Mixotrophics sulfur disproportionation enhables rapid and low-N2O denitrification in sulfur-packed systems through a novel symbiotic network

Mixotrophics sulfur disproportionation enhables rapid and low-N2O denitrification in sulfur-packed systems through a novel symbiotic network | 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 Mixotrophics sulfur disproportionation enhables rapid and low-N2O denitrification in sulfur-packed systems through a novel symbiotic network chuan chen, Yu Zhang, Wei Wang, Xueting Wang, Lei Zhao, Xijun Xu, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8740407/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 Elemental sulfur (S0) supports low-carbon denitrification in natural sediments and engineered reactors, yet nitrate reduction rates frequently far exceed those expected from S0’s slow abiotic dissolution. This kinetic discrepancy is often attributed to enzymatic sulfur disproportionation (SD). However, SD is considered as a predominantly autotrophic process and potentially suppressed by organic carbon in the prevalent mixotrophic conditions. This attribution appears insufficient to explain the pervasive occurrence of this kinetic discrepancy. Furthermore, the sulfide generated during SD process could inhibit nitrous oxide reductase (NosZ), promoting high emissions of N2O. Nevertheless, this effect is not always accompanied by a measurable N₂O accumulation, implying that additional regulatory mechanisms may be involved. Here, we combine long‑term reactor operation, targeted batch assays, DNA-stable isotope probing (DNA‑SIP) and genome-resolved metagenomics to identify the SD microbial mechanism under mixotrophy. A sulfur‑packed bed reactor operated for >300 days achieved >99% nitrate removal at a hydraulic retention time of 2 h, while accumulating both sulfate and sulfide and producing measurable ammonium, indicating concurrent cryptic sulfur cycling and DNRA signals. DNA‑SIP links carbon assimilation to distinct functional guilds and enriches sulfur oxidation, DNRA and N₂O‑reduction genes in isotopically heavy fractions. Metagenomics of active fractions reveal a novel thriving set of facultative mixotrophic sulfur-disproportionating denitrifiers (FMSDs) whose genomes consolidate SD, complete denitrification, and dissimilatory nitrate reduction to ammonium (DNRA) within a single cellular framework. This unique integration facilitates an intrinsic dual-detoxification mechanism: the internal DNRA module acts as a sink for nitrite, mitigating the accumulation of inhibitory free nitrous acid (FNA), while robust sulfide oxidation modules concurrently detoxify the sulfide produced by SD’s reductive branch. By collectively safeguarding the terminal nitrous oxide reductase enzyme, this self-regulating network ensures profound N2O mitigation. This discovery redefines the microbial ecology of S-N coupling and provides a new blueprint for designing resilient, climate-friendly biotechnologies for water reclamation. Biological sciences/Microbiology/Environmental microbiology/Water microbiology Biological sciences/Biotechnology/Environmental biotechnology Mixotrophic denitrification Sulfur-disproportionating Nitrous oxide mitigation Dissimilatory nitrate reduction to ammonium DNA-stable isotope probing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The accelerating accumulation of reactive nitrogen in global aquatic ecosystems constitutes a profound anthropogenic perturbation of planetary biogeochemical cycles, driving eutrophication and exacerbating greenhouse gas emissions 1-4 . In this context, sulfur-driven denitrification has emerged as a promising low-carbon pathway for nitrogen removal, exploiting the geochemically abundant elemental sulfur (S 0 ) as an electron donor to reduce nitrate to dinitrogen (N 2 ) gas. This process is central to the diverse natural environments and engineered systems, from marine sediments to engineered bioreactors. Yet, its mechanistic foundation is fundamentally constrained by a long-standing biogeochemical paradox: observed rates of nitrate reduction in sulfur-mediated systems consistently and dramatically exceed the theoretical maxima plausible from the slow, abiotic dissolution of S 0 , a notoriously inert substrate 5,6 . For instance, sulfur-packed bed reactors frequently achieve near-complete nitrate removal at rates that are orders of magnitude greater than those supported by chemical dissolution alone, pointing towards a cryptic biological mechanism that profoundly enhances sulfur’s bioavailability and unlocks its bioenergetic potential. The identity and function of this mechanism, however, have remained elusive 7-11 . Enzymatic sulfur disproportionation (SD), the simultaneous microbial oxidation and reduction of intermediate-valence sulfur species, has been widely proposed as the central mechanism to explain this kinetic acceleration. By catalysing the transformation of solid-phase S 0 into a continuous flux of soluble and highly reactive sulfur species, such as sulfide and polysulfides, SD effectively bypasses the kinetic bottleneck of substrate dissolution. This enables a dynamic, high-rate coupling between the sulfur and nitrogen cycles 6,12-14 . Extensive research has characterised this process primarily under autotrophic conditions, identifying specialised microorganisms that utilise the modest energy yield from SD to support carbon fixation. This conventional understanding, however, encounters a significant thermodynamic paradox when applied to most anthropogenically impacted ecosystems. Classical bioenergetic principles predict that SD, as a low-energy-yielding metabolic pathway, should be competitively suppressed by the presence of organic carbon, which provides a more favourable energy source 15,16 . Given the ubiquity of organic matter in environments such as wastewater treatment plants and eutrophic water bodies—which establish prevalent mixotrophic conditions—attributing rapid sulfur cycling solely to obligate autotrophs seems insufficient to explain the pervasive nature of this kinetic anomaly. Furthermore, a second paradox emerges when considering climate implications. The reductive branch of the SD process inherently generates sulfide, a well-documented and potent inhibitor of nitrous oxide reductase (NosZ), the terminal enzyme responsible for converting the powerful greenhouse gas nitrous oxide (N 2 O) to N 2 17,18 . This inhibition theoretically predisposes sulfur-driven systems to high N 2 O emissions, potentially undermining their environmental benefits. However, a perplexing contradiction emerges from field and operational data. Many long-term, high-efficiency sulfur-based denitrification systems, including full-scale bioreactors, exhibit remarkably low N 2 O emission factors, successfully achieving complete denitrification without the anticipated accumulation of N 2 O 2,13,19-21 . This pronounced discrepancy between the predicted inhibitory effects of sulfide and the observed climate-neutral performance strongly implies the existence of robust, yet unidentified, biological self-regulation mechanisms that actively mitigate sulfide toxicity and safeguard the integrity of the denitrification pathway. Resolving these interconnected kinetic, thermodynamic, and functional paradoxes calls for a fundamental reassessment of the integrated metabolic strategies of the key microbial players under realistic, mixotrophic conditions. Who orchestrates high-rate sulfur disproportionation in the presence of organic carbon, and how is the complete reduction of nitrate to N 2 gas achieved without significant N 2 O leakage? Here, we address these questions by combining long-term, high-performance reactor operation with an integrated suite of advanced molecular tools, including DNA-stable isotope probing (DNA-SIP) and genome-resolved metagenomics, to dissect the underlying microbial ecology. Through this integrated analytical approach, we have identified key functional microbial groups and uncovered a novel metabolic paradigm, thereby providing a new blueprint for developing high-rate and climate-friendly nitrogen removal technologies. Results High-rate denitrification is coupled to anomalous sulfur and nitrogen cycling The long-term operation of the sulfur-packed bed reactor (S 0 PBR) under low C/N, mixotrophic conditions established a remarkably efficient and stable nitrogen removal platform. Over 300 days, the system consistently achieved near-complete nitrate removal (>99%), even when challenged with an exceptionally short hydraulic retention time (HRT) of 2 hours, resulting in negligible nitrite or soluble COD accumulation in the effluent (Fig. 1a). This high-rate performance, however, was accompanied by two profound biogeochemical anomalies that challenge classical denitrification models. First, we observed a significant and supra-stoichiometric accumulation of both sulfate (avg. 75.5 mg S L -1 ) and, critically, sulfide (avg. 68.8 mg S L -1 ). These concentrations far exceed the theoretical yields from canonical sulfur-oxidizing denitrification, strongly suggesting a cryptic sulfur cycle was operating. Second, we detected consistent ammonium production (1.18–5.83 mg N L -1 ), providing unequivocal evidence for active dissimilatory nitrate reduction to ammonium (DNRA). The sustained activity of DNRA was particularly perplexing, as this pathway is typically outcompeted by denitrification under the non-limiting nitrate and available organic carbon conditions present in our system 22-26 . To probe the microbial basis for these observations, we characterized the reactor’s microbial community architecture. 16S rRNA gene amplicon sequencing revealed a highly specialized consortium dominated by genera implicated in sulfur and nitrogen cycles (Fig. 1b, c). This community included established denitrifiers such as Thiobacillus (7.46%) and Thauera (8.99%), but was co-dominated by a significant population of known sulfur-disproportionating (SD) taxa, such as Desulfocapsa (1.861%) 27 , Sulfurovum (6.46%) 28 and Sulfurimonas (12.69%) 29 .. Furthermore, the DNRA-associated genus Trichlorobacter was a stable and significant member of the community (3.29%) 30 . A microbial co-occurrence network analysis provided further insight, revealing strong, statistically significant positive correlations between the key denitrifiers ( Thauera , Thiobacillus ), the SD taxa ( Sulfurimonas , Sulfurovum ), and the DNRA bacterium ( Trichlorobacter ) (Fig. 1d). This pattern strongly suggested a deeply integrated functional coupling rather than resource competition. The confluence of these findings—anomalous sulfur geochemistry and the unexpected prominence of DNRA—prompted the central hypothesis that an active sulfur disproportionation cycle was fundamentally reshaping the reactor’s biogeochemistry and ecosystem function. Mixotrophic sulfur disproportionation underpins low N 2 O emissions To mechanistically deconstruct the complex interplay between heterotrophic and autotrophic processes observed in the long-term reactor, we conducted a series of controlled batch experiments simulating distinct metabolic regimes (Fig. 2a, b, Fig.S1, S2). The multi-layered evidence from these assays unequivocally establishes that enzymatic sulfur disproportionation (SD) functions as the central catabolic engine, even in the presence of organic carbon, fundamentally redefining the established models of electron flow in sulfur-based systems 31-33 . Physiological data revealed that the mixotrophic group (+C+N+S) exhibited markedly superior performance. The nitrogen removal rate in this group reached 3.33 mg N L -1 h -1 , 2.4 times higher than the the 1.39 mg N L -1 h -1 observed in the purely heterotrophic group (+C+N) (Fig. 2c). This kinetic enhancement was accompanied by the same stoichiometric anomaly seen in the reactor: the concurrent production of sulfide and sulfate far exceeded the yields predicted by conventional models of direct sulfur-oxidizing denitrification 34 . This strongly implies that SD—a process where elemental sulfur concomitantly serves as both an electron donor and acceptor—was the dominant sulfur transformation pathway. This process generates a continuous flux of highly reactive and bioavailable sulfur intermediates (e.g., polysulfides, thiosulfate), which circumvent the kinetic limitations of solid sulfur dissolution and facilitate rapid electron transfer to the denitrification pathway 33-35 . Critically, this SD-driven metabolism resulted in a profound suppression of nitrous oxide (N 2 O) accumulation, which was reduced by approximately 68% compared to the heterotrophic control (Fig. 2b), underscoring the role of SD in creating biogeochemical conditions that favor complete denitrification to benign N 2 gas. The microbial capacity for SD was unequivocally confirmed in the +C+S group, where substantial sulfide and sulfate generation occurred in the absence of nitrate, proving this pathway is not inhibited by organic carbon availability. Molecular and microbiological analyses provided robust corroboration for these physiological trends. Quantitative PCR (qPCR) showed that the copy numbers of key functional genes— soxB (sulfur oxidation), nrfA (DNRA), and nosZ (N 2 O reduction) were all significantly upregulated in the mixotrophic group (Fig. 2d). The concurrent enrichment of these genes provides direct genetic evidence for a coupled metabolic network where SD establishes a microenvironment that simultaneously promotes diverse nitrogen transformations. This functional synergy was directly reflected in the microbial community structure (Fig. 2e), which was selectively enriched for known SD specialists, including Sulfurimonas and Sulfurovum , whose relative abundances were 1.3 to 7.3 times higher than in the heterotrophic group. This demonstrates how the unique biogeochemical niche created by SD activity directly shapes a specialized microbial community. DNA-SIP identifies functionally distinct mixotrophic and autotrophic guilds To link microbial identity to metabolic function, we performed DNA-stable isotope probing (DNA-SIP) using dual ¹³C-labeled inorganic ( 13 C i ) and organic ( 13 C o ) carbon sources. This provided quantitative evidence of distinct carbon assimilation patterns among the key microbial guilds (Fig. 3). Across all treatments, a clear shift in DNA buoyant density from ~1.70 to 1.75 g mL -1 confirmed the incorporation of 13 C into the DNA of active microorganisms. The most pronounced shift occurred in the mixotrophic (+C+N+S) treatment, where heavy DNA fractions accounted for 32.6 ± 4.8% of total 16S rRNA gene copies—approximately 1.4-fold higher than in the heterotrophic (+C+N) microcosm—indicating strong metabolic activation under sulfur-driven mixotrophic conditions. Taxonomic profiling of these heavy fractions revealed substrate-specific enrichment patterns that delineated the community’s functional roles. Under 13 Cₒ labeling, canonical heterotrophic denitrifiers including Thauera and Azoarcus were actively assimilating organic carbon. In striking contrast, 13 C i labeling highlighted the dominance of autotrophic sulfur oxidizers, with Thiobacillus , Sulfurimonas , and Sulfurovum showing significant enrichments of 14.4% to 66.9% in the heavy fractions. This confirmed their preferential use of inorganic carbon via sulfur-dependent chemolithoautotrophic pathways. Critically, some taxa, notably Pseudomonas , demonstrated clear mixotrophic potential by showing enrichment in both 13 C o - and 13 C i -labeled treatments, hinting at a more complex, flexible metabolic strategy. Functional gene distribution across the density gradients provided the strongest evidence of functional coupling (Fig. 4, Fig. S3). The heavy DNA fractions of the mixotrophic microcosm showed a significant co-enrichment of soxB (thiosulfate oxidation), nrfA (DNRA), and nosZ (N 2 O reduction) genes. Specifically, the abundance of nosZ in the active biomass increased by 2.6-fold compared with the unlabeled control. The statistically significant co-occurrence of these distinct functional genes within the same isotopically heavy fractions was a critical finding, strongly indicating that these key biogeochemical processes were being carried out by either tightly associated or, more provocatively, the very same microorganisms. Genomes reveal a novel guild of facultative mixotrophic sulfur-disproportionating denitrifiers (FMSDs) To resolve the genetic basis for the observed metabolic versatility and functional coupling, we conducted deep metagenomic sequencing on the functionally active ‘heavy’ DNA fractions recovered from our SIP experiments. This genome-resolved approach yielded 55 high- and medium-quality metagenome-assembled genomes (MAGs), revealing a phylogenetically diverse community (Fig. 5). Within this collection, we identified a remarkable cohort of 23 MAGs that possess the complete genetic repertoire for both sulfur disproportionation (SD) and denitrification. Based on this unique combination of genomic potential and their demonstrated mixotrophic activity in SIP assays, we define these organisms as a novel functional guild: Facultative Mixotrophic Sulfur-Disproportionating Denitrifiers (FMSDs) (Fig. 6). The genomic architecture of these FMSDs provides a clear blueprint for their ecological success. Their nitrogen-cycling machinery is comprehensive, featuring genes for the complete denitrification pathway from nitrate to N 2 . Critically, we observed a particularly high abundance and copy number of the nitrous oxide reductase gene ( nosZ ) in many FMSDs, such as Azoarcus (bin.50) and bin.257, providing a direct genetic explanation for the system’s profound N 2 O sink capacity. The defining feature that distinguishes FMSDs from conventional denitrifiers, however, is the seamless integration of this nitrogen pathway with a sophisticated toolkit for sulfur metabolism. These MAGs encoded enzymes for sulfide detoxification ( sqr ) and the oxidation of various sulfur compounds via the sox gene cluster, alongside the widespread detection of SD-associated genes ( psrA, dsrE, tusA ), which confirms that sulfur disproportionation is a core, genetically encoded metabolic strategy. This extraordinary functional overlap—further emphasized by the presence of genes for Dissimilatory Nitrate Reduction to Ammonium (DNRA) ( nrfAH, nirBD ) in several key MAGs—provides the definitive genetic foundation for the deeply synergistic coupling of the sulfur and nitrogen cycles. Strikingly, representative FMSDs, including Sulfurovum (bin.303) and Sulfurimicrobium (bin.48), largely lacked conventional autotrophic carbon fixation pathways while encoding extensive machinery for organic matter degradation. This, combined with their enrichment in 13 C o -labeled treatments, affirms their primary reliance on a flexible mixotrophic or heterotrophic lifestyle, solidifying their identity as a novel and ecologically significant functional guild. FMSD genomes encode a self-regulating, multifunctional metabolic network Our multi-omics investigation culminates in a transformative conclusion: the system’s robust performance originates not from a symbiotic partnership between separate guilds, but from the unprecedented internal metabolic architecture of the FMSDs themselves. The genomic blueprints recovered from the active microbial fraction demonstrate that these single organisms function as autonomous, self-regulating biogeochemical hubs, capable of managing a complex network of catabolic and detoxification pathways internally (Fig. 7). This remarkable capability is rooted in the co-location of multiple, seemingly competing pathways within a single cellular framework. The sulfur disproportionation (SD) pathway acts as an internal engine, breaking down elemental sulfur to generate a continuous flux of both oxidized (sulfate) and reduced (sulfide) sulfur species. The electrons liberated from the oxidative branch of this process fuel their own respiratory denitrification. The key to the system’s profound N 2 O mitigation lies in an intrinsic, dual-detoxification mechanism encoded within the FMSD genome. First, the organism actively manages its own metabolic waste by protecting itself from sulfide toxicity. The sulfide generated from the reductive branch of SD is immediately detoxified by versatile sulfide oxidation modules (s qr, sox ) encoded on the same genome, maintaining it at concentrations below the nosZ inhibition threshold³⁴. Second, for those members harboring the requisite genes, the DNRA module ( nrfAH ) can function as a complementary internal sink for nitrite. By rapidly shunting nitrite to ammonium, it offers an additional mechanism to prevent the accumulation of free nitrous acid (FNA), a potent inhibitor of the copper-dependent NosZ enzyme³³, thus further safeguarding the final, critical step of denitrification. Beyond this core S-N network, the FMSDs exhibit profound metabolic flexibility that ensures their ecological dominance. Their genomes are replete with extensive enzymatic machinery for degrading a wide array of complex organic compounds. This allows them to switch seamlessly between lithotrophy (using sulfur as an energy source) and organotrophy (using organic carbon), adapting instantly to fluctuating nutrient availability. This intricate, self-contained metabolic network—where a single organism generates its own electron donors, internally detoxifies its own inhibitory byproducts, and flexibly utilizes available organic matter—establishes a highly resilient and efficient system that achieves high-rate nitrogen removal with minimal greenhouse gas emissions. Discussion This study resolves a long-standing paradox in sulfur-driven nitrogen transformation: the discrepancy between the sluggish dissolution kinetics of solid elemental sulfur (S 0 ) and the rapid nitrate reduction rates frequently observed in natural and engineered systems 5,6 . Classical models assuming the direct oxidation of S 0 to sulfate fail to explain the observed stoichiometric imbalances and high activity 34 . Through a multi-omics approach, we demonstrate that enzymatic sulfur disproportionation (SD) acts as the central catabolic engine, even under mixotrophic conditions. This process unlocks the cryptic bioenergetic potential of sulfur, fundamentally reshaping the bioenergetic landscape of anoxic ecosystems and redefining the coupling between the sulfur and nitrogen cycles. Contrary to classical thermodynamic predictions that organic carbon suppresses SD 16,36 , our integrated DNA-SIP and metagenomic analyses reveal that SD remains the dominant metabolic strategy among a novel guild of facultative mixotrophic sulfur-disproportionating denitrifiers (FMSDs). This conclusion is supported by multiple, convergent lines of evidence: (1) the significant enrichment of SD-related genes ( psrA, dsrE, tusA ) in FMSD metagenome-assembled genomes (MAGs); (2) the active assimilation of 13 C-labeled inorganic carbon despite the presence of abundant organic substrates; and (3) the supra-stoichiometric accumulation of both oxidized and reduced sulfur products. These findings challenge classical inhibition models and reframe SD as a powerful form of niche construction. By converting a solid-phase, kinetically inert resource (S 0 ) into a continuous flux of soluble, reactive intermediates, these organisms actively engineer their environment to create a sustained source of accessible electron donors. This provides the metabolic fuel for multiple nitrogen-transforming pathways and sustains high nitrate turnover, even under challenging low C/N conditions 37,38 . Our genomic evidence further reveals a paradigm shift away from the traditional view of microbial division of labor. We demonstrate that SD, denitrification, and possible dissimilatory nitrate reduction to ammonium (DNRA) are not necessarily executed by separate guilds but can be consolidated within single microbial genomes. The FMSD MAGs recovered—including Pseudomonas (bin.27) and Sulfurimicrobium (bin.113)—encode integrated functional modules for SD, sulfide oxidation, complete denitrification, and the potential for DNRA. This unique configuration establishes these microorganisms as autonomous redox hubs, capable of internally balancing sulfur transformations while dynamically partitioning electrons among multiple nitrogen sinks. The co-occurrence of nosZ and nrfAH confers an intrinsic regulatory plasticity, allowing them to switch between N 2 O reduction and ammonium formation in response to redox status and carbon availability—a critical adaptive trait for thriving in fluctuating anoxic environments. Moreover, the diverse FMSD lineages identified here, such as Sulfurimicrobium (bin.48) and Sulfurovum (bin.303), exhibit a remarkable genetic potential for nitrous oxide (N 2 O) reduction, often carrying multiple copies of the nosZ gene. Their genomes uniquely couple heterotrophic respiration with a complete sulfur-driven denitrification pathway. Concurrently, the conspicuous absence of traditional carbon fixation pathways in these key MAGs underscores their reliance on organic carbon and confirms their facultative mixotrophic nature. This metabolic plasticity, combining robust denitrification capacity with flexible substrate utilization, reveals a vast and previously underappreciated functional diversity within global sulfur-cycling communities, conferring a significant competitive advantage in ecosystems characterized by fluctuating nutrient inputs, such as tidally influenced sediments and stratified water columns. The mitigation of nitrous oxide (N 2 O), a potent greenhouse gas, remains a critical environmental challenge 39,40 . The copper-dependent nitrous oxide reductase (NosZ) represents a key point of vulnerability in denitrification, as even micromolar sulfide concentrations can chelate its catalytic centers and halt N 2 O reduction 41 . This extreme sensitivity explains the high N 2 O emissions commonly observed in conventional denitrifying systems when exposed to sulfide 13,42,43 . In stark contrast, the FMSDs identified here possess an elegant, intrinsic dual-detoxification mechanism that ensures complete denitrification. Within the framework of a single genome, the integration of sulfur disproportionation (SD), denitrification, and the possible Dissimilatory Nitrate Reduction to Ammonium (DNRA) capacity forms a coordinated intracellular network that minimizes inhibitory intermediates. In members harboring this potential, the DNRA module (nrfAH) can serve as an internal scavenging system, consuming nitrite to prevent the accumulation of free nitrous acid (FNA) 44-46 , a potent NosZ inhibitor. Concurrently, the same organism employs its robust sulfide oxidation modules ( sqr, sox ) to rapidly detoxify the sulfide generated from SD’s reductive branch, maintaining it below inhibitory thresholds 47 . This remarkable capacity for self-protection resolves the fundamental trade-off between high activity and low emissions, explaining how complete denitrification can persist in biogeochemical hotspots that are otherwise considered inhibitory. The principles uncovered here have profound ecological implications. By integrating these multiple metabolic functions, FMSDs act as keystone species that fundamentally regulate biogeochemical stability in dynamic anoxic ecosystems. In environments like coastal sediments, wetlands, and oxygen minimum zones, where periodic sulfidic incursions from sulfate reduction would otherwise decouple the nitrogen cycle, these organisms ensure the continuity and resilience of nitrogen removal. Their ability to switch between organotrophy and lithotrophy provides a form of metabolic insurance, a physiological buffer that confers stability not just to the individual cell but to the entire ecosystem function against perturbations like eutrophication-driven organic matter pulses or redox swings 48 . We propose that this integrated metabolic strategy is not an anomaly but a fundamental rule governing the stability and efficiency of cryptic S-N cycling in many of the planet’s most important biogeochemical hotspots. This discovery provides a definitive mechanistic basis for the cryptic yet highly efficient nitrogen cycling that has long been observed in these critical habitats. From an applied perspective, the metabolic organization of FMSDs represents a naturally evolved framework for self-regulating microbial processes, ideally suited for sustainable biotechnology. By consolidating SD, denitrification, and the potential for DNRA within single cells, these microorganisms achieve efficient electron utilization and intrinsic control over N 2 O emissions, offering a solution to the persistent challenge of greenhouse gas production in wastewater treatment. This discovery redefines the microbial ecology of sulfur-based nitrogen cycling and provides a mechanistic framework for understanding the persistence and efficiency of nitrogen removal in both natural and engineered ecosystems. Future research should move beyond simple quantification and aim to decipher the regulatory logic that governs the dynamic partitioning of electrons between these integrated pathways. Exploring the evolutionary trajectory and biogeographic distribution of this multifunctional metabolic architecture across global biomes will be crucial for refining predictive models of coupled biogeochemical cycles. Ultimately, these insights will guide the development of next-generation biotechnologies that mimic the inherent resilience and efficiency of these natural microbial systems, truly integrating ecological theory into environmental engineering. Methods Reactor Setup and Long-Term Operation A laboratory-scale elemental sulfur-packed bed reactor (S 0 PBR) with a total volume of 5.0 L and a working volume of 3.5 L was constructed from polymethyl methacrylate. Elemental sulfur flakes (S 0 , 2–5 mm particle size) functioned as the sole electron donor and biofilm support material. The inoculum was prepared by blending sediments from a nitrate-contaminated aquifer and activated sludge from a municipal wastewater treatment plant to enhance microbial diversity. To establish a model microbial community suitable for mixotrophic conditions, we operated a continuously fed bioreactor using synthetic wastewater. The inoculum for this reactor was a mixture of activated sludge from the Wenchang-Taiping Municipal Wastewater Treatment Plant and estuarine sediments. This design was intended to encompass a broad diversity of microbial lineages from both engineered and natural ecosystems. The basal medium comprised the following constituents per liter: 40.0 mg N (as KNO₃), and a certain amount of glucose and CH₃COONa (sodium acetate), resulting in a carbon-to-nitrogen (C/N) ratio of 0.8, 4.0 mg KH₂PO₄, along with essential minerals and trace elements. The pH was maintained at 7.2 ± 0.2 through automated addition of a 5% (w/v) NaHCO₃ solution. Operational conditions included a hydraulic retention time (HRT) ranging from 20 hours to 2 hours and a constant temperature of 25 ± 1 °C. Steady-state performance and microbial community stability were achieved following over 300 days of operation (Test S1). Liquid samples were periodically collected from influent and effluent ports for comprehensive quantification of nitrogen species (NO 3 ⁻-N, NO 2 ⁻-N, NH 4 ⁺-N, N 2 O-N) and sulfur species (SO 4 ²⁻, S²⁻, S 2 O 3 ²⁻) to evaluate reactor efficiency and nitrogen mass balance. Batch Activity Assays Isotopic incubation assays and DNA-based stable isotope probing (DNA-SIP) To elucidate microbial metabolic pathways inferred from continuous reactor operation, a series of controlled batch assays were conducted. Isotopic incubation experiments were first carried out to identify microorganisms actively assimilating inorganic versus organic carbon under distinct metabolic scenarios, followed by DNA-stable isotope probing (DNA-SIP) for validation. Isotopic incubations were performed in 150 mL serum bottles with a 100 mL working volume. Each bottle was sealed with a butyl rubber stopper and an aluminum crimp seal, and the headspace was flushed with ultra-pure helium (99.999%) for 30 minutes to establish stringent anaerobic conditions. Nine experimental conditions were set up in triplicate using a dual 13 C-labeling strategy ( 13 C i : inorganic carbon; 13 C o : organic carbon) to simultaneously trace assimilation of both carbon sources: amendment with S⁰ and NO₃⁻ (to probe sulfur-mediated mixotrophic denitrification), including ( 13 C i + 12 C o )+S+N, ( 12 C i + 13 C o )+S+N, and an unlabeled control ( 12 C i + 12 C o )+S+N; amendment with NO₃⁻ only (conventional heterotrophic denitrification), including ( 13 C i + 12 C o )+N, ( 12 C i + 13 C o )+N, and an unlabeled control ( 12 C i + 12 C o )+N; and amendment with S 0 only (sulfur disproportionation), including ( 13 C i + 12 C o )+S, ( 12 C i + 13 C o )+S, and an unlabeled control ( 12 C i + 12 C o )+S (Table S1). Labeled carbon sources were supplied as NaH 13 CO 3 ( 13 C i ) and a ¹³C-acetate-glucose mixture ( 13 C o ). Inoculum was derived from mixed liquor collected from a steady-state sulfur-packed bed reactor (S 0 PBR), which was centrifuged and washed twice with phosphate-buffered saline (PBS, pH 7.2). Incubations took place at 25 °C with shaking at 150 rpm for 18 days (approximately three microbial generations). Time-series liquid samples were collected using gas-tight syringes for monitoring concentrations of nitrate, nitrite, N 2 O, sulfide, thiosulfate, and sulfate. After incubation, total DNA was extracted from each microcosm using the DNeasy PowerSoil Pro Kit (QIAGEN, Germany). For each condition, approximately 3 µg of DNA was mixed with cesium chloride (CsCl) solution to a final volume of 5.1 mL and a target buoyant density of 1.729 g·mL⁻¹. Isopycnic centrifugation was performed using a Beckman Coulter Optima XPN-100 ultracentrifuge equipped with a VT190 vertical rotor at 408,500 × g for 44 hours at 20 °C. After centrifugation, gradients were fractionated into 12 equal volumes (~425 µL each) by sterile water displacement. Buoyant density was determined via refractive index measurement (Reichert, USA). DNA from each fraction was recovered by polyethylene glycol (PEG) 6000 precipitation, washed with 70% ethanol, and resuspended in 30 µL of TE buffer for downstream molecular analyses. Quantification of Functional Genes and Microbial Community Analysis The distributions of bacterial 16S rRNA genes and key functional genes across all 12 density fractions from each SIP treatment were quantified via quantitative PCR (qPCR). Targeted functional genes included: soxB (thiosulfate oxidation), napA (periplasmic nitrate reduction), nosZ (nitrous oxide reduction), nrfA (dissimilatory nitrate reduction to ammonium, DNRA) (Table S2). qPCR assays were performed in triplicate on a QuantStudio 6 Pro Real-Time PCR System (Applied Biosystems, USA) using TB Green Premix Ex Taq II (Takara, Japan). Standard curves were generated from serial dilutions of plasmid DNA harboring cloned target genes. Fractions demonstrating peak gene copy numbers in 13 C-labeled treatments, compared to 12 C controls, were identified as 'heavy' ( 13 C-DNA) and 'light' ( 12 C-DNA). Microbial community composition was further characterized by 16S rRNA gene amplicon sequencing of pre-centrifugation samples and pooled heavy/light DNA fractions from selected treatments. The V3–V4 hypervariable region was amplified with primers 341F (CCTACGGGNGGCWGCAG) and 806R (GGACTACHVGGGTWTCTAAT), and sequenced on an Illumina MiSeq platform (2 × 300 bp). Sequence processing—encompassing quality filtering, denoising, amplicon sequence variant (ASV) calling, and taxonomic assignment—was executed using QIIME2 (v2022.11) with the SILVA (v138) database as reference. Metagenomic Sequencing, Assembly, and Binning To obtain genome-resolved insights into metabolically active microorganisms, DNA from heavy fractions of pertinent SIP treatments (e.g., ¹³Cᵢ- or ¹³Cₒ-labeled samples under sulfur-mediated mixotrophic denitrification, ¹³Cᵢ- or ¹³Cₒ-labeled samples under S⁰-only amendment (sulfur disproportionation), and the unlabeled control (ck)) was subjected to shotgun metagenomic sequencing. Owing to low DNA yields, fractions with buoyant densities > 1.735 g mL⁻¹ from replicate microcosms were pooled to constitute composite samples per treatment. Metagenomic libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit and sequenced on an Illumina NovaSeq 6000 platform (PE150). Raw reads underwent quality control and adapter trimming using Fastp (v0.23.2). High-quality reads were assembled de novo using MEGAHIT (v1.2.9). Contigs exceeding 2,500 bp were binned into metagenome-assembled genomes (MAGs) employing MetaBAT2, MaxBin2, and CONCOCT integrated within the MetaWRAP (v1.3.2) pipeline. Bins were refined via the ‘bin_refinement’ module to yield high-quality MAGs, which were evaluated for completeness (>80%) and contamination (<5%) using CheckM (v1.1.3). High-quality MAGs were taxonomically classified with GTDB-Tk (v2.1.1) and functionally annotated against the KEGG, NCBI-nr, and Pfam databases using Prokka (v1.14.6) and Diamond (v2.0.15) ((Table S3). Analytical Techniques Nitrate (NO₃⁻-N) and nitrite (NO₂⁻-N) concentrations were quantified by ion chromatography (Dionex ICS-3000, USA). Ammonium (NH₄⁺-N) was determined spectrophotometrically using Nessler’s reagent. Sulfate (SO₄²⁻) and thiosulfate (S₂O₃²⁻) were analyzed via ion chromatography. 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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-8740407","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":586550587,"identity":"a1979d5d-300e-4603-8729-72e789ddfcad","order_by":0,"name":"chuan 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Microbial community succession (Day 50: A, Day 100: B, Day 150: C, Day 200: D, Day 250: E, Day 300: F): (b) Microbial community composition at the genus level; (c) Species diversity profiles at each stage; (d) Co-occurrence network analysis of dominant genera: node sizes are normalized to degree values, and the connection strength is represented by the width of the curves.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8740407/v1/89abee4ad6e7021642c95a19.png"},{"id":102205562,"identity":"541c5b63-b968-45ed-b26f-35674f14c304","added_by":"auto","created_at":"2026-02-09 11:42:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1900879,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of isotope microcosm incubation process. (a) Schematic diagram of the S\u003csup\u003e0\u003c/sup\u003ePBR reactor; (b) Nitrogen and sulfur transformation patterns in the batch incubation system designed to investigate dual-isotope assimilation under three incubation treatments: (a) +C+N+S (mixotrophic denitrification) treatment, (b) +C+N (heterotrophic denitrification) treatment, and (c) +C+S (sulfur disproportionation) treatment. Herein,\u0026nbsp;C\u0026nbsp;denotes the simultaneous provision of inorganic carbon (C\u003csub\u003ei\u003c/sub\u003e) and organic carbon (C\u003csub\u003eo\u003c/sub\u003e);\u0026nbsp;C\u003csub\u003ei\u0026nbsp;\u003c/sub\u003e(inorganic carbon) refers to the supplied bicarbonate, while\u0026nbsp;C\u003csub\u003eo\u003c/sub\u003e\u0026nbsp;(organic carbon) refers to the supplied glucose and acetate;\u0026nbsp;N\u0026nbsp;and\u0026nbsp;S\u0026nbsp;represent the addition of nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e) and elemental sulfur (S\u003csup\u003e0\u003c/sup\u003e), respectively; (c) Total nitrogen removal efficiency of mixotrophic denitrification and heterotrophic denitrification; (d) Copy numbers of functional genes involved in nitrogen and sulfur transformation processes; (e) Heatmap showing the relative abundance of the top 20 microbial genera (at the genus level) based on 16S rRNA gene sequencing under different isotope incubation conditions; (f) Workflow of isotope microcosm incubation and DNA-stable isotope probing (DNA-SIP).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8740407/v1/98ba731c5c9c029247837bee.png"},{"id":102205563,"identity":"30c552d2-c0da-447e-83f7-dec5f8e7ea2f","added_by":"auto","created_at":"2026-02-09 11:42:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":505166,"visible":true,"origin":"","legend":"\u003cp\u003eDNA-stable isotope probing (DNA-SIP) profiles showing the buoyant density distribution of community 16S rRNA genes (determined by quantitative polymerase chain reaction, qPCR) across three incubation treatments: (a) +C+N+S treatment, (b) +C+N treatment, and (c) +C+S treatment. Herein,\u0026nbsp;C\u0026nbsp;denotes the simultaneous provision of inorganic carbon (C\u003csub\u003ei\u003c/sub\u003e) and organic carbon (C\u003csub\u003eo\u003c/sub\u003e);\u0026nbsp;C\u003csub\u003ei\u0026nbsp;\u003c/sub\u003e(inorganic carbon) refers to the supplied bicarbonate, while\u0026nbsp;C\u003csub\u003eo\u003c/sub\u003e\u0026nbsp;(organic carbon) refers to the supplied glucose and acetate;\u0026nbsp;N\u0026nbsp;and\u0026nbsp;S\u0026nbsp;represent the addition of nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e) and elemental sulfur (S\u003csup\u003e0\u003c/sup\u003e), respectively. (d) Composition of the top 20 microbial genera (at the genus level) in the heavy (H) and light (L) DNA fractions across the three incubation treatments.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8740407/v1/cd029dbf1a265caad0060e3b.png"},{"id":102205498,"identity":"24ed76f1-e8ee-41a7-8f99-14e4287aa78c","added_by":"auto","created_at":"2026-02-09 11:42:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":778569,"visible":true,"origin":"","legend":"\u003cp\u003eComplementary gradient gene quantification of key biomarkers in nitrogen and sulfur metabolisms. a-d, Dynamic abundance of functional genes along the DNA density gradient in the \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e+12C\u003csub\u003eo\u003c/sub\u003e incubation microcosms. e-h, Dynamic abundance of functional genes along the DNA density gradient in the \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003ei+\u003c/sub\u003e\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e treatment incubation microcosms. i-l, Dynamic abundance of functional genes along the DNA density gradient in the \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e+\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e incubation microcosms. For each buoyant density fraction, the gene abundance is normalized as a percentage of its copy number relative to the total DNA copy number (summed copies across all density fractions). m-p, Comparison of total copy numbers of functional genes among the three microcosms (based on three technical replicates, with a 95% confidence interval). All copy numbers were determined via absolute quantitative PCR amplification. \u003cem\u003esoxB\u003c/em\u003e, S-sulfosulfanyl-L-cysteine sulfohydrolase; \u003cem\u003enapA\u003c/em\u003e, cytochrome nitrate reductase; \u003cem\u003enosZ\u003c/em\u003e, nitrous oxide reductase; and \u003cem\u003enrfA\u003c/em\u003e, nitrite reductase.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8740407/v1/2032a9d6b0b5f0e40089eafa.png"},{"id":102205520,"identity":"0455a2d6-b213-4060-afd7-986e3e465280","added_by":"auto","created_at":"2026-02-09 11:42:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2228231,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree, relative abundance, and gene content of metagenome-assembled genomes (MAGs). (a) Phylogenetic placement of 55 representative MAGs assembled in the present study. (b) Relative abundances of the assembled MAGs in the enrichment cultures from 300-day continuous-flow operation and the heavy DNA fractions of DNA-SIP experiments. (c) Presence/absence of genes involved in carbon, nitrogen, and sulfur metabolism. Solid/hollow circles indicate the presence/absence of the target metabolic pathways.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8740407/v1/bd368ffbba971e0f44b742b0.png"},{"id":102205513,"identity":"fb1dd5fa-94eb-4bbd-affc-ee3c2f5936af","added_by":"auto","created_at":"2026-02-09 11:42:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":317605,"visible":true,"origin":"","legend":"\u003cp\u003eGene annotation of representative facultative mixotrophic sulfur-disproportionating denitrifier strains identified from heavy DNA fractions. The left panel shows the phylogenetic tree of 23 representative metagenome-assembled genomes (MAGs) assembled in the present study. The predicted functional genes on the right panel are classified into functional features, including denitrification, dissimilatory nitrate reduction to ammonium (DNRA), sulfur oxidation, sulfur reduction, and sulfur disproportionation.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8740407/v1/2e89b543468da4fa192efd31.png"},{"id":102205515,"identity":"a2905cd7-a7f4-4640-82a2-3dfe066d9bd6","added_by":"auto","created_at":"2026-02-09 11:42:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2094526,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of metabolic potentials of facultative mixotrophic sulfur-disproportionating denitrifier genomes in heavy DNA fractions. Relevant metabolic pathways are shown based on genomic information from three metagenome-assembled genomes (MAGs): bin.27 (\u003cem\u003ePseudomonas\u003c/em\u003e), bin.113 (\u003cem\u003eSulfurimicrobium\u003c/em\u003e) and bin.187 (\u003cem\u003eUnclassified Bacteria\u003c/em\u003e). Detected pathways and genes related to the following processes are presented: organic matter fermentation, glycolysis, acetate oxidation, tricarboxylic acid (TCA) cycle, denitrification, dissimilatory nitrate reduction to ammonium (DNRA), sulfur oxidation, sulfur reduction, and sulfur disproportionation.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8740407/v1/37c9086b8f94065ff46f8aff.png"},{"id":105904398,"identity":"be4aef31-4ba9-4c07-b919-c57f08a95de7","added_by":"auto","created_at":"2026-04-01 10:08:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8468479,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8740407/v1/87522e4e-eef4-4830-b4e2-7c7416e64d10.pdf"},{"id":102205514,"identity":"1b59f2f5-187f-4c01-94eb-2226da60ba10","added_by":"auto","created_at":"2026-02-09 11:42:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1233931,"visible":true,"origin":"","legend":"Supplementary and Additional Material","description":"","filename":"SupplementaryandAdditionalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8740407/v1/d75b50351c04c9e2d8a5d5d7.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Mixotrophics sulfur disproportionation enhables rapid and low-N2O denitrification in sulfur-packed systems through a novel symbiotic network","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe accelerating accumulation of reactive nitrogen in global aquatic ecosystems constitutes a profound anthropogenic perturbation of planetary biogeochemical cycles, driving eutrophication and exacerbating greenhouse gas emissions\u003csup\u003e1-4\u003c/sup\u003e. In this context, sulfur-driven denitrification has emerged as a promising low-carbon pathway for nitrogen removal, exploiting the geochemically abundant elemental sulfur (S\u003csup\u003e0\u003c/sup\u003e) as an electron donor to reduce nitrate to dinitrogen (N\u003csub\u003e2\u003c/sub\u003e) gas. This process is central to the diverse natural environments and engineered systems, from marine sediments to engineered bioreactors. Yet, its mechanistic foundation is fundamentally constrained by a long-standing biogeochemical paradox: observed rates of nitrate reduction in sulfur-mediated systems consistently and dramatically exceed the theoretical maxima plausible from the slow, abiotic dissolution of S\u003csup\u003e0\u003c/sup\u003e, a notoriously inert substrate\u003csup\u003e5,6\u003c/sup\u003e. For instance, sulfur-packed bed reactors frequently achieve near-complete nitrate removal at rates that are orders of magnitude greater than those supported by chemical dissolution alone, pointing towards a cryptic biological mechanism that profoundly enhances sulfur\u0026rsquo;s bioavailability and unlocks its bioenergetic potential. The identity and function of this mechanism, however, have remained elusive\u003csup\u003e7-11\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eEnzymatic sulfur disproportionation (SD), the simultaneous microbial oxidation and reduction of intermediate-valence sulfur species, has been widely proposed as the central mechanism to explain this kinetic acceleration. By catalysing the transformation of solid-phase S\u003csup\u003e0\u003c/sup\u003e into a continuous flux of soluble and highly reactive sulfur species, such as sulfide and polysulfides, SD effectively bypasses the kinetic bottleneck of substrate dissolution. This enables a dynamic, high-rate coupling between the sulfur and nitrogen cycles\u003csup\u003e6,12-14\u003c/sup\u003e. Extensive research has characterised this process primarily under autotrophic conditions, identifying specialised microorganisms that utilise the modest energy yield from SD to support carbon fixation. This conventional understanding, however, encounters a significant thermodynamic paradox when applied to most anthropogenically impacted ecosystems. Classical bioenergetic principles predict that SD, as a low-energy-yielding metabolic pathway, should be competitively suppressed by the presence of organic carbon, which provides a more favourable energy source\u003csup\u003e15,16\u003c/sup\u003e. Given the ubiquity of organic matter in environments such as wastewater treatment plants and eutrophic water bodies\u0026mdash;which establish prevalent mixotrophic conditions\u0026mdash;attributing rapid sulfur cycling solely to obligate autotrophs seems insufficient to explain the pervasive nature of this kinetic anomaly.\u003c/p\u003e\n\u003cp\u003eFurthermore, a second paradox emerges when considering climate implications. The reductive branch of the SD process inherently generates sulfide, a well-documented and potent inhibitor of nitrous oxide reductase (NosZ), the terminal enzyme responsible for converting the powerful greenhouse gas nitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO) to N\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e17,18\u003c/sup\u003e. This inhibition theoretically predisposes sulfur-driven systems to high N\u003csub\u003e2\u003c/sub\u003eO emissions, potentially undermining their environmental benefits. However, a perplexing contradiction emerges from field and operational data. Many long-term, high-efficiency sulfur-based denitrification systems, including full-scale bioreactors, exhibit remarkably low N\u003csub\u003e2\u003c/sub\u003eO emission factors, successfully achieving complete denitrification without the anticipated accumulation of N\u003csub\u003e2\u003c/sub\u003eO \u003csup\u003e2,13,19-21\u003c/sup\u003e. This pronounced discrepancy between the predicted inhibitory effects of sulfide and the observed climate-neutral performance strongly implies the existence of robust, yet unidentified, biological self-regulation mechanisms that actively mitigate sulfide toxicity and safeguard the integrity of the denitrification pathway.\u003c/p\u003e\n\u003cp\u003eResolving these interconnected kinetic, thermodynamic, and functional paradoxes calls for a fundamental reassessment of the integrated metabolic strategies of the key microbial players under realistic, mixotrophic conditions. Who orchestrates high-rate sulfur disproportionation in the presence of organic carbon, and how is the complete reduction of nitrate to N\u003csub\u003e2\u003c/sub\u003e gas achieved without significant N\u003csub\u003e2\u003c/sub\u003eO leakage?\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere, we address these questions by combining long-term, high-performance reactor operation with an integrated suite of advanced molecular tools, including DNA-stable isotope probing (DNA-SIP) and genome-resolved metagenomics, to dissect the underlying microbial ecology. Through this integrated analytical approach, we have identified key functional microbial groups and uncovered a novel metabolic paradigm, thereby providing a new blueprint for developing high-rate and climate-friendly nitrogen removal technologies.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHigh-rate denitrification is coupled to anomalous sulfur and nitrogen cycling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe long-term operation of the sulfur-packed bed reactor (S\u003csup\u003e0\u003c/sup\u003ePBR) under low C/N, mixotrophic conditions established a remarkably efficient and stable nitrogen removal platform. Over 300 days, the system consistently achieved near-complete nitrate removal (\u0026gt;99%), even when challenged with an exceptionally short hydraulic retention time (HRT) of 2 hours, resulting in negligible nitrite or soluble COD accumulation in the effluent (Fig. 1a). This high-rate performance, however, was accompanied by two profound biogeochemical anomalies that challenge classical denitrification models. First, we observed a significant and supra-stoichiometric accumulation of both sulfate (avg. 75.5 mg S L\u003csup\u003e-1\u003c/sup\u003e) and, critically, sulfide (avg. 68.8 mg S L\u003csup\u003e-1\u003c/sup\u003e). These concentrations far exceed the theoretical yields from canonical sulfur-oxidizing denitrification, strongly suggesting a cryptic sulfur cycle was operating. Second, we detected consistent ammonium production (1.18\u0026ndash;5.83 mg N L\u003csup\u003e-1\u003c/sup\u003e), providing unequivocal evidence for active dissimilatory nitrate reduction to ammonium (DNRA). The sustained activity of DNRA was particularly perplexing, as this pathway is typically outcompeted by denitrification under the non-limiting nitrate and available organic carbon conditions present in our system\u003csup\u003e22-26\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo probe the microbial basis for these observations, we characterized the reactor\u0026rsquo;s microbial community architecture. 16S rRNA gene amplicon sequencing revealed a highly specialized consortium dominated by genera implicated in sulfur and nitrogen cycles (Fig. 1b, c). This community included established denitrifiers such as \u003cem\u003eThiobacillus\u003c/em\u003e (7.46%) and \u003cem\u003eThauera\u003c/em\u003e (8.99%), but was co-dominated by a significant population of known sulfur-disproportionating (SD) taxa, such as \u003cem\u003eDesulfocapsa \u0026nbsp;\u003c/em\u003e(1.861%)\u003csup\u003e27\u003c/sup\u003e, \u003cem\u003eSulfurovum\u003c/em\u003e (6.46%)\u003csup\u003e28\u003c/sup\u003e and \u003cem\u003eSulfurimonas\u003c/em\u003e (12.69%)\u003csup\u003e29\u003c/sup\u003e.. Furthermore, the DNRA-associated genus \u003cem\u003eTrichlorobacter\u003c/em\u003e was a stable and significant member of the community (3.29%)\u003csup\u003e30\u003c/sup\u003e. A microbial co-occurrence network analysis provided further insight, revealing strong, statistically significant positive correlations between the key denitrifiers (\u003cem\u003eThauera\u003c/em\u003e, \u003cem\u003eThiobacillus\u003c/em\u003e), the SD taxa (\u003cem\u003eSulfurimonas\u003c/em\u003e, \u003cem\u003eSulfurovum\u003c/em\u003e), and the DNRA bacterium (\u003cem\u003eTrichlorobacter\u003c/em\u003e) (Fig. 1d). This pattern strongly suggested a deeply integrated functional coupling rather than resource competition. The confluence of these findings\u0026mdash;anomalous sulfur geochemistry and the unexpected prominence of DNRA\u0026mdash;prompted the central hypothesis that an active sulfur disproportionation cycle was fundamentally reshaping the reactor\u0026rsquo;s biogeochemistry and ecosystem function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMixotrophic sulfur disproportionation underpins low N\u003csub\u003e2\u003c/sub\u003eO emissions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo mechanistically deconstruct the complex interplay between heterotrophic and autotrophic processes observed in the long-term reactor, we conducted a series of controlled batch experiments simulating distinct metabolic regimes (Fig. 2a, b, Fig.S1, S2). The multi-layered evidence from these assays unequivocally establishes that enzymatic sulfur disproportionation (SD) functions as the central catabolic engine, even in the presence of organic carbon, fundamentally redefining the established models of electron flow in sulfur-based systems\u003csup\u003e31-33\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePhysiological data revealed that the mixotrophic group (+C+N+S) exhibited markedly superior performance. The nitrogen removal rate in this group reached 3.33 mg N L\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e, 2.4 times higher than the \u0026nbsp;the 1.39 mg N L\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e observed in the purely heterotrophic group (+C+N) (Fig. 2c). This kinetic enhancement was accompanied by the same stoichiometric anomaly seen in the reactor: the concurrent production of sulfide and sulfate far exceeded the yields predicted by conventional models of direct sulfur-oxidizing denitrification\u003csup\u003e34\u003c/sup\u003e. This strongly implies that SD\u0026mdash;a process where elemental sulfur concomitantly serves as both an electron donor and acceptor\u0026mdash;was the dominant sulfur transformation pathway. This process generates a continuous flux of highly reactive and bioavailable sulfur intermediates (e.g., polysulfides, thiosulfate), which circumvent the kinetic limitations of solid sulfur dissolution and facilitate rapid electron transfer to the denitrification pathway\u003csup\u003e33-35\u003c/sup\u003e. Critically, this SD-driven metabolism resulted in a profound suppression of nitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO) accumulation, which was reduced by approximately 68% compared to the heterotrophic control (Fig. 2b), underscoring the role of SD in creating biogeochemical conditions that favor complete denitrification to benign N\u003csub\u003e2\u003c/sub\u003e gas. The microbial capacity for SD was unequivocally confirmed in the +C+S group, where substantial sulfide and sulfate generation occurred in the absence of nitrate, proving this pathway is not inhibited by organic carbon availability.\u003c/p\u003e\n\u003cp\u003eMolecular and microbiological analyses provided robust corroboration for these physiological trends. Quantitative PCR (qPCR) showed that the copy numbers of key functional genes\u0026mdash;\u003cem\u003esoxB\u003c/em\u003e (sulfur oxidation), \u003cem\u003enrfA\u003c/em\u003e (DNRA), and \u003cem\u003enosZ\u003c/em\u003e (N\u003csub\u003e2\u003c/sub\u003eO reduction) were all significantly upregulated in the mixotrophic group (Fig. 2d). The concurrent enrichment of these genes provides direct genetic evidence for a coupled metabolic network where SD establishes a microenvironment that simultaneously promotes diverse nitrogen transformations. This functional synergy was directly reflected in the microbial community structure (Fig. 2e), which was selectively enriched for known SD specialists, including\u003cem\u003e\u0026nbsp;Sulfurimonas\u003c/em\u003e and \u003cem\u003eSulfurovum\u003c/em\u003e, whose relative abundances were 1.3 to 7.3 times higher than in the heterotrophic group. This demonstrates how the unique biogeochemical niche created by SD activity directly shapes a specialized microbial community.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA-SIP identifies functionally distinct mixotrophic and autotrophic guilds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo link microbial identity to metabolic function, we performed DNA-stable isotope probing (DNA-SIP) using dual \u0026sup1;\u0026sup3;C-labeled inorganic (\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e) and organic (\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e) carbon sources. This provided quantitative evidence of distinct carbon assimilation patterns among the key microbial guilds (Fig. 3). Across all treatments, a clear shift in DNA buoyant density from ~1.70 to 1.75 g mL\u003csup\u003e-1\u003c/sup\u003e confirmed the incorporation of \u003csup\u003e13\u003c/sup\u003eC into the DNA of active microorganisms. The most pronounced shift occurred in the mixotrophic (+C+N+S) treatment, where heavy DNA fractions accounted for 32.6\u0026nbsp;\u0026plusmn;\u0026nbsp;4.8% of total 16S rRNA gene copies\u0026mdash;approximately 1.4-fold higher than in the heterotrophic (+C+N) microcosm\u0026mdash;indicating strong metabolic activation under sulfur-driven mixotrophic conditions.\u003c/p\u003e\n\u003cp\u003eTaxonomic profiling of these heavy fractions revealed substrate-specific enrichment patterns that delineated the community\u0026rsquo;s functional roles. Under \u003csup\u003e13\u003c/sup\u003eCₒ\u0026nbsp;labeling, canonical heterotrophic denitrifiers including \u003cem\u003eThauera\u003c/em\u003e and \u003cem\u003eAzoarcus\u003c/em\u003e were actively assimilating organic carbon. In striking contrast, \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e labeling highlighted the dominance of autotrophic sulfur oxidizers, with \u003cem\u003eThiobacillus\u003c/em\u003e, \u003cem\u003eSulfurimonas\u003c/em\u003e, and \u003cem\u003eSulfurovum\u003c/em\u003e showing significant enrichments of 14.4% to 66.9% in the heavy fractions. This confirmed their preferential use of inorganic carbon via sulfur-dependent chemolithoautotrophic pathways. Critically, some taxa, notably \u003cem\u003ePseudomonas\u003c/em\u003e, demonstrated clear mixotrophic potential by showing enrichment in both \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e- and \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e-labeled treatments, hinting at a more complex, flexible metabolic strategy.\u003c/p\u003e\n\u003cp\u003eFunctional gene distribution across the density gradients provided the strongest evidence of functional coupling (Fig. 4, Fig. S3). The heavy DNA fractions of the mixotrophic microcosm showed a significant co-enrichment of \u003cem\u003esoxB\u003c/em\u003e (thiosulfate oxidation), \u003cem\u003enrfA\u003c/em\u003e (DNRA), and \u003cem\u003enosZ\u003c/em\u003e (N\u003csub\u003e2\u003c/sub\u003eO reduction) genes. Specifically, the abundance of \u003cem\u003enosZ\u003c/em\u003e in the active biomass increased by 2.6-fold compared with the unlabeled control. The statistically significant co-occurrence of these distinct functional genes within the same isotopically heavy fractions was a critical finding, strongly indicating that these key biogeochemical processes were being carried out by either tightly associated or, more provocatively, the very same microorganisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenomes reveal a novel guild of facultative mixotrophic sulfur-disproportionating denitrifiers (FMSDs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo resolve the genetic basis for the observed metabolic versatility and functional coupling, we conducted deep metagenomic sequencing on the functionally active \u0026lsquo;heavy\u0026rsquo; DNA fractions recovered from our SIP experiments. This genome-resolved approach yielded 55 high- and medium-quality metagenome-assembled genomes (MAGs), revealing a phylogenetically diverse community (Fig. 5). Within this collection, we identified a remarkable cohort of 23 MAGs that possess the complete genetic repertoire for both sulfur disproportionation (SD) and denitrification. Based on this unique combination of genomic potential and their demonstrated mixotrophic activity in SIP assays, we define these organisms as a novel functional guild:\u0026nbsp;Facultative Mixotrophic Sulfur-Disproportionating Denitrifiers (FMSDs)\u0026nbsp;(Fig. 6).\u003c/p\u003e\n\u003cp\u003eThe genomic architecture of these FMSDs provides a clear blueprint for their ecological success. Their nitrogen-cycling machinery is comprehensive, featuring genes for the complete denitrification pathway from nitrate to N\u003csub\u003e2\u003c/sub\u003e. Critically, we observed a particularly high abundance and copy number of the nitrous oxide reductase gene (\u003cem\u003enosZ\u003c/em\u003e) in many FMSDs, such as\u0026nbsp;Azoarcus\u0026nbsp;(bin.50) and bin.257, providing a direct genetic explanation for the system\u0026rsquo;s profound N\u003csub\u003e2\u003c/sub\u003eO sink capacity. The defining feature that distinguishes FMSDs from conventional denitrifiers, however, is the seamless integration of this nitrogen pathway with a sophisticated toolkit for sulfur metabolism. These MAGs encoded enzymes for sulfide detoxification (\u003cem\u003esqr\u003c/em\u003e) and the oxidation of various sulfur compounds via the \u003cem\u003esox\u003c/em\u003e gene cluster, alongside the widespread detection of SD-associated genes (\u003cem\u003epsrA,\u0026nbsp;dsrE,\u0026nbsp;tusA\u003c/em\u003e), which confirms that sulfur disproportionation is a core, genetically encoded metabolic strategy. This extraordinary functional overlap\u0026mdash;further emphasized by the presence of genes for Dissimilatory Nitrate Reduction to Ammonium (DNRA) (\u003cem\u003enrfAH,\u0026nbsp;nirBD\u003c/em\u003e) in several key MAGs\u0026mdash;provides the definitive genetic foundation for the deeply synergistic coupling of the sulfur and nitrogen cycles. Strikingly, representative FMSDs, including \u003cem\u003eSulfurovum\u003c/em\u003e (bin.303) and \u003cem\u003eSulfurimicrobium\u003c/em\u003e (bin.48), largely lacked conventional autotrophic carbon fixation pathways while encoding extensive machinery for organic matter degradation. This, combined with their enrichment in \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e-labeled treatments, affirms their primary reliance on a flexible mixotrophic or heterotrophic lifestyle, solidifying their identity as a novel and ecologically significant functional guild.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFMSD genomes encode a self-regulating, multifunctional metabolic network\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur multi-omics investigation culminates in a transformative conclusion: the system\u0026rsquo;s robust performance originates not from a symbiotic partnership between separate guilds, but from the\u0026nbsp;unprecedented internal metabolic architecture of the FMSDs themselves. The genomic blueprints recovered from the active microbial fraction demonstrate that these single organisms function as autonomous, self-regulating biogeochemical hubs, capable of managing a complex network of catabolic and detoxification pathways internally (Fig. 7).\u003c/p\u003e\n\u003cp\u003eThis remarkable capability is rooted in the co-location of multiple, seemingly competing pathways within a single cellular framework. The sulfur disproportionation (SD) pathway acts as an internal engine, breaking down elemental sulfur to generate a continuous flux of both oxidized (sulfate) and reduced (sulfide) sulfur species. The electrons liberated from the oxidative branch of this process fuel their own respiratory denitrification. The key to the system\u0026rsquo;s profound N\u003csub\u003e2\u003c/sub\u003eO mitigation lies in an\u0026nbsp;intrinsic, dual-detoxification mechanism encoded within the FMSD genome. First, the organism actively manages its own metabolic waste by protecting itself from sulfide toxicity. The sulfide generated from the reductive branch of SD is immediately detoxified by versatile sulfide oxidation modules (s\u003cem\u003eqr,\u0026nbsp;sox\u003c/em\u003e) encoded on the same genome, maintaining it at concentrations below the \u003cem\u003enosZ\u003c/em\u003e inhibition threshold\u0026sup3;⁴. Second, for those members harboring the requisite genes, the DNRA module (\u003cem\u003enrfAH\u003c/em\u003e) can function as a complementary internal sink for nitrite. By rapidly shunting nitrite to ammonium, it offers an additional mechanism to prevent the accumulation of free nitrous acid (FNA), a potent inhibitor of the copper-dependent NosZ enzyme\u0026sup3;\u0026sup3;, thus further safeguarding the final, critical step of denitrification.\u003c/p\u003e\n\u003cp\u003eBeyond this core S-N network, the FMSDs exhibit profound metabolic flexibility that ensures their ecological dominance. Their genomes are replete with extensive enzymatic machinery for degrading a wide array of complex organic compounds. This allows them to switch seamlessly between lithotrophy (using sulfur as an energy source) and organotrophy (using organic carbon), adapting instantly to fluctuating nutrient availability. This intricate, self-contained metabolic network\u0026mdash;where a single organism generates its own electron donors, internally detoxifies its own inhibitory byproducts, and flexibly utilizes available organic matter\u0026mdash;establishes a highly resilient and efficient system that achieves high-rate nitrogen removal with minimal greenhouse gas emissions.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study resolves a long-standing paradox in sulfur-driven nitrogen transformation: the discrepancy between the sluggish dissolution kinetics of solid elemental sulfur (S\u003csup\u003e0\u003c/sup\u003e) and the rapid nitrate reduction rates frequently observed in natural and engineered systems\u003csup\u003e5,6\u003c/sup\u003e. Classical models assuming the direct oxidation of S\u003csup\u003e0\u003c/sup\u003e to sulfate fail to explain the observed stoichiometric imbalances and high activity\u003csup\u003e34\u003c/sup\u003e. Through a multi-omics approach, we demonstrate that enzymatic sulfur disproportionation (SD) acts as the central catabolic engine, even under mixotrophic conditions. This process unlocks the cryptic bioenergetic potential of sulfur, fundamentally reshaping the bioenergetic landscape of anoxic ecosystems and redefining the coupling between the sulfur and nitrogen cycles.\u003c/p\u003e\n\u003cp\u003eContrary to classical thermodynamic predictions that organic carbon suppresses SD\u003csup\u003e16,36\u003c/sup\u003e, our integrated DNA-SIP and metagenomic analyses reveal that SD remains the dominant metabolic strategy among a novel guild of facultative mixotrophic sulfur-disproportionating denitrifiers (FMSDs). This conclusion is supported by multiple, convergent lines of evidence: (1) the significant enrichment of SD-related genes (\u003cem\u003epsrA, dsrE, tusA\u003c/em\u003e) in FMSD metagenome-assembled genomes (MAGs); (2) the active assimilation of \u003csup\u003e13\u003c/sup\u003eC-labeled inorganic carbon despite the presence of abundant organic substrates; and (3) the supra-stoichiometric accumulation of both oxidized and reduced sulfur products. These findings challenge classical inhibition models and reframe SD as a powerful form of niche construction. By converting a solid-phase, kinetically inert resource (S\u003csup\u003e0\u003c/sup\u003e) into a continuous flux of soluble, reactive intermediates, these organisms actively engineer their environment to create a sustained source of accessible electron donors. This provides the metabolic fuel for multiple nitrogen-transforming pathways and sustains high nitrate turnover, even under challenging low C/N conditions\u003csup\u003e37,38\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur genomic evidence further reveals a paradigm shift away from the traditional view of microbial division of labor. We demonstrate that SD, denitrification, and\u0026nbsp;possible dissimilatory nitrate reduction to ammonium (DNRA) are not necessarily executed by separate guilds but can be consolidated within single microbial genomes. The FMSD MAGs recovered\u0026mdash;including \u003cem\u003ePseudomonas\u003c/em\u003e (bin.27) and \u003cem\u003eSulfurimicrobium\u003c/em\u003e (bin.113)\u0026mdash;encode integrated functional modules for SD, sulfide oxidation, complete denitrification, and the potential for DNRA. This unique configuration establishes these microorganisms as autonomous redox hubs, capable of internally balancing sulfur transformations while dynamically partitioning electrons among multiple nitrogen sinks. The co-occurrence of \u003cem\u003enosZ\u003c/em\u003e and \u003cem\u003enrfAH\u003c/em\u003e confers an intrinsic regulatory plasticity, allowing them to switch between N\u003csub\u003e2\u003c/sub\u003eO reduction and ammonium formation in response to redox status and carbon availability\u0026mdash;a critical adaptive trait for thriving in fluctuating anoxic environments. Moreover, the diverse FMSD lineages identified here, such as \u003cem\u003eSulfurimicrobium\u0026nbsp;\u003c/em\u003e(bin.48) and Sulfurovum (bin.303), exhibit a remarkable genetic potential for nitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO) reduction, often carrying multiple copies of the \u003cem\u003enosZ\u003c/em\u003e gene. Their genomes uniquely couple heterotrophic respiration with a complete sulfur-driven denitrification pathway. Concurrently, the conspicuous absence of traditional carbon fixation pathways in these key MAGs underscores their reliance on organic carbon and confirms their facultative mixotrophic nature. This metabolic plasticity, combining robust denitrification capacity with flexible substrate utilization, reveals a vast and previously underappreciated functional diversity within global sulfur-cycling communities, conferring a significant competitive advantage in ecosystems characterized by fluctuating nutrient inputs, such as tidally influenced sediments and stratified water columns.\u003c/p\u003e\n\u003cp\u003eThe mitigation of nitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO), a potent greenhouse gas, remains a critical environmental challenge\u003csup\u003e39,40\u003c/sup\u003e. The copper-dependent nitrous oxide reductase (NosZ) represents a\u0026nbsp;key point of vulnerability\u0026nbsp;in denitrification, as even micromolar sulfide concentrations can chelate its catalytic centers and halt N\u003csub\u003e2\u003c/sub\u003eO reduction\u003csup\u003e41\u003c/sup\u003e. This extreme sensitivity explains the high N\u003csub\u003e2\u003c/sub\u003eO emissions commonly observed in conventional denitrifying systems when exposed to sulfide\u003csup\u003e13,42,43\u003c/sup\u003e. In stark contrast, the FMSDs identified here possess an elegant, intrinsic dual-detoxification mechanism that ensures complete denitrification. \u0026nbsp;Within the framework of a single genome, the integration of sulfur disproportionation (SD), denitrification, and the possible Dissimilatory Nitrate Reduction to Ammonium (DNRA) capacity forms a coordinated intracellular network that minimizes inhibitory intermediates. In members harboring this potential, the DNRA module (nrfAH) can serve as an internal scavenging system, consuming nitrite to prevent the accumulation of free nitrous acid (FNA)\u003csup\u003e44-46\u003c/sup\u003e, a potent \u003cem\u003eNosZ\u003c/em\u003e inhibitor. Concurrently, the same organism employs its robust sulfide oxidation modules (\u003cem\u003esqr,\u0026nbsp;sox\u003c/em\u003e) to rapidly detoxify the sulfide generated from SD\u0026rsquo;s reductive branch, maintaining it below inhibitory thresholds\u003csup\u003e47\u003c/sup\u003e. This remarkable capacity for self-protection resolves the fundamental trade-off between high activity and low emissions, explaining how complete denitrification can persist in biogeochemical hotspots that are otherwise considered inhibitory.\u003c/p\u003e\n\u003cp\u003eThe principles uncovered here have profound ecological implications. By integrating these multiple metabolic functions, FMSDs act as\u0026nbsp;keystone species that fundamentally regulate\u0026nbsp;biogeochemical stability in dynamic anoxic ecosystems. In environments like coastal sediments, wetlands, and oxygen minimum zones, where periodic sulfidic incursions from sulfate reduction would otherwise decouple the nitrogen cycle, these organisms ensure the continuity and resilience of nitrogen removal. Their ability to switch between organotrophy and lithotrophy provides a form of\u0026nbsp;metabolic insurance, a physiological buffer that confers stability not just to the individual cell but to the entire ecosystem function against perturbations like eutrophication-driven organic matter pulses or redox swings\u003csup\u003e48\u003c/sup\u003e. We propose that this integrated metabolic strategy is not an anomaly but a fundamental rule governing the stability and efficiency of cryptic S-N cycling in many of the planet\u0026rsquo;s most important biogeochemical hotspots. This discovery provides a definitive mechanistic basis for the cryptic yet highly efficient nitrogen cycling that has long been observed in these critical habitats.\u003c/p\u003e\n\u003cp\u003eFrom an applied perspective, the metabolic organization of FMSDs represents a\u0026nbsp;naturally evolved framework for self-regulating microbial processes, ideally suited for sustainable biotechnology. By consolidating SD, denitrification, and\u0026nbsp;the potential for\u0026nbsp;DNRA within single cells, these microorganisms achieve efficient electron utilization and intrinsic control over N\u003csub\u003e2\u003c/sub\u003eO emissions, offering a solution to the persistent challenge of greenhouse gas production in wastewater treatment. This discovery redefines the microbial ecology of sulfur-based nitrogen cycling and provides a mechanistic framework for understanding the persistence and efficiency of nitrogen removal in both natural and engineered ecosystems. Future research should move beyond simple quantification and aim to decipher the regulatory logic that governs the dynamic partitioning of electrons between these integrated pathways. Exploring the evolutionary trajectory and biogeographic distribution of this multifunctional metabolic architecture across global biomes will be crucial for refining predictive models of coupled biogeochemical cycles. Ultimately, these insights will guide the development of next-generation biotechnologies that mimic the inherent resilience and efficiency of these natural microbial systems, truly integrating ecological theory into environmental engineering.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eReactor Setup and Long-Term Operation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA laboratory-scale elemental sulfur-packed bed reactor (S\u003csup\u003e0\u003c/sup\u003ePBR) with a total volume of 5.0 L and a working volume of 3.5 L was constructed from polymethyl methacrylate. Elemental sulfur flakes (S\u003csup\u003e0\u003c/sup\u003e, 2\u0026ndash;5 mm particle size) functioned as the sole electron donor and biofilm support material. The inoculum was prepared by blending sediments from a nitrate-contaminated aquifer and activated sludge from a municipal wastewater treatment plant to enhance microbial diversity.\u003c/p\u003e\n\u003cp\u003eTo establish a model microbial community suitable for mixotrophic conditions, we operated a continuously fed bioreactor using synthetic wastewater. The inoculum for this reactor was a mixture of activated sludge from the Wenchang-Taiping Municipal Wastewater Treatment Plant and estuarine sediments. This design was intended to encompass a broad diversity of microbial lineages from both engineered and natural ecosystems. The basal medium comprised the following constituents per liter: 40.0 mg N (as KNO₃), and a certain amount of glucose and CH₃COONa (sodium acetate), resulting in a carbon-to-nitrogen (C/N) ratio of 0.8, 4.0 mg KH₂PO₄, along with essential minerals and trace elements. The pH was maintained at 7.2 \u0026plusmn; 0.2 through automated addition of a 5% (w/v) NaHCO₃ solution. Operational conditions included a hydraulic retention time (HRT) ranging from 20 hours to 2 hours and a constant temperature of 25 \u0026plusmn; 1 \u0026deg;C. Steady-state performance and microbial community stability were achieved following over 300 days of operation (Test S1).\u003c/p\u003e\n\u003cp\u003eLiquid samples were periodically collected from influent and effluent ports for comprehensive quantification of nitrogen species (NO\u003csub\u003e3\u003c/sub\u003e⁻-N, NO\u003csub\u003e2\u003c/sub\u003e⁻-N, NH\u003csub\u003e4\u003c/sub\u003e⁺-N, N\u003csub\u003e2\u003c/sub\u003eO-N) and sulfur species (SO\u003csub\u003e4\u003c/sub\u003e\u0026sup2;⁻, S\u0026sup2;⁻, S\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u0026sup2;⁻) to evaluate reactor efficiency and nitrogen mass balance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBatch Activity Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsotopic incubation assays and DNA-based stable isotope probing (DNA-SIP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate microbial metabolic pathways inferred from continuous reactor operation, a series of controlled batch assays were conducted. Isotopic incubation experiments were first carried out to identify microorganisms actively assimilating inorganic versus organic carbon under distinct metabolic scenarios, followed by DNA-stable isotope probing (DNA-SIP) for validation.\u003c/p\u003e\n\u003cp\u003eIsotopic incubations were performed in 150 mL serum bottles with a 100 mL working volume. Each bottle was sealed with a butyl rubber stopper and an aluminum crimp seal, and the headspace was flushed with ultra-pure helium (99.999%) for 30 minutes to establish stringent anaerobic conditions. Nine experimental conditions were set up in triplicate using a dual \u003csup\u003e13\u003c/sup\u003eC-labeling strategy (\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e: inorganic carbon; \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e: organic carbon) to simultaneously trace assimilation of both carbon sources: amendment with S⁰ and NO₃⁻ (to probe sulfur-mediated mixotrophic denitrification), including (\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e+\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e)+S+N, (\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e+\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e)+S+N, and an unlabeled control (\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e+\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e)+S+N; amendment with NO₃⁻ only (conventional heterotrophic denitrification), including (\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e+\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e)+N, (\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e+\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e)+N, and an unlabeled control (\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e+\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e)+N; and amendment with S\u003csup\u003e0\u003c/sup\u003e only (sulfur disproportionation), including (\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e+\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e)+S, \u0026nbsp;(\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e+\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e)+S, and an unlabeled control (\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e+\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e)+S (Table S1). Labeled carbon sources were supplied as NaH\u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e3\u003c/sub\u003e (\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003ei\u003c/sub\u003e) and a \u0026sup1;\u0026sup3;C-acetate-glucose mixture (\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003eo\u003c/sub\u003e). Inoculum was derived from mixed liquor collected from a steady-state sulfur-packed bed reactor (S\u003csup\u003e0\u003c/sup\u003ePBR), which was centrifuged and washed twice with phosphate-buffered saline (PBS, pH 7.2). Incubations took place at 25 \u0026deg;C with shaking at 150 rpm for 18 days (approximately three microbial generations). Time-series liquid samples were collected using gas-tight syringes for monitoring concentrations of nitrate, nitrite, N\u003csub\u003e2\u003c/sub\u003eO, sulfide, thiosulfate, and sulfate.\u003c/p\u003e\n\u003cp\u003eAfter incubation, total DNA was extracted from each microcosm using the DNeasy PowerSoil Pro Kit (QIAGEN, Germany). For each condition, approximately 3 \u0026micro;g of DNA was mixed with cesium chloride (CsCl) solution to a final volume of 5.1 mL and a target buoyant density of 1.729 g\u0026middot;mL⁻\u0026sup1;. Isopycnic centrifugation was performed using a Beckman Coulter Optima XPN-100 ultracentrifuge equipped with a VT190 vertical rotor at 408,500 \u0026times; g for 44 hours at 20 \u0026deg;C. After centrifugation, gradients were fractionated into 12 equal volumes (~425 \u0026micro;L each) by sterile water displacement. Buoyant density was determined via refractive index measurement (Reichert, USA). DNA from each fraction was recovered by polyethylene glycol (PEG) 6000 precipitation, washed with 70% ethanol, and resuspended in 30 \u0026micro;L of TE buffer for downstream molecular analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of Functional Genes and Microbial Community Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe distributions of bacterial 16S rRNA genes and key functional genes across all 12 density fractions from each SIP treatment were quantified via quantitative PCR (qPCR). Targeted functional genes included: \u003cem\u003esoxB\u003c/em\u003e (thiosulfate oxidation), \u003cem\u003enapA\u003c/em\u003e (periplasmic nitrate reduction), \u003cem\u003enosZ\u003c/em\u003e (nitrous oxide reduction), \u003cem\u003enrfA\u0026nbsp;\u003c/em\u003e(dissimilatory nitrate reduction to ammonium, DNRA) (Table S2). qPCR assays were performed in triplicate on a QuantStudio 6 Pro Real-Time PCR System (Applied Biosystems, USA) using TB Green Premix Ex Taq II (Takara, Japan). Standard curves were generated from serial dilutions of plasmid DNA harboring cloned target genes. Fractions demonstrating peak gene copy numbers in \u003csup\u003e13\u003c/sup\u003eC-labeled treatments, compared to \u003csup\u003e12\u003c/sup\u003eC controls, were identified as \u0026apos;heavy\u0026apos; (\u003csup\u003e13\u003c/sup\u003eC-DNA) and \u0026apos;light\u0026apos; (\u003csup\u003e12\u003c/sup\u003eC-DNA).\u003c/p\u003e\n\u003cp\u003eMicrobial community composition was further characterized by 16S rRNA gene amplicon sequencing of pre-centrifugation samples and pooled heavy/light DNA fractions from selected treatments. The V3\u0026ndash;V4 hypervariable region was amplified with primers 341F (CCTACGGGNGGCWGCAG) and 806R (GGACTACHVGGGTWTCTAAT), and sequenced on an Illumina MiSeq platform (2 \u0026times; 300 bp). Sequence processing\u0026mdash;encompassing quality filtering, denoising, amplicon sequence variant (ASV) calling, and taxonomic assignment\u0026mdash;was executed using QIIME2 (v2022.11) with the SILVA (v138) database as reference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetagenomic Sequencing, Assembly, and Binning\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo obtain genome-resolved insights into metabolically active microorganisms, DNA from heavy fractions of pertinent SIP treatments (e.g., \u0026sup1;\u0026sup3;Cᵢ- or \u0026sup1;\u0026sup3;Cₒ-labeled samples under sulfur-mediated mixotrophic denitrification, \u0026sup1;\u0026sup3;Cᵢ- or \u0026sup1;\u0026sup3;Cₒ-labeled samples under S⁰-only amendment (sulfur disproportionation), and the unlabeled control (ck)) was subjected to shotgun metagenomic sequencing. Owing to low DNA yields, fractions with buoyant densities \u0026gt; 1.735 g mL⁻\u0026sup1; from replicate microcosms were pooled to constitute composite samples per treatment.\u003c/p\u003e\n\u003cp\u003eMetagenomic libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit and sequenced on an Illumina NovaSeq 6000 platform (PE150). Raw reads underwent quality control and adapter trimming using Fastp (v0.23.2). High-quality reads were assembled de novo using MEGAHIT (v1.2.9). Contigs exceeding 2,500 bp were binned into metagenome-assembled genomes (MAGs) employing MetaBAT2, MaxBin2, and CONCOCT integrated within the MetaWRAP (v1.3.2) pipeline. Bins were refined via the \u0026lsquo;bin_refinement\u0026rsquo; module to yield high-quality MAGs, which were evaluated for completeness (\u0026gt;80%) and contamination (\u0026lt;5%) using CheckM (v1.1.3). High-quality MAGs were taxonomically classified with GTDB-Tk (v2.1.1) and functionally annotated against the KEGG, NCBI-nr, and Pfam databases using Prokka (v1.14.6) and Diamond (v2.0.15) ((Table S3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalytical Techniques\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNitrate (NO₃⁻-N) and nitrite (NO₂⁻-N) concentrations were quantified by ion chromatography (Dionex ICS-3000, USA). Ammonium (NH₄⁺-N) was determined spectrophotometrically using Nessler\u0026rsquo;s reagent. Sulfate (SO₄\u0026sup2;⁻) and thiosulfate (S₂O₃\u0026sup2;⁻) were analyzed via ion chromatography. Dissolved sulfide (S\u0026sup2;⁻) was measured employing the methylene blue method. Dissolved nitrous oxide (N₂O) was analyzed by gas chromatography (Agilent 7890B, USA) equipped with an electron capture detector (ECD) after headspace equilibration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequence data of 16S rRNA amplicons, metagenomes, and metagenome-assembled genomes generated in this study have been deposited in the NCBI Sequence Read Archive under Bioproject PRJNA1356331. The detailed information of genomes is provided in Supplementary Table 3.\u003c/p\u003e\n\u003cp id=\"_Toc11287\"\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by the National Natural Science Foundation of China (No. 52321005, No. 52400025, No. 52300155, No. 32570108), National Key Research and Development Program of China (No. 2023YFC3207203), China Postdoctoral Science Foundation (No. 2024M754204, 2023M740917), Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (No. QA202432).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKuypers, M. M. M., Marchant, H. K. \u0026amp; Kartal, B. 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A Thiosulfate Shunt in the Sulfur Cycle of Marine Sediments. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e249\u003c/strong\u003e, 152-154, doi:10.1126/science.249.4965.152 (1990).\u003c/li\u003e\n\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","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":"Mixotrophic denitrification, Sulfur-disproportionating, Nitrous oxide mitigation, Dissimilatory nitrate reduction to ammonium, DNA-stable isotope probing","lastPublishedDoi":"10.21203/rs.3.rs-8740407/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8740407/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Elemental sulfur (S0) supports low-carbon denitrification in natural sediments and engineered reactors, yet nitrate reduction rates frequently far exceed those expected from S0’s slow abiotic dissolution. This kinetic discrepancy is often attributed to enzymatic sulfur disproportionation (SD). However, SD is considered as a predominantly autotrophic process and potentially suppressed by organic carbon in the prevalent mixotrophic conditions. This attribution appears insufficient to explain the pervasive occurrence of this kinetic discrepancy. Furthermore, the sulfide generated during SD process could inhibit nitrous oxide reductase (NosZ), promoting high emissions of N2O. Nevertheless, this effect is not always accompanied by a measurable N₂O accumulation, implying that additional regulatory mechanisms may be involved. Here, we combine long‑term reactor operation, targeted batch assays, DNA-stable isotope probing (DNA‑SIP) and genome-resolved metagenomics to identify the SD microbial mechanism under mixotrophy. A sulfur‑packed bed reactor operated for \u003e300 days achieved \u003e99% nitrate removal at a hydraulic retention time of 2 h, while accumulating both sulfate and sulfide and producing measurable ammonium, indicating concurrent cryptic sulfur cycling and DNRA signals. DNA‑SIP links carbon assimilation to distinct functional guilds and enriches sulfur oxidation, DNRA and N₂O‑reduction genes in isotopically heavy fractions. Metagenomics of active fractions reveal a novel thriving set of facultative mixotrophic sulfur-disproportionating denitrifiers (FMSDs) whose genomes consolidate SD, complete denitrification, and dissimilatory nitrate reduction to ammonium (DNRA) within a single cellular framework. This unique integration facilitates an intrinsic dual-detoxification mechanism: the internal DNRA module acts as a sink for nitrite, mitigating the accumulation of inhibitory free nitrous acid (FNA), while robust sulfide oxidation modules concurrently detoxify the sulfide produced by SD’s reductive branch. By collectively safeguarding the terminal nitrous oxide reductase enzyme, this self-regulating network ensures profound N2O mitigation. This discovery redefines the microbial ecology of S-N coupling and provides a new blueprint for designing resilient, climate-friendly biotechnologies for water reclamation.","manuscriptTitle":"Mixotrophics sulfur disproportionation enhables rapid and low-N2O denitrification in sulfur-packed systems through a novel symbiotic network","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-09 11:40:14","doi":"10.21203/rs.3.rs-8740407/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","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":"1c993fd5-b3da-47a0-9150-ff135758db5a","owner":[],"postedDate":"February 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":62407974,"name":"Biological sciences/Microbiology/Environmental microbiology/Water microbiology"},{"id":62407975,"name":"Biological sciences/Biotechnology/Environmental biotechnology"}],"tags":[],"updatedAt":"2026-03-30T20:20:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-09 11:40:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8740407","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8740407","identity":"rs-8740407","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}