Genomic structure predicts metabolite dynamics in microbial communities.

The metabolic activities of microbial communities play a defining role in the evolution and persistence of life on Earth, driving redox reactions that give rise to global biogeochemical cycles. Community metabolism emerges from a hierarchy of processes, including gene expression, ecological interactions, and environmental factors. In wild communities, gene content is correlated with environmental context, but predicting metabolite dynamics from genomes remains elusive. Here, we show, for the process of denitrification, that metabolite dynamics of a community are predictable from the genes each member of the community possesses. A simple linear regression reveals a sparse and generalizable mapping from gene content to metabolite dynamics for genomically diverse bacteria. A consumer-resource model correctly predicts community metabolite dynamics from single-strain phenotypes. Our results demonstrate that the conserved impacts of metabolic genes can predict community metabolite dynamics, enabling the prediction of metabolite dynamics from metagenomes, designing denitrifying communities, and discovering how genome evolution impacts metabolism.

Diversity and evolution of nitric oxide reduction in bacteria and archaea.

Nitrous oxide is a potent greenhouse gas whose production is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) enzyme superfamily. We identified several previously uncharacterized HCO families, four of which (eNOR, sNOR, gNOR, and nNOR) appear to perform NO reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple NO reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from the bacterium Rhodothermus marinus and found that it performs NO reduction. These recently identified NORs exhibited broad phylogenetic and environmental distributions, greatly expanding the diversity of microbes in nature capable of NO reduction. Phylogenetic analyses further demonstrated that NORs evolved multiple times independently from oxygen reductases, supporting the view that complete denitrification evolved after aerobic respiration.

Denitrification in foraminifera has an ancient origin and is complemented by associated bacteria.

Benthic foraminifera are unicellular eukaryotes that inhabit sediments of aquatic environments. Several foraminifera of the order Rotaliida are known to store and use nitrate for denitrification, a unique energy metabolism among eukaryotes. The rotaliid Globobulimina spp. has been shown to encode an incomplete denitrification pathway of bacterial origin. However, the prevalence of denitrification genes in foraminifera remains unknown, and the missing denitrification pathway components are elusive. Analyzing transcriptomes and metagenomes of 10 foraminiferal species from the Peruvian oxygen minimum zone, we show that denitrification genes are highly conserved in foraminifera. We infer the last common ancestor of denitrifying foraminifera, which enables us to predict the ability to denitrify for additional foraminiferal species. Additionally, an examination of the foraminiferal microbiota reveals evidence for a stable interaction with Desulfobacteraceae, which harbor genes that complement the foraminiferal denitrification pathway. Our results provide evidence that foraminiferal denitrification is complemented by the foraminifera-associated microbiome. The interaction of foraminifera with their resident bacteria is at the basis of foraminiferal adaptation to anaerobic environments that manifested in ecological success in oxygen depleted habitats.

Tuning Local Atomic Structures in MoS2 Based Catalysts for Electrochemical Nitrate Reduction.

In recent years, there has been a substantial surge in the investigation of transition-metal dichalcogenides such as MoS2 as a promising electrochemical catalyst. Inspired by denitrification enzymes such as nitrate reductase and nitrite reductase, the electrochemical nitrate reduction catalyzed by MoS2 with varying local atomic structures is reported. It is demonstrated that the hydrothermally synthesized MoS2 containing sulfur vacancies behaves as promising catalysts for electrochemical denitrification. With copper doping at less than 9% atomic ratio, the selectivity of denitrification to dinitrogen in the products can be effectively improved. X-ray absorption characterizations suggest that two sulfur vacancies are associated with one copper dopant in the MoS2 skeleton. DFT calculation confirms that copper dopants replace three adjacent Mo atoms to form a trigonal defect-enriched region, introducing an exposed Mo reaction center that coordinates with Cu atom to increase N2 selectivity. Apart from the higher activity and selectivity, the Cu-doped MoS2 also demonstrates remarkably improved tolerance toward oxygen poisoning at high oxygen concentration. Finally, Cu-doped MoS2 based catalysts exhibit very low specific energy consumption during the electrochemical denitrification process, paving the way for potential scale-up operations.

deepNEC: a novel alignment-free tool for the identification and classification of nitrogen biochemical network-related enzymes using deep learning.

Nitrogen is essential for life and its transformations are an important part of the global biogeochemical cycle. Being an essential nutrient, nitrogen exists in a range of oxidation states from +5 (nitrate) to -3 (ammonium and amino-nitrogen), and its oxidation and reduction reactions catalyzed by microbial enzymes determine its environmental fate. The functional annotation of the genes encoding the core nitrogen network enzymes has a broad range of applications in metagenomics, agriculture, wastewater treatment and industrial biotechnology. This study developed an alignment-free computational approach to determine the predicted nitrogen biochemical network-related enzymes from the sequence itself. We propose deepNEC, a novel end-to-end feature selection and classification model training approach for nitrogen biochemical network-related enzyme prediction. The algorithm was developed using Deep Learning, a class of machine learning algorithms that uses multiple layers to extract higher-level features from the raw input data. The derived protein sequence is used as an input, extracting sequential and convolutional features from raw encoded protein sequences based on classification rather than traditional alignment-based methods for enzyme prediction. Two large datasets of protein sequences, enzymes and non-enzymes were used to train the models with protein sequence features like amino acid composition, dipeptide composition (DPC), conformation transition and distribution, normalized Moreau-Broto (NMBroto), conjoint and quasi order, etc. The k-fold cross-validation and independent testing were performed to validate our model training. deepNEC uses a four-tier approach for prediction; in the first phase, it will predict a query sequence as enzyme or non-enzyme; in the second phase, it will further predict and classify enzymes into nitrogen biochemical network-related enzymes or non-nitrogen metabolism enzymes; in the third phase, it classifies predicted enzymes into nine nitrogen metabolism classes; and in the fourth phase, it predicts the enzyme commission number out of 20 classes for nitrogen metabolism. Among all, the DPC + NMBroto hybrid feature gave the best prediction performance (accuracy of 96.15% in k-fold training and 93.43% in independent testing) with an Matthews correlation coefficient (0.92 training and 0.87 independent testing) in phase I; phase II (accuracy of 99.71% in k-fold training and 98.30% in independent testing); phase III (overall accuracy of 99.03% in k-fold training and 98.98% in independent testing); phase IV (overall accuracy of 99.05% in k-fold training and 98.18% in independent testing), the DPC feature gave the best prediction performance. We have also implemented a homology-based method to remove false negatives. All the models have been implemented on a web server (prediction tool), which is freely available at http://bioinfo.usu.edu/deepNEC/.

Transcriptomic profiling of haloarchaeal denitrification through RNA-Seq analysis.

Denitrification, a crucial biochemical pathway prevalent among haloarchaea in hypersaline ecosystems, has garnered considerable attention in recent years due to its ecological implications. Nevertheless, the underlying molecular mechanisms and genetic regulation governing this respiration/detoxification process in haloarchaea remain largely unexplored. In this study, RNA-sequencing was used to compare the transcriptomes of the haloarchaeon Haloferax mediterranei under oxic and denitrifying conditions, shedding light on the intricate metabolic alterations occurring within the cell, such as the accurate control of the metal homeostasis. Furthermore, the investigation identifies several genes encoding transcriptional regulators and potential accessory proteins with putative roles in denitrification. Among these are bacterioopsin-like transcriptional activators, proteins harboring a domain of unknown function (DUF2249), and cyanoglobin. In addition, the study delves into the genetic regulation of denitrification, finding a regulatory motif within promoter regions that activates numerous denitrification-related genes. This research serves as a starting point for future molecular biology studies in haloarchaea, offering a promising avenue to unravel the intricate mechanisms governing haloarchaeal denitrification, a pathway of paramount ecological importance.IMPORTANCEDenitrification, a fundamental process within the nitrogen cycle, has been subject to extensive investigation due to its close association with anthropogenic activities, and its contribution to the global warming issue, mainly through the release of N2O emissions. Although our comprehension of denitrification and its implications is generally well established, most studies have been conducted in non-extreme environments with mesophilic microorganisms. Consequently, there is a significant knowledge gap concerning extremophilic denitrifiers, particularly those inhabiting hypersaline environments. The significance of this research was to delve into the process of haloarchaeal denitrification, utilizing the complete denitrifier haloarchaeon Haloferax mediterranei as a model organism. This research led to the analysis of the metabolic state of this microorganism under denitrifying conditions and the identification of regulatory signals and genes encoding proteins potentially involved in this pathway, serving as a valuable resource for future molecular studies.

Novel Alphaproteobacteria transcribe genes for nitric oxide transformation at high levels in a marine oxygen-deficient zone.

Marine oxygen-deficient zones (ODZs) are portions of the ocean where intense nitrogen loss occurs primarily via denitrification and anammox. Despite many decades of study, the identity of the microbes that catalyze nitrogen loss in ODZs is still being elucidated. Intriguingly, high transcription of genes in the same family as the nitric oxide dismutase (nod) gene from Methylomirabilota has been reported in the anoxic core of ODZs. Here, we show that the most abundantly transcribed nod genes in the Eastern Tropical North Pacific ODZ belong to a new order (UBA11136) of Alphaproteobacteria, rather than Methylomirabilota as previously assumed. Gammaproteobacteria and Planctomycetia also transcribe nod, but at lower relative abundance than UBA11136 in the upper ODZ. The nod-transcribing Alphaproteobacteria likely use formaldehyde and formate as a source of electrons for aerobic respiration, with additional electrons possibly from sulfide oxidation. They also transcribe multiheme cytochrome (here named ptd) genes for a putative porin-cytochrome protein complex of unknown function, potentially involved in extracellular electron transfer. Molecular oxygen for aerobic respiration may originate from nitric oxide dismutation via cryptic oxygen cycling. Our results implicate Alphaproteobacteria order UBA11136 as a significant player in marine nitrogen loss and highlight their potential in one-carbon, nitrogen, and sulfur metabolism in ODZs.IMPORTANCEIn marine oxygen-deficient zones (ODZs), microbes transform bioavailable nitrogen to gaseous nitrogen, with nitric oxide as a key intermediate. The Eastern Tropical North Pacific contains the world's largest ODZ, but the identity of the microbes transforming nitric oxide remains unknown. Here, we show that highly transcribed nitric oxide dismutase (nod) genes belong to Alphaproteobacteria of the novel order UBA11136, which lacks cultivated isolates. These Alphaproteobacteria show evidence for aerobic respiration, using oxygen potentially sourced from nitric oxide dismutase, and possess a novel porin-cytochrome protein complex with unknown function. Gammaproteobacteria and Planctomycetia transcribe nod at lower levels. Our results pinpoint the microbes mediating a key step in marine nitrogen loss and reveal an unexpected predicted metabolism for marine Alphaproteobacteria.

Nitrous oxide reduction by two partial denitrifying bacteria requires denitrification intermediates that cannot be respired.

Denitrification is a form of anaerobic respiration wherein nitrate (NO3-) is sequentially reduced via nitrite (NO2-), nitric oxide, and nitrous oxide (N2O) to dinitrogen gas (N2) by four reductase enzymes. Partial denitrifying bacteria possess only one or some of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here, we tested the denitrifying capabilities of two purple nonsulfur bacteria, Rhodopseudomonas palustris CGA0092 and Rhodobacter capsulatus SB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO2- to N2, and SB1003 reduced N2O to N2. For each bacterium, N2O reduction could be used both for electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in darkness. However, N2O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO3- served as a stable, non-catalyzable molecule that was sufficient to activate N2O reduction. Using a ß-galactosidase reporter, we found that NO3- acted, at least in part, by stimulating N2O reductase gene expression. In SB1003, NO2- but not NO3- activated N2O reduction, but NO2- was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use.IMPORTANCEDenitrification is a form of microbial respiration wherein nitrate is converted via several nitrogen oxide intermediates into harmless dinitrogen gas. Partial denitrifying bacteria, which individually have some but not all denitrifying enzymes, can achieve complete denitrification as a community by cross-feeding nitrogen oxide intermediates. However, the last intermediate, nitrous oxide (N2O), is a potent greenhouse gas that often escapes, motivating efforts to understand and improve the efficiency of denitrification. Here, we found that at least some partial denitrifying N2O reducers can sense and respond to nitrogen oxide intermediates that they cannot otherwise use. The regulatory effects of nitrogen oxides on partial denitrifiers are thus an important consideration in understanding and applying denitrifying bacterial communities to combat greenhouse gas emissions.

Can plants build their niche through modulation of soil microbial activities linked with nitrogen cycling? A test with Arabidopsis thaliana.

In natural systems, different plant species have been shown to modulate specific nitrogen (N) cycling processes so as to meet their N demand, thereby potentially influencing their own niche. This phenomenon might go beyond plant interactions with symbiotic microorganisms and affect the much less explored plant interactions with free-living microorganisms involved in soil N cycling, such as nitrifiers and denitrifiers. Here, we investigated variability in the modulation of soil nitrifying and denitrifying enzyme activities (NEA and DEA, respectively), and their ratio (NEA : DEA), across 193 Arabidopsis thaliana accessions. We studied the genetic and environmental determinants of such plant-soil interactions, and effects on plant biomass production in the next generation. We found that NEA, DEA, and NEA : DEA varied c. 30-, 15- and 60-fold, respectively, among A. thaliana genotypes and were related to genes linked with stress response, flowering, and nitrate nutrition, as well as to soil parameters at the geographic origin of the analysed genotypes. Moreover, plant-mediated N cycling activities correlated with the aboveground biomass of next-generation plants in home vs away nonautoclaved soil, suggesting a transgenerational impact of soil biotic conditioning on plant performance. Altogether, these findings suggest that nutrient-based plant niche construction may be much more widespread than previously thought.

Insights into the evolutionary and ecological adaption strategies of nirS- and nirK-type denitrifying communities.

Denitrification is a crucial process in the global nitrogen cycle, in which two functionally equivalent genes, nirS and nirK, catalyse the critical reaction and are usually used as marker genes. The nirK gene can function independently, whereas nirS requires additional genes to encode nitrite reductase and is more sensitive to environmental factors than nirK. However, the ecological differentiation mechanisms of those denitrifying microbial communities and their adaptation strategies to environmental stresses remain unclear. Here, we conducted metagenomic analysis for sediments and bioreactor samples from Lake Donghu, China. We found that nirS-type denitrifying communities had a significantly lower horizontal gene transfer frequency than that of nirK-type denitrifying communities, and nirS gene phylogeny was more congruent with taxonomy than that of nirK gene. Metabolic reconstruction of metagenome-assembled genomes further revealed that nirS-type denitrifying communities have robust metabolic systems for energy conservation, enabling them to survive under environmental stresses. Nevertheless, nirK-type denitrifying communities seemed to adapt to oxygen-limited environments with the ability to utilize various carbon and nitrogen compounds. Thus, this study provides novel insights into the ecological differentiation mechanism of nirS and nirK-type denitrifying communities, as well as the regulation of the global nitrogen cycle and greenhouse gas emissions.

Species pool and local assembly processes drive ß diversity of ammonia-oxidizing and denitrifying microbial communities in rivers along a latitudinal gradient.

Both regional species pool and local community assembly mechanism drive the microbial diversity patterns across geographical gradients. However, little has been done to separate their effects on the ß diversity patterns of microbial communities involved in nitrogen (N) cycling in river ecosystems. Here, we use high-throughput sequencing of the archaeal amoA, bacterial amoA, nirK, and nirS genes, null model, and neutral community model to distinguish the relative importance of species pool and local assembly processes for ammonia-oxidizing and denitrifying communities in river wetlands along a latitudinal gradient in eastern China. Results indicated that the ß diversity of the nirS-type denitrifying community co-varied with γ diversity and environmental heterogeneity, implying that regional species pool and heterogeneous selection explained variation in ß diversity. However, the ß diversity of ammonia-oxidizing and nirK-type denitrifying communities did not correlate with γ diversity and environmental heterogeneity. The continuous hump distribution of ß deviation along the latitudinal gradient and the lower species dispersal rate indicated that the dispersal limitation shaped the variation in ß diversity of ammonia-oxidizing and nirK-type denitrifying communities. Additionally, biotic interactions drove ammonia-oxidizing and nirS-type denitrifying communities by influencing species co-occurrence patterns. Our study highlights the importance of regional species pool and local community assembly processes in shaping geographical patterns of N-cycling microorganisms and extends knowledge of their adaptability to a continuously changing environment on a large scale.

Revisiting the metal sites of nitrous oxide reductase in a low-dose structure from Marinobacter nauticus.

Copper-containing nitrous oxide reductase catalyzes a 2-electron reduction of the green-house gas N2O to yield N2. It contains two metal centers, the binuclear electron transfer site CuA, and the unique, tetranuclear CuZ center that is the site of substrate binding. Different forms of the enzyme were described previously, representing variations in oxidation state and composition of the metal sites. Hypothesizing that many reported discrepancies in the structural data may be due to radiation damage during data collection, we determined the structure of anoxically isolated Marinobacter nauticus N2OR from diffraction data obtained with low-intensity X-rays from an in-house rotating anode generator and an image plate detector. The data set was of exceptional quality and yielded a structure at 1.5 Å resolution in a new crystal form. The CuA site of the enzyme shows two distinct conformations with potential relevance for intramolecular electron transfer, and the CuZ cluster is present in a [4Cu:2S] configuration. In addition, the structure contains three additional types of ions, and an analysis of anomalous scattering contributions confirms them to be Ca2+, K+, and Cl-. The uniformity of the present structure supports the hypothesis that many earlier analyses showed inhomogeneities due to radiation effects. Adding to the earlier description of the same enzyme with a [4Cu:S] CuZ site, a mechanistic model is presented, with a structurally flexible CuZ center that does not require the complete dissociation of a sulfide prior to N2O binding.

Modeling denitrification nitrogen losses in China's rice fields based on multiscale field-experiment constraints.

Denitrification plays a critical role in soil nitrogen (N) cycling, affecting N availability in agroecosystems. However, the challenges in direct measurement of denitrification products (NO, N2 O, and N2 ) hinder our understanding of denitrification N losses patterns across the spatial scale. To address this gap, we constructed a data-model fusion method to map the county-scale denitrification N losses from China's rice fields over the past decade. The estimated denitrification N losses as a percentage of N application from 2009 to 2018 were 11.8 ± 4.0% for single rice, 12.4 ± 3.7% for early rice, and 11.6 ± 3.1% for late rice. The model results showed that the spatial heterogeneity of denitrification N losses is primarily driven by edaphic and climatic factors rather than by management practices. In particular, diffusion and production rates emerged as key contributors to the variation of denitrification N losses. These findings humanize a 38.9 ± 4.8 kg N ha-1 N loss by denitrification and challenge the common hypothesis that substrate availability drives the pattern of N losses by denitrification in rice fields.

Biological mitigation of soil nitrous oxide emissions by plant metabolites.

Plant metabolites significantly affect soil nitrogen (N) cycling, but their influence on nitrous oxide (N2O) emissions has not been quantitatively analyzed on a global scale. We conduct a comprehensive meta-analysis of 173 observations from 42 articles to evaluate global patterns of and principal factors controlling N2O emissions in the presence of root exudates and extracts. Overall, plant metabolites promoted soil N2O emissions by about 10%. However, the effects of plant metabolites on N2O emissions from soils varied with experimental conditions and properties of both metabolites and soils. Primary metabolites, such as sugars, amino acids, and organic acids, strongly stimulated soil N2O emissions, by an average of 79%, while secondary metabolites, such as phenolics, terpenoids, and flavonoids, often characterized as both biological nitrification inhibitors (BNIs) and biological denitrification inhibitors (BDIs), reduced soil N2O emissions by an average of 41%. The emission mitigation effects of BNIs/BDIs were closely associated with soil texture and pH, increasing with increasing soil clay content and soil pH on acidic and neutral soils, and with decreasing soil pH on alkaline soils. We furthermore present soil incubation experiments that show that three secondary metabolite types act as BNIs to reduce N2O emissions by 32%-45%, while three primary metabolite classes possess a stimulatory effect of 56%-63%, confirming the results of the meta-analysis. Our results highlight the potential role and application range of specific secondary metabolites in biomitigation of global N2O emissions and provide new biological parameters for N2O emission models that should help improve the accuracy of model predictions.

Ecotoxicity characterization assisted performance assessment of electro-bioremediation reactors for nitrate and arsenite elimination.

The performance of combined reduction of nitrate (NO3 - ) to dinitrogen gas (N2 ) and oxidation of arsenite (As[III]) to arsenate (As[V]) by a bioelectrochemical system was assessed, supported by ecotoxicity characterization. For the comprehensive toxicity characterization of the untreated model groundwater and the treated reactor effluents, a problem-specific ecotoxicity test battery was established. The performance of the applied technology in terms of toxicity and target pollutant elimination was compared and analyzed. The highest toxicity attenuation was achieved under continuous flow mode with hydraulic retention time (HRT) = 7.5 h, with 95%, nitrate removal rate and complete oxidation of arsenite to arsenate. Daphnia magna proved to be the most sensitive test organism. The results of the D. magna lethality test supported the choice of the ideal operational conditions based on chemical data analysis. The outcomes of the study demonstrated that the applied technology was able to improve the groundwater quality in terms of both chemical and ecotoxicological characteristics. The importance of ecotoxicity evaluation was also highlighted, given that significant target contaminant elimination did not necessarily lower the environmental impact of the initial, untreated medium, in addition, anomalies might occur during the technology operational process which in some instances, could result in elevated toxicity levels.

Biofilm formation and EPS production enhances the bioremediation potential of Pseudomonas species: a novel study from eutrophic waters of Dal lake, Kashmir, India.

The present study was conducted with the aim of isolation and identification of the biofilm-forming denitrifying Pseudomonas bacterial strains from eutrophic waters of Dal lake, India, followed by the study of inter-relation of biofilm formation and denitrification potential of Pseudomonas strains. The bacterial strains were characterized by morphological observations and identified using 16S rDNA sequencing followed by the quantification of biofilm formation of these st by crystal violet (CV) assay using 96-well microtiter plate and extracellular polymeric substance (EPS) extraction. Lastly, the nitrate-reducing potential of all Pseudomonas species was studied. Our evaluation revealed that four different Pseudomonas species were observed to have the biofilm-forming potential and nitrate-reducing properties and the species which showed maximum biofilm-forming potential and maximum EPS production exhibited higher nitrate-removing capacity. Moreover, P. otitis was observed to have the highest denitrification capacity (89%) > P. cedrina (83%) > P. azotoform (79%) and the lowest for P. peli (70%). These results clearly signify a positive correlation of biofilm-forming capacity and nitrate-removing ability of Pseudomonas species. This study has for the first time successfully revealed the bioremediation potential of P. otitis, P. cedrina, P. azotoform, and P. peli species, thus contributing to the growing list of known nitrate-reducing Pseudomonas species. Based upon the results, these strains can be extrapolated to nitrate-polluted water systems for combating water pollution.

Transcriptome analysis of the degradation process of organic nitrogen by two heterotrophic nitrifying and aerobic denitrifying bacteria.

The heterotrophic nitrification aerobic denitrification bacteria (HNDS) can perform nitrification and denitrification at the same time. Two HNDS strains, Achromobacter sp. HNDS-1 and Enterobacter sp. HNDS-6 which exhibited an amazing ability to solution nitrogen (N) removal have been successfully isolated from paddy soil in our lab. When peptone or ammonium sulfate as sole N source, no significant difference in gene expression related to nitrification and denitrification of the strains was found according to the transcriptome analysis. The expression of phosphomethylpyrimidine synthase (thiC), ABC transporter substrate-binding protein, branched-chain amino acid ABC transporter substrate-binding protein, and RNA polymerase (rpoE) in HNDS-1 were significantly upregulated when used peptone as N source, while the expression of exopolysaccharide production protein (yjbE), RNA polymerase (rpoC), glutamate synthase (gltD) and ABC-type branched-chain amino acid transport systems in HNDS-6 were significantly upregulated. This indicated that these two strains are capable of using organic N and converting it into NH4+-N, then utilizing NH4+-N to synthesize amino acids and proteins for their own growth, and strain HNDS-6 can also remove NH4+-N through nitrification and denitrification.

Parasedimentitalea denitrificans sp. nov., a novel denitrifying bacteria isolated from the Yellow Sea and transfer of Zongyanglinia huanghaiensis and Zongyanglinia marina to the genus Parasedimentitalea as Parasedimentitalea huanghaiensis comb. nov. and Parasedimentitalea maritima nom. nov.

A Gram-stain-negative and rod-shaped bacterium, designated strain CY04T, was isolated from a sediment sample collected from the Yellow Sea. CY04T exhibited the highest 16S rRNA gene sequence similarity of 98.7 % to Zongyanglinia huanghaiensis CY05T, followed by the similarities of 98.6 %, 98.0 and 98.0 % to Zongyanglinia marina DSW4-44T, Parasedimentitalea marina W43T and Parasedimentitalea psychrophila QS115T respectively. Phylogenetic analysis based on 16S rRNA gene and phylogenomic analysis based on genome sequences revealed that CY04T formed a robust cluster with Z. huanghaiensis CY05T, Z. marina DSW4-44T, P. marina W43T and P. psychrophila QS115T. Calculated digital DNA-DNA hybridisation and average nucleotide identity values between CY04T and its closely related species were 22.2-23.7 % and 79.0-81.2 % respectively. Cells of CY04T were strictly aerobic, non-motile and positive for catalase, oxidase and denitrification. CY04T harboured a set of genes encoding the enzymes involved in denitrification. Growth occurred at 10-30 °C (optimum, 20 °C), at pH 6.5-9.5 (optimum, pH 8.0) and with 1-6 % (w/v) (optimum, 2.5 %,) NaCl. The major component of the fatty acids was summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The isoprenoid quinone was Q-10. Results of the phenotypic, chemotaxonomic and molecular study indicate that strain CY04T represents a novel species of the genus Parasedimentitalea, for which the name Parasedimentitalea denitrificans sp. nov. is proposed. The type strain is CY04T (=MCCC 1K08635T=KCTC 62199T). It is also proposed that Zongyanglinia huanghaiensis and Zongyanglinia marina should be reclassified as Parasedimentitalea huanghaiensis comb. nov. and Parasedimentitalea maritima nom. nov. An emended description of the genus Parasedimentitalea is also proposed.

Nitrogen Removal Performance and Microbial Community Structure of IMTA Ponds (Apostistius japonicus-Penaeus japonicus-Ulva).

Denitrification and anaerobic ammonium oxidation (anammox) are key processes for nitrogen removal in aquaculture, reducing the accumulated nitrogen nutrients to nitrogen gas or nitrous oxide gas. Complete removal of nitrogen from aquaculture systems is an important measure to solve environmental pollution. In order to evaluate the nitrogen removal potential of marine aquaculture ponds, this study investigated the denitrification and anammox rates, the flux of nitrous oxide (N2O) at the water-air interface, the sediment microbial community structure, and the gene expression associated with the nitrogen removal process in integrated multi-trophic aquaculture (IMTA) ponds (Apostistius japonicus-Penaeus japonicus-Ulva) with different culture periods. The results showed that the denitrification and anammox rates in sediments increased with the increase of cultivation periods and depth, and there was no significant difference in nitrous oxide gas flux at the water-air interface between different cultivation periods (p > 0.05). At the genus and phylum levels, the abundance of microorganisms related to nitrogen removal reactions in sediments changed significantly with the increase of cultivation period and depth, and was most significantly affected by the concentration of particulate organic nitrogen (PON) in sediments. The expression of denitrification gene (narG, nirS, nosZ) in surface sediments was significantly higher than that in deep sediments (p < 0.05), and was negatively correlated with denitrification rate. All samples had a certain anammox capacity, but no known anammox bacteria were found in the microbial diversity detection, and the expression of gene (hzsB) related to the anammox process was extremely low, which may indicate the existence of an unknown anammox bacterium. The data of this study showed that the IMTA culture pond had a certain potential for nitrogen removal, and whether it could make a contribution to reducing the pollution of culture wastewater still needed additional practice and evaluation, and also provided a theoretical basis for the nitrogen removal research of coastal mariculture ponds.

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.