Simultaneous aerobic nitrogen and phosphorus removal by novel halotolerant fungus Mucor circinelloides SNDM1: Function and metabolism pathway.

Fungi capable of simultaneous nitrogen and phosphorus removal from wastewater is rarely found. Here, a novel fungal strain (SNDM1) performing heterotrophic nitrification, aerobic denitrification, and phosphate removal was isolated and identified as Mucor circinelloides. The favorable nutrient removal conditions by the strain using glucose were C/N ratios of 25-30, salinities of 0 %-3 %, and pH of 7.5. Strain SNDM1 achieved ammonium, nitrite, nitrate, and phosphate removal rates of 5.23, 10.08, 4.88, and 0.97 mg/L/h. Nitrogen balance indicated that gaseous (18.60 %-24.55 %) and intracellular nitrogen (43.76 %-70.63 %) were primary fate of initial nitrogen. Enzyme activity revealed that ammonium removal occurred through heterotrophic nitrification and aerobic denitrification. Removed phosphorus was mainly transformed into cell membranes (56 %-64 %) and extracellular polymeric substances (20 %-26 %). Orthophosphate was the major intracellular phosphorus species, while polyphosphate and pyrophosphate existed extracellularly. These findings highlight the potential of this fungal strain for bioremediating polluted wastewater.

Effect of carbon source on carbon and nitrogen metabolism of common heterotrophic nitrification-aerobic denitrification pathway.

Pseudomonas sp. ZHL02, removing nitrogen via ammonia nitrogen (NH4+) → hydroxylamine (HN2OH) → nitrite (NO2-) → nitrate (NO3-) → NO2- → nitric oxide (NO) → nitrous oxide (N2O) pathway was employed for getting in-depth information on the heterotrophic nitrification-aerobic denitrification (HNAD) pathway from carbon oxidation, nitrogen conversion, electron transport process, enzyme activity, as well as gene expression while sodium succinate, sodium citrate, and sodium acetate were utilized as the carbon sources. The nitrogen balance analysis results demonstrated that ZHL02 mainly removed NH4+-N through assimilation. The carbon source metabolism resulted in the discrepancies in electron transport chain and nitrogen removal between different HNAD bacteria. Moreover, the prokaryotic strand-specific transcriptome method showed that, amo and hao were absent in ZHL02, and unknown genes may be involved in ZHL02 during the HNAD process. As a fascinating process for removing nitrogen, the HNAD process is still puzzling, and the relationship between carbon metabolism and nitrogen metabolism among different HNAD pathways should be studied further.

Enhancing nitrogen removal in constructed wetlands: The role of influent substrate concentrations in integrated vertical-flow systems.

Recent advancements in constructed wetlands (CWs) have highlighted the imperative of enhancing nitrogen (N) removal efficiency. However, the variability in influent substrate concentrations presents a challenge in optimizing N removal strategies due to its impact on removal efficiency and mechanisms. Here we show the interplay between influent substrate concentration and N removal processes within integrated vertical-flow constructed wetlands (IVFCWs), using wastewaters enriched with NO3--N and NH4+-N at varying carbon to nitrogen (C/N) ratios (1, 3, and 6). In the NO3--N enriched systems, a positive correlation was observed between the C/N ratio and total nitrogen (TN) removal efficiency, which markedly increased from 13.46 ± 2.23% to 87.00 ± 2.37% as the C/N ratio escalated from 1 to 6. Conversely, in NH4+-N enriched systems, TN removal efficiencies in the A-6 setup (33.69 ± 4.83%) were marginally 1.25 to 1.29 times higher than those in A-3 and A-1 systems, attributed to constraints in dissolved oxygen (DO) levels and alkalinity. Microbial community analysis and metabolic pathway assessment revealed that anaerobic denitrification, microbial N assimilation, and dissimilatory nitrate reduction to ammonium (DNRA) predominated in NO3--N systems with higher C/N ratios (C/N ≥ 3). In contrast, aerobic denitrification and microbial N assimilation were the primary pathways in NH4+-N systems and low C/N NO3--N systems. A mass balance approach indicated denitrification and microbial N assimilation contributed 4.12-47.12% and 8.51-38.96% in NO3--N systems, respectively, and 0.55-17.35% and 7.83-33.55% in NH4+-N systems to TN removal. To enhance N removal, strategies for NO3--N dominated systems should address carbon source limitations and electron competition between denitrification and DNRA processes, while NH4+-N dominated systems require optimization of carbon utilization pathways, and ensuring adequate DO and alkalinity supply.

Comparison of moving bed biofilm reactor and bio-contact oxidation reactor start-up with heterotrophic nitrification-aerobic denitrification bacteria and activated sludge inoculation under high ammonia nitrogen conditions.

To overcome poor ammonia tolerance and removal performance of bio-contact oxidation (BCO) reactor inoculated with activated sludge for high-ammonia nitrogen (NH4+-N) chemical wastewater treatment, this study compared inoculating heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria in moving bed biofilm reactor (MBBR) with activated sludge inoculation in BCO reactor under simulated high NH4+-N conditions. Results revealed that MBBR achieved faster biofilm formation (20 days vs. 100 days for BCO) with notable advantages: 27.6 % higher total nitrogen (TN) and 29.9 % higher NH4+-N removal efficiency than BCO. Microbial analysis indicated optimal enrichment of the key nitrogen removal (NR) bacterium Alcaligenes, leading to increased expression of NR enzymes hydroxylamine reductase, ensuring the superior NR efficiency of the MBBR. Additionally, functional enzymes and genes analysis speculated that the NR pathway in MBBR was: NH4+-N â†’ NH2OH â†’ NO3--N â†’ NO2--N â†’ NO â†’ N2O â†’ N2. This research offers a practical and theoretical foundation for extending HN-AD bacteria-inoculated MBBR processes.

Nitrogen removal characteristics of heterotrophic nitrification-aerobic denitrification bacterium Acinetobacter ZQ-A1 and community characteristics analysis of its application in pig farm wastewater.

A heterotrophic nitrifying aerobic denitrifying (HN-AD) strain ZQ-A1 with excellent denitrification performance, identified as Acinetobacter, was isolated from simultaneous nitrification and denitrification (SND) craft. ZQ-A1 was capable of removing NH4+, NO2-, and NO3-; the 21-hour removal rates were 84.84%, 87.13%, and 92.63%. ZQ-A1 has the ability to treat mixed nitrogen sources. In addition, ZQ-A1 can be well applied to actual sewage. According to the analysis of microbial community characteristics, the relative abundance of Acinetobacter in the experimental group increased from 0.06% to 2.38%, which is an important reason for the removal rate of NH4+ exceeding 99% within 30 days. The results of KEGG function prediction showed that with the addition of ZQ-A1, the relative abundance of pathways related to bacterial metabolism, such as tricarboxylic acid cycle metabolism, was higher. The research expanded the thinking of HN-AD bacteria in actual production and laid a foundation for its application in sewage treatment.

Systematic investigation of simultaneous copper biosorption and nitrogen removal from wastewater by an aerobic denitrifying bacterium of auto-aggregation.

Finding effective methods for simultaneous removal of eutrophic nutrients and heavy metals has attracted increasing concerns for the environmental remediation. Herein, a novel auto-aggregating aerobic denitrifying strain (Aeromonas veronii YL-41) was isolated with capacities for copper tolerance and biosorption. The denitrification efficiency and nitrogen removal pathway of the strain were investigated by nitrogen balance analysis and amplification of key denitrification functional genes. Moreover, the changes in the auto-aggregation properties of the strain caused by extracellular polymeric substances (EPS) production were focused on. The biosorption capacity and mechanisms of copper tolerance during denitrification were further explored by measuring changes in copper tolerance and adsorption indices, as well as by variations in extracellular functional groups. The strain showed extremely strong total nitrogen removal ability, with 67.5%, 82.08% and 78.48% of total nitrogen removal when NH4+-N, NO2--N, and NO3--N were used as the only initial nitrogen source, respectively. The successful amplification of napA, nirK, norR, and nosZ genes further demonstrated that the strain accomplished nitrate removal through a complete aerobic denitrification pathway. The production of protein-rich EPS of up to 23.31 mg/g and an auto-aggregation index of up to 76.42% may confer a strong biofilm-forming potential to the strain. Under the stress of 20 mg/L copper ions, the removal of nitrate-nitrogen was still as high as 71.4%. In addition, the strain could achieve an efficient removal of 96.9% of copper ions at an initial concentration of 80 mg/L. Scanning electron microscopy and deconvolution analysis of characteristic peaks confirmed that the strains encapsulate heavy metals by secreting EPS and, meanwhile, form strong hydrogen bonding structures to enhance intermolecular forces to resist copper ion stress. This study provides an innovative and effective biological approach for the synergistic bioaugmentation removal of eutrophic substances and heavy metals from aquatic environments.

Dual-edged effects and mechanisms of hydroxylamine in partial denitrification-anaerobic ammonium oxidation system.

The combination of partial denitrification (PD) and anaerobic ammonium oxidation (anammox) is a novel and promising nitrogen removal process. Regulating the synergistic reaction between denitrifiers and anammox bacteria (AnAOB) is the key to achieving stable and efficient PD-anammox performance. In this study, 10 mg/L of hydroxylamine (NH2OH) was considered to efficiently promote the bacterial activity, microbial energy flow, and the synergy of functional microflora. As a result, the nitrogen removal rate (NRR) significantly increased from 0.05 to 0.30 g N/L/d in parallel with an increase in the nitrogen loading rate (NLR) from 0.10 to 0.40 g N/L/d. However, the dual-edged effect of NH2OH was also confirmed. The long-term presence of NH2OH caused overgrowth of complete-denitrifying bacteria and decreased the NRR to 0.11 g N/L/d. Additionally, NH2OH enhanced nitrous oxide (N2O) emissions via chemical pathways as well as enhanced denitrification Fortunately, the inhibition caused by NH2OH was reversible by stopping the dosing, the reactor restored to stable operation with an NRR of 0.27 g N/L/d. Analysis of metabolic intensity and pathways revealed the effecting process and mechanism of NH2OH on the PD-anammox system. This study verified the dual-edged effects and mechanisms of NH2OH, therefore proving a theoretical basis and technical reference for the application of PD-anammox.

Efficient nitrogen removal of a novel Pseudomonas chengduensis strain BF6 mainly through assimilation in the recirculating aquaculture systems.

Biological nitrogen removal has received increasing attention in wastewater treatment. A bacterium with excellent nitrogen removal performance was isolated from biofilters of recirculating aquaculture systems (RAS) and identified as Pseudomonas chengduensis BF6. It was indicated that inorganic nitrogen is transformed into gaseous and biological nitrogen by the metabolic pathways of denitrification, anammox, and assimilation, which is the main nitrogen removal pathway of strain BF6. The strain BF6 could effectively remove nitrogen within 24 h under the conditions of ammonia, nitrate, nitrite, and mixed nitrogen sources with maximum total nitrogen removal efficiencies reaching 97.00 %, 61.40 %, 79.10 %, and 84.98 %, respectively. The strain BF6 exhibited total nitrogen removal efficiency of 91.14 %, altered the microbial diversity and enhanced the relative abundance of Pseudomonas in the RAS biofilter. These findings demonstrate that Pseudomonas sp. BF6 is a highly efficient nitrogen-removing bacterium with great potential for application in aquaculture wastewater remediation.

Insight into the roles of microalgae on simultaneous nitrification and denitrification in microalgal-bacterial sequencing batch reactors: Nitrogen removal, extracellular polymeric substances, and microbial communities.

This study explored the influence and mechanism of microalgae on simultaneous nitrification and denitrification (SND) in microalgal-bacterial sequencing batch reactors (MB-SBR). It particularly focused on nitrogen transformation in extracellular polymeric substances (EPS) and functional groups associated with nitrogen removal. The results showed that MB-SBR achieved more optimal performance than control, with an SND efficiency of 68.01% and total nitrogen removal efficiency of 66.74%. Further analyses revealed that microalgae changed compositions and properties of EPS by increasing EPS contents and improving transfer, conversion, and storage capacity of nitrogen in EPS. Microbial community analysis demonstrated that microalgae promoted the enrichment of functional groups and genes related to SND and introduced diverse nitrogen removal pathways. Moreover, co-occurrence network analysis elucidated the interactions between communities of bacteria and microalgae and the promotion of SND by microalgae as keystone connectors in the MB-SBR. This study provides insights into the roles of microalgae for enhanced SND.

Genomics and nitrogen metabolic characteristics of a novel heterotrophic nitrifying-aerobic denitrifying bacterium Acinetobacter oleivorans AHP123.

A novel aerobic strain of Acinetobacter oleivorans AHP123 was isolated from activated sludge, which could conduct heterotrophic nitrification and denitrification simultaneously. This strain has excellent NH4+-N removal ability, with 97.93% removal rate at 24-hour. To identify the metabolic pathways of this novel strain, genes of gam, glnA, gdhA, gltB, nirB, nasA, nar, nor, glnK and amt were detected by genome analysis. Through RT-qPCR, it was found that the expression of key genes confirmed two possible ways of nitrogen removal in strain AHP123: nitrogen assimilation and heterotrophic nitrification aerobic denitrification (HNAD). However, the absence of some common HNAD genes (amo, nap and nos) suggested that strain AHP123 might have a different HNAD pathway from other HNAD bacteria. Nitrogen balance analysis revealed that strain AHP123 assimilated most of the external nitrogen sources into intracellular nitrogen.

Overlooked pathways of endogenous simultaneous nitrification and denitrification in anaerobic/aerobic/anoxic sequencing batch reactors with organic supplementation.

The anaerobic/aerobic/anoxic (A/O/A) process is a promising biotechnology to intensify denitrification in low carbon/nitrogen (C/N) wastewater treatment, but the neglected typical rate-limiting step-nitrification-would hinder its wider application. Heterotrophic nitrification driven by intracellular carbon (PHAs) could enhance nitrification and achieve endogenous simultaneous nitrification and denitrification (ESND) in the A/O/A process, but its feasibility remains unexamined. Here we established four A/O/A-SBRs at different C/N ratios (3, 7.5, 12, and 16.5) to address the above-mentioned knowledge gaps. The results showed that organic supplementation promoted both nitrification and denitrification (performance and relevant enzymatic activities) until organic overdose (C/N = 16.5) exacerbated niche competitions from other non-functional heterotrophs. qPCR and batch tests indicated that high C/N ratios inhibited autotrophic nitrifiers, and heterotrophic nitrifiers (HNB) dominated in the enhanced nitrification. Given the high HNB contribution (43.7%) and low COD variation (< 10 mg L-1) in the SND (76.4%) of CN12, we proposed a potential SND pathway based on heterotrophic nitrification and denitrification driven by PHAs and verified it with batch tests. Microbial and functional analyses suggested that CN12 favored the intracellular carbon transformation and harbored the minimum autotrophic nitrifiers, supporting the dominance of ESND in the enhanced SND. Our findings expand the understanding of the relationships between intracellular carbon transformation and SND and provide a novel nitrogen removal pathway for the practical application of the A/O/A process.

Isolation and characteristics of two heterotrophic nitrifying and aerobic denitrifying bacteria, Achromobacter sp. strain HNDS-1 and Enterobacter sp. strain HNDS-6.

In order to solve nitrogen pollution in environmental water, two heterotrophic nitrifying and aerobic denitrifying strains isolated from acid paddy soil were identified as Achromobacter sp. strain HNDS-1 and Enterobacter sp. strain HNDS-6 respectively. Strain HNDS-1 and strain HNDS-6 exhibited amazing ability to nitrogen removal. When (NH4)2SO4, KNO3, NaNO2 were used as nitrogen resource respectively, the NH4+-N, NO3--N, NO2--N removal efficiencies of strain HNDS-1 were 93.31%, 89.47%, and 100% respectively, while those of strain HNDS-6 were 82.39%, 96.92%, and 100%. And both of them could remove mixed nitrogen effectively in low C/N (C/N = 5). Strain HNDS-1 could remove 76.86% NH4+-N and 75.13% NO3--N. And strain HNDS-6 can remove 65.07% NH4+-N and 78.21% NO3--N. A putative ammonia monooxygenase, nitrite reductase, nitrate reductase, assimilatory nitrate reductase, nitrate/nitrite transport protein and nitric oxide reductase of strain HNDS-1, while hydroxylamine reductase, nitrite reductase, nitrate reductase, assimilatory nitrate reductase, nitrate/nitrite transport protein, and nitric oxide reductase of strain HNDS-6 were identified by genomic analysis. DNA-SIP analysis showed that genes Nxr, narG, nirK, norB, nosZ were involved in nitrogen removal pathway, which indicates that the denitrification pathway of strain HNDS-1 and strain HNDS-6 was NO3-→NO2-→NO→N2O→N2 during NH4+-N removal process. And the nitrification pathway of strain HNDS-1 and strain HNDS-6 was NO2-→NO3-, but the nitrification pathway of NH4+→ NO2- needs further studies.

A novel Pseudomonas aeruginosa strain performs simultaneous heterotrophic nitrification-aerobic denitrification and aerobic phosphate removal.

Nitrogen and phosphate removal from wastewater relies on different functional bacteria. In this study, a novel strain affiliated with Pseudomonas aeruginosa was isolated from activated sludge by gradient dilution and performed heterotrophic nitrification-aerobic denitrification and aerobic phosphate removal (HNADPR). The strain showed an ammonium removal efficiency of 87% and a phosphate removal efficiency of 97% under optimal conditions, such as C/N ratio of 10, P/N ratio of 0.1, temperature of 30°C, and pH of 7.5-8.5. The modified Gompertz model could fit well the heterotrophic ammonium nitrification, aerobic nitrite/nitrate denitrification, and aerobic phosphate removal processes. Functional gene amplification indicated that ammonium removal followed the complete HN-AD pathway (NH4+ â†’ NH2OH â†’ NO2- â†’ NO3- â†’ NO2- â†’ NO â†’ N2O â†’ N2). Phosphate removal only occurred under aerobic conditions and ceased under anaerobic conditions. In successive aerobic cycles, the strain persistently took up phosphate. In wastewater, phosphate was aerobically converted into cell membrane, intracellular and extracellular polymeric substrates (EPS). Phosphorus in the form of phosphate monoester was pooled in EPS. A hypothetic aerobic phosphate removal model for strain SNDPR-01 is proposed to improve our understanding of the novel bacterial function of HNADPR.

Nitrogen removal by a novel heterotrophic nitrification and aerobic denitrification bacterium Acinetobacter calcoaceticus TY1 under low temperatures.

A new bacterial strain, Acinetobacter calcoaceticus TY1, was identified in activated sludge. This strain efficiently metabolized nitrogen from ammonium at low temperatures, utilizing NH4+-N, NO3--N, and NO2--N as nitrogen sources. Of these, NH4+-N was superior in terms of both assimilation and heterotrophic nitrification at 8 °C. The nitrogen metabolism-associated genes amoA, nirK, and nosZ were identified in TY1. Optimal requirements for growth and nitrogen removal were pH 7, shaking speed of 90 rpm, a C/N ratio of 10, and sodium citrate for the carbon supply. The ability to denitrify at low temperature suggests TY1's potential for wastewater management.

Microbial composition and nitrogen removal pathways in a novel sequencing batch reactor integrated with semi-fixed biofilm carrier: evidence from a pilot study for low- and high-strength sewage treatment.

The sequencing batch reactor (SBR) activated sludge process is a well-established technology for sewage treatment. One of the drawbacks of SBRs, however, total nitrogen (TN) removals is insufficient. By means of introducing four improvements, including semi-fixed biofilm carrier, sludge elevation mixing and change for the mode of influent and effluent, compliant standard for TN discharge was obtained in this novel SBR configuration during low- and high-strength sewage load. To illustrate the microbial compositions and functions of the attached biofilm on semi-fixed carrier and the suspended aggregates, as well as the nitrogen removal pathway, high throughput 16S rRNA gene amplicon sequencing, PICRUSt2 algorithm, and KEGG database were applied. The results revealed that (i) the microbial communities from suspended aggregates and biofilm samples were significantly different from each other; (ii) during low-strength sewage loads, TN removal was mainly by nitrification-denitrification. The suspended aggregates was responsible for denitrification, while the biofilm was focused on ammonium oxidation; (iii) during high-strength sewage loads, function of nitrate reductase from suspended aggregates was faded, and anammox and N assimilation by biofilm became dominant. Meanwhile, TN removal referring to the formation of L-glutamine via assimilation was the main pathway.

Heterotrophic nitrification and aerobic denitrification by a novel Acinetobacter sp. TAC-1 at low temperature and high ammonia nitrogen.

A novel strain was isolated from swinewastewater and identified as Acinetobacter sp. TAC-1 based on its phylogenetic and phenotypic characteristics. The strain TAC-1 was found to have a high ability to metabolize ammonium-N under low temperature condition. The strain TAC-1 could remove approximately 94.6% of ammonium-N (400 mg/L), 93.3% of nitrate-N (400 mg/L) and 42.4% of nitrite-N (400 mg/L) at 5 °C. The functional genes nitrate reductase gene (narG) and nitrite reductase gene (nirK, nirS) were successfully amplified by qPCR, further evidencing the heterotrophic nitrification and aerobic denitrification capability of Acinetobacter sp. TAC-1. The transcriptome data confirmed that the membrane transport protein and unsaturated fatty acid dehydrogenase-related genes of the strain TAC-1 were significantly up-regulated at 5 °C, enabling it to survive low temperatures. The high nitrogen removal ability at 5 °C makes this strain have a good application prospect.

Pathways regulating nitrogen removal in constructed ditch wetlands: effects of different inflow ratios and artificial aeration.

Constructed ditch wetland (CDW) is a combination of idle ditch and constructed wetland, which is typically used in rural areas to remove pollutants from domestic wastewater. However, its low total nitrogen (TN) removal remains a pressing issue. To enhance total nitrogen removal, an approach of supplying water at two locations in the CDW at different influent flow ratios, combined with artificial aeration, was proposed to adjust carbon and oxygen distribution in the system. The highest average TN removal was achieved at low influent concentration (CDW4; influent flow ratio 1:2). The removal of TN in winter and spring were 58.93 and 83.26%, respectively. The distribution of carbon sources in the back zone enhanced denitrification. Of the high influent concentration treatments, CDW2 (2:1) achieved 16.97% more TN removal on average compared with CDW1 (3:0), after extra artificial aeration was applied in the front zone. However, nitrification was a limiting step in the system, which became the primary problem preventing pollutant purification. Moreover, nitrifying bacteria abundance was negatively correlated to the influent flow ratio and autotrophic denitrifying bacterial abundance was positively correlated to the influent flow ratios. Graphical abstract.

[Construction and Denitrification Performance of A2/O Based on Partial Nitrification Coupled with an ANAMMOX System].

To resolve the issue of existing municipal wastewater treatment plants (WWTPs) in China with an insufficient influent carbon source, a bench-scale A2/O process based on partial nitrification coupled with ANAMMOX was constructed by controlling aeration partition ratio, dissolved oxygen (DO) concentration, and sludge retention time (SRT). In this study, the nitrogen removal performance, nitrogen removal pathway, and microbial community structure of the system under different conditions were investigated. The results showed that the system had excellent nitrogen removal efficiency at low-C/N influent (C/N=5). The A2/O reactor had experienced the co-culture stage (Phase 1), screening stage (Phase 2-3), and enrichment stage (Phase 4) successively during the 140-day experiment, and the nitrogen removal pathway changed from nitrification and denitrification to partial nitrification coupled ANAMMOX in the end. The optimal removal efficiencies of 97.69% for NH4+-N and 87.83% for TN were obtained in the enrichment stage (Phase 4), and the effluent concentration of NH4+-N and TN were 1.20 mg·L-1 and 7.03 mg·L-1, respectively. Illumina MiSeq sequencing results showed that the enrichment of AOB including Nitrosomonas and Nitrosospira and the elimination of NOB including Nitrospira, Nitrococcus, and Nitrobacter were the main causes of achieving partial nitrification in the system. The enrichment of AnAOB including Candidatus Kuenenia and Candidatus Jettenia was the key point for the occurrence of ANAMMOX in the system, and thus, played an important role in the achievement of advanced nitrogen removal.

Effects of COD/TN ratio on nitrogen removal efficiency, microbial community for high saline wastewater treatment based on heterotrophic nitrification-aerobic denitrification process.

High salinity and high organic concentration impose negative impacts on autotrophic nitrification, which hinders efficient nitrogen removal. To achieve efficient nitrogen removal, high saline wastewater nitrogen removal systems with different COD/TN ratios based on heterotrophic nitrification-aerobic denitrification (HN-AD) process were established in this study. Results demonstrated that the COD/TN ratio had significant effects on nitrogen removal efficiency, microbial community structures and metabolic pathways. The optimal COD/TN ratio was 25 for nitrogen removal, with NH4+-N and TN removal rates of 11.86 mg·L-1·h-1 and 11.50 mg·L-1·h-1, respectively (3.65 and 3.31 times higher than those COD/TN ratio of 10). 16S rRNA high-throughput sequencing revealed that HN-AD functional bacteria (Pseudomonas, Bacillus, Paracoccus) with highest abundance (7.61%) played a key role in high saline wastewater treatment. And Halomonas, Nitrincola and Oceanimonas participated in the denitrification process. Moreover, the abundance of genes related to nitrogen removal was the highest (1.90%) revealed by functional genes prediction.

Overlooked pathways of denitrification in a sulfur-based denitrification system with organic supplementation.

Elemental sulfur-driven autotrophic denitrification (SADN) is a cost-effective approach for treating secondary effluent from wastewater treatment plants (WWTPs). Additional organics are generally supplemented to promote total nitrogen (TN) removal, reduce nitrite accumulation and sulfate production, and balance the pH decrease induced by SADN. However, understanding of the impacts of organic supplementation on microbial communities, nitrogen metabolism, denitrifier activity, and SADN rates in sulfur-based denitrification reactors is still limited. Here, a sulfur-based denitrification reactor was continuously operated for 272 days during which six different C/N ratios were tested successively (2.7, 1.5, 0.7, 0.5, 0.25, and 0). Organic supplementation improved TN removal and decreased NO2- accumulation, but reduced the relative abundance of denitrifiers and the contribution of autotrophic nitrate-reducing bacteria (aNRB) to TN removal during the long-term operation of reactor. Predictive functional profiling showed that nitrogen metabolism potential increased with decreasing C/N ratios. SADN was the predominant removal process when the C/N ratio was ≤0.7 (achieving 60% contribution when C/N = 0.7). Although organic supplementation weakened the dominant role of aNRB in denitrification, batch tests for the first time demonstrated that it could accelerate the SADN rate, attributed to the improvement of sulfur bioavailability, likely via the formation of polysulfide. A possible nitrogen removal pathway with multiple electron donors (i.e., sulfur, organics, sulfide, and polysulfide) in a sulfur-based denitrification reactor with organic supplementation was therefore proposed. However, supplementation with a high level of organics could increase the operational cost and effluent concentrations of sulfide and organics as well as enrich heterotrophic denitrifiers. Moreover, microbial community had substantial changes at C/N ratios of >0.5. Accordingly, an optimal C/N ratio of 0.25-0.5 was suggested, which could simultaneously minimize the additional operating cost associated with organic supplementation and maximize TN removal and SADN rates.