Exploring the influence of atmospheric CO2 and O2 levels on the utility of nitrogen isotopes as proxy for biological N2 fixation.

Biological N2 fixation (BNF) is traced to the Archean. The nitrogen isotopic fractionation composition (δ15N) of sedimentary rocks is commonly used to reconstruct the presence of ancient diazotrophic ecosystems. While δ15N has been validated mostly using organisms grown under present-day conditions; it has not under the pre-Cambrian conditions, when atmospheric pO2 was lower and pCO2 was higher. Here, we explore δ15N signatures under three atmospheres with (i) elevated CO2 and no O2 (Archean), (ii) present-day CO2, and O2 and (iii) future elevated CO2, in marine and freshwater, heterocytous cyanobacteria. Additionally, we augment our data set from literature for more generalized dependencies of δ15N and the associated fractionation factor epsilon (ε = δ15Nbiomass - δ15NN2) during BNF in Archaea and Bacteria, including cyanobacteria, and habitats. The ε ranges between 3.70‰ and -4.96‰ with a mean ε value of -1.38 ± 0.95‰, for all bacteria, including cyanobacteria, across all tested conditions. The expanded data set revealed correlations of isotopic fractionation of BNF with CO2 concentrations, toxin production, and light, although within 1‰. Moreover, correlation showed significant dependency of ε to species type, C/N ratios and toxin production in cyanobacteria, albeit it within a small range (-1.44 ± 0.89‰). We therefore conclude that δ15N is likely robust when applied to the pre-Cambrian-like atmosphere, stressing the strong cyanobacterial bias. Interestingly, the increased fractionation (lower ε) observed in the toxin-producing Nodularia and Nostoc spp. suggests a heretofore unknown role of toxins in modulating nitrogen isotopic signals that warrants further investigation.IMPORTANCENitrogen is an essential element of life on Earth; however, despite its abundance, it is not biologically accessible. Biological nitrogen fixation is an essential process whereby microbes fix N2 into biologically usable NH3. During this process, the enzyme nitrogenase preferentially uses light 14N, resulting in 15N depleted biomass. This signature can be traced back in time in sediments on Earth, and possibly other planets. In this paper, we explore the influence of pO2 and pCO2 on this fractionation signal. We find the signal is stable, especially for the primary producers, cyanobacteria, with correlations to CO2, light, and toxin-producing status, within a small range. Unexpectedly, we identified higher fractionation signals in toxin-producing Nodularia and Nostoc species that offer insight into why some organisms produce these N-rich toxic secondary metabolites.

Development of Modular Expression Across Phylogenetically Distinct Diazotrophs.

Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs closely associate with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression by testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrated genome editing in Klebsiella michiganensis M5al.

Phylogenetic diversity of Rhizobium species recovered from nodules of common beans (Phaseolus vulgaris L.) in fields in Uganda: R. phaseoli, R. etli, and R. hidalgonense.

A total of 75 bacterial isolates were obtained from nodules of beans cultivated across 10 sites in six agro-ecological zones in Uganda. Using recA gene sequence analysis, 66 isolates were identified as members of the genus Rhizobium, while nine were related to Agrobacterium species. In the recA gene tree, most Rhizobium strains were classified into five recognized species. Phylogenetic analysis based on six concatenated sequences (recA-rpoB-dnaK-glnII-gyrB-atpD) placed 32 representative strains into five distinct Rhizobium species, consistent with the species groups observed in the recA gene tree: R. phaseoli, R. etli, R. hidalgonense, R. ecuadorense, and R. sophoriradicis, with the first three being the predominant. The rhizobial strains grouped into three nodC subclades within the symbiovar phaseoli clade, encompassing strains from distinct phylogenetic groups. This pattern reflects the conservation of symbiotic genes, likely acquired through horizontal gene transfer among diverse rhizobial species. The 32 representative strains formed symbiotic relationships with host beans, while the Agrobacterium strains did not form nodules and lacked symbiotic genes. Multivariate analysis revealed that species distribution was influenced by the environmental factors of the sampling sites, emphasizing the need to consider these factors in future effectiveness studies to identify effective nitrogen-fixing strains for specific locations.

Soil diazotrophs sustain nitrogen fixation under high nitrogen enrichment via adjustment of community composition.

Biological nitrogen (N) fixation, an important pathway of N, inputs from the atmosphere to Earth's ecosystems, is well demonstrated to decline under N input. However, it remains unclear why N fixers sustain N fixation in many forests under high atmospheric N deposition. To address this knowledge gap, we analyzed the response of the diazotroph community to low N loads (short-term and low N addition; 3-year N addition at the rates of 25-50 kg N ha-1 year-1) vs high loads (chronic and high N addition; 9-year N addition at the rate of 150 kg N ha-1 year-1) in forest soils using high-throughput sequencing. Rates of N fixation decreased under low and high N loads (by 13%-27% and 10%-12%, respectively). Richness and alpha diversity (ACE and Chao1) of the soil diazotroph community decreased under low but not high N loads. Approximately 67.1%-74.4% of the nifH gene sequences at the OTU level overlapped between the control and low N loads, but only 52.0%-53.6% of those overlapped between the control and high N loads, indicating a larger shift of diazotroph community composition under high N loads. Low N loads increased soil NH4+ concentrations, which decreased diazotroph community richness, diversity, and N fixation rates, whereas the increased soil NH4+ concentrations under high N loads did not have negative impacts on the structure and function of the diazotroph community. These findings indicate that diazotrophs sustain N fixation under high N deposition via adjustment of their community composition in forest soils. IMPORTANCE: This study examined the changes in soil diazotroph community under different loads of simulated N deposition and analyzed its relationship with N fixation rates in in five forests using high-throughput sequencing. The magnitudes of N fixation rates reduced by low N loads were higher than those by high N loads. Low N loads decreased richness and diversity of diazotroph community, whereas diazotroph community structure remained stable under high N loads. Compared with low N loads, high N loads resulted in a less similarity and overlap of nifH gene sequences among the treatments and a larger adjustment of diazotroph community. Low N loads increased soil NH4+ concentrations, which decreased diazotroph community richness, diversity, and N fixation rates, whereas the increased soil NH4+ under high N loads did not have negative impacts on diazotroph community structure and N fixation. Based on these findings, it is urgently needed to incorporate the loads of N deposition and the composition of diazotroph community into terrestrial N-cycling models for accurate understanding of N inputs in forest ecosystems.

Metagenomic insights into nitrogen cycling functional gene responses to nitrogen fixation and transfer in maize-peanut intercropping.

The fixation and transfer of biological nitrogen from peanuts to maize in maize-peanut intercropping systems play a pivotal role in maintaining the soil nutrient balance. However, the mechanisms through which root interactions regulate biological nitrogen fixation and transfer remain unclear. This study employed a 15N isotope labelling method to quantify nitrogen fixation and transfer from peanuts to maize, concurrently elucidating key microorganisms and genera in the nitrogen cycle through metagenomic sequencing. The results revealed that biological nitrogen fixation in peanut was 50 mg and transfer to maize was 230 mg when the roots interacted. Moreover, root interactions significantly increased nitrogen content and the activities of protease, dehydrogenase (DHO) and nitrate reductase in the rhizosphere soil. Metagenomic analyses and structural equation modelling indicated that nrfC and nirA genes played important roles in regulating nitrogen fixation and transfer. Bradyrhizobium was affected by soil nitrogen content and DHO, indirectly influencing the efficiency of nitrogen fixation and transfer. Overall, our study identified key bacterial genera and genes associated with nitrogen fixation and transfer, thus advancing our understanding of interspecific interactions and highlighting the pivotal role of soil microorganisms and functional genes in maintaining soil ecosystem stability from a molecular ecological perspective.

Developed Rhizobium Strains Enhance Soil Fertility and Yield of Legume Crops in Haryana, India.

Three strains of Gram-negative bacterium, Rhizobium, were developed by gamma (γ)-irradiation random mutagenesis. The developed strains were evaluated for their augmented features for symbiotic association, nitrogen fixation, and crop yield of three leguminous plants-chickpea, field-pea, and lentil-in agricultural fields of the northern Indian state of Haryana. Crops treated with developed mutants exhibited significant improvement in plant features and the yield of crops when compared to the control-uninoculated crops and crops grown with indigenous or commercial crop-specific strains of Rhizobium. This improvement was attributed to generated mutants, MbPrRz1 (on chickpea), MbPrRz2 (on lentil), and MbPrRz3 (on field-pea). Additionally, the cocultured symbiotic response of MbPrRz1 and MbPrRz2 mutants was found to be more pronounced on all three crops. The statistical analysis using Pearson's correlation coefficients revealed that nodulation and plant biomass were the most related parameters of crop yield. Among the effectiveness of developed mutants, MbPrRz1 yielded the best results for all three tested crops. Moreover, the developed mutants enhanced macro- and micronutrients of the experimental fields when compared with fields harboring the indigenous rhizobial community. These developed mutants were further genetically characterized, predominantly expressing nitrogen fixation marker, nifH, and appeared to belong to Mesorhizobium ciceri (MbPrRz1) and Rhizobium leguminosarum (both MbPrRz2 and MbPrRz3). In summary, this study highlights the potential of developed Rhizobium mutants as effective biofertilizers for sustainable agriculture, showcasing their ability to enhance symbiotic relationships, crop yield, and soil fertility.

Nitrogen-fixing bacteria promote growth and bioactive components accumulation of Astragalus mongholicus by regulating plant metabolism and rhizosphere microbiota.

BACKGROUND: The excessive application of chemical fertilizers in the cultivation of Astragalus mongholicus Bunge results in a reduction in the quality of the medicinal plant and compromises the sustainable productivity of the soil. PGPB inoculant is a hot topic in ecological agriculture research. In the cultivation of Astragalus mongholicus, the screened nitrogen-fixing bacteria can promote plant growth, however, whether it can promote the accumulation of main bioactive components remains unknown. In this study, mixed inoculants containing 5 strains of growth promoting bacteria (Rhizobium T16 , Sinorhizobium T21 , Bacillus J1 , Bacillus G4 and Arthrobacter J2) were used in the field experiment. The metabolic substances in the root tissues of Astragalus mongholicus were identified during the harvest period by non-targeted metabolomics method, and the differential metabolites between groups were identified by statistical analysis. Meanwhile, high-throughput sequencing was performed to analyze the changes of rhizosphere soil and endophytic microbial community structure after mixed microbial treatment. RESULTS: The results of non-targeted metabolism indicated a significant increase in the levels of 26 metabolites after treatment including 13 flavonoids, 3 saponins and 10 other components. The contents of three plant hormones (abscisic acid, salicylic acid and spermidine) also increased after treatment, which presumed to play an important role in regulating plant growth and metabolism. Studies on endosphere and rhizosphere bacterial communities showed that Rhzobiaceae, Micromonosporaceae, and Hypomicrobiaceae in endophytic, and Oxalobactereae in rhizosphere were significantly increased after treatment. These findings suggest their potential importance in plant growth promotion and secondary metabolism regulation. CONCLUSIONS: This finding provides a basis for developing nitrogen-fixing bacteria fertilizer and improving the ecological planting efficiency of Astragalus mongholicus.

Response of N, P, and metal ions in deep soil layers to long-term cultivation of rubber and rubber-based agroforestry systems.

The growing demand for natural rubber products has driven the expansion of rubber plantations in recent decades. While much attention has been given to studying the long-term effects of rubber and rubber-based agroforestry systems on surface soil properties, there has been a tendency to overlook changes in soil properties in deeper layers. Our study addresses this gap by examining alterations in nitrogen (N), phosphorus (P), and metal ion levels in deep soil layers resulting from the prolonged cultivation of rubber and rubber-based agroforestry systems. We found notable shifts in soil NH4+ and NO3- concentrations within the 0-30 cm soil layer across different-aged rubber and rubber-based agroforestry systems. Particularly in mature systems, NO3- and available P levels were close to zero below 30 cm soil depth. Introducing Flemingia macrophylla into young rubber plantations increased soil NH4+ and NO3- in the 0-90 cm soil layer and available P in the 0-10 cm soil layer. Over the long term, cultivation of rubber plantations increased the depletion of total P in the 0-50 cm soil layer, available iron (Fe) and manganese (Mn) in the 30-90 cm soil layer, available copper (Cu) and zinc (Zn) in the 0-90 cm soil layer, accompanied by a decrease in soil pH and increase in exchangeable aluminum (Al) in the 0-90 cm soil layer. Notably, soil exchangeable Al levels exceeding 2.0 cmol kg-1 appeared to induce aluminum toxicity. Furthermore, soil pH below 5.2 triggered a sharp release of exchangeable Al within the 0-90 cm soil layer of rubber plantations, with soil available P nearing zero when exchangeable Al levels assed 7.3 cmol kg-1. Our findings underscore the profound impact of long-term rubber plantation cultivation on surface and deep soil properties. Addressing soil degradation in these deep soil layers poses significant challenges for future soil restoration efforts.

Sulfate-reducing consortium HQ23 stabilizes metal(loid)s and activates biological N-fixation in mixed heavy metal-contaminated soil.

Sulfate-reducing bacteria (SRB) are used in the remediation of mine pollution; however, the mechanism of stabilizing multiple heavy metal(loid)s by the SRB consortium under low oxygen conditions needs further study. Indigenous microflora were extracted from non-ferrous metal-contaminated soil co-inoculated with enriched SRB consortium and assembled as the HQ23 consortium. The presence of Desulfovibrio (SRB) in HQ23 was confirmed by 16S rRNA sequencing and qPCR. The effects of culture media, dissolved oxygen (DO), SO42¯, and pH on the HQ23 growth rate, and the SO42¯-reducing activity were examined. Data indicates that the HQ23 sustained SRB function under low DO conditions (3.67 ± 0.1 mg/L), but the SRB activity was inhibited at high DO content (5.75 ± 0.39 mg/L). The HQ23 can grow from pH 5 to pH 9 and can decrease mobile or bioavailable Cr, Cu, and Zn concentrations in contaminated soil samples. FTIR revealed that Cu and Cr adsorbed to similar binding sites on bacteria, likely decreasing bacterial Cu toxicity. Increased abundances of DSV (marker for Desulfovibrio) and nifH (N-fixation) genes were observed, as well as an accumulation of nitrate-N content in soils suggesting that HQ23 stimulates the biological N-fixation in soils. This study strongly supports the future application of SRB for the bioremediation of heavy metal-polluted sites.

Nitroplasts suggest the creation of artificial nitrogen-fixing eukaryotes.

It is believed that nitrogen-fixing eukaryotes do not exist in nature, and constructing such eukaryotes is extremely challenging. Coale et al., however, have identified the first eukaryote capable of fixing nitrogen through a nitroplast organelle. Understanding the eukaryotic nitrogen-fixing machinery may advance the development of artificial nitrogen-fixing crops and industrial yeasts.

Differential Mechanisms of Microbial As(III) and Sb(III) Oxidation and Their Contribution to Tailings Reclamation.

Mine tailings are extremely oligotrophic environments frequently contaminated with elevated As and Sb, making As(III) and Sb(III) oxidation potentially important energy sources for the tailing microbiome. Although they have been proposed to share similar metabolic pathways, a systemic comparison of the As(III) and Sb(III) oxidation mechanisms and energy utilization efficiencies requires further elucidation. In this study, we employed a combination of physicochemical, molecular, and bioinformatic analyses to compare the kinetic and genetic mechanisms of As(III) and Sb(III) oxidation as well as their respective energy efficiencies for fueling the key nutrient acquisition metabolisms. Thiobacillus and Rhizobium spp. were identified as functional populations for both As(III) and Sb(III) oxidation in mine tailings by DNA-stable isotope probing. However, these microorganisms mediated As(III) and Sb(III) oxidation via different metabolic pathways, resulting in preferential oxidation of Sb(III) over As(III). Notably, both As(III) and Sb(III) oxidation can facilitate nitrogen fixation and phosphate solubilization in mine tailings, with Sb(III) oxidation being more efficient in powering these processes. Thus, this study provided novel insights into the microbial As(III) and Sb(III) oxidation mechanisms and their respective nutrient acquisition efficiencies, which may be critical for the reclamation of mine tailings.

Combined dynamic transcriptome and flavonoid metabolome reveal the role of Mo nanoparticles in the nodulation process in soybean.

Symbiotic nitrogen fixation can reduce the impact of agriculture on the environment by reducing fertilizer input. The rapid development of nanomaterials in agriculture provides a new prospect for us to improve the biological nitrogen fixation ability of leguminous crops. Molybdenum is an important component of nitrogenase, and the potential application of MoO3NPs in agriculture is largely unexplored. In this study, on the basis of verifying that MoO3NPs can improve the nitrogen fixation ability of soybean, the effects of MoO3NPs on the symbiotic nitrogen fixation process of soybean were investigated by using dynamic transcriptome and targeted metabolome techniques. Here we showed that compared with conventional molybdenum fertilizer, minute concentrations of MoO3NPs (0.01-0.1 mg kg-1) could promote soybean growth and nitrogen fixation efficiency. The nodules number, fresh nodule weight and nitrogenase activity of 0.1 mg kg-1 were increased by 17 %, 14 % and 27 %, and plant nitrogen accumulation increased by 17 %. Compared with conventional molybdenum fertilizer, MoO3NPs had a greater effect on apigenin, kaempferol and other flavonoid, and the expression of nodulation related genes such as ENOD93, F3'H. Based on WGCNA analysis, we identified a core gene GmCHS9 that was positively responsive to molybdenum and was highly expressed during MoO3NPs induced nodulation. MoO3NPs could improve the nitrogen fixation ability of soybean by promoting the secretion of flavonoids and the expression of key genes. This study provided a new perspective for the nano-strengthening strategy of nodules development and flavonoid biosynthesis by molybdenum.

Estuarine mangrove niches select cultivable heterotrophic diazotrophs with diverse metabolic potentials-a prospective cross-dialog for functional diazotrophy.

Introduction: Biological nitrogen fixation (BNF), an unparalleled metabolic novelty among living microorganisms on earth, globally contributes ~88-101 Tg N year-1 to natural ecosystems, ~56% sourced from symbiotic BNF while ~22-45% derived from free-living nitrogen fixers (FLNF). The success of symbiotic BNF is largely dependent on its interaction with host-plant, however ubiquitous environmental heterotrophic FLNFs face many limitations in their immediate ecological niches to sustain unhindered BNF. The autotrophic FLNFs like cyanobacteria and oceanic heterotrophic diazotrophs have been well studied about their contrivances acclimated/adapted by these organisms to outwit the environmental constraints for functional diazotrophy. However, FLNF heterotrophs face more adversity in executing BNF under stressful estuarine/marine/aquatic habitats. Methods: In this study a large-scale cultivation-dependent investigation was accomplished with 190 NCBI accessioned and 45 non-accessioned heterotrophic FLNF cultivable bacterial isolates (total 235) from halophilic estuarine intertidal mangrove niches of Indian Sundarbans, a Ramsar site and UNESCO proclaimed World Heritage Site. Assuming ~1% culturability of the microbial community, the respective niches were also studied for representing actual bacterial diversity via cultivation-independent next-generation sequencing of V3-V4 rRNA regions. Results: Both the studies revealed a higher abundance of culturable Gammaproteobacteria followed by Firmicutes, the majority of 235 FLNFs studied belonging to these two classes. The FLNFs displayed comparable selection potential in media for free nitrogen fixers and iron-oxidizing bacteria, linking diazotrophy with iron oxidation, siderophore production, phosphorus solubilization, phosphorus uptake and accumulation as well as denitrification. Discussion: This observation validated the hypothesis that under extreme estuarine mangrove niches, diazotrophs are naturally selected as a specialized multidimensional entity, to expedite BNF and survive. Earlier metagenome data from mangrove niches demonstrated a microbial metabolic coupling among C, N, P, S, and Fe cycling in mangrove sediments, as an adaptive trait, evident with the co-abundant respective functional genes, which corroborates our findings in cultivation mode for multiple interrelated metabolic potential facilitating BNF in a challenging intertidal mangrove environment.

Recent progress and potential future directions to enhance biological nitrogen fixation in faba bean (Vicia faba L.).

The necessity for sustainable agricultural practices has propelled a renewed interest in legumes such as faba bean (Vicia faba L.) as agents to help deliver increased diversity to cropped systems and provide an organic source of nitrogen (N). However, the increased cultivation of faba beans has proven recalcitrant worldwide as a result of low yields. So, it is hoped that increased and more stable yields would improve the commercial success of the crop and so the likelihood of cultivation. Enhancing biological N fixation (BNF) in faba beans holds promise not only to enhance and stabilize yields but also to increase residual N available to subsequent cereal crops grown on the same field. In this review, we cover recent progress in enhancing BNF in faba beans. Specifically, rhizobial inoculation and the optimization of fertilizer input and cropping systems have received the greatest attention in the literature. We also suggest directions for future research on the subject. In the short term, modification of crop management practices such as fertilizer and biochar input may offer the benefits of enhanced BNF. In the long term, natural variation in rhizobial strains and faba bean genotypes can be harnessed. Strategies must be optimized on a local scale to realize the greatest benefits. Future research must measure the most useful parameters and consider the economic cost of strategies alongside the advantages of enhanced BNF.

Overexpression of GmPAP4 Enhances Symbiotic Nitrogen Fixation and Seed Yield in Soybean under Phosphorus-Deficient Condition.

Legume crops establish symbiosis with nitrogen-fixing rhizobia for biological nitrogen fixation (BNF), a process that provides a prominent natural nitrogen source in agroecosystems; and efficient nodulation and nitrogen fixation processes require a large amount of phosphorus (P). Here, a role of GmPAP4, a nodule-localized purple acid phosphatase, in BNF and seed yield was functionally characterized in whole transgenic soybean (Glycine max) plants under a P-limited condition. GmPAP4 was specifically expressed in the infection zones of soybean nodules and its expression was greatly induced in low P stress. Altered expression of GmPAP4 significantly affected soybean nodulation, BNF, and yield under the P-deficient condition. Nodule number, nodule fresh weight, nodule nitrogenase, APase activities, and nodule total P content were significantly increased in GmPAP4 overexpression (OE) lines. Structural characteristics revealed by toluidine blue staining showed that overexpression of GmPAP4 resulted in a larger infection area than wild-type (WT) control. Moreover, the plant biomass and N and P content of shoot and root in GmPAP4 OE lines were also greatly improved, resulting in increased soybean yield in the P-deficient condition. Taken together, our results demonstrated that GmPAP4, a purple acid phosphatase, increased P utilization efficiency in nodules under a P-deficient condition and, subsequently, enhanced symbiotic BNF and seed yield of soybean.

Deciphering Microbial Community and Nitrogen Fixation in the Legume Rhizosphere.

Nitrogen is the most limiting factor in crop production. Legumes establish a symbiotic relationship with rhizobia and enhance nitrogen fixation. We analyzed 1,624 rhizosphere 16S rRNA gene samples and 113 rhizosphere metagenomic samples from three typical legumes and three non-legumes. The rhizosphere microbial community of the legumes had low diversity and was enriched with nitrogen-cycling bacteria (Sphingomonadaceae, Xanthobacteraceae, Rhizobiaceae, and Bacillaceae). Furthermore, the rhizosphere microbiota of legumes exhibited a high abundance of nitrogen-fixing genes, reflecting a stronger nitrogen-fixing potential, and Streptomycetaceae and Nocardioidaceae were the predominant nitrogen-fixing bacteria. We also identified helper bacteria and confirmed through metadata analysis and a pot experiment that the synthesis of riboflavin by helper bacteria is the key factor in promoting nitrogen fixation. Our study emphasizes that the construction of synthetic communities of nitrogen-fixing bacteria and helper bacteria is crucial for the development of efficient nitrogen-fixing microbial fertilizers.

Biochar addition and reduced irrigation modulates leaf morpho-physiology and biological nitrogen fixation in faba bean-ryegrass intercropping.

Intercropping legume with grass has potential to increase biomass and protein yield via biological N2-fixation (BNF) benefits, whereas the joint effects of biochar (BC) coupled with deficit irrigation on intercropping systems remain elusive. A 15N isotope-labelled experiment was implemented to investigate morpho-physiological responses of faba bean-ryegrass intercrops on low- (550 °C, LTBC) or high-temperature BC (800 °C, HTBC) amended sandy-loam soil under full (FI), deficit (DI) and partial root-zone drying irrigation (PRD). LTBC and HTBC significantly reduced intrinsic water-use efficiency (WUE) by 12 and 14 %, and instantaneous WUE by 8 and 16 %, respectively, in faba bean leaves, despite improved photosynthetic (An) and transpiration rate (Tr), and stomatal conductance (gs). Compared to FI, DI and PRD lowered faba bean An, gs and Tr, but enhanced leaf-scale and time-integrated WUE as proxied by the diminished shoots Δ13C. PRD enhanced WUE as lower gs, Tr and guard cell length than DI-plants. Despite higher carbon ([C]) and N concentration ([N]) in faba bean shoots amended by BC, the aboveground C- and N-pool of faba bean were reduced, while these pools increased for ryegrass. The N-use efficiency (NUE) in faba bean shoots was reduced by 9 and 14 % for LTBC and HTBC, respectively, but not for ryegrass. Interestingly, ryegrass shoots had 52 % higher NUE than faba bean shoots. The N derived from atmosphere (% Ndfa) was increased by 2 and 9 % under LTBC and HTBC, respectively, while it decreased slightly by reduced irrigation. Quantity of BNF in faba bean aboveground biomass decreased with HTBC coupled with reduced irrigation, mainly towards decreased biomass and soil N uptake by faba bean. Therefore, HTBC might not be a feasible option to improve WUE and BNF in faba bean-ryegrass intercropping, but PRD is permissible as the clear trade-off between BC and PRD.

Phototrophic Nitrogen Fixation, a Neglected Biogeochemical Process in Mine Tailings?

Biological nitrogen fixation (BNF) has important ecological significance in mine tailing by contributing to the initial accumulation of nitrogen. In addition to chemolithotrophic and heterotrophic BNF, light may also fuel BNF in oligotrophic mine tailings. However, knowledge regarding the occurrence and ecological significance of this biogeochemical process in mine tailings remains ambiguous. The current study observed phototrophic BNF in enrichment cultures established from three primary successional stages (i.e., original tailings, biological crusts, and pioneer plants) of tailings. Notably, phototrophic BNF in tailings may be more active at vegetation stages (i.e., biological crusts and pioneering plants) than in bare tailings. DNA-stable isotope probing identified Roseomonas species as potential aerobic anoxygenic phototrophs responsible for phototrophic BNF. Furthermore, metagenomic binning as well as genome mining revealed that Roseomonas spp. contained essential genes involved in nitrogen fixation, anoxygenic photosynthesis, and carbon fixation, suggesting their genetic potential to mediate phototrophic BNF. A causal inference framework equipped with the structural causal model suggested that the enrichment of putative phototrophic diazotrophic Roseomonas may contribute to an elevated total nitrogen content during primary succession in these mine tailings. Collectively, our findings suggest that phototrophic diazotrophs may play important roles in nutrient accumulation and hold the potential to facilitate ecological succession in tailings.

Ferrihydrite-mediated methanotrophic nitrogen fixation in paddy soil under hypoxia.

Biological nitrogen fixation (BNF) by methanotrophic bacteria has been shown to play an important role in maintaining fertility. However, this process is still limited to aerobic methane oxidation with sufficient oxygen. It has remained unknown whether and how methanotrophic BNF proceeds in hypoxic environments. Herein, we incubated paddy soils with a ferrihydrite-containing mineral salt medium to enrich methanotrophic bacteria in the presence of methane (20%, v/v) under oxygen constraints (0.27%, v/v). The resulting microcosms showed that ferrihydrite-dependent aerobic methane oxidation significantly contributed (81%) to total BNF, increasing the 15N fixation rate by 13-fold from 0.02 to 0.28 µmol 15N2 (g dry weight soil) -1 d-1. BNF was reduced by 97% when ferrihydrite was omitted, demonstrating the involvement of ferrihydrite in methanotrophic BNF. DNA stable-isotope probing indicated that Methylocystis, Methylophilaceae, and Methylomicrobium were the dominant methanotrophs/methylotrophs that assimilated labeled isotopes (13C or 15N) into biomass. Metagenomic binning combined with electrochemical analysis suggested that Methylocystis and Methylophilaceae had the potential to perform methane-induced BNF and likely utilized riboflavin and c-type cytochromes as electron carriers for ferrihydrite reduction. It was concluded that ferrihydrite mediated methanotrophic BNF by methanotrophs/methylotrophs solely or in conjunction with iron-reducing bacteria. Overall, this study revealed a previously overlooked yet pronounced coupling of iron-dependent aerobic methane oxidation to BNF and improves our understanding of methanotrophic BNF in hypoxic zones.

Ridge with no-tillage facilitates microbial N2 fixation associated with methane oxidation in rice soil.

Aerobic methane-oxidizing bacteria (MOB) play a crucial role in mitigating the greenhouse gas methane emission, particularly prevalent in flooded wetlands. The implementation of ridge with no-tillage practices within a rice-rape rotation system proves effective in overcoming the restrictive redox conditions associated with waterlogging. This approach enhances capillary water availability from furrows, especially during periods of low rainfall, thereby supporting plant growth on the ridges. However, the microbe-mediated accumulation of soil organic carbon and nitrogen remains insufficiently understood under this agricultural practice, particularly concerning methane oxidation, which holds ecological and agricultural significance in the rice fields. In this study, the ridge and ditch soils from a 28-year-old ridge with no-tillage rice field experiment were utilized for incubation with 13C-CH4 and 15NN2 to estimate the methane-oxidizing and N2-fixing potentials. Our findings reveal a significantly higher net production of fresh soil organic carbon in the ridge compared to the ditch soil during methane oxidation, with values of 626 and 543 µg 13C g-1 dry weight soil, respectively. Additionally, the fixed 15N exhibited a twofold increase in the ridge soil (14.1 µg 15N g-1 dry weight soil) compared to the ditch soil. Interestingly, the result of DNA-based stable isotope probing indicated no significant differences in active MOB and N2 fixers between ridge and ditch soils. Both Methylocystis-like type II and Methylosarcina/Methylomonas-like type I MOB catalyzed methane into organic biomass carbon pools. Soil N2-fixing activity was associated with the 15N-labeling of methane oxidizers and non-MOB, such as methanol oxidizers (Hyphomicrobium) and conventional N2 fixers (Burkholderia). Methane oxidation also fostered microbial interactions, as evidenced by co-occurrence patterns. These results underscore the dual role of microbial methane oxidation - not only as a recognized sink for the potent greenhouse gas methane but also as a source of soil organic carbon and bioavailable nitrogen. This emphasizes the pivotal role of microbial methane metabolism in contributing to soil carbon and nitrogen accumulation in ridge with no-tillage systems.