N2 Fixation Rates Research Articles

Enhancement of nitrogen fixation and diazotrophs by long-term polychlorinated biphenyl contamination in paddy soil

Biological nitrogen fixation (BNF) driven by diazotrophs is a major means of increasing available nitrogen (N) in paddy soil, in addition to anthropogenic fertilization. However, the influence of long-term polychlorinated biphenyl (PCB) contamination on the diazotrophic community and nitrogen fixation in paddy soil is poorly understood. In this study, samples were collected from paddy soil subjected to > 30 years of PCB contamination, and the soil diazotrophic community and N2 fixation rate were evaluated by Illumina MiSeq sequencing and acetylene reduction assays, respectively. The results indicated that high PCB contamination increased diazotrophic abundance and the N2 fixation rate, and altered diazotrophic community structure in the paddy soil. The random forest model demonstrated that the β-diversity of the diazotrophic community was the most significant predictor of the N2 fixation rate. Structure equation modeling identified a specialized keystone diazotrophic ecological cluster, predominated by Bradyrhizobium, Desulfomonile, and Cyanobacteria, as the key driver of N2 fixation. Overall, our findings indicated that long-term PCB contamination enhanced the N2 fixation rate by altering diazotrophic community abundance and structure, which may deepen our understanding of the ecological function of diazotrophs in organic-contaminated soil.

Construction of g-C3N4 with three coordinated nitrogen (N3C) vacancies for excellent photocatalytic activities of N2 fixation and H2O2 production

Nitrogen defect engineering has been confirmed to be an effective strategy to improve photocatalytic performance, and nitrogen vacancies at different positions show different effects on photocatalytic performance. Here, we prepare g-C3N4 with three-coordinate nitrogen (N3C) vacancies (g-C3N4-N3C) by a simple in situ copyrolysis method. The introduction of N3C vacancies helps to narrow the band gap, enhance the light-harvesting efficiency, accelerate the separation and transfer of photogenerated carriers, and provide more active centers. Interestingly, g-C3N4-N3C exhibits photocatalytic performance for the production of NH3 and H2O2. The optimized g-C3N4-N3C-0.3 exhibits the best photocatalytic N2 fixation rate (1915 μmol h−1 g−1) and photocatalytic H2O2 production rate (1098 μmol h−1 g−1), with corresponding apparent quantum efficiency (AQE) of 7.79 % and 12.43 % at λ = 370 nm, respectively, which are superior to most known photocatalysts. Meanwhile, density functional theory (DFT) calculations indicate that g-C3N4-N3C makes the activation and further reduction of *N2 and *O2 more thermodynamically favorable than pure g-C3N4. This work deepens the understanding of the role of nitrogen defect engineering in enhancing the photocatalytic performance of g-C3N4, and provides a new way for the design and preparation of efficient and stable photocatalysts.

Nonconservative behavior of dissolved molybdenum and its potential role in nitrogen cycling in the Bohai and Yellow Seas

Molybdenum plays an important role in marine biological activity, especially in nitrogen cycling as a cofactor for N2 fixation and nitrate reductase. However, the dissolved Mo (dMo) behavior and its interaction with N cycling in the coastal waters is still unclear. In this study, the dMo concentrations and parameters related to Mo distribution and N cycling in surface and bottom seawaters of the Bohai (BS) and Yellow Seas (YS) were examined. The results showed that dMo concentrations ranged from 36.4 nmol L-1 to 125.0 nmol L-1, most of which deviated significantly from the conservative line, indicating nonconservative behavior of Mo relative to salinity. The highest dMo concentrations occurring in 36°N section of north of the South YS (SYS), were close to conservative value (105 nmol L-1). Significant depletion up to 40-50 nmol L-1 of dMo mainly appeared in the BS, NYS and south of the SYS, suggesting the possible removal of dMo by biological utilization and particle adsorption. Particularly, the increasing dMo concentrations away the Yellow River estuary indicated that freshwater dilution was one of reasons for dMo distributions in the BS. The similar spatial distribution of dMo and dissolved Mn concentrations suggested the possible scavenging by MnOx phases for Mo removal. The negative correlation between dMo and chlorophyll-a (Chl-a) concentrations in surface seawaters suggested that biological uptake was involved in dMo removal. The depleted dMo in most of sites corresponded with the higher nitrite concentrations, implying the possible involvement of nitrate reduction process. Although the highest N2 fixation rates and relative abundances of cyanobacteria appeared in 36°N section, corresponding with the conservative dMo, suggesting that Mo may play a minor role in N2 fixation process there. The ten-folds of relative abundance of bacteria with nitrate reduction function than that with N2 fixation function suggested that dMo seems to play more important role in nitration reduction than nitrogen fixation in the BS and YS.

Tailoring Surface Frustrated Lewis Pairs of Mo‐Doped Porous Ultrathin Carbon Nitride for Photocatalytic Nitrogen Fixation

Frustrated Lewis pairs (FLPs), new catalysts with pairs of spatially frustrated Lewis acid and Lewis bases within or between molecules, have aroused high attention due to their strong activation ability for inert small molecules such as CO2 and N2. Herein, Mo is introduced as a Lewis acid site to endow porous ultrathin carbon nitride (g‐C3N4) with FLPs‐like properties. The as‐prepared Mo‐doped porous ultrathin carbon nitride (Mo(0.005)–CN) exhibits N2 fixation rate as high as 479 μmol g−1 h−1, which is seven times more than that of bulk g‐C3N4. This is mainly attributed to the synergistic effect of two aspects. First, Mo(0.005)–CN has an excellent light utilization ability. Second, the charge transfer efficiency between the d orbital electrons of metallic Mo and the g‐C3N4 energy band is enhanced in Mo(0.005)–CN. This work paves the way for designing g‐C3N4 from the perspective of FLPs for high photocatalytic N2 fixation efficiency.

The response of diazotrophs to nutrient amendment in the South China Sea and western North Pacific

Abstract. The availability of iron (Fe) and phosphorus (P) has been shown to be a key factor regulating rates of nitrogen fixation in the western subtropical Pacific. However, the relative importance of Fe and P at finer spatial scales between the northern South China Sea (NSCS) and the western boundary of the North Pacific is poorly constrained. Furthermore, nutrient limitation of specific diazotroph types has not yet been assessed. Here we investigated these unknowns by (i) carrying out measurements of finer-scale spatial variabilities in N2 fixation rates and diazotroph nifH gene abundances throughout these regions and (ii) conducting eight additional Fe and phosphate addition bioassay experiments where both changes in N2 fixation rates and the nifH gene abundances of specific diazotrophs were measured. Overall, nitrogen fixation rates and nifH gene abundances were lower in the NSCS than around the Luzon Strait and the western North Pacific. The nutrient addition bioassay experiments demonstrated that N2 fixation rates in the central NSCS were co-limited by Fe and P, whereas at the western boundary of the North Pacific they were P-limited. Changes in the abundances of nifH in response to nutrient addition varied in how well they correlated with changes in N2 fixation rates, and in six out of eight experiments the largest responses in nifH gene abundances were dominated by either Trichodesmium or UCYN-B (unicellular diazotrophic cyanobacteria group B). In general, nutrient addition had a relatively restricted impact on the composition of the six phylotypes that we surveyed apart from on UCYN-B. This unicellular cyanobacterium group showed increased contribution to the total nifH gene abundance following P addition at sites where N2 fixation rates were P-limited. Our study provides comprehensive evidence of nutrient controls on N2 fixation biogeography in the margin of the western North Pacific. Future research that more accurately constrains nutrient supply rates to this region would be beneficial for resolving what controls diazotroph community structure.

Seasonal dynamic of diazotrophic activity and environmental variables affecting it in the Gulf of Riga, Baltic Sea.

The semi-enclosed Baltic Sea experiences regular summer blooms of diazotrophic cyanobacteria. Previously, it has been conclusively demonstrated that in open nitrogen-limited parts of the Baltic Sea, cyanobacteria successfully fix atmospheric N2. At the same time, diazotrophic activity is still poorly understood in Baltic Sea sub-regions where nitrogen and phosphorus are co-limiting primary production. To address this gap in research, we used the15N tracer method for in situ incubations and measured the N2-fixation rate of heterocyst-forming cyanobacteria and picocyanobacteria in the Gulf of Riga, Baltic Sea, from April to September. Physicochemical variables and phytoplankton community composition were also determined. Our results show that the dominant species of cyanobacteria for this region (Aphanizomenon flosaquae) was present in the phytoplankton community during most of the study period. We also establish that the N2-fixation rate has a strong correlation with the proportion of A. flosaquae biomass containing heterocysts (r = 0.80). Our findings highlight the importance of a heterocyst-focused approach for an accurate diazotrophic activity evaluation that is one of the foundations for future management and protection of the Baltic Sea.

Native Rhizobia Improve Plant Growth, Fix N2, and Reduce Greenhouse Emissions of Sunnhemp More than Commercial Rhizobia Inoculants in Florida Citrus Orchards.

Sunnhemp (Crotalaria juncea L.) is an important legume cover crop used in tree cropping systems, where there is increased interest by growers to identify rhizobia to maximize soil nitrogen (N) inputs. We aimed to isolate and identify native rhizobia and compare their capabilities with non-native rhizobia from commercial inoculants to fix atmospheric dinitrogen (N2), produce and reduce nitrous oxide (N2O), and improve plant growth. Phylogenetic analyses of sequences of the 16S rRNA and recA, atpD, and glnII genes showed native rhizobial strains belonged to Rhizobium tropici and the non-native strain to Bradyrhizobium japonicum. Plant nodulation tests, sequencing of nodC and nifH genes, and the acetylene-dependent ethylene production assay confirmed the capacity of all strains to nodulate sunnhemp and fix N2. Inoculation with native rhizobial strains resulted in significant increases in root and shoot weight and total C and N contents in the shoots, and showed greater N2-fixation rates and lower emissions of N2O compared to the non-native rhizobium. Our results suggest that native rhizobia improve plant growth, fix N2, and reduce greenhouse emissions of sunnhemp more than commercial rhizobia inoculants in Florida citrus orchards.

Nitrogen fixation contribution to nitrogen cycling during cyanobacterial blooms in Utah Lake.

Nitrogen (N) cycling is an essential process in lake systems and N-fixation is an important component of it. Recent studies have also found that nitrate reduction through heterotrophic denitrification in lake systems did not prevent harmful cyanobacterial blooms, but instead, may have favored the dominance of N2-fixing cyanobacteria. The overall objective of this study was to estimate nitrogen fixation rates and the expressions of associated nitrogenase (nif gene) functional gene at several sites at different occasions in freshwater Utah Lake. For comparison purposes, one time sampling was also conducted in the brackish Farmington Bay of Great Salt Lake (GSL). The microbial ecology of the top 20-cm of surface water was investigated to assess the dominant cyanobacterial communities and N-related metabolisms. Our study revealed that Dolichospermum and Nodularia were potential N2-fixers for Utah Lake and brackish Farmington Bay, respectively. The in situ N2-fixation rates were 0-0.73nmolN hr-1L-1 for Utah Lake and 0-0.85nmolN hr-1L-1 for Farmington Bay, and these rates positively correlated with the abundance and expressions of the nif gene. In addition, nitrate reduction was measured in sediment (0.002-0.094mgN VSS-1 hr-1). Significantly positive correlations were found among amoA, nirS and nirK abundance (R=0.56-0.87, p<0.05, Spearman) in both lakes. An exception was the lower nirK gene abundance detected at one site in Farmington Bay where high ammonium retentions were also detected. Based on a mass balance approach, we concluded that the amount of inorganic N loss through denitrification still exceeded the N input by N2-fixation, much like in most lakes, rivers, and marine ecosystems. This indicates that N cycling processes such as denitrification mediated by heterotrophic bacteria contributes to N-export from the lakes resulting in N limitations.

Construction of a 2D/2D g-C3N5/NiCr-LDH Heterostructure to Boost the Green Ammonia Production Rate under Visible Light Illumination.

Photocatalytic N2 fixation has emerged as one of the most useful ways to produce NH3, a useful asset for chemical industries and a carbon-free energy source. Recently, significant progress has been made toward designing efficient photocatalysts to achieve this objective. Here, we introduce a highly active type-II heterojunction fabricated via integrating two-dimensional (2D) nanosheets of exfoliated g-C3N5 with nickel-chromium layered double hydroxide (NiCr-LDH). With an optimized loading of NiCr-LDH on exfoliated g-C3N5, excellent performance is realized for green ammonia synthesis under ambient conditions without any noble metal cocatalyst(s). Indeed, the g-C3N5/NiCr-LDH heterostructure with 2 wt % of NiCr-LDH (CN-NCL-2) exhibits an ammonia yield of about 2.523 mmol/g/h, which is about 7.51 and 2.86 times higher than that of solo catalysts, i.e., NiCr-LDH (NC-L) and exfoliated g-C3N5 (CN-5), respectively, where methanol is used as a sacrificial agent. The enhancement of NH3 evolution by the g-C3N5/NiCr-LDH heterostructure can be attributed to the efficient charge transfer, a key factor to the photocatalytic N2 fixation rate enhancement. Additionally, N2 vacancies present in the system help adsorb N2 on the surface, which improves the ammonia production rate further. The best-performing heterostructure also shows long-term stability with the NH3 production rate remaining nearly constant over 20 h, demonstrating the excellent robustness of the photocatalyst.

Contrasting seasonal patterns and factors regulating biocrust N2-fixation in two Florida agroecosystems.

Biocrusts are communities of microorganisms within the top centimeter of soil, often dominated by phototrophic dinitrogen-fixing (N2-fixing) organisms. They are common globally in arid ecosystems and have recently been identified in agroecosystems. However, unlike natural ecosystem biocrusts, agroecosystem biocrusts receive regular fertilizer and irrigation inputs. These inputs could influence seasonal biocrust N2-fixation and their relationship with soil nutrients in perennial agroecosystems, which is of particular interest given crop management requirements. In this study, biocrust and adjacent bare soil N2-fixation activity was measured in the field during the summer, fall, spring, and winter seasons in a Florida citrus orchard and vineyard using both acetylene reduction assays and 15N2 incubations. Samples were analyzed for microbial and extractable carbon (MBC, EC), nitrogen (MBN, EN), and phosphorus (MBP, EP). In both agroecosystems, biocrusts had greater microbial biomass and extractable nutrients compared to bare soil. The citrus and grape biocrusts were both actively fixing N2, despite crop fertilization, with rates similar to those found in natural arid and mesic systems, from 0.1 to 142 nmol of C2H4 g–1 of biocrust dry weight h–1 (equivalent to 1–401 μmol m–2h–1). Lower soil temperatures and higher EC:EN ratios were associated with higher N2-fixation rates in citrus biocrusts, while higher soil moisture and higher EP were associated with higher N2-fixation rates in grape biocrusts. The N2-fixation activity of these agroecosystem biocrusts indicates the possibility of biocrusts to enhance N cycling in perennial agroecosystems, with potential benefits for crop production.

Contrasting Roles of DOP as a Source of Phosphorus and Energy for Marine Diazotrophs

The oceanic dissolved organic phosphorus (DOP) pool is mainly composed of P-esters and, to a lesser extent, equally abundant phosphonate and P-anhydride molecules. In phosphate-limited ocean regions, diazotrophs are thought to rely on DOP compounds as an alternative source of phosphorus (P). While both P-esters and phosphonates effectively promote dinitrogen (N2) fixation, the role of P-anhydrides for diazotrophs is unknown. Here we explore the effect of P-anhydrides on N2 fixation at two stations with contrasting biogeochemical conditions: one located in the Tonga trench volcanic arc region (“volcano,” with low phosphate and high iron concentrations), and the other in the South Pacific Gyre (“gyre,” with moderate phosphate and low iron). We incubated surface seawater with AMP (P-ester), ATP (P-ester and P-anhydride), or 3polyP (P-anhydride) and determined cell-specific N2 fixation rates, nifH gene abundance, and transcription in Crocosphaera and Trichodesmium. Trichodesmium did not respond to any DOP compounds added, suggesting that they were not P-limited at the volcano station and were outcompeted by the low iron conditions at the gyre station. Conversely, Crocosphaera were numerous at both stations and their specific N2 fixation rates were stimulated by AMP at the volcano station and slightly by 3polyP at both stations. Heterotrophic bacteria responded to ATP and 3polyP additions similarly at both stations, despite the contrasting phosphate and iron availability. The use of 3polyP by Crocosphaera and heterotrophic bacteria at both low and moderate phosphate concentrations suggests that this compound, in addition to being a source of P, can be used to acquire energy for which both groups compete. P-anhydrides may thus leverage energy restrictions to diazotrophs in the future stratified and nutrient-impoverished ocean.

Introduction of exotic species Sonneratia apetala alters diazotrophic community and stimulates nitrogen fixation in mangrove sediments

Mangrove restoration by exotic mangrove species Sonneratia apetala has been practiced as a strategy to reconstruct mangrove wetlands. Diazotrophs in mangrove sediment are nitrogen suppliers for mangrove ecosystems. However, the response of sediment diazotrophs, especially their nitrogen fixation rates, to the introduction of exotic S. apetala remains unclear. In the present study, we compared the variations in sediment physicochemical properties, N2 fixation rate, and the composition of total bacterial and diazotrophic communities in mangrove sediments between exotic species (S. apetala) and three native species (i.e., Aegiceras corniculatum, Lumnitzera racemosa, and Rhizophora apiculata). Our results indicated that the introduction of exotic S. apetala significantly (P < 0.05) increased nutrient levels (including TC, TN, and TS) and improved the N2 fixation rate in sediments. Though S. apetala introduction had not caused significant changes in bacterial and diazotrophic alpha diversity, their community structure and composition were altered. The dominant diazotrophic groups were sulfur-oxidizing (SOB) and sulfate-reducing (SRB) bacteria. The relative abundance of Chromatiaceae (affiliated with SOB) significantly increased (P < 0.05), while the relative abundance of Desulfuromonadaceae (affiliated with SRB) decreased in S. apetala sediments. In addition, several SOB and SRB groups were identified as putative keystone taxa by network analysis, suggesting that SOB/SRB groups potentially played a central role in the biogeochemical cycles involving nitrogen (N) and sulfur (S) of the mangrove ecosystem. Redundancy analysis revealed that C/N and NH4+-N were the key factors (P < 0.05) driving sediment diazotrophic communities. This study provides new insights into the ecological impact of exotic S. apetala introduction, which has important implications for the sustainable management and restoration of mangrove ecosystems.

Chemical Stimulation of Heterocyte Differentiation by the Feather Moss Hylocomium splendens: a Potential New Step in Plant-Cyanobacteria Symbioses.

Cyanobacteria associated with mosses play a key role in the nitrogen (N) cycle in unpolluted ecosystems. Mosses have been found to release molecules that induce morphophysiological changes in epiphytic cyanobionts. Nevertheless, the extent of moss influence on these microorganisms remains unknown. To evaluate how mosses or their metabolites influence N2 fixation rates by cyanobacteria, we assessed the nitrogenase activity, heterocyte frequency and biomass of a cyanobacterial strain isolated from the feather moss Hylocomium splendensand a non-symbiotic strain when they were either growing by themselves, together with H. splendens or exposed to H. splendens water, acetone, ethanol, or isopropanol extracts. The same cyanobacterial strains were added to another moss (Taxiphyllum barbieri) and a liverwort (Monosolenium tenerum) to assess if these bryophytes affect N2 fixation differently. Although no significant increases in nitrogenase activity by the cyanobacteria were observed when in contact with H. splendens shoots, both the symbiotic and non-symbiotic cyanobacteria increased nitrogenase activity as well as heterocyte frequency significantly upon exposure to H. splendens ethanol extracts. Contact with T. barbieri shoots, on the other hand, did lead to increases in nitrogenase activity, indicating low host-specificity to cyanobacterial activity. These findings suggest that H. splendens produces heterocyte-differentiating factors (HDFs) that are capable of stimulating cyanobacterial N2 fixation regardless of symbiotic competency. Based on previous knowledge about the chemical ecology and dynamics of moss-cyanobacteria interactions, we speculate that HDF expression by the host takes place in a hypothetical new step occurring after plant colonization and the repression of hormogonia.

Effects of applying maize stover on soil diazotroph community diminish with depth

Biological nitrogen fixation (BNF) is a vital approach to replenishing soil nitrogen (N) pools by converting atmospheric N2. Agronomic practice of growing legumes frequently includes stover return, which generally induces the growth of N2-fixers (i.e., diazotrophs) and stimulates BNF; however, there is uncertainty regarding the stover return effects along soil depth. Here, soil samples were collected from 0 to 10, 10–20 and 20–30 cm in the peanut (Arachis hypogaea) field experiment (established in 2013) comprising treatments with no fertilizer (CK), maize (Zea mays) stover (MS), and MS + chemical fertilizers (SNPK). The abundance of 16S rRNA and nifH genes was determined by real-time PCR, the rate of N2 fixation in soil (RNfix) was measured by acetylene reduction assay, and diazotroph community structure was explored by high-throughput sequencing. Results showed that maize stover application alone increased nifH gene abundance in the 0–10 cm layer, and combined with chemical fertilizer application increased RNfix in the 0–20 cm layers, affecting diazotroph community structure succession in the 0–20 cm layers. This is consistent with the effects of stover on soil properties such as dissolved organic carbon and nitrogen, soil organic matter (SOM) and carbon-to‑nitrogen ratio, with SOM playing a dominant role in governing the abundance, structure and activity of diazotroph communities in the soil. In addition, total nitrogen (TN) was the key factor in shaping the vertical stratification of diazotroph community structure, the abundance of diazotrophs and bacteria as well as the structure and RNfix of the diazotroph community were highly correlated. This study demonstrated that maize stover incorporation caused significant positive changes in the vertical stratification of soil diazotroph communities; although these effects decreased with soil depth and were minor when stover was combined with chemical fertilizers, they still highlighted the crucial role of stover return in enhancing BNF in soil where legumes were present.

Sinking Trichodesmium fixes nitrogen in the dark ocean.

The photosynthetic cyanobacterium Trichodesmium is widely distributed in the surface low latitude ocean where it contributes significantly to N2 fixation and primary productivity. Previous studies found nifH genes and intact Trichodesmium colonies in the sunlight-deprived meso- and bathypelagic layers of the ocean (200-4000 m depth). Yet, the ability of Trichodesmium to fix N2 in the dark ocean has not been explored. We performed 15N2 incubations in sediment traps at 170, 270 and 1000 m at two locations in the South Pacific. Sinking Trichodesmium colonies fixed N2 at similar rates than previously observed in the surface ocean (36-214 fmol N cell-1 d-1). This activity accounted for 40 ± 28% of the bulk N2 fixation rates measured in the traps, indicating that other diazotrophs were also active in the mesopelagic zone. Accordingly, cDNA nifH amplicon sequencing revealed that while Trichodesmium accounted for most of the expressed nifH genes in the traps, other diazotrophs such as Chlorobium and Deltaproteobacteria were also active. Laboratory experiments simulating mesopelagic conditions confirmed that increasing hydrostatic pressure and decreasing temperature reduced but did not completely inhibit N2 fixation in Trichodesmium. Finally, using a cell metabolism model we predict that Trichodesmium uses photosynthesis-derived stored carbon to sustain N2 fixation while sinking into the mesopelagic. We conclude that sinking Trichodesmium provides ammonium, dissolved organic matter and biomass to mesopelagic prokaryotes.

(Invited, Digital Presentation) Using Electricity to Enhance Microbial Fixation of Nitrogen Gas and Generation of Ammonium

There is a growing need to replace the Haber-Bosch process of converting nitrogen gas (N2) into ammonia (NH3) for fertilizers and fuels. To generate the 190 million tons of NH3 each year through this process, roughly 2–3% of global energy is consumed and over 740 million tons of CO2 released. Microbial processes may provide a low-energy and sustainable alternative. The nitrogenase enzymes in many free-living microorganisms convert N2 into ammonium (NH4 +), generating fixed nitrogen for cellular growth. Attempts to increase NH4 + yields from microorganisms through genetic engineering are seeing signs of success, but there is a lack of methods to increase NH4 + generation rates. For aerobic microorganisms, providing sufficient oxygen gas as a terminal electron acceptor can increase these rates; however, oxygen gas can also shut down the nitrogenase enzyme. To overcome this limitation, we are exploring the ability of electricity to drive N2 fixation in anaerobic, exoelectrogenic bacteria. These bacteria have the unique ability to respire on electrodes in microbial electrochemical technologies (METs). Applying a voltage to METs increases the respiration rates of exoelectrogens. Since the most abundant exoelectrogenic bacteria in METs are affiliated with the Geobacter genus and many known Geobacter species are N2 fixers in soils and sediments, we hypothesized that 1) anode biofilms would exhibit N2 fixation activity, 2) N2 fixation rates would respond to an applied voltage, and 3) NH3 could be generated by inhibiting NH4 + uptake pathways. To test our hypothesis, we set up lab-scale microbial electrolysis cells (MECs) and promoted the growth of a Geobacter-rich anode biofilm using defined medium and operating conditions. We found that MET anode biofilms are capable of fixing N2 while maintaining high current densities over several months. N2 fixation rates increased more than three times to a max of 26 [nmol-ethylene (a proxy for N2 fixation) / min] when the applied voltage increased from 0.7 V to 1.0 V. By adding an NH4 + uptake inhibitor, NH3 was generated and recovered from the MET. Using a pure culture of G. sulfurreducens, we also conducted transcriptomics under N2 fixation conditions in METs operated at two different fixed anode potentials. This approach allowed us to identify what genes were up- and down-regulated under the two conditions. The presence of NH4 + decreased the expression of genes associated with N2 fixation which was expected due to the known sensitivity of nitrogenases to NH4 +. On the other hand, the two anode potentials had a dramatic and unexpected impact on the expression levels of N2 fixation genes. At the low anode potential [−0.15 V vs. standard hydrogen electrode (SHE)], nitrogenase genes were significantly up-regulated, as were genes associated with NH4 + uptake and transport, such as glutamine and glutamate synthetases. Considering that −0.15 V provides less energy to the cells relative to +0.15 V (based on thermodynamic predictions), the results suggest that the cells responded to this highly energy-constrained environment by increasing expression of N2 fixation pathways. Based on our new knowledge of how G. sulfurreducens regulates N2 fixation and NH4 + production, we are currently developing new strains of G. sulfurreducens using the CRISPR platform that can excrete NH4 + during N2 fixation.

High-throughput screening of soybean di-nitrogen fixation and seed nitrogen content using spectral sensing

Symbiotic di-nitrogen fixation of grain legumes has a substantial impact on crop performance, harvest product quality, and nitrogen (N) balance of crop rotations, particularly under organic management regimes. In soybean breeding, selection for increased nitrogen fixation is desirable for improving seed protein content and N balance of cropping systems. However, the lack of high-throughput screening methods for direct measurement of N2 fixation rates prohibits practical breeding efforts. Therefore, hyperspectral canopy reflectance measurement as a field-based phenotyping method was evaluated in three environments for indirect estimation of N fixation and uptake of soil nitrogen in a set of early maturity soybean genotypes exhibiting a wide range in seed protein content. Reflectance spectra were collected in repeated measurements during flowering and early seed filling stages. Subsequently, various spectral reflectance indices (SRIs) were calculated for characterizing nitrogen accumulation of individual genotypes. Moreover, prediction models for seed protein content as an end-of-season target trait were developed utilizing full spectral information in partial-least-square regression (PLSR) models. A number of N-related SRIs calculated from spectral reflectance data recorded at the beginning of the seed filling stage were significantly correlated to seed protein content. The best prediction of seed protein content, however, was achieved in PLSR models (validation R2 = 0.805 across all three environments). Environments lower in initial soil mineral N content appeared as more favorable selection sites in terms of prediction accuracy, because N fixation is not masked by soil N uptake in such environments. Hyperspectral reflectance data proved to be a valuable method for determining genetic variation in crop N accumulation, which might be implemented in high-throughput screening protocols for N fixation in plant breeding programs.

High Daily and Year-Round Variability in Denitrification and Nitrogen Fixation in a Northern Temperate River

Rates of nitrogen (N) cycling processes like denitrification and dinitrogen (N2) fixation, which together are the primary contributors to N2 flux rates from surface waters, may change at different time scales from seasons to weeks to days. Yet, we know little about the magnitude, mechanisms or drivers of these temporal changes, especially at shorter daily and weekly timescales. Quantifying variation in rates and drivers across temporal scales is essential to understand how nutrient cycling processes operate in aquatic ecosystems and predict how they may respond to shifting seasonal dynamics caused by global change (i.e., earlier snowmelt and extreme weather events). This study quantified denitrification and N2 fixation rates seasonally and daily in a northern temperate river, and explored how environmental conditions such as discharge, light, and N and phosphorus (P) concentrations were related to that variation at different time scales. We measured denitrification and N2 fixation rates on biweekly and daily intervals at a single 20-m long sampling reach in the Pilgrim River in Michigan's Upper Peninsula from May 2017 through May 2019. We found high rates of daily change (difference in rate from one day to the next) for both processes in all seasons (maximum daily change 5,690 μg N/m2/h for denitrification and 38 μg N/m2/h for N2 fixation). No detectable differences in rates among seasons were detected using Multiple Response Permutation Procedure (MRPP). Day-to-day variation did not change before and after elevated discharge events, including a 1,000-year flood that occurred in June 2018. Partial least squares (PLS) regression identified total dissolved N, dissolved organic N, and ammonium as important predictors of denitrification and N2 fixation, but explained only 15–28% of the variation in all measured rates. The unexpectedly high daily variation and lack of seasonal difference in rates found in this study demonstrate the need to use caution when studying these processes and/or extrapolating rates across time scales, as discrete and infrequent measurements may be misleading.

N2 fixation is less sensitive to changes in soil water content than carbon and net nitrogen mineralization

Biogeochemical processes catalyzed by soil microorganisms depend on the soil water content (SWC). Yet, little is known about the sensitivity of non-symbiotic N2 fixation to changes in SWC in comparison to carbon (C) and net nitrogen (N) mineralization. Therefore, we determined the rates of N2 fixation, C mineralization, and net N mineralization in soils at five SWCs that were created by water addition to the field-moist soil (rewetting), resulting in SWCs of 20, 40, 60 and 80% of the soil water holding capacity (WHC) which correspond to 14, 28, 42, and 57% water-filled pore space (WFPS). The soils originated from an arid, semiarid, Mediterranean, and humid site located in the Coastal Cordillera of Chile. The N2 fixation rate was measured immediately after the adjustment of the SWC and additionally after a five-day pre-incubation period. We found that the rates of all three processes where higher in the rewetted soils compared to the field-moist soils, but the sensitivity to changes in SWC differed between the processes. N2 fixation tended not to increase with increasing SWC beyond 20% WHC. Furthermore, the N2 fixation rate increased faster after rewetting in the soils of the semiarid and Mediterranean zone than in the soil of the humid zone. In contrast, C mineralization reached the highest rate at 80% WHC in the soil of the humid zone and at 60% and 80% WHC in the soils of the other three climate zones. The net N mineralization rate was highest at 40% and 60% WHC in the soils of the semiarid and Mediterranean zone, and was significantly decreased at 80% WHC compared to 60% WHC. Our study shows, first, that N2 fixation is less sensitive to changes in SWC than C and net N mineralization and tends to be already at its maximum in a given soil at low SWC, i.e., 20% WHC. This is likely because the diffusion of N2 in soil does not increase with the SWC, in contrast to the diffusion of organic solutes which are the substrate of C and N mineralization. Second, our results indicate that the N2 fixation rate increases more quickly in response to rewetting of soils of the more arid climate zones than of the humid zone, which might indicate a microbial adaptation to the climate conditions. The results have important implications since they suggest that the N2 fixation rate is likely less affected than the C and net N mineralization rates by decreases in the SWC that might occur more frequently in the future due to climate change.

Dark Diazotrophy during the Late Summer in Surface Waters of Chile Bay, West Antarctic Peninsula.

Although crucial for the addition of new nitrogen in marine ecosystems, dinitrogen (N2) fixation remains an understudied process, especially under dark conditions and in polar coastal areas, such as the West Antarctic Peninsula (WAP). New measurements of light and dark N2 fixation rates in parallel with carbon (C) fixation rates, as well as analysis of the genetic marker nifH for diazotrophic organisms, were conducted during the late summer in the coastal waters of Chile Bay, South Shetland Islands, WAP. During six late summers (February 2013 to 2019), Chile Bay was characterized by high NO3− concentrations (~20 µM) and an NH4+ content that remained stable near 0.5 µM. The N:P ratio was approximately 14.1, thus close to that of the Redfield ratio (16:1). The presence of Cluster I and Cluster III nifH gene sequences closely related to Alpha-, Delta- and, to a lesser extent, Gammaproteobacteria, suggests that chemosynthetic and heterotrophic bacteria are primarily responsible for N2 fixation in the bay. Photosynthetic carbon assimilation ranged from 51.18 to 1471 nmol C L−1 d−1, while dark chemosynthesis ranged from 9.24 to 805 nmol C L−1 d−1. N2 fixation rates were higher under dark conditions (up to 45.40 nmol N L−1 d−1) than under light conditions (up to 7.70 nmol N L−1 d−1), possibly contributing more than 37% to new nitrogen-based production (≥2.5 g N m−2 y−1). Of all the environmental factors measured, only PO43- exhibited a significant correlation with C and N2 rates, being negatively correlated (p < 0.05) with dark chemosynthesis and N2 fixation under the light condition, revealing the importance of the N:P ratio for these processes in Chile Bay. This significant contribution of N2 fixation expands the ubiquity and biological potential of these marine chemosynthetic diazotrophs. As such, this process should be considered along with the entire N cycle when further reviewing highly productive Antarctic coastal waters and the diazotrophic potential of the global marine ecosystem.