Characterization of the redox-active extracellular polymeric substances in an anaerobic methanotrophic consortium.

Anaerobic oxidation of methane (AOM) is a microbial process of importance in the global carbon cycle. AOM is predominantly mediated by anaerobic methanotrophic archaea (ANME), the physiology of which is still poorly understood. Here we present a new addition to the current physiological understanding of ANME by examining, for the first time, the biochemical and redox-active properties of the extracellular polymeric substances (EPS) of an ANME enrichment culture. Using a 'Candidatus Methanoperedens nitroreducens'-dominated methanotrophic consortium as the representative, we found it can produce an EPS matrix featuring a high protein-to-polysaccharide ratio of ∼8. Characterization of EPS using FTIR revealed the dominance of protein-associated amide I and amide II bands in the EPS. XPS characterization revealed the functional group of C-(O/N) from proteins accounted for 63.7% of total carbon. Heme-reactive staining and spectroscopic characterization confirmed the distribution of c-type cytochromes in this protein-dominated EPS, which potentially enabled its electroactive characteristic. Redox-active c-type cytochromes in EPS mediated the EET of 'Ca. M. nitroreducens' for the reduction of Ag+ to metallic Ag, which was confirmed by both ex-situ experiments with extracted soluble EPS and in-situ experiments with pristine EPS matrix surrounding cells. The formation of nanoparticles in the EPS matrix during in-situ extracellular Ag + reduction resulted in a relatively lower intracellular Ag distribution fraction, beneficial for alleviating the Ag toxicity to cells. The results of this study provide the first biochemical information on EPS of anaerobic methanotrophic consortia and a new insight into its physiological role in AOM process.

Microbial Stratification Affects Conversions of Nitrogen and Methane in Biofilms Coupling Anammox and n-DAMO Processes.

A methane-based membrane biofilm reactor (MBfR) has a suitable configuration to incorporate anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) processes because of its high gas-transfer efficiency and efficient biomass retention. In this study, the spatial distribution of microorganisms along with the biofilm depth in methane-based MBfRs was experimentally revealed, showing the dominance of anammox bacteria, n-DAMO bacteria, and n-DAMO archaea in the outer layer, middle layer, and inner layer of biofilms, respectively. The long-term and short-term experimental investigations in conjunction with mathematical modeling collectively revealed that microorganisms living in the outer layer of biofilms tend to use substrates from wastewater, while microorganisms inhabiting the inner layer of biofilms tend to use substrates originating from biofilm substratum. Specifically, anammox bacteria dominating the biofilm surface preferentially removed the nitrite provided from wastewater, while n-DAMO bacteria mostly utilized the nitrite generated from n-DAMO archaea as these two methane-related populations spatially clustered together inside the biofilm. Likewise, the methane supplied from the membrane was mostly consumed by n-DAMO archaea, while the dissolved methane in wastewater would be primarily utilized by n-DAMO bacteria. This study offers novel insights into the impacts of microbial stratification in biofilm systems, not only expanding the fundamental understanding of biofilms and microbial interactions therein but also providing a rationale for the potential applications of methane-based MBfRs in sewage treatment.

Dynamic modeling of anaerobic methane oxidation coupled to sulfate reduction: role of elemental sulfur as intermediate.

The process dynamics of anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR), and the potential role of elemental sulfur as intermediate are presented in this paper. Thermodynamic screening and experimental evidence from the literature conclude that a prominent model to describe AOM-SR is based on the concept that anaerobic methane oxidation proceeds through the production of the intermediate elemental sulfur. Two microbial groups are involved in the process: (a) anaerobic methanotrophs (ANME-2) and (b) Desulfosarcina/Desulfococcus sulfur reducers cluster (DSS). In this work, a dynamic model was developed to explore the interactions between biotic and abiotic processes to simulate the microbial activity, the chemical composition and speciation of the liquid phase, and the gas phase composition in the reactor headspace. The model includes the microbial kinetics for the symbiotic growth of ANME-2 and DSS, mass transfer phenomena between the gas and liquid phase for methane, hydrogen sulfide, and carbon dioxide and acid-base reactions for bicarbonate, sulfide, and ammonium. A data set from batch experiments, running for 250 days in artificial seawater inoculated with sediment from Marine Lake Grevelingen (The Netherlands) was used to calibrate the model. The inherent characteristics of AOM-SR make the identification of the kinetic parameters difficult due to the high correlation between them. However, by meaningfully selecting a set of kinetic parameters, the model simulates successfully the experimental data for sulfate reduction and sulfide production. The model can be considered as the basic structure for simulating continuous flow three-phase engineered systems based on AOM-SR.

Improved PCR primers to amplify 16S rRNA genes from NC10 bacteria.

Anaerobic oxidation of methane (AOM) coupled to nitrite reduction (AOM-NIR) is ecologically significant for mitigating the methane-induced greenhouse effect. The microbes responsible for this reaction, NC10 bacteria, have been widely detected in diverse ecosystems. However, some defects were discovered in the commonly used NC10-specific primers, 202F and qP1F. In the present work, the primers were redesigned and improved to overcome the defects found in the previous primers. A new nested PCR method was developed using the improved primers to amplify 16S ribosomal RNA (rRNA) genes from NC10 bacteria. In the new nested PCR method, the qP1mF/1492R and 1051F/qP2R primer sets were used in the first and second rounds, respectively. The PCR products were sequenced, and more operational taxonomic units (OTUs) of the NC10 phylum were obtained using the new primers compared to the previous primers. The sensitivity of the new nested PCR was tested by the serial dilution method, and the limit of detection was approximately 10(3) copies g(-1) dry sed. for the environmental samples compared to approximately 10(5) copies g(-1) dry sed. by the previous method. Finally, the improved primer, qP1mF, was used in quantitative PCR (qPCR) to determine the abundance of NC10 bacteria, and the results agreed well with the activity of AOM-NIR measured by isotope tracer experiments. The improved primers are able to amplify NC10 16S rRNA genes more efficiently than the previous primers and useful to explore the microbial community of the NC10 phylum in different systems.

Anaerobic oxidation of methane driven by different electron acceptors: A review.

Methane, the most significant reduced form of carbon on Earth, acts as a crucial fuel and greenhouse gas. Globally, microbial methane sinks encompass both aerobic oxidation of methane (AeOM), conducted by oxygen-utilizing methanotrophs, and anaerobic oxidation of methane (AOM), performed by anaerobic methanotrophs employing various alternative electron acceptors. These electron acceptors involved in AOM include sulfate, nitrate/nitrite, humic substances, and diverse metal oxides. The known anaerobic methanotrophic pathways comprise the internal aerobic oxidation pathway found in NC10 bacteria and the reverse methanogenesis pathway utilized by anaerobic methanotrophic archaea (ANME). Diverse anaerobic methanotrophs can perform AOM independently or in cooperation with symbiotic partners through several extracellular electron transfer (EET) pathways. AOM has been documented in various environments, including seafloor methane seepages, coastal wetlands, freshwater lakes, soils, and even extreme environments like hydrothermal vents. The environmental activities of AOM processes, driven by different electron acceptors, primarily depend on the energy yields, availability of electron acceptors, and environmental adaptability of methanotrophs. It has been suggested that different electron acceptors driving AOM may occur across a wider range of habitats than previously recognized. Additionally, it is proposed that methanotrophs have evolved flexible metabolic strategies to adapt to complex environmental conditions. This review primarily focuses on AOM, driven by different electron acceptors, discussing the associated reaction mechanisms and the habitats where these processes are active. Furthermore, it emphasizes the pivotal role of AOM in mitigating methane emissions.

Active anaerobic methane oxidation in the groundwater table fluctuation zone of rice paddies.

Rice paddies are globally important sources of methane emissions and also active regions for methane consumption. However, the impact of fluctuating groundwater levels on methane cycling has received limited attention. In this study, we delved into the activity and microbial mechanisms underlying anaerobic oxidation of methane (AOM) in paddy fields. A comprehensive approach was employed, including 13C stable isotope assays, inhibition experiments, real-time quantitative reverse transcription PCR, metagenomic sequencing, and binning technology. Geochemical profiles revealed the abundant coexistence of both methane and electron acceptors in the groundwater table fluctuation (GTF) zone, at a depth of 40-60 cm. Notably, the GTF zone exhibited the highest rate of AOM, potentially linked to the reduction of iron oxides and nitrate. Within this zone, Candidatus Methanoperedens (belonging to the ANME-2d group) dominated the Archaea population, accounting for a remarkable 85.4 %. Furthermore, our results from inhibition experiments, RT-qPCR, and metagenome-assembled genome (MAG) analysis highlighted the active role of Ca. Methanoperedens GTF50 in the GTF zone. This microorganism could independently mediate AOM process through the intriguing "reverse methanogenesis" pathway. Considering the similarity in geochemical conditions across different paddy fields, it is likely that Ca. Methanoperedens-mediated AOM is prevalent in the GTF zones.

Humic substances as electron acceptor for anaerobic oxidation of methane (AOM) and electron shuttle in Mn (IV)-dependent AOM.

Anaerobic methanotrophic archaea (ANME) belonging to the family Methanoperedenaceae are crucial for the global carbon cycle and different biogeochemical processes, owing to their metabolic versatility to couple anaerobic oxidation of methane (AOM) with different electron acceptors. A universal feature of Methanoperedenaceae is the abundant genes encoded in their genomes associated with extracellular electron transfer (EET) pathways. Candidatus. 'Methanoperedens manganicus', an archaeon belonging to the family Methanoperedenaceae, was recently enriched in a bioreactor performing AOM coupled with Mn (IV) reduction. Using this EET-capable ANME, we tested the hypothesis in this study that ANME can catalyse the humic-dependent AOM for growth. A two-year incubation showed that AOM activity can be sustained by Ca. 'M. manganicus' consortium in a bioreactor fed only with humic acids and methane. An isotopic mass balance batch test confirmed that the observed AOM was coupled to the reduction of humic acids. The increase of relative abundance of Ca. 'M. manganicus', and the total archaea population in the microbial community suggested that Ca. 'M. manganicus' can grow on methane and humic acids. The observation of humic-dependent AOM led to a subsequent hypothesis that humic acids could be used as the electron shuttle to mediate the EET in dissimilatory Mn (IV) reduction by Ca. 'M. manganicus'. We tested this hypothesis by adding humic acids to a Ca. 'M. manganicus' dominated-culture, which showed that the AOM rate was doubled by the addition of humic acids. X-ray photoelectron spectroscopy (XPS) showed that quinone moieties were consumed when humic acids worked as electron acceptors while remaining stable when functioning as a shuttle for electron transfer. The results of our study suggest that humic acids may serve as electron shuttles to allow ANME to access more electron acceptors through long-range EET.

Long-term effects of soluble and insoluble ferric irons on anaerobic oxidation of methane in paddy soil.

Iron-dependent anaerobic oxidation of methane (Fe-AOM) is an important process to reduce methane emissions into the atmosphere. It is well known that iron bioavailability largely influences microbial iron reduction, but the long-term effects of different ferric irons on soil Fe-AOM remain unknown. In this work, paddy soil in the ferruginous zone was collected and inoculated with insoluble ferrihydrite and soluble EDTA-Fe(III) for 420 days. Stable isotope experiments, activity inhibition tests, and molecular biological techniques were performed to reveal the activity, microbial community, and possible mechanism of paddy soil Fe-AOM. The results showed that ferrihydrite was a better electron acceptor for long-term Fe-AOM cultivation. Although EDTA-Fe(III) is highly bioavailable and could stimulate Fe-AOM activity for a short time, it restricted the activity increase in the long term. The abundances of archaea, iron-reducing bacteria (IRB), and gene mcrA largely increased after cultivation, indicating the important roles of mcrA-carrying archaea and IRB. Remarkably, archaeal communities were similar, but bacteria were totally different with different ferric irons. The results of the microbial community and activity inhibition suggested that Fe-AOM was performed likely by the cooperation between archaea (Methanomassiliicoccaceae or pGrfC26) and IRB in the cultures.

Salinity effect on an anaerobic methane- and ammonium-oxidising consortium: Shifts in activity, morphology, osmoregulation and syntrophic relationship.

Nitrate-dependent anaerobic methane oxidation (AOM) is a microbial process of both ecological significance for global methane mitigation and application potential for wastewater treatment. It is mediated by organisms belonging to the archaeal family 'Candidatus Methanoperedenaceae', which have so far mainly been found in freshwater environments. Their potential distribution in saline environments and their physiological responses to salinity variation were still poorly understood. In this study, the responses of the freshwater 'Candidatus Methanoperedens nitroreducens'-dominated consortium to different salinities were investigated using short- and long-term setups. Short-term exposure to salt stress significantly affected nitrate reduction and methane oxidation activities over the tested concentration range of 15-200‰ NaCl, and 'Ca. M. nitroreducens' showed the higher tolerance to high salinity stress than its partner of anammox bacteria. At high salinity concentration, near marine conditions of 37‰, the target organism 'Ca. M. nitroreducens' showed stabilized nitrate reduction activity of 208.5 µmol day-1 gCDW-1 in long-term bioreactors over 300 days, in comparison to 362.9 and 334.3 µmol day-1 gCDW-1 under low-salinity conditions (1.7‰ NaCl) and control conditions (∼15‰ NaCl). Different partners of 'Ca.  M. nitroreducens' evolved in the consortia with three different salinity conditions, suggesting the different syntrophic mechanisms shaped by changes in salinity. A new syntrophic relationship between 'Ca. M. nitroreducens' and Fimicutes and/or Chloroflexi denitrifying populations was identified under the marine salinity condition. Metaproteomic analysis shows that the salinity changes lead to higher expression of response regulators and selective ion (Na+/H+) channeling proteins that can regulate the osmotic pressure between the cell and its environment. The reverse methanogenesis pathway was, however, not impacted. The finding of this study has important implications for the ecological distribution of the nitrate-dependent AOM process in marine environments and the potential of this biotechnological process for the treatment of high-salinity industrial wastewater.

Polyhydroxyalkanoate-driven current generation via acetate by an anaerobic methanotrophic consortium.

Anaerobic oxidation of methane (AOM) is an important microbial process mitigating methane (CH4) emission from natural sediments. Anaerobic methanotrophic archaea (ANME) have been shown to mediate AOM coupled to the reduction of several compounds, either directly (i.e. nitrate, metal oxides) or in consortia with syntrophic bacterial partners (i.e. sulfate). However, the mechanisms underlying extracellular electron transfer (EET) between ANME and their bacterial partners or external electron acceptors are poorly understood. In this study, we investigated electron and carbon flow for an anaerobic methanotrophic consortium dominated by 'Candidatus Methanoperedens nitroreducens' in a CH4-fed microbial electrolysis cell (MEC). Acetate was identified as a likely intermediate for the methanotrophic consortium, which stimulated the growth of the known electroactive genus Geobacter. Electrochemical characterization, stoichiometric calculations of the system, along with stable isotope-based assays, revealed that acetate was not produced from CH4 directly. In the absence of CH4, current was still generated and the microbial community remained largely unchanged. A substantial portion of the generated current in the absence of CH4 was linked to the oxidation of the intracellular polyhydroxybutyrate (PHB) and the breakdown of extracellular polymeric substances (EPSs). The ability of 'Ca. M. nitroreducens' to use stored PHB as a carbon and energy source, and its ability to donate acetate as a diffusible electron carrier expands the known metabolic diversity of this lineage that likely underpins its success in natural systems.

Humic substances as electron acceptors for anaerobic oxidation of methane driven by ANME-2d.

Humic substances (humics) are ubiquitous in terrestrial and aquatic environments where they can serve as electron acceptors for anaerobic oxidation of organic compounds. Methane is a powerful greenhouse gas, as well as the least reactive organic molecule. Anaerobic oxidation of methane (AOM) coupled to microbial reduction of various electron acceptors plays a crucial role in mitigating methane emissions. Here, we reported that humics could serve as terminal electron acceptors for AOM using enriched nitrate-reducing AOM microorganisms. AOM coupled to the reduction of humics was demonstrated based on the production of 13C-labelled carbon dioxide, and AOM activity was evaluated with different methane partial pressures and electron acceptor concentrations. After three-cycle reduction, both AOM activity and copy numbers of the archaea 16S rRNA and mcrA genes were the highest when anthraquinone-2,6-disulfonic acid and anthraquinone-2-sulfonic acid were electron acceptors. The high-throughput sequencing results suggested that ANME-2d were the dominant methane oxidation archaea after humics reduction, although the partner bacteria NC10 trended downward, other reported humics reduction bacteria (Geobactor and Anammox) appeared. The potential electron transfer models from ANME-2d to humics were proposed. These results enable a better understanding of available electron acceptors for AOM in natural environments and broaden our insight into the significant role of ANME-2d.

A Critical Look at the Combined Use of Sulfur and Oxygen Isotopes to Study Microbial Metabolisms in Methane-Rich Environments.

Separating the contributions of anaerobic oxidation of methane and organoclastic sulfate reduction in the overall sedimentary sulfur cycle of marine sediments has benefited from advances in isotope biogeochemistry. Particularly, the coupling of sulfur and oxygen isotopes measured in the residual sulfate pool (δ18OSO4 vs. δ34SSO4). Yet, some important questions remain. Recent works have observed patterns that are inconsistent with previous interpretations. We differentiate the contributions of oxygen and sulfur isotopes to separating the anaerobic oxidation of methane and organoclastic sulfate reduction into three phases; first evidence from conventional high methane vs. low methane sites suggests a clear relationship between oxygen and sulfur isotopes in porewater and the metabolic process taking place. Second, evidence from pure cultures and organic matter rich sites with low levels of methane suggest the signatures of both processes overlap and cannot be differentiated. Third, we take a critical look at the use of oxygen and sulfur isotopes to differentiate metabolic processes (anaerobic oxidation of methane vs. organoclastic sulfate reduction). We identify that it is essential to develop a better understanding of the oxygen kinetic isotope effect, the degree of isotope exchange with sulfur intermediates as well as establishing their relationships with the cell-specific metabolic rates if we are to develop this proxy into a reliable tool to study the sulfur cycle in marine sediments and the geological record.

Enrichment of ANME-2 dominated anaerobic methanotrophy from cold seep sediment in an external ultrafiltration membrane bioreactor.

Anaerobic oxidation of methane (AOM) coupled to sulfate reduction is a microbially mediated unique natural phenomenon with an ecological relevance in the global carbon balance and potential application in biotechnology. This study aimed to enrich an AOM performing microbial community with the main focus on anaerobic methanotrophic archaea (ANME) present in sediments from the Ginsburg mud volcano (Gulf of Cadiz), a known site for AOM, in a membrane bioreactor (MBR) for 726 days at 22 (± 3)°C and at ambient pressure. The MBR was equipped with a cylindrical external ultrafiltration membrane, fed a defined medium containing artificial seawater and operated at a cross flow velocity of 0.02 m/min. Sulfide production with simultaneous sulfate reduction was in equimolar ratio between days 480 and 585 of MBR operation, whereas methane consumption was in oscillating trend. At the end of the MBR operation (day 726), the enriched biomass was incubated with 13C labeled methane, 13C labeled inorganic carbon was produced and the AOM rate based on 13C-inorganic carbon was 1.2 µmol/(gdw d). Microbial analysis of the enriched biomass at 400 and 726 days of MBR operation showed that ANME-2 and Desulfosarcina type sulfate reducing bacteria were enriched in the MBR, which formed closely associated aggregates. The major relevance of this study is the enrichment of an AOM consortium in a MBR system which can assist to explore the ecophysiology of ANME and provides an opportunity to explore the potential application of AOM.

Decoupling of DAMO archaea from DAMO bacteria in a methane-driven microbial fuel cell.

Anaerobic oxidation of methane (AOM) contributes significantly to the global methane sink. Previously, studies of anaerobic methanotrophic (ANME) archaea have been limited as they have not been separable from their bacterial partners during the AOM process because of their dependence on the bacteria. A microbial fuel cell (MFC) is a device capable of directly transforming chemical energy to electrical energy via electrochemical reactions involving biochemical pathways. In this study, decoupling of denitrifying anaerobic methane oxidation (DAMO) archaea and DAMO bacteria was investigated in an microbial fuel cell (MFC) using methane as the fuel. The DAMO fuel cell worked successfully but demonstrated weak electrogenic capability with around 25 mV production. After 45 days' enrichment, the sequencing and fluorescence in situ hybridization results showed the DAMO archaea percentage had increased from 26.96% (inoculum) to 65.77% (electrode biofilm), while the DAMO bacteria percentage decreased from 24.39% to 2.07%. Moreover, the amount of ANME-2d had doubled in the electrode biofilm compared with the inoculum. The sequencing results also showed substantial enrichment of the Ignavibacterium and Geobacter genera. The roles of Ignavibacterium and Geobacter in the MFC system need to be further investigated. Nevertheless, these results illustrate that an MFC device may provide a possible approach to separate DAMO archaea from DAMO bacteria.