N2-fixing Microorganisms Research Articles

Biodegradation of poly-3-hydroxybutyrate after soil inoculation with microbial consortium: Soil microbiome and plant responses to the changed environment

Biodegradable plastics play a vital role in addressing global plastics disposal challenges. Poly-3-hydroxybutyrate (P3HB) is a biodegradable bacterial intracellular storage polymer with substantial usage potential in agriculture. Poly-3-hydroxybutyrate and its degradation products are non-toxic; however, previous studies suggest that P3HB biodegradation negatively affects plant growth because the microorganisms compete with plants for nutrients. One possible solution to this issue could be inoculating soil with a consortium of plant growth-promoting and N-fixing microorganisms. To test this hypothesis, we conducted a pot experiment using lettuce (Lactuca sativa L. var. capitata L.) grown in soil amended with two doses (1 % and 5 % w/w) of P3HB and microbial inoculant (MI). We tested five experimental variations: P3HB 1 %, P3HB 1 % + MI, P3HB 5 %, P3HB 5 % + MI, and MI, to assess the impact of added microorganisms on plant growth and P3HB biodegradation. The efficient P3HB degradation, which was directly dependent on the amount of bioplastics added, was coupled with the preferential utilization of P3HB as a carbon (C) source. Due to the increased demand for nutrients in P3HB-amended soil by microbial degraders, respiration and enzyme activities were enhanced. This indicated an increased mineralisation of C as well as nitrogen (N), sulphur (S), and phosphorus (P). Microbial inoculation introduced specific bacterial taxa that further improved degradation efficiency and nutrient turnover (N, S, and P) in P3HB-amended soil. Notably, soil acidification related to P3HB was not the primary factor affecting plant growth inhibition. However, despite plant growth-promoting rhizobacteria and N2-fixing microorganisms originating from MI, plant biomass yield remained limited, suggesting that these microorganisms were not entirely successful in mitigating the growth inhibition caused by P3HB.

Enhancing Nitrogen Fixating Species as Potential for Crop Nutrition and Yield Stability in Agriculture

Biological nitrogen fixation (BNF) alludes to a microbial intervened process based on an enzymatic "Nitrogenase" change of atmospheric nitrogen (N2) into ammonium promptly absorbable by roots. N2-fixing microorganisms altogether named as "diazotrophs" can fix organically N2 in associate with plant roots.

Electricity-driven bioproduction from CO2 and N2 feedstocks using enriched mixed microbial culture

Microbial electrosynthesis (MES) is an emerging technology with the potential to reduce carbon emissions by converting CO2 and renewable power into chemicals. Here, MES of acetate using gaseous N2 as a nitrogen source is investigated as an approach to substitute fixed nitrogen (ammonia) in the process. At an applied cathode potential of − 0.995 V vs. SHE, the mixed microbial community with a few known N2 and CO2 fixing microorganisms produced 463.5 ± 14 mg/l acetate using gaseous N2 and CO2 feedstock along with traces of ammonia (0.80 ± 0.06 mg/l) confirming the simultaneous fixation of these gases. Up to 85%, 0.5%, and 4.9% electrons were recovered in acetate, ammonia, and biomass, respectively, with a maximum of 39% energetic efficiency. A preliminary analysis suggested the possibility of bringing down a considerable amount of CO2 from going into the environment both by fixing CO2 into acetate and avoiding the use of fixed ammonia, the production of which is associated with the CO2 emissions. Thus, the demonstrated process circumvents the conventional fixed nitrogen (ammonia) usage in MES while still being able to produce considerable acetate from CO2.

Terrestrial-type nitrogen-fixing symbiosis between seagrass and a marine bacterium

Symbiotic N2-fixing microorganisms have a crucial role in the assimilation of nitrogen by eukaryotes in nitrogen-limited environments1–3. Particularly among land plants, N2-fixing symbionts occur in a variety of distantly related plant lineages and often involve an intimate association between host and symbiont2,4. Descriptions of such intimate symbioses are lacking for seagrasses, which evolved around 100 million years ago from terrestrial flowering plants that migrated back to the sea5. Here we describe an N2-fixing symbiont, ‘Candidatus Celerinatantimonas neptuna’, that lives inside seagrass root tissue, where it provides ammonia and amino acids to its host in exchange for sugars. As such, this symbiosis is reminiscent of terrestrial N2-fixing plant symbioses. The symbiosis between Ca. C. neptuna and its host Posidonia oceanica enables highly productive seagrass meadows to thrive in the nitrogen-limited Mediterranean Sea. Relatives of Ca. C. neptuna occur worldwide in coastal ecosystems, in which they may form similar symbioses with other seagrasses and saltmarsh plants. Just like N2-fixing microorganisms might have aided the colonization of nitrogen-poor soils by early land plants6, the ancestors of Ca. C. neptuna and its relatives probably enabled flowering plants to invade nitrogen-poor marine habitats, where they formed extremely efficient blue carbon ecosystems7.

Evidence that nano-TiO2 induces acute cytotoxicity to the agronomically beneficial nitrogen-fixing bacteria Sinorhizobium meliloti.

When nano-sized titanium dioxide (nano-TiO2) absorbs ultra-violet (UV-A) radiation, it produces reactive oxygen species that can be toxic to bacteria. We used the agronomically beneficial nitrogen-fixing bacterium Sinorhizobium meliloti strain 1021 as a model microorganism to detect nano-TiO2 toxicity. Sinorhizobium meliloti was exposed to aqueous dispersions of micrometer-sized TiO2 (micron-TiO2, 44 μm) or nanometer-sized TiO2 (nano-TiO2, 21 nm) at nominal concentrations of 0, 100, 300, 600, 900, and 1800 mg TiO2/L. There were fewer viable S. meliloti cells after exposure to nano-TiO2 under dark and UV-A light conditions. Nano-TiO2 was more toxic to S. meliloti with UV-A irradiation (100% mortality at 100 mg TiO2/L) than under dark conditions (100% mortality at 900 mg TiO2/L). Micron-TiO2 concentrations less than 300 mg TiO2/L had no effect on S. meliloti viability under dark or UV-A light conditions. Exposure to 600 mg/L or more of micron-TiO2 under UV-A light could also photo-kill S. meliloti cells (100% mortality). Further studies are needed to ascertain whether nano-TiO2 interferes with the growth of N2-fixing microorganisms in realistic agricultural environments.

Purple sulfur bacteria fix N2 via molybdenum-nitrogenase in a low molybdenum Proterozoic ocean analogue

Biological N2 fixation was key to the expansion of life on early Earth. The N2-fixing microorganisms and the nitrogenase type used in the Proterozoic are unknown, although it has been proposed that the canonical molybdenum-nitrogenase was not used due to low molybdenum availability. We investigate N2 fixation in Lake Cadagno, an analogue system to the sulfidic Proterozoic continental margins, using a combination of biogeochemical, molecular and single cell techniques. In Lake Cadagno, purple sulfur bacteria (PSB) are responsible for high N2 fixation rates, to our knowledge providing the first direct evidence for PSB in situ N2 fixation. Surprisingly, no alternative nitrogenases are detectable, and N2 fixation is exclusively catalyzed by molybdenum-nitrogenase. Our results show that molybdenum-nitrogenase is functional at low molybdenum conditions in situ and that in contrast to previous beliefs, PSB may have driven N2 fixation in the Proterozoic ocean.

Composition and Activity of N2-Fixing Microorganisms in Mangrove Forest Soils

Mangrove forests are considered to be a highly productive ecosystem, but they are also generally nitrogen (N)-limited. Thus, soil N2 fixation can be important for the stability of both mangrove ecosystem functions and upland N supply. This study evaluates the N2 fixation activity and composition of relevant microorganisms in two coastal mangrove forests—the Guandu mangrove in an upstream estuary and the Bali mangrove in a downstream estuary—using the acetylene reduction method, real-time polymerase chain reaction, and next-generation sequencing. The results demonstrated that ambient nitrogenase activity was higher in downstream mangrove forests (13.2–15.6 nmol h−1 g−1 soil) than in upstream mangrove forests (0.2–1.4 nmol h−1 g−1 soil). However, both the maximum potential nitrogenase activity and nitrogenase gene (nifH gene) copy number were found to be higher in the upstream than in the downstream mangrove forests, implying that the nitrogenase activity and diazotrophic abundance may not necessarily be positively correlated. In addition, amended MoO4 (which inhibits the activity of sulfate-reducing bacteria in N2-fixation) yielded low nitrogenase activity, and sulfate-reducing bacteria made up 20–50% of the relative diazotrophic abundance in the mangrove forests, indicating that these bacteria might be the major active diazotrophs in this environment.

Nitrogen Fixing Azotobacter Species as Potential Soil Biological Enhancers for Crop Nutrition and Yield Stability.

Biological nitrogen fixation (BNF) refers to a microbial mediated process based upon an enzymatic “Nitrogenase” conversion of atmospheric nitrogen (N2) into ammonium readily absorbable by roots. N2-fixing microorganisms collectively termed as “diazotrophs” are able to fix biologically N2 in association with plant roots. Specifically, the symbiotic rhizobacteria induce structural and physiological modifications of bacterial cells and plant roots into specialized structures called nodules. Other N2-fixing bacteria are free-living fixers that are highly diverse and globally widespread in cropland. They represent key natural source of nitrogen (N) in natural and agricultural ecosystems lacking symbiotic N fixation (SNF). In this review, the importance of Azotobacter species was highlighted as both important free-living N2-fixing bacteria and potential bacterial biofertilizer with proven efficacy for plant nutrition and biological soil fertility. In addition, we described Azotobacter beneficial plant promoting traits (e.g., nutrient use efficiency, protection against phytopathogens, phytohormone biosynthesis, etc.). We shed light also on the agronomic features of Azotobacter that are likely an effective component of integrated plant nutrition strategy, which contributes positively to sustainable agricultural production. We pointed out Azotobacter based-biofertilizers, which possess unique characteristics such as cyst formation conferring resistance to environmental stresses. Such beneficial traits can be explored profoundly for the utmost aim to research and develop specific formulations based on inoculant Azotobacter cysts. Furthermore, Azotobacter species still need to be wisely exploited in order to address specific agricultural challenges (e.g., nutrient deficiencies, biotic and abiotic constraints) taking into consideration several variables including their biological functions, synergies and multi-trophic interactions, and biogeography and abundance distribution.

Diazotroph Paenibacillus triticisoli BJ-18 Drives the Variation in Bacterial, Diazotrophic and Fungal Communities in the Rhizosphere and Root/Shoot Endosphere of Maize.

Application of diazotrophs (N2-fixing microorganisms) can decrease the overuse of nitrogen (N) fertilizer. Until now, there are few studies on the effects of diazotroph application on microbial communities of major crops. In this study, the diazotrophic and endospore-forming Paenibacillus triticisoli BJ-18 was inoculated into maize soils containing different N levels. The effects of inoculation on the composition and abundance of the bacterial, diazotrophic and fungal communities in the rhizosphere and root/shoot endosphere of maize were evaluated by sequencing the 16S rRNA, nifH gene and ITS (Inter Transcribed Spacer) region. P. triticisoli BJ-18 survived and propagated in all the compartments of the maize rhizosphere, root and shoot. The abundances and diversities of the bacterial and diazotrophic communities in the rhizosphere were significantly higher than in both root and shoot endospheres. Each compartment of the rhizosphere, root and shoot had its specific bacterial and diazotrophic communities. Our results showed that inoculation reshaped the structures of the bacterial, diazotrophic and fungal communities in the maize rhizosphere and endosphere. Inoculation reduced the interactions of the bacteria and diazotrophs in the rhizosphere and endosphere, while it increased the fungal interactions. After inoculation, the abundances of Pseudomonas, Bacillus and Paenibacillus in all three compartments, Klebsiella in the rhizosphere and Paenibacillus in the root and shoot were significantly increased, while the abundances of Fusarium and Giberella were greatly reduced. Paenibacillus was significantly correlated with plant dry weight, nitrogenase, N2-fixing rate, P solubilization and other properties of the soil and plant.

FeMo3S4 for Efficient Nitrogen Reduction Reaction

In nature, the N2-fixing microorganisms mainly utilize the nitrogenase enzymes to achieve biological N2 fixation, where the FeMoS cofactor functions as the key active site in the enzymatic nitrogen...

Changing perspectives in marine nitrogen fixation.

Nitrogen fixation, the reduction of atmospheric dinitrogen gas (N2) to ammonia, is critical for biological productivity but is difficult to study in the vast expanse of the global ocean. Decades of field studies and the infusion of molecular biological, genomic, isotopic, and geochemical modeling approaches have led to new paradigms and questions. The discovery of previously unknown N2-fixing (diazotrophic) microorganisms and unusual physiological adaptations, combined with diagnostic distributions of nutrients and their isotopes as well as measured and modeled biogeographic patterns, have revolutionized our understanding of marine N2 fixation and its role in the global nitrogen cycle. Anthropogenic upper-ocean warming, increased dissolved carbon dioxide, and acidification will affect the distribution and relative importance of specific subgroups of N2 fixers in the sea; these changes have implications for foodwebs and biogeochemical cycles.

Un-culturable Microbial Community Analysis of Paddy Ecosystem at Different Depths using Nif H Gene DGGE Analysis Approach

Background: Biological N2 fixation is gaining importance in rice ecosystem, because of current concern on the environmental and soil health that are caused by the continuous use of nitrogenous fertilizers and the need for improved sustainable rice productivity. BNF is the conversion of atmospheric nitrogen by certain microorganism’s viz., N2 fixing microorganisms or diazotrophs into forms that plants can use. A substantial molecular diversity of N2 fixing bacteria has been detected in the rice field soil based on retrieval of nif H gene fragment from DNA by molecular approach, i.e., DGGE (Denaturant Gradient Gel Electrophoresis). The present investigation aimed to assess the diversity of N2 fixing bacteria by sequencing and phylogenetic analysis of nif H gene of the metagenomic DNA collected from different depth of flooded rice soil. Methods: In this laboratory investigation, soil samples were collected from the paddy field at different depths viz., rhizosphere region, 5 cm, 10 cm, 15 cm depth using the core sampler during active tillering stage. In the laboratory, the collected soil samples were analyzed by molecular methods involving the use of primer sets based on nif H sequences (nitrogenase reductase). Result: Our investigations in the genetic diversity of diazotrophs associated with rice field soil using nif H revealed the existence of seven discrete bands. The sequencing results showed all sequences were classified as uncultured bacterium clone nitrogenase iron protein (nif H) gene, partial cds. The present works based on the applications of molecular methods have facilitated the study of individual ecosystem and their specific activity.

Effect of mesoscale eddies on diazotroph community structure and nitrogen fixation rates in the South China Sea

Mesoscale eddies play a key role in ocean biogeochemistry, yet their effects on dinitrogen (N2) fixation are not well understood. Using quantitative polymerase chain reaction (qPCR) and dinitrogen-15 (15N2) tracer methods, we examined the abundance of nifH genes in N2-fixing microorganisms and N2 fixation rates in the upper water column (0–100 m) of mesoscale eddies in the northeastern South China Sea (SCS). High N2 fixation rates affected by an anticyclonic eddy were associated with high abundances of unicellular nitrogen-fixing cyanobacteria in groups A and B (UCYN-A, UCYN-B), while low nitrogen fixation was found in both cyclonic eddy and reference stations. The estimated N2 fixation rates met 9% ± 5% of the nitrogen demand for primary production affected by the anticyclonic eddy. Our results indicate that mesoscale eddies can affect nitrogen fixation through affecting nutrient availability and eddy transport, and play a crucial role in regulating N2 fixation in the oligotrophic SCS.

Quantifying Oxygen Management and Temperature and Light Dependencies of Nitrogen Fixation by Crocosphaera watsonii.

Crocosphaera is a major dinitrogen (N2)-fixing microorganism, providing bioavailable nitrogen (N) to marine ecosystems. The N2-fixing enzyme nitrogenase is deactivated by oxygen (O2), which is abundant in marine environments. Using a cellular scale model of Crocosphaera sp. and laboratory data, we quantify the role of three O2 management strategies by Crocosphaera sp.: size adjustment, reduced O2 diffusivity, and respiratory protection. Our model predicts that Crocosphaera cells increase their size under high O2 Using transmission electron microscopy, we show that starch granules and thylakoid membranes are located near the cytoplasmic membranes, forming a barrier for O2 The model indicates a critical role for respiration in protecting the rate of N2 fixation. Moreover, the rise in respiration rates and the decline in ambient O2 with temperature strengthen this mechanism in warmer water, providing a physiological rationale for the observed niche of Crocosphaera at temperatures exceeding 20°C. Our new measurements of the sensitivity to light intensity show that the rate of N2 fixation reaches saturation at a lower light intensity (∼100 μmol m-2 s-1) than photosynthesis and that both are similarly inhibited by light intensities of >500 μmol m-2 s-1 This suggests an explanation for the maximum population of Crocosphaera occurring slightly below the ocean surface.IMPORTANCECrocosphaera is one of the major N2-fixing microorganisms in the open ocean. On a global scale, the process of N2 fixation is important in balancing the N budget, but the factors governing the rate of N2 fixation remain poorly resolved. Here, we combine a mechanistic model and both previous and present laboratory studies of Crocosphaera to quantify how chemical factors such as C, N, Fe, and O2 and physical factors such as temperature and light affect N2 fixation. Our study shows that Crocosphaera combines multiple mechanisms to reduce intracellular O2 to protect the O2-sensitive N2-fixing enzyme. Our model, however, indicates that these protections are insufficient at low temperature due to reduced respiration and the rate of N2 fixation becomes severely limited. This provides a physiological explanation for why the geographic distribution of Crocosphaera is confined to the warm low-latitude ocean.

Experimental warming alters the community composition, diversity, and N2 fixation activity of peat moss (Sphagnum fallax) microbiomes.

Sphagnum‐dominated peatlands comprise a globally important pool of soil carbon (C) and are vulnerable to climate change. While peat mosses of the genus Sphagnum are known to harbor diverse microbial communities that mediate C and nitrogen (N) cycling in peatlands, the effects of climate change on Sphagnum microbiome composition and functioning are largely unknown. We investigated the impacts of experimental whole‐ecosystem warming on the Sphagnum moss microbiome, focusing on N2 fixing microorganisms (diazotrophs). To characterize the microbiome response to warming, we performed next‐generation sequencing of small subunit (SSU) rRNA and nitrogenase (nifH) gene amplicons and quantified rates of N2 fixation activity in Sphagnum fallax individuals sampled from experimental enclosures over 2 years in a northern Minnesota, USA bog. The taxonomic diversity of overall microbial communities and diazotroph communities, as well as N2 fixation rates, decreased with warming (p < 0.05). Following warming, diazotrophs shifted from a mixed community of Nostocales (Cyanobacteria) and Rhizobiales (Alphaproteobacteria) to predominance of Nostocales. Microbiome community composition differed between years, with some diazotroph populations persisting while others declined in relative abundance in warmed plots in the second year. Our results demonstrate that warming substantially alters the community composition, diversity, and N2 fixation activity of peat moss microbiomes, which may ultimately impact host fitness, ecosystem productivity, and C storage potential in peatlands.

Urban traffic changes the biodiversity, abundance, and activity of phyllospheric nitrogen-fixing bacteria.

The phyllosphere provides appropriate conditions for colonization by microorganisms, including diazotrophic bacteria. However, a poor understanding of the effects of the atmospheric environment on the phyllospheric diazotrophic communities persists. We detected the biodiversity, abundance, and activity of nitrogen-fixing bacteria in the phyllospheres of two evergreen shrubs, Nerium indicum Mill. and Osmanthus sp., sampled from urban areas with heavy traffic, a college campus, and a forest. Quantitative PCR analysis indicated that the copy numbers of nifH sequences were highest in the phyllospheres of both plants in heavy-traffic urban areas and correlated with the recorded nitrogenase activities of the phyllospheres. Similarly, the phyllosphere from heavy-traffic urban areas also possessed the highest biodiversity indices of diazotrophic communities from both the two plants. Pyrosequencing analysis revealed a diversity of nifH sequences in phyllosphere that were mostly uniquely found in the phyllosphere, and many of these were proteobacteria-like and cyanobacteria-like. Members of the Proteobacteria, mostly of which were not closely related to unknown organisms, were detected exclusively in the phyllosphere and represented substantial fractions of their associated diazotrophic communities. Our study provides initial insight into the shifts in the biodiversity and community structure of N2-fixing microorganisms in the phyllospheres of different atmospheric environments.

In Situ Diazotroph Population Dynamics Under Different Resource Ratios in the North Pacific Subtropical Gyre.

Major advances in understanding the diversity, distribution, and activity of marine N2-fixing microorganisms (diazotrophs) have been made in the past decades, however, large gaps in knowledge remain about the environmental controls on growth and mortality rates. In order to measure diazotroph net growth rates and microzooplankton grazing rates on diazotrophs, nutrient perturbation experiments and dilution grazing experiments were conducted using free-floating in situ incubation arrays in the vicinity of Station ALOHA in March 2016. Net growth rates for targeted diazotroph taxa as well as Prochlorococcus, Synechococcus and photosynthetic picoeukaryotes were determined under high (H) and low (L) nitrate:phosphate (NP) ratio conditions at four depths in the photic zone (25, 45, 75, and 100 m) using quantitative PCR and flow cytometry. Changes in the prokaryote community composition in response to HNP and LNP treatments were characterized using 16S rRNA variable region tag sequencing. Microzooplankton grazing rates on diazotrophs were measured using a modified dilution technique at two depths in the photic zone (15 and 125 m). Net growth rates for most of the targeted diazotrophs after 48 h were not stimulated as expected by LNP conditions, rather enhanced growth rates were often measured in HNP treatments. Interestingly, net growth rates of the uncultivated prymnesiophyte symbiont UCYN-A1 were stimulated in HNP treatments at 75 and 100 m, suggesting that N used for growth was acquired through continuing to fix N2 in the presence of nitrate. Net growth rates for UCYN-A1, UCYN-C, Crocosphaera sp. (UCYN-B) and the diatom symbiont Richelia (associated with Rhizosolenia) were uniformly high at 45 m (up to 1.6 ± 0.5 d-1), implying that all were growing optimally at the onset of the experiment at that depth. Differences in microzooplankton grazing rates on UCYN-A1 and UCYN-C in 15 m waters indicate that the grazer assemblage preyed preferentially on UCYN-A1. Deeper in the water column (125 m), both diazotrophs were grazed at substantial rates, suggesting grazing pressure may increase with depth in the photic zone. Constraining in situ diazotroph growth and mortality rates are important steps for improving parameterization for diazotrophs in global ecosystem models.

Bacterial Diversity and Nitrogen Utilization Strategies in the Upper Layer of the Northwestern Pacific Ocean.

Nitrogen (N) is a primary limiting nutrient for bacterial growth and productivity in the ocean. To better understand bacterial community and their N utilization strategy in different N regimes of the ocean, we examined bacterial diversity, diazotrophic diversity, and N utilization gene expressions in the northwestern Pacific Ocean (NWPO) using a combination of high-throughput sequencing and real-time qPCR methods. 521 and 204 different operational taxonomic units (OTUs) were identified in the 16s rRNA and nifH libraries from nine surface samples. Of the 16s rRNA gene OTUs, 11.9% were observed in all samples while 3.5 and 15.9% were detected only in N-sufficient and N-deficient samples. Proteobacteria, Cyanobacteria and Bacteroidetes dominated the bacterial community. Prochlorococcus and Pseudoalteromonas were the most abundant at the genus level in N-deficient regimes, while SAR86, Synechococcus and SAR92 were predominant in the Kuroshio-Oyashio confluence region. The distribution of the nifH gene presented great divergence among sampling stations: Cyanobacterium_UCYN-A dominated the N-deficient stations, while clusters related to the Alpha-, Beta-, and Gamma-Proteobacteria were abundant in other stations. Temperature was the main factor that determined bacterial community structure and diversity while concentration of NOX-N was significantly correlated with structure and distribution of N2-fixing microorganisms. Expression of the ammonium transporter was much higher than that of urea transporter subunit A (urtA) and ferredoxin-nitrate reductase, while urtA had an increased expression in N-deficient surface water. The predicted ammonium transporter and ammonium assimilation enzymes were most abundant in surface samples while urease and nitrogenase were more abundant in the N-deficient regions. These findings underscore the fact that marine bacteria have evolved diverse N utilization strategies to adapt to different N habitats, and that urea metabolism is of vital ecological importance in N-deficient regimes.

Functional Single-Cell Approach to Probing Nitrogen-Fixing Bacteria in Soil Communities by Resonance Raman Spectroscopy with 15N2 Labeling.

Nitrogen (N) fixation is the conversion of inert nitrogen gas (N2) to bioavailable N essential for all forms of life. N2-fixing microorganisms (diazotrophs), which play a key role in global N cycling, remain largely obscure because a large majority are uncultured. Direct probing of active diazotrophs in the environment is still a major challenge. Herein, a novel culture-independent single-cell approach combining resonance Raman (RR) spectroscopy with 15N2 stable isotope probing (SIP) was developed to discern N2-fixing bacteria in a complex soil community. Strong RR signals of cytochrome c (Cyt c, frequently present in diverse N2-fixing bacteria), along with a marked 15N2-induced Cyt c band shift, generated a highly distinguishable biomarker for N2 fixation. 15N2-induced shift was consistent well with 15N abundance in cell determined by isotope ratio mass spectroscopy. By applying this biomarker and Raman imaging, N2-fixing bacteria in both artificial and complex soil communities were discerned and imaged at the single-cell level. The linear band shift of Cyt c versus 15N2 percentage allowed quantification of N2 fixation extent of diverse soil bacteria. This single-cell approach will advance the exploration of hitherto uncultured diazotrophs in diverse ecosystems.

Impact of fertilization regimes on diazotroph community compositions and N2-fixation activity in paddy soil

Abstract Nutrient status in soil is crucial for the growth and activity of resident microorganisms, which in turn regulate nitrogen transformation in the terrestrial biosphere. Biological nitrogen fixation (BNF) is an important N input in agricultural ecosystems, but the influence of fertilization on the structural and functional behavior of diazotrophs is not clear. In this study, we assessed the nutrient limitations on the abundance and N2-fixation activity of diazotrophs in a long term (20 years) fertilization experiment. Although both phosphorus (P) deficiency and potassium (K) deficiency resulted in significant decreases in nifH gene expression and N2-fixation activity, P deficiency exhibited more restrictive effects. Assessments of community structures based upon transcripts also indicated that the active diazotroph populations were more sensitive to P deficiency than K deficiency, and some diazotroph groups were detected in the P deficiency treatment. Long-term rice straw addition significantly increased diazotroph abundance, but in contrast, sharply reduced nifH gene expression and N2-fixation activity. The close relationship between N2-fixation activity and nifH gene expression rather than its copy number suggests that the nifH gene transcript level is a suitable indicator for predicting the N2-fixation activity of N2-fixing microorganisms in paddy soil of various fertility status.