Nitrite Reductase Gene Research Articles

Efficient partial denitrification-anammox process enabled by a novel denitrifier with truncated nitrite reduction pathway

Partial denitrification coupled with anaerobic ammonium oxidation (PD-anammox) is a promising technology for cost-effective nitrogen removal from wastewater. Nitrite availability is crucial to anammox performance but often limited by the slow partial denitrification process. Here we report an efficient PD-anammox system driven by the novel denitrifier Bacillus velezensis C1–3 with truncated nitrite reduction pathway. Whole-genome sequencing analysis revealed that the lack of nitrite reductase genes nirS/nirK and norBC in strain C1–3 enabled nitrite accumulation without the need for precise control of carbon dosage. By coupling it with anammox sludge, over 79 % total nitrogen (TN) removal was stably achieved, under a TN loading rate of 660 mg/L/d and a carbon/nitrogen ratio below 1.0. Mechanism explorations indicate that the niche differentiation of C1–3 and anammox bacteria facilitated their mutualism while avoiding nitrite competition. This study demonstrates a novel strategy for establishing efficient PD-anammox process by harnessing the unique metabolic deficiency of denitrifiers, shedding light on the development of stable and sustainable biological nitrogen removal technologies with minimal carbon footprint.

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.

Metabolic engineering of the substrate utilization pathway in Escherichia coli increases L-lysine production

L-lysine is an essential amino acid with broad applications in the animal feed, human food, and pharmaceutical industries. The fermentation production of L-lysine by Escherichia coli has limitations such as poor substrate utilization efficiency and low saccharide conversion rates. We deleted the global regulatory factor gene mlc and introduced heterologous genes, including the maltose phosphotransferase genes (malAP) from Bacillus subtilis, to enhance the use efficiency of disaccharides and trisaccharides. The engineered strain E. coli XC3 demonstrated improved L-lysine production, yield, and productivity, which reached 160.00 g/L, 63.78%, and 4.44 g/(L‧h), respectively. Furthermore, we overexpressed the glutamate dehydrogenase gene (gdhA) and assimilated nitrate reductase genes (BsnasBC) from B. subtilis, along with nitrite reductase genes (EcnirBD) from E. coli, in strain E. coli XC3. This allowed the construction of E. coli XC4 with a nitrate assimilation pathway. The L-lysine production, yield, and productivity of E. coli XC4 were elevated to 188.00 g/L, 69.44%, and 5.22 g/(L‧h), respectively. After optimization of the residual sugar concentration and carbon to nitrogen ratio, the L-lysine production, yield, and productivity were increased to 204.00 g/L, 72.32%, and 5.67 g/(L‧h), respectively, in a 5 L fermenter. These values represented the increases of 40.69%, 20.03%, and 40.69%, respectively, compared with those of the starting strain XC1. By engineering the substrate utilization pathway, we successfully constructed a high-yield L-lysine producing strain, laying a solid foundation for the industrial production of L-lysine.

Microplastic exposure inhibits nitrate uptake and assimilation in wheat plants

Microplastic (MP) contamination in soil severely impairs plant growth. However, mechanisms underlying the effects of MPs on plant nutrient uptake remain largely unknown. In this study, we revealed that NO3− content was significantly decreased in shoots and roots of wheat plants exposed to high concentrations (50–100 mg L−1) of MPs (1 μm and 0.1 μm; type: polystyrene) in the hydroponic solution. Isotope labeling experiments demonstrated that MP exposure led to a significant inhibition of NO3− uptake in wheat roots. Further analysis indicated that the presence of MPs markedly inhibited root growth and caused oxidative damage to the roots. Additionally, superoxide dismutase and peroxidase activities in wheat roots decreased under all MP treatments, whereas catalase and ascorbate peroxidase activities significantly increased under the 100 mg L−1 MP treatment. The transcription levels of most nitrate transporters (NRTs) in roots were significantly downregulated by MP exposure. Furthermore, exposure to MPs distinctly suppressed the activity of nitrate reductase (NR) and nitrite reductase (NiR), as well as the expression levels of their coding genes in wheat shoots. These findings indicate that a decline in root uptake area and root vitality, as well as in the expression of NRTs, NR, and NiR genes caused by MP exposure may have adverse effects on NO3− uptake and assimilation, consequently impairing normal growth of plants.

Genome streamlining in Parcubacteria transitioning from soil to groundwater

BackgroundTo better understand the influence of habitat on the genetic content of bacteria, with a focus on members of Candidate Phyla Radiation (CPR) bacteria, we studied the effects of transitioning from soil via seepage waters to groundwater on genomic composition of ultra-small Parcubacteria, the dominating CPR class in seepage waters, using genome resolved metagenomics.ResultsBacterial metagenome-assembled genomes (MAGs), (318 total, 32 of Parcubacteria) were generated from seepage waters and compared directly to groundwater counterparts. The estimated average genome sizes of members of major phyla Proteobacteria, Bacteroidota and Cand. Patescibacteria (Candidate Phyla Radiation – CPR bacteria) were significantly higher in soil-seepage water as compared to their groundwater counterparts. Seepage water Parcubacteria (Paceibacteria) exhibited 1.18-fold greater mean genome size and 2-fold lower mean proportion of pseudogenes than those in groundwater. Bacteroidota and Proteobacteria also showed a similar trend of reduced genomes in groundwater compared to seepage. While exploring gene loss and adaptive gains in closely related CPR lineages in groundwater, we identified a membrane protein, and a lipoglycopeptide resistance gene unique to a seepage Parcubacterium genome. A nitrite reductase gene was also identified and was unique to the groundwater Parcubacteria genomes, likely acquired from other planktonic microbes via horizontal gene transfer.ConclusionsOverall, our data suggest that bacteria in seepage waters, including ultra-small Parcubacteria, have significantly larger genomes and higher metabolic enrichment than their groundwater counterparts, highlighting possible genome streamlining of the latter in response to habitat selection in an oligotrophic environment.

N2O emission in temperate seagrass meadows: Fluxes, pathway and molecular mechanism

Seagrass meadows act as filters for nitrogen in coastal areas, but whether they are a source or sink for N2O has been still controversy. Additionally, the production pathways of N2O as well as the microbial driving mechanism in seagrass meadows are seldom reported. In this study, the air-sea fluxes, sediment release potential, and production pathway of N2O in a temperate Zostera marina and Z. japonica mixed meadow were investigated by using gas chromatography and 15N isotopic tracing methods. The qPCR and metagenome sequencing were used to compare the difference in functional gene abundance and expression between seagrass vegetated and non-grass sediments. The results showed that the N2O air-sea fluxes in the meadow ranged from −1.97 to −1.77 nmol m⁻2 h⁻1, which was slightly lower in the seagrass region than in the adjacent bare region. Seagrass sediment N2O release potential dramatically increased after warming and nitrogen enrichment treatments. Heterotrophic nitrification was firstly investigated in seagrass meadows, and the process (26.80%–62.41%) and denitrification (37.55%–72.83%) contributed significantly to N2O production in the meadow, affected deeply by sediment organic content, while the contribution of autotrophic nitrification can be neglected. Compared with the bare sediments, the ammonia monooxygenase genes amoA, amoB and amoC, and nitrite oxidoreductase genes nxrA and nxrB, as well as nitrite reductase gene nirS and nitric oxide reductase gene norB were down-regulated, while the nitrous oxide reductase gene nosZ was up-regulated in the seagrass sediments, explaining less N2O emission in seagrass regions from the perspective of molecular. The nosZII-bearing bacteria like Bacteroidia, Polyangia, Anaerolineae, and Verrucomicrobiae could play important roles in N2O reduction in the seagrass meadow. The result is of great significance for highlighting the ability of seagrass meadows to mitigate climate changes.

Dietary crude protein and protein solubility manipulation enhances intestinal nitrogen absorption and mitigates reactive nitrogen emissions through gut microbiota and metabolome reprogramming in sheep

Dietary nutrient manipulation (e.g. protein fractions) could lower the environmental footprints of ruminants, especially reactive nitrogen (N). This study investigated the impacts of dietary soluble protein (SP) levels with decreased crude protein (CP) on intestinal N absorption, hindgut N metabolism, fecal microbiota and metabolites, and their linkage with N metabolism phenotype. Thirty-two male Hu sheep, with an age of six months and an initial BW of 40.37 ± 1.18 kg, were randomly assigned to four dietary groups. The control diet (CON), aligning with NRC standards, maintained a CP content of 16.7% on a dry matter basis. Conversely, the experimental diets (LPA, LPB, and LPC) featured a 10% reduction in CP compared with CON, accompanied by SP adjustments to 21.2%, 25.9%, and 29.4% of CP, respectively. Our results showed that low-protein diets led to significant reductions in the concentrations of plasma creatinine, ammonia, urea N, and fecal total short-chain fatty acids (SCFA) (P < 0.05). Notably, LPB and LPC exhibited increased total SCFA and propionate concentrations compared with LPA (P < 0.05). The enrichment of the Prevotella genus in fecal microbiota associated with energy metabolism and amino acid (AA) biosynthesis pathways was evident with SP levels in low-protein diets of approximately 25% to 30%. Moreover, LPB and LPC diets demonstrated a decrease in fecal NH4+–N and NO2−–N contents as well as urease activity, compared with CON (P < 0.05). Concomitantly, reductions in fecal glutamic acid dehydrogenase gene (gdh), nitrite reductase gene (nirS), and nitric oxide reductase gene (norB) abundances were observed (P < 0.05), pointing towards a potential reduction in reactive N production at the source. Of significance, the up-regulation of mRNA abundance of AA and peptide transporters in the small intestine (duodenum, jejunum, and ileum) and the elevated concentration of plasma AA (e.g. arginine, methionine, aspartate, glutamate, etc.) underscored the enhancement of N absorption and N efficiency. In summary, a 10% reduction in CP, coupled with an SP level of approximately 25% to 30%, demonstrated the potential to curtail reactive N emissions through fecal Prevotella enrichment and improve intestinal energy and N utilization efficiency.

A novel mechanism for dissimilatory nitrate reduction to ammonium in Acididesulfobacillus acetoxydans.

The biological route of nitrate reduction has important implications for the bioavailability of nitrogen within ecosystems. Nitrate reduction via nitrite, either to ammonium (ammonification) or to nitrous oxide or dinitrogen (denitrification), determines whether nitrogen is retained within the system or lost as a gas. The acidophilic sulfate-reducing bacterium (aSRB) Acididesulfobacillus acetoxydans can perform dissimilatory nitrate reduction to ammonium (DNRA). While encoding a Nar-type nitrate reductase, A. acetoxydans lacks recognized nitrite reductase genes. In this study, A. acetoxydans was cultivated under conditions conducive to DNRA. During cultivations, we monitored the production of potential nitrogen intermediates (nitrate, nitrite, nitric oxide, hydroxylamine, and ammonium). Resting cell experiments were performed with nitrate, nitrite, and hydroxylamine to confirm their reduction to ammonium, and formed intermediates were tracked. To identify the enzymes involved in DNRA, comparative transcriptomics and proteomics were performed with A. acetoxydans growing under nitrate- and sulfate-reducing conditions. Nitrite is likely reduced to ammonia by the previously undescribed nitrite reductase activity of the NADH-linked sulfite reductase AsrABC, or by a putatively ferredoxin-dependent homolog of the nitrite reductase NirA (DEACI_1836), or both. We identified enzymes and intermediates not previously associated with DNRA and nitrosative stress in aSRB. This increases our knowledge about the metabolism of this type of bacteria and helps the interpretation of (meta)genome data from various ecosystems on their DNRA potential and the nitrogen cycle.IMPORTANCENitrogen is crucial to any ecosystem, and its bioavailability depends on microbial nitrogen-transforming reactions. Over the recent years, various new nitrogen-transforming reactions and pathways have been identified, expanding our view on the nitrogen cycle and metabolic versatility. In this study, we elucidate a novel mechanism employed by Acididesulfobacillus acetoxydans, an acidophilic sulfate-reducing bacterium, to reduce nitrate to ammonium. This finding underscores the diverse physiological nature of dissimilatory reduction to ammonium (DNRA). A. acetoxydans was isolated from acid mine drainage, an extremely acidic environment where nitrogen metabolism is poorly studied. Our findings will contribute to understanding DNRA potential and variations in extremely acidic environments.

Enhanced adaptability of pyrite-based constructed wetlands for low carbon to nitrogen ratio wastewater treatments: Modulation of nitrogen removal mechanisms and reduction of carbon emissions

Pyrite-based constructed wetlands (CWs) stimulated nitrate removal performance at low carbon to nitrogen (C/N) ratio has been gaining widely attention. However, the combined effects of pyrite and C/N on the nitrate removal mechanisms and greenhouse gases (GHGs) reduction were ignored. This study found that pyrite-based CWs significantly enhanced nitrate removal in C/N of 0, 1.5 and 3 by effectively driving autotrophic denitrification with high abundance of autotrophs denitrifiers (Rhodanobacter) and nitrate reductase (EC 1.7.7.2), while the enhancement was weakened in C/N of 6 by combined effect of mixotrophic denitrification and dissimilatory nitrate reduction to ammonium (DNRA) with high abundance of organic carbon-degrading bacteria (Stenotrophobacter) and DNRA-related nitrite reductase genes (nrf). Moreover, pyrite addition significantly reduced GHGs emissions from CWs in all stages with the occurrence of iron-coupled autotrophic denitrification. The study shed light on the potential mechanism for pyrite-based CWs for treating low C/N ratio wastewater.

Distinct microbial characteristics of the robust single-stage coupling system during the conversion from anammox-denitritation to anammox-denitratation patterns

Simultaneous anammox-denitrification is effectively operated in two types, i.e., the anammox-denitritation (SAD pattern) and the anammox-denitratation (PDA pattern). The nitrate derived from inevitable nitrite oxidization likely determines the practical operational pattern of the coupling system, while little information is available regarding the microbial characteristics during the pattern conversion. Here, the single-stage bioreactor coupling anammox with denitrification was operated under conditions with a changed ratio of influent nitrite and nitrate. Results showed that the bioreactor exhibited a robust performance during the conversion from SAD to PDA patterns, corresponding with the total nitrogen removal efficiency ranging from 89.5% to 92.4%. Distinct community structures were observed in two patterns, while functional bacteria including the genera Denitratisoma, Thauera, Candidatus Brocadia, and Ca. Jettenia steadily co-existed. Meanwhile, the high transcription of hydrazine synthase genes demonstrated a stable anammox process, while the up-regulated transcription of nitrite and nitrous oxide reductase genes indicated that the complete denitrification process was enhanced for total nitrogen removal during the PDA pattern. Ecologically, stochastic processes dominantly governed the community assembly in two patterns. The PDA pattern improved the interconnectivity of communities, especially for the cooperative behaviors between dominant denitrifying bacteria and low-abundant species. These findings deepen our understanding of the microbial mechanism underlying the different patterns of the coupling system and potentially expand its engineering application.

PH selects for distinct N2O-reducing microbiomes in tropical soil microcosms.

Nitrous oxide (N2O), a greenhouse gas with ozone destruction potential, is mitigated by the microbial reduction to dinitrogen catalyzed by N2O reductase (NosZ). Bacteria with NosZ activity have been studied at circumneutral pH but the microbiology of low pH N2O reduction has remained elusive. Acidic (pH < 5) tropical forest soils were collected in the Luquillo Experimental Forest in Puerto Rico, and microcosms maintained with low (0.02mM) and high (2mM) N2O assessed N2O reduction at pH4.5 and 7.3. All microcosms consumed N2O, with lag times of up to 7months observed in microcosms with 2mM N2O. Comparative metagenome analysis revealed that Rhodocyclaceae dominated in circumneutral microcosms under both N2O feeding regimes. At pH4.5, Peptococcaceae dominated in high-N2O, and Hyphomicrobiaceae in low-N2O microcosms. Seventeen high-quality metagenome-assembled genomes (MAGs) recovered from the N2O-reducing microcosms harbored nos operons, with all eight MAGs derived from acidic microcosms carrying the Clade II type nosZ and lacking nitrite reductase genes (nirS/K). Five of the eight MAGs recovered from pH4.5 microcosms represent novel taxa indicating an unexplored N2O-reducing diversity exists in acidic tropical soils. A survey of pH3.5-5.7 soil metagenome datasets revealed that nosZ genes commonly occur, suggesting broad distribution of N2O reduction potential in acidic soils.

Fungal diversity and key functional gene abundance in Iowa bioretention cells: implications for stormwater remediation potential.

Stormwater bioretention cells are green stormwater infrastructure systems that can help mitigate flooding and remove contaminants. Plants and bacteria improve nutrient removal and degrade organic contaminants; however, the roles of fungi in bioretention cells are less known. Although mycorrhizal fungi aid in plant growth/improve nutrient uptake, there is a notable lack of research investigating fungal diversity in bioretention cells. Other types of fungi could benefit bioretention cells (e.g., white rot fungi degrade recalcitrant contaminants). This study addresses the knowledge gap of fungal function and diversity within stormwater bioretention cells. We collected multiple soil samples from 27 different bioretention cells in temperate-climate eastern Iowa USA, characterized soil physicochemical parameters, sequenced the internal transcribed spacer (ITS) amplicon to identify fungal taxa from extracted DNA, and measured functional gene abundances for two fungal laccases (Cu1, Cu1A) and a fungal nitrite reductase gene (nirKf). Fungal biodegradation functional genes were present in bioretention soils (mean copies per g: 7.4 × 105nirKf, 3.2 × 106Cu1, 4.0 × 108Cu1A), with abundance of fungal laccase and fungal nitrite reductase genes significantly positively correlated with soil pH and organic matter (Pearson's R: >0.39; rho < 0.05). PERMANOVA analysis determined soil characteristics were not significant explanatory variables for community composition (beta diversity). In contrast, planting specifications significantly impacted fungal diversity; the presence/absence of a few planting types and predominant vegetation type in the cell explained 89% of variation in fungal diversity. These findings further emphasize the importance of plants and media as key design parameters for bioretention cells, with implications for fungal bioremediation of captured stormwater contaminants.

Temporal differentiation in the adaptation of functional bacteria to low-temperature stress in partial denitrification and anammox system

Despite reliable nitrite supply through partial denitrification, the adaptation of denitrifying bacteria to low temperatures remains elusive in partial denitrification and anammox (PDA) systems. Here, temporal differentiations of the structure, activity, and relevant cold-adaptation mechanism of functional bacteria were investigated in a lab-scale PDA bioreactor at decreased temperature. Although distinct denitrifying bacteria dominated after low-temperature stress, both short- and long-term stresses exerted differential selectivity towards the species with close phylogenetic distance. Species Azonexus sp.149 showed high superiority over Azonexus sp.384 under short-term stress, and long-term stress improved the adaptation of Aquabacterium sp.93 instead of Aquabacterium sp.184. The elevated transcription of nitrite reductase genes suggested that several denitrifying bacteria (e.g., Azonexus sp.149) could compete with anammox bacteria for nitrite. Species Rivicola pingtungensis and Azonexus sp.149 could adapt through various adaptation pathways, such as the two-component system, cold shock protein (CSP), membrane alternation, and electron transport chain. By contrast, species Zoogloea sp.273 and Aquabacterium sp.93 mainly depended on the CSP and oxidative stress response. This study largely deepens our understanding of the performance deterioration in PDA systems during cold shock and provides several references for efficient adaptation to seasonal temperature fluctuation.

Phyloecology of nitrate ammonifiers and their importance relative to denitrifiers in global terrestrial biomes

Nitrate ammonification is important for soil nitrogen retention. However, the ecology of ammonifiers and their prevalence compared with denitrifiers, being competitors for nitrate, are overlooked. Here, we screen 1 million genomes for nrfA and onr, encoding ammonifier nitrite reductases. About 40% of ammonifier assemblies carry at least one denitrification gene and show higher potential for nitrous oxide production than consumption. We then use a phylogeny-based approach to recruit gene fragments of nrfA, onr and denitrification nitrite reductase genes (nirK, nirS) in 1861 global terrestrial metagenomes. nrfA outnumbers the nearly negligible onr counts in all biomes, but denitrification genes dominate, except in tundra. Random forest modelling teases apart the influence of the soil C/N on nrfA-ammonifier vs denitrifier abundance, showing an effect of nitrate rather than carbon content. This study demonstrates the multiple roles nitrate ammonifiers play in nitrogen cycling and identifies factors ultimately controlling the fate of soil nitrate.

Nitrogen fertilizer alleviates rice yield loss without increasing global warming potential under elevated temperatures

The adverse impacts of climate warming on rice production have become increasingly evident. In this regard, although our previous field studies enabled us to establish the positive impacts of nitrogen fertilizer on rice grain yield during warming, it remains necessary to clarify whether nitrogen fertilizer under elevated temperatures has any adverse effects on the environment during the grain-filling stage. To further investigate the impacts of nitrogen fertilizer on rice yield and the associated emissions of methane (CH4) and nitrous oxide (N2O) under elevated temperatures, we conducted a 2-year paddy field study using a free-air temperature enhancement (FATE) system. FATE system had a favorable warming effect on the canopy and soil, and greatly decreased grain yield by 23 % on average. In two trial years, elevated temperatures significantly increased in CH4 emissions by 17.7 kg ha−1and 18.9 kg ha−1, but did not affect N2O emissions. Consequently, we detected significant increases in global warming potential (GWP) by 52 % and 19 % and greenhouse gas intensity (GHGI) by 94 % and 56 % under elevated temperatures in 2019 and 2020, respectively. Although nitrogen fertilizer had no significant influence on CH4 emissions under elevated temperatures, N2O emissions significantly increased by 1.5 kg ha−1 and 1.4 kg ha−1 in 2019 and 2020. Nevertheless, nitrogen fertilizer did not affect GWP and significantly decreased GHGI by 10 % under elevated temperatures in 2020. Additionally, elevated temperatures significantly increased the abundance of methanogens (mcrA), ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and nitrous oxide reductase (nosZ) genes. Whereas, nitrogen fertilizer significantly increased nitrite reductase (nirS) gene abundance under elevated temperatures. In conclusion, our findings indicated that 60 kg N ha−1 at the heading stage could effectively alleviate the reduction in grain yield attributable to elevated temperatures, without increasing GWP, thereby enhancing the economic and environmental sustainability of paddy field system.

Identification of a Strain Degrading Ammonia Nitrogen, Optimization of Ammonia Nitrogen Degradation Conditions, and Gene Expression of Key Degrading Enzyme Nitrite Reductase

In recent years, nitrogen pollutants have become one of the main causes of water pollution and eutrophication; thus, it is very important to increase the research on nitrogen removal in wastewater. In this study, a bacterium with outstanding ammonia nitrogen degradation capability was isolated from piggery wastewater and identified as Bacillus tequilensis (designated as A2). The ammonia nitrogen degradation rate of A2 reached the highest level (95%) when the incubation temperature was 42 °C, the initial pH was 7, the seed volume was 5%, the rotation speed was 160 r·min−1, the C/N was 10:1, and the carbon source was sodium citrate. A new nitrite reductase gene was successfully expressed in E. coli BL21 (DE3), and the result showed that the enzyme gene contained 2418 bp and 805 encoding amino acids, the recombinant enzyme was purified through an Ni2+ affinity chromatography column, it had a molecular weight of about 94 kDa, it displayed the maximum enzyme activity at 40 °C and pH 6.0, it exhibited good stability in the range of 25 °C to 35 °C, and it showed a pH of 6.0 to 7.0. A 1 mM concentration of Fe3+ promoted the enzyme activity, followed by a 1 mM concentration of Fe2+ and Mg2+. The kinetic parameters of Km, Kcat, and the Vmax of NiR-A2 were calculated to be 1.37 μmol·mL−1, 4.9 × 102 s−1, and 23.75 μmol·mg−1·min−1, respectively. This strain shows good prospects for wastewater treatment, especially in the treatment of high concentration ammonia nitrogen and nitrite degradation, because of its tolerance to and high degradation rate of high concentrations of ammonia nitrogen and high nitrite.

Woronichinia naegeliana: A common nontoxigenic component of temperate freshwater cyanobacterial blooms with 30% of its genome in transposons

Monitoring in the U.S. state of Washington across the period 2007–2019 showed that Woronichinia has been present in many lakes state-wide. This cyanobacterium was commonly dominant or sub-dominant in cyanobacterial blooms in the wet temperate region west of the Cascade Mountains. In these lakes, Woronichinia often co-existed with Microcystis, Dolichospermum and Aphanizomenon flos-aquae and the cyanotoxin microcystin has often been present in those blooms, although it has not been known whether Woronichinia is a toxin producer. We report the first complete genome of Woronichinia naegeliana WA131, assembled from the metagenome of a sample collected from Wiser Lake, Washington, in 2018. The genome contains no genes for cyanotoxin biosynthesis or taste-and-odor compounds, but there are biosynthetic gene clusters for other bioactive peptides, including anabaenopeptins, cyanopeptolins, microginins and ribosomally produced, post-translationally modified peptides. Genes for photosynthesis, nutrient acquisition, vitamin synthesis and buoyancy that are typical of bloom-forming cyanobacteria are present, although nitrate and nitrite reductase genes are conspicuously absent. However, the 7.9 Mbp genome is 3–4 Mbp larger than those of the above-mentioned frequently co-existing cyanobacteria. The increased genome size is largely due to an extraordinary number of insertion sequence elements (transposons), which account for 30.3% of the genome and many of which are present in multiple copies. The genome contains a relatively large number of pseudogenes, 97% of which are transposase genes. W. naegeliana WA131 thus seems to be able to limit the potentially deleterious effects of high rates of recombination and transposition to the mobilome fraction of its genome.

Effect of tillage state of paddy soils with heavy metal pollution on the nosZ gene of N2O reductase

Paddy soils are an important source of atmospheric nitrous oxide (N2O). However, numerous studies have focused on N2O production during the soil tillage period, neglecting the N2O production during the dry fallow period. In this study, we conducted an incubation experiment using the acetylene inhibition technique to investigate N2O emission and reduction rates of paddy soil profiles (0-1 m) from Guangdong Province and Jinlin Province in China, with different heavy-metal pollution levels. The abundance and community structures of denitrifying bacteria were determined via quantitative-PCR and Illumina MiSeq sequencing of nosZ, nirK, and nirS genes. Our results showed that the potential N2O emission rate, N2O production rate, and denitrification rate have decreased with increasing soil vertical depth and heavy-metal pollution. More importantly, we found that the functional gene type of N2O reductase switched with the tillage state of paddy soils, which clade Ⅱ nosZ genes were the dominant gene during the tillage period, while clade Ⅰ nosZ genes were the dominant gene during the dry fallow period. The heavy-metal pollution has less effect on the niche differentiation of the nosZ gene. The N2O emission rate was significantly regulated by the genus Bradyhizobium, which contains both N2O reductase and nitrite reductase genes. Our findings suggests that the nosZ gene of N2O reductase can significantly impact the N2O emission from paddy soils.

Comparative investigation on heterotrophic denitrification driven by different biodegradable polymers for nitrate removal in mariculture wastewater: Organic carbon release, denitrification performance, and microbial community.

Heterotrophic denitrification is widely studied to purify freshwater wastewater, but its application to seawater wastewater is rarely reported. In this study, two types of agricultural wastes and two types of synthetic polymers were selected as solid carbon sources in denitrification process to explore their effects on the purification capacity of low-C/N marine recirculating aquaculture wastewater (NO3 --N 30 mg/L, salinity 32‰). The surface properties of reed straw (RS), corn cob (CC), polycaprolactone (PCL) and poly3-hydroxybutyrate-hydroxypropionate (PHBV) were evaluated by Brunauer-Emmett-Teller, Scanning electron microscope and Fourier-transform infrared spectroscopy. Short-chain fatty acids, dissolved organic carbon (DOC), and chemical oxygen demand (COD) equivalents were used to analyze the carbon release capacity. Results showed that agricultural waste had higher carbon release capacity than PCL and PHBV. The cumulative DOC and COD of agricultural waste were 0.56-12.65 and 1.15-18.75 mg/g, respectively, while those for synthetic polymers were 0.07-1.473 and 0.045-1.425 mg/g, respectively. The removal efficiency of nitrate nitrogen (NO3 --N) was CC 70.80%, PCL 53.64%, RS 42.51%, and PHBV 41.35%. Microbial community analysis showed that Proteobacteria and Firmicutes were the most abundant phyla in agricultural wastes and biodegradable natural or synthetic polymers. Quantitative real-time PCR indicated the conversion from nitrate to nitrogen was achieved in all four carbon source systems, and all six genes had the highest copy number in CC. The contents of medium nitrate reductase, nitrite reductase and nitrous oxide reductase genes in agricultural wastes were higher than those in synthetic polymers. In summary, CC is an ideal carbon source for denitrification technology to purify low C/N recirculating mariculture wastewater.

Spatial and temporal conversion of nitrogen using Arthrobacter sp. 24S4-2, a strain obtained from Antarctica.

According to average nucleotide identity (ANI) analysis of the complete genomes, strain 24S4-2 isolated from Antarctica is considered as a potential novel Arthrobacter species. Arthrobacter sp. 24S4-2 could grow and produce ammonium in nitrate or nitrite or even nitrogen free medium. Strain 24S4-2 was discovered to accumulate nitrate/nitrite and subsequently convert nitrate to nitrite intracellularly when incubated in a nitrate/nitrite medium. In nitrogen-free medium, strain 24S4-2 not only reduced the accumulated nitrite for growth, but also secreted ammonia to the extracellular under aerobic condition, which was thought to be linked to nitrite reductase genes nirB, nirD, and nasA by the transcriptome and RT-qPCR analysis. A membrane-like vesicle structure was detected in the cell of strain 24S4-2 by transmission electron microscopy, which was thought to be the site of intracellular nitrogen supply accumulation and conversion. This spatial and temporal conversion process of nitrogen source helps the strain maintain development in the absence of nitrogen supply or a harsh environment, which is part of its adaption strategy to the Antarctic environment. This process may also play an important ecological role, that other bacteria in the environment would benefit from its extracellular nitrogen source secretion and nitrite consumption characteristics.