Microbial functions and metabolism are intrinsic drivers of pollutant removal in mixotrophic denitrification systems. Four pyrite-based mixotrophic denitrifying biofilters were constructed and monitored for 304 days. Variations in pollutant characteristics indicated that the hot zones of heterotrophic denitrification, autotrophic denitrification, and sulfate reduction were located in the bottom, middle-lower, and upper parts of biofilters, respectively. These hot zones corresponded to preferential enrichment of heterotrophic denitrifying, S-based mixotrophic denitrifying, and sulfate-reducing bacteria, respectively, highlighting microbial spatial stratification. Differential functional gene analysis for S reduction revealed that only a dissimilated sulfate reduction process could consistently provide biogenic S0 as a new electron donor via the flavocytochrome c sulfide dehydrogenase (Fcc) enzyme and extracellular polymeric substance protection systems, enhancing the denitrification process. X-ray photoelectron spectroscopy confirmed the accumulation of biogenic S0 . Untargeted metabolomic analysis suggested that vitamin B12 and tryptophan might be the key metabolites for realizing synergistic promotion of autotrophic and heterotrophic denitrification. The microbe-metabolite network indicated that dominant bacteria (e.g., Thiothrix and unclassified\_f\_Rhodocyclaceae) were specialists with less cross-feeding metabolism, while rare species (e.g., Thiobacillus and Desulfobacter) were generalists with complex cross-feeding metabolism in the constructed mixotrophic denitrification systems. The electron transfer pattern indicated that most of the electrons released from S, C, and Fe oxidation were utilized in denitrification processes as the dominant nitrogen removal pathway, including S2- /S0-based autotrophic, fermentation acetic acid production-heterotrophic, and Fe(II)-based autotrophic denitrification. Some electrons were utilized for coupling dissimilatory nitrate reduction to ammonia (DNRA) and anammox processes as an auxiliary pathway for systemic nitrogen removal. The findings of this study advance our understanding of the deeper intrinsic drivers of nitrogen removal by pyrite-based mixotrophic denitrifying biofilters, facilitating their optimization. (c) 2025 THE AUTHORS. Published by Elsevier LTD on behalf of Chinese Academy of Engineering and Higher Education Press Limited Company. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

The reactive substance consisting manganese oxides (MnOx) and solid carbon have been reported to be effective in polishing secondary wastewater; however, the treatment characteristics and mechanism remains limited. In this study, MnOx/carbon (Mn-C) composites were applied in biofilters to evaluate simultaneous removal of nitrate and sulfamethoxazole (SMX), with the single carbon composites as control. Results showed that the effluent concentrations of NO3 –N and SMX were below 2.87 mg L- 1 and 7.97 mu g L- 1 under hydraulic retention time (HRT) of 6 h. The intermittent aeration optimized Mn cycle with treatment performance improved under lower HRT and Mn(II) accumulation decreased. Mn-C composites could reduce the emission of N2O, CO2 and CH4. The dominant genera gradually evolved from fermentation to glycogen aggregation, and from heterotrophic/sulfur autotrophic to heterotrophic denitrifiers by intracellular substance and manganese autotrophic/heterotrophic bacteria. Microbial network analysis indicated higher antagonism, lower modularity and shorter average path among microbes in Mn-C biofilters, which highlighted microbial differentiation and faster electron transfer. Improved functions of denitrification and Mn respiration, and the increasing genes encoding electron transfer chain, including NADH dehydrogenase, Cytc and ubiquinone, further elucidated the superiority of Mn-C com- posites. These results improved our understanding of Mn-C composites application in low-carbon wastewater treatment.

This study developed a two-stage biofilter utilizing pyrite/sawdust composites to treat actual secondary effluent with high dissolved oxygen (DO) concentrations (3-8.5 mg/L) over a period of 169 days. The findings demonstrated that the two-stage pyrite-based biofilters achieved advanced purification of the real secondary effluent, maintaining effluent concentrations of total nitrogen (TN) and total phosphorus (TP) below 2.0 mg/L and 0.5 mg/L, respectively, with an influent TN of 10 mg/L and a temperature of >21 degrees C. The Rhodocyclaceae family is the predominant mixotrophic denitrifying bacteria in both the first-class (FC) and second-class (SC) systems. Sulfate-reducing bacteria (i.e., Desulfrispora and Desulfatirhabdium) might be keystone species in the SC system, underscoring that the sulfate reduction process enhanced denitrification under low DO conditions. Differential functional gene analysis exposed that high DO might suppress the activity of Complex III, NAR, NIR, NOR, and NOS, leading to slow-unstable electron transport and consumption in the denitrification process. Moreover, the diminished expression of S and Fe cycling genes (soxA/B/Z/X, aprA/B, dsrA/B, ABC.FEV<middle dot>S, and korA/B/C) in the FC system indicated that high DO predominantly might inhibit the Sox pathway, dissimilatory sulfate reduction process, Fe2+/Fe3+ transfer, and biological Fe2+ oxidation system. The dormancy of the S and Fe cycles induced by high DO levels primarily accounted for the diminished performance of the pyrite-based biofilters. This study offers novel insights into the extensive application of pyrite-based composites and enhances the understanding of DO effects on S and Fe cycles in the nitrogen removal process of municipal tailwater.

The complexity and variability of actual secondary effluent from wastewater treatment plants (WWTPs) pose significant treatment challenges. In this study, a two-stage biofilter packed with ZVI/Poly-3-hydroxybutyrate-cohyroxyvelate/sawdust (ZPS) composites was innovatively constructed to treat actual secondary effluent with high influent dissolved oxygen (DO) concentrations (3-8.5 mg/L) for 143 days in a WWTP. Results showed that advanced purification of real secondary effluent was achieved, and the effluent concentrations of TN and TP were stable below 2.0 mg/L and 0.1 mg/L, respectively, at influent TN < 10 mg/L. Microbial community analysis identified unclassified\_f\_\_Rhodocyclaceae as the dominant denitrifiers in both first-class (FC) and second-class (SC) systems. The shift in dominant Fe-related bacteria from Ferritrophicum to Clostridium sensu stricto\_7 from the FC to SC system with DO decreased suggested that ZVI's triple role in oxygen-capturing reagent, denitrification and organic matter decomposition. Co-occurrence network analysis deciphered that Thermomonas and Clostridium\_sensu\_stricto\_10 were key genera in SC system, which formed an obvious Fe redox cycle process that bolsters denitrification under low DO levels. Differential functional gene analysis revealed that high DO could inhibit the activity of Cyt c, NOR and NOS, resulting in a slow and unstable electron transport and consumption in denitrification process. Furthermore, the down-regulation iron cycling genes (feoA and ABC.FEV.S) in FC system suggested that high DO mainly inhibited the Fe2+/Fe3+ transfer system. An inactive Fe cycle at high DO levels highlights the important role of Fe cycle in iron-based denitrification process. These findings advanced the understanding of effected mechanism of DO on nitrogen removal mediated by ZPS composites in actual tailwater treatment. Additionally, the novel ZPS composites can be combined with the removal of antibiotics, and other toxic or harmful substances within wastewater to expand their application.

Further reducing total nitrogen (TN) and total phosphorus (TP) in the secondary effluent needs to be realized effectively and in an eco-friendly manner. Herein, four pyrite/sawdust composite-based biofilters were established to treat simulated secondary effluent for 304 days. The results demonstrated that effluent TN and TP concentrations from biofilters under the optimal hydraulic retention time (HRT) of 3.5 h were stable at <2.0 and 0.1 mg/L, respectively, and no significant differences were observed between inoculated sludge sources. The pyrite/sawdust composite-based biofilters had low N2O, CH4, and CO2 emissions, and the effluent's DOM was mainly composed of five fluorescence components. Moreover, mixotrophic denitrifiers (Thiothrix) and sulfate-reducing bacteria (Desulfosporosinus) contributing to microbial nitrogen and sulfur cycles were enriched in the biofilm. Co-occurrence network analysis deciphered that Chlorobaculum and Desulfobacterales were key genera, which formed an obvious sulfur cycle process that strengthened the denitrification capacity. The higher abundances of genes encoding extracellular electron transport (EET) chains/mediators revealed that pyrite not only functioned as an electron conduit to stimulate direct interspecies electron transfer by flagella but also facilitated EET-associated enzymes for denitrification. This study comprehensively evaluates the water-gas-biofilm phases of pyrite/sawdust composite-based biofilters during a long-term study, providing an in-depth understanding of boosted electron transfer in pyrite-based mixotrophic denitrification systems.

The precise structure and assembly process of pyrite-based biofilms remain poorly understood. The polysaccharides (PN), proteins (PS), and extracellular DNA were enriched in the soluble extracellular polymeric substance (EPS), loosely bound EPS, and tightly bound EPS, respectively, indicating a significant stratified structure of biofilms. The tryptophan facilitated mixotrophic metabolic processes. Both dominant (>1%) and rare species (<0.01 %) harbored core bacteria, including sulfur autotrophic bacteria, sulfate-reducing bacteria, and heterotrophic bacteria. Furthermore, partial least-squares path modeling quantified the contributions of total phosphorus (TP) (2 = 0.32), dissolved organic matter (DOC) (2 = 0.29), and NH4+-N (2 = 0.26) to variations in the microbial community. Nonmetric multidimensional scaling analysis revealed three distinct stages in biofilm development: colonization (0-36 d), succession (36-149 d), and maturation/old (149-215 d). Furthermore, neutral community model indicated that stochastic processes drove the colonization and maturation/old stages, while deterministic processes dominated the succession stage. This study offered valuable insights into the regulation of pyrite-based engineered ecosystems.

Fine-grained pyrites are difficult to be denitrified under natural environment due to chemical oxidation with O2. In this study, the pyrite/PHBV composites were synthesized through high-temperature melting and realized nitrogen and phosphate removal under natural aerobic conditions. Results showed that pyrite/PHBV-40 composites had the highest denitrification rate of 0.61 mg NO3–N /(LCh) with low SO42-production, and its removal efficiency of nitrogen and phosphorus was 98% and 41%, respectively. Microbial community structure analysis revealed that the enrich sulfate-reducing bacteria (SRB) on the pyrite/PHBV-40 composites demonstrated that the sulfate reduction driven by SRB enhanced denitrification process, and thereby the S cycle could underpin the potential self-sustainability of pyrite/PHBV-40 composites. Co-occurrence network analysis showed that Fe oxidizers/reducers (e.g., Ferruginibacter/Geobacter) and SRB (e.g., Desulfovibrio) were the keystone species in microbial community. Bugbase analysis showed that formed biofilms mainly consisted of aerobic and facultative anaerobic strains, which was corresponding to structure of biofilm including aerobic and anoxic layer. Partial mantel test revealed the total Fe and nutrients (e.g., N and P) are the drivers in OTU and phenotype composition, respectively. Metabolic pathway analysis suggested that pyrite/PHBV composites may not only accelerate glycolysis with rapid hydrolysis of PHBV, but also enhance the TCA cycle with high production of ATP and NADH. The final product of nitrate reduction is N2O or NO, and the cysJ gene play an important role in sulfate reduction in pyrite/PHBV systems. Overall, the novel synthesized pyrite/PHBV composites are an ideal functional material with high denitrification rate, no secondary pollution and long service life. Our study highlights pyrite/PHBV-induced strength in microbiota dynamics and C, N, S transformation, therein, the sulfate reduction process cannot be overlooked.

In this study, Mn-C composites using different MnO2 contents and solid carbon material were prepared to explore the synchronous removal performance of nutrients and SMX. Higher nitrate removal performance (97-98 %) with quickest nitrate removal rate (4.97 mg N L -1h- 1) was obtained in Mn\_20 systems. The increased Mn content and Mn-P compound were observed via surface characteristics, indicating the involvement of MnOx in pollutants removal, particularly for higher phosphorus removal (84-89 %) via Mn-P precipitation and BioMnOx adsorption. Nevertheless, compared to systems based on Mn\_0 composites (74 %), systems with Mn-C composites presented lower SMX reduction efficiency (34-51 %), which might be attributed to the large Mn(II) accumulation, impairing certain microbes and lower the MnOx function. Higher abundance of genera affiliated to Bacter-oidetes\_vadinHA17 and Rhodocyclaceae was observed in the Mn-C composites, as well as the gathering of Geo-bacter and Desulfovibrio as keystone taxa, responsible for the removal of nitrate and SMX and microbial interactions. Besides, the increase of sulfonamide ARGs was closely related to the predominant microbes in the Mn-C composites, which acted as the hosts of ARGs. This study broadens the knowledge of Mn-C composites in synergetic removal of nutrients and organics, and supports the potential application of manganese oxide in wastewater treatment.

In this study, an integrated treatment system was proposed and applied in situ, including detention tank, multistage constructed wetlands (CWs) and wastewater treatment plants (WWTPs), preventing nutrients flowing into Dianchi Lake, in which the treatment performance of multistage CWs were evaluated principally. Results skillfully realized the bypass purification of upstream river at dry reasons, as well as the effective management and treatment of the collected diffuse pollution at rainy reasons. The purified water flowing into water bodies could satisfy the Grade III of environmental quality standards for surface water in China with the average effluent concentrations of COD, NH4+-N, TN and TP decreased to 10 (51.2-72.7%), 0.5 (67.2-83.0%), 1.0 (71.2-79.6%) and 0.15 (72.3-89.4%) mg L-1, respectively. High-throughput sequencing results indicated that the application of poly-3-hydroxybutyrate-cohyroxyvelate-sawdust (PS) blends could enrich norank\_f\_Anaerolineaceae (7.95%) and Bradyrhizobium (10.2%), which were distinct from the dominant genera of Pleurocapsa (13.0%) in gravel -based CWs. Functional genes and metabolism analysis uncovered that the heterotrophic denitrification was the main pathway of nitrogen removal with the abundance of genes encoding TCA cycle, glycolysis and deni-trification process up-regulated. In addition, molecular ecological network (MEN) analysis suggested the deni-trification genes were positively correlated with the predominant microbes in PS-based CWs, favorable for denitrifiers to transfer and utilize electron donors during denitrification process. This study proved that the developed PS blends as carbon supplies in CWs and the proposed integrated treatment system are effective methods for watershed management, providing valuable reference to low-pollution wastewater treatment in practical engineering projects.

Manganese oxides (MnOx) are recognized as a strongest oxidant and adsorbent, of which composites have been proved to be effective in the removal of contaminants from wastewater. This review provides a comprehensive analysis of Mn biochemistry in water environment including Mn oxidation and Mn reduction. The recent research on the application of MnOx in the wastewater treatment was summarized, including the involvement of organic micropollutant degradation, the transformation of nitrogen and phosphorus, the fate of sulfur and the methane mitigation. In addition to the adsorption capacity, the Mn cycling mediated by Mn(II) oxidizing bacteria and Mn(IV) reducing bacteria is the driving force for the MnOx utilization. The common category, characteristics and functions of Mn microorganisms in recent studies were also reviewed. Finally, the discussion on the influence factors, microbial response, reaction mechanism and potential risk of MnOx application in pollutants’ transformation were proposed, which might be the promising opportunities for the future investigation of MnOx application in wastewater treatment.

The solid carbon source (poly-3-hydroxybutyrate co 3 hyroxyvalerate, PHBV) and manganese oxide mineral (Mn ore) were proposed firstly as co-substrates for eliminating nutrients and sulfamethoxazole (SMX) in this study. Results showed that high-rate nitrate and phosphate removal could be achieved in PHBV/Mn ore systems with the average efficiencies of 90% and 66.7%, respectively, although the addition of SMX decreased denitrification performance by 4.5-10.5%. SMX was removed mainly via biodegradation of enriched denitrifying microbes, with the average removal efficiency of 20-50% in PHBV/Mn ore systems, which was higher than that in PHBV systems. The existence of Mn ore markedly shaped the microbial community structure, leading to the dominant bacteria transforming from Microscillaceae to Sporomusaceae. The genera of Geobactor, Desulfovibrio and Anaerovorax were found to maintain the stability of microbial system as keystone species. Surprisingly, large amount of Mn(II) was accumulated, which not only verify the involvement of Mn cycling in decontamination process, but also might explain the propagation of ARGs (tnpA-04 and tnpA-05) in host microorganisms. Therefore, the optimized mixture proportion of PHBV and Mn ore should be further estimated avoiding Mn (II) accumulation in the effluent. On the whole, these results might shed light on new insight for advanced treatment of nutrients and emerging pollutants in biofilm reactors.

Effective control of nitrogen and phosphorus simultaneously is of great significance to satisfy the strict requirement of the ecological health of receiving waters. In this study, PHBV/ZVI composites made from solid carbon (poly-3-hydroxybutyrate-cohyroxyvelate, PHBV) and zero-valent iron (ZVI) were proposed to be functional fillers in biofilters for advanced wastewater treatment. Results showed that high-rate treatment performance was obtained with nitrate and phosphorus removal efficiencies of 79-97% and 97-98% in the biofilters packed with PHBV/ZVI composites. Lower N2O and CH4 emission (56.3-129.2 mu g m(-2) h(-1)) were also achieved simultaneously, further indicating the superiority of PHBV/ZVI composites applied in the wastewater treatment. High-throughput quantitative-PCR (HT-qPCR) results uncovered that the existence of ZVI could enrich carbon degradation genes (manA, gam and mxa) and facilitate denitrifier utilize organic matters more efficiently, as evidenced by up-regulations of genes involved in nitrate reduction (nirS and nosZ). Meanwhile, higher Fe concentration and less functional genes inducing lower activities of phosphate metabolism and in PHBV/ZVI systems indicated ZVI corrosion and coprecipitation were the main pathway of phosphorus removal. Network and redundancy analysis highlighted the role of ZVI in the removal of pollutants with keystone genes changed (pox and napA) and genes distribution remodeled compared to single PHBV fillers. Further, the activities of dehydrogenase (DHA) and nitrite reductase (Nir) enzymes also increased by the modulation of microbes, which explicitly interpreted the synergistic promotion of PHBV and ZVI on the denitrification process. These findings provided an alternative for the advanced treatment of wastewater and improve the understanding of C, N and P cycling in the co-occurrence of PHBV and ZVI.

In this study, a biofilter was developed with a ZVI/PHBV/sawdust (ZPS) composite for treating simulative secondary effluent from wastewater treatment plants. Results showed that effluent concentrations of NO3–N and TP in the ZPS biofilter were stable below 2.0 mg/L and 0.1 mg/L, corresponding to 95% NO3–N removal and 99% TP removal, respectively. Microbial community analysis revealed that the transformation of dominant taxa from Dechloromonas to Clostridium sensu stricto\_7 from 30 d to 120 d suggested that the ZVI-induced succession of dominant fermentation bacteria ensured the stable carbon supply for denitrification. Co-occurrence network analysis showed that the ZVI directly enhanced the interaction of microbial community. Fe-related bacteria occupied a key position in the rare species, which might maintain the function of iron-mediated organic matter decomposition and denitrification. These findings provide an alternative for advanced removal of nitrogen and phosphorus in biofilters packed with ZPS composites.

ABSTRACT: A higher denitrification rate was realized via controlling the mass ratio of pyrite and poly(3-hydroxybutyrate-hydroxyvalerate) (PHBV) under natural aerobic conditions. The results showed that the suitable mass ratio of PHBV and pyrite could be 1:2 with its removal efficiency of nitrogen and phosphorus of 99.7 and 53.4%, respectively. The PHBV/pyrite system has formed the spatial patterns of the biofilm community, such as Dechloromons attached to the pyrite surface, Rhodocyclaceae attached to the PHBV surface, and Acidovorax attached to the suppled sludge, which highlighted that the autotrophic??? heterotrophic synergy was achieved. The difference analysis among functional genes detected by high-throughput quantitative polymerase chain reaction indicated that the surface of pyrite in the pyrite/PHBV system is the hot area of methane production, the denitrifying process, and phosphorus removal. Network analysis indicated that there was a closer connection among functional genes on the pyrite surface, also supporting the speculation that pyrite was the hot area for the interaction of various genes in the pyrite/PHBV system. The key gene co-occurrence revealed that lig, nirS, and aspA are the keystone genes for cellulose degradation, denitrification, and S cycling, respectively. These results suggested that the pyrite surface was the hot area for denitrification, phosphorus removal in the blending system with pyrite and PHBV for nitrogen and phosphorus removal.

With the advancement of water ecological protection and water control standard, it is the general trend to upgrade the wastewater treatment plants (WWTPs). The simultaneous removal of nitrogen and phosphorus is the key to improve the water quality of secondary effluent of WWTPs to prevent the eutrophication. Therefore, it is urgent to develop the applicable technologies for simultaneous biological removal of nitrogen and phosphorus from secondary effluent. In this review, the composition of secondary effluent from municipal WWTPs were briefly introduced firstly, then the three main treatment processes for simultaneous nitrogen and phosphorus removal, i.e., the enhanced denitrifying phosphorus removal filter, the pyrite-based autotrophic denitrification and the microalgae biological treatment system were summarized, their performances and mechanisms were analyzed. The influencing factors and microbial community structure were discussed. The advanced removal of nitrogen and phosphorus by different technologies were also compared and summarized in terms of performance, operational characteristics, disadvantage and cost. Finally, the challenges and future prospects of simultaneous removal of nitrogen and phosphorus technologies for secondary effluent were proposed. This review will deepen to understand the principles and applications of the advanced removal of nitrogen and phosphorus and provide some valuable information for upgrading the treatment process of WWTPs.

In this study, batch experiments were conducted to investigate the immobilization of HMs (Cr and Pb) by DOM derived from biochar in the presence and absence of zero-valent iron (Fe) in nitrate and HMs co-contaminated groundwater. Both Cr and Pb were removed effectively in biochar-Fe aqueous systems, while only Pb could be mitigated in biochar systems. Excitation-emission spectrophotometry combined with parallel factor analysis (EEM-PARAFAC) revealed that DOM released from biochar mainly contained human-like and tryptophan-like substances. Moreover, the fluorescence of hemic-like components could be quenched differently by the complexation of HMs, which proved the different removal efficiencies of Cr and Pb in biochar aqueous phase. In biocharFe aqueous systems, Fe-C micro-electrolysis was formed in prior to the complexation of DOM-Fe hydroxides. Thus, the chemical reduction was the primary way to removal HMs in batch-Fe systems, which was corresponding with the less variation of DOM components when adding Cr and Pb into aqueous systems. Besides, the observed DOM components with higher aromaticity and humification after adding Cr and Pb, further indicated the complexation of DOM-HMs through the analysis of adsorption and fluorescence indices. These results will provide new insights into the HMs retention on biochar, particularly for the role of Fe on the complexation process. (c) 2021 Elsevier B.V. All rights reserved.

The pilot-scale solid-phase denitrification systems supporting with poly(3-hydroxybutyrateco-3-hydroxyvalerate) (PHBV) and PHBV-sawdust were constructed for advanced nitrogen removal from wastewater treatment plants (WWTPs) effluent, and the impacts of biomass blended carbon source on microbial community structure, functions and metabolic pathways were analyzed by metagenomic sequencing. PHBV-sawdust system achieved the optimal denitrification performance with higher NO3- - N removal efficiency (96.58%), less DOC release (9.00 +/- 4.16 mg L–(1)) and NH4+-N accumulation (0.37 +/- 0.32 mg L (- 1)) than PHBV system. Metagenomic analyses verified the significant differences in the structure of microbial community between systems and the presence of four anaerobic anammox bacteria. Compared with PHBV, the utilization of PHBV-sawdust declined the relative abundance of genes encoding enzymes for NH4+-N generation and increased the relative abundance of genes encoding enzymes involved in anammox, which contributed to the reduction of NH4+-N in effluent. What's more, the encoding gene for electrons generation in glycolysis metabolism obtained higher relative abundance in PHBV-sawdust system. A variety of lignocellulase encoding genes were significantly enriched in PHBV-sawdust system, which guaranteed the stable carbon supply and continuous operation of system. The results of this study are expected to provide theoretical basis and data support for the promotion of solid-phase denitrification. (C) 2021 Elsevier Ltd. All rights reserved.