D-Ring Modifications of Tetracyclines Determine Their Ability to Induce Resistance Genes in the Environment.

The widespread utilization of tetracyclines (TCs) in agriculture and medicine has led to the borderless spread of tetracycline resistance in humans, animals, and the environment, posing huge risks to both the ecosystem and human society. Changes in the functional group modifications resulted in a higher bacteriostatic efficacy of the new generation of TCs, but their effect on the emergence and evolution of antibiotic resistance genes (ARGs) is not yet known. To this end, four TCs from three generations were chosen to compare their structural effects on influencing the evolution of ARGs in soil microbial communities. The findings revealed that low-generation TCs, such as tetracycline and oxytetracycline, exhibited a greater propensity to stimulate the production and proliferation of ARGs than did high-generation tigecycline. Molecular docking analysis demonstrated that modifications of the D-ring functional group determined the binding capacity of TCs to the substrate-binding pocket of transcriptional regulators and efflux pumps mainly involved in drug resistance. This can be further evidenced by reverse transcription-quantitative polymerase chain reaction quantification and intracellular antibiotic accumulation assessment. This study sheds light on the mechanism of the structural effect of antibiotic-induced ARG production from the perspective of compound-protein binding, therefore providing theoretical support for controlling the dissemination of antibiotic resistance.

Fungi play a crucial role in sustaining microbial networks and accelerating organic matter mineralization and humification during thermophilic phase of composting.

Fungi play an important role in the mineralization and humification of refractory organic matter such as lignocellulose during composting. However, limited research on the ecological role of fungi in composting system hindered the development of efficient microbial agents. In this study, six groups of lab-scale composting experiments were conducted to reveal the role of fungal community in composting ecosystems by comparing them with bacterial community. The findings showed that the thermophilic phase was crucial for organic matter degradation and humic acid formation. The Richness index of the fungal community peaked at 1165 during this phase. PCoA analysis revealed a robust thermal stability in the fungal community. Despite temperature fluctuations, the community structure, predominantly governed by Pichia and Candida, remained largely unaltered. The stability of fungal community and the complexity of ecological networks were 1.26 times and 5.15 times higher than those observed in bacterial community, respectively. Fungi-bacteria interdomain interaction markedly enhanced network complexity, contributing to maintain microbial ecological functions. The core fungal species belonging to the family Saccharomycetaceae drove interdomain interaction during thermophilic phase. This study demonstrated the key role of fungi in the composting system, which would provide theoretical guidance for the development of high efficiency composting agents to strengthen the mineralization and humification of organic matter.

Unveiling the unique role of iron in the metabolism of methanogens: A review.

Methanogens are the main participants in the carbon cycle, catalyzing five methanogenic pathways. Methanogens utilize different iron-containing functional enzymes in different methanogenic processes. Iron is a vital element in methanogens, which can serve as a carrier or reactant in electron transfer. Therefore, iron plays an important role in the growth and metabolism of methanogens. In this paper, we cast light on the types and functions of iron-containing functional enzymes involved in different methanogenic pathways, and the roles iron play in energy/substance metabolism of methanogenesis. Furthermore, this review provides certain guiding significance for lowering CH4 emissions, boosting the carbon sink capacity of ecosystems and promoting green and low-carbon development in the future.

Deterministic Effect of pH on Shaping Soil Resistome Revealed by Metagenomic Analysis.

Soil is recognized as the major reservoir of antibiotic resistance genes (ARGs), harboring the most diverse naturally evolved ARGs on the planet. Multidrug resistance genes are a class of ARGs, and their high prevalence in natural soil ecosystems has recently raised concerns. Since most of these genes express proton motive force (PMF) driven efflux pumps, studying whether soil pH is a determinant for the selection of multidrug efflux pump genes and thus shaping the soil resistome are of great interest. In this study, we collected 108 soils with pH values ranging from 4.37 to 9.69 from multiple ecosystems and profiled the composition of ARGs for metagenomes and metagenome-assembled genomes. We observed the multidrug efflux pump genes enriched in the acidic soil resistome, and their abundances have significant soil pH dependence. This reflects the benefits of high soil proton activity on the multidrug efflux pump genes, especially for the PMF-driven inner membrane transferase. In addition, we preliminary indicate the putative microbial participants in pH shaping the soil resistome by applying ecological analyzing tools such as stepwise regression and random forest model fitting. The decisive influence of proton activity on shaping the resistome is more impactful than any other examined factors, and as the consequence, we revisited the influence of edaphic factors on the soil resistome; i.e., the deterministic selection of resistance mechanisms by edaphic factors could lead to the bottom-up shaping of the ARG composition. Such natural developing mechanisms of the resistome are herein suggested to be considered in assessing human-driven ARG transmissions.

Composition identification and functional verification of bacterial community in disease-suppressive soils by machine learning.

It has been widely reported that probiotic consortia in the rhizosphere can enhance the plant resistance to pathogens. However, the general composition and functional profiles of bacterial community in soils which suppress multiple diseases for various plants remain largely unknown. Here, we combined metadata analysis with machine learning to identify the general patterns of bacterial-community composition in disease-suppressive soils. Disease-suppressive soils significantly enriched Firmicutes and Actinobacteria but showed a decrease in Proteobacteria and Bacteroidetes. Our machine-learning models accurately identified the disease-conducive and -suppressive soils with 54 biomarker genera, 28 of which were potentially beneficial. We further carried out a successive passaging experiment with the susceptible rps2 mutant of Arabidopsis thaliana invaded by Pseudomonas syringae pv. tomato DC3000 (avrRpt2) for functional verification of potential beneficial bacteria. The disease-suppressive ability of Kribbella, Nocardioides and Bacillus was confirmed, and they positively activated the pathogen-associated molecular patterns-triggered immunity pathway. Results also showed that chemical control by pesticides in agricultural production decreased the disease-suppressive ability of soil. This study provides a method for accurately predicting the occurrence of multiple diseases in soil and identified potential beneficial bacteria to guide a wide range of multiple-strain biological control strategies in agricultural management.

Development of a machine-learning model to identify the impacts of pesticides characteristics on soil microbial communities from high-throughput sequencing data.

High-throughput sequencing (HTS) of soil environmental DNA provides an advanced insight into the effects of pesticides on soil microbial systems. However, the association between the properties of the pesticide and its ecological impact remains methodically challenging. Risks associated with pesticide use can be minimized if pesticides with optimal structural traits were applied. For this purpose, we merged the 20 independent HTS studies, to reveal that pesticides significantly reduced beneficial bacteria associated with soil and plant immunity, enhanced the human pathogen and weaken the soil's ecological stability. Through the machine-learning approach, correlating these impacts with the physicochemical properties of the pesticides yielded a random forest model with good predictive capabilities. The models revealed that physical pesticide properties such as the dissociation constant (pKa), the molecular weight and water solubility, determined the ecological impact of pesticides to a large extent. Moreover, this study identified that eco-friendly pesticides should possess a value of pKa > 5 and a molecular weight in the range of 200-300 g/mol, which were found to be conducive to bacteria related to plant immunity promotion and exerted the lowest fluctuation of human opportunistic pathogen and keystone species. This guides the design of pesticides for which the impacts on soil biota are minimized.

Microbial interaction promotes desulfurization efficiency under high pH condition.

The existence of H2S in biogas may cause equipment corrosion and considerable SO2 emission. Commonly used biotrickling filters may cause biogas dilution or generation of explosive mixtures. Compared with biotrickling filters, two-step process such as bioscrubber filters can overcome these shortages. However, its removal efficiency was still limited due to low microbial activity under high pH condition. Here, a bioreactor filter was carried out under pH 9.0. Removal efficiency higher than 99% was achieved under sulfide loading rate reaching 4.24 kg S m-3d-1. Results of network and high throughput sequencing showed that Thiobacillus acted as both dominant species (accounting for 75%) and unique kinless hub in this bioreactor. Other bacteria (accounting for 25%) contributed 75% to the network, which implied the intensive interaction between Thiobacillus and others. Sulfide removal ability and pH tolerance of pure bacteria and mixed culture were considered to verify how microbial interaction influenced them. Compared with pure bacteria, mixed culture had better performance under high pH condition, which confirmed that microbial interaction promoted desulfurization efficiency under high pH condition. These results showed that intensive microbial interaction might be the key to enhance sulfide removal efficiency under high pH condition.

Pollutant toxicology with respect to microalgae and cyanobacteria.

Microalgae and cyanobacteria are fundamental components of aquatic ecosystems. Pollution in aquatic environment is a worldwide problem. Toxicological research on microalgae and cyanobacteria can help to establish a solid foundation for aquatic ecotoxicological assessments. Algae and cyanobacteria occupy a large proportion of the biomass in aquatic environments; thus, their toxicological responses have been investigated extensively. However, the depth of toxic mechanisms and breadth of toxicological investigations need to be improved. While existing pollutants are being discharged into the environment daily, new ones are also being produced continuously. As a result, the phenomenon of water pollution has become unprecedentedly complex. In this review, we summarize the latest findings on five kinds of aquatic pollutants, namely, metals, nanomaterials, pesticides, pharmaceutical and personal care products (PPCPs), and persistent organic pollutants (POPs). Further, we present information on emerging pollutants such as graphene, microplastics, and ionic liquids. Efforts in studying the toxicological effects of pollutants on microalgae and cyanobacteria must be increased in order to better predict the potential risks posed by these materials to aquatic ecosystems as well as human health.

Formation of biofilms from new pipelines at both ends of the drinking water distribution system and comparison of disinfection by-products formation potential.

The gradual updating of the water supply network is one of the crucial ways to ensure the safety of drinking water all over the world. The phenomenon and regularity of the biological risk and chemical risk of biofilms of the new pipes in drinking water distribution system (DWDS) is inadequate researched by now. In order to explore the biochemical risks of biofilms after new pipes are used, this paper studied the growth of biofilms, the content of disinfection by-products (DBPs) and the potential for disinfection by-products (DBPsFP) after 2-year use by establishing a pilot test platform at both ends of the DWDS in City S. The results showed that the total bacterial count in new pipelines was between 1.38 × 108-9.97 × 108/cm2; the DBPsFP at the front end and at the back end was subtly different. The overall DBPsFP of biofilms was the highest, followed by the ductile cast iron pipe and the galvanized pipe whereas the stainless steel pipe was the lowest. The HPC content of the 2-year-old pipe (1.68 × 105-7.09 × 106 CFU/cm2) was remarkably higher than that of the 1-year-old pipe (1.04 × 105-8.76 × 105 CFU/cm2), and the generation DBPsFP was about 50% higher. When a new pipeline was put into use in the urban drinking water distribution system, biofilms with certain biological hazards and risks of DBPs disinfection by-products would form in a short period of time.

Temporal discrepancy of airborne total bacteria and pathogenic bacteria between day and night.

As the most abundant microbes in the atmosphere, airborne bacteria are closely involved in affecting human health, regional climate and ecological balance. The mobility of airborne microorganisms makes it necessary to study the community dynamic in short cycle. Nevertheless, it remains obscure how the airborne bacteria especially the pathogenic bacteria vary on the small time scale of day and night. To investigate the nycterohemeral discrepancy of airborne total bacteria and pathogenic bacteria, PM2.5 samples were collected in Hangzhou between day and night. Microbial taxonomic information was obtained through 16S rRNA gene sequencing and "human pathogens database" screening. Further analyses based on Multiple Regression Matrices (MRM) approach and Concentration Weighted Trajectory (CWT) model were conducted to elucidate the effect of local environmental factors and long-range transport. The community composition of total bacteria tended to be similar in the daytime while pathogenic bacteria turned out to be homogeneous in the nighttime. To be vigilant, the diversity of airborne pathogenic bacteria echoed the frequency of anthropogenic activities with the pathogen inhalation rate roughly at 428 copies/h and 235 copies/h respectively in daytime and nighttime. The nycterohemeral discrepancy of total bacteria was principally driven by the filtering of environmental factors, i.e., CO and NO2, indicating that anthropogenic activities brought about the homogeneity. Airborne pathogenic bacteria coupled with the strong resistances of environmental filtering stood out from their non-pathogenic counterpart, which enabled the long-range transport. Indeed, the nycterohemeral discrepancy of pathogenic bacteria was shaped by the transport of air masses. This research filled the gaps in temporal variance of airborne microorganisms on the small time scale of day and night, providing crucial foundation for precisely predicting ecological and health effects of bioaerosols.

Effects of environmental factors on the removal of heavy metals by sulfide-modified nanoscale zerovalent iron.

Sulfide-modified nanoscale zerovalent iron (S-nZVI) has excellent reducing performance for heavy metals in water. The influence of environmental factors on the reactivity can be used to explore the practical feasibility of S-nZVI and analyze the reaction mechanism in depth. This study compared the removal effect and mechanism of Cu2+ and Ni2+ by nanoscale zerovalent iron (nZVI), S-nZVI, and carboxymethyl cellulose-modified nanoscale zerovalent iron (CMC-nZVI). The results show that the pseudo-first-order kinetic constant of Cu2+ removal by nZVI, S-nZVI, and CMC-nZVI was 1.384, 1.919, and 2.890 min-1, respectively, and the rate of Ni2+ removal was 0.304, 0.931, and 0.360 min-1, respectively. The removal mechanism of S-nZVI was similar to that of nZVI and CMC-nZVI. Specifically, Cu2+ was predominantly removed by reduction, while Ni2+ removal included adsorption and reduction. Environmental factors had a specific inhibitory effect on the removal of Cu2+ but had a negligible impact on Ni2+. The condition of low pH, the presence of Cl- and humic acid (HA) promoted the corrosion consumption of Fe0, in which H+ directly corroded Fe0 at low pH. At the same time, Cl- and HA inhibited the adsorption or binding of heavy metal ions on the particle surface, thereby reducing the electron transfer and utilization efficiency. The passivation of NO3- reduced the anaerobic corrosion of the material in water but suppressed the release of electrons, thereby reducing the reduction efficiency of the three types of materials. The anaerobic corrosion of S-nZVI was less affected by environmental factors, and it can still maintain more than 80% of the electronic utilization efficiency under different environmental factors, which illustrates that S-nZVI has broad prospects for practical applications in heavy metal polluted water.

Spatial-Temporal Pattern of Sulfate-Dependent Anaerobic Methane Oxidation in an Intertidal Zone of the East China Sea.

Methane is a primary greenhouse gas which is responsible for global warming. The sulfate-dependent anaerobic methane oxidation (S-AOM) process catalyzed by anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB) is a vital link connecting the global carbon and sulfur cycles, and it is considered to be the overriding methane sink in marine ecosystem. However, there have been few studies regarding the role of S-AOM process and the distribution of ANME archaea in intertidal ecosystem. The intertidal zone is a buffer zone between sea and land and plays an important role in global geochemical cycle. In the present study, the abundance, potential methane oxidation rate, and community structure of ANME archaea in the intertidal zone were studied by quantitative PCR, stable isotope tracing method and high-throughput sequencing. The results showed that the potential S-AOM activity ranged from 0 to 0.77 nmol 13CO2 g-1 (dry sediment) day-1 The copy number of 16S rRNA gene of ANME archaea reached 106 ∼ 107 copies g-1 (dry sediment). The average contribution of S-AOM to total anaerobic methane oxidation was up to 34.5%, while denitrifying anaerobic methane oxidation accounted for the rest, which implied that S-AOM process was an essential methane sink that cannot be overlooked in intertidal ecosystem. The simulated column experiments also indicated that ANME archaea were sensitive to oxygen and preferred anaerobic environmental conditions. This study will help us gain a better understanding of the global carbon-sulfur cycle and greenhouse gas emission reduction and introduce a new perspective into the enrichment of ANME archaea.IMPORTANCE The sulfate-dependent anaerobic methane oxidation (S-AOM) process catalyzed by anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB) is a vital link connecting the global carbon and sulfur cycles. We conducted a research into the spatial-temporal pattern of S-AOM process and the distribution of ANME archaea in coastal sediments collected from the intertidal zone. The results implied that S-AOM process was a methane sink that cannot be overlooked in the intertidal ecosystem. We also found that ANME archaea were sensitive to oxygen and preferred anaerobic environmental conditions. This study will help us gain a better understanding of the global carbon-sulfur cycle and greenhouse gas emission reduction and introduce a new perspective into the enrichment of ANME archaea.

Ecological Success of the Nitrosopumilus and Nitrosospira Clusters in the Intertidal Zone.

The intertidal zone is an important buffer and a nitrogen sink between land and sea. Ammonia oxidation is the rate-limiting step of nitrification, conducted by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). However, it remains a debatable issue regarding dominant ammonia oxidizers in this region, and environmental factors driving their spatiotemporal niche differentiation have yet to be identified. In this study, intertidal and subtidal zones of Zhoushan Islands were selected for seasonal sampling. Ammonia-oxidizing activity, quantitative PCR, and 454 high-throughput sequencing were performed to study the nitrification potential, abundance, and community structure of ammonia-oxidizing archaea and bacteria. AOA and AOB amoA abundance (107-108amoA gene copies/g dry weight sediment) varied spatiotemporally independently of environmental factors. AOA surpassed AOB in most samples, driven by sediment temperature, moisture, and total nitrogen. The diversity of both AOA and AOB differed spatiotemporally. The Nitrosopumilus and Nitrosospira clusters accounted for an absolutely dominant percentage of AOA (> 99%) and AOB (> 99%) respectively, indicating a negligible contribution of other clusters to ammonia oxidation. However, there was no significant correlation between nitrification potential and the abundance of AOA or AOB. Overall, the present study showed that AOA dominated over AOB spatiotemporally in the intertidal zone of Zhoushan Islands due to fluctuations in environmental factors, and the Nitrosopumilus and Nitrosospira clusters ecologically succeeded in the intertidal zone of Zhoushan Islands.

Denitrifying Anaerobic Methane Oxidation: A Previously Overlooked Methane Sink in Intertidal Zone.

The intertidal zone is an open ecosystem rich in organic matter and plays an important role in global biogeochemical cycles. It was previously considered that methane was mainly removed by sulfate-dependent anaerobic methane oxidation (sulfate-AOM) process in marine ecosystems while other anaerobic methane oxidation processes were ignored. Recent researches have demonstrated that denitrifying anaerobic methane oxidation (DAMO), consisting of nitrite-dependent anaerobic methane oxidation (nitrite-AOM) and nitrate-dependent anaerobic methane oxidation (nitrate-AOM), can also oxidize methane. In this work, the community structure, quantity and potential methane oxidizing rate of DAMO archaea and bacteria in the intertidal zone were studied by high-throughput sequencing, qPCR and stable isotope tracing method. The results showed that nitrate-AOM and nitrite-AOM were both active in the intertidal zone and showed approximate methane oxidation rates. The copy number of 16S rRNA gene of DAMO archaea and DAMO bacteria were 104 ∼ 105 copies g-1 (dry sediment), whereas NC10 bacteria were slightly higher. The contribution rate of DAMO process to total anaerobic methane removal in the intertidal zone reached 65.6% ∼ 100%, which indicates that DAMO process is an important methane sink in intertidal ecosystem. Laboratory incubations also indicated that DAMO archaea were more sensitive to oxygen and preferred a more anoxic environment. These results help us draw a more complete picture of methane and nitrogen cycles in natural habitats.

Spatial and temporal distribution of nitrite-dependent anaerobic methane-oxidizing bacteria in an intertidal zone of the East China Sea.

Nitrite-dependent anaerobic methane oxidation (N-DAMO), which couples anaerobic methane oxidation and nitrite reduction, is a recently discovered bioprocess coupling microbial nitrogen and carbon cycles. The discovery of this microbial process challenges the traditional knowledge of global methane sinks and nitrogen losses. In this study, the abundance and activity of N-DAMO bacteria were investigated and their contributions to methane sink and nitrogen loss were estimated in different seasons and different partitions of an intertidal zone of the East China Sea. The results showed that N-DAMO bacteria were extensively and continuously present in the intertidal zone, with the number of cells ranging from 5.5 × 104 to 2.8 × 105 copy g-1 soil and the potential activity ranging from 0.52 to 5.7 nmol CO2 g-1 soil day-1, contributing 5.0-36.6% of nitrite- and sulfate-dependent anaerobic methane oxidation in the intertidal zone. The N-DAMO activity and its contribution to the methane consumption were highest in the spring and in the low intertidal zone. These findings showed that the N-DAMO process is an important methane and nitrogen sink in the intertidal zone and varies with the seasons and the partitions of the intertidal zone.

Bacterial community radial-spatial distribution in biofilms along pipe wall in chlorinated drinking water distribution system of East China.

Biofilms in the pipe wall may lead to water quality deterioration and biological instability in drinking water distribution systems (DWDSs). In this study, bacterial community radial-spatial distribution in biofilms along the pipe wall in a chlorinated DWDS of East China was investigated. Three pipes of large diameter (300, 600, and 600 mm) were sampled in this DWDS, including a ductile cast iron pipe (DCIP) with pipe age of 11 years and two gray cast iron pipes (GCIP) with pipe ages of 17 and 19 years, and biofilms in the upper, middle, and lower parts of each pipe wall were collected. Real-time quantitative polymerase chain reaction (qPCR) and culture-based method were used to quantify bacteria. 454 pyrosequencing was used for bacterial community analysis. The results showed that the biofilm density and total solid (TS) and volatile solid (VS) contents increased gradually from the top to the bottom along the pipe wall. Microorganisms were concentrated in the upper and lower parts of the pipe wall, together accounting for more than 80 % of the total biomass in the biofilms. The bacterial communities in biofilms were significantly different in different areas of the pipe wall and had no strong interaction. Compared with the upper and lower parts of the pipe wall, the bacterial community in the middle of the pipe wall was distributed evenly and had the highest diversity. The 16S rRNA genes of various possible pathogens, including Escherichia coli, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Salmonella enterica, were detected in the biofilms, and the abundances of these possible pathogens were highest in the middle of the pipe wall among three areas. The detachment of the biofilms is the main reason for the deterioration of the water quality in DWDSs. The results of this study suggest that the biofilms in the middle of the pipe wall have highly potential risk for drinking water safety, which provides new ideas for the study of the microbial ecology in DWDS.

Evidence for nitrite-dependent anaerobic methane oxidation as a previously overlooked microbial methane sink in wetlands.

The process of nitrite-dependent anaerobic methane oxidation (n-damo) was recently discovered and shown to be mediated by "Candidatus Methylomirabilis oxyfera" (M. oxyfera). Here, evidence for n-damo in three different freshwater wetlands located in southeastern China was obtained using stable isotope measurements, quantitative PCR assays, and 16S rRNA and particulate methane monooxygenase gene clone library analyses. Stable isotope experiments confirmed the occurrence of n-damo in the examined wetlands, and the potential n-damo rates ranged from 0.31 to 5.43 nmol CO2 per gram of dry soil per day at different depths of soil cores. A combined analysis of 16S rRNA and particulate methane monooxygenase genes demonstrated that M. oxyfera-like bacteria were mainly present in the deep soil with a maximum abundance of 3.2 × 10(7) gene copies per gram of dry soil. It is estimated that ∼0.51 g of CH4 m(-2) per year could be linked to the n-damo process in the examined wetlands based on the measured potential n-damo rates. This study presents previously unidentified confirmation that the n-damo process is a previously overlooked microbial methane sink in wetlands, and n-damo has the potential to be a globally important methane sink due to increasing nitrogen pollution.

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 methane oxidation coupled to nitrite reduction can be a potential methane sink in coastal environments.

In the current study, we investigated nitrite-dependent anaerobic methane oxidation (N-DAMO) as a potential methane sink in the Hangzhou Bay and the adjacent Zhoushan sea area. The potential activity of the N-DAMO process was primarily observed in Hangzhou Bay by means of (13)C-labeling experiments, whereas very low or no potential N-DAMO activity could be detected in the Zhoushan sea area. The measured potential N-DAMO rates ranged from 0.2 to 1.3 nmol (13)CO2 g(-1) (dry sediment) day(-1), and the N-DAMO potentially contributed 2.0-9.4 % to the total microbial methane oxidation in the examined sediments. This indicated that the N-DAMO process may be an alternative pathway in the coastal methane cycle. Phylogenetic analyses confirmed the presence of Candidatus Methylomirabilis oxyfera-like bacteria in all the examined sediments, while the group A members (the dominant bacteria responsible for N-DAMO) were found mainly in Hangzhou Bay. Quantitative PCR showed that the 16S rRNA gene abundance of Candidatus M. oxyfera-like bacteria varied from 5.4 × 10(6) to 5.0 × 10(7) copies g(-1) (dry sediment), with a higher abundance observed in Hangzhou Bay. In addition, the overlying water NO3 (-) concentration and salinity were identified as the most important factors influencing the abundance and potential activity of Candidatus M. oxyfera-like bacteria in the examined sediments. This study showed the evidence of N-DAMO in coastal environments and indicated the importance of N-DAMO as a potential methane sink in coastal environments.

Anaerobic Oxidation of Methane Coupled to Nitrite Reduction by Halophilic Marine NC10 Bacteria.

Anaerobic oxidation of methane (AOM) coupled to nitrite reduction is a novel AOM process that is mediated by denitrifying methanotrophs. To date, enrichments of these denitrifying methanotrophs have been confined to freshwater systems; however, the recent findings of 16S rRNA and pmoA gene sequences in marine sediments suggest a possible occurrence of AOM coupled to nitrite reduction in marine systems. In this research, a marine denitrifying methanotrophic culture was obtained after 20 months of enrichment. Activity testing and quantitative PCR (qPCR) analysis were then conducted and showed that the methane oxidation activity and the number of NC10 bacteria increased correlatively during the enrichment period. 16S rRNA gene sequencing indicated that only bacteria in group A of the NC10 phylum were enriched and responsible for the resulting methane oxidation activity, although a diverse community of NC10 bacteria was harbored in the inoculum. Fluorescence in situ hybridization showed that NC10 bacteria were dominant in the enrichment culture after 20 months. The effect of salinity on the marine denitrifying methanotrophic culture was investigated, and the apparent optimal salinity was 20.5‰, which suggested that halophilic bacterial AOM coupled to nitrite reduction was obtained. Moreover, the apparent substrate affinity coefficients of the halophilic denitrifying methanotrophs were determined to be 9.8 ± 2.2 µM for methane and 8.7 ± 1.5 µM for nitrite.