Impact of organic carbon on sulfide-driven autotrophic denitrification: Insights from isotope fractionation and functional genes.

Additional organics are generally supplemented in the sulfide-driven autotrophic denitrification system to accelerate the denitrification rate and reduce sulfate production. In this study, different concentrations of sodium acetate (NaAc) were added to the sulfide-driven autotrophic denitrification reactor, and the S0 accumulation increased from 7.8% to 100% over a 120-day operation period. Batch experiments revealed a threefold increase in total nitrogen (TN) removal rate at an Ac--C/N ratio of 2.8 compared to a ratio of 0.5. Addition of organic carbon accelerated denitrification rate and nitrite consumption, which shortened the emission time of N2O, but increased the N2O production rate. The lowest N2O emissions were achieved at the Ac--C/N ratio of 1.3. Stable isotope fractionation is a powerful tool for evaluating different reaction pathways, with the 18ε/15ε values in nitrate reduction ranging from 0.5 to 1.0. This study further confirmed that isotope fractionation can reveal denitrifying nutrient types, with the 18ε (isotopic enrichment factor of oxygen)/15ε (isotopic enrichment factor of nitrogen) value approaching 1.0 for autotrophic denitrification and 0.5 for heterotrophic denitrification. Additionally, the 18ε/15ε values can indicate changes in nitrate reductase. There is a positive correlation between the 18ε/15ε values and the abundance of the functional gene napA, and a negative correlation with the abundance of the gene narG. Moreover, 18ε and 15ε were associated with changes in kinetic parameters during nitrate reduction. In summary, the combination of functional gene analysis and isotope fractionation effectively revealed the complexities of mixotrophic denitrification systems, providing insights for optimizing denitrification processes.

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.

Revealing the ammonia oxidation process and shortcut nitrification performance using nitrogen and oxygen isotope fractionation effect.

Natural abundance isotope fractionation properties have become the most effective way to explore nitrogen transformations of biological nitrogen removal from wastewater. The migration and transformation characteristics of N and O elements in the shortcut nitrification were analyzed using the N and O dual isotopic fractionation technique. The effects of dissolved oxygen (DO) and temperature changes on the performance of shortcut nitrification and isotopic fractionation were investigated. The fractionation characteristics of N and O elements during shortcut nitrification were explored by adjusting DO concentration (0.2-0.4, 1-1.2 and 3-4 mg/L) and temperature (33 ± 1 °C, 25 ± 1 °C and 18 ± 1 °C). Both δ15NNO2 and δ18ONO2 showed a gradually increasing trend with the accumulation of NO2--N, and the fractionation effects induced by temperature were significantly higher than those by DO. The higher the temperature, the more significant the increase in δ15NNO2; the higher the DO, the more remarkable the increase in δ18ONO2, while δ15NNO2: δ18ONO2 was maintained at 0.77-6.45. The 18O-labeled H2O was successfully transferred to NO2--N, and the replacement of O element was as high as 100 %, indicating that DO and H2O simultaneously participated in the shortcut nitrification process. The dynamic changes in isotope fractionation effects can be successfully applied to reveal the performance and mechanism of shortcut nitrification.

The successful application of light to the system of simultaneous nitrification/endogenous denitrification and phosphorus removal: Promotion of partial nitrification and glycogen accumulation metabolism.

Partial nitrification (PN) and high glycogen accumulating metabolism (GAM) activity are the basis for efficient nitrogen (N) and phosphorus (P) removal in simultaneous nitrification endogenous denitrification and phosphorus removal (SNDPR) systems. However, achieving these processes in practical operations is challenging. This study proposes that light irradiation is a novel strategy to enhance the nutrient removal performance of the SNDPR system with low carbon to nitrogen ratios (C/N of 3.3-4.1) domestic wastewater. Light energy densities (Es) of 55-135 J/g VSS were found to promote the activity of ammonia-oxidizing bacteria (AOB) and GAM, while inhibiting the activity of nitrite-oxidizing bacteria (NOB) and polyphosphate accumulating metabolism (PAM). Long-term exposure to different light patterns at Es of 55-135 J/g VSS revealed that continuous light rapidly achieved PN by inhibiting NOB activity and promoted the growth of glycogen accumulating organisms (GAOs), allowing the removal of above 82 % N and below 80 % P. Intermittent light maintained stable PN by inhibiting the activity and growth of NOB and promoted the growth of polyphosphate accumulating organisms (PAOs) with high GAM activity (Accmulibacer IIC-ii and IIC-iii), allowing the removal of above 82 % N and 95 % P. Flow cytometry and enzyme activity assays showed that light promoted GAM-related enzyme activity and the metabolic activity of partial Accmulibacer II over other endogenous denitrifying bacteria, while inhibiting NOB translation activity. These findings provide a new approach for enhancing nutrient removal, especially for achieving PN and promoting GAM activity, in SNDPR systems treating low C/N ratio domestic wastewater using light irradiation.

Simultaneous nitrogen and sulfur removal through synergy of sulfammox, anammox and sulfur-driven autotrophic denitrification in a modified bioreactor enhanced by activated carbon.

Anaerobic ammonium (NH4+ - N) oxidation coupled with sulfate (SO42-) reduction (sulfammox) is a new pathway for the autotrophic removal of nitrogen and sulfur from wastewater. Sulfammox was achieved in a modified up-flow anaerobic bioreactor filled with granular activated carbon. After 70 days of operation, the NH4+ - N removal efficiency almost reached 70%, with activated carbon adsorption and biological reaction accounting for 26% and 74%, respectively. Ammonium hydrosulfide (NH4SH) was found in sulfammox by X-ray diffraction analysis for the first time, which confirmed that hydrogen sulfide (H2S) was one of the sulfammox products. Microbial results indicated that NH4+ - N oxidation and SO42- reduction in sulfammox were carried out by Crenothrix and Desulfobacterota, respectively, in which activated carbon may operate as electron shuttle. In the 15NH4+ labeled experiment, 30N2 were produced at a rate of 34.14 µmol/(g sludge·h) and no 30N2 was detected in the chemical control group, proving that sulfammox was present and could only be induced by microorganisms. The 15NO3- labeled group produced 30N2 at a rate of 88.77 µmol/(g sludge·h), demonstrating the presence of sulfur-driven autotrophic denitrification. In the adding 14NH4+ and 15NO3- group, it was confirmed that NH4+ - N was removed by the synergy of sulfammox, anammox and sulfur-driven autotrophic denitrification, where the main product of sulfammox was nitrite (NO2-) and anammox was the main cause of nitrogen loss. The findings showed that SO42- as a non-polluting species to environment may substitute NO2- to create a new "anammox" process.

Simultaneous nitrate and phosphate removal based on thiosulfate-driven autotrophic denitrification biofilter filled with volcanic rock and sponge iron.

This study constructed two thiosulfate-driven autotrophic denitrification biofilters filled with volcanic rock (VR-BF), sponge iron and volcanic rock (SIVR-BF), respectively. The nitrate removal load (3200 g/m3/d) and efficiency (98 %) of SIVR-BF were higher than those of VR-BF. The removal of phosphate in SIVR-BF was mainly through forming FePO4 and Fe3(PO4)2(OH)2. Sulfur and iron cycles in SIVR-BF contributed to Fe (II)/Fe (III) electron shuttle, as well as S2-, S0, Sn2- electron buffer and energy storage, which improved nitrate removal and electron utilization. The formation of multi-path collaborative denitrification dominated by sulfur autotrophic denitrification (64.2 âˆ¼ 89.6 %) in SIVR-BF. The other denitrification pathways, such as iron autotrophic denitrification, which buffered pH and reduced sulfate production. Thiobacillus (38.6 %) and Ferritrophicum (25.3 %) were the dominant genus of VR-BF and SIVR-BF, respectively, which played crucial roles in autotrophic denitrification of iron and sulfur. SIVR-BF was a promising process to realize iron-sulfur coupling autotrophic denitrification and phosphate removal.

Achieving deep autotrophic nitrogen removal in aerated biofilter driven by sponge iron: Performance and mechanism.

Different from anammox, the combination of Fe (III) reduction coupled to anaerobic ammonium oxidation (Feammox) and nitrate/nitrite dependent ferrous oxidation (NDFO) do not require to control nitrite accumulation. Furthermore, sponge iron can avoid continuous iron supplementation in practice and is a good iron source for the occurrence of Feammox and NDFO in wastewater treatment. Therefore, a biofilter using sponge iron as carrier treating low nitrogen wastewater was built. In this study, the performances of nitrogen removal were explored under different hydraulic retention times (HRT) and gas-water ratios in sponge iron biofilter. And the pathways of nitrogen removal were analyzed by activity tests. The results showed ammonia removal efficiency reached 94.1% and total inorganic nitrogen removal efficiency was up to 70.6% at HRT of 19 h and gas-water ratio of 18. Compared to nitrogen removal by adsorption under non-aeration, the activity tests showed that total inorganic nitrogen loss was caused by Feammox and NDFO after aeration. The results of microbial communities showed that appearances of nitrifier-Nitrosomonadaceae, Feammox bacteria-Clostridiaceae and NDFO bacteria-Gallionellaceae resulted in deep nitrogen removal after aeration, in which Nitrosomonadaceae and Clostridiaceae contributed to ammonia removal and Gallionellaceae contributed to nitrite/nitrate reduction to nitrogen gas. Therefore, it was feasible to achieve deep autotrophic nitrogen removal and Fe (II) and Fe (III) cycle in sponge iron biofilter.

Effect of S2O32--S addition on Anammox coupling sulfur autotrophic denitrification and mechanism analysis using N and O dual isotope effects.

Anaerobic ammonia oxidation (Anammox) coupling sulfur autotrophic denitrification is an effective method for the advanced nitrogen removal from the wastewater with limited carbon source. The influence of S2O32--S addition on Anammox coupling sulfur autotrophic denitrification was investigated by adding different concentrations of S2O32--S (0, 39, 78, 156 and 312 mg/L) to the Anammox system. The contribution of sulfur autotrophic denitrification and Anammox to nitrogen removal at S2O32--S concentrations of 156 mg/L was 75% ∼83% and 17%∼25%, respectively, and the mixed system achieved completely nitrogen removal. However, Anammox bioactivity was completely inhibited at S2O32--S concentrations up to 312 mg/L, and only sulfur autotrophic denitrification occurred. The isotopic effects of NO2--N (δ15NNO2 and δ18ONO2) and NO3--N (δ15NNO3 and δ18ONO3) during Anammox coupling sulfur autotrophic denitrification showed a gradual decrease trend with the increase of S2O32--S addition. The ratios of δ15NNO2:δ18ONO2 and δ15NNO3:δ18ONO3 was maintained at 1.30-2.41 and 1.36-2.52, respectively, which revealed that Anammox was dominant nitrogen removal pathway at S2O32--S concentrations less than 156 mg/L. Microbial diversity gradually decreased with the increase of S2O32--S. The S2O32--S addition enhanced the S2O32--driven autotrophic denitrification and weakened the Anammox, leading to a gradually decreasing trend of the proportion of Candidatus Brocadia as Anammox bacteria from the initial 27% to 4% (S2O32--S of 156 mg/L). Yet Norank-f-Hydrogenophilaceae (more than 50%) and Thiobacillus (54%) as functional bacteria of autotrophic denitrification obviously increased. The appropriate amount of S2O32--S addition promoted the performance of Anammox coupling sulfur autotrophic denitrification achieved completely nitrogen removal.

Analysis of nitrite oxidation process and nitrification performance by nitrogen and oxygen isotope fractionation effect.

The N and O isotope fractionation effects in NO2--N oxidation and nitrification performance of an activated sludge system treating municipal wastewater are unknown. The nitrifying sludge was cultured under different temperature (33 ± 1 °C, 25 ± 1 °C,and 18 ± 1 °C) and dissolved oxygen (DO: 0.5-1 mg/L, 3-4 mg/L, and 7-8 mg/L). The inverse kinetic isotope effects of N and O (15εNO2 and 18εNO2) were -0.62‰ to -7.08‰ and -0.87‰ to -1.68‰ in the process of NO2--N oxidation, respectively. 15εNO3 gradually increased with increasing of temperature (15εNO3-33°C (14.49‰) > 15εNO3-25°C (10.43‰) > 15εNO3-18°C (7.3‰)), while the 15εNO3:18εNO3 was maintained at 1.02-5.32. The increase of temperature improved the nitrification activity, which promoted the fractionation effect, but the change of DO did not highlight this difference. The exchange of NO2--N and H2O (XNOB) was 32.5 ± 1.5%, and the kinetic isotope effect of H2O participating in the reaction (18εk, H2O, 2) was 22.57 ± 1.79‰, indicating that H2O was involved in the NO2--N oxidation rather than DO. In summary, the elevated temperature enhanced the fractionation effect of NO2--N oxidation. This study provides a new perspective to reveal the mechanism of NO2--N oxidation, optimize the process of nitrogen removal from wastewater and further control water eutrophication.

Nitrogen removal performance of sulfur autotrophic denitrification under different S2O32- additions using isotopic fractionation of nitrogen and oxygen.

The dual isotope fractionation of nitrogen (N) and oxygen (O) is an effective way to track the transformation of NO3--N in biological denitrification process. The Sulfur autotrophic denitrification combined with the different concentrations of S2O32- was investigated using the dual isotope fractionation of nitrogen (N) and oxygen (O) to reveal the nitrogen removal mechanism of the activated sludge. Based on successful autotrophic denitrification incubation, the modified Logistic model responded to the short-term effects of S2O32- addition on NO3--N removal and SO42- generation. Under the S2O32- addition of 0.5, 1, 2 and 4 times of the incubation stage (49.29 mg/L-394.32 mg/L), the fractionation effect of N in NO3--N (15εNO3) decreased from 8.74 ± 1.81‰ to 2.08 ± 0.06‰, and the fractionation effect of O in NO3--N (18εNO3) declined from 11.34 ± 0.46‰ to 5.48 ± 0.46‰. The 15εNO3/18εNO3 was maintained at 0.46-0.94, indicating a negative correlation between addition amount and isotope effect, and the addition of high concentrations of S2O32- was not suitable for system stabilization. Moreover, the 18O-labeled H2O (δ18OH2O) tests significantly proved the presence of O exchange between NO2--N/NO3--N and H2O (67%/97%) during the nitrogen removal process, while the reoxidation of NO2--N was explored in the autotrophic denitrification. The kinetic models coupled with isotope fractionation effectively revealed the nitrogen removal characteristics in the autotrophic denitrification systems, and verified the difference between the activated sludge-based wastewater treatment process and the natural ecosystem.