Simultaneous nitrification-denitrification (SND) is a promising process for low C/N wastewater treatment. A hybrid activated sludge-membrane aerated biofilm reactor (H-MABR) capable of achieving two‑stage SND was developed and exhibited satisfactory nitrogen removal performance; however, the underlying mechanisms remain unclear. To address this, an improved H-MABR was developed and integrated metagenomic and metaproteomic analyses were performed to systematically elucidate the spatial distribution of microbial communities and their functions in the reactor. The results showed that the improved H-MABR achieved removal efficiencies of 94.4 ± 0.4% for chemical oxygen demand, 92.9 ± 0.7% for total nitrogen, and 97.8 ± 0.8% for ammonia nitrogen. Multi‑omics analysis identified seven dominant microbial genera and three highly expressed nitrogen metabolic pathways. The outer ring was the primary nitrogen removal zone, where the aerobic zone mainly mediated nitrification and the anoxic zone exhibited significantly upregulated activities of NosZ, NirK, NirS, and NorC (ORA vs. ORO), strongly supporting the denitrification function in this zone. The inner ring served as an important auxiliary nitrogen removal unit, showing particularly strong ammonia nitrogen removal performance, which was attributed to the highly expressed hydroxylamine dehydrogenase. This study provides a foundation for advancing the understanding and application of SND systems.

Perfluorobutanoic acid (PFBA), a representative short-chain perfluoroalkyl carboxylic acid, is increasingly detected in aquatic environments, yet its impacts on anaerobic nitrogen cycling remain poorly understood. Here, a laboratory-scale simulated river sediment system was established to investigate the concentration-dependent effects of PFBA (1 ng/L-50 mg/L) on Anammox performance, microbial communities, functional genes, and PFBA transformation. Low PFBA concentrations (≤1 μg/L) stimulated nitrogen removal, with NH4[+]-N conversion and NO2[-]-N removal increasing by 30.8% and 38.9%, respectively. In contrast, inhibition emerged at 1 mg/L and became pronounced at ≥ 10 mg/L. At 50 mg/L, NO2[-]-N and TN removal rates decreased to 7.58 and 5.22 μg/(g·h), respectively, and remained suppressed during recovery. High PFBA exposure reduced extracellular polymeric substances (EPS) -associated protein content and key Anammox enzyme activities, accompanied by downregulation of hao, hdh, and hzs and increased relative abundance of nrf, indicating a shift from autotrophic Anammox toward heterotrophic dissimilatory nitrate reduction to ammonium (DNRA). Notably, 91.7% of PFBA was apparently transformed at 50 mg/L, with shorter-chain products such as perfluoropropanoic acid and trifluoroacetic acid being qualitatively identified, suggesting sequential dealkylation and decarboxylation. However, a complete mass balance was not established because the transformation products were not quantified. These findings demonstrate that PFBA exerts concentration-dependent effects on sedimentary nitrogen cycling by disrupting Anammox metabolism and reshaping microbial functional pathways, providing new insights into per- and polyfluoroalkyl substances (PFAS) ecological risks and sediment bioremediation.

Nitrification is mediated by ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and complete ammonia oxidizers (comammox), yet their functional partitioning across habitats with contrasting salinity and redox conditions remains poorly resolved. Here, we integrated amoA gene abundance, amoA transcript abundance, and dual-inhibitor-derived potential ammonia oxidation rates to compare the three guilds in intertidal sediments, saline soils, and agricultural soils. In high-salinity intertidal sediments, comammox had the highest gene and transcript abundances and accounted for approximately 54-85% of estimated ammonia oxidation, indicating strong agreement among population-, transcription-, and process-level indicators. In saline soils, AOA contributed the largest proportion of ammonia oxidation at all depths, accounting for approximately 47-59%, despite greater AOB gene abundance. Agricultural soils showed pronounced depth-dependent functional partitioning. Comammox contributed most at 0-5 cm at approximately 45%, AOA dominated at 5-10 cm at approximately 50%, and AOA and AOB contributed comparably at 10-15 cm at approximately 39% and 37%, respectively. Cross-level decoupling also differed among habitats. Intertidal AOB showed high transcript abundance relative to its small gene pool, whereas agricultural-soil AOA showed high gene abundance but comparatively low transcript abundance. Salinity and oxidation-reduction potential were associated with shifts in abundance, transcription, and functional contribution, together with variation in pH, organic carbon, and inorganic-N availability. These findings show that contrasting salinity and redox settings were associated with habitat-dependent reorganization, rather than uniform suppression, of ammonia oxidation. Integrating DNA-, RNA-, and process-level evidence provides a more reliable framework for identifying functionally important ammonia oxidizers in coastal transition zones and salt-affected soils.

Hydroxylamine (NH2OH, HA), a highly reactive intermediate of ammonia oxidation, shows great potential for modulating multiple nitrogen removal pathways in activated sludge systems. However, the effects of repeated low-dose HA addition on nitrogen fate and the potential coupling of nitrogen removal pathways remain unclear. In this study, a step-feed sequencing batch reactor (SBR) was operated under progressively carbon-limited conditions to investigate HA-mediated nitrogen removal. At an HA dose of 5 mg/L and an overall C/N ratio of 3.61 ± 0.06, the HA-amended reactor achieved an effluent total nitrogen concentration of 3.57 ± 0.70 mg/L and a TN removal efficiency of 91.96 ± 1.57%, compared with 11.74 ± 0.84 mg/L and 71.42 ± 2.05%, respectively, in the control reactor. Cycle profiles and batch tests showed enhanced nitrite retention, inhibited nitrite-oxidizing bacterial activity, and delayed initial COD decline. Notably, the second feeding step promoted transient NH4[+]-NO2[-] coexistence, creating conditions that could favor nitrite-based denitrification and potential anammox activity. Microbial community and functional gene analyses provided complementary evidence of shifts in nitrogen transformation potential. Specifically, the relative abundance of Nitrospira decreased, whereas Alcaligenes, Candidatus Competibacter, and functional genes associated with nitrogen removal were enriched under HA exposure. Overall, low-dose HA enhanced nitrogen removal by reshaping nitrogen fate and microbial community functions in activated sludge. These findings offer new insights into achieving efficient biological nitrogen removal in wastewater treatment under low C/N conditions.

Upflow anaerobic sludge blanket (UASB) reactors are widely used for treating high-strength organic wastewater but are vulnerable to short-term complex shocks caused by increased organic loading, hydraulic retention time regulation, and partial sludge loss. At the same time, the role of homoacetogenic bioaugmentation in improving UASB recovery after such disturbances remains poorly understood. This study evaluated the potential of a novel homoacetogenic strain, Clostridium magnum WY-4 (WY-4), to enhance UASB resilience. Two laboratory-scale UASB reactors were operated for 184 days, and reactor performance, volatile fatty acids (VFA), extracellular polymeric substances (EPS), N-acyl-homoserine lactones (AHL), and microbial community dynamics were analyzed. Under stable conditions, WY-4 had only a limited effect on reactor performance. During and after the shock, however, bioaugmentation markedly accelerated recovery. The maximum effluent VFA concentration in the WY-4 reactor during the shock reached 190 mg/L, 53% lower than in the control. Propionate degradation was enhanced, with only trace amounts remaining in the bottom zone of the WY-4 reactor, whereas acetate and propionate continued to accumulate in the control. During the first nine days of recovery, methane yield was approximately 20% higher in the WY-4 reactor. Principal component analysis showed a faster recovery of AHL profiles, accompanied by altered AHL-EPS relationships. Although dominant microbial populations changed little, the WY-4 reactor maintained greater microbial richness and diversity. The homoacetogenic bioaugmentation with WY-4 enhanced UASB resilience by reducing VFA accumulation, accelerating methane recovery, and promoting restoration of AHL-mediated quorum sensing following short-term complex shock.

In situ assimilation of off-gases (H2/CO2) into liquid metabolites during anaerobic fermentation presents a promising approach to maximize waste biomass valorization. However, mechanisms governing the interaction between microbial carbon fixation and chain elongation (CE) through headspace dynamics remain unclear. This study explored gas-liquid metabolic flux and microbial response by varying headspace pressure/composition in batch CE bioreactors. Autogenic pressure reshaped the physicochemical microenvironment, which potentially induced specific biological inhibition and constrained ethanol utilization. Accordingly, caproic acid production decreased by 40.50-51.68%, whereas acetic acid accumulation increased by 70.67-104.44% under both N2 and H2/CO2 headspace. This alteration reflected metabolic flux redistribution between CE and acetic acid-producing pathways driven mainly by electron donor oxidation with homoacetogenesis as a potential secondary contributor. Meanwhile, an initial H2/CO2 headspace curbed off-gas emissions and induced a final 17.62 kPa (abs) vacuum, enhancing soluble metabolite carbon retention. Consequently, liquid-phase carbon recovery increased to 84.29% under autogenic pressure and peaked at 89.68% following further H2/CO2 headspace replacement. Microbial profiling revealed that self-pressurized conditions suppressed Oscillibacter from 15.14% to 1.93%, whereas H2/CO2 exposure selectively enriched Caproiciproducens to 30.52% as the dominant CE driver. Metatranscriptomics demonstrated that autogenic pressure markedly upregulated the Wood-Ljungdahl pathway genes by up to 28.62-fold, accompanied by induction of stress-responsive genes for physiological adaptation. Additionally, the initial H2/CO2 headspace promoted the expression of gene korA, suggesting a potential partial reductive tricarboxylic acid (rTCA) pathway for carbon fixation. These insights provide a mechanistic framework for optimizing carbon recovery and product selectivity in engineered CE systems, advancing sustainable waste-to-biochemical platforms.

Tetracycline (TC) contamination poses a potential threat to the stability of anammox-based wastewater treatment systems, yet effective strategies for accelerating functional recovery remain poorly understood. This study explored the inhibitory effects of 5 mg/L TC on a single-stage partial nitritation-anammox (SPNA) system, and further unraveled its functional potential and underlying mechanisms of exogenous riboflavin for restoring system performance. TC exposure substantially impaired nitrogen removal, with total nitrogen removal efficiency decreasing from 53.39 % to 34.44 %, accompanied by a pronounced decline in anammox activity. Supplementation with 0.5 mg/L riboflavin accelerated system recovery, partially restoring total nitrogen removal efficiency to 48.15 %, by enhancing anammox activity and suppressing nitrite-oxidizing bacteria proliferation. Riboflavin alleviated these disturbances by enhancing electron transfer, mitigating oxidative stress, and restoring nitrogen metabolism-related functional genes. Metagenomic analysis further revealed that riboflavin recovered extracellular electron transfer and amino acid and nucleotide metabolism pathways disrupted by TC stress, while promoting the enrichment of tetracycline resistance and degradation-related genes and putative tetracycline-degrading bacteria, including Sphingobacterium and Bacillus. These responses were accompanied by enhanced tetracycline removal, suggesting potential mechanisms of improved microbial adaptation to antibiotic stress.

Anaerobic digestion (AD) is a promising method for treating organic waste, but it requires thorough management. While it is known that organic loading fluctuations affect anaerobic digestion, their specific impact has not been verified. Therefore, this study hypothesized that the response to overload differs depending on the organic loading rate (OLR) strategy to which anaerobic digesters are adapted. To test this hypothesis, sequencing batch reactors were operated using whey as the substrate. Five operational conditions were compared under three fixed OLRs of 0.25, 1.0, and 2.0 g COD/L∙day (i.e., low-L, medium-M, and high-H, respectively) and two dynamic OLR regimes of stepwise oscillation (SO; M-L-M-H conditions) and drastic oscillation (DO; L-H conditions). After the initial operation period, all reactors were subjected to the same overload, recovery, and treatment (C-control, B-bioaugmentation). The LB and SOB reactors showed marked pH decreases and caproic acid accumulation, whereas the HB and DOB reactors showed delayed propanoic acid degradation. In contrast, the MB reactor exhibited superior performance in terms of volatile fatty acid (VFA) removal and pH recovery on D159. The microbial community changed dynamically according to the OLR history. PICRUSt2 analysis suggested that syntrophic VFA degradation and amino acid metabolism were affected by the OLR histories. Network analysis revealed putative associations between minor syntrophic acetate-oxidizing bacteria and Methanosarcina during recovery. Additionally, the connection of protein-related consortia (Aminobacterium-Methanomassiliicoccus) was inferred by network analysis. Overall, the results of this study indicate that prior OLR history shapes overload resilience by altering VFA degradation trajectories and microbial recovery patterns.

Cadmium contamination in acidic environments remains a critical challenge due to enhanced metal mobility and the limited efficiency of conventional remediation technologies. Microbially induced carbonate precipitation (MICP) is a promising sustainable approach for heavy metal immobilization; however, its application under acidic conditions is severely restricted by reduced microbial activity and insufficient carbonate generation. Modified biochar was used as a functional carrier to develop adsorption-based microcapsules (AM) and entrapment-based microcapsules (EM) loaded with ureolytic bacteria for Cd[2+] immobilization in acidic environments. Results showed that EM effectively protected bacteria under acid stress and maintained high urease activity, and Cd[2+] removal achieved over 99.7 % in aqueous solutions, much greater than other groups at pH 3. The unconfined compressive strength of solidified soil increased with curing age and pH. At pH 7 and 14 curing days, AM exhibited the highest strength, whereas EM showed lower strength due to the limited mechanical stiffness of sodium alginate hydrogel beads. Both microcapsules reduced Cd[2+] leaching, EM achieving the greatest reduction, 37.3 % lower than the control group. Cadmium immobilization was attributed to exchangeable Cd transformed into carbonate-bound and residual fractions through surface adsorption, complexation, and carbonate coprecipitation. The results of this study offer valuable insights for the remediation of heavy metal contaminated sites, especially in acidic environments.

Lignin is an abundant, renewable aromatic biopolymer that remains largely underutilized despite its rich chemical potential. In this work, lignin is valorized through a mild electrochemical functionalization process using chloride ions as a redox mediator on a boron-doped diamond electrode. This halide-mediated oxidation introduces oxygen-containing functional groups, while simultaneously inducing particle fragmentation and increasing surface accessibility. These structural modifications significantly enhance the hydrophilicity and interfacial interaction of lignin with gold electrodes. The electrochemically oxidized lignin is combined with polyvinylpyrrolidone (PVP) and subsequently integrated as the active sensing layer in a resistive humidity sensor fabricated on flexible Au/Cr interdigitated electrodes. The resulting sensor achieves a high humidity response of up to 2.0 × 10[7]% over a broad relative humidity range (33-90% RH). The device exhibits an optimal dynamic operating range between 40% and 90% RH, featuring fast response and recovery times of 0.2 s. Kinetic limitations are observed at the lower humidity (ca. 200 s at 33% RH) as the isolated water monolayers lack continuous hydrogen-bonding required to facilitate rapid Grotthuss proton transport. Through this approach, we demonstrate an effective pathway for advanced biomass valorization in functional, moisture-responsive flexible electronics.

Translating natural, successional wood decomposition into an engineered two-stage fungal pretreatment offers a promising strategy for biomass deconstruction, yet how pioneer white-rot fungal modifications facilitate subsequent brown-rot decay remains unclear. Scots pine (Pinus sylvestris L.) was pretreated with Trametes versicolor (Tv) and Phanerochaete chrysosporium (Pc) and subsequently exposed to Gloeophyllum trabeum (Gt). After 15-day BRF incubation, second-stage mass loss increased by 40.9% and 126.2% in the Tv- and Pc-pretreated groups, respectively, relative to the control without WRF pretreatment. FTIR, chemical-composition and qualitative HSQC-NMR analyses collectively indicated that this accelerated degradation of Scots pine was associated with the WRF-induced modification of the lignin-carbohydrate matrix. Furthermore, this substrate-mediated facilitative effect was also observed in red oak (Quercus rubra L.), for which second-stage mass loss reached 13.1% and 14.7% after P. chrysosporium and T. versicolor pretreatment, respectively, compared with 6.7% in the control. Under the fungal combinations and laboratory conditions tested, these results show that WRF-induced substrate modification can facilitate subsequent brown-rot deconstruction and provide a basis for further exploring sequential fungal treatments in woody biomass.

Organophosphate esters (OPEs) are widespread soil contaminants, yet their effects on microbially driven phosphorus (P) cycling remain poorly understood. Here, we conducted 90-day microcosm experiments using agricultural soil spiked with triphenyl phosphate (TPhP, an aryl-OPE) or tris(1-chloro-2-propyl) phosphate (TCPP, a halogenated OPE) to investigate their effects on microbially mediated P cycling. After 90 days, TCPP decreased available P by 12-26% compared to CK and exerted stronger microbial suppression than TPhP at high concentrations, disrupting co-occurrence networks, causing keystone taxa niche loss, and broadly suppressing C-P cycling genes. High-concentration TCPP significantly perturbed microbial lipid metabolism, specifically linoleic acid metabolism and unsaturated fatty acid biosynthesis. In contrast, TPhP, especially at low exposure levels, enriched key P-cycling taxa (e.g., Massilia, Sphingomonas, and Cladosporium) and phosphatase-encoding genes, thereby accelerating available P release. Bioaugmentation experiment indicated that the SynCom treatment consisting of two indigenous keystone degraders, Sphingomonas sp. OP1 and Pseudomonas sp. OP2, increased TPhP and TCPP removal by 26.1% and 16.1%, respectively, compared with the corresponding non-inoculated treatment at 60 days. These findings demonstrate that OPEs regulate soil P cycling through concentration- and structure-dependent mechanisms and identify indigenous SynComs as a promising bioremediation strategy for OPE-contaminated soils.

Hydrochar retains abundant oxygen-containing functional groups but little porosity, and thermal upgrading opens pores at the expense of surface chemistry. Ball milling requires no external heating, so its structural outcome is decided by how freshly fragmented particles reorganize. Here, dry and water-mediated ball milling of corn-straw hydrochar were compared to resolve how the milling medium directs this reorganization, with 600℃ biochar as a thermal reference. Without a liquid phase, fragments recontacted and densified: the specific surface area fell from 11.23 to 2.91 m[2]·g[-1], the total pore volume from 0.0818 to 0.0179 cm[3]·g[-1], and the CO proportion from 26.2 % to 19.0 %, so pore closure and carbonyl loss occurred together. In water, dispersion suppressed reagglomeration, increasing the specific surface area, total pore volume, and CO proportion to 34.26 m[2]·g[-1], 0.1903 cm[3]·g[-1], and 33.3 %, respectively. HTC-WBM adsorbed 2.53, 2.41, and 3.15 times as much TC as HTC, HTC-DBM, and BC, respectively. Although BC had the highest specific surface area (54.99 m[2]·g[-1]), its total pore volume and BET-equivalent average pore diameter were only 0.0439 cm[3]·g[-1] and 3.19 nm, compared with 0.1903 cm[3]·g[-1] and 22.22 nm for HTC-WBM. Open meso-/macropores facilitated TC transport to retained oxygen-containing sites, where hydrogen bonding and electrostatic interactions contributed to adsorption. Spectroscopic evidence and SHAP analysis were consistent with this coupled pore-accessibility/functionality effect. Medium-controlled milling therefore couples pore opening with functionality retention.

The effect of freeze-thaw pretreatment on the artificial humification of rice straw via hydrothermal carbonization was evaluated in this work. The physical-chemical characteristics of hydrochar were characterized using XRD, XPS, FTIR, SEM, and the liquid was analyzed by FT-ICR-MS. Results showed that one cycle of freeze-thaw at -20 °C for 24h increased total humic acid yield by 7.2 % compared to untreated straw, with a 6.8 % enhancement under KOH-assisted hydrothermal humification conditions (KOH-to-straw mass ratio of 1:5, 200 °C with reaction time of 2h). The pretreatment reduced crystallinity from 20.53 % to 12.85 %, exposing amorphous cellulose for preferential hydrolysis. FT-ICR-MS reveals that freeze-thaw more than doubles carbohydrate diversity in dissolved organic matter while increasing aliphatic species by 18 %. XPS confirms the emergence of carboxyl groups (4.65 % of surface carbon) and a 7.5 % increase in surface oxygen content. These cryogenic with modifications promoted oxidative condensation, yielding lower-molecular-weight humic substances with enhanced aromaticity.

Integrating multi-walled carbon nanotubes (MWCNTs) into anaerobic ammonium oxidation (anammox) systems offers a promising nitrogen removal strategy, yet the balance between conductive stimulation and nanotoxicity remains poorly understood. This study elucidated the dose-dependent mechanisms of MWCNTs on anammox consortia. A distinct biphasic response was observed: low doses (optimal 30 mg·L[-1]) enhanced specific anammox activity by 18.82 ± 1.73% (p < 0.001), whereas high doses (≥ 100 mg·L[-1]) caused severe inhibition. Mechanistically, 30 mg·L[-1] MWCNTs stimulated EPS to physically intercept nanoparticles while triggering an adaptive antioxidant response. Metatranscriptomic revealed that MWCNTs acted as "conductive bridges" to reprogram microbial electron flow, thereby selectively favoring AnAOB, promoting endogenous electron production and transfer, and ultimately enhancing the turnover of key terminal enzymes (hzs, hdh). Conversely, excessive exposure overwhelmed EPS and antioxidant defenses, causing reactive oxygen species accumulation and cellular disruption. Long-term operation confirmed sustained performance, expanding the AnAOB ecological niche (21.72 ± 9.62% increase) while suppressing competing denitrifiers. These findings provide a comprehensive molecular framework for precisely regulating nanomaterial dosing to optimize advanced wastewater treatment while mitigating ecological risks.