly capable cultures. The present results will facilitate practical applications in bioremediation and research on the molecular mechanisms underlying benzene activation and syntrophic interactions in benzene mineralization.

Detoxification of Ciprofloxacin in an Anaerobic Bioprocess Supplemented with Magnetic Carbon Nanotubes: Contribution of Adsorption and Biodegradation Mechanisms.

In anaerobic bioreactors, the electrons produced during the oxidation of organic matter can potentially be used for the biological reduction of pharmaceuticals in wastewaters. Common electron transfer limitations benefit from the acceleration of reactions through utilization of redox mediators (RM). This work explores the potential of carbon nanomaterials (CNM) as RM on the anaerobic removal of ciprofloxacin (CIP). Pristine and tailored carbon nanotubes (CNT) were first tested for chemical reduction of CIP, and pristine CNT was found as the best material, so it was further utilized in biological anaerobic assays with anaerobic granular sludge (GS). In addition, magnetic CNT were prepared and also tested in biological assays, as they are easier to be recovered and reused. In biological tests with CNM, approximately 99% CIP removal was achieved, and the reaction rates increased ≈1.5-fold relatively to the control without CNM. In these experiments, CIP adsorption onto GS and CNM was above 90%. Despite, after applying three successive cycles of CIP addition, the catalytic properties of magnetic CNT were maintained while adsorption decreased to 29 ± 3.2%, as the result of CNM overload by CIP. The results suggest the combined occurrence of different mechanisms for CIP removal: adsorption on GS and/or CNM, and biological reduction or oxidation, which can be accelerated by the presence of CNM. After biological treatment with CNM, toxicity towards Vibrio fischeri was evaluated, resulting in ≈ 46% detoxification of CIP solution, showing the advantages of combining biological treatment with CNM for CIP removal.

Genomic and transcriptomic insights into methanogenesis potential of novel methanogens from mangrove sediments.

BACKGROUND: Methanogens are crucial to global methane budget and carbon cycling. Methanogens from the phylum Euryarchaeota are currently classified into one class and seven orders, including two novel methanogen taxa, Methanofastidiosa and Methanomassiliicoccales. The relative importance of the novel methanogens to methane production in the natural environment is poorly understood. RESULTS: Here, we used a combined metagenomic and metatranscriptomic approach to investigate the metabolic activity of methanogens in mangrove sediments in Futian Nature Reserve, Shenzhen. We obtained 13 metagenome-assembled genomes (MAGs) representing one class (Methanofastidiosa) and five orders (Methanomassiliicoccales, Methanomicrobiales, Methanobacteriales, Methanocellales, and Methanosarcinales) of methanogens, including the two novel methanogens. Comprehensive annotation indicated the presence of an H2-dependent methylotrophic methanogenesis pathway in Methanofastidiosa and Methanomassiliicoccales. Based on the functional gene analysis, hydrogenotrophic and methylotrophic methanogenesis are the dominant pathways in mangrove sediments. MAG mapping revealed that hydrogenotrophic Methanomicrobiales were the most abundant methanogens and that methylotrophic Methanomassiliicoccales were the most active methanogens in the analyzed sediment profile, suggesting their important roles in methane production. CONCLUSIONS: Partial or near-complete genomes of two novel methanogen taxa, Methanofastidiosa and Methanomassiliicoccales, in natural environments were recovered and analyzed here for the first time. The presented findings highlight the ecological importance of the two novel methanogens and complement knowledge of how methane is produced in mangrove ecosystem. This study implies that two novel methanogens play a vital role in carbon cycle. Video Abstract.

Biogas from Tannery Solid Waste Anaerobic Digestion Is Driven by the Association of the Bacterial Order Bacteroidales and Archaeal Family Methanosaetaceae.

The search for renewable energies has been one of the biggest challenges of the last decades. Sludge and solid wastes of many sources have been used to produce biogas of high calorific value. Thus, this work aimed to evaluate the biogas production of solid waste originating from a tannery that uses chromium salts as a tanning agent and to characterize the physicochemical parameters and microbial composition of the biogas-producing biomass. Wastes were collected and the parameters were evaluated at the initial and final time points of the anaerobic incubation process. At the end of 150 days, there was a production of 26.1 mL g-1 VSS of biogas with 52% of methane. The highest amount of biomethane observed was related to the archaeal family Methanosaetaceae and bacterial order Bacteroidales. Knowledge about changes in the microbial composition can provide tools for manipulation, isolation, and inoculation of the microorganisms inside the bioreactors to maximize methane production.

Methane emission suppression in flooded soil from Amazonia.

The coupling between ferrous iron and methane production has important global implications, with iron ions acting as electron acceptors for anaerobic oxidation of methane (AOM) and inhibitors of methanogenesis in different environments, including floodplain soils. In this sense, we analyzed the relationship between Fe(II) concentration and methane production in soil layers collected at 0-15 cm and 15-30 cm from flooded-forest and -agroforestry in Amazonian clear water floodplain incubated in anaerobic batch reactors using acetate, formate and glucose as organic sources. High throughput sequencing of archaeal and bacterial 16S rRNA genes was employed to assess the abundance and composition of the active methanogenic and methanotrophic microbial groups potentially involved in Fe(III)-dependent AOM in the soil used as inoculum. Positive correlation was revealed between Fe(II) concentration and methane production, with higher accumulation of Fe(II) in incubated soil layer collected at 0-15 cm in both forest and agroforestry sites for all the three organic sources. The accumulation of Fe(II) in the incubated soil evidenced the oxidation of Fe(III) potentially by Methanobacterium, Desulfobulbus and 'Candidatus methanoperedens nitroreducens' living in anaerobic condition at this soil layer. The results point out to the microbial ferric iron reduction as an important potential pathway for anaerobic organic matter decomposition in Amazonian floodplain, evidencing methanogenesis suppression by Fe(III) reduction in flooded-forest and -agroforestry in Amazonian clear water river floodplain.

Anaerobic methane oxidation coupled to manganese reduction by members of the Methanoperedenaceae.

Anaerobic oxidation of methane (AOM) is a major biological process that reduces global methane emission to the atmosphere. Anaerobic methanotrophic archaea (ANME) mediate this process through the coupling of methane oxidation to different electron acceptors, or in concert with a syntrophic bacterial partner. Recently, ANME belonging to the archaeal family Methanoperedenaceae (formerly known as ANME-2d) were shown to be capable of AOM coupled to nitrate and iron reduction. Here, a freshwater sediment bioreactor fed with methane and Mn(IV) oxides (birnessite) resulted in a microbial community dominated by two novel members of the Methanoperedenaceae, with biochemical profiling of the system demonstrating Mn(IV)-dependent AOM. Genomic and transcriptomic analyses revealed the expression of key genes involved in methane oxidation and several shared multiheme c-type cytochromes (MHCs) that were differentially expressed, indicating the likely use of different extracellular electron transfer pathways. We propose the names "Candidatus Methanoperedens manganicus" and "Candidatus Methanoperedens manganireducens" for the two newly described Methanoperedenaceae species. This study demonstrates the ability of members of the Methanoperedenaceae to couple AOM to the reduction of Mn(IV) oxides, which suggests their potential role in linking methane and manganese cycling in the environment.

Extracellular electron uptake in Methanosarcinales is independent of multiheme c-type cytochromes.

The co-occurrence of Geobacter and Methanosarcinales is often used as a proxy for the manifestation of direct interspecies electron transfer (DIET) in the environment. Here we tested eleven new co-culture combinations between methanogens and electrogens. Previously, only the most electrogenic Geobacter paired by DIET with Methanosarcinales methanogens, namely G. metallireducens and G. hydrogenophilus. Here we provide additional support, and show that five additional Methanosarcinales paired with G. metallireducens, while a strict hydrogenotroph could not. We also show that G. hydrogenophilus, which is incapable to grow with a strict hydrogenotrophic methanogen, could pair with a strict non-hydrogenotrophic Methanosarcinales. Likewise, an electrogen outside the Geobacter cluster (Rhodoferrax ferrireducens) paired with Methanosarcinales but not with strict hydrogenotrophic methanogens. The ability to interact with electrogens appears to be conserved among Methanosarcinales, the only methanogens with c-type cytochromes, including multihemes (MHC). Nonetheless, MHC, which are often linked to extracellular electron transfer, were neither unique nor universal to Methanosarcinales and only two of seven Methanosarcinales tested had MHC. Of these two, one strain had an MHC-deletion knockout available, which we hereby show is still capable to retrieve extracellular electrons from G. metallireducens or an electrode suggesting an MHC-independent strategy for extracellular electron uptake.

Calcium effect on microbial activity and biomass aggregation during anaerobic digestion at high salinity.

The potential effect of different Ca2+ additions (150, 300, 450, 600 and 1000 mg/L) on microbial activity and aggregation, during anaerobic digestion at moderate (8 g/L Na+) and high salinity (20 g/L Na+) has been investigated. Batch tests were carried out in duplicate serum bottles and operated for 30 days at 37 °C. At 8 g/L Na+, methanogenic activity and protein degradation were comparable from 150 to 450 mg/L Ca2+, and a significant inhibition was only observed at a Ca2+concentration of 1000 mg/L. In contrast, at 20 g/L Na+, 150 to 300 mg/L were the only Ca2+ concentrations to maintain chemical oxygen demand (COD) removal, protein hydrolysis and methane production. Overall, increasing Ca2+ concentrations had a larger impact on acetotrophic methanogenesis at 20 g/L than at 8 g/L Na+. Increasing Ca2+ had a negative effect on the aggregation behaviour of the dominant methanogen Methanosaeta when working at 8 g/L Na+. At 20 g/L Na+ the aggregation of Methanosaeta was less affected by addition of Ca2+ than at 8 g/L Na+. The negative effect appeared to be connected with Ca2+ precipitation and its impact on cell-to cell communication. The results highlight the importance of ionic balance for microbial aggregation at high salinity, bringing to the forefront the effect on Methanosaeta cells, known to be important to obtain anaerobic granules.

Presence of diverse nitrate-dependent anaerobic methane oxidizing archaea in sewage sludge.

AIM: The aim of this study was to explore the community diversity and abundance of nitrate-dependent anaerobic methane oxidizing archaea, Candidatus Methanoperedens nitroreducens, in sewage sludge from wastewater treatment plants. METHODS AND RESULTS: Seasonal sampling of the sewage sludge was carried out from two wastewater treatment plants (WWTPs) located in the northern and southern parts of China. Through amplicon sequencing using our newly designed primers, a large number of Candidatus Methanoperedens nitroreducens-like (M. nitroreducens) archaeal sequences (638 743) were generated. These sequences were assigned into 742 operational protein units (OPUs) at 90% cut-off level and classified as Group B member of M. nitroreducens archaea in the phylogenetic tree. More than 80% of the OPUs were not shared between these two WWTPs, showing the M. nitroreducens-like archaeal community in each WWTP was unique. Quantitative PCR assays also confirmed the presence of M. nitroreducens-like archaea and revealed a higher abundance in autumn and winter than other seasons, indicating that the environmental attributes in these seasons might favour the growth of this archaea. Further redundancy analysis revealed that volatile solid and pH were the significant environmental attributes (P < 0·05) in shaping the M. nitroreducens-like archaeal community based on variance inflation factor selection and Monte Carlo permutation test. CONCLUSIONS: The results confirmed the presence of diverse M. nitroreducens-like archaea in sewage sludge using Illumina-based mcrA gene sequencing and quantitative PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study revealed the ecological characteristics of M. nitroreducens-like archaea in sewage sludge that improved our understanding of nitrate-dependent anaerobic methane oxidation process and may be the basis for future application of M. nitroreducens-like archaea for new nitrogen removal in WWTPs.

Biotransformation of hexachlorocyclohexanes contaminated biomass for energetic utilization demonstrated in continuous anaerobic digestion system.

Lindane, the γ-hexachlorocyclohexane (HCH) isomer, was among the most used pesticides worldwide. Although it was banned in 2009, residues of Lindane and other HCH-isomers are still found with high concentrations in contaminated fields. For clean-up, phytoremediation combined with anaerobic digestion (AD) of contaminated biomass to produce biogas and fertilizer could be a promising strategy and was tested in two 15 L laboratory-scale continuous stirred tank reactors. During operation over one year by adding HCH isomers (γ, α and ß) consecutively, no negative influence on conventional reactor parameters was observed. The γ- and α-HCH isomers were transformed to chlorobenzene and benzene, and transformation became faster along with time, while ß-HCH was not removed. Genus Methanosaeta and order Clostridiales, showing significant enhancement on abundance with HCH addition, may be used as bioindicators for HCH dehalogenation in AD process. The potential for HCH degradation in AD system was restricted to axial Cl atoms of HCH and it showed slight enantioselective preference towards transformation of (+) α-HCH. Moreover, metabolite benzene was mineralized to CO2 and methane, deducing from tracer experiments with benzene-13C6. Overall, AD appears to be a feasible option for treatment of γ and α-HCHs contaminated biomass.

Specific affinity and relative abundance of methanogens in acclimated anaerobic sludge treating low-strength wastewater.

Kinetic parameters affecting effluent water quality including half saturation constant (Ks), maximum specific growth rate (µmax), and specific affinity ([Formula: see text], defined as µmax/Ks) were investigated using three types of anaerobic sludge (raw anaerobic digestion sludge referred to as unacclimated sludge, unacclimated sludge after endogenous decay, and sludge acclimated to low-strength wastewater in an anaerobic membrane bioreactor (AnMBR) for 360 days). Long-term acclimation to low-strength wastewater resulted in sludge with high specific affinity (1.6 × 10-3 L/mg COD/day for acclimated sludge compared to 4.1 × 10-4 L/mg COD/day for unacclimated sludge). The µmax values for unacclimated sludge and acclimated sludge were 0.08 and 0.07 day-1, respectively. The Ks values for unacclimated sludge and acclimated sludge were 194 ± 81 mg COD/L and 45 ± 13 mg COD/L, respectively. Although the Ks of unacclimated sludge after endogenous decay increased to 772 ± 74 mg COD/L, µmax increased to 0.35 day-1 as well, resulting in no statistically significant difference of [Formula: see text] between the two types of unacclimated sludge. Overall, [Formula: see text] is a better indicator than µmax or Ks alone for determining effluent water quality, as effluent substrate concentration is approximately inversely proportional to the specific affinity. 16S rRNA sequencing data analysis indicated a high abundance (85.8% of total archaea) of Methanosaeta in the microbial community after long-term acclimation. High [Formula: see text] associated with the enrichment of Methanosaeta appears to ensure successful anaerobic treatment of low-strength wastewater.

Acetotrophic Activity Facilitates Methanogenesis from LCFA at Low Temperatures: Screening from Mesophilic Inocula.

The inoculum source plays a crucial role in the anaerobic treatment of wastewaters. Lipids are present in various wastewaters and have a high methanogenic potential, but their hydrolysis results in the production of long chain fatty acids (LCFAs) that are inhibitory to anaerobic microorganisms. Screening of inoculum for the anaerobic treatment of LCFA-containing wastewaters has been performed at mesophilic and thermophilic conditions. However, an evaluation of inocula for producing methane from LCFA-containing wastewater has not yet been conducted at low temperatures and needs to be undertaken. In this study, three inocula (one granular sludge and two municipal digester sludges) were assessed for methane production from LCFA-containing synthetic dairy wastewater (SDW) at low temperatures (10 and 20°C). A methane yield (based on mL-CH4/g-CODadded) of 86-65% with acetate and 45-20% with SDW was achieved within 10 days using unacclimated granular sludge, whereas the municipal digester sludges produced methane only at 20°C but not at 10°C even after 200 days of incubation. The acetotrophic activity in the inoculum was found to be crucial for methane production from LCFA at low temperatures, highlighting the role of Methanosaeta (acetoclastic archaea) at low temperatures. The presence of bacterial taxa from the family Syntrophaceae (Syntrophus and uncultured taxa) in the inoculum was found to be important for methane production from SDW at 10°C. This study suggests the evaluation of acetotrophic activity and the initial microbial community characteristics by high-throughput amplicon sequencing for selecting the inoculum for producing methane at low temperatures (up to 10°C) from lipid-containing wastewaters.

Growth kinetics of Candidatus 'Methanoperedens nitroreducens' enriched in a laboratory reactor.

Recently it has been shown that Candidatus 'Methanoperedens nitroreducens', an anaerobic methanotrophic archaea (ANME), can reduce nitrate to nitrite using electrons derived from anaerobic oxidation of methane. In this study, the growth kinetics of 'M. nitroreducens' enriched in a laboratory reactor were studied. In the experimental concentration range (up to 16 mg CH4 L-1), anaerobic oxidation of methane by 'M. nitroreducens' was found to comply with first order kinetic model with a rate constant of 0.019 ±â€¯0.006 h-1 and a biomass-specific rate constant of 0.04-0.14 L h-1 g-1VSS. Meanwhile, the nitrate reduction to nitrite was well described by the Monod-type kinetic model with an affinity constant for nitrate of 2.1 ±â€¯0.4 mg N L-1, which is slightly higher than, but comparable to, that of most known denitrifying bacteria. This is the first time that the growth kinetics of 'M. nitroreducens' have been experimentally studied. The applicability of the kinetic model reported herein to this organism or similar organisms in natural or engineering systems requires further investigation.

Single and combined inhibition of Methanosaeta concilii by ammonia, sodium ion and hydrogen sulfide.

Single and combined inhibition of lag time λ and specific methanogenic activity RCH4 of Methanosaeta concilii by NH3, Na+ and H2S were investigated using inhibition tests with a single inhibitor and a 33 full-factorial experiment of NH3, Na+ and H2S concentrations (1.5 ≤ total ammonia nitrogen (TAN)/L ≤ 4.5 g, 1 ≤ Na+/L ≤ 4.3 g, 14.2 ≤ total hydrogen sulfide sulfur (THSS)/L ≤ 836 mg). All three inhibitors significantly increased λ and reduced RCH4 of M. concilii. The half-maximal inhibitory concentrations of NH3, Na+ and H2S for M. concilii were 6.4 g TAN/L, 5.2 g Na+/L and 1.6 g THSS/L. Partial cubic models adequately approximated the corresponding response surfaces of λ and RCH4 from the 33 full-factorial experiment. The inhibitors inhibited RCH4 synergistically, but inhibited λ in a complex manner. The combination of NH3 and Na+ showed the strongest synergistic inhibition of both λ and RCH4.