{"paper_id":"372a7d0c-e040-4bbf-bf3d-8e43e3122b09","body_text":"Synergistic interaction between microbial nitrogen fixation and iron reduction in the environment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Synergistic interaction between microbial nitrogen fixation and iron reduction in the environment Ping Li, Xiaohan Liu, Keman Bao, Yaqi Wang, Helin Wang, Yanhong Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5306474/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Nitrogen (N) and iron (Fe) are essential but often limiting nutrients in ecosystems. Microbial nitrogen fixation (MNF) by diazotrophs and dissimilatory ferric iron (Fe(Ⅲ)) reduction (DIR) are environmentally friendly processes that sustain N and Fe availability. However, the interactions between these processes remain unclear. This study demonstrates a synergistic relationship between MNF and DIR in both laboratory and field settings. N fixation significantly increased heterotrophic Fe(Ⅲ)-reducing rates in diazotrophic DIR bacteria (DIRB) Klebsiella sp. N7 and Geobacter sulfurreducens PCA by 14.7- and 3.3-fold, respectively, while Fe(Ⅲ) reduction enhanced 15 N fixation by up to 100%. Similar synergies were found between diazotroph Azospirillum humicireducens SgZ-5T and DIRB Shewanella oneidensis MR-1. Transcriptomic analysis revealed that N fixation upregulated genes associated with anaerobic respiration, accelerating Fe(Ⅲ) reduction through N supply. Simultaneously, Fe(Ⅲ) reduction provided the energy and electrons required for N fixation derived from the oxidation of organic carbon. These findings, validated across environmental samples from aquifers, hot springs, marine sediments, and soils, provide new insights into the coupled N, Fe, and C cycles in natural ecosystems. Earth and environmental sciences/Biogeochemistry/Element cycles Biological sciences/Ecology/Microbial ecology Nitrogen fixation iron reduction 15N isotope tracing transcriptomics metagenomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Nitrogen (N) is essential for all organisms and affects the habitability of the Earth 1 . Although N₂ is the most abundant and accessible form of N, it becomes bioavailable only through N fixation 2 . Presently, approximately 40% of the global population relies on artificial N fixation, which consumes more than 1–2% of the world’s total energy output and releases over 300 million metric tons of CO 2 3,4 . Additionally, the application of N-based fertilizers and fossil fuel combustion has driven an estimated 20% increase in atmospheric nitrous oxide (N 2 O) emissions since 1750 5 . In contrast, microbial nitrogen fixation (MNF) by diazotrophs naturally converts N 2 to ammonia (NH 3 ) without polluting the environment 6 , providing about half of the globally fixed N 7 and playing an important role in supporting bioavailable N and sustaining biogeochemical cycles within ecosystems. MNF, driven by diazotrophs, requires large amounts of high-potential electrons and adenosine triphosphate (ATP) 8 , yet it is widespread in natural environments, including marine sediments 9 , estuaries 10 , soils 11 , mine tailings 12 , and groundwater 13 . This distribution suggests that diazotrophs may utilize alternative catabolic pathways for energy generation, interconnected with other biochemical cycles 14 . Enhanced insight into the mechanisms and regulatory factors governing MNF is crucial for advancing our understanding of diazotrophic metabolic networks and ecological impacts. MNF often couples with sulfur (S) and carbon (C) metabolism in nature. For example, N fixation linked to sulfate reduction or sulfur oxidation occurs widely in marine sediments 15 , rhizospheres 16 , seagrass meadows 17 , soils and coastal ecosystems 18 . Studies have shown that microbial S reduction and N fixation frequently co-occurred in organic-rich environments 19 , with organic matter (OM) bioavailability as a major limiting factor for MNF 20 . Positive correlations have also been found between N fixation rates and dissolved organic carbon (DOC) concentrations in coastal seawater 21 . Methane (CH 4 ) serves as an additional C source for aerobic methanotrophic MNF, with evidence showing N fixation coupled to methanogenesis and CH 4 oxidation in both terrestrial and aquatic environments, such as deep-sea CH 4 seeps 22 and paddy soils 23 . Furthermore, S-oxidizers/-reducers 16,24 , methanogens 25 , and methanotrophs 26 possess the nifH gene and actively fix N 2 , suggesting that diazotrophs exhibit both phylogenetic and metabolic diversity 8 . Recent studies also suggest a close association between ferric iron (Fe(Ⅲ)) and N fixation, though the precise interactions remain unclear. Fe is abundant in natural environments, and microbial Fe(Ⅲ) reduction is regarded as central to many biogeochemical processes, influencing C and N transformation in ecosystems 27 . In marine and fresh waters, Fe availability can significantly shape diazotrophic communities 28 , 29 . Several dissimilatory Fe(Ⅲ)-reducing bacteria (DIRBs) fix N, such as Geobacter , Geomonas , Anaeromyxobacter , Magnetospirillum , Azotobacter , Bacillus , Pseudomonas , and Klebsiella 30 , 31 , contribute to soil N fertility and rice growth yield 30 . Respiratory and fermentative DIRBs are instrumental in OM transformation, with Fe(Ⅲ) reduction estimated to account for 5–109% of anaerobic OM oxidation in coastal wetlands 32 . This transformation may, in turn, enhance the activity of heterotrophic N-fixers in the environment. Studies have shown that adding Fe(Ⅲ) (oxyhydr)oxides increases nitrogenase activity in paddy soils 33 , and high-throughput sequencing of bacterial 16S rRNA genes suggests a link between diazotrophs and DIRBs 6 . Our recent metagenomic analysis indicated that N fixation might be associated with Fe(Ⅱ) transport and Fe(Ⅲ) reduction in groundwater 13 . The widespread occurrence of MNF and Fe(Ⅲ) reduction across diverse ecosystems highlights the need for further investigation into potential interactions between these processes. However, direct evidence of synergy between N fixation and Fe(Ⅲ) reduction remains lacking, and the mechanisms of underlying potential interactions are not fully understood. To fill this knowledge gap, two types of diazotrophic DIRBs, Klebsiella sp. N7 (fermentative type) and Geobacter sulfurreducens PCA (respiratory type), as well as two model stains, i.e. the diazotroph Azospirillum humicireducens SgZ-5T and DIRB Shewanella oneidensis MR-1, were selected to explore the synergy between N fixation and Fe(Ⅲ) reduction. Microcosms were established with environmental samples from aquifers, hot springs, soils, and marine sediments to assess the occurrence of diazotroph-synergized Fe(Ⅲ) reduction. Quantitative RT-PCR (RT-qPCR), transcriptomic, and metagenomic analyses of pure cultures and environmental samples across ecosystems were used to elucidate the synergy mechanisms. 2 Results 2.1 Effects of nitrogen fixation on Fe(Ⅲ) reduction The intraspecies and interspecies effects of N fixation on Fe(Ⅲ) reduction were detected using batch pure cultures. The intraspecies results of two treatments, including N 2 + Fe and Ar + Fe, showed that N 2 significantly promoted the growth (OD 600 , with 269.4% and 172.2%, respectively) and Fe(Ⅲ) reduction (maximum rate increased by 771.3% and 175.8%, respectively) of strains Klebsiella sp. N7 and G . sulfurreducens PCA (Fig. 1A, B and Fig. S4A, B). After 7 days, approximately 5 mM Fe(Ⅲ) was fully reduced to Fe(Ⅱ) in the N 2 group, while the increase of Fe(Ⅱ) in the Ar group was only 1.9 mM, with none in the control group at day 14. Strain N7 could also rapidly grow and reduce ferrihydrite in the N 2 group, producing 1.8 mM Fe(Ⅱ) at day 14 which was 6 times higher than that in the Ar group (Fig. S5A, B). Our results showed that strain N7 also exhibited high nitrogenase activities under a concentration of 0.5 mM NH 4 + (Fig. S6), indicating that N fixation enhances both growth and Fe(Ⅲ) reduction across in situ groundwater concentrations of NH 4 + -N (7.14–271.43 µM) and NO 3 − -N (3.57–507.85 µM) (Fig. S5A, B and Fig. S7). Similarly, G. sulfurreducens PCA reduced Fe(Ⅲ) by 55.9 mM in the N₂ group, 1.7 times that of the Ar control (Fig. S4B). The strong N 2 dependence of growth and Fe(Ⅲ) reduction indicated that N fixation plays an important role in the metabolism of the strain. For interspecies interactions, the model diazotroph A. humicireducens SgZ-5T and model DIRB S . oneidensis MR-1 were selected to detect the effect of N fixation on Fe(Ⅲ) reduction. The growth characteristics and Fe(Ⅲ) reduction of A. humicireducens SgZ-5T, S. oneidensis MR-1, and co-culture are shown in Fig. 1C, D. Co-cultures showed significant synergistic growth and Fe(Ⅲ) reduction (complete 5 mM reduction within 7 days) compared to the respective monocultures (30% and 14% Fe(Ⅲ) reduction for MR-1 and SgZ-5T, respectively). These results suggest that diazotrophs significantly enhance DIRB Fe(Ⅲ) reduction. 2.2 Effects of Fe(Ⅲ) reduction on diazotrophic activity The effect of Fe(Ⅲ) reduction on N fixation within species was investigated using strains N7 and PCA under conditions with or without Fe(Ⅲ) amendment. Cultures amended with ferric citrate or ferrihydrite exhibited significantly higher nitrogenase activities (711.5 ± 29.2 µmol L − 1 ) than those without Fe(Ⅲ) reduction (456.5 ± 24.1 µmol L − 1 ) after 3 days of incubation (Fig. 2A and Fig. S5C). Furthermore, the concentration of fixed 15 N (134.61 ± 8.9 µmol L − 1 ) was significantly higher in Fe(Ⅲ)-amended cultures compared to those without Fe(Ⅲ) (75.69 ± 22.9 µmol L − 1 ) (Fig. 2B and Fig. S5D). Additionally, the total amino acid concentration (N-fixing products) in Fe(Ⅲ)-amended cultures was approximately 1.5 times higher than in the non-amended treatments (Fig. 2C). The interspecies effects of DIRB on diazotroph N fixation during Fe(Ⅲ) reduction were also explored. No nitrogenase activity was detected in the monoculture of S. oneidensis MR-1 (Fig. S8). However, the co-culture of S. oneidensis MR-1 and A. humicireducens SgZ-5T demonstrated enhanced N fixation activity, particularly during the middle and late stages, as indicated by the acetylene reduction and 15 N isotope tracing (Fig. 2D, E). In contrast, no enhancement was observed in a co-culture lacking Fe(Ⅲ) (Fig. S9), suggesting that Fe(Ⅲ) reduction may play an important role in promoting N fixation. Furthermore, the extracellular fixed 15 N concentration in co-cultures was significantly lower than that in the A. humicireducens SgZ-5T monoculture (Fig. 2F). The diazotroph A. humicireducens SgZ-5T released 378.72 µM of amino acids during N fixation in monoculture, which was significantly higher than in co-culture ( P < 0.001) (Fig. 3E). These findings suggest that S. oneidensis MR-1 may utilize amino acids secreted by A. humicireducens SgZ-5T as an N source for growth and Fe(Ⅲ) reduction, thereby promoting N fixation. 2.3 Transcriptional response of species to nitrogen fixation and Fe(Ⅲ) reduction To investigate the mechanisms underlying the synergy between N fixation and Fe(Ⅲ) reduction within and between species, transcriptomics analyses were performed on strain N7 cultures under Ar + Fe(Ⅲ), N 2 + Fe(Ⅲ), and N 2 conditions, as well as on monocultures and co-cultures of A. humicireducens SgZ-5T and S. oneidensis MR-1 (Table S2). During N fixation in strain N7, 908 genes were significantly upregulated (Fig. 3C). GO enrichment analysis revealed that upregulated DEGs were mainly involved in key metabolic pathways, including amino acid metabolism, ATP biosynthetic process, carbohydrate metabolism, N compound transport, and nucleotide metabolism (Fig. S10A). KEGG Orthologs annotation showed that nif genes and N assimilation were highly expressed in the presence of N 2 , with increases of up to 3.6- and 2.7-fold, respectively, compared to the Ar group (Fig. S11). Energy-yielding processes, including glycolysis, the TCA cycle, NADH-quinone oxidoreductases ( nuo ) of complex I, cytochrome c oxidase ( cyo ) of complex IV, and V-type ATPase ( atp ) were upregulated by 2.9-, 2.9-, 4.9-, 3.7-, and 3.7-fold, respectively, during N fixation (Fig. 3D). RT-qPCR also revealed significant upregulation of the 16S rRNA and electron transfer genes ( cymA and mtrA ) of DIRB S. oneidensis MR-1 in co-culture ( P < 0.05) (Fig. 3F). Consistently, increased C catabolism during N fixation accelerated DOC depletion and CO 2 production (Fig. 3A, B). These results indicate that N fixation enhances bacterial anaerobic respiration and biomass production by supplying N, thereby accelerating Fe(Ⅲ) reduction. Bacterial transcriptional responses to Fe(Ⅲ) reduction were also examined. GO analysis of DEGs revealed that 520 upregulated genes in the N 2 + Fe(Ⅲ) condition were associated with NADH dehydrogenase (quinone) activity, electron transfer activity, Fe-S cluster binding, amino acid metabolism, and membrane protein complexes (Fig. 4C and Fig. S10B). KEGG analysis identified upregulation of genes encoding components of electron transport chains, including NADH-quinone oxidoreductase ( nuo ), succinate-dehydrogenase ( frdA ), cytochromes bd ( cydAB ), and ATP synthase ( atp ) (Fig. 4D) ( P < 0.05, Log 2 FC > 0.5). Genes related to anaerobic fermentation, such as pflB and nifJ , were also upregulated by 58.5% and 50.1%, respectively, under Fe(Ⅲ)-reducing conditions (Fig. 4D). Correspondingly, increased C decomposition, CO 2 production, and ATP yield were observed under Fe(Ⅲ) reduction (Fig. 4A, B, E, F). Meanwhile, the respiratory DIRB G. sulfurreducens PCA directly utilizes Fe(Ⅲ) as an electron acceptor for growth and N fixation promotion (Fig. S4C). For interspecies interactions, respiratory DIRB S. oneidensis MR-1, which lacks N fixation capability, showed significant upregulation of the electron transfer genes ( cymA and mtrA ) in co-culture, along with increased ATP production and OM consumption (Fig. 4E, F). This pattern was consistent with intraspecific interactions. As a result, diazotroph nifH gene expression was significantly upregulated by Fe(Ⅲ) reduction ( P < 0.05) (Fig. 4D, G). These results demonstrate that the synergy between N fixation and Fe(Ⅲ) reduction occurs both within and between species (Fig. 5). 2.4. Nitrogen fixation and Fe(Ⅲ) reduction in different ecosystems. To validate the above findings, microcosm experiments were conducted using environmental samples collected from aquifers, hot springs, soil, and marine ecosystems. The results indicated that Fe(Ⅲ) reduction significantly enhanced N fixation activity in samples from aquifer waters/sediments ( P < 0.001), marine sediments ( P < 0.001), and soils ( P < 0.001) (Fig. 6A and Fig. S12). After the end of incubation, significantly higher nitrogenase activities (1.1–8106.9 µmol L − 1 ) were detected in Fe(Ⅲ)-amended cultures compared to those without Fe(Ⅲ) (0.3–884.9 µmol L − 1 ) (Fig. S12). Simultaneously, Fe(Ⅲ) reduction rates were higher in N 2 cultures than in no-N 2 controls (Fig. 6A and Fig. S12). N fixation accelerated Fe(Ⅲ) reduction in marine and hot spring sediments early in incubation, with Fe(Ⅱ) concentrations increasing by 15.5–40.6% ( P < 0.05). In aquifer sediments and soils, Fe(Ⅲ) reduction rates were significantly higher than in the Ar group during the mid-incubation stage, with Fe(Ⅱ) production increasing by 14.8–38.2% ( P < 0.05). Overall, the synergy between N fixation and Fe(Ⅲ) reduction occurs across various ecosystems. To elucidate these findings, metagenomic analysis was conducted on samples from aquifer waters/sediments, hot spring sediments, soils, and marine sediments to assess the metabolic potential for N fixation and Fe(Ⅲ) reduction. Key functional genes involved in N fixation ( nif ) and Fe(Ⅲ) reduction ( omc , cym , and mtr ) were identified using the KEGG database and FeGenie. Nitrogenase ( nifHDK and nifENB ) gene abundance positively correlated with Fe(Ⅲ) reduction genes (traditional multi-heme c-type cytochromes (MHCs): mtrABC and omcSZ ) in all ecosystems ( P < 0.05) (Fig. 6B). Additionally, nif clusters were strongly correlated with the genes encoding N transport ( amt ), N assimilation ( glt , gln , and gdh ), electron transport ( nuo , cyd , fix , and kor ), F-type ATPases ( atp ), and Fe transport ( feo ) ( P < 0.05) (Fig. S12). After metagenomic binning, medium-quality metagenome-assembled genomes (MAGs) (completeness > 50%, contamination < 10%) containing key genes involved in N fixation ( nif ) and Fe(Ⅲ) reduction (MHCs: omc , cym , and mtr ) were retrieved from aquifer, soil, and marine environments. These included GW-bin 16 ( Thermodesulfovibrionia ), S-bin 31 ( Kapabacteria ), soil2-bin 12 ( Bacteria ), soil4-bin 12 ( Bacteria ), and sea8-bin 14 ( Bacteria ) (Fig. 6C). Although the diazotrophic genomes from hot springs lacked MHCs, genes encoding pathways for redox molecule biosynthesis and electron transport were identified, such as riboflavin/menaquinone biosynthesis and quinone oxidoreductase (Fig. 6C). Based on KEGG Module analysis, genes related to C metabolism (e.g., glycolysis, pyruvate oxidation, and citrate cycle), electron transport (e.g., NADH dehydrogenase, quinone oxidoreductase, cytochrome c oxidase), ATP synthesis (F-type ATPase), electron shuttle biosynthesis (riboflavin/menaquinone biosynthesis), N fixation( nif ), and Fe(Ⅲ) reduction ( omc , cym , and mtr ) were identified in the retrieved MAGs from aquifer, hot springs, soil, and marine sediments (Fig. 6C). Thus, microorganisms in these environments possess the metabolic potential to facilitate the synergy between N fixation and Fe(Ⅲ) reduction across diverse ecosystems. 3. Discussion 3.1 Evidence and mechanism of the synergy between nitrogen fixation and Fe(Ⅲ) reduction Our integrated pure culture studies and field investigations provide substantial evidence of the synergistic interaction between N fixation and Fe(Ⅲ) reduction. Multiple microorganisms have been shown to mediate N fixation and Fe(Ⅲ) reduction simultaneously 6 . In our study, fermentative and respiratory diazotrophic DIRBs displayed accelerated growth and Fe(Ⅲ) reduction under N fixation conditions. In turn, Fe(Ⅲ) reduction facilitated MNF. Additionally, our findings indicate that diazotrophs feed on DIRB by secreting N-fixed products, and DIRB provides energy for diazotrophs, enhancing fixed N in co-culture. Previous studies have speculated on the correlation between diazotrophs and DIRBs in soils 6 , coastal sediments 10 , and wetlands 34 , and our recent research extends this to groundwater systems, revealing positive interactions between N fixation and Fe(Ⅲ) reduction 13 . In this study, microcosm experiments and metagenomic analyses across various habitats further validate this synergy. The reduction of 1 mmol Fe(III) is estimated to contribute 1.42–75.58 µmol N fixation, representing a potential pathway for N input in the environment (Table S6). Moreover, N fixation increased by 4.1–36.2% of Fe(Ⅱ) production in these sediments (Fig. S12A). Intraspecies, interspecies, and environmental observations consistently support the hypothesis of synergistic N fixation and Fe(Ⅲ) reduction, improving our understanding of coupled N and Fe cycles in nature. Despite the high energy demand of MNF, diazotrophs link various catabolic processes to fix N in natural environments 14 . The availability of electron donors and energy sources are critical factors driving N fixation 20 . In oligotrophic mine tailings, dominant chemolithotrophic diazotrophs utilize S, arsenic (As), and antimony (Sb) as electron donors 12 , 35 , whereas heterotrophic diazotrophs oxidize organic compounds to produce ATP for N fixation in diverse terrestrial and aquatic systems 21 , 36 . In this study, Fe(Ⅲ) acted as a reservoir for excess reducing equivalents in fermentative DIRBs 37 , enhancing organic fermentation, electron transport, ATP synthesis, and biomass production, thereby facilitating N fixation (Fig. 4, Fig. S11, and Fig. S13). Respiratory diazotrophic DIRBs use Fe(Ⅲ) as a terminal electron acceptor to generate the energy necessary for N fixation, and their inability to fix N in the absence of Fe(Ⅲ) demonstrates the critical role of Fe(Ⅲ) in this process. Interestingly, in interspecific interactions, non-N-fixing respiratory DIRBs indirectly promote N fixation in diazotrophs through Fe(Ⅲ)-dependent anaerobic respiration. N fixation, in turn, accelerates Fe(Ⅲ) reduction by enhancing microbial growth and carbon metabolic pathways, creating a positive feedback loop (Fig. 5) 38 . This synergy is further evidenced by the strong correlation between nitrogenase, carbon metabolism, and Fe(Ⅲ) reduction genes in various ecosystems. These findings indicate a bidirectional interaction between N fixation and Fe(Ⅲ) reduction, which enhances the flow of nutrients and energy in natural environments. 3.2 Environment implications of the synergy between nitrogen fixation and Fe(Ⅲ) reduction Fe(Ⅲ) reduction and Fe(Ⅱ) oxidation occur simultaneously or cyclically in many environments. DIRBs reduce Fe(Ⅲ) to Fe(Ⅱ) under anoxic conditions, whereas Fe(Ⅱ) can be biotically oxidized by nitrate-reducing Fe(Ⅱ)-oxidizing microbes or abiotically oxidized back to Fe(Ⅲ) by atmospheric oxygen 39 . Studies have demonstrated that Fe cycling is closely coupled with OM transformation under fluctuating oxygen conditions 40 . Under such alternating redox conditions, the reduction and oxidation of Fe continuously fuel N fixation, while N fixation can, in turn, enhance microbial growth and Fe cycling. The impact of N fixation on Fe(III) reduction and Fe(II) transport may significantly contribute to phytoplankton productivity production and benthic nutrient fluxes (Fig. S11) 41 . Consequently, N fixation serves as a crucial nutrient source for microbial-mediated C-Fe cycles, whereas Fe redox cycling has the potential to support substantial N pools in natural environments by promoting N fixation 42 . Under these redox fluctuations, N fixation may enhance microbial resistance to reactive oxygen species (ROS) (Fig. S11) 43 . In laboratory settings, N 2 is commonly used to create anoxic environments for incubating DIRBs, providing a potential N source for diazotrophic DIRBs. Moreover, studies suggest that coupled N fixation and Fe(Ⅲ) reduction could regulate CH 4 emissions 23 . Recently, N 2 O fixation by diazotrophs has been proposed as an alternative sink for N 2 O, potentially accounting for 60% of total N 2 O reduction in the Pacific Ocean 44 , 45 . In our study, we found that microbial N 2 O fixation also promoted the growth of diazotrophic DIRB and Fe(Ⅲ) reduction, with Fe(Ⅲ) reduction significantly accelerating N 2 O consumption (Fig. S14). These findings suggest that Fe(Ⅲ) application could be a feasible strategy for reducing N 2 O emissions in the environment. We propose that cryptic Fe cycles under redox fluctuations, such as those caused by alternating soil wetting and drying, groundwater-surface water interactions, and oceanic vertical stratification, may contribute to nutrient flow and climate gas mitigation in natural ecosystems. Theoretically, N fixation is more likely to occur in oligotrophic habitats. However, studies have shown that diazotrophs are widely distributed in both oligotrophic and eutrophic environments 46 , 47 . In this study, N fixation activity was detected across different ecosystems. Serval diazotrophs lack N assimilation or reduction genes, leaving N fixation as the only bioavailable N source in certain ecosystems 20 . Moreover, the synergy between N fixation and Fe(Ⅲ) reduction is expected to be more pronounced in eutrophic environments rich in OM, such as freshwater sediments 46 and paddy soils 48 . In these systems, coupled Fe-C cycles may stimulate diazotrophs and DIRBs, contributing to substantial N and Fe(Ⅱ) inputs. In marine environments, particles serve as an OM source for heterotrophic diazotrophs and DIRBs 49 . We speculate that N depletion by DIRBs in particle-associated microenvironments creates N-deficient niches conducive to diazotrophs. Consequently, aquatic environments with high particle fluxes are hotspots for N fixation 50 . In extreme oligotrophic environments, such as hot springs, C-fixing microorganisms or algae residues may provide C sources for heterotrophs (our unpublished data) 51 , supporting the synergistic occurrence of N fixation and Fe(Ⅲ) reduction. Interestingly, diazotrophs may fix N not only to meet N demands but also to maintain an optimal intracellular redox state 20 . Thus, N fixation could serve as a mechanism to balance redox states in natural environments by eliminating excess electrons generated from the oxidation of OM, Fe, and H 2 . In summary, the effective synergy between N fixation and Fe(Ⅲ) reduction, accompanied by C decomposition, may partly explain the widespread occurrence of N fixation in eutrophic environments. The interaction between MNF and Fe(Ⅲ) reduction is an overlooked process that enhances N supply, biomass yield, and Fe/C cycling across various ecosystems. Our study provides important insights into the mechanisms and prevalence of synergistic MNF and Fe(Ⅲ) reduction, which may help mitigate greenhouse gas emissions linked to agricultural fertilizers. Further studies are warranted to assess the role of this synergistic interaction in sustaining bioavailable N and Fe/C cycling in natural ecosystems. 4. Materials and methods 4.1 Strains, environmental samples, and media The strain Klebsiella sp. N7 (N7), a facultative anaerobic N-fixing bacterium, was isolated from groundwater as described in a previous study (Fig. S1) (accession number PRJNA1160161). Strain N7 can reduce dissolved Fe(Ⅲ) (Fe(Ⅲ)-citrate) and Fe(Ⅲ) mineral (ferrihydrite) in N-free Burk’s medium. The diazotrophic DIRB G. sulfurreducens PCA was purchased from the American Type Culture Collection (Manassas, VA, United States) (ATCC 51573). A. humicireducens SgZ-5T (CCTCC AB 2012021), which can fix N in Burk’s medium, was purchased from the China Center for Type Culture Collection (Wuhan University, China). The DIRB S. oneidensis MR-1 (ATCC 700550), which cannot fix N but can reduce Fe(Ⅲ), was also obtained from ATCC (number: 700550). To activate cultures, Luria Broth (LB) medium containing (per liter) 10 g NaCl, 5 g yeast extract, and 10 g tryptone was used to grow Klebsiella sp. N7 and S. oneidensis MR-1 at 30°C. A. humicireducens SgZ-5T was cultured in Nutrient Broth (NB) medium containing (per liter) 5 g NaCl, 5 g peptone, and 3 g beef extract at 30°C. Modified anaerobic Burk’s medium was used to cultivate strains for both N fixation and Fe(Ⅲ) reduction at 30°C. The medium contained the following components (per liter): 20 g mannitol, 0.2 g KH 2 PO 4 , 0.8 g·K 2 HPO 4 , 0.2 g MgSO 4 ·7H 2 O, 0.1 g CaSO 4 ·2H 2 O, and trace amounts of Na 2 MoO 4 ·2H 2 O and FeCl 3 , at pH 7.0. Fe(Ⅲ) reduction was conducted in this medium supplemented with Fe(Ⅲ)-citrate or ferrihydrite. Ferrihydrite was prepared following previously published methods 31 . G . sulfurreducens PCA was cultured in an N-free FWAFC medium for N fixation and Fe(Ⅲ) reduction analyses 52 . Representative aquifer sediments were collected from two boreholes at a depth of 20 m in the recharge and discharge zones of the Hetao Plain, Inner Mongolia, China 53 . Hot spring sediments were sampled from two drainage channels in Qamdo, eastern Tibet, designed as DB-A and DB-B 54 . Surface soil samples were taken from areas near the Han River and southeastern Jianghan Plain, Hubei Province, China 55 . Subsurface paddy soils were also collected from Xiazhaijin Village, Zhejiang Province, China. Marine sediments were obtained from the surface 0–5 cm layer of a box core in the East China Sea. The basic information of these samples is provided in Table S3. All fresh sediments and soils were sealed in 50 mL sterile polyethylene tubes, stored on dry ice, and transported to the laboratory for homogenization and microcosmic culturing as soon as possible. Additionally, metagenomic analysis was conducted on samples from various habitats, including aquifer waters/sediments (our study) 53 , hot spring sediments (our unpublished data), marine waters/sediments 56,57 , and paddy soils 58 , 59 , 60 . The geographic distribution of environmental sampling sites for microcosm and metagenomic data is presented in Fig. S2. The method for metagenome data processing and metagenomic binning analysis are detailed in our recent publication 13 . 4.2 Monoculture and interspecies assays Strain Klebsiella sp. N7 was routinely cultured in 150 mL of LB medium at 30°C with shaking at 150 rpm. Cells were harvested by centrifugation (8,000 rpm, 10 min, 4°C), washed three times with phosphate-buffered saline (PBS), and resuspended in sterilized anaerobic N/Fe-free Burk’s medium. The bacterial suspension's optical density at 600 nm (OD 600 ) was measured and adjusted to a final OD 600 of 0.05 by inoculating it into fresh Burk’s medium. To investigate the synergistic interaction between N fixation and Fe(Ⅲ) reduction, four experimental groups were established: Burk, Burk + Fe, N 2 + Fe, and Ar + Fe. The N 2 + Fe and Ar + Fe groups, designed to assess the impact of N fixation on Fe(Ⅲ) reduction, were supplemented with 5 mM ferric citrate or ferrihydrite and degassed with N 2 and Ar, respectively. The Burk and Burk + Fe groups were purged with N 2 gas. To simulate environmentally relevant Fe concentrations, 0.2 mM ferric citrate or ferrihydrite was added to the Burk + Fe group to examine the influence of Fe(Ⅲ) reduction on N fixation. A non-inoculated control group served as the blank control. G. sulfurreducens PCA was cultured in N-free FWAFC medium under anaerobic conditions. At the early stationary phase (OD 600 ≈ 0.5), 10 mL of culture was inoculated into 100 mL N-free FWAFC medium. The N 2 + Fe and Ar + Fe groups were degassed with 80% N 2 -20% CO 2 and 80% Ar-20% CO 2 , respectively. To assess the effect of Fe(Ⅲ) reduction on N fixation in PCA, treatments with and without ferric citrate as the electron acceptor were established. All cultures were incubated at 30°C in the dark and in triplicate. A co-culture system consisting of diazotroph A. humicireducens SgZ-5T and DIRB S. oneidensis MR-1 was constructed to explore interspecies interactions between N fixation and Fe(Ⅲ) reduction. Active cells from both strains were harvested, washed three times with PBS, and resuspended in sterilized anaerobic N/Fe-free Burk’s medium. The suspensions of diazotroph and DIRB were inoculated either individually or in combination (at a 1:1 volume ratio, each with an OD 600 of 0.02). The non-inoculated treatment served as the blank control. All groups were supplemented with 5 mM ferric citrate, degassed with N 2 , and incubated at 30°C with shaking at 150 rpm in the dark. 4.3 Characterization of nitrogen fixation and Fe(Ⅲ) reduction Nitrogenase activity was characterized using acetylene-reduction assays as previously described 61 . Briefly, 3 mL headspace air (10% of headspace) was extracted from the anoxic medium and replaced with 3 mL acetylene. The ethylene concentration was measured by a gas chromatograph (GC-4000A, EWAI) equipped with a flame ionization detector and a Porpack N column. Isotopic enrichment of 15 N in various treatment groups was analyzed using a membrane inlet mass spectrometer (MIMS, Hiden HPR-40, UK) via 15 N isotope tracing 10 , 62 . Specifically, 60 mL serum bottles containing 30 mL of medium were degassed with Ar for 40 min, after which the headspace was replaced with 30 mL 15 N 2 (Cambridge Isotope Laboratories, > 98 atom%). Bottles were incubated inverted at 30°C with shaking at 150 rpm. After incubation, the cultures were autoclaved at 121°C for 30 min and purged with Ar for 40 min to eliminate background 15 N 2 . The fixed 15 N-labeled products were analyzed by MIMS after oxidation with hypobromite iodine solution to 29 N 2 and/or 30 N 2 62 . A standard calibration curve was generated using a concentration gradient of 15 NH 4 Cl (Cambridge Isotope Laboratories, 99%). The linear relationship between 15 N concentrations and the total produced 29 N 2 plus 30 N 2 signal is shown in Fig. S3. Initial sample bottles were processed immediately after 15 N 2 tracer addition. For Fe(Ⅱ) and total Fe quantification, samples were collected under anoxic conditions in a glovebox, mixed with an equal volume of 1 M HCl, and analyzed using a ferrozine assay 31 . DOC was measured by filtering the bacterial suspension through 0.22-µm membrane filters (Millipore) and analyzed with a TOC analyzer (TOC-L, Shimadzu, Japan). The CO 2 content in the headspace was determined using a gas chromatograph (Shimadzu GC-2014). Amino Acid (AA) production during N fixation was quantified using an AA Assay Kit (Sangon, China). Intracellular adenosine triphosphate (ATP) levels were measured following the manufacturer’s instructions with an ATP Content Assay Kit (Solarbio Science & Technology Co., Ltd) 63 . Mannitol content was determined using spectrophotometry 64 . 4.4. RNA extraction, RT-qPCR, and transcriptomic analysis To investigate the gene expression of strains during N fixation and Fe(Ⅲ) reduction, three treatment groups of intraspecific assays were analyzed via transcriptomics (N7): Burk, Burk + Fe, and Burk + Fe + Ar. Both Burk and Burk + Fe groups were purged with N 2 gas, while 0.2 mM Fe(Ⅲ)-citrate was added to the Burk + Fe group. The Burk + Fe + Ar group, also treated with 0.2 mM Fe(Ⅲ)-citrate, was degassed with Ar. For cross-feeding experiments, three treatment groups were used for gene expression profiling: monocultures of A. humicireducens SgZ-5T and S. oneidensis MR-1, as well as a co-culture. Following incubation, cells in all treatments were collected during the log phase via centrifugation (8,000 rpm, 20 min, 4°C). Total RNA was extracted using RNAiso Plus (TaKaRa, Bio) following the manufacturer’s protocol, and residual DNA in RNA was removed with thePrimeScript™ RT re-agent Kit with gDNA Eraser (TaKaRa, Bio) for 2 min at 42°C. RNA integrity was assessed via 1.5% agarose gel electrophoresis (Biowest, Riverside, MO, USA), and RNA concentrations were determined using a NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For cDNA synthesis, RNA was diluted and reverse transcribed using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Bio). Quantitative RT-PCR (RT-qPCR) was then performed (Applied Biosystems, Waltham, MA, USA) with the TB Green™ Premix Ex TaqTM Ⅱ (TaKaRa, Bio) qPCR kit. Primer sequences and amplification conditions are listed in Table S1. The bacterial 16S rRNA gene was used as an endogenous reference, and transcript levels of Shewanella 16S rRNA, nifH , cymA , and mtrA were quantified using the 2 −ΔΔCT method 65 . RNA samples for sequencing were stored at − 80°C and sent to Magigene Technology (Guangzhou, China) as soon as possible for RNA-Seq library preparation (ALFA-SEQ RNA Library Prep Kit Ⅱ) and sequenced using the Illumina NovaSeq 6000 platform. Raw reads were processed to remove low-quality data using Trimmomatic (Version 0.36) to generate clean reads 66 . These were subsequently mapped to the reference genome of Klebsiella grimontⅡ from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using Bowtie2 (Version 2.4.5) 67 . Quantification of read counts was performed using Salmon (Version 1.9.0), and differential expression analysis was conducted with DESeq 2 (Version 1.30.1) to identify differentially expressed genes (DEGs) 68 , 69 . DEGs were considered significant with a P -adjust value < 0.05 and |log 2 fold change| ≥ 0.5. The transcriptomic results are presented in Table S2. Gene annotation was performed using the KEGG database, while Gene Ontology (GO) enrichment analysis followed the protocol from our previous study 70 . 4.5 Sediment microcosms and metagenomic analysis To investigate the widespread occurrence of the synergy between N fixation and Fe(Ⅲ) reduction, microcosm cultures were established using environmental samples from various habitats, including aquifers, hot springs, marine waters/sediments, and soils. Triplicate microcosm incubations were conducted in 120 mL sterile serum bottles, each containing 50 mL of homogenized slurries prepared by mixing sediment and sterile water at a sediment/water weight ratio of 1:5. The N 2 and Ar groups were purged with N 2 and Ar gases for 50 min to assess the impact of N fixation on Fe(Ⅲ) reduction. Two additional treatments were set up by amending Fe(Ⅲ)-citrate (at concentrations reflecting near-field water Fe levels) (Table S3) to evaluate nitrogenase activity driven by Fe(Ⅲ) reduction. Microcosms of hot spring sediments were incubated at 60°C, while the other cultures were incubated at 25°C in the dark. Nitrogenase activity was determined using acetylene-reduction assays, as described in section 4.3 of Materials and Methods. Fe(Ⅱ) samples were collected inside an anoxic glovebox, centrifuged at 1,2000 rpm for 5 min, and the supernatant was diluted in 1 M HCl for quantification via the ferrozine assay. Sediments were extracted with 0.5 M HCl, and the supernatant was stabilized in 0.5 M HCl for Fe(Ⅱ) analysis. 4.6 Statistical analyses and data availability Spearman correlations among the abundances of functional genes were computed using R v4.1.2. Data visualization, fitting, and evaluation were performed using GraphPad Prism v10 (GraphPad Software, San Diego, CA, USA). Declarations Data availability Raw RNA-Seq data have been deposited in the NCBI database (accession number: PRJNA1172356). Metagenome sequencing reads from the aquifer and hot spring samples are available from our previous study under NCBI BioProject accession numbers PRJNA882225 and PRJNA943127. Acknowledgments This work is supported by the National Natural Science Foundation of China (Nos. 42177068 [P.L.], 42025703 [S.Y.], Nos. 41772260 [P.L.], and 41976043 [Y.Y.]). Marine surface sediment and environmental parameters were collected onboard, implementing the open research cruises, NORC2022-03 and NORC2023-06. We thank Prof. Liang Shi from the China University of Geosciences (Wuhan) for providing the iron-reducing bacteria Geobacter sulfurreducens PCA. Author contributions P.L. and X.H.L. conceived the research. X.H.L. performed most of the experiments. K.M.B., Y.Q.W., and H.L.W. supported the methodology for nitrogen fixation characterization. P.L., X.H.L., H.L.W., Y.H.W., Z.J., and Y.Y. contributed to sediment sample collection and analysis. X.H.L. and P.L. wrote and revised the manuscript. S.H.Y., A. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-5306474\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":372700922,\"identity\":\"ec9e81ef-30c8-49ae-ad0e-b0c995cd41db\",\"order_by\":0,\"name\":\"Ping 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Growth curve and (b) ferric citrate reduction of \\u003cem\\u003eKlebsiella\\u003c/em\\u003e sp. N7 under N\\u003csub\\u003e2\\u003c/sub\\u003e and Ar conditions. (c) Growth curve and (d) ferric citrate reduction of \\u003cem\\u003eAzospirillum humicireducens\\u003c/em\\u003e SgZ-5T and \\u003cem\\u003eShewanella\\u003c/em\\u003e \\u003cem\\u003eoneidensis\\u003c/em\\u003e MR-1 in monocultures and co-culture under nitrogen-free conditions. The blank presents the abiotic control.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5306474/v1/dc46acda00e264a0af46433d.png\"},{\"id\":68348112,\"identity\":\"267586e0-5d86-4f1e-9e9b-18f607e1f53e\",\"added_by\":\"auto\",\"created_at\":\"2024-11-06 10:03:46\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":333355,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eNitrogen fixation activity in pure culture incubations. \\u003c/strong\\u003e(a) Nitrogenase activity, (b) \\u003csup\\u003e15\\u003c/sup\\u003eN products, and (c) amino acid concentrations in \\u003cem\\u003eKlebsiella\\u003c/em\\u003e sp. N7 with or without ferric citrate. (d) Nitrogenase activity, (e) total \\u003csup\\u003e15\\u003c/sup\\u003eN products, and (f) extracellular \\u003csup\\u003e15\\u003c/sup\\u003eN products in \\u003cem\\u003eAzospirillum humicireducens\\u003c/em\\u003e SgZ-5T monoculture and co-culture. *, **, and *** represent \\u003cem\\u003ep \\u003c/em\\u003e\\u0026lt; 0.05, 0.01, and 0.001, respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5306474/v1/008ea368993d8e50fa63fabe.png\"},{\"id\":68348113,\"identity\":\"e764cf2c-bab7-4dd0-9894-71bcdc89ba06\",\"added_by\":\"auto\",\"created_at\":\"2024-11-06 10:03:46\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":594927,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCharacterization of the effect of nitrogen fixation on microbial cultures. \\u003c/strong\\u003eResponse of \\u003cem\\u003eKlebsiella\\u003c/em\\u003e sp. N7 to nitrogen fixation: (a) DOC consumption, (b) CO\\u003csub\\u003e2\\u003c/sub\\u003e production, (c) volcano plots of differentially expressed genes with or without added N\\u003csub\\u003e2\\u003c/sub\\u003e, and (d) upregulated gene expression measured by log 2-fold change (Log2FC) under N\\u003csub\\u003e2\\u003c/sub\\u003e compared to Ar incubation (\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.05). (e) extracellular amino acid concentrations and (f) fold-change in gene expression related to electron transfer in \\u003cem\\u003eShewanella\\u003c/em\\u003e \\u003cem\\u003eoneidensis\\u003c/em\\u003e MR-1 in co-culture compared to monoculture.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5306474/v1/acaa8f697a1e4fcd4a51408d.png\"},{\"id\":68349059,\"identity\":\"47ed3fa4-8c7f-4e2b-ab14-5cca30b1612f\",\"added_by\":\"auto\",\"created_at\":\"2024-11-06 10:11:46\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":658608,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eBacterial response to ferric iron reduction.\\u003c/strong\\u003e (a) CO\\u003csub\\u003e2\\u003c/sub\\u003e production, (b) ATP concentration, (c) volcano plots of differentially expressed genes with or without ferric iron, and (d) log 2-fold change (Log\\u003csub\\u003e2\\u003c/sub\\u003eFC) in gene expression with or without ferric iron in \\u003cem\\u003eKlebsiella\\u003c/em\\u003e sp. N7 (\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.05). (e) Mannitol consumption, (f) ATP concentration, and (g) fold change in \\u003cem\\u003enifH\\u003c/em\\u003e gene expression in \\u003cem\\u003eAzospirillum humicireducens\\u003c/em\\u003e SgZ-5T in co-culture compared to monoculture.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5306474/v1/1ad73c9479871321ce567e79.png\"},{\"id\":68348114,\"identity\":\"00cf3357-430e-425e-b7b1-90a94b973794\",\"added_by\":\"auto\",\"created_at\":\"2024-11-06 10:03:46\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":92945,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSchematic illustration of the synergistic interaction between nitrogen fixation and ferric iron reduction.\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5306474/v1/6249c7646e33e6b64feedcae.png\"},{\"id\":68348115,\"identity\":\"4516db4d-b27e-4c2e-84dd-fd3f1a2a3c73\",\"added_by\":\"auto\",\"created_at\":\"2024-11-06 10:03:46\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":897953,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSynergistic nitrogen fixation and ferric iron reduction across different ecosystems. \\u003c/strong\\u003e(a) Nitrogenase activity and ferric iron reduction in microcosms from various environmental samples. Grey and red dots represent nitrogenase activity in Burk and Burk+Fe(Ⅲ), respectively. All treatments were conducted in triplicate (N = 3) to assess ferrous iron production kinetics in N\\u003csub\\u003e2\\u003c/sub\\u003e and Ar groups. (b) Spearman’s correlations between functional genes involved in nitrogen fixation and ferric iron reduction. (c) Metagenome-assembled genomes (MAGs) with nitrogen fixation and ferric iron reduction potential from different environments.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5306474/v1/8c9aeb0146798cd084bb730f.png\"},{\"id\":68350394,\"identity\":\"305285ff-98d7-443c-8c9b-2afc55509562\",\"added_by\":\"auto\",\"created_at\":\"2024-11-06 10:27:47\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3899678,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5306474/v1/a7518425-d842-4f2a-b449-be4d22ba2604.pdf\"},{\"id\":68348109,\"identity\":\"4ee6c54b-c142-49e2-b09b-c853db6ec0ce\",\"added_by\":\"auto\",\"created_at\":\"2024-11-06 10:03:46\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":2294216,\"visible\":true,\"origin\":\"\",\"legend\":\"Supplementary materials\",\"description\":\"\",\"filename\":\"Supplementarymaterials.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5306474/v1/e7d2ef66d0b8961e04817d49.docx\"}],\"financialInterests\":\"There is \\u003cb\\u003eNO\\u003c/b\\u003e Competing Interest.\",\"formattedTitle\":\"Synergistic interaction between microbial nitrogen fixation and iron reduction in the environment\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eNitrogen (N) is essential for all organisms and affects the habitability of the Earth\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. Although N₂ is the most abundant and accessible form of N, it becomes bioavailable only through N fixation\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e. Presently, approximately 40% of the global population relies on artificial N fixation, which consumes more than 1\\u0026ndash;2% of the world\\u0026rsquo;s total energy output and releases over 300\\u0026nbsp;million metric tons of CO\\u003csub\\u003e2\\u003c/sub\\u003e\\u003csup\\u003e3,4\\u003c/sup\\u003e. Additionally, the application of N-based fertilizers and fossil fuel combustion has driven an estimated 20% increase in atmospheric nitrous oxide (N\\u003csub\\u003e2\\u003c/sub\\u003eO) emissions since 1750\\u003csup\\u003e5\\u003c/sup\\u003e. In contrast, microbial nitrogen fixation (MNF) by diazotrophs naturally converts N\\u003csub\\u003e2\\u003c/sub\\u003e to ammonia (NH\\u003csub\\u003e3\\u003c/sub\\u003e) without polluting the environment\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e, providing about half of the globally fixed N\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e and playing an important role in supporting bioavailable N and sustaining biogeochemical cycles within ecosystems. MNF, driven by diazotrophs, requires large amounts of high-potential electrons and adenosine triphosphate (ATP)\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e, yet it is widespread in natural environments, including marine sediments\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e, estuaries\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e, soils\\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e, mine tailings\\u003csup\\u003e\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e, and groundwater\\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e. This distribution suggests that diazotrophs may utilize alternative catabolic pathways for energy generation, interconnected with other biochemical cycles\\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e. Enhanced insight into the mechanisms and regulatory factors governing MNF is crucial for advancing our understanding of diazotrophic metabolic networks and ecological impacts.\\u003c/p\\u003e \\u003cp\\u003eMNF often couples with sulfur (S) and carbon (C) metabolism in nature. For example, N fixation linked to sulfate reduction or sulfur oxidation occurs widely in marine sediments\\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003e, rhizospheres\\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e, seagrass meadows\\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e, soils and coastal ecosystems\\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e. Studies have shown that microbial S reduction and N fixation frequently co-occurred in organic-rich environments\\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e, with organic matter (OM) bioavailability as a major limiting factor for MNF\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. Positive correlations have also been found between N fixation rates and dissolved organic carbon (DOC) concentrations in coastal seawater\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. Methane (CH\\u003csub\\u003e4\\u003c/sub\\u003e) serves as an additional C source for aerobic methanotrophic MNF, with evidence showing N fixation coupled to methanogenesis and CH\\u003csub\\u003e4\\u003c/sub\\u003e oxidation in both terrestrial and aquatic environments, such as deep-sea CH\\u003csub\\u003e4\\u003c/sub\\u003e seeps\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u003c/sup\\u003e and paddy soils\\u003csup\\u003e\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e. Furthermore, S-oxidizers/-reducers\\u003csup\\u003e16,24\\u003c/sup\\u003e, methanogens\\u003csup\\u003e\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e, and methanotrophs\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e possess the \\u003cem\\u003enifH\\u003c/em\\u003e gene and actively fix N\\u003csub\\u003e2\\u003c/sub\\u003e, suggesting that diazotrophs exhibit both phylogenetic and metabolic diversity\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e. Recent studies also suggest a close association between ferric iron (Fe(Ⅲ)) and N fixation, though the precise interactions remain unclear.\\u003c/p\\u003e \\u003cp\\u003eFe is abundant in natural environments, and microbial Fe(Ⅲ) reduction is regarded as central to many biogeochemical processes, influencing C and N transformation in ecosystems\\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e. In marine and fresh waters, Fe availability can significantly shape diazotrophic communities\\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e. Several dissimilatory Fe(Ⅲ)-reducing bacteria (DIRBs) fix N, such as \\u003cem\\u003eGeobacter\\u003c/em\\u003e, \\u003cem\\u003eGeomonas\\u003c/em\\u003e, \\u003cem\\u003eAnaeromyxobacter\\u003c/em\\u003e, \\u003cem\\u003eMagnetospirillum\\u003c/em\\u003e, \\u003cem\\u003eAzotobacter\\u003c/em\\u003e, \\u003cem\\u003eBacillus\\u003c/em\\u003e, \\u003cem\\u003ePseudomonas\\u003c/em\\u003e, and \\u003cem\\u003eKlebsiella\\u003c/em\\u003e\\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e, contribute to soil N fertility and rice growth yield\\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e. Respiratory and fermentative DIRBs are instrumental in OM transformation, with Fe(Ⅲ) reduction estimated to account for 5\\u0026ndash;109% of anaerobic OM oxidation in coastal wetlands\\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e. This transformation may, in turn, enhance the activity of heterotrophic N-fixers in the environment. Studies have shown that adding Fe(Ⅲ) (oxyhydr)oxides increases nitrogenase activity in paddy soils\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e, and high-throughput sequencing of bacterial 16S rRNA genes suggests a link between diazotrophs and DIRBs\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. Our recent metagenomic analysis indicated that N fixation might be associated with Fe(Ⅱ) transport and Fe(Ⅲ) reduction in groundwater\\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e. The widespread occurrence of MNF and Fe(Ⅲ) reduction across diverse ecosystems highlights the need for further investigation into potential interactions between these processes. However, direct evidence of synergy between N fixation and Fe(Ⅲ) reduction remains lacking, and the mechanisms of underlying potential interactions are not fully understood.\\u003c/p\\u003e \\u003cp\\u003eTo fill this knowledge gap, two types of diazotrophic DIRBs, \\u003cem\\u003eKlebsiella\\u003c/em\\u003e sp. N7 (fermentative type) and \\u003cem\\u003eGeobacter sulfurreducens\\u003c/em\\u003e PCA (respiratory type), as well as two model stains, i.e. the diazotroph \\u003cem\\u003eAzospirillum humicireducens\\u003c/em\\u003e SgZ-5T and DIRB \\u003cem\\u003eShewanella oneidensis\\u003c/em\\u003e MR-1, were selected to explore the synergy between N fixation and Fe(Ⅲ) reduction. Microcosms were established with environmental samples from aquifers, hot springs, soils, and marine sediments to assess the occurrence of diazotroph-synergized Fe(Ⅲ) reduction. Quantitative RT-PCR (RT-qPCR), transcriptomic, and metagenomic analyses of pure cultures and environmental samples across ecosystems were used to elucidate the synergy mechanisms.\\u003c/p\\u003e\"},{\"header\":\"2 Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 Effects of nitrogen fixation on Fe(Ⅲ) reduction\\u003c/h2\\u003e \\u003cp\\u003eThe intraspecies and interspecies effects of N fixation on Fe(Ⅲ) reduction were detected using batch pure cultures. The intraspecies results of two treatments, including N\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026thinsp;+\\u0026thinsp;Fe and Ar\\u0026thinsp;+\\u0026thinsp;Fe, showed that N\\u003csub\\u003e2\\u003c/sub\\u003e significantly promoted the growth (OD\\u003csub\\u003e600\\u003c/sub\\u003e, with 269.4% and 172.2%, respectively) and Fe(Ⅲ) reduction (maximum rate increased by 771.3% and 175.8%, respectively) of strains \\u003cem\\u003eKlebsiella\\u003c/em\\u003e sp. N7 and \\u003cem\\u003eG\\u003c/em\\u003e. \\u003cem\\u003esulfurreducens\\u003c/em\\u003e PCA (Fig.\\u0026nbsp;1A, B and Fig. S4A, B). After 7 days, approximately 5 mM Fe(Ⅲ) was fully reduced to Fe(Ⅱ) in the N\\u003csub\\u003e2\\u003c/sub\\u003e group, while the increase of Fe(Ⅱ) in the Ar group was only 1.9 mM, with none in the control group at day 14. Strain N7 could also rapidly grow and reduce ferrihydrite in the N\\u003csub\\u003e2\\u003c/sub\\u003e group, producing 1.8 mM Fe(Ⅱ) at day 14 which was 6 times higher than that in the Ar group (Fig. S5A, B). Our results showed that strain N7 also exhibited high nitrogenase activities under a concentration of 0.5 mM NH\\u003csub\\u003e4\\u003c/sub\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e (Fig. S6), indicating that N fixation enhances both growth and Fe(Ⅲ) reduction across \\u003cem\\u003ein situ\\u003c/em\\u003e groundwater concentrations of NH\\u003csub\\u003e4\\u003c/sub\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e-N (7.14\\u0026ndash;271.43 \\u0026micro;M) and NO\\u003csub\\u003e3\\u003c/sub\\u003e\\u003csup\\u003e\\u0026minus;\\u003c/sup\\u003e-N (3.57\\u0026ndash;507.85 \\u0026micro;M) (Fig. S5A, B and Fig. S7). Similarly, \\u003cem\\u003eG. sulfurreducens\\u003c/em\\u003e PCA reduced Fe(Ⅲ) by 55.9 mM in the N₂ group, 1.7 times that of the Ar control (Fig. S4B). The strong N\\u003csub\\u003e2\\u003c/sub\\u003e dependence of growth and Fe(Ⅲ) reduction indicated that N fixation plays an important role in the metabolism of the strain.\\u003c/p\\u003e \\u003cp\\u003eFor interspecies interactions, the model diazotroph \\u003cem\\u003eA. humicireducens\\u003c/em\\u003e SgZ-5T and model DIRB \\u003cem\\u003eS\\u003c/em\\u003e. \\u003cem\\u003eoneidensis\\u003c/em\\u003e MR-1 were selected to detect the effect of N fixation on Fe(Ⅲ) reduction. The growth characteristics and Fe(Ⅲ) reduction of \\u003cem\\u003eA. humicireducens\\u003c/em\\u003e SgZ-5T, \\u003cem\\u003eS. oneidensis\\u003c/em\\u003e MR-1, and co-culture are shown in Fig.\\u0026nbsp;1C, D. Co-cultures showed significant synergistic growth and Fe(Ⅲ) reduction (complete 5 mM reduction within 7 days) compared to the respective monocultures (30% and 14% Fe(Ⅲ) reduction for MR-1 and SgZ-5T, respectively). These results suggest that diazotrophs significantly enhance DIRB Fe(Ⅲ) reduction.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2 Effects of Fe(Ⅲ) reduction on diazotrophic activity\\u003c/h2\\u003e \\u003cp\\u003eThe effect of Fe(Ⅲ) reduction on N fixation within species was investigated using strains N7 and PCA under conditions with or without Fe(Ⅲ) amendment. Cultures amended with ferric citrate or ferrihydrite exhibited significantly higher nitrogenase activities (711.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;29.2 \\u0026micro;mol L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) than those without Fe(Ⅲ) reduction (456.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;24.1 \\u0026micro;mol L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) after 3 days of incubation (Fig.\\u0026nbsp;2A and Fig. S5C). Furthermore, the concentration of fixed \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003eN (134.61\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;8.9 \\u0026micro;mol L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) was significantly higher in Fe(Ⅲ)-amended cultures compared to those without Fe(Ⅲ) (75.69\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;22.9 \\u0026micro;mol L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) (Fig.\\u0026nbsp;2B and Fig. S5D). Additionally, the total amino acid concentration (N-fixing products) in Fe(Ⅲ)-amended cultures was approximately 1.5 times higher than in the non-amended treatments (Fig.\\u0026nbsp;2C).\\u003c/p\\u003e \\u003cp\\u003eThe interspecies effects of DIRB on diazotroph N fixation during Fe(Ⅲ) reduction were also explored. No nitrogenase activity was detected in the monoculture of \\u003cem\\u003eS. oneidensis\\u003c/em\\u003e MR-1 (Fig. S8). However, the co-culture of \\u003cem\\u003eS. oneidensis\\u003c/em\\u003e MR-1 and \\u003cem\\u003eA. humicireducens\\u003c/em\\u003e SgZ-5T demonstrated enhanced N fixation activity, particularly during the middle and late stages, as indicated by the acetylene reduction and \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003eN isotope tracing (Fig.\\u0026nbsp;2D, E). In contrast, no enhancement was observed in a co-culture lacking Fe(Ⅲ) (Fig. S9), suggesting that Fe(Ⅲ) reduction may play an important role in promoting N fixation. Furthermore, the extracellular fixed \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003eN concentration in co-cultures was significantly lower than that in the \\u003cem\\u003eA. humicireducens\\u003c/em\\u003e SgZ-5T monoculture (Fig.\\u0026nbsp;2F). The diazotroph \\u003cem\\u003eA. humicireducens\\u003c/em\\u003e SgZ-5T released 378.72 \\u0026micro;M of amino acids during N fixation in monoculture, which was significantly higher than in co-culture (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) (Fig.\\u0026nbsp;3E). These findings suggest that \\u003cem\\u003eS. oneidensis\\u003c/em\\u003e MR-1 may utilize amino acids secreted by \\u003cem\\u003eA. humicireducens\\u003c/em\\u003e SgZ-5T as an N source for growth and Fe(Ⅲ) reduction, thereby promoting N fixation.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3 Transcriptional response of species to nitrogen fixation and Fe(Ⅲ) reduction\\u003c/h2\\u003e \\u003cp\\u003eTo investigate the mechanisms underlying the synergy between N fixation and Fe(Ⅲ) reduction within and between species, transcriptomics analyses were performed on strain N7 cultures under Ar\\u0026thinsp;+\\u0026thinsp;Fe(Ⅲ), N\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026thinsp;+\\u0026thinsp;Fe(Ⅲ), and N\\u003csub\\u003e2\\u003c/sub\\u003e conditions, as well as on monocultures and co-cultures of \\u003cem\\u003eA. humicireducens\\u003c/em\\u003e SgZ-5T and \\u003cem\\u003eS. oneidensis\\u003c/em\\u003e MR-1 (Table S2). During N fixation in strain N7, 908 genes were significantly upregulated (Fig.\\u0026nbsp;3C). GO enrichment analysis revealed that upregulated DEGs were mainly involved in key metabolic pathways, including amino acid metabolism, ATP biosynthetic process, carbohydrate metabolism, N compound transport, and nucleotide metabolism (Fig. S10A). KEGG Orthologs annotation showed that \\u003cem\\u003enif\\u003c/em\\u003e genes and N assimilation were highly expressed in the presence of N\\u003csub\\u003e2\\u003c/sub\\u003e, with increases of up to 3.6- and 2.7-fold, respectively, compared to the Ar group (Fig. S11). Energy-yielding processes, including glycolysis, the TCA cycle, NADH-quinone oxidoreductases (\\u003cem\\u003enuo\\u003c/em\\u003e) of complex I, cytochrome c oxidase (\\u003cem\\u003ecyo\\u003c/em\\u003e) of complex IV, and V-type ATPase (\\u003cem\\u003eatp\\u003c/em\\u003e) were upregulated by 2.9-, 2.9-, 4.9-, 3.7-, and 3.7-fold, respectively, during N fixation (Fig.\\u0026nbsp;3D). RT-qPCR also revealed significant upregulation of the 16S rRNA and electron transfer genes (\\u003cem\\u003ecymA\\u003c/em\\u003e and \\u003cem\\u003emtrA\\u003c/em\\u003e) of DIRB \\u003cem\\u003eS. oneidensis\\u003c/em\\u003e MR-1 in co-culture (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) (Fig.\\u0026nbsp;3F). Consistently, increased C catabolism during N fixation accelerated DOC depletion and CO\\u003csub\\u003e2\\u003c/sub\\u003e production (Fig.\\u0026nbsp;3A, B). These results indicate that N fixation enhances bacterial anaerobic respiration and biomass production by supplying N, thereby accelerating Fe(Ⅲ) reduction.\\u003c/p\\u003e \\u003cp\\u003eBacterial transcriptional responses to Fe(Ⅲ) reduction were also examined. GO analysis of DEGs revealed that 520 upregulated genes in the N\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026thinsp;+\\u0026thinsp;Fe(Ⅲ) condition were associated with NADH dehydrogenase (quinone) activity, electron transfer activity, Fe-S cluster binding, amino acid metabolism, and membrane protein complexes (Fig.\\u0026nbsp;4C and Fig. S10B). KEGG analysis identified upregulation of genes encoding components of electron transport chains, including NADH-quinone oxidoreductase (\\u003cem\\u003enuo\\u003c/em\\u003e), succinate-dehydrogenase (\\u003cem\\u003efrdA\\u003c/em\\u003e), cytochromes bd (\\u003cem\\u003ecydAB\\u003c/em\\u003e), and ATP synthase (\\u003cem\\u003eatp\\u003c/em\\u003e) (Fig.\\u0026nbsp;4D) (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, Log\\u003csub\\u003e2\\u003c/sub\\u003eFC\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.5). Genes related to anaerobic fermentation, such as \\u003cem\\u003epflB\\u003c/em\\u003e and \\u003cem\\u003enifJ\\u003c/em\\u003e, were also upregulated by 58.5% and 50.1%, respectively, under Fe(Ⅲ)-reducing conditions (Fig.\\u0026nbsp;4D). Correspondingly, increased C decomposition, CO\\u003csub\\u003e2\\u003c/sub\\u003e production, and ATP yield were observed under Fe(Ⅲ) reduction (Fig.\\u0026nbsp;4A, B, E, F). Meanwhile, the respiratory DIRB \\u003cem\\u003eG. sulfurreducens\\u003c/em\\u003e PCA directly utilizes Fe(Ⅲ) as an electron acceptor for growth and N fixation promotion (Fig. S4C). For interspecies interactions, respiratory DIRB \\u003cem\\u003eS. oneidensis\\u003c/em\\u003e MR-1, which lacks N fixation capability, showed significant upregulation of the electron transfer genes (\\u003cem\\u003ecymA\\u003c/em\\u003e and \\u003cem\\u003emtrA\\u003c/em\\u003e) in co-culture, along with increased ATP production and OM consumption (Fig.\\u0026nbsp;4E, F). This pattern was consistent with intraspecific interactions. As a result, diazotroph \\u003cem\\u003enifH\\u003c/em\\u003e gene expression was significantly upregulated by Fe(Ⅲ) reduction (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) (Fig.\\u0026nbsp;4D, G). These results demonstrate that the synergy between N fixation and Fe(Ⅲ) reduction occurs both within and between species (Fig.\\u0026nbsp;5).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4. Nitrogen fixation and Fe(Ⅲ) reduction in different ecosystems.\\u003c/h2\\u003e \\u003cp\\u003eTo validate the above findings, microcosm experiments were conducted using environmental samples collected from aquifers, hot springs, soil, and marine ecosystems. The results indicated that Fe(Ⅲ) reduction significantly enhanced N fixation activity in samples from aquifer waters/sediments (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001), marine sediments (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001), and soils (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) (Fig.\\u0026nbsp;6A and Fig. S12). After the end of incubation, significantly higher nitrogenase activities (1.1\\u0026ndash;8106.9 \\u0026micro;mol L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) were detected in Fe(Ⅲ)-amended cultures compared to those without Fe(Ⅲ) (0.3\\u0026ndash;884.9 \\u0026micro;mol L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) (Fig. S12). Simultaneously, Fe(Ⅲ) reduction rates were higher in N\\u003csub\\u003e2\\u003c/sub\\u003e cultures than in no-N\\u003csub\\u003e2\\u003c/sub\\u003e controls (Fig.\\u0026nbsp;6A and Fig. S12). N fixation accelerated Fe(Ⅲ) reduction in marine and hot spring sediments early in incubation, with Fe(Ⅱ) concentrations increasing by 15.5\\u0026ndash;40.6% (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). In aquifer sediments and soils, Fe(Ⅲ) reduction rates were significantly higher than in the Ar group during the mid-incubation stage, with Fe(Ⅱ) production increasing by 14.8\\u0026ndash;38.2% (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Overall, the synergy between N fixation and Fe(Ⅲ) reduction occurs across various ecosystems.\\u003c/p\\u003e \\u003cp\\u003eTo elucidate these findings, metagenomic analysis was conducted on samples from aquifer waters/sediments, hot spring sediments, soils, and marine sediments to assess the metabolic potential for N fixation and Fe(Ⅲ) reduction. Key functional genes involved in N fixation (\\u003cem\\u003enif\\u003c/em\\u003e) and Fe(Ⅲ) reduction (\\u003cem\\u003eomc\\u003c/em\\u003e, \\u003cem\\u003ecym\\u003c/em\\u003e, and \\u003cem\\u003emtr\\u003c/em\\u003e) were identified using the KEGG database and FeGenie. Nitrogenase (\\u003cem\\u003enifHDK\\u003c/em\\u003e and \\u003cem\\u003enifENB\\u003c/em\\u003e) gene abundance positively correlated with Fe(Ⅲ) reduction genes (traditional multi-heme c-type cytochromes (MHCs): \\u003cem\\u003emtrABC\\u003c/em\\u003e and \\u003cem\\u003eomcSZ\\u003c/em\\u003e) in all ecosystems (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) (Fig.\\u0026nbsp;6B). Additionally, \\u003cem\\u003enif\\u003c/em\\u003e clusters were strongly correlated with the genes encoding N transport (\\u003cem\\u003eamt\\u003c/em\\u003e), N assimilation (\\u003cem\\u003eglt\\u003c/em\\u003e, \\u003cem\\u003egln\\u003c/em\\u003e, and \\u003cem\\u003egdh\\u003c/em\\u003e), electron transport (\\u003cem\\u003enuo\\u003c/em\\u003e, \\u003cem\\u003ecyd\\u003c/em\\u003e, \\u003cem\\u003efix\\u003c/em\\u003e, and \\u003cem\\u003ekor\\u003c/em\\u003e), F-type ATPases (\\u003cem\\u003eatp\\u003c/em\\u003e), and Fe transport (\\u003cem\\u003efeo\\u003c/em\\u003e) (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) (Fig. S12). After metagenomic binning, medium-quality metagenome-assembled genomes (MAGs) (completeness\\u0026thinsp;\\u0026gt;\\u0026thinsp;50%, contamination\\u0026thinsp;\\u0026lt;\\u0026thinsp;10%) containing key genes involved in N fixation (\\u003cem\\u003enif\\u003c/em\\u003e) and Fe(Ⅲ) reduction (MHCs: \\u003cem\\u003eomc\\u003c/em\\u003e, \\u003cem\\u003ecym\\u003c/em\\u003e, and \\u003cem\\u003emtr\\u003c/em\\u003e) were retrieved from aquifer, soil, and marine environments. These included GW-bin 16 (\\u003cem\\u003eThermodesulfovibrionia\\u003c/em\\u003e), S-bin 31 (\\u003cem\\u003eKapabacteria\\u003c/em\\u003e), soil2-bin 12 (\\u003cem\\u003eBacteria\\u003c/em\\u003e), soil4-bin 12 (\\u003cem\\u003eBacteria\\u003c/em\\u003e), and sea8-bin 14 (\\u003cem\\u003eBacteria\\u003c/em\\u003e) (Fig.\\u0026nbsp;6C). Although the diazotrophic genomes from hot springs lacked MHCs, genes encoding pathways for redox molecule biosynthesis and electron transport were identified, such as riboflavin/menaquinone biosynthesis and quinone oxidoreductase (Fig.\\u0026nbsp;6C). Based on KEGG Module analysis, genes related to C metabolism (e.g., glycolysis, pyruvate oxidation, and citrate cycle), electron transport (e.g., NADH dehydrogenase, quinone oxidoreductase, cytochrome c oxidase), ATP synthesis (F-type ATPase), electron shuttle biosynthesis (riboflavin/menaquinone biosynthesis), N fixation(\\u003cem\\u003enif\\u003c/em\\u003e), and Fe(Ⅲ) reduction (\\u003cem\\u003eomc\\u003c/em\\u003e, \\u003cem\\u003ecym\\u003c/em\\u003e, and \\u003cem\\u003emtr\\u003c/em\\u003e) were identified in the retrieved MAGs from aquifer, hot springs, soil, and marine sediments (Fig.\\u0026nbsp;6C). Thus, microorganisms in these environments possess the metabolic potential to facilitate the synergy between N fixation and Fe(Ⅲ) reduction across diverse ecosystems.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Discussion\",\"content\":\"\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1 Evidence and mechanism of the synergy between nitrogen fixation and Fe(Ⅲ) reduction\\u003c/h2\\u003e \\u003cp\\u003eOur integrated pure culture studies and field investigations provide substantial evidence of the synergistic interaction between N fixation and Fe(Ⅲ) reduction. Multiple microorganisms have been shown to mediate N fixation and Fe(Ⅲ) reduction simultaneously\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. In our study, fermentative and respiratory diazotrophic DIRBs displayed accelerated growth and Fe(Ⅲ) reduction under N fixation conditions. In turn, Fe(Ⅲ) reduction facilitated MNF. Additionally, our findings indicate that diazotrophs feed on DIRB by secreting N-fixed products, and DIRB provides energy for diazotrophs, enhancing fixed N in co-culture. Previous studies have speculated on the correlation between diazotrophs and DIRBs in soils\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e, coastal sediments\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e, and wetlands\\u003csup\\u003e\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e, and our recent research extends this to groundwater systems, revealing positive interactions between N fixation and Fe(Ⅲ) reduction\\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e. In this study, microcosm experiments and metagenomic analyses across various habitats further validate this synergy. The reduction of 1 mmol Fe(III) is estimated to contribute 1.42\\u0026ndash;75.58 \\u0026micro;mol N fixation, representing a potential pathway for N input in the environment (Table S6). Moreover, N fixation increased by 4.1\\u0026ndash;36.2% of Fe(Ⅱ) production in these sediments (Fig. S12A). Intraspecies, interspecies, and environmental observations consistently support the hypothesis of synergistic N fixation and Fe(Ⅲ) reduction, improving our understanding of coupled N and Fe cycles in nature.\\u003c/p\\u003e \\u003cp\\u003eDespite the high energy demand of MNF, diazotrophs link various catabolic processes to fix N in natural environments\\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e. The availability of electron donors and energy sources are critical factors driving N fixation\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. In oligotrophic mine tailings, dominant chemolithotrophic diazotrophs utilize S, arsenic (As), and antimony (Sb) as electron donors\\u003csup\\u003e\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e, whereas heterotrophic diazotrophs oxidize organic compounds to produce ATP for N fixation in diverse terrestrial and aquatic systems\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u003c/sup\\u003e. In this study, Fe(Ⅲ) acted as a reservoir for excess reducing equivalents in fermentative DIRBs\\u003csup\\u003e\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u003c/sup\\u003e, enhancing organic fermentation, electron transport, ATP synthesis, and biomass production, thereby facilitating N fixation (Fig.\\u0026nbsp;4, Fig. S11, and Fig. S13). Respiratory diazotrophic DIRBs use Fe(Ⅲ) as a terminal electron acceptor to generate the energy necessary for N fixation, and their inability to fix N in the absence of Fe(Ⅲ) demonstrates the critical role of Fe(Ⅲ) in this process. Interestingly, in interspecific interactions, non-N-fixing respiratory DIRBs indirectly promote N fixation in diazotrophs through Fe(Ⅲ)-dependent anaerobic respiration. N fixation, in turn, accelerates Fe(Ⅲ) reduction by enhancing microbial growth and carbon metabolic pathways, creating a positive feedback loop (Fig.\\u0026nbsp;5)\\u003csup\\u003e\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e. This synergy is further evidenced by the strong correlation between nitrogenase, carbon metabolism, and Fe(Ⅲ) reduction genes in various ecosystems. These findings indicate a bidirectional interaction between N fixation and Fe(Ⅲ) reduction, which enhances the flow of nutrients and energy in natural environments.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2 Environment implications of the synergy between nitrogen fixation and Fe(Ⅲ) reduction\\u003c/h2\\u003e \\u003cp\\u003eFe(Ⅲ) reduction and Fe(Ⅱ) oxidation occur simultaneously or cyclically in many environments. DIRBs reduce Fe(Ⅲ) to Fe(Ⅱ) under anoxic conditions, whereas Fe(Ⅱ) can be biotically oxidized by nitrate-reducing Fe(Ⅱ)-oxidizing microbes or abiotically oxidized back to Fe(Ⅲ) by atmospheric oxygen\\u003csup\\u003e\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u003c/sup\\u003e. Studies have demonstrated that Fe cycling is closely coupled with OM transformation under fluctuating oxygen conditions\\u003csup\\u003e\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e\\u003c/sup\\u003e. Under such alternating redox conditions, the reduction and oxidation of Fe continuously fuel N fixation, while N fixation can, in turn, enhance microbial growth and Fe cycling. The impact of N fixation on Fe(III) reduction and Fe(II) transport may significantly contribute to phytoplankton productivity production and benthic nutrient fluxes (Fig. S11)\\u003csup\\u003e\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e. Consequently, N fixation serves as a crucial nutrient source for microbial-mediated C-Fe cycles, whereas Fe redox cycling has the potential to support substantial N pools in natural environments by promoting N fixation\\u003csup\\u003e\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e\\u003c/sup\\u003e. Under these redox fluctuations, N fixation may enhance microbial resistance to reactive oxygen species (ROS) (Fig. S11)\\u003csup\\u003e\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e\\u003c/sup\\u003e. In laboratory settings, N\\u003csub\\u003e2\\u003c/sub\\u003e is commonly used to create anoxic environments for incubating DIRBs, providing a potential N source for diazotrophic DIRBs. Moreover, studies suggest that coupled N fixation and Fe(Ⅲ) reduction could regulate CH\\u003csub\\u003e4\\u003c/sub\\u003e emissions\\u003csup\\u003e\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e. Recently, N\\u003csub\\u003e2\\u003c/sub\\u003eO fixation by diazotrophs has been proposed as an alternative sink for N\\u003csub\\u003e2\\u003c/sub\\u003eO, potentially accounting for 60% of total N\\u003csub\\u003e2\\u003c/sub\\u003eO reduction in the Pacific Ocean\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e\\u003c/sup\\u003e. In our study, we found that microbial N\\u003csub\\u003e2\\u003c/sub\\u003eO fixation also promoted the growth of diazotrophic DIRB and Fe(Ⅲ) reduction, with Fe(Ⅲ) reduction significantly accelerating N\\u003csub\\u003e2\\u003c/sub\\u003eO consumption (Fig. S14). These findings suggest that Fe(Ⅲ) application could be a feasible strategy for reducing N\\u003csub\\u003e2\\u003c/sub\\u003eO emissions in the environment. We propose that cryptic Fe cycles under redox fluctuations, such as those caused by alternating soil wetting and drying, groundwater-surface water interactions, and oceanic vertical stratification, may contribute to nutrient flow and climate gas mitigation in natural ecosystems.\\u003c/p\\u003e \\u003cp\\u003eTheoretically, N fixation is more likely to occur in oligotrophic habitats. However, studies have shown that diazotrophs are widely distributed in both oligotrophic and eutrophic environments\\u003csup\\u003e\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e\\u003c/sup\\u003e. In this study, N fixation activity was detected across different ecosystems. Serval diazotrophs lack N assimilation or reduction genes, leaving N fixation as the only bioavailable N source in certain ecosystems\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. Moreover, the synergy between N fixation and Fe(Ⅲ) reduction is expected to be more pronounced in eutrophic environments rich in OM, such as freshwater sediments\\u003csup\\u003e\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e\\u003c/sup\\u003e and paddy soils\\u003csup\\u003e\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e\\u003c/sup\\u003e. In these systems, coupled Fe-C cycles may stimulate diazotrophs and DIRBs, contributing to substantial N and Fe(Ⅱ) inputs. In marine environments, particles serve as an OM source for heterotrophic diazotrophs and DIRBs\\u003csup\\u003e\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e\\u003c/sup\\u003e. We speculate that N depletion by DIRBs in particle-associated microenvironments creates N-deficient niches conducive to diazotrophs. Consequently, aquatic environments with high particle fluxes are hotspots for N fixation\\u003csup\\u003e\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e\\u003c/sup\\u003e. In extreme oligotrophic environments, such as hot springs, C-fixing microorganisms or algae residues may provide C sources for heterotrophs (our unpublished data)\\u003csup\\u003e\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e\\u003c/sup\\u003e, supporting the synergistic occurrence of N fixation and Fe(Ⅲ) reduction. Interestingly, diazotrophs may fix N not only to meet N demands but also to maintain an optimal intracellular redox state\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. Thus, N fixation could serve as a mechanism to balance redox states in natural environments by eliminating excess electrons generated from the oxidation of OM, Fe, and H\\u003csub\\u003e2\\u003c/sub\\u003e. In summary, the effective synergy between N fixation and Fe(Ⅲ) reduction, accompanied by C decomposition, may partly explain the widespread occurrence of N fixation in eutrophic environments.\\u003c/p\\u003e \\u003cp\\u003eThe interaction between MNF and Fe(Ⅲ) reduction is an overlooked process that enhances N supply, biomass yield, and Fe/C cycling across various ecosystems. Our study provides important insights into the mechanisms and prevalence of synergistic MNF and Fe(Ⅲ) reduction, which may help mitigate greenhouse gas emissions linked to agricultural fertilizers. Further studies are warranted to assess the role of this synergistic interaction in sustaining bioavailable N and Fe/C cycling in natural ecosystems.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.1 Strains, environmental samples, and media\\u003c/h2\\u003e \\u003cp\\u003eThe strain \\u003cem\\u003eKlebsiella\\u003c/em\\u003e sp. N7 (N7), a facultative anaerobic N-fixing bacterium, was isolated from groundwater as described in a previous study (Fig. S1) (accession number PRJNA1160161). Strain N7 can reduce dissolved Fe(Ⅲ) (Fe(Ⅲ)-citrate) and Fe(Ⅲ) mineral (ferrihydrite) in N-free Burk\\u0026rsquo;s medium. The diazotrophic DIRB \\u003cem\\u003eG. sulfurreducens\\u003c/em\\u003e PCA was purchased from the American Type Culture Collection (Manassas, VA, United States) (ATCC 51573). \\u003cem\\u003eA. humicireducens\\u003c/em\\u003e SgZ-5T (CCTCC AB 2012021), which can fix N in Burk\\u0026rsquo;s medium, was purchased from the China Center for Type Culture Collection (Wuhan University, China). The DIRB \\u003cem\\u003eS. oneidensis\\u003c/em\\u003e MR-1 (ATCC 700550), which cannot fix N but can reduce Fe(Ⅲ), was also obtained from ATCC (number: 700550). To activate cultures, Luria Broth (LB) medium containing (per liter) 10 g NaCl, 5 g yeast extract, and 10 g tryptone was used to grow \\u003cem\\u003eKlebsiella\\u003c/em\\u003e sp. N7 and \\u003cem\\u003eS. oneidensis\\u003c/em\\u003e MR-1 at 30\\u0026deg;C. \\u003cem\\u003eA. humicireducens\\u003c/em\\u003e SgZ-5T was cultured in Nutrient Broth (NB) medium containing (per liter) 5 g NaCl, 5 g peptone, and 3 g beef extract at 30\\u0026deg;C. Modified anaerobic Burk\\u0026rsquo;s medium was used to cultivate strains for both N fixation and Fe(Ⅲ) reduction at 30\\u0026deg;C. The medium contained the following components (per liter): 20 g mannitol, 0.2 g KH\\u003csub\\u003e2\\u003c/sub\\u003ePO\\u003csub\\u003e4\\u003c/sub\\u003e, 0.8 g\\u0026middot;K\\u003csub\\u003e2\\u003c/sub\\u003eHPO\\u003csub\\u003e4\\u003c/sub\\u003e, 0.2 g MgSO\\u003csub\\u003e4\\u003c/sub\\u003e\\u0026middot;7H\\u003csub\\u003e2\\u003c/sub\\u003eO, 0.1 g CaSO\\u003csub\\u003e4\\u003c/sub\\u003e\\u0026middot;2H\\u003csub\\u003e2\\u003c/sub\\u003eO, and trace amounts of Na\\u003csub\\u003e2\\u003c/sub\\u003eMoO\\u003csub\\u003e4\\u003c/sub\\u003e\\u0026middot;2H\\u003csub\\u003e2\\u003c/sub\\u003eO and FeCl\\u003csub\\u003e3\\u003c/sub\\u003e, at pH 7.0. Fe(Ⅲ) reduction was conducted in this medium supplemented with Fe(Ⅲ)-citrate or ferrihydrite. Ferrihydrite was prepared following previously published methods \\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e. \\u003cem\\u003eG\\u003c/em\\u003e. \\u003cem\\u003esulfurreducens\\u003c/em\\u003e PCA was cultured in an N-free FWAFC medium for N fixation and Fe(Ⅲ) reduction analyses\\u003csup\\u003e\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eRepresentative aquifer sediments were collected from two boreholes at a depth of 20 m in the recharge and discharge zones of the Hetao Plain, Inner Mongolia, China\\u003csup\\u003e\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e\\u003c/sup\\u003e. Hot spring sediments were sampled from two drainage channels in Qamdo, eastern Tibet, designed as DB-A and DB-B\\u003csup\\u003e\\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e\\u003c/sup\\u003e. Surface soil samples were taken from areas near the Han River and southeastern Jianghan Plain, Hubei Province, China\\u003csup\\u003e\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e\\u003c/sup\\u003e. Subsurface paddy soils were also collected from Xiazhaijin Village, Zhejiang Province, China. Marine sediments were obtained from the surface 0\\u0026ndash;5 cm layer of a box core in the East China Sea. The basic information of these samples is provided in Table S3. All fresh sediments and soils were sealed in 50 mL sterile polyethylene tubes, stored on dry ice, and transported to the laboratory for homogenization and microcosmic culturing as soon as possible. Additionally, metagenomic analysis was conducted on samples from various habitats, including aquifer waters/sediments (our study)\\u003csup\\u003e\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e\\u003c/sup\\u003e, hot spring sediments (our unpublished data), marine waters/sediments\\u003csup\\u003e56,57\\u003c/sup\\u003e, and paddy soils\\u003csup\\u003e\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e\\u003c/sup\\u003e. The geographic distribution of environmental sampling sites for microcosm and metagenomic data is presented in Fig. S2. The method for metagenome data processing and metagenomic binning analysis are detailed in our recent publication\\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.2 Monoculture and interspecies assays\\u003c/h2\\u003e \\u003cp\\u003eStrain \\u003cem\\u003eKlebsiella\\u003c/em\\u003e sp. N7 was routinely cultured in 150 mL of LB medium at 30\\u0026deg;C with shaking at 150 rpm. Cells were harvested by centrifugation (8,000 rpm, 10 min, 4\\u0026deg;C), washed three times with phosphate-buffered saline (PBS), and resuspended in sterilized anaerobic N/Fe-free Burk\\u0026rsquo;s medium. The bacterial suspension's optical density at 600 nm (OD\\u003csub\\u003e600\\u003c/sub\\u003e) was measured and adjusted to a final OD\\u003csub\\u003e600\\u003c/sub\\u003e of 0.05 by inoculating it into fresh Burk\\u0026rsquo;s medium. To investigate the synergistic interaction between N fixation and Fe(Ⅲ) reduction, four experimental groups were established: Burk, Burk\\u0026thinsp;+\\u0026thinsp;Fe, N\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026thinsp;+\\u0026thinsp;Fe, and Ar\\u0026thinsp;+\\u0026thinsp;Fe. The N\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026thinsp;+\\u0026thinsp;Fe and Ar\\u0026thinsp;+\\u0026thinsp;Fe groups, designed to assess the impact of N fixation on Fe(Ⅲ) reduction, were supplemented with 5 mM ferric citrate or ferrihydrite and degassed with N\\u003csub\\u003e2\\u003c/sub\\u003e and Ar, respectively. The Burk and Burk\\u0026thinsp;+\\u0026thinsp;Fe groups were purged with N\\u003csub\\u003e2\\u003c/sub\\u003e gas. To simulate environmentally relevant Fe concentrations, 0.2 mM ferric citrate or ferrihydrite was added to the Burk\\u0026thinsp;+\\u0026thinsp;Fe group to examine the influence of Fe(Ⅲ) reduction on N fixation. A non-inoculated control group served as the blank control. \\u003cem\\u003eG. sulfurreducens\\u003c/em\\u003e PCA was cultured in N-free FWAFC medium under anaerobic conditions. At the early stationary phase (OD\\u003csub\\u003e600\\u003c/sub\\u003e\\u0026thinsp;\\u0026asymp;\\u0026thinsp;0.5), 10 mL of culture was inoculated into 100 mL N-free FWAFC medium. The N\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026thinsp;+\\u0026thinsp;Fe and Ar\\u0026thinsp;+\\u0026thinsp;Fe groups were degassed with 80% N\\u003csub\\u003e2\\u003c/sub\\u003e-20% CO\\u003csub\\u003e2\\u003c/sub\\u003e and 80% Ar-20% CO\\u003csub\\u003e2\\u003c/sub\\u003e, respectively. To assess the effect of Fe(Ⅲ) reduction on N fixation in PCA, treatments with and without ferric citrate as the electron acceptor were established. All cultures were incubated at 30\\u0026deg;C in the dark and in triplicate.\\u003c/p\\u003e \\u003cp\\u003eA co-culture system consisting of diazotroph \\u003cem\\u003eA. humicireducens\\u003c/em\\u003e SgZ-5T and DIRB \\u003cem\\u003eS. oneidensis\\u003c/em\\u003e MR-1 was constructed to explore interspecies interactions between N fixation and Fe(Ⅲ) reduction. Active cells from both strains were harvested, washed three times with PBS, and resuspended in sterilized anaerobic N/Fe-free Burk\\u0026rsquo;s medium. The suspensions of diazotroph and DIRB were inoculated either individually or in combination (at a 1:1 volume ratio, each with an OD\\u003csub\\u003e600\\u003c/sub\\u003e of 0.02). The non-inoculated treatment served as the blank control. All groups were supplemented with 5 mM ferric citrate, degassed with N\\u003csub\\u003e2\\u003c/sub\\u003e, and incubated at 30\\u0026deg;C with shaking at 150 rpm in the dark.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.3 Characterization of nitrogen fixation and Fe(Ⅲ) reduction\\u003c/h2\\u003e \\u003cp\\u003eNitrogenase activity was characterized using acetylene-reduction assays as previously described\\u003csup\\u003e\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e\\u003c/sup\\u003e. Briefly, 3 mL headspace air (10% of headspace) was extracted from the anoxic medium and replaced with 3 mL acetylene. The ethylene concentration was measured by a gas chromatograph (GC-4000A, EWAI) equipped with a flame ionization detector and a Porpack N column. Isotopic enrichment of \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003eN in various treatment groups was analyzed using a membrane inlet mass spectrometer (MIMS, Hiden HPR-40, UK) via \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003eN isotope tracing\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e\\u003c/sup\\u003e. Specifically, 60 mL serum bottles containing 30 mL of medium were degassed with Ar for 40 min, after which the headspace was replaced with 30 mL \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003eN\\u003csub\\u003e2\\u003c/sub\\u003e (Cambridge Isotope Laboratories, \\u0026gt;\\u0026thinsp;98 atom%). Bottles were incubated inverted at 30\\u0026deg;C with shaking at 150 rpm. After incubation, the cultures were autoclaved at 121\\u0026deg;C for 30 min and purged with Ar for 40 min to eliminate background \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003eN\\u003csub\\u003e2\\u003c/sub\\u003e. The fixed \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003eN-labeled products were analyzed by MIMS after oxidation with hypobromite iodine solution to \\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003eN\\u003csub\\u003e2\\u003c/sub\\u003e and/or \\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003eN\\u003csub\\u003e2\\u003c/sub\\u003e\\u003csup\\u003e62\\u003c/sup\\u003e. A standard calibration curve was generated using a concentration gradient of \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003eNH\\u003csub\\u003e4\\u003c/sub\\u003eCl (Cambridge Isotope Laboratories, 99%). The linear relationship between \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003eN concentrations and the total produced \\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003eN\\u003csub\\u003e2\\u003c/sub\\u003e plus \\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003eN\\u003csub\\u003e2\\u003c/sub\\u003e signal is shown in Fig. S3. Initial sample bottles were processed immediately after \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003eN\\u003csub\\u003e2\\u003c/sub\\u003e tracer addition.\\u003c/p\\u003e \\u003cp\\u003eFor Fe(Ⅱ) and total Fe quantification, samples were collected under anoxic conditions in a glovebox, mixed with an equal volume of 1 M HCl, and analyzed using a ferrozine assay\\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e. DOC was measured by filtering the bacterial suspension through 0.22-\\u0026micro;m membrane filters (Millipore) and analyzed with a TOC analyzer (TOC-L, Shimadzu, Japan). The CO\\u003csub\\u003e2\\u003c/sub\\u003e content in the headspace was determined using a gas chromatograph (Shimadzu GC-2014). Amino Acid (AA) production during N fixation was quantified using an AA Assay Kit (Sangon, China). Intracellular adenosine triphosphate (ATP) levels were measured following the manufacturer\\u0026rsquo;s instructions with an ATP Content Assay Kit (Solarbio Science \\u0026amp; Technology Co., Ltd)\\u003csup\\u003e\\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e\\u003c/sup\\u003e. Mannitol content was determined using spectrophotometry\\u003csup\\u003e\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.4. RNA extraction, RT-qPCR, and transcriptomic analysis\\u003c/h2\\u003e \\u003cp\\u003eTo investigate the gene expression of strains during N fixation and Fe(Ⅲ) reduction, three treatment groups of intraspecific assays were analyzed via transcriptomics (N7): Burk, Burk\\u0026thinsp;+\\u0026thinsp;Fe, and Burk\\u0026thinsp;+\\u0026thinsp;Fe\\u0026thinsp;+\\u0026thinsp;Ar. Both Burk and Burk\\u0026thinsp;+\\u0026thinsp;Fe groups were purged with N\\u003csub\\u003e2\\u003c/sub\\u003e gas, while 0.2 mM Fe(Ⅲ)-citrate was added to the Burk\\u0026thinsp;+\\u0026thinsp;Fe group. The Burk\\u0026thinsp;+\\u0026thinsp;Fe\\u0026thinsp;+\\u0026thinsp;Ar group, also treated with 0.2 mM Fe(Ⅲ)-citrate, was degassed with Ar. For cross-feeding experiments, three treatment groups were used for gene expression profiling: monocultures of \\u003cem\\u003eA. humicireducens\\u003c/em\\u003e SgZ-5T and \\u003cem\\u003eS. oneidensis\\u003c/em\\u003e MR-1, as well as a co-culture. Following incubation, cells in all treatments were collected during the log phase via centrifugation (8,000 rpm, 20 min, 4\\u0026deg;C). Total RNA was extracted using RNAiso Plus (TaKaRa, Bio) following the manufacturer\\u0026rsquo;s protocol, and residual DNA in RNA was removed with thePrimeScript\\u0026trade; RT re-agent Kit with gDNA Eraser (TaKaRa, Bio) for 2 min at 42\\u0026deg;C. RNA integrity was assessed via 1.5% agarose gel electrophoresis (Biowest, Riverside, MO, USA), and RNA concentrations were determined using a NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For cDNA synthesis, RNA was diluted and reverse transcribed using PrimeScript\\u0026trade; RT reagent Kit with gDNA Eraser (TaKaRa, Bio). Quantitative RT-PCR (RT-qPCR) was then performed (Applied Biosystems, Waltham, MA, USA) with the TB Green\\u0026trade; Premix Ex TaqTM Ⅱ (TaKaRa, Bio) qPCR kit. Primer sequences and amplification conditions are listed in Table S1. The bacterial 16S rRNA gene was used as an endogenous reference, and transcript levels of \\u003cem\\u003eShewanella\\u003c/em\\u003e 16S rRNA, \\u003cem\\u003enifH\\u003c/em\\u003e, \\u003cem\\u003ecymA\\u003c/em\\u003e, and \\u003cem\\u003emtrA\\u003c/em\\u003e were quantified using the 2\\u003csup\\u003e\\u0026minus;ΔΔCT\\u003c/sup\\u003e method\\u003csup\\u003e\\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e65\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eRNA samples for sequencing were stored at \\u0026minus;\\u0026thinsp;80\\u0026deg;C and sent to Magigene Technology (Guangzhou, China) as soon as possible for RNA-Seq library preparation (ALFA-SEQ RNA Library Prep Kit Ⅱ) and sequenced using the Illumina NovaSeq 6000 platform. Raw reads were processed to remove low-quality data using Trimmomatic (Version 0.36) to generate clean reads\\u003csup\\u003e\\u003cspan citationid=\\\"CR66\\\" class=\\\"CitationRef\\\"\\u003e66\\u003c/span\\u003e\\u003c/sup\\u003e. These were subsequently mapped to the reference genome of \\u003cem\\u003eKlebsiella grimontⅡ\\u003c/em\\u003e from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using Bowtie2 (Version 2.4.5)\\u003csup\\u003e\\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e67\\u003c/span\\u003e\\u003c/sup\\u003e. Quantification of read counts was performed using Salmon (Version 1.9.0), and differential expression analysis was conducted with DESeq 2 (Version 1.30.1) to identify differentially expressed genes (DEGs)\\u003csup\\u003e\\u003cspan citationid=\\\"CR68\\\" class=\\\"CitationRef\\\"\\u003e68\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR69\\\" class=\\\"CitationRef\\\"\\u003e69\\u003c/span\\u003e\\u003c/sup\\u003e. DEGs were considered significant with a \\u003cem\\u003eP\\u003c/em\\u003e-adjust value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 and |log\\u003csub\\u003e2\\u003c/sub\\u003e fold change| \\u0026ge; 0.5. The transcriptomic results are presented in Table S2. Gene annotation was performed using the KEGG database, while Gene Ontology (GO) enrichment analysis followed the protocol from our previous study\\u003csup\\u003e\\u003cspan citationid=\\\"CR70\\\" class=\\\"CitationRef\\\"\\u003e70\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.5 Sediment microcosms and metagenomic analysis\\u003c/h2\\u003e \\u003cp\\u003eTo investigate the widespread occurrence of the synergy between N fixation and Fe(Ⅲ) reduction, microcosm cultures were established using environmental samples from various habitats, including aquifers, hot springs, marine waters/sediments, and soils. Triplicate microcosm incubations were conducted in 120 mL sterile serum bottles, each containing 50 mL of homogenized slurries prepared by mixing sediment and sterile water at a sediment/water weight ratio of 1:5. The N\\u003csub\\u003e2\\u003c/sub\\u003e and Ar groups were purged with N\\u003csub\\u003e2\\u003c/sub\\u003e and Ar gases for 50 min to assess the impact of N fixation on Fe(Ⅲ) reduction. Two additional treatments were set up by amending Fe(Ⅲ)-citrate (at concentrations reflecting near-field water Fe levels) (Table S3) to evaluate nitrogenase activity driven by Fe(Ⅲ) reduction. Microcosms of hot spring sediments were incubated at 60\\u0026deg;C, while the other cultures were incubated at 25\\u0026deg;C in the dark. Nitrogenase activity was determined using acetylene-reduction assays, as described in section \\u003cspan refid=\\\"Sec13\\\" class=\\\"InternalRef\\\"\\u003e4.3\\u003c/span\\u003e of Materials and Methods. Fe(Ⅱ) samples were collected inside an anoxic glovebox, centrifuged at 1,2000 rpm for 5 min, and the supernatant was diluted in 1 M HCl for quantification via the ferrozine assay. Sediments were extracted with 0.5 M HCl, and the supernatant was stabilized in 0.5 M HCl for Fe(Ⅱ) analysis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.6 Statistical analyses and data availability\\u003c/h2\\u003e \\u003cp\\u003eSpearman correlations among the abundances of functional genes were computed using R v4.1.2. Data visualization, fitting, and evaluation were performed using GraphPad Prism v10 (GraphPad Software, San Diego, CA, USA).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eRaw RNA-Seq data have been deposited in the NCBI database (accession number: PRJNA1172356). Metagenome sequencing reads from the aquifer and hot spring samples are available from our previous study under NCBI BioProject accession numbers PRJNA882225 and PRJNA943127.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work is supported by the National Natural Science Foundation of China\\u0026nbsp;(Nos. 42177068 [P.L.], 42025703 [S.Y.], Nos. 41772260 [P.L.], and 41976043 [Y.Y.]). Marine surface sediment and environmental parameters were collected onboard, implementing the open research cruises, NORC2022-03 and NORC2023-06. We thank Prof. Liang Shi from the China University of Geosciences (Wuhan) for providing the iron-reducing bacteria \\u003cem\\u003eGeobacter sulfurreducens\\u0026nbsp;\\u003c/em\\u003ePCA.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eP.L. and X.H.L. conceived the research. X.H.L.\\u0026nbsp;performed most of the experiments. K.M.B., Y.Q.W., and H.L.W.\\u0026nbsp;supported the methodology for nitrogen fixation characterization. P.L., X.H.L.,\\u0026nbsp;H.L.W., Y.H.W., Z.J., and Y.Y. contributed to sediment sample collection and analysis.\\u0026nbsp;X.H.L. and P.L.\\u0026nbsp;wrote and revised the manuscript. S.H.Y., A. K., and Y.X.W. reviewed and edited the paper.\\u0026nbsp;P.L., S.Y., and Y.Y.\\u0026nbsp;funded the research.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eXiaohan Liu: xiaohanl@cug.edu.cn\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003ePing Li: pli@cug.edu.cn\\u003c/p\\u003e\\n\\u003cp\\u003eKeman Bao: kemanbao@163.com\\u003c/p\\u003e\\n\\u003cp\\u003eYaqi Wang: yqwang059@163.com\\u003c/p\\u003e\\n\\u003cp\\u003eHelin Wang: whl@cug.edu.cn\\u003c/p\\u003e\\n\\u003cp\\u003eYanhong Wang: wangyh@cug.edu.cn\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eZhou Jiang: jiangzhou@cug.edu.cn\\u003c/p\\u003e\\n\\u003cp\\u003eYi Yang:\\u0026nbsp;yiyang@cug.edu.cn\\u003c/p\\u003e\\n\\u003cp\\u003eSonghu Yuan: yuansonghu622@cug.edu.cn\\u003c/p\\u003e\\n\\u003cp\\u003eAndreas Kappler: andreas.kappler@uni-tuebingen.de\\u003c/p\\u003e\\n\\u003cp\\u003eYanxin Wang: yx.wang@cug.edu.cn\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eHuang W et al (2024) Inefficient nitrogen transport to the lower mantle by sediment subduction. 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Nat Methods 9:357\\u0026ndash;359\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePatro R, Duggal G, Love MI, Irizarry RA, Kingsford C (2017) Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14:417\\u0026ndash;419\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLove MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYuan C, Li P, Qing C, Kou Z, Jiang Z (2022) Transcriptomic and genomic profiling revealed the unique cellular response mechanism involved in arsenite stress in \\u003cem\\u003eThermus tengchongensis\\u003c/em\\u003e. Int Biodeterior Biodegrad 175:105504\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":true,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Nitrogen fixation, iron reduction, 15N isotope tracing, transcriptomics, metagenomics\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5306474/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5306474/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eNitrogen (N) and iron (Fe) are essential but often limiting nutrients in ecosystems. Microbial nitrogen fixation (MNF) by diazotrophs and dissimilatory ferric iron (Fe(Ⅲ)) reduction (DIR) are environmentally friendly processes that sustain N and Fe availability. However, the interactions between these processes remain unclear. This study demonstrates a synergistic relationship between MNF and DIR in both laboratory and field settings. N fixation significantly increased heterotrophic Fe(Ⅲ)-reducing rates in diazotrophic DIR bacteria (DIRB) \\u003cem\\u003eKlebsiella\\u003c/em\\u003e sp. N7 and \\u003cem\\u003eGeobacter sulfurreducens\\u003c/em\\u003e PCA by 14.7- and 3.3-fold, respectively, while Fe(Ⅲ) reduction enhanced \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003eN fixation by up to 100%. Similar synergies were found between diazotroph \\u003cem\\u003eAzospirillum humicireducens\\u003c/em\\u003e SgZ-5T and DIRB \\u003cem\\u003eShewanella oneidensis\\u003c/em\\u003e MR-1. Transcriptomic analysis revealed that N fixation upregulated genes associated with anaerobic respiration, accelerating Fe(Ⅲ) reduction through N supply. Simultaneously, Fe(Ⅲ) reduction provided the energy and electrons required for N fixation derived from the oxidation of organic carbon. These findings, validated across environmental samples from aquifers, hot springs, marine sediments, and soils, provide new insights into the coupled N, Fe, and C cycles in natural ecosystems.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Synergistic interaction between microbial nitrogen fixation and iron reduction in the environment\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-11-06 10:03:41\",\"doi\":\"10.21203/rs.3.rs-5306474/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"c9be9c52-6d6c-4521-9780-327c967dd01d\",\"owner\":[],\"postedDate\":\"November 6th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":39673064,\"name\":\"Earth and environmental sciences/Biogeochemistry/Element cycles\"},{\"id\":39673065,\"name\":\"Biological sciences/Ecology/Microbial ecology\"}],\"tags\":[],\"updatedAt\":\"2024-11-06T10:03:41+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-11-06 10:03:41\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5306474\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5306474\",\"identity\":\"rs-5306474\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}