nifH Gene Research Articles

Nitrogen fixation and diazotroph diversity in groundwater systems.

Biological nitrogen fixation (BNF), the conversion of N2 into bioavailable nitrogen (N), is the main process for replenishing N loss in the biosphere. However, BNF in groundwater systems remains poorly understood. In this study, we examined the activity, abundance, and community composition of diazotrophs in groundwater in the Hetao Plain of Inner Mongolia using 15N tracing methods, reverse transcription qPCR (RT-qPCR), and metagenomic/metatranscriptomic analyses. 15N2 tracing incubation of near in situ groundwater (9.5-585.4 nmol N L-1 h-1) and N2-fixer enrichment and isolates (13.2-1728.4 nmol N g-1 h-1, as directly verified by single-cell resonance Raman spectroscopy), suggested that BNF is a non-negligible source of N in groundwater in this region. The expression of nifH genes ranged from 3.4 × 103 to 1.2 × 106 copies L-1 and was tightly correlated with dissolved oxygen (DO), Fe(II), and NH4+. Diazotrophs in groundwater were chiefly aerobes or facultative anaerobes, dominated by Stutzerimonas, Pseudomonas, Paraburkholderia, Klebsiella, Rhodopseudomonas, Azoarcus, and additional uncultured populations. Active diazotrophs, which prefer reducing conditions, were more metabolically diverse and potentially associated with nitrification, sulfur/arsenic mobilization, Fe(II) transport, and CH4 oxidation. Our results highlight the importance of diazotrophs in subsurface geochemical cycles.

Biological soil health with conventional and qPCR based indicators under conservation agriculture based rice-wheat cropping system in Indo-Gangetic Plain

Conservation agriculture (CA) entails resource sustainability, crop productivity and climate benefits. We assessed biological soil health index (BSHI) using both conventional and state-of-the-art indicators for a long-term rice-wheat system under a regime of CA-practices in Indo-Gangetic Plains (IGP). The practices include zero till direct seeded rice (ZTDSR)–zero till wheat (ZTW), ZTDSR + wheat residue (WR)-ZTW + rice residue (RR), ZTDSR + WR + sesbania brown manuring (SBM)–ZTW + RR, ZTDSR–ZTW–zero till mungbean (ZTMB), ZTDSR + mungbean residue (MR)–ZTW + RR-ZTMB + WR, transplanted rice (TPR)-conventional till wheat (CTW)–conventional till mungbean (CTMB). Collected soil samples (0–5 cm depth) were analysed for 8 labile organic carbon pools, 8 soil enzyme activities, population of 7 microbes viz., bacteria, fungi and actinomycetes, and two microbial functions. Further, abundance of different phylogenetic groups and nutrient cycling genes was quantified by molecular based qPCR technique. In general, triple/double ZT + crop residues caused an increase in most of the pools of carbon, enzymes, and microbial population including qPCR-led genes in soils. Specifically, inclusion of mungbean residues in triple ZT and sesbania brown manuring in double ZT improved nifH gene abundance over other double and triple ZT treatments. Of the analysed parameters, β-glucosidase, Bacterial amoA, Archaeal 16S rRNA, Bacteroidetes 16S rRNA, Bacterial 16S rRNA, and mineralizable C were screened out as the key indicators of BSHI; its value was maximised under triple ZT with residues (ZTDSR + MR–ZTW + RR–ZTMB + WR) treatment. Attempt may be made to use the screened indicators for assessment of BSHI and upscale the identified practice for rice-wheat system in IGP.

Antioxidant CeO2 doped with carbon dots enhance ammonia production by an electroactive Azospirillum humicireducens SgZ-5T

Microbial nitrogen fixation is a fundamental process in the nitrogen cycle, providing a continuous supply of biologically available nitrogen essential for life. In this study, we combined cerium oxide-doped carbon dots (CeO2/CDs) with electroactive nitrogen-fixing bacterium Azospirillum humicireducens SgZ-5T to enhance nitrogen fixation through ammonium production. Our research demonstrates that treatment of SgZ-5T cells with CeO2/CDs (0.2 mg mL−1) resulted in a 265.70% increase in ammonium production compared to SgZ-5T cells alone. CeO2/CDs facilitate electron transfer in the biocatalytic process, thereby enhancing nitrogenase activity. Additionally, CeO2/CDs reduce the concentration of reactive oxygen species in SgZ-5T cells, leading to increased ammonium production. The upregulation of nifD, nifH and nifK gene expression upon incorporation of CeO2/CDs (0.2 mg mL−1) into SgZ-5T cells supports this observation. Our findings not only provide an economical and environmentally friendly approach to enhance biological nitrogen fixation but also hold potential for alleviating nitrogen fertilizer scarcity.

Characteristics of Microbial Abundance in Rhizosphere and Non-Rhizosphere Soils of Permafrost Peatland, Northeast China

The rhizosphere microenvironment is crucial to plant–soil physiological processes. The differences among microbial communities in the rhizosphere and non-rhizosphere peatland topsoil (0–15 cm) and subsoil (15–30 cm) in five plant communities dominated by Carex schmidtii, Chamaedaphne calyculata, Ledum palustre, Betula fruticosa, and Vaccinium uliginosum, as well as non-rhizosphere soil in discontinuous and continuous permafrost regions, were studied. We found that the bacteria and nifH gene abundances in the C. calyculata rhizosphere soil in the discontinuous permafrost region were higher than those in continuous permafrost region, while the nirK and nifH gene abundances in the non-rhizosphere soil of the discontinuous permafrost region were lower than those in the continuous permafrost region. The ratio of bacteria to fungi decreased and that of nirK to nirS increased significantly from the discontinuous to the continuous permafrost region, indicating that permafrost degradation can change soil microbial community composition. Fungal abundance was higher in the rhizosphere than the non-rhizosphere soils, suggesting that plant roots provide a more suitable environment for fungi. Moreover, the abundances of the topsoil bacteria; the fungi; and the nirK, nirS, and nifH genes were higher than those in the subsoil because of the organic matter from plant litter as a source of nutrients. The microbial abundance in the subsoil was also more affected by nutrient availability. To sum up, the microbial abundance varied among the different types of rhizosphere and non-rhizosphere soils, and the carbon and nitrogen cycling processes mediated by soil microorganisms may be greatly altered due to permafrost degradation under climate warming.

Global oceanic diazotroph database version 2 and elevated estimate of global oceanic N2 fixation

Abstract. Marine diazotrophs convert dinitrogen (N2) gas into bioavailable nitrogen (N), supporting life in the global ocean. In 2012, the first version of the global oceanic diazotroph database (version 1) was published. Here, we present an updated version of the database (version 2), significantly increasing the number of in situ diazotrophic measurements from 13 565 to 55 286. Data points for N2 fixation rates, diazotrophic cell abundance, and nifH gene copy abundance have increased by 184 %, 86 %, and 809 %, respectively. Version 2 includes two new data sheets for the nifH gene copy abundance of non-cyanobacterial diazotrophs and cell-specific N2 fixation rates. The measurements of N2 fixation rates approximately follow a log-normal distribution in both version 1 and version 2. However, version 2 considerably extends both the left and right tails of the distribution. Consequently, when estimating global oceanic N2 fixation rates using the geometric means of different ocean basins, version 1 and version 2 yield similar rates (43–57 versus 45–63 Tg N yr−1; ranges based on one geometric standard error). In contrast, when using arithmetic means, version 2 suggests a significantly higher rate of 223±30 Tg N yr−1 (mean ± standard error; same hereafter) compared to version 1 (74±7 Tg N yr−1). Specifically, substantial rate increases are estimated for the South Pacific Ocean (88±23 versus 20±2 Tg N yr−1), primarily driven by measurements in the southwestern subtropics, and for the North Atlantic Ocean (40±9 versus 10±2 Tg N yr−1). Moreover, version 2 estimates the N2 fixation rate in the Indian Ocean to be 35±14 Tg N yr−1, which could not be estimated using version 1 due to limited data availability. Furthermore, a comparison of N2 fixation rates obtained through different measurement methods at the same months, locations, and depths reveals that the conventional 15N2 bubble method yields lower rates in 69 % cases compared to the new 15N2 dissolution method. This updated version of the database can facilitate future studies in marine ecology and biogeochemistry. The database is stored at the Figshare repository (https://doi.org/10.6084/m9.figshare.21677687; Shao et al., 2022).

Metagenomics reveals taxon-specific responses of soil nitrogen cycling under different fertilization regimes in heavy metal contaminated soil

Soil deficiency, cyclic erosion, and heavy metal pollution have led to fertility loss and ecological function decline in mining areas. Fertilization is an important way to rapidly replenish soil nutrients, which have a major influence on the soil nitrogen cycling process, but different fertilization regimes have different impacts on soil properties and microbial functional potentials. Here, metagenomic sequencing was used to investigate the different responses of key functional genes of microbial nitrogen cycling to fertilization regimes and explore the potential effects of soil physicochemical properties on the key functional genes. The results indicated that AC-HH (ammonium chloride-high frequency and concentration) treatment significantly increased the gene abundance of norC (13.40-fold), nirK (5.46-fold), and napA (5.37-fold). U-HH (urea-high frequency and concentration) treatment significantly increased the gene abundance of hao (6.24-fold), pmoA-amoA (4.32-fold) norC (7.00-fold), nosZ (3.69-fold), and nirK (6.88-fold). Functional genes were distributed differently among the 10 dominant phyla. The nifH and nifK genes were distributed only in Proteobacteria. The hao gene was distributed in Gemmatimonadetes, Nitrospirae and Proteobacteria. Fertilization regimes caused changes in functional redundancy in soil, and nirK and nirB, which are involved in denitrification, were present in different genera. Fertilization regimes with high frequency and high concentration were more likely to increase the gene abundance at the genus level. In summary, this study provides insights into the taxon-specific response of soil nitrogen cycling under different fertilization regimes, where changes in fertilization regimes affect microbial nitrogen cycling by altering soil physicochemical properties in a complex dynamic environment.

Bacillus paralicheniformis 2R5 and its impact on canola growth and N-cycle genes in the rhizosphere.

Chemical fertilization has a negative impact on natural environment. Plant growth-promoting (PGP) rhizobacterial biofertilizers can be a safer alternative to synthetic agrochemicals. In this research, a culture-based method was used to assess the population size of rhizobacteria at the vegetative, flowering, and maturity stages of canola. Rhizobacteria were then isolated from each of the canola growth stages, and their seven PGP traits were determined. The highest abundance of culturable bacteria was found at vegetative stage of the plants. Furthermore, four out of seven PGP traits were produced by the highest % of isolates at vegetative stage. In the greenhouse experiment that included six rhizobacterial strains with best PGP traits, the greatest canola growth promotion ability under sterile conditions was observed after the introduction of Bacillus paralicheniformis 2R5. Moreover, under non-sterile conditions, 2R5 significantly increased canola growth. The presence of trpA, B, C, D, E, F and pstA, S genes in the 2R5 genome could be associated with canola growth promotion abilities. chiA and mbtH genes could contribute to 2R5 antifungal activity against fungal pathogens. Moreover, the introduction of 2R5 significantly increased the abundance of narG, nosZ, nifH, and nirS genes, which can prove that 2R5 strain may be an important member of soil bacterial community.

Improvement of soil acidification and ammonium nitrogen content in tea plantation by long-term use of organic fertilizer.

Soil acidification was common in Chinese tea plantations, which seriously affected the growth of tea trees, and it was of great significance to explore soil remediation in acidified tea plantations for the sustainable development of tea industry. We sought to determine how different fertilizers improve acidified soil and their nitrogen transformation in tea plantation. Different fertilizers were used to treat acidified soils in tea plantations for 4 consecutive years from 2017 to 2021, and the changes in soil pH, indexes related to soil nitrogen transformation and tea yield were analyzed to construct interaction networks of the above indexes and obtain the indexes with the greatest influence on the fertilization effect. Long-term use of sheep manure fertilizer in tea plantation improved soil acidification, increased the soil pH value, enhanced the number and intensity of nitrogen-fixing bacteria and ammonifying bacteria, urease, protease, asparaginase and N-acetamide glucose ribosidase activities, nifH gene expression, reduced the number and intensity of soil nitrifying bacteria and denitrifying bacteria, nitrate reductase and nitrite reductase activities, and the expressions of amoA-AOA, nirK, nirS, narG and nosZ genes, which in turn increased ammonium nitrogen content of the soil, reduced nitrate nitrogen content, and enhanced tea yield. Topsis index weight analysis showed that ammonium nitrogen content in soil had the greatest impact on the fertilization effect. Long-term use of sheep manure fertilizer was beneficial to restore the balance of micro-ecosystem of acidified soil. This study provided an important practical basis for soil remediation and fertilizer management in acidified tea plantation soils.

Endophytic Bacteria; Diversity, Characterization and Role in Agriculture

Entophytic bacteria have an important role in the growth process and health of the plant host. Nevertheless, also some endophytic bacteria are existing in seeds and have not been studied yet. In addition, some Entophytic bacteria are important in plant tolerance to environmental stresses. They can colonize the internal tissues of the host and are able to use a variety of different relations including symbiotic, mutualism, communalistic, and trophobiotic. They have the ability for plant hormone production like auxin, indole acetic acid, and gibberellin; also some endophytic bacteria have the ability for siderophore creation, phosphate solubilization, nitrogen fixation, protease, and hydrogen cyanide formation.. Moreover, they produce compounds that could have possible usage in drug, agriculture or engineering. They have the ability to removesoil toxins thus, improving phytoremediation and soil fertility. Further, most of endophytic bacteria are diazotrophs and associated with the Proteobacteria, and a varied range has been detected agreeing to the nifH gene which codes for nitrogenase enzyme, structures recovered from plant materials, however a limited part of these genes looks to be stated. The endophytes discussed in this review are isolated from surface-disinfested plant tissue, and that do not damage the plant. Moreover, endophytes appear to be in-between saprophytic bacteria and plant pathogens, they are either saprophytes growing to be pathogens, or extremely grown plant pathogens with protective accommodation and nutrient provisions, but not killing their host. Generally, endophytic bacteria are partial under biotic and abiotic influences, with the plant itself being one of the main prompting influences.

Stochastic Processes Dominate in the Water Mass-Based Segregation of Diazotrophs in a High Arctic Fjord (Svalbard).

Nitrogen-fixing or diazotrophic microbes fix atmospheric nitrogen (N2) to ammonia (NH3+) using nitrogenase enzyme and play a crucial role in regulating marine primary productivity and carbon dioxide sequestration. However, there is a lack of information about the diversity, structure, and environmental regulations of the diazotrophic communities in the high Arctic fjords, such as Kongsfjorden. Here, we employed nifH gene sequencing to clarify variations in composition, community structure, and assembly mechanism among the diazotrophs of the salinity-driven stratified waters of Kongsfjorden. The principal environmental and ecological drivers of the observed variations were identified. The majority of the nifH gene sequences obtained in the present study belonged to cluster I and cluster III nifH phylotypes, accounting for 65% and 25% of the total nifH gene sequences. The nifH gene diversity and composition, irrespective of the size fractions (free-living and particle attached), showed a clear separation among water mass types, i.e., Atlantic-influenced versus glacier-influenced water mass. Higher nifH gene diversity and relative abundances of non-cyanobacterial nifH OTUs, affiliated with uncultured Rhizobiales, Burkholderiales, Alteromonadaceae, Gallionellaceae (cluster I) and uncultured Deltaproteobacteria including Desulfuromonadaceae (cluster III), were prevalent in GIW while uncultured Gammaproteobacteria and Desulfobulbaceae were abundant in AIW. The diazotrophic community assembly was dominated by stochastic processes, principally ecological drift, and to lesser degrees dispersal limitation and homogeneous dispersal. Differences in the salinity and dissolved oxygen content lead to the vertical segregation of diazotrophs among water mass types. These findings suggest that water column stratification affects the composition and assembly mechanism of diazotrophic communities and thus could affect nitrogen fixation in the Arctic fjord.

Trade-off strategy for the usage of thiencarbazone-methyl·isoxaflutole on maize fields: Nitrogen-associated microbial response and environmental implications

Due to the lack of knowledge on the mechanism of the effect of new herbicides on nitrogen-functioning microorganisms and nitrogen use efficiency, a balancing strategy for the application of new herbicides in maize fields is rarely proposed. By studying the effects of thiencarbazone-methyl·isoxaflutole (TMI) on nitrogen transformation in soil and nitrogen agronomic efficiency (NAE) of maize, the mechanism of nitrogen functional groups and genes involved was revealed, and controlling factors affecting soil nitrogen transformation were identified. Results showed that on day 18, the nitrate nitrogen (NO3−-N) content increased but ammonium nitrogen (NH4+-N) content decreased with the TMI application. This can be further verified by the positive correlations (p < 0.05) among isoxaflutole residues, “aerobic_ammonia_oxidation” and NO3−-N content. The NH4+-N content increased significantly with TMI application at 10 times the recommended dose (412.5 ml hm−2) on days 30 and 45, which explains the high value of NAE at the high-dose application of TMI. Random forest model and redundancy analysis confirmed the significant impacts of the nifH gene on NH4+-N content and TMI residues on NO3−-N content (p < 0.05). Comprehensively considering the disturbance of nitrogen-related microorganisms and the effects on the NAE of maize, this research proposed a trade-off strategy for the application of new pre-emergence herbicide in maize fields, involving background investigation, experimental design, analytical methods and trade-off indicators. This may be further popularised and achieve a balance between economic and environmental benefits.

Methane consumption under the influence of different nitrogen sources in a tropical soil ecosystem

In order to minimize methane (CH4) concentration in the atmosphere we need to better understand CH4 consumption in agricultural soils. Nitrogen application to agriculture is predicted to increase significantly in the coming years to meet food security needs. However, the interaction between soil nitrogen and CH4 consumption is poorly understood. Experiments were carried out to evaluate CH4 consumption under the influence of the three nitrogen sources comprising N2(at ambient+5% and ambient+10%),NO3-N (at 10 mM and 20 mM) and NH4-N (at 10 mM and 20 mM). CH4 consumption was evaluated at different CH4 concentrations and feeding cycles to validate the effect of nitrogen. Among different N sources, N2 stimulated CH4 consumption potential by about 1.11–1.71 times over that in the absence of additional nitrogen (control), while N in the form of both NO3 and NH4 inhibited CH4 consumption by 1.14–2.18 times than in the control. CH4 consumption rate increased with CH4 feeding cycles. The effect of N sources on CH4 consumption followed similar trends irrespective of the N rate added.N2 stimulated the abundance of both nifH and pmoA genes. Abundance of methanotrophs pmoA gene copies and nitrifiers amoA gene copies were more in NH4-Namended soil than NO3-N.Available NO3 content in soil increased 9–30% with CH4 driven N2 fixation. This study concludes that N2 stimulated CH4 consumption while nitrogen in the form of NO3 and NH4 inhibited CH4 consumption in a tropical vertisol.

Cloning, Prokaryotic Expression and Functional Characterization of NifH Gene from the Associative Nitrogen-Fixing Bacteria Klebsiella Variicola DX120E.

Biological nitrogen fixation (BNF) is a unique mechanism in which microorganisms utilize the nitrogenase enzyme to catalyze the conversion of atmospheric nitrogen (N2) to ammonia (NH3). Fe protein, encoded by the nifH gene, is an essential component of the nitrogenase in Klebsiella variicola DX120E. However, the function of this gene in regulating nitrogen fixing activity is still unclear. The objective of this study was to reveal the function of nifH gene in associative nitrogen-fixing bacteria Klebsiella variicola DX120E and micro-sugarcane system by immunoassay and gene editing. In the current investigation, the nifH gene was cloned in a pET-30a (+) vector and expressed in Escherichia coli. The NifH protein was purified and used to immunize rabbit, and then the serum was collected and purified to obtain rabbit anti-NifH polyclonal antibodies. The CRISPR-Cas9 system was applied to produce nifH mutant strains, and the nitrogen-fixing enzyme activity, gene, and protein expression were analyzed. Both in vitro and in vivo NifH proteins were detected by Western blotting, which were 43 and 32 kDa respectively. The expression of nifD and nifK genes was decreased, and nitrogenase activity was reduced in the nifH mutant strain. The nifH gene mutant weakened the nitrogenase activity by regulating the expression of Fe protein, which suggests a potential strategy to study the nitrogen fixation-related genes and the interactions between endophytic nitrogen-fixing bacteria and sugarcane.

Response of rare and abundant rhizosphere microbial communities to inoculated rhizobium in cadmium-contaminated soil phytoremediation

Soil microorganisms play an important role in maintaining ecosystem balance and exerting ecological functions. The taxonomic and functional changes in rare and abundant communities during in bioremediation of cadmium-contaminated soils and their contributions to the remediation of ecosystem functions remain elusive despite the significance of rare and abundant microbial taxa in maintaining soil ecological function. Mesorhizobiumloti HZ76-inoculated seedlings of the Robinia pseudoacacia were used to create a symbiotic system. The effects of rhizobium inoculation on rare and abundant rhizosphere microbial populations were investigated in pot experiments. Despite having minimal effects on the α-diversity of rare and abundant microbial communities, inoculation altered microbial community composition and functional gene abundance. For example, abundant phyla such as Proteobacteria were enriched following inoculation, whereas rare phyla such as Ascomycota were depleted. After inoculation, the abundance of nifH gene increased, while the abundance of amoA, amoB, nirS, nirK and nosZ gene decreased. Rare bacterial populations were more susceptible to the inoculated rhizobium's environmental alterations. In rare communities compared to abundance communities, the α-diversity was higher. Stochastic processes predominated in the communities of uncommon and abundant microorganisms. Furthermore, keystone taxa, particularly rare bacterial taxa (Gemmatimonadaceae and Saprospiraceae) and rare fungal taxa (Ascomycota), more mediates nitrogen cycle processes and is directly associated to the abundance of nitrogen functional genes. Together, our findings demonstrate the different ways that rare and abundant microbial species react to rhizobia inoculation and their connections to the nitrogen cycle.

Reasonable deep application of sheep manure fertilizer to alleviate soil acidification to improve tea yield and quality.

Soil acidification in Chinese tea plantations is widespread, and it has significantly affected the growth of tea trees; it was important to explore soil remediation of acidified tea plantations in depth for the sustainable development of tea industry. In this study, the effects of sheep manure fertilizer with different application depths on soil acidification, tea yield and quality, and soil nitrogen transformation in tea plantations were analyzed for five consecutive years from 2018 to 2022. The results showed that long-term use of sheep manure fertilizer significantly reduced soil acidification (P< 0.05) in tea plantations, improved soil pH and soil ammonium nitrogen content, enhanced root activity and root nitrogen uptake capacity of tea trees, and thus improved tea yield and quality. The effect of different application depths of sheep manure fertilizer on tea yield and quality was mainly reflected in the transformation ability of soil ammonium nitrogen and nitrate nitrogen, which showed that high transformation ability of soil ammonium nitrogen and high ammonium nitrogen content were beneficial to high tea yield and vice versa, and the best effect was achieved when sheep manure was applied at a depth of 50 cm and 70 cm. The topsis analysis confirmed that sheep manure fertilization had a greater effect on root activity, ammonium nitrogen, ammonia intensity, and nifH gene. This study provided an important practical basis for the restoration of acidified tea plantation soil through sheep manure fertilizer management.

Glacial meltwater and seasonality influence community composition of diazotrophs in Arctic coastal and open waters.

The Arctic Ocean is particularly affected by climate change with unknown consequences for primary productivity. Diazotrophs-prokaryotes capable of converting atmospheric nitrogen to ammonia-have been detected in the often nitrogen-limited Arctic Ocean but distribution and community composition dynamics are largely unknown. We performed amplicon sequencing of the diazotroph marker gene nifH from glacial rivers, coastal, and open ocean regions and identified regionally distinct Arctic communities. Proteobacterial diazotrophs dominated all seasons, epi- to mesopelagic depths and rivers to open waters and, surprisingly, Cyanobacteria were only sporadically identified in coastal and freshwaters. The upstream environment of glacial rivers influenced diazotroph diversity, and in marine samples putative anaerobic sulphate-reducers showed seasonal succession with highest prevalence in summer to polar night. Betaproteobacteria (Burkholderiales, Nitrosomonadales, and Rhodocyclales) were typically found in rivers and freshwater-influenced waters, and Delta- (Desulfuromonadales, Desulfobacterales, and Desulfovibrionales) and Gammaproteobacteria in marine waters. The identified community composition dynamics, likely driven by runoff, inorganic nutrients, particulate organic carbon, and seasonality, imply diazotrophy a phenotype of ecological relevance with expected responsiveness to ongoing climate change. Our study largely expands baseline knowledge of Arctic diazotrophs-a prerequisite to understand underpinning of nitrogen fixation-and supports nitrogen fixation as a contributor of new nitrogen in the rapidly changing Arctic Ocean.

Soil phosphorus drives variation in diazotrophic communities in a subtropical nitrogen-rich forest

Although nitrogen (N)-fixing bacteria (diazotrophs) play a vital role in the biosphere's N cycle. However, large uncertainty exists regarding how atmospheric N deposition regulates biological N fixation (BNF) in soils and which diazotrophic taxa are responsible for this process. We targeted the nifH gene to investigate the effects of long-term N addition on the abundance and composition of diazotrophic communities in subtropical N-rich forests, using real-time quantitative polymerase chain reaction (q-PCR) and MiSeq sequencing. We conducted a field trial for seven years and included three N treatment levels (+0, +50, +150 kg N ha−1 yr−1; CK, LN, HN). With increasing N addition, the average total P concentration in the whole soil profile and the topsoil P availability decreased significantly, further reducing nitrogenase activity and nifH abundance significantly, while increasing the diversity of the diazotrophic community. After seven years of N addition, the relative abundance of uncultured Cyanobacteria significantly decreased, while those of Rhizobiales and Desulfobulbaceae significantly increased. The diazotrophic communities transitioned from free-living N fixers (uncultured Cyanobacteria-dominated) to symbiotic N fixers (Rhizobiales-dominated; HN treatment). The relative abundance of heterotrophic diazotrophic bacteria and the occasional diazotrophic species increased, resulting in diazotrophic bacteria still maintaining high competitiveness in the N-rich and/or P-limited soils. Proteobacteria (e.g., Rhizobiales) had the highest potential to participate in microbial mechanisms triggered by P scarcity. Variation partitioning analyses showed that diazotrophic community composition and diversity were mainly correlated with the soil P availability, with soil AP having the highest individual effect. Overall, this study will broaden our understanding of N fixation mechanisms and the diazotrophic community variation in N-rich forests, while also highlighting soil P as a key factor driving changes in diazotrophic communities.

The key role of denitrification and dissimilatory nitrate reduction in nitrogen pollution along vertical landfill profiles from metagenomic perspective

Landfill are persistent sources of nitrogen (N) pollution even in the decades after closure. However, the biological pathways of N-pollution, particularly N2O and NH4+, at different landfill depths have received little attention. In this study, metagenomic analysis was conducted on landfill refuse from vertical reservoir profiles in two closed landfills named XT and MT. NH4+ concentrations were found to be higher in deeper layers of MT, while greater potential for N2O emissions occurred in XT and the shallow layers of MT. Furthermore, the community structure and function of N-metabolizing microbes were more strongly defined by landfill depth than landfill type. Denitrification, involving abundant nirK and norB genes, was identified as the major pathway for N2O production in both XT and MT-shallow, while dissimilatory nitrate reduction with abundant nirBD genes was identified as the major pathway for NH4+ accumulation. Microbes of norB-type and nirBD-type were positively affected by NO3− in XT, whereas negatively affected by contents of organic material and moisture in MT-shallow. The mechanism by which nitrogen fixation, with abundant nifH genes, contributes to NH4+ accumulation in MT-deep should be further elucidated. These findings can provide a theoretical basis for governing scientific N-pollution control strategies throughout the entire landfill process.

Soil development following glacier retreat shapes metagenomic and metabolomic functioning associated with asynchronous C and N accumulation

Glacier retreat caused by global warming may result in the variation of soil organic carbon and nutrient cycling. Yet, the dynamic change of soil microbial functional profiles, especially C metabolism-related, with soil development following glacier retreat are still unclear. In the present study, we investigated the soil microbial communities, metagenomic functioning, and metabolomic profiles along the Hailuogou Glacier forefield representing a 120-year chronosequence. The alpha diversity indices of soil bacteria, protozoa and nifH genes showed an upward trend with increased soil ages, and the beta diversity of soil archaea, bacteria, fungi, protozoa, nifH and nirS genes were significantly correlated with soil ages, in which increasing soil C and P while decreased C/N and pH significantly contributed to the differences of soil microbial communities among the analyzed environmental variables. The metagenomic functional genes related to the metabolisms of Glycogen and Cellulosome, Iron Acquisition and Metabolism were significantly decreased with chronosequence, while the utilization of Xylose and Lactate, Potassium Metabolism, Sulfur Metabolism showing an upward trend with soil ages, in which soil C/N ratios and pH were the most influential factors. In addition, soil C and C/N ratios were also significantly correlated to metabolomic compositions, in which the complexity of the metabolite structure increased with soil ages. Our results indicate that glacier retreat may lead to the asynchronous C and N accumulation along the chronosequence, thereby affecting the metagenomic and metabolomic functioning of soil microbial communities related to C metabolisms during soil development following glacier retreat.

Low nitrogen fertilization enriches nitrogen‐fixing bacteria in the Brassica seed microbiome of subsequent generations

AbstractIntroductionThe consequences of overfertilization on agricultural and native ecosystems are well‐documented, but the impact of reduced fertilization on plant–microbe interactions is less understood. Here, we analysed the impact of different nitrogen (N) fertilization regimes over two subsequent growing seasons on the seed microbiome of winter oilseed rape (Brassica napus L.).Materials and MethodsSeed microbial diversity, composition, and abundance were analysed by a combined approach of amplicon sequencing, quantitative real‐time PCR, and microscopy. A germination assay was conducted to quantify bacteria and nifH genes in roots.ResultsTwo consecutive years of either low or high N fertilization resulted in a significant shift in seed microbiota; nine discriminating bacterial orders and 32 bacterial features were identified. Among them, potentially N‐fixing bacteria (Rhizobiales and Rhizobium) were identified as biomarkers for low N fertilization. Moreover, quantitative measurements revealed that 16S ribosomal RNA (rRNA) and bacterial nifH gene counts were significantly higher under lower N availability. Relative to total 16S rRNA genes, seeds and roots contained 0.002% and 0.3% nifH genes on average, respectively. The alternation of N levels between the first and the second growing season (high to low, or low to high) had, however, no impact on seed microbiota, suggesting that a low N availability for at least two generations is required to enrich N‐fixing taxa in seeds.ConclusionOur study revealed a flexible, environmentally influenced seed microbiome assembly and suggests innovative microbiome management via biological nitrogen fixation to optimize fertilization regimes in future agriculture.