New molecular method to detect denitrifying anaerobic methane oxidation bacteria from different environmental niches

The denitrifying anaerobic methane oxidation is an ecologically important process for reducing the potential methane emission into the atmosphere.The responsible bacterium for this process was Candidatus Methylomirabilis oxyfera belonging to the bacterial phylum of NC10.In this study,a new pair of primers targeting all the five groups of NC10 bacteria was designed to amplify NC10 bacteria from different environmental niches.The results showed that the group A was the dominant NC10 phylum bacteria from the sludges and food waste digestate while in paddy soil samples,group A and group B had nearly the same proportion.Our results also indicated that NC10 bacteria could exist in a high p H environment（pH 9.24）from the food waste treatment facility.The Pearson relationship analysis showed that the p H had a significant positive relationship with the NC10 bacterial diversity（p0.05）.The redundancy analysis further revealed that the p H,volatile solid and nitrite nitrogen were the most important factors in shaping the NC10 bacterial structure（p=0.01）based on the variation inflation factors selection and Monte Carlo test（999 times）.Results of this study extended the existing molecular tools for studying the NC10 bacterial community structures and provided new information on the ecological distributions of NC10 bacteria.

The influence of coupling mode of methane leakage and debris input on anaerobic oxidation of methane

Anaerobic oxidation of methane(AOM) is an important biogeochemical process, which has important scientific significance for global climate change and atmospheric evolution. This research examined the δ^(34)S, terrigenous clastic indices of TiO_(2) and Al_(2)O_(3), and times for formation of the Ba front at site SH1, site SH3 and site 973-4 in the South China Sea. Three different coupling mechanisms of deposition rate and methane flux were discovered. The different coupling mechanisms had different effects on the role of AOM. At site 973-4, a high deposition rate caused a rapid vertical downward migration of the sulphate–methane transition zone(SMTZ), and the higher input resulted in mineral dissolution. At site SH3, the deposition rate and methane flux were basically in balance,so the SMTZ and paleo-SMTZ were the most stable of any site, and these were in a slow process of migration. At site SH1, the methane flux dominated the coupled mode, so the movement of the SMTZ at site SH1 was consistent with the general understanding. Understanding the factors influencing the SMTZ is important for understanding the early diagenesis process.

Interactions of Microplastics and Methane Seepage in the Deep-Sea Environment

Microplastics(MPs)are important exempla of the Anthropocene and are exerting an increasing impact on Earth’s carbon cycle.The huge imbalance between the MPs floating on the marine surface and those that are estimated to have been introduced into the ocean necessitates a detailed assessment of marine MP sinks.Here,we demonstrate that cold seep sediments,which are characterized by methane fluid seepage and a chemosynthetic ecosystem,effectively capture and accommodate small-scale(<100μm)MPs,with 16 types of MPs being detected.The abundance of MPs in the surface of the sediment is higher in methane-seepage locations than in non-seepage areas.Methane seepage is beneficial to the accumulation,fragmentation,increased diversity,and aging of MPs.In turn,the rough surfaces of MPs contribute to the sequestration of the electron acceptor ferric oxide,which is associated with the anaerobic oxidation of methane(AOM).The efficiency of the AOM determines whether the seeping methane(which has a greenhouse effect 83 times greater than that of CO_(2)over a 20-year period)can enter the atmosphere,which is important to the global methane cycle,since the deep-sea environment is regarded as the largest methane reservoir associated with natural gas hydrates.

Sulfate-Methane Transition Depths and Its Implication for Gas Hydrate

The biological removal of CH4 by methanotrophic(CH4-oxidizing)archaea always occurs at a distinct zone which is known as sulfate-methane transition zone(SMTZ).It is an important indication for high methane flux and gas hydrate occurrence.In this study,we collected pore-water data from South China Sea,Carolina Rise and Blake Ridge to analyze the relationship between CH4,SO42−concentration and depth.We found that below the SMTZ,the methane concentration increases continuously with depth and sulfate concentration decrease linearly to zero.In addition,the geochemical data taken from all these sites show that SMTZ is relatively shallow(less than 20 m),which may indicate that these areas have high methane flux.

Pore-water geochemistry in methane-seep sediments of the Makran accretionary wedge off Pakistan:Possible link to subsurface methane hydrate

Cold seeps are pervasive along the continental margin worldwide,and are recognized as hotspots for elemental cycling pathway on Earth.In this study,analyses of pore water geochemical compositions of one-400 cm piston core(S3)and the application of a mass balance model are conducted to assess methane-associated biogeochemical reactions and uncover the relationship of methane in shallow sediment with gas hydrate reservoir at the Makran accretionary wedge off Pakistan.The results revealed that approximately 77%of sulfate is consumed by the predominant biogeochemical process of anaerobic oxidation of methane.However,the estimated sulfate-methane interface depth is-400 cm below sea floor with the methane diffusive flux of 0.039 mol/(m^(2)·a),suggesting the activity of methane seepage.Based on the δ^(13)C_(DIC) mass balance model combined with the contribution proportion of different dissolved inorganic carbon sources,this study calculated the δ^(13)C of the exogenous methane to be-57.9‰,indicating that the exogenous methane may be a mixture source,including thermogenic and biogenic methane.The study of pore water geochemistry at Makran accretionary wedge off Pakistan may have considerable implications for understanding the specific details on the dynamics of methane in cold seeps and provide important evidence for the potential occurrence of subsurface gas hydrate in this area.

Combining biological denitrification and electricity generation in methane-powered microbial fuel cells

Methane has been demonstrated to be a feasible substrate for electricity generation in microbial fuel cells(MFCs)and denitrifying anaerobic methane oxidation(DAMO).However,these two processes were evaluated separately in previous studies and it has remained unknown whether methane is able to simultaneously drive these processes.Here we investigated the co-occurrence and performance of these two processes in the anodic chamber of MFCs.The results showed that methane successfully fueled both electrogenesis and denitrification.Importantly,the maximum nitrate removal rate was significantly enhanced from(1.4±0.8)to(18.4±1.2)mg N/(L·day)by an electrogenic process.In the presence of DAMO,the MFCs achieved a maximum voltage of 610 mV and a maximum power density of 143±12 mW/m^(2).Electrochemical analyses demonstrated that some redox substances(e.g.riboflavin)were likely involved in electrogenesis and also in the denitrification process.High-throughput sequencing indicated that the methanogen Methanobacterium,a close relative of Methanobacterium espanolae,catalyzed methane oxidation and cooperated with both exoelectrogens and denitrifiers(e.g.,Azoarcus).This work provides an effective strategy for improving DAMO in methane-powered MFCs,and suggests that methanogens and denitrifiers may jointly be able to provide an alternative to archaeal DAMO for methane-dependent denitrification.

Authigenic Gypsum in Gas-Hydrate Associated Sediments from the East Coast of India (Bay of Bengal)

Authigenic gypsum crystals, along with pyrite and carbonate mineralization, predominantly calcites were noticed in distinct intervals in a 32 m long piston core, collected in the gas hydratebearing sediments in the northern portion of the Krishna-Godavari basin, eastern continental margin of India at a water depth of 1691 m. X-ray diffraction and energy dispersive spectrum studies confirm presence of pyrite, gypsum, calcite, and other mineral aggregates. The occurrence of gypsum in such deep sea environment is intriguing, because gypsum is a classical evaporite mineral and is under saturated with respect to sea water. Sedimentological, geochemical evidences point to diagenetic formation of the gypsum due to oxidation of sulphide minerals （i.e. pyrite）. Euhedral, transparent gypsum crystals, with pyrite inclusions are cemented with authigenic carbonates, possibly indicating that they were formed authigenically in situ in the gas hydrate-influenced environment due to late burial diagenesis involving sulphate reduction and anaerobic oxidation of methane （AOM）. Therefore, the authigenic gypsums found in sediments of the Krishna-Godavari and Mahanadi offshore regions could be seen as one of the parameters to imply the presence of high methane flux possibly from gas hydrate at depth.

Biomarker assessments of sources and environmental implications of organic matter in sediments from potential cold seep areas of the northeastern South China Sea

Multi-biomarker indexes were analyzed for two piston cores from potential cold seep areas of the South China Sea off southwestern Taiwan. Total organic carbon（TOC） normalized terrestrial（n-alkanes） and marine（brassicasterol, dinosterol, alkenones and iso-GDGTs） biomarker contents and ratios（TMBR, 1/Pmar-aq, BIT） were used to evaluate the contributions of terrestrial and marine organic matter（TOM and MOM respectively） to the sedimentary organic matter, indicating that MOM dominated the organic sources in Core MD052911 and the sedimentary organic matter in Core ORI-_（86）0-22 was mainly derived from terrestrial inputs, and different morphologies were the likely reason for TOM percentage differences. BIT results suggested that river-transported terrestrial soil organic matter was not a major source of TOM of sedimentary organic matter around these settings.Diagnostic biomarkers for methane-oxidizing archaea（MOA） were only detected in one sample at 172 cm depth of Core ORI-_（86）0-22, with abnormally high iso-GDGTs content and Methane Index（MI） value（0.94）. These results indicated high anaerobic oxidation of methane（AOM） activities at or around 172 cm in Core ORI-_（86）0-22.However in Core MD052911, MOA biomarkers were not detected and MI values were lower（0.19–0.38）, indicated insignificant contributions of iso-GDGTs from methanotrophic archaea and the absence of significant AOM activities. Biomarker results thus indicated that the discontinuous upward methane seepage and insufficient methane flux could not induce high AOM activities in our sampling sites. In addition, the different patterns of TEX_（86） and U_（37）^（K′） temperature in two cores suggested that AOM activities affected TEX_（86）37 temperature estimates with lower values in Core ORI-_（86）0-22, but not significantly on TEX_（86） temperature estimates in Core MD052911.

Microbial Mats in the Mesoproterozoic Carbonates of the North China Platform and Their Potential for Hydrocarbon Generation

The well-preserved Mesoproterozoic succession in the North China platform consists mainly of three iithological associations including peritidal quartz sandstone, shallow marine and lagoonal dark to black shales, and shallow epeiric carbonates, with a total thickness of up to 8 000 m. In addition to well-documented microplants, macroalgae, and microbial buildups, abundant microbially induced sedimentary structures （MISS） and mat-related sediments have been recognized in these rocks. Intensive microbial mat layers and MISS are especially well preserved in the carbonates of the upper Gaoyuzhuang （高于庄） （ca. 1.5 Ga） and lower Wumishan （雾迷山） （ca. 1.45 Ga） formations, indicating diversified microbial activities and a high organic production. In these petrified biomats, putative microbial fossils （both coccoidal and filamentous） and framboidal pyrites have been identified. The abundance of authigenic carbonate minerals in the host rocks, such as, acicular aragonites, rosette barites, radial siderites, ankerites, and botryoidal carbonate cements, suggests authigenic carbonate precipitation from anaerobic oxidation of methane （AOM） under anoxic/euxinic conditions. Warm climate and anoxic/euxinic conditions in the Mesoproterozoic oceans may have facilitated high microbial productivity and organic burial in sediments. Although authigenic carbonate cements may record carbonate precipitation from anaerobic methane oxidation, gas blister （or dome） structures may indicate gas release from active methanogenesis during shallow burial. Bituminous fragments in mat-related carbonates also provide evidence for hydrocarbon generation. Under proper conditions, the Mesoproterozoic mat-rich carbonates will have the potential for hydrocarbon generation and serve as source rocks. On the basis of petrified biomats, a rough estimation suggests that the Mesoproterozoic carbonates of the North China platform might have a hydrocarbon production potential in the order of 10 ×10^8 t.

Petrology and geochemistry of cold seep carbonates from the northern Okinawa Trough,East China Sea:implications to early diagenesis

Carbonate samples were collected from the northern Okinawa Trough in the East China Sea in 2013.The petrology,mineralogy,carbon and oxygen isotopes,and rare earth elements(REEs)of these samples were analyzed.Aragonite,high-Mg calcite,and dolomite were the main carbonate minerals,the contents of which varied greatly among the carbonate samples.Petrological observations revealed the common occurrence of framboidal pyrites.Theδ^(13)C values of carbonates varied from-53.7‰to-39.3‰(average of-47.3‰based on Vienna Pee Dee Belemnite(V-PDB),n=9),and theδ^(18)O values ranged from 0.6‰to 3.4‰(average of 1.9‰;V-PDB,n=9).The carbon and oxygen isotope characteristics indicated that the carbonates precipitated during the anaerobic oxidation of methane.The carbon source was a mixture of thermogenic methane and biogenic methane,possibly with a greater contribution from the former.The oxygen isotope data showed that gas hydrate dissociation occurred during carbonate precipitation.The Ce anomalies suggested that the carbonates precipitated in an anoxic environment.A slight enrichment of middle REEs(MREEs)could be attributable to the early diagenesis.The structures,minerals,oxygen isotopes,and MREEs all indicated that the carbonates experienced some degree of early diagenesis.Therefore,the infl uence of early diagenesis should be considered when using geological and geochemical proxies to reconstruct original methane seepage environments.

The crucial role of deep-sourced methane in maintaining the subseafloor sulfate budget

Methane(CH4)is a powerful greenhouse gas and its largest reservoir on Earth is held in marine sediments.CH4 in marine sediments is mainly stored in gas-hydrate reservoirs and deep sedimentary strata along continental margins,where large amounts of deep-sourced CH4 ascend to different degrees toward the seafloor.However,the amount of deep-sourced CH4 and its role in subseafloor carbon and sulfur cycling remains poorly constrained.We analyzed sulfate(SO_(4)^(2-))profiles of 157 sites along with previous published 85 sites to determine the regional distribution and amount of SO_(4)^(2-) reduction for an area of 1.23×10^(5) km^(2) of the northern South China Sea.Then we compared these obtained results with estimates based on sedimentation rates from the same area.Significantly higher regional SO_(4)^(2-) flux estimates based on SO_(4)^(2-) profiles(4.26×10^(-3)Tmol a^(-1)),compared to lower estimates based on sedimentation rates(1.23×10^(-3)Tmol a^(-1)),reflect abundant ascending deep-sourced CH4.The difference of the regional SO_(4)^(2-) flux estimates(3.03×10^(-3)Tmol a^(-1))represents the amount of SO_(4)^(2-) reduced by CH_(4) through the anaerobic oxidation of CH_(4)(AOM).Deep-sourced CH_(4) contributes 71%to total SO_(4)^(2-) consumption in the study area,largely exceeding SO_(4)^(2-) consumption by organoclastic sulfate reduction.Our findings substantiate that deep-sourced CH4 governs subseafloor carbon and sulfur cycling to a previously underrated extent,fueling extensive chemosynthesis-based ecosystems along continental slope and rise.

Acid Orange 7 degradation using methane as the sole carbon source and electron donor

Azo dyes are widely applied in the textile industry but are not entirely consumed during the dyeing process and can thus be discharged to the environment in wastewater.However,azo dyes can be degraded using various electron donors,and in this paper,Acid Orange 7(AO7)degradation performance is investigated using methane(CH4)as the sole electron donor.Methane has multiple sources and is readily available and inexpensive.Experiments using ^(13)C-labeled isotopes showed that AO7 degradation was coupled with anaerobic oxidation of methane(AOM)and,subsequently,affected by the initial concentrations of AO7.Higher concentrations of AO7 could inhibit the activity of microorganisms,which was confirmed by the long-term performance of AO7 degradation,with maximum removal rates of 8.94 mg/(L·d)in a batch reactor and 280 mg/(L·d)in a hollow fiber membrane bioreactor(HfMBR).High-throughput sequencing using 16S rRNA genes showed that Candidatus Methanoperedens,affiliated to ANME-2d,dominated the microbial community in the batch reactor and HfMBR.Additionally,the relative abundance of Proteobacteria bacteria(Phenylobacterium,Pseudomonas,and Geothermobacter)improved after AO7 degradation.This outcome suggested that ANME-2d alone,or acting synergistically with partner bacteria,played a key role in the process of AO7 degradation coupled with AOM.

The main anammox-based processes, the involved microbes and the novel process concept from the application perspective

Anammox technology has been widely researched over the past 40-year from the laboratory-scale to full-scale. It is well-known that in actual applications, the solo application of anammox is not feasible. Since both ammonium and nitrite are prerequisites based on the reaction mechanism, the pre-treatment of wastewater is necessary. With the combination of anammox process and other pre-treatment processes to treat the actual wastewater, many types of anammox-based processes have been developed with distinct nitrogen removal performance. Thus, in order to heighten the awareness of researchers to the developments and accelerate the application of these processes to the treatment of actual wastewater, the main anammox-based processes are reviewed in this paper. It includes the partial nitritation/anammox process, the denitratation/anammox (PD/A) process, the denitrifying anaerobic methane oxidation/anammox (DAMO/A) process, and more complex deuterogenic processes. These processes have made the breakthroughs in the application of the anammox technology, such as the combination of nitrification and PD/A process can achieve stability and reliability of nitrogen removal in the treatment of mainstream wastewater, the PD/A process and the DAMO/A have brought about further improvements in the total nitrogen removal efficiency of wastewater. The diversity of functional microbe characteristics under the specific condition indicate the wide application potential of anammox-based processes, and further exploration is necessary. A whole waste treatment system concept is proposed through the effective allocation of above mentioned processes, with the maximum recovery of energy and resources, and minimal environmental impact.