Treatment of high ammonia anaerobically digested molasses wastewater using aerobic granular sludge reactor.

This study addressed the treatment of high ammonia, low biodegradable chemical oxygen demand (bCOD) anaerobically digested molasses wastewater, utilizing an aerobic granular sludge (AGS) reactor. The AGS achieved 99 % ammonia removal regardless of the bCOD supplementation. By adding low ammonia (<60 mg/L), high bCOD raw molasses wastewater (before anaerobic digestion) as a carbon source, enhanced nitrogen removal, increasing from 10 % to 97 %, and improved sludge settleability via bio-induced calcite precipitation were observed. Functional genes prediction suggested two potential denitrification pathways, including heterotrophic denitrification by Paracoccus and Thauera, and autotrophic denitrification, specifically sulfide-oxidizing autotrophic denitrification by Thiobacillus. An increase in the relative abundance of microorganisms involved in heterotrophic denitrification was observed with the addition of high bCOD raw molasses wastewater. Consequently, incorporating raw molasses wastewater into the AGS presents a sustainable approach to achieve mixotrophic denitrification, maintain stable granular sludge and ensure stable treatment performance when treating anaerobically digested molasses wastewater.

Effect of pyrite particle size on the denitrification performance of autotrophic or split-mixotrophic bioreactors supported by pyrite/polycaprolactone.

In this study, a pyrite-based autotrophic denitrification (PAD) system, a polycaprolactone (PCL)-supported heterotrophic denitrification (PHD) system, and a pyrite+PCL-based split-mixotrophic denitrification (PPMD) system were constructed. The pyrite particle size was controlled in 1-3, 3-5, or 5-8 mm in both the PAD and PPMD systems to investigate the effect of pyrite particle size on the denitrification performance of autotrophic or split-mixotrophic bioreactors. It was found that the PAD system achieved the best denitrification efficiency with an average removal rate of 98.98% in the treatment of 1- to 3-mm particle size, whereas it was only 19.24% in the treatment of 5- to 8-mm particle size. At different phases of the whole experiment, the nitrate removal rates of both the PHD and PPMD systems remained stable at a high level (>94%). Compared with the PAD or PHD system, the PPMD system reduced the concentrations of sulfate and chemical oxygen demand in the final effluent efficiently. The interconnection network diagram explained the intrinsic metabolic pathways of nitrogen, sulfur, and carbon in the three denitrification systems at different phases. In addition, the microbial community analysis showed that the PPMD system was beneficial for the enrichment of Firmicutes. Finally, the impact mechanism of pyrite particle size on the performance of the PPMD system was proposed. PRACTITIONER POINTS: The reduction of pyrite particle size was beneficial for improving the efficiency of the PAD process. The change in particle size had an effect on NO2 --N accumulation in the PAD system. The accumulation of NH4 +-N in the PPMD system increased with the decrease in particle size. The reduction of pyrite particle size increased the production of SO4 2- in the PAD and PPMD systems. The correlations among the effluent indicators of the PAD and PPMD systems could be well explained.

Significantly Enhanced Nitrate and Phosphorus Removal by Pyrite/Sawdust Composite-Driven Mixotrophic Denitrification with Boosted Electron Transfer: Comprehensive Evaluation of Water-Gas-Biofilm Phases during a Long-Term Study.

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.

Enhancing nitrogen removal in the partial denitrification/anammox processes for SO4- - Rich wastewater treatment: Insights into autotrophic and mixotrophic strategies.

The investigation of partial denitrification/anammox (PD/anammox) processes was conducted under autotrophic (N-S cycle) and mixotrophic (N-S-C cycle) conditions over 180 days. Key findings revealed the remarkable capability of SO42--dependent systems to produce NO2- effectively, supporting anaerobic NH4+ oxidation. Additionally, SO42- served as an additional electron acceptor in sulfate reduction ammonium oxidation (SRAO). Increasing influent SO42- concentrations notably improved ammonia utilization rates (AUR) and NH4+ and total nitrogen (TN) utilization efficiencies, peaking at 57% for SBR1 and nearly 100% for SBR2. Stoichiometric analysis showed a 7.5-fold increase in AUR (SRAO and anammox) in SBR1 following SO42- supplementation. However, the analysis for SBR2 indicated a shift towards SRAO and mixotrophic denitrification, with anammox disappearing entirely by the end of the study. Comparative assessments between SBR1 and SBR2 emphasized the impact of organic compounds (CH3COONa) on transformations within the N-S-C cycle. SBR1 performance primarily involved anammox, SRAO and other SO42- utilization pathways, with minimal S-dependent autotrophic denitrification (SDAD) involvement. In contrast, SBR2 performance encompassed SRAO, mixotrophic denitrification, and other pathways for SO42- production. The SRAO process involved two dominant genera, such as Candidatus Brocadia and PHOS-HE36.

The role of the combined nitrogen-sulfur-carbon cycles for efficient performance of anammox-based systems.

The combined anammox/mixotrophic denitrification process was conducted in two granular sequencing batch reactors (SBRs) during a 200-day operation. Both reactors were fed with synthetic medium, but SBR2 was enriched with additional sulfate (SO42-) which influenced sulfate reduction ammonium oxidation (SRAO) and heterotrophic reduction of SO42- by sulfate reducing bacteria. It was hypothesized that the addition of SO42- could positively impact the removal rates of N-S-C compounds. A low C/N ratio (0.4-1.6) was maintained to prevent inhibition of anaerobic ammonium oxidizing bacteria (AnAOB), and alternating chemical oxygen demand (COD) on/off conditions were used to regenerate AnAOB during COD-off phases and heterotrophic denitrifiers during COD-on phases. Stoichiometric analysis showed that introducing SO42- in SBR2 enhanced the ammonium utilization rate, which was approximately 10 % higher compared to SBR1 in the final stage of the experiment (25.8 vs. 22.8 mg N/(g VSS·h)). The total nitrogen removal efficiencies ranged from 62 % to 99 % in both reactors, with SBR2 consistently exhibiting approximately 4 % higher efficiency than SBR1. In SBR2, the maximum overall SO42- utilization efficiency reached 27 % under COD-off conditions, while overall COD utilization was almost complete under COD-on conditions. A strong correlation (R2 = 0.98) was observed between SO42- production and COD utilization. The key players responsible for N and S transformations in response to SO42- addition were Candidatus Brocadia and Chloroflexi - Anaerolineae. This study highlights the potential to enhance the overall efficiency of N-S-C removal by implementing an integrated anammox/mixotrophic denitrification process. The combination of cycles emerges as a sustainable approach for treating wastewater rich in N-S-C compounds.

Deciphering the influencing mechanism of hydraulic retention time on purification performance of a mixotrophic system from the perspective of reaction kinetics.

At present, eutrophication is increasingly serious, so it is necessary to effectively reduce nitrogen and phosphorus in water bodies. In this study, a pyrite/polycaprolactone-based mixotrophic denitrification (PPMD) system using pyrite and polycaprolactone (PCL) as electron donors was developed and compared with pyrite-based autotrophic denitrification (PAD) system and PCL-based heterotrophic denitrification (PHD) system through continuous flow experiment. The removal efficiency of NO3--N (NRE) and PO43--P (PRE) and the contribution proportion of PAD in the PPMD system were significantly increased by prolonging hydraulic retention time (HRT, from 1 to 48 h). When HRT was equal to 24 h, the PPMD system conformed to the zero-order kinetic model, so NRE and PRE were mainly limited by the PAD process. When HRT was equal to 48 h, the PPMD system met the first-order kinetic model with NRE and PRE reaching 98.9 ± 1.1% and 91.8 ± 4.5%, respectively. When HRT = 48 h, the NRE and PRE by PAD system were 82.7 ± 9.1% and 88.5 ± 4.7%, respectively, but the effluent SO42- concentration was as high as 152.1 ± 13.7 mg/L (the influent SO42- concentration was 49.2 ± 3.3 mg/L); the NRE by PHD system was 98.5 ± 1.7%, but the PO43--P could not be removed ideally. The concentrations of NO3--N, total nitrogen, PO43--P, and SO42- in the PPMD system also showed distinct changes along the reactor column. In addition, the microbial diversity analysis showed that prolonging HRT (from 24 to 48 h) increased the abundance of autotrophic denitrifying microorganisms in the PPMD system, ultimately increasing the contribution proportion of PAD.

Uncovering interactions among ternary electron donors of organic carbon source, thiosulfate and Fe0 in mixotrophic advanced denitrification: Proof of concept from simulated to authentic secondary effluent.

To offset the imperfections of higher cost and emission of CO2 greenhouse gas in heterotrophic denitrification (HDN) as well as longer start-up time in autotrophic denitrification (ADN), we synergized the potential ternary electron donors of organic carbon source, thiosulfate and zero-valent iron (Fe0) to achieve efficient mixotrophic denitrification (MDN) of oligotrophic secondary effluent. When the influent chemical oxygen demand to nitrogen (COD/N) ratio ascended gradually in the batch operation with sufficient sulfur to nitrogen (S/N) ratio, the MDN with thiosulfate and Fe0 added achieved the highest TN removal for treating simulated and authentic secondary effluents. The external carbon is imperative for initiating MDN, while thiosulfate is indispensable for promoting TN removal efficiency. Although Fe0 hardly donated electrons for denitrification, the suitable circumneutral environment for denitrification was implemented by OH- released from Fe0 corrosion, which neutralized H+generated during thiosulfate-driven ADN. Meanwhile, Fe0 corrosion consumed the dissolved oxygen (DO) and created the low DO environment suitable for anoxic denitrification. This process was further confirmed by the continuous flow operation for treating authentic secondary effluent. The TN removal efficiency achieved its maximum under the combination condition of influent COD/N ratio of 3.1-3.5 and S/N ratio of 2.0-2.1. Whether in batch or continuous flow operation, the coordination of thiosulfate and Fe0 maintained the dominance of Thiobacillus for ADN, with the dominant heterotrophic denitrifiers (e.g., Plasticicumulans, Terrimonas, Rhodanobacter and KD4-96) coexisting in MDN system. The interaction insights of ternary electron donors in MDN established a pathway for realizing high-efficiency nitrogen removal of secondary effluent.

Ultra-stable mixotrophic denitrification coupled with anammox under organic stress for mainstream municipal wastewater treatment.

Sulfur-based autotrophic denitrification (SAD) coupled with anammox is a promising process for autotrophic nitrogen removal in view of the stable nitrite accumulation during SAD. In this study, a mixotrophic nitrogen removal system integrating SAD, anammox and heterotrophic denitrification was established in a single-stage reactor. The long-term nitrogen removal performance was investigated under the intervention of organic carbon sources in real municipal wastewater. With the shortening of hydraulic retention time, the nitrogen removal rate of the mixotrophic system dominated by the autotrophic subsystem reached 0.46 Kg N/m³/d at an organic loading rate of 0.57 Kg COD/m³/d, with COD and total nitrogen removal efficiencies of 82.5 % and 94 %, respectively, realizing an ideal combination of autotrophic and heterotrophic systems. The 15NO3--N isotope labeling experiments indicated that thiosulfate-driven autotrophic denitrification was the main pathway for nitrite supply accounting for 80.6 %, while anammox exhibited strong competitiveness for nitrite under the dual electron supply of sulfur and organic carbon sources and contributed to 65.1 % of nitrogen removal. Sludge granulation created differential functional distributions in different forms of sludge, with SAD showing faster reaction rate as well as higher nitrite accumulation rate in floc sludge, while anammox was more active in granular sludge. Real-time quantitative PCR, RT-PCR and high-throughput sequencing results revealed a dynamically changing community composition at the gene and transcription levels. The decrease in heterotrophic denitrification bacteria abundance indicated the effectiveness of the operational strategy for introduction of thiosulfate and maintaining the dominance of SAD in denitrification process in suppressing the excessive growth of heterotrophic bacteria in the mixotrophic system. The high transcriptional expression of sulfur-oxidizing bacteria (SOB) (Thiobacillus and Sulfurimonas) and anammox bacteria (Candaditus_Brocadia and Candidatus_Kuenenia) played a crucial role in the stable nitrogen removal.

Mixotrophic denitrification process driven by lime sulfur and butanediol: Denitrification performance and metagenomic analysis.

Heterotrophic sulfur-based autotrophic denitrification is a promising biological denitrification technology for low COD/TN (C/N) wastewater due to its high efficiency and low cost. Compared to the conventional autotrophic denitrification process driven by elemental sulfur, the presence of polysulfide in the system can promote high-speed nitrogen removal. However, autotrophic denitrification mediated by polysulfide has not been reported. This study investigated the denitrification performance and microbial metabolic mechanism of heterotrophic denitrification, sulfur-based autotrophic denitrification, and mixotrophic denitrification using lime sulfur and butanediol as electron donors. When the influent C/N was 1, the total nitrogen removal efficiency of the mixotrophic denitrification process was 1.67 and 1.14 times higher than that of the heterotrophic and sulfur-based autotrophic denitrification processes, respectively. Microbial community alpha diversity and principal component analysis indicated different electron donors lead to different evolutionary directions in microbial communities. Metagenomic analysis showed the enriched denitrifying bacteria (Thauera, Pseudomonas, and Pseudoxanthomonas), dissimilatory nitrate reduction to ammonia bacteria (Hydrogenophaga), and sulfur oxidizing bacteria (Thiobacillus) can stably support nitrate reduction. Analysis of metabolic pathways revealed that complete denitrification, dissimilatory nitrate reduction to ammonia, and sulfur disproportionation are the main pathways of the N and S cycle. This study demonstrates the feasibility of a mixotrophic denitrification process driven by a combination of lime sulfur and butanediol as a cost-effective solution for treating nitrogen pollution in low C/N wastewater and elucidates the N and S metabolic pathways involved.

A novel sulfur autotrophic denitrification in-situ coupled sequencing batch reactor system to treat low carbon to nitrogen ratio municipal wastewater: Performance, niche equilibrium and pollutant removal mechanisms.

A novel integrated sulfur fixed-film activated sludge in SBR system (IS0FAS-SBR) was proposed to treat the low C/N ratio municipal wastewater. The effluent total inorganic nitrogen (TIN) and PO43--P decreased from 17 mg/L and 3.5 mg/L to 8.5 mg/L and 0.5 mg/L, and higher nitrogen removal efficiency was contributed by the autotrophic denitrification. Microbial response characteristics showed that catalase (CAT), reduced nicotinamide adenine dinucleotide (NADH) and extracellular polymeric substance (EPS) alleviated the oxidative stress of sulfur carrier to maintain cell activity, while metabolic activity analysis indicated that the electron transfer rate was enhanced to improve mixotrophic denitrification efficiency. Meanwhile, the increased key enzyme activities further facilitated nitrogen removal and sulfur oxidation process. Additionally, the microbial community, functional proteins and genes revealed a niche equilibrium of C, N, S metabolic bacteria. Sulfur autotrophic in-situ coupled SBR system enlarged a promising strategy for treatment of low C/N ratio municipal wastewater.

The enrichment of more functional microbes induced by the increasing hydraulic retention time accounts for the increment of autotrophic denitrification performance.

With pyrite (FeS2) and polycaprolactone (PCL) as electron donors, three denitrification systems, namely FeS2-based autotrophic denitrification (PAD) system, PCL-supported heterotrophic denitrification (PHD) system and split-mixotrophic denitrification (PPMD) system, were constructed and operated under varying hydraulic retention times (HRT, 1-48 h). Compared with PAD or PHD, the PPMD system could achieve higher removals of NO3--N and PO43--P, and the effluent SO42- concentration was greatly reduced to 7.28 mg/L. Similarly, the abundance of the dominant genera involved in the PAD (Thiobacillus, Sulfurimonas, and Ferritrophicum, etc.) or PHD (Syntrophomonas, Desulfomicrobium, and Desulfovibrio, etc.) process all increased in the PPMD system. Gene prediction completed by PICRUSt2 showed that the abundance of the functional genes involved in denitrification and sulfur oxidation all increased with the increase of HRT. This also accounted for the increased contribution of autotrophic denitrification to total nitrogen removal in the PPMD system. In addition, the analysis of metabolic pathways disclosed the specific conversion mechanisms of nitrogen and sulfur inside the reactor.

Organic carbon source excites extracellular polymeric substances to boost Fe0-mediated autotrophic denitrification in mixotrophic system.

Fe0-mediated autotrophic denitrification (ADN) can be suppressed by iron oxide coverage resulting from Fe0 corrosion. The mixotrophic denitrification (MDN) coupling Fe0-mediated ADN with heterotrophic denitrification (HDN) can circumvent the weakening of Fe0-mediated ADN over operation time. But the interaction between HDN and Fe0-mediated ADN for nitrogen removal of secondary effluent with deficient bioavailable organics remains unclear. When the influent COD/NO3--N ratio increased from 0.0 to 1.8-2.1, the TN removal efficiency was promoted significantly. The increased carbon source did not inhibit ADN, but promoted ADN and HDN synchronously. The formation of extracellular polymeric substances (EPS) was also facilitated concomitantly. Protein (PN) and humic acid (HA) in EPS increased significantly, which capable of accelerating electron transfer of denitrification. Due to that the electron transfer of HDN occurs intracellularly, the EPS with the capacity of accelerating electron transfer had a negligible influence on HDN. But for Fe0-mediated ADN, the increased EPS as well as corresponding PN and HA facilitated TN and NO3--N removal significantly, while accelerated the electron release originating from Fe0 corrosion. The bioorganic-Fe complexes were generated on Fe0 surface after used, meaning that the soluble EPS and soluble microbial products (SMP) participated in the electron transfer of Fe0-mediated ADN. The coexistence of HDN and ADN denitrifiers demonstrated the synchronous enhancement of HDN and ADN by the external carbon source. From the perspective of EPS and related SMP, the insight of enhancing Fe0-mediated ADN by external carbon source is beneficial to implement high-efficiency MDN for organics-deficient secondary wastewater.

Microbial mixotrophic denitrification using iron(II) as an assisted electron donor.

Mixotrophic denitrification processes have a great potential in nitrogen removal in biological wastewater treatment processes. However, so far, few studies have focused on the mixotrophic denitrification system using Fe(II) as an exclusively assisted electron donors and the underlying mechanisms in such a process remain unclear. Furthermore, the mechanisms by which microorganisms cover carbon, nitrogen, phosphorus and iron in an iron-assisted mixotrophic system remain unrevealed. In this work, we explore the feasibility of using Fe(II) as an assisted electron donor for enhancing simultaneous nitrogen and phosphorus removal via long-term reactor operation and batch tests. The results show that Fe(II) could provide electrons for efficient nitrate reduction and that biological reactions played a predominant role in these systems. In these systems Thermomonas, a strain of nitrate-reduction Fe(II)-oxidation bacterium, was enriched and accounted for a maximum abundance of 60.2%. These findings indicate a great potential of the Fe(II)-assisted mixotrophic denitrification system for practical use as an efficient simultaneous nitrogen and phosphorus removal process.

Start-up phase optimization of pyrite-intensified hybrid sequencing batch biofilm reactor (PIHSBBR): Mixotrophic denitrification performance and mechanism.

Pyrite-based autotrophic denitrification (PAD) is an emerging biological process to diminish nitrate pollution, but the relatively low NO3--N removal rate limits its practical application. In this research, a pyrite-intensified hybrid sequencing batch biofilm reactor (PIHSBBR) was designed to treat low C/N ratio domestic wastewater. The results showed that PIHSBBR could achieve optimal removal of COD, NH4+-N, and TN under the aeration rate of 1.0 L/L∙min and the hydraulic retention time (HRT) of 8 h, with removal rates of 69.67 ± 4.37%, 77.04 ± 4.84%, and 63.92 ± 6.66%, respectively. The PAD efficiency in PIHSBBR during the stable operation was not high (13.05-31.01%), and the main nitrogen removal pathway in PIHSBBR, especially in the aerobic zone, was simultaneous nitrification and denitrification (SND). High-throughput sequencing analysis unraveled that Planctomycetota (3.65%) had a high abundance in the anoxic zone of PIHSBBR, implying that anaerobic ammonium oxidation (anammox) might have occurred in the anoxic zone. In addition, the nitrogen cycle function gene with the highest abundance was nirBD, indicating the possible presence of dissimilatory nitrate reduction to ammonium (DNRA) within the system (aerobic and anoxic zones). Our research can provide useful information for the improvement and future application of PIHSBBR.

A comprehensive review on mixotrophic denitrification processes for biological nitrogen removal.

Biological denitrification is the most widely used method for nitrogen removal in water treatment. Compared with heterotrophic and autotrophic denitrification, mixotrophic denitrification is later studied and used. Because mixotrophic denitrification can overcome some shortcomings of heterotrophic and autotrophic denitrification, such as a high carbon source demand for heterotrophic denitrification and a long start-up time for autotrophic denitrification. It has attracted extensive attention of researchers and is increasingly used in biological nitrogen removal processes. However, so far, a comprehensive review is lacking. This paper aims to review the current research status of mixotrophic denitrification and provide guidance for future research in this field. It is shown that mixotrophic denitrification processes can be divided into three main kinds based on different kinds of electron donors, mainly including sulfur-, hydrogen-, and iron-based reducing substances. Among them, sulfur-based mixotrophic denitrification is the most widely studied. The most concerned influencing factors of mixotrophic denitrification processes are hydraulic retention times (HRT) and ratio of chemical oxygen demand (COD) to total inorganic nitrogen (C/N). The dominant functional bacteria of sulfur-based mixotrophic denitrification system are Thiobacillus, Azoarcus, Pseudomonas, and Thauera. At present, mixotrophic denitrification processes are mainly applied for nitrogen removal in drinking water, groundwater, and wastewater treatment. Finally, challenges and future research directions are discussed.

Mixotrophic denitrification improvement in ecological floating bed: Interaction between iron scraps and plant biomass.

In this study, an iron scrap (IS)-based ecological floating bed was constructed to couple with plant biomass (FeB-EFB) for treating low-polluted water, and the nitrogen removal performance and mechanism were explored. The results showed that the nitrogen could be effectively removed in FeB-EFB, and the nitrate removal efficiency was 29.14 ± 8.06% even at a low temperature (13.9 ± 2.2 °C). After the temperature rose to 20.0 ± 0.9 °C, the denitrification rate was increased by 0.63 ± 0.16-0.81 ± 0.27 g/(m2 d) due to the synergistic effect of ISs and plant biomass. Plant biomass could promote the ISs release efficiency, while ISs could facilitate plant biomass availability by promoting cellulose decomposition. High-throughput sequencing analysis revealed that the iron-oxidizing bacteria Pseudomonas were the dominant genus in FeB-EFB. Meanwhile, the existence of plant biomass could increase the abundance of iron-related bacteria and enrich heterotrophic and facultative denitrifying bacteria (e.g., Hydrogenophaga, Comamonas) as well, improving iron-mediated denitrification and heterotrophic denitrification simultaneously. Therefore, mixotrophic denitrification improvement played a major role in promoting nitrogen removal of FeB-EFB. These results indicated that coupling iron scraps with plant biomass may be an effective way to improve the nitrogen removal performance of EFB.

Coupling mixotrophic denitrification and electroactive anodic nitrification by nitrate addition for promoting current generation and nitrogen removal.

Nitrate promotes anodic denitrification and fasts organic matter removal in microbial fuel cells (MFCs). However, it suffers from poor total nitrogen (TN) removal and current recovery. In this study, some novel electroactive nitrifying/denitrifying bacteria (ENDB) were introduced in a single chambered air-cathode MFC to investigate the performance of this device and the microbial community shift by adding nitrate. Results showed a similar disturbance in current output by adding nitrate during a short-term operation. However, a stable and reproducible current increase was achieved in the continuous experiment. A maximum current of 0.76 A m-3 and a maximum TN removal of >99 % were accomplished. The corresponding corrected coulombic efficiency was approximately 18 %. Under repeatable batches, a sharp decrease in chemical oxygen demand (COD) with feeding nitrate confirmed the temporary competition on electron donors through heterotrophic denitrification. The later current increase and nitrite detection occurring without metabolized COD could be considered evidence of electroactive anodic nitrification. The ENDB biofilm successfully coupled mixotrophic denitrification and electroactive anodic nitrification. It eventually promoted TN removal. In the process, genera Pseudoxanthomonas, Thauera, and Pseudomonas were enriched in the anodic ENDB biofilms. Cyclic voltammetry data confirmed the promotion of the electron transfer process by biofilms. The bacterial function predication revealed that the genes related to nitrogen removal and electron transfer were upregulated. Therefore, mixotrophic denitrification and electroactive anodic nitrification processes facilitated power recovery with the high efficiency of pollutant removal, finally ensuring water body security.

Effect of hydraulic retention time on performance of autotrophic, heterotrophic, and split-mixotrophic denitrification systems supported by polycaprolactone/pyrite: Difference and potential explanation.

Biological denitrification is still the most important pathway to purifying nitrate-containing wastewater. In this study, pyrite (FeS2 ) and polycaprolactone (PCL) were used as electron donors to construct sole or combined denitrification systems, that is, pyrite-based autotrophic denitrification (PAD) system, PCL-supported heterotrophic denitrification (PHD) system, and split-mixotrophic denitrification system (combined PAD + PHD), all of which were operated under five different hydraulic retention times (HRTs) for 150 days. The results showed that the removal rates (RE) of nitrate (NO3 - -N) and inorganic phosphorus (PO4 3- -P) by PAD were 91% and 94%, respectively, but the effluent sulfate (SO4 2- ) concentration was as high as 168.2 mg/L; the removal rate of NO3 - -N by PHD was higher than 99%, but the PO4 3- -P could not be removed ideally; the removal rates of NO3 - -N and PO4 3- -P by PAD + PHD were higher than 95% and 99%, respectively, and the effluent SO4 2- concentration was only 7.2 mg/L. Through the analysis of the surface scanning electron microscope (SEM) images of the two kinds of media before and after use, it was found that the coupled mode of PAD + PHD was more favorable for biofilm formation than the sole PAD or PHD process, and the microorganisms in the PAD + PHD mode made more full use of electron donors. Moreover, the biomass of the PAD + PHD mode was lower than that of the PAD or PHD process, but the denitrification efficiency of the coupled mode was more efficient, indicating that the functional microorganisms in the PAD + PHD mode might have a certain synergistic effect. PRACTITIONER POINTS: Removal rates of NO3 -, PO4 3 -, and SO4 2 - by PAD were 91%, 94%, and -233%, respectively. Removal rate of NO3 - by PHD exceeded 99%, but PO4 3 - could not be removed ideally. Removal rates of NO3 -, PO4 3 -, and SO4 2 - by PAD + PHD were 95%, 99%, and 86%, respectively. The coupled mode was more favorable for biofilm formation than the sole PAD or PHD. The coupled mode had lower biomass but got more excellent denitrification efficiency.

The nitrogen removal performance and microbial community on mixotrophic denitrification process.

Sulfur autotrophic denitrifiers and heterotrophic denitrifiers widely exist in aquatic ecosystem, however, the response of sulfide to the microbial community structure in mixotrophic denitrification ecosystem is unknown yet. In this study, the denitrification performance and microbial community were explored by changing the molar ratio of influent C/N/S. From the level of genus, the joint action of Thauera, Pacacoccus, Fusibacter Pseudoxanthomonas, Thiobacillus, Sulfurovum and Sulfurimonas brought about the efficient denitrification performance in the mixotrophic system. Thauera increased from from 0.97% to more than 13%, and the relative abundances of Thiobacillus and Sulfurimonas were about 4.14% and 3.89% separately after adding S2-. The results of this study showed that the denitrification performance could be indeed intensified in the mixotrophic system, among which provided a theoretical basis for establishing an efficient biological nitrogen removal system.

Effect of electric current intensity on performance of polycaprolactone/FeS2-based mixotrophic biofilm-electrode reactor.

In this study, a bioelectrochemical system consisting of pyrite-based autotrophic denitrification (PAD) and heterotrophic denitrification (HD) was established to polish nitrate wastewater. The loading of electric current (EC) could stimulate the dissolution of pyrite. Appropriate EC (I ≤ 30 mA) was conducive to nitrate removal, too high EC (I = 40 mA) would inhibit nitrate removal and lead to an obvious accumulation of NO2--N and NH4+-N. Microbial analysis revealed that the increase of EC could inhibit the diversity of heterotrophic microbes, but appropriate EC (I = 10 mA) could increase the diversity of autotrophic microbes. The EC loading was conducive to the enrichment of iron autotrophic denitrifiers (Ferritrophicum), pyrite-oxidizing bacteria (Thiobacillus, Sulfurimonas), and sulfur autotrophic denitrifiers (Dechloromonas, Thiobacillus, and Arenimonas). The EC loading enlarged the contribution of PAD, making PAD a dominant pathway in denitrification.