Soil Biological Nitrogen Fixation Research Articles

Intensive N2 fixation accelerates microbial turnover in cropland soils

Biological nitrogen fixation (BNF) is strongly affected by the carbon (C) and nitrogen (N) stoichiometry in soil and depends on the input of organic C. Due to the high metabolic costs of nitrogenase activity, however, the response of BNF to organic C input and its impact on microbial turnover remain unclear. To address this knowledge gap, we combined 15N2 tracing with high-throughput sequencing by adding glucose or glucose plus mineral N fertilizer for a 12-day incubation in three cropland soils. Glucose addition alone strongly changed the BNF activity (0.76–2.51 mg N kg−1 d−1), while BNF was completely absent after mineral N fertilization. This switch-on of BNF by glucose addition supported equally high rates of microbial growth and organic C mineralization compared with the direct mineral N assimilation by microorganisms. Glucose-induced BNF was predominantly catalyzed by Azotobacter-affiliated free-living diazotrophs (>50 % of the total nifH genes), which increased with diverse nondiazotrophs such as Nitrososphaera, Bacillus and Pseudoxanthomonas. Structural equation models (SEMs) and random forest (RF) analyses consistently revealed that the soil C:N ratio and Azotobacter-affiliated diazotrophic abundances were the key factors affecting glucose-induced BNF. Our findings emphasize the importance of free-living diazotrophs for microbial turnover of organic C in soil.

Effects of Rhizophagus intraradices and Acinetobacter calcoaceticus on Soybean Growth and Carbendazim Residue

In agricultural production, carbendazim and other pesticides are used to prevent soybean root rot. However, carbendazim degrades slowly and affects a series of biochemical processes such as soil biological nitrogen fixation and soil nutrient activation in the natural environment. This study mainly investigated the effects of Rhizophagus intraradices, Acinetobacter calcoaceticus, and carbendazim on soybean biomass, the incidence of root rot, the total number of bacterial colonies and phosphorus-solubilizing bacteria in rhizosphere soil, and carbendazim residue in soybean grains and rhizosphere soil. The results showed that the arbuscular mycorrhizal fungi (AMF) spore density, AMF infection rate, soybean biomass, nodule number, total bacterial colonies, and phosphorus-solubilizing bacteria colonies in the soybean rhizosphere soil were the highest in the R. intraradices and A. calcoaceticus treatment groups under natural soil conditions. Moreover, the incidence of root rot and carbendazim residue in soybean grains and rhizosphere soil were the lowest in the R. intraradices and A. calcoaceticus treatment group under natural soil conditions. This result indicated that R. intraradices and A. calcoaceticus can effectively reduce carbendazim residue in soybean grains and rhizosphere soil. This study provided theoretical support for the development of microbial fertilizer and microbial degradation of pesticide residues and improved the practical basis for ensuring food safety.

Synergistic effects of diazotrophs and arbuscular mycorrhizal fungi on soil biological nitrogen fixation after three decades of fertilization.

Biological nitrogen (N) fixation (BNF) via diazotrophs is an important ecological process for the conversion of atmospheric N to biologically available N. Although soil diazotrophs play a dominant role in BNF and arbuscular mycorrhizal fungi (AMF) serve as helpers to favor BNF, the response of soil BNF and diazotrophic communities to different long-term fertilizations and the role of AMF in diazotrophs-driven BNF are poorly understood. Herein, a 33-year fertilization experiment in a wheat-maize intercropping system was conducted to investigate the changes in soil BNF rates, diazotrophic and AMF communities, and their interactions after long-term representative fertilization (chemical fertilizer, cow manure, wheat straw, and green manure). We found a remarkable increase in soil BNF rates after more than three decades of fertilization compared with nonfertilized soil, and the green manure treatment rendered the highest enhancement. The functionality strengthening was mainly associated with the increase in the absolute abundance of diazotrophs and AMF and the relative abundance of the key ecological cluster of Module #0 (gained from the co-occurrence network of diazotrophic and AMF species) with dominant diazotrophs such as Skermanella and Azospirillum. Furthermore, although the positive correlations between diazotrophs and AMF were reduced under long-term organic fertilization regimes, green manuring could reverse the decline within Module #0, and this had a positive relationship with the BNF rate. This study suggests that long-term fertilization could promote N fixation and select specific groups of N fixers and their helpers in certain areas. Our work provides solid evidence that N fixation and certain groups of diazotrophic and AMF taxa and their interspecies relationship will be largely favored after the fertilized strategy of green manure.

Compound-specific amino acid 15N-stable isotope probing for the quantification of biological nitrogen fixation in soils

Biological nitrogen fixation (BNF) performed by diazotrophs is vital to our understanding of ecosystem functions, as plant nitrogen (N) is commonly a limiting nutrient for primary productivity. However, significant limitations have remained in our knowledge of the controls and rates of this process, due to technical difficulties in directly quantifying nitrogen (N2) fixation rates. To address this, we developed a novel compound-specific 15N-stable isotope probing method involving analysis of acid hydrolysable soil amino acids (AAs) by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the quantification of BNF in soils. By analysing 15N-enriched AAs (as N-acetyl, O-isopropyl derivatives), this new approach aimed to provide greater specificity compared to existing methods, and to contribute previously unobtainable quantitative information on the capture and flow of N2 fixed in soils. Laboratory incubations using 15N2 gas were carried out on surface peat over 15 days to obtain quantitative measures of N2 fixation by the microbial community. Longer incubations with the addition of a glucose energy source significantly increased the level of 15N enrichment, i.e. N fixed. The enhanced detection limits of 15N-AAs by GC-C-IRMS, compared to bulk soil δ15N value determinations, was key to assessments of N2 fixation. Valuable insights into the assimilation pathway of the applied 15N2-substrate were revealed; for peat soils, 15N incorporation into glutamate dominated over other AAs.

Alpine meadow degradation depresses soil nitrogen fixation by regulating plant functional groups and diazotrophic community composition

Biological nitrogen fixation (BNF), a function performed by diazotrophic microbes, plays an essential role in nitrogen (N) bioavailability in terrestrial ecosystems. However, little is known about the effects of meadow degradation on soil BNF and diazotrophic communities in alpine meadows. We investigated changes in soil BNF and their potential drivers in alpine meadows along a degradation gradient on the Tibetan Plateau (non-degraded, lightly degraded, moderately degraded, and severely degraded meadows) using real-time quantitative PCR and amplicon sequencing. Soil BNF rates decreased significantly along the meadow degradation gradient with a range of 17.34–79.84 nmol C2H4 g−1 dry soil d−1 across all sites. The highest BNF rate in the non-degraded meadow was 1.5–4.6-fold higher than that in the degraded meadows. The abundance and diversity of diazotrophs measured by nifH abundance and Shannon diversity was also decreased in the degraded meadows, accompanied by decreases in plant biomass, soil moisture, and nutrient content (C, N). Soil BNF rate was correlated with plant biomass, soil nutrient content, and diazotrophic abundance (including Nostoc, Scytonema, Rhodopseudomonas, and unidentified genera within the Rhizobiales and Proteobacteria). The community composition of diazotrophs differed markedly among sites with different levels of degradation, with both autotrophic (Cyanobacteria) and heterotrophic (Proteobacteria) diazotrophs contributing significantly to BNF. The plant functional groups, particularly the sedge family, were the primary drivers of soil BNF rates via mediating soil moisture, nutrient content (dissolved organic C and N), nifH gene abundance, and diazotrophic community composition. Our results reveal the main drivers of decreased BNF during alpine meadow degradation and emphasize the importance of plant functional groups in shaping the diazotrophic community and regulating the BNF rate. This information can be applied to the restoration of degraded meadow ecosystems.

High Resistance of a Sludge Enriched with Nitrogen-Fixing Bacteria to Ammonium Salts and Its Potential as a Biofertilizer.

The increasing use of chemical fertilizers causes the loss of natural biological nitrogen fixation in soils, water eutrophication and emits more than 300 Mton CO2 per year. It also limits the success of external bacterial inoculation in the soil. Nitrogen fixing bacteria can be inhibited by the presence of ammonia as its presence can inhibit biological nitrogen fixation. Two aerobic sludges from wastewater treatment plants (WWTP) were exposed to high ammonium salts concentrations (>450 mg L−1 and >2 dS m−1). Microbial analysis after treatment through 16S pyrosequencing showed the presence of Fluviicola sp. (17.70%), a genus of the Clostridiaceae family (11.17%), and Azospirillum sp. (10.42%), which were present at the beginning with lower abundance. Denaturing gradient gel electrophoresis (DGGE) analysis based on nifH genes did not show changes in the nitrogen-fixing population. Nitrogen-Fixing Bacteria (NFB) were identified and associated with other microorganisms involved in the nitrogen cycle, presumably for survival at extreme conditions. The potential use of aerobic sludges enriched with NFB is proposed as an alternative to chemical fertilizer as this bacteria could supplement nitrogen to the plant showing competitive results with chemical fertilization.

Molybdenum, phosphorus, and pH do not constrain nitrogen fixation in a tropical forest in the southeastern Amazon.

High rates of biological nitrogen fixation (BNF) are commonly reported for tropical forests, but most studies have been conducted in regions that receive substantial inputs of molybdenum (Mo) from atmospheric dust and sea-salt aerosols. Even in these regions, the low availability of Mo can constrain free-living BNF catalyzed by heterotrophic bacteria and archaea. We hypothesized that in regions where atmospheric inputs of Mo are low and soils are highly weathered, such as the southeastern Amazon, Mo would constrain BNF. We also hypothesized that the high soil acidity, characteristic of the Amazon Basin, would further constrain Mo availability and therefore soil BNF. We conducted two field experiments across the wet and dry seasons, adding Mo, phosphorus (P), and lime alone and in combination to the forest floor in the southeastern Amazon. We sampled soils and litter immediately, and then weeks and months after the applications, and measured Mo and P availability through resin extractions and BNF with the acetylene reduction assay. The experimental additions of Mo and P increased their availability and the lime increased soil pH. While the combination of Mo and P increased BNF at some time points, BNF rates did not increase strongly or consistently across the study as a whole, suggesting that Mo, P, and soil pH are not the dominant controls over BNF. In a separate short-term laboratory experiment, BNF did not respond strongly to Mo and P even when labile carbon was added. We postulate that high nitrogen (N) availability in this area of the Amazon, as indicated by the stoichiometry of soils and vegetation and the high nitrate soil stocks, likely suppresses BNF at this site. These patterns may also extend across highly weathered soils with high N availability in other topographically stable regions of the tropics.

Depression of soil nitrogen fixation by drying soil in a degraded alpine peatland

Soil biological nitrogen fixation (BNF) represents a major pathway through which nitrogen enters pristine peatlands. Many peatlands have been undergoing human-induced changes in environmental factors, while environmental changes dramatically affect the community composition and activity of nitrogen-fixing prokaryotes (i.e., diazotrophs). However, the impact of peatland degradation on soil BNF remains unclear. By carrying out a field campaign, we examined how soil BNF varies along a natural gradient from pristine marshes to moderately-degraded meadows and sandy meadows on the Zoige Plateau. Plant and topsoil samples from four pristine marshes, three moderately-degraded meadows, and three sandy meadows were collected to determine the potential rate of nitrogen fixation (RNfix), abundance of the nifH gene, diazotrophic community composition, and soil and plant characteristics. Our results showed that topsoil RNfix varied in the range 0.018–3.00 μmol N g d.w.−1 day−1 (i.e. 21.74–1632.37 mg N m−2 day−1) across the ten sites, being lowest in sandy meadows and highest in pristine marshes. Topsoil RNfix and diazotrophic abundance were positively correlated with soil water content, sedge cover, plant biomass, soil organic carbon content, and total nitrogen and phosphorus contents. Soil water content, which affected most plant and soil characteristics, had dominant influences on the abundance and community structure of diazotrophs. The RNfix was closely correlated with the abundance of dominant diazotroph groups. The community composition of diazotrophs differed markedly among sites of different degradation levels. Proteobacteria was the most abundant diazotrophic phylum across the ten sites. Heterotrophic diazotrophs acted as the major contributor to BNF, especially in pristine marshes and moderately-degraded meadows. We conclude that soil water content was the main factor driving the depressed soil BNF during peatland degradation in Zoige, due to soil water effects on plant cover and biomass, soil organic carbon and total phosphorus, and the abundance and community structure of diazotrophs.

Impact of pyrochar and hydrochar on soybean (Glycine max L.) root nodulation and biological nitrogen fixation

AbstractThe aim of this study was to identify effects of carbonized organic material (“biochar”) on soybean growth, root nodulation and biological nitrogen fixation, and to elucidate possible underlying mechanisms.Soybean (Glycine max L.) was grown in four arable soils amended with carbonized organic material produced from wood or maize as feedstocks, by pyrolysis (“pyrochar”) or hydrothermal carbonization (“hydrochar”). Nodulation by Bradyrhizobium, biological nitrogen fixation (BNF) assessed by 15N techniques, plant growth, nutrient uptake and changes in chemical soil properties after soil amendment were determined. Data were analyzed by means of a three way ANOVA on the factors soil, carbonization technique and feedstock. It turned out that soybean root nodulation and BNF was influenced by the carbonization technique used to prepare the soil amendment. Hydrochar, in average and across all soils, increased nodule dry matter and BNF by factors of 3.4 and 2.3, respectively, considerably more than pyrochar, which led to 1.8 and 1.2 fold increases, respectively. Nodule dry matter and BNF correlated positively with available soil sulfur and negatively with available soil nitrogen. Hydrochars provided more available sulfur than pyrochars, and hydrochars caused a decrease in nitrogen availability in the soil solution, thereby exerting a positive influence on nodulation and BNF. Pyrochar amendment increased soil pH but had no effect on nodulation and BNF. Plant growth was affected by the soil and by the feedstock used for the “biochar”, and increased slightly more in treatments with pyrochar and hydrochar made from maize, which was richer in nitrogen and potassium.The results show that carbonized organic materials, and specifically hydrochar, have the capacity to increase BNF in soils. We suggest that this enhancement in BNF in response to soil amendments with carbonized organic materials is due to an increase in available sulfur and a reduction of available soil nitrogen.

Effect of Humic Acids on Nodulation and Dry Matter Production of Soybean, Peanut, and Clover

AbstractHumic substances have been reported to increase plant growth. A stimulating effect on the growth of legumes may improve biological nitrogen fixation in soils. The present investigation studied dry matter production, nodulation and N content in nodules of soybean (Glycine max L., ‘Bragg’), peanut (Arachis hypogaea L., ‘Florunner’), and clover (Trifolium vesiculosum, ‘Arrow leaf’), grown in sand cultures containing a fertilizer mixture and 0 to 800 mg kg−1 (ppm) of fulvic acid (FA) or humic acid (HA). The results indicated that FA and HA were capable of stimulating dry matter production in the plants. Dry weights of roots and nodules showed a tendency to increase over the control at 100 to 400 mg kg−1 (ppm) of FA or HA. The increases in total dry matter production were statistically significant at 800 mg kg−1 (ppm) of FA or HA, and a positive correlation was noticed between increases in dry weights of shoots, roots, and nodules and FA and HA concentrations. Fewer nodules were produced by plants receiving FA and HA treatments than the control, but the nodule mass was greater with the FA and HA treatments. The N content of nodules was little affected by the treatments, although a tendency can be noticed that 100 mg kg−1 (ppm) of FA had decreased the N content in the nodules of both soybean and peanut plants.