Temperature differentially regulates estuarine microbial N2O production along a salinity gradient.

Nitrous oxide (N2O) is atmospheric trace gas that contributes to climate change and affects stratospheric and ground-level ozone concentrations. Ammonia oxidizers and denitrifiers contribute to N2O emissions in estuarine waters. However, as an important climate factor, how temperature regulates microbial N2O production in estuarine water remains unclear. Here, we have employed stable isotope labeling techniques to demonstrate that the N2O production in estuarine waters exhibited differential thermal response patterns between nearshore and offshore regions. The optimal temperatures (Topt) for N2O production rates (N2OR) were higher at nearshore than offshore sites. 15N-labeled nitrite (15NO2-) experiments revealed that at the nearshore sites dominated by ammonia-oxidizing bacteria (AOB), the thermal tolerance of 15N-N2OR increases with increasing salinity, suggesting that N2O production by AOB-driven nitrifier denitrification may be co-regulated by temperature and salinity. Metatranscriptomic and metagenomic analyses of enriched water samples revealed that the denitrification pathway of AOB is the primary source of N2O, while clade II N2O-reducers dominated N2O consumption. Temperature regulated the expression patterns of nitrite reductase (nirK) and nitrous oxide reductase (nosZ) genes from different sources, thereby influencing N2O emissions in the system. Our findings contribute to understanding the sources of N2O in estuarine waters and their response to global warming.

Four years of climate warming reduced dark carbon fixation in coastal wetlands.

Dark carbon fixation (DCF), conducted mainly by chemoautotrophs, contributes greatly to primary production and the global carbon budget. Understanding the response of DCF process to climate warming in coastal wetlands is of great significance for model optimization and climate change prediction. Here, based on a 4-yr field warming experiment (average annual temperature increase of 1.5°C), DCF rates were observed to be significantly inhibited by warming in coastal wetlands (average annual DCF decline of 21.6%, and estimated annual loss of 0.08-1.5 Tg C yr-1 in global coastal marshes), thus causing a positive climate feedback. Under climate warming, chemoautotrophic microbial abundance and biodiversity, which were jointly affected by environmental changes such as soil organic carbon and water content, were recognized as significant drivers directly affecting DCF rates. Metagenomic analysis further revealed that climate warming may alter the pattern of DCF carbon sequestration pathways in coastal wetlands, increasing the relative importance of the 3-hydroxypropionate/4-hydroxybutyrate cycle, whereas the relative importance of the dominant chemoautotrophic carbon fixation pathways (Calvin-Benson-Bassham cycle and W-L pathway) may decrease due to warming stress. Collectively, our work uncovers the feedback mechanism of microbially mediated DCF to climate warming in coastal wetlands, and emphasizes a decrease in carbon sequestration through DCF activities in this globally important ecosystem under a warming climate.

Global Atlas of Methane Metabolism Marker Genes in Soil.

Methane, a greenhouse gas, plays a pivotal role in the global carbon cycle, influencing the Earth's climate. Only a limited number of microorganisms control the flux of biologically produced methane in nature, including methane-oxidizing bacteria, anaerobic methanotrophic archaea, and methanogenic archaea. Although previous studies have revealed the spatial and temporal distribution characteristics of methane-metabolizing microorganisms in local regions by using the marker genes pmoA or mcrA, their biogeographical patterns and environmental drivers remain largely unknown at a global scale. Here, we used 3419 metagenomes generated from georeferenced soil samples to examine the global patterns of methane metabolism marker gene abundances in soil, which generally represent the global distribution of methane-metabolizing microorganisms. The resulting maps revealed notable latitudinal trends in the abundances of methane-metabolizing microorganisms across global soils, with higher abundances in the sub-Arctic, sub-Antarctic, and tropical rainforest regions than in temperate regions. The variations in global abundances of methane-metabolizing microorganisms were primarily governed by vegetation cover. Our high-resolution global maps of methane-metabolizing microorganisms will provide valuable information for the prediction of biogenic methane emissions under current and future climate scenarios.

Acidification Offset Warming-Induced Increase in N2O Production in Estuarine and Coastal Sediments.

Global warming and acidification, induced by a substantial increase in anthropogenic CO2 emissions, are expected to have profound impacts on biogeochemical cycles. However, underlying mechanisms of nitrous oxide (N2O) production in estuarine and coastal sediments remain rarely constrained under warming and acidification. Here, the responses of sediment N2O production pathways to warming and acidification were examined using a series of anoxic incubation experiments. Denitrification and N2O production were largely stimulated by the warming, while N2O production decreased under the acidification as well as the denitrification rate and electron transfer efficiency. Compared to warming alone, the combination of warming and acidification decreased N2O production by 26 ± 4%, which was mainly attributed to the decline of the N2O yield by fungal denitrification. Fungal denitrification was mainly responsible for N2O production under the warming condition, while bacterial denitrification predominated N2O production under the acidification condition. The reduced site preference of N2O under acidification reflects that the dominant pathways of N2O production were likely shifted from fungal to bacterial denitrification. In addition, acidification decreased the diversity and abundance of nirS-type denitrifiers, which were the keystone taxa mediating the low N2O production. Collectively, acidification can decrease sediment N2O yield through shifting the responsible production pathways, partly counteracting the warming-induced increase in N2O emissions, further reducing the positive climate warming feedback loop.

Active dark carbon fixation evidenced by 14C isotope assimilation and metagenomic data across the estuarine-coastal continuum.

Estuaries, as important land-ocean transitional zones across the Earth's surface, are hotspots of microbially driven dark carbon fixation (DCF), yet understanding of DCF process remains limited across the estuarine-coastal continuum. This study explored DCF activities and associated chemoautotrophs along the estuarine and coastal environmental gradients, using radiocarbon labelling and molecular techniques. Significantly higher DCF rates were observed at middle- and high-salinity regions (0.65-2.31 and 0.66-2.82 mmol C m-2 d-1, respectively), compared to low-salinity zone (0.07-0.19 mmol C m-2 d-1). Metagenomic analysis revealed relatively stable DCF pathways along the estuarine-coastal continuum, primarily dominated by Calvin-Benson-Bassham (CBB) cycle and Wood-Ljungdahl (WL) pathway. Nevertheless, chemoautotrophic communities driving DCF exhibited significant spatial variations. It is worth noting that although CBB cycle played an important role in DCF in estuarine sediments, WL pathway might play a more significant role, which has not been previously recognized. Overall, this study highlights that DCF activities coincide with the genetic potential of chemoautotrophy and the availability of reductive substrates across the estuarine-coastal continuum, and provides an important scientific basis for accurate quantitative assessment of global estuarine carbon sink.

Role of n-DAMO in Mitigating Methane Emissions from Intertidal Wetlands Is Regulated by Saltmarsh Vegetations.

Coastal wetlands are hotspots for methane (CH4) production, reducing their potential for global warming mitigation. Nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) plays a crucial role in bridging carbon and nitrogen cycles, contributing significantly to CH4 consumption. However, the role of n-DAMO in reducing CH4 emissions in coastal wetlands is poorly understood. Here, the ecological functions of the n-DAMO process in different saltmarsh vegetation habitats as well as bare mudflats were quantified, and the underlying microbial mechanisms were explored. Results showed that n-DAMO rates were significantly higher in vegetated habitats (Scirpus mariqueter and Spartina alterniflora) than those in bare mudflats (P < 0.05), leading to an enhanced contribution to CH4 consumption. Compared with other habitats, the contribution of n-DAMO to the total anaerobic CH4 oxidation was significantly lower in the Phragmites australis wetland (15.0%), where the anaerobic CH4 oxidation was primarily driven by ferric iron (Fe3+). Genetic and statistical analyses suggested that the different roles of n-DAMO in various saltmarsh wetlands may be related to divergent n-DAMO microbial communities as well as environmental parameters such as sediment pH and total organic carbon. This study provides an important scientific basis for a more accurate estimation of the role of coastal wetlands in mitigating climate change.

Natural chalcopyrite mitigates nitrous oxide emissions in sediment from coastal wetlands.

Coastal wetlands are one of the most important natural sources of nitrous oxide (N2O). Previous studies have shown that copper-containing chemicals are able to reduce N2O emissions from these ecosystems. However, these chemicals may harm organisms present in coastal waters and sediment, and disturb the ecological balance of these areas. Here, we first investigated the physiological characteristics and genetic potential of denitrifying bacteria isolated from coastal wetlands. Based on an isolated denitrifier carrying a complete denitrification pathway, we tested the effect of the natural mineral chalcopyrite on N2O production by the bacteria. The results demonstrated that chalcopyrite addition lowers N2O emissions from the bacteria while increasing its N2 production rate. Among the four denitrification genes of the isolate, only nosZ gene expression was significantly upregulated following the addition of 2 mg L-1 chalcopyrite. Furthermore, chalcopyrite was applied to coastal wetland sediments. The N2O flux was significantly reduced in 50-100 mg L-1 chalcopyrite-amended sets relative to the controls. Notably, the dissolved Cu concentration in chalcopyrite-amended sediment remained within the limit set by the National Sewage Treatment Discharge Standard. qPCR and metagenomic analysis revealed that the abundance of N2O-reducing bacteria with the nosZ or nirK + nosZ genotype increased significantly in the chalcopyrite-amended groups relative to the controls, suggesting their active involvement in the reduction of N2O emissions. Our findings offer valuable insights for the use of natural chalcopyrite in large-scale field applications to reduce N2O emissions.

Invasive Spartina alterniflora accelerates the increase in microbial nitrogen fixation over nitrogen removal in coastal wetlands of China.

Salt marsh plants play a vital role in mediating nitrogen (N) biogeochemical cycle in estuarine and coastal ecosystems. However, the effects of invasive Spartina alterniflora on N fixation and removal, as well as how these two processes balance to determine the N budget, remain unclear. Here, simultaneous quantifications of N fixation and removal via 15N tracing experiment with native Phragmites australis, invasive S. alterniflora, and bare flats as well as corresponding functional gene abundance by qPCR were carried out to explore the response of N dynamics to S. alterniflora invasion. Our results showed that N fixation and removal rates ranged from 0.77 ± 0.08 to 16.12 ± 1.13 nmol/(g·h) and from 1.42 ± 0.14 to 16.35 ± 1.10 nmol/(g·h), respectively, and invasive S. alterniflora generally facilitated the two processes rates. Based on the difference between N removal and fixation rates, net N2 fluxes were estimated in the range of -0.39 ± 0.14 to 8.24 ± 2.23 nmol/(g·h). Estimated net N2 fluxes in S. alterniflora stands were lower than those in bare flats and P. australis stands, indicating that the increase in N removal caused by S. alterniflora invasion may be more than offset by N fixation process. Random forest analysis revealed that functional microorganisms were the most important factor associated with the corresponding N transformation process. Overall, our results highlight the importance of N fixation in evaluating N budget of estuarine and coastal wetlands, providing valuable insights into the ecological effect of S. alterniflora invasion.

Active pathways of anaerobic methane oxidization in deep-sea cold seeps of the South China Sea.

IMPORTANCE: Cold seeps occur in continental margins worldwide and are deep-sea oases. Anaerobic oxidation of methane is an important microbial process in the cold seeps and plays an important role in regulating methane content. This study elucidates the diversity and potential activities of major microbial groups in dependent anaerobic methane oxidation and sulfate-dependent anaerobic methane oxidation processes and provides direct evidence for the occurrence of nitrate-/nitrite-dependent anaerobic methane oxidation (Nr-/N-DAMO) as a previously overlooked microbial methane sink in the hydrate-bearing sediments of the South China Sea. This study provides direct evidence for occurrence of Nr-/N-DAMO as an important methane sink in the deep-sea cold seeps.

Tidal dynamics regulates potential coupling of carbon­nitrogen­sulfur cycling microbes in intertidal flats.

Tide-driven hydrodynamic process causes significant geochemical gradients that influence biogeochemical cycling and ecological functioning of estuarine and coastal ecosystems. However, the effects of tidal dynamics on microbial communities, particularly at the functional gene level, remain unclear even though microorganisms play critical roles in biogeochemical carbon (C), nitrogen (N) and sulfur (S) cycling. Here, we used 16S rRNA gene amplicon sequencing and microarray-based approach to reveal the stratification of microorganisms related to C, N and S cycles along vertical redox gradients in intertidal wetlands. Alpha-diversity of bacteria and archaea was generally higher at the deep groundwater-sediment interface. Microbial compositions were markedly altered along the sediment profile, and these shifts were largely due to changes in nutrient availability and redox potential. Furthermore, functional genes exhibited redox partitioning between interfaces and transition layer, with abundant genes involved in C decomposition, methanogenesis, heterotrophic denitrification, sulfite reduction and sulfide oxidation existed in the middle anoxic zone. The influence of tidal dynamics on sediment function was highly associated with redox state, sediment texture, and substrates availability, leading to distinct distribution pattern of metabolic coupling of microbes involved in energy flux and elemental cycling in intertidal wetlands. These results indicate that tidal cycles are critical in determining microbial community and functional structure, and they provide new insights into sediment microbe-mediated biogeochemical cycling in intertidal habitats.

Novel Gene Clusters for Natural Product Synthesis Are Abundant in the Mangrove Swamp Microbiome.

Natural microbial communities produce a diverse array of secondary metabolites with ecologically and biotechnologically relevant activities. Some of them have been used clinically as drugs, and their production pathways have been identified in a few culturable microorganisms. However, since the vast majority of microorganisms in nature have not been cultured, identifying the synthetic pathways of these metabolites and tracking their hosts remain a challenge. The microbial biosynthetic potential of mangrove swamps remains largely unknown. Here, we examined the diversity and novelty of biosynthetic gene clusters in dominant microbial populations in mangrove wetlands by mining 809 newly reconstructed draft genomes and probing the activities and products of these clusters by using metatranscriptomic and metabolomic techniques. A total of 3,740 biosynthetic gene clusters were identified from these genomes, including 1,065 polyketide and nonribosomal peptide gene clusters, 86% of which showed no similarity to known clusters in the Minimum Information about a Biosynthetic Gene Cluster (MIBiG) repository. Of these gene clusters, 59% were harbored by new species or lineages of Desulfobacterota-related phyla and Chloroflexota, whose members are highly abundant in mangrove wetlands and for which few synthetic natural products have been reported. Metatranscriptomics revealed that most of the identified gene clusters were active in field and microcosm samples. Untargeted metabolomics was also used to identify metabolites from the sediment enrichments, and 98% of the mass spectra generated were unrecognizable, further supporting the novelty of these biosynthetic gene clusters. Our study taps into a corner of the microbial metabolite reservoir in mangrove swamps, providing clues for the discovery of new compounds with valuable activities. IMPORTANCE At present, the majority of known clinical drugs originated from cultivated species of a few bacterial lineages. It is vital for the development of new pharmaceuticals to explore the biosynthetic potential of naturally uncultivable microorganisms using new techniques. Based on the large numbers of genomes reconstructed from mangrove wetlands, we identified abundant and diverse biosynthetic gene clusters in previously unsuspected phylogenetic groups. These gene clusters exhibited a variety of organizational architectures, especially for nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), implying the presence of new compounds with valuable activities in the mangrove swamp microbiome.

Potential response of dark carbon fixation to global warming in estuarine and coastal waters.

Dark carbon fixation (DCF), through which chemoautotrophs convert inorganic carbon to organic carbon, is recognized as a vital process of global carbon biogeochemical cycle. However, little is known about the response of DCF processes in estuarine and coastal waters to global warming. Using radiocarbon labelling method, the effects of temperature on the activity of chemoautotrophs were investigated in benthic water of the Yangtze estuarine and coastal areas. A dome-shaped thermal response pattern was observed for DCF rates (i.e., reduced rates at lower or higher temperatures), with the optimum temperature (Topt ) varying from about 21.9 to 32.0°C. Offshore sites showed lower Topt values and were more vulnerable to global warming compared with nearshore sites. Based on temperature seasonality of the study area, it was estimated that warming would accelerate DCF rate in winter and spring but inhibit DCF activity in summer and fall. However, at an annual scale, warming showed an overall promoting effect on DCF rates. Metagenomic analysis revealed that the dominant chemoautotrophic carbon fixation pathways in the nearshore area were Calvin-Benson-Bassham (CBB) cycle, while the offshore sites were co-dominated by CBB and 3-hydroxypropionate/4-hydroxybutyrate cycles, which may explain the differential temperature response of DCF along the estuarine and coastal gradients. Our findings highlight the importance of incorporating DCF thermal response into biogeochemical models to accurately estimate the carbon sink potential of estuarine and coastal ecosystems in the context of global warming.

Microbial necromass carbon in estuarine tidal wetlands of China: Influencing factors and environmental implication.

Microbial necromass is an important component of the stable soil organic carbon (SOC) pool. However, little is known about the spatial and seasonal patterns of soil microbial necromass and their influencing environmental factors in estuarine tidal wetlands. In the present study, amino sugars (ASs) as biomarkers of microbial necromass were investigated along the estuarine tidal wetlands of China. Microbial necromass carbon (C) contents were in the range of 1.2-6.7 mg g-1 (3.6 ± 2.2 mg g-1, n = 41) and 0.5-4.4 mg g-1 (2.3 ± 1.5 mg g-1, n = 41), which accounted for 17.3-66.5 % (44.8 % ± 16.8 %) and 8.9-45.0 % (31.0 % ± 13.7 %) of the SOC pool in the dry (March to April) and wet (August to September) seasons, respectively. At all sampling sites, fungal necromass C predominated over bacterial necromass C as a component of microbial necromass C. Compared to bacterial necromass C, fungal necromass C showed a stronger connection with ferrous oxides (Fe2+) and total Fe concentrations. Both fungal and bacterial necromass C contents revealed large spatial heterogeneity and declined in the estuarine tidal wetlands with the increase in latitude. Statistical analyses showed that the increases in salinity and pH in the estuarine tidal wetlands suppressed the accumulation of soil microbial necromass C.

Effects of acidification on nitrification and associated nitrous oxide emission in estuarine and coastal waters.

In the context of an increasing atmospheric carbon dioxide (CO2) level, acidification of estuarine and coastal waters is greatly exacerbated by land-derived nutrient inputs, coastal upwelling, and complex biogeochemical processes. A deeper understanding of how nitrifiers respond to intensifying acidification is thus crucial to predict the response of estuarine and coastal ecosystems and their contribution to global climate change. Here, we show that acidification can significantly decrease nitrification rate but stimulate generation of byproduct nitrous oxide (N2O) in estuarine and coastal waters. By varying CO2 concentration and pH independently, an expected beneficial effect of elevated CO2 on activity of nitrifiers ("CO2-fertilization" effect) is excluded under acidification. Metatranscriptome data further demonstrate that nitrifiers could significantly up-regulate gene expressions associated with intracellular pH homeostasis to cope with acidification stress. This study highlights the molecular underpinnings of acidification effects on nitrification and associated greenhouse gas N2O emission, and helps predict the response and evolution of estuarine and coastal ecosystems under climate change and human activities.

Increased Nitrogen Loading Facilitates Nitrous Oxide Production through Fungal and Chemodenitrification in Estuarine and Coastal Sediments.

Estuarine and coastal environments are assumed to contribute to nitrous oxide (N2O) emissions under increasing nitrogen loading. However, isotopic and molecular mechanisms underlying N2O production pathways under elevated nitrogen concentration remain poorly understood. Here we used microbial inhibition, isotope mass balance, and molecular approaches to investigate N2O production mechanisms in estuarine and coastal sediments through a series of anoxic incubations. Site preference of the N2O molecule increased due to increasing nitrate concentration, suggesting the changes in N2O production pathways. Enhanced N2O production under high nitrate concentration was not mediated by bacterial denitrification, but instead was mainly regulated by fungal denitrification. Elevated nitrate concentration increased the contribution of fungal denitrification to N2O production by 11-25%, whereas it decreased bacterial N2O production by 16-33%. Chemodenitrification was also enhanced by high nitrate concentration, contributing to 13-28% of N2O production. Elevated nitrate concentration significantly mediated nirK-type denitrifiers structure and abundance, which are the keystone taxa driving N2O production. Collectively, these results suggest that increasing nitrate concentration can shift N2O production pathways from bacterial to fungal and chemodenitrification, which are mainly responsible for the enhanced N2O production and have widespread implications for N2O projections under ongoing nitrogen pollution in estuarine and coastal ecosystems.

Effects of periodic drying-wetting on microbial dynamics and activity of nitrite/nitrate-dependent anaerobic methane oxidizers in intertidal wetland sediments.

Nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) plays an important role in methane (CH4) consumption in intertidal wetlands. However, little is known about the responses of n-DAMO in intertidal wetlands to periodic drying-wetting caused by tidal cycling. Here, comparative experiments (waterlogged, desiccated, reflooded) with the Yangtze estuarine intertidal sediments were performed to examine the effects of periodic tidal changes on n-DAMO microbial communities, abundances, and potential activities. Functional gene sequencing indicated the coexistence of n-DAMO bacteria and archaea in the tide-fluctuating environments and generally higher biodiversity under reflooded conditions than consecutive inundation or emersion. The n-DAMO microbial abundance and associated activity varied significantly during alternative exposure and inundation, with higher abundance and activity under the waterlogged than desiccated conditions. Reflooding of intertidal wetlands might intensify n-DAMO activities, indicating the resilience of n-DAMO microbial metabolisms to the wetting-drying events. Structural equation modeling and correlation analysis showed that n-DAMO activity was highly related to n-DAMO microbial abundance and substrate availability under inundation, whereas salt accumulation in sediment was the primary factor restraining n-DAMO activity under the desiccation. Overall, this study reveals tidal-induced shifts of n-DAMO activity and associated contribution to mitigating CH4, which may help accurately project CH4 emission from intertidal wetlands under different tidal scenarios.

Spatiotemporal Dynamics of Bacterial Taxonomic and Functional Profiles in Estuarine Intertidal Soils of China Coastal Zone.

Bacteria play an important role in regulating carbon (C), nitrogen (N), and sulfur (S) in estuarine intertidal wetlands. To gain insights into the ecological and metabolic modes possessed by bacteria in estuarine intertidal wetlands, a total of 78 surface soil samples were collected from China's coastal intertidal wetlands to examine the spatial and seasonal variations of bacterial taxonomic composition, assembly processes, and ecological system functions through shotgun metagenomic and 16S rRNA gene sequencing. Obvious spatiotemporal dynamic patterns in the bacterial community structure were identified, with more pronounced seasonal rather than spatial variations. Dispersion limitation was observed to act as a critical factor affecting community assembly, explaining approximately half of the total variation in the bacterial community. Functional bacterial community structure exhibited a more significant latitudinal change than seasonal variability, highlighting that functional stability of the bacterial communities differed with their taxonomic variability. Identification of biogeochemically related links between C, N, and S cycles in the soils showed the adaptive routed metabolism of the bacterial communities and the strong interactions between coupled metabolic pathways. Our study broadens the insights into the taxonomic and functional profiles of bacteria in China's estuarine intertidal soils and helps us understand the effects exerted by environmental factors on the ecological health and microbial diversity of estuarine intertidal flats.

Nitrification Regulates the Spatiotemporal Variability of N2O Emissions in a Eutrophic Lake.

Nitrous oxide (N2O) emissions from lakes exhibit significant spatiotemporal heterogeneity, and quantitative identification of the different N2O production processes is greatly limited, causing the role of nitrification to be undervalued or ignored in models of a lake's N2O emissions. Here, the contributions of nitrification and denitrification to N2O production were quantitatively assessed in the eutrophic Lake Taihu using molecular biology and isotope mapping techniques. The N2O fluxes ranged from -41.48 to 28.84 µmol m-2 d-1 in the lake, with lower N2O concentrations being observed in spring and summer and significantly higher N2O emissions being observed in autumn and winter. The 15N site preference and relevant isotopic evidence demonstrated that denitrification contributed approximately 90% of the lake's gross N2O production during summer and autumn, 27-83% of which was simultaneously eliminated via N2O reduction. Surprisingly, nitrification seemed to act as a key process promoting N2O production and contributing to the lake as a source of N2O emissions. A combination of N2O isotopocule-based approaches and molecular techniques can be used to determine the precise characteristics of microbial N2O production and consumption in eutrophic lakes. The results of this study provide a basis for accurately assessing N2O emissions from lakes at the regional and global scales.

Expanding the phylogenetic distribution of cytochrome b-containing methanogenic archaea sheds light on the evolution of methanogenesis.

Methane produced by methanogenic archaea has an important influence on Earth's changing climate. Methanogenic archaea are phylogenetically diverse and widespread in anoxic environments. These microorganisms can be divided into two subgroups based on whether or not they use b-type cytochromes for energy conservation. Methanogens with b-type cytochromes have a wider substrate range and higher growth yields than those without them. To date, methanogens with b-type cytochromes were found exclusively in the phylum "Ca. Halobacteriota" (formerly part of the phylum Euryarchaeota). Here, we present the discovery of metagenome-assembled genomes harboring methyl-coenzyme M reductase genes reconstructed from mesophilic anoxic sediments, together with the previously reported thermophilic "Ca. Methylarchaeum tengchongensis", representing a novel archaeal order, designated the "Ca. Methylarchaeales", of the phylum Thermoproteota (formerly the TACK superphylum). These microorganisms contain genes required for methyl-reducing methanogenesis and the Wood-Ljundahl pathway. Importantly, the genus "Ca. Methanotowutia" of the "Ca. Methylarchaeales" encode a cytochrome b-containing heterodisulfide reductase (HdrDE) and methanophenazine-reducing hydrogenase complex that have similar gene arrangements to those found in methanogenic Methanosarcinales. Our results indicate that members of the "Ca. Methylarchaeales" are methanogens with cytochromes and can conserve energy via membrane-bound electron transport chains. Phylogenetic and amalgamated likelihood estimation analyses indicate that methanogens with cytochrome b-containing electron transfer complexes likely evolved before diversification of Thermoproteota or "Ca. Halobacteriota" in the early Archean Eon. Surveys of public sequence databases suggest that members of the lineage are globally distributed in anoxic sediments and may be important players in the methane cycle.

High importance of coupled nitrification-denitrification for nitrogen removal in a large periodically low-oxygen estuary.

The coupling between nitrification and denitrification/anammox (nitrate/nitrite used in denitrification/anammox derives from nitrification) is a significant process of reactive nitrogen (N) removal that has attracted much attention. However, the dynamics of coupled nitrification-denitrification/anammox in the periodically low-oxygen estuaries and coasts remain unclear. Here, continuous-flow experiments combined with isotope tracing techniques were conducted in periodically low-oxygen areas of the Yangtze Estuary to reveal the changes in benthic sediment denitrification and anammox as well as their coupling with nitrification. Our results showed that denitrification increased but anammox decreased during low-oxygen summer. The occurrence of low oxygen also promoted coupled nitrification-denitrification but decreased coupled nitrification-anammox. These results implied that decreased dissolved oxygen in summer did not largely restrict nitrification activity, and anaerobic denitrification/anammox regulated the magnitude of coupled nitrification-denitrification/anammox rates. Denitrification (74.95-100 %) was the dominant process in total N removal, while coupled nitrification-denitrification accounted for a higher proportion (45.68-97.05 %) of denitrification, indicating that coupling between nitrification and denitrification played a dominant role in N removal. In addition to dissolved oxygen levels, carbon and N substrate availabilities were also important variables to regulate N transformations. Overall, this study advanced our knowledge of the distribution patterns and controlling factors of N removal processes and highlighted that coupled nitrification-denitrification might have a significant but neglected role in N removal from periodically low-oxygen estuaries.