Sulfide Toxicity as Key Control on Anaerobic Oxidation of Methane in Eutrophic Coastal Sediments.

Coastal zones account for 75% of marine methane emissions, despite covering only 15% of the ocean surface area. In these ecosystems, the tight balance between methane production and oxidation in sediments prevents most methane from escaping into seawater. However, anthropogenic activities could disrupt this balance, leading to an increased methane escape from coastal sediments. To quantify and unravel potential mechanisms underlying this disruption, we used a suite of biogeochemical and microbiological analyses to investigate the impact of anthropogenically induced redox shifts on methane cycling in sediments from three sites with contrasting bottom water redox conditions (oxic-hypoxic-euxinic) in the eutrophic Stockholm Archipelago. Our results indicate that the methane production potential increased under hypoxia and euxinia, while anaerobic oxidation of methane was disrupted under euxinia. Experimental, genomic, and biogeochemical data suggest that the virtual disappearance of methane-oxidizing archaea at the euxinic site occurred due to sulfide toxicity. This could explain a near 7-fold increase in the extent of escape of benthic methane at the euxinic site relative to the hypoxic one. In conclusion, these insights reveal how the development of euxinia could disrupt the coastal methane biofilter, potentially leading to increased methane emissions from coastal zones.

Enhanced Extracellular Electron Transfer in Magnetite-Mediated Anaerobic Oxidation of Methane Coupled to Humic Substances Reduction: The Pivotal Role of Membrane-Bound Electron Transfer Proteins.

Humic substances are organic substances prevalent in various natural environments, such as wetlands, which are globally important sources of methane (CH4) emissions. Extracellular electron transfer (EET)-mediated anaerobic oxidation of methane (AOM)-coupled with humic substances reduction plays an important role in the reduction of methane emissions from wetlands, where magnetite is prevalent. However, little is known about the magnetite-mediated EET mechanisms in AOM-coupled humic substances reduction. This study shows that magnetite promotes the reduction of the AOM-coupled humic substances model compound, anthraquinone-2,6-disulfonate (AQDS). 13CH4 labeling experiments further indicated that AOM-coupled AQDS reduction occurred, and acetate was an intermediate product of AOM. Moreover, 13CH313COONa labeling experiments showed that AOM-generated acetate can be continuously reduced to methane in a state of dynamic equilibrium. In the presence of magnetite, the EET capacity of the microbial community increased, and Methanosarcina played a key role in the AOM-coupled AQDS reduction. Pure culture experiments showed that Methanosarcina barkeri can independently perform AOM-coupled AQDS reduction and that magnetite increased its surface protein redox activity. The metatranscriptomic results indicated that magnetite increased the expression of membrane-bound proteins involved in energy metabolism and electron transfer in M. barkeri, thereby increasing the EET capacity. This phenomenon potentially elucidates the rationale as to why magnetite promoted AOM-coupled AQDS reduction.

Role and environmental regulation of iron-driven anaerobic methane oxidation in riverine sediment.

Iron is an abundant element in the environment and acts as a thermodynamically favorable electron acceptor driving the anaerobic oxidation of methane (AOM). Presently, the role and environmental regulation of iron-driven AOM in rivers, an important source of methane emission, are nearly unknown. Here, we provided direct evidence for iron-driven AOM activity in sediment of a mountainous river (Wuxijiang River, China) through 13C-labeled isotopic experiment. The potential rate of iron-driven AOM ranged between 0.40 and 1.84 nmol 13CO2 g (sediment) d-1, which contributed 36% on average to total AOM activity when combined the potential nitrate- and nitrite-driven AOM rates measured previously. There were significant variations in iron-driven AOM rates among different reaches (upper, middle, and lower) and between seasons (summer and winter). Sediment temperature, pH, and nitrate content were closely associated with the dynamic of AOM activity. Our results indicate that iron-driven AOM has great potential for reducing methane emissions from riverine ecosystems, and suggest the necessity of taking both spatial and temporal scales into account to evaluate the quantitative role of this AOM process.

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.

Coupled anaerobic methane oxidation and metal reduction in soil under elevated CO2.

Continued current emissions of carbon dioxide (CO2 ) and methane (CH4 ) by human activities will increase global atmospheric CO2 and CH4 concentrations and surface temperature significantly. Fields of paddy rice, the most important form of anthropogenic wetlands, account for about 9% of anthropogenic sources of CH4 . Elevated atmospheric CO2 may enhance CH4 production in rice paddies, potentially reinforcing the increase in atmospheric CH4 . However, what is not known is whether and how elevated CO2 influences CH4 consumption under anoxic soil conditions in rice paddies, as the net emission of CH4 is a balance of methanogenesis and methanotrophy. In this study, we used a long-term free-air CO2 enrichment experiment to examine the impact of elevated CO2 on the transformation of CH4 in a paddy rice agroecosystem. We demonstrate that elevated CO2 substantially increased anaerobic oxidation of methane (AOM) coupled to manganese and/or iron oxides reduction in the calcareous paddy soil. We further show that elevated CO2 may stimulate the growth and metabolism of Candidatus Methanoperedens nitroreducens, which is actively involved in catalyzing AOM when coupled to metal reduction, mainly through enhancing the availability of soil CH4 . These findings suggest that a thorough evaluation of climate-carbon cycle feedbacks may need to consider the coupling of methane and metal cycles in natural and agricultural wetlands under future climate change scenarios.

Pyrogenic Carbon Promotes Anaerobic Oxidation of Methane Coupled with Iron Reduction via the Redox-Cycling Mechanism.

Pyrogenic carbon (PC) can mediate electron transfer and thus catalyze biogeochemical processes to impact greenhouse gas (GHG) emissions. Here, we demonstrate that PC can contribute to mitigating GHG emissions by promoting the Fe(III)-dependent anaerobic oxidation of methane (AOM). It was found that the amendment PCs in microcosms dominated by Methanoperedenaceae performing Fe(III)-dependent AOM simultaneously promoted the rate of AOM and Fe(III) reduction with a consistent ratio close to the theoretical stoichiometry of 1:8. Further correlation analysis showed that the AOM rate was linearly correlated with the electron exchange capacity, but not the conductivity, of added PC materials, indicating the redox-cycling electron transfer mechanism to promote the Fe(III)-dependent AOM. The mass content of the C═O moiety from differentially treated PCs was well correlated with the AOM rate, suggesting that surface redox-active quinone groups on PCs contribute to facilitating Fe(III)-dependent AOM. Further microbial analyses indicate that PC likely shuttles direct electron transfer from Methanoperedenaceae to Fe(III) reduction. This study provides new insight into the climate-cooling impact of PCs, and our evaluation indicates that the PC-facilitated Fe(III)-dependent AOM could have a significant contribution to suppressing methane emissions from the world's reservoirs.

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.

Community Structure and Microbial Associations in Sediment-Free Methanotrophic Enrichment Cultures from a Marine Methane Seep.

Syntrophic consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) consume large amounts of methane and serve as the foundational microorganisms in marine methane seeps. Despite their importance in the carbon cycle, research on the physiology of ANME-SRB consortia has been hampered by the slow growth and complex physicochemical environment the consortia inhabit. Here, we report successful sediment-free enrichment of ANME-SRB consortia from deep-sea methane seep sediments in the Santa Monica Basin, California. Anoxic Percoll density gradients and size-selective filtration were used to separate ANME-SRB consortia from sediment particles and single cells to accelerate the cultivation process. Over a 3-year period, a subset of the sediment-associated ANME and SRB lineages, predominantly comprised of ANME-2a/2b ("Candidatus Methanocomedenaceae") and their syntrophic bacterial partners, SEEP-SRB1/2, adapted and grew under defined laboratory conditions. Metagenome-assembled genomes from several enrichments revealed that ANME-2a, SEEP-SRB1, and Methanococcoides in different enrichments from the same inoculum represented distinct species, whereas other coenriched microorganisms were closely related at the species level. This suggests that ANME, SRB, and Methanococcoides are more genetically diverse than other members in methane seeps. Flow cytometry sorting and sequencing of cell aggregates revealed that Methanococcoides, Anaerolineales, and SEEP-SRB1 were overrepresented in multiple ANME-2a cell aggregates relative to the bulk metagenomes, suggesting they were physically associated and possibly interacting. Overall, this study represents a successful case of selective cultivation of anaerobic slow-growing microorganisms from sediments based on their physical characteristics, introducing new opportunities for detailed genomic, physiological, biochemical, and ecological analyses. IMPORTANCE Biological anaerobic oxidation of methane (AOM) coupled with sulfate reduction represents a large methane sink in global ocean sediments. Methane consumption is carried out by syntrophic archaeal-bacterial consortia and fuels a unique ecosystem, yet the interactions in these slow-growing syntrophic consortia and with other associated community members remain poorly understood. The significance of this study is the establishment of sediment-free enrichment cultures of anaerobic methanotrophic archaea and sulfate-reducing bacteria performing AOM with sulfate using selective cultivation approaches based on size, density, and metabolism. By reconstructing microbial genomes and analyzing community composition of the enrichment cultures and cell aggregates, we shed light on the diversity of microorganisms physically associated with AOM consortia beyond the core syntrophic partners. These enrichment cultures offer simplified model systems to extend our understanding of the diversity of microbial interactions within marine methane seeps.

Temperature sensitivity of anaerobic methane oxidation versus methanogenesis in paddy soil: Implications for the CH4 balance under global warming.

The global methane (CH4 ) budget is based on a sensitive balance between methanogenesis and CH4 oxidation (aerobic and anaerobic). The response of these processes to climate warming, however, is not quantified. This largely reflects our lack of knowledge about the temperature sensitivity (Q10 ) of the anaerobic oxidation of CH4 (AOM)-a ubiquitous process in soils. Based on a 13 CH4 labeling experiment, we determined the rate, Q10 and activation energy of AOM and of methanogenesis in a paddy soil at three temperatures (5, 20, 35°C). The rates of AOM and of methanogenesis increased exponentially with temperature, whereby the AOM rate was significantly lower than methanogenesis. Both the activation energy and Q10 of AOM dropped significantly from 5-20 to 20-35°C, indicating that AOM is a highly temperature-dependent microbial process. Nonetheless, the Q10 of AOM and of methanogenesis were similar at 5-35°C, implying a comparable temperature dependence of AOM and methanogenesis in paddy soil. The continuous increase of AOM Q10 over the 28-day experiment reflects the successive utilization of electron acceptors according to their thermodynamic efficiency. The basic constant for Q10 of AOM was calculated to be 0.1 units for each 3.2 kJ mol-1 increase of activation energy. We estimate the AOM in paddy soils to consume 2.2~5.5 Tg CH4 per year on a global scale. Considering these results in conjunction with literature data, the terrestrial AOM in total consumes ~30% of overall CH4 production. Our data corroborate a similar Q10 of AOM and methanogenesis. As the rate of AOM in paddy soils is lower than methanogenesis, however, it will not fully compensate for an increased methane production under climate warming.

Characterization of anaerobic oxidation of methane and microbial community in landfills with aeration.

Landfills are the third largest source of anthropogenic CH4 emissions. Anaerobic oxidation of methane (AOM) activity and communities of methane-oxidizing bacteria were investigated in three informal landfills in this study, namely, BJ, CH and SZ landfills, among which BJ and CH represent traditional anaerobic landfills, while the SZ landfill was subjected to aeration to accelerate waste stabilization. The AOM rates of the investigated landfilled wastes ranged from 3.66 to 23.91 nmol g-1 h-1. Among the three landfills, the AOM rate was highest in the SZ-1-Top sample, which was closest to the aeration pipe. Among the possible electron acceptors for AOM, including NO3-, NO2-, SO42- and Fe3+, the NO2--N content was the only variable that was positively correlated with the AOM rate. Compared with α-Proteobacteria methanotrophs, γ-Proteobacteria methanotrophs were more abundant in the landfilled waste, especially Methylobacter, which was detected in nearly all samples. Members of the family Methylomirabilaceae, including Candidatus Methylomirabilis, were also detected in the SZ-1 and SZ-2-Bot samples. The relative abundance of the main methanotrophs in the families Methylomonadaceae, Methylococcaceae, Rokubacteriales and Methylomirabilaceae, the genus Methylocystis and the phylum NC10 were all positive correlations with the contents of NO2--N in the landfilled waste samples. Additionally, significantly positive correlations were observed between the AOM rates and the relative abundance of the main methanotrophs except for the family Methylococcaceae. This indicated that aeration could enhance the conversion of nitrogen compounds in the landfilled waste, in which the high contents of NO2--N could stimulate the growth of methanotrophs and increase AOM rate. These findings are helpful for understanding the mechanisms of CH4 oxidation in landfills and for taking effective measures to mitigate CH4 emissions from landfills.

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.

The role and related microbial processes of Mn-dependent anaerobic methane oxidation in reducing methane emissions from constructed wetland-microbial fuel cell.

Anaerobic oxidation of methane (AOM) plays an important role in global carbon cycle and greenhouse gas emission reduction. In this study, an effective green technology to reduce methane emissions was proposed by introducing Mn-dependent anaerobic oxidation of methane (Mn-AOM) and microbial fuel cell (MFC) technology into constructed wetland (CW). The results indicate that the combination of biological methods and bioelectrochemical methods can more effectively control the methane emission from CW than the reported methods. The role of dissimilated metal reduction in methane control in CW and the biochemical process associated with Mn-AOM were also investigated. The results demonstrated that using Mn ore as the matrix and operating MFC effectively reduced methane emissions from CW, and higher COD removal rate was obtained in CW-MFC (Mn) during the 200 days of operation. Methane emission from CW-MFC (Mn) (53.76 mg/m2/h) was 55.61% lower than that of CW (121.12 mg/m2/h). The highest COD removal rate (99.85%) in CW-MFC (Mn) was obtained. As the dissimilative metal-reducing microorganisms, Geobacter (5.10%) was found enriched in CW-MFC (Mn). The results also showed that the presence of Mn ore was beneficial to the biodiversity of CW-MFCs and the growth of electrochemically active bacteria (EAB) including Proteobacteria (35.32%), Actinobacteria (2.38%) and Acidobacteria (2.06%), while the growth of hydrogenotrophic methanogens Methanobacterium was effectively inhibited. This study proposed an effective way to reduce methane from CW. It also provided reference for low carbon technology of wastewater treatment.

Diversity, enrichment, and genomic potential of anaerobic methane- and ammonium-oxidizing microorganisms from a brewery wastewater treatment plant.

Anaerobic wastewater treatment offers several advantages; however, the effluent of anaerobic digesters still contains high levels of ammonium and dissolved methane that need to be removed before these effluents can be discharged to surface waters. The simultaneous anaerobic removal of methane and ammonium by denitrifying (N-damo) methanotrophs in combination with anaerobic ammonium-oxidizing (anammox) bacteria could be a potential solution to this challenge. After a molecular survey of a wastewater plant treating brewery effluent, indicating the presence of both N-damo and anammox bacteria, we started an anaerobic bioreactor with a continuous supply of methane, ammonium, and nitrite to enrich these anaerobic microorganisms. After 14 months of operation, a stable enrichment culture containing two types of 'Candidatus Methylomirabilis oxyfera' bacteria and two strains of 'Ca. Brocadia'-like anammox bacteria was achieved. In this community, anammox bacteria converted 80% of the nitrite with ammonium, while 'Ca. Methylomirabilis' contributed to 20% of the nitrite consumption. The analysis of metagenomic 16S rRNA reads and fluorescence in situ hybridization (FISH) correlated well and showed that, after 14 months, 'Ca. Methylomirabilis' and anammox bacteria constituted approximately 30 and 20% of the total microbial community. In addition, a substantial part (10%) of the community consisted of Phycisphaera-related planctomycetes. Assembly and binning of the metagenomic sequences resulted in high-quality draft genome of two 'Ca. Methylomirabilis' species containing the marker genes pmoCAB, xoxF, and nirS and putative NO dismutase genes. The anammox draft genomes most closely related to 'Ca. Brocadia fulgida' included the marker genes hzsABC, hao, and hdh. Whole-reactor and batch anaerobic activity measurements with methane, ammonium, nitrite, and nitrate revealed an average anaerobic methane oxidation rate of 0.12 mmol h-1 L-1 and ammonium oxidation rate of 0.5 mmol h-1 L-1. Together, this study describes the enrichment and draft genomes of anaerobic methanotrophs from a brewery wastewater treatment plant, where these organisms together with anammox bacteria can contribute significantly to the removal of methane and ammonium in a more sustainable way. KEY POINTS: • An enrichment culture containing both N-damo and anammox bacteria was obtained. • Simultaneous consumption of ammonia, nitrite, and methane under anoxic conditions. • In-depth metagenomic biodiversity analysis of inoculum and enrichment culture.

Anaerobic oxidation of methane with denitrification in sediments of a subtropical estuary: Rates, controlling factors and environmental implications.

Anaerobic oxidation of methane with denitrification (DAMO), as an important microbial process regulating methane emission, has been widely reported in freshwater ecosystems. However, the DAMO process and associated biogeochemical controls in estuaries remain poorly understood. Here, we used 13C- and 15N-labelling experiments to quantify the potential rates of DAMO and determined the crucial factors controlling the DAMO rates in the sediment of Yangtze Estuary. Potential rates of DAMO varied greatly across the estuary, ranging from 0.07 to 0.28 nmol CO2 g-1 d-1. Salinity negatively affected the DAMO and also showed an indirectly negative influence on DAMO process by high salinity inhibition on NO3- availability and denitrification. Nitrate concentrations were significantly correlated with the DAMO rates. Denitrification rates showed positive correlation with DAMO rates, implying that nitrate reduction drives the DAMO process. Sediment total organic carbon and NH4+ had important effects on DAMO rates. These results together indicate that DAMO process can occur and the DAMO rates were mainly controlled by sediment NO3- and denitrification in estuary. We further conclude that increasing NO3- load can drive the DAMO process with more important implications on methane sink in estuarine ecosystems.

Kinetics of anaerobic methane oxidation coupled to denitrification in the membrane biofilm reactor.

Anaerobic oxidation of methane coupled to denitrification (AOM-D) in a membrane biofilm reactor (MBfR), a platform used for efficiently coupling gas delivery and biofilm development, has attracted attention in recent years due to the low cost and high availability of methane. However, experimental studies have shown that the nitrate-removal flux in the CH4 -based MBfR (<1.0 g N/m2 -day) is about one order of magnitude smaller than that in the H2 -based MBfR (1.1-6.7 g N/m2 -day). A one-dimensional multispecies biofilm model predicts that the nitrate-removal flux in the CH4 -based MBfR is limited to <1.7 g N/m2 -day, consistent with the experimental studies reported in the literature. The model also determines the two major limiting factors for the nitrate-removal flux: The methane half-maximum-rate concentration (K2 ) and the specific maximum methane utilization rate of the AOM-D syntrophic consortium (kmax2 ), with kmax2 being more important. Model simulations show that increasing kmax2 to >3 g chemical oxygen demand (COD)/g cell-day (from its current 1.8 g COD/g cell-day) and developing a new membrane with doubled methane-delivery capacity (Dm ) could bring the nitrate-removal flux to ≥4.0 g N/m2 -day, which is close to the nitrate-removal flux for the H2 -based MBfR. Further increase of the maximum nitrate-removal flux can be achieved when Dm and kmax2 increase together.

Archaea catalyze iron-dependent anaerobic oxidation of methane.

Anaerobic oxidation of methane (AOM) is crucial for controlling the emission of this potent greenhouse gas to the atmosphere. Nitrite-, nitrate-, and sulfate-dependent methane oxidation is well-documented, but AOM coupled to the reduction of oxidized metals has so far been demonstrated only in environmental samples. Here, using a freshwater enrichment culture, we show that archaea of the order Methanosarcinales, related to "Candidatus Methanoperedens nitroreducens," couple the reduction of environmentally relevant forms of Fe3+ and Mn4+ to the oxidation of methane. We obtained an enrichment culture of these archaea under anaerobic, nitrate-reducing conditions with a continuous supply of methane. Via batch incubations using [13C]methane, we demonstrated that soluble ferric iron (Fe3+, as Fe-citrate) and nanoparticulate forms of Fe3+ and Mn4+ supported methane-oxidizing activity. CO2 and ferrous iron (Fe2+) were produced in stoichiometric amounts. Our study connects the previous finding of iron-dependent AOM to microorganisms detected in numerous habitats worldwide. Consequently, it enables a better understanding of the interaction between the biogeochemical cycles of iron and methane.

Persistence times of refractory materials in landfills: A review of rate limiting conditions by mass transfer and reaction kinetics.

Monitoring programs at closed landfills show that transformation of plastics, wood, and metals continue long after the active decomposition of the waste fractions are considered as complete. Studies conducted in natural anaerobic environments (e.g., marine sediments and rocks) provide insight for slow degradation mechanisms involving coupling of thermodynamically favorable and unfavorable reactions and biochemical transformations by microbial consortia. These transformations occur at much slower rates through more complex and less obvious mechanisms and are not evident until after the primary decomposition mechanisms become less significant. This study presents a review of the conditions that limit the mass transfer and reaction kinetics for anaerobic transformations in landfills and provides new insights for reaction mechanisms (e.g., anaerobic oxidation and anaerobic corrosion) that occur at relatively slow rates in mature landfills. Conditions and mechanisms of slow transformations by microbial and chemical activities with relatively small energy yields and availability of electron acceptors (e.g., inorganics, plastics) and/or diffusion of gas and moisture into the previously isolated areas in waste deposits were discussed. Time scales for mass transfer and reaction kinetics were compared under anaerobic conditions for different waste components deposited at municipal solid waste landfills. Half-lives of different materials under anaerobic conditions were estimated and compared. Emergence of syntrophic methanogenic communities and conditions for triboelectric effects were evaluated as possible electron transfer mechanisms between waste layers for occurrence of extremely slow transformations of wastes deposited in landfills.

Response of the Anaerobic Methanotroph "Candidatus Methanoperedens nitroreducens" to Oxygen Stress.

"Candidatus Methanoperedens nitroreducens" is an archaeon that couples the anaerobic oxidation of methane to nitrate reduction. In natural and man-made ecosystems, this archaeon is often found at oxic-anoxic interfaces where nitrate, the product of aerobic nitrification, cooccurs with methane produced by methanogens. As such, populations of "Ca Methanoperedens nitroreducens" could be prone to regular oxygen exposure. Here, we investigated the effect of 5% (vol/vol) oxygen exposure in batch activity assays on a "Ca Methanoperedens nitroreducens" culture, enriched from an Italian paddy field. Metagenome sequencing of the DNA extracted from the enrichment culture revealed that 83% of 16S rRNA gene reads were assigned to a novel strain, "Candidatus Methanoperedens nitroreducens Verserenetto." RNA was extracted, and metatranscriptome sequencing upon oxygen exposure revealed that the active community changed, most notably in the appearance of aerobic methanotrophs. The gene expression of "Ca Methanoperedens nitroreducens" revealed that the key genes encoding enzymes of the methane oxidation and nitrate reduction pathways were downregulated. In contrast to this, we identified upregulation of glutaredoxin, thioredoxin family/like proteins, rubrerythrins, peroxiredoxins, peroxidase, alkyl hydroperoxidase, type A flavoproteins, FeS cluster assembly protein, and cysteine desulfurases, indicating the genomic potential of "Ca Methanoperedens nitroreducens Verserenetto" to counteract the oxidative damage and adapt in environments where they might be exposed to regular oxygen intrusion.IMPORTANCE "Candidatus Methanoperedens nitroreducens" is an anaerobic archaeon which couples the reduction of nitrate to the oxidation of methane. This microorganism is present in a wide range of aquatic environments and man-made ecosystems, such as paddy fields and wastewater treatment systems. In such environments, these archaea may experience regular oxygen exposure. However, "Ca Methanoperedens nitroreducens" is able to thrive under such conditions and could be applied for the simultaneous removal of dissolved methane and nitrogenous pollutants in oxygen-limited systems. To understand what machinery "Ca Methanoperedens nitroreducens" possesses to counteract the oxidative stress and survive, we characterized the response to oxygen exposure using a multi-omics approach.

Harnessing a methane-fueled, sediment-free mixed microbial community for utilization of distributed sources of natural gas.

Harnessing the metabolic potential of uncultured microbial communities is a compelling opportunity for the biotechnology industry, an approach that would vastly expand the portfolio of usable feedstocks. Methane is particularly promising because it is abundant and energy-rich, yet the most efficient methane-activating metabolic pathways involve mixed communities of anaerobic methanotrophic archaea and sulfate reducing bacteria. These communities oxidize methane at high catabolic efficiency and produce chemically reduced by-products at a comparable rate and in near-stoichiometric proportion to methane consumption. These reduced compounds can be used for feedstock and downstream chemical production, and at the production rates observed in situ they are an appealing, cost-effective prospect. Notably, the microbial constituents responsible for this bioconversion are most prominent in select deep-sea sediments, and while they can be kept active at surface pressures, they have not yet been cultured in the lab. In an industrial capacity, deep-sea sediments could be periodically recovered and replenished, but the associated technical challenges and substantial costs make this an untenable approach for full-scale operations. In this study, we present a novel method for incorporating methanotrophic communities into bioindustrial processes through abstraction onto low mass, easily transportable carbon cloth artificial substrates. Using Gulf of Mexico methane seep sediment as inoculum, optimal physicochemical parameters were established for methane-oxidizing, sulfide-generating mesocosm incubations. Metabolic activity required >∼40% seawater salinity, peaking at 100% salinity and 35 °C. Microbial communities were successfully transferred to a carbon cloth substrate, and rates of methane-dependent sulfide production increased more than threefold per unit volume. Phylogenetic analyses indicated that carbon cloth-based communities were substantially streamlined and were dominated by Desulfotomaculum geothermicum. Fluorescence in situ hybridization microscopy with carbon cloth fibers revealed a novel spatial arrangement of anaerobic methanotrophs and sulfate reducing bacteria suggestive of an electronic coupling enabled by the artificial substrate. This system: 1) enables a more targeted manipulation of methane-activating microbial communities using a low-mass and sediment-free substrate; 2) holds promise for the simultaneous consumption of a strong greenhouse gas and the generation of usable downstream products; and 3) furthers the broader adoption of uncultured, mixed microbial communities for biotechnological use.

Metabolic engineering of Methanosarcina acetivorans for lactate production from methane.

We previously demonstrated anaerobic conversion of the greenhouse gas methane into acetate using an engineered archaeon that produces methyl-coenzyme M reductase (Mcr) from unculturable microorganisms from a microbial mat in the Black Sea to create the first culturable prokaryote that reverses methanogenesis and grows anaerobically on methane. In this work, we further engineered the same host with the goal of converting methane into butanol. Instead, we discovered a process for converting methane to a secreted valuable product, L-lactate, with sufficient optical purity for synthesizing the biodegradable plastic poly-lactic acid. We determined that the 3-hydroxybutyryl-CoA dehydrogenase (Hbd) from Clostridium acetobutylicum is responsible for lactate production. This work demonstrates the first metabolic engineering of a methanogen with a synthetic pathway; in effect, we produce a novel product (lactate) from a novel substrate (methane) by cloning the three genes for Mcr and one for Hbd. We further demonstrate the utility of anaerobic methane conversion with an increased lactate yield compared to aerobic methane conversion to lactate. Biotechnol. Bioeng. 2017;114: 852-861. © 2016 Wiley Periodicals, Inc.