Multiple electron acceptor-mediated sulfur autotrophic denitrification: Nitrogen source competition, long-term performance and microbial community evolution.

Sulfur-driven autotrophic denitrification (SDAD) is feasible for the treatment of low-C/N-ratio and sulfur-laden wastewaters. The nitrite accumulated in SDAD will affect the performance and stability of the system but can be a potential electron acceptor. Thus, single- and multiple-electron acceptor-mediated SDAD systems were investigated. Batch assays revealed that nitrite and nitrate were the preferential options in the SDAD system with single and multiple electron acceptors, respectively. Synchronous nitrogen and sulfur removal was successfully achieved in continuous flow experiments with multiple electron acceptors, and the system could adapt well to high concentrations of sulfide, nitrate and nitrite (i.e., 720, 108 and 64.8 mg L-1, respectively), with the predominant genera shifting from Thiobacillus (48.88%) at the initial stage to unclassified_p_Firmicute (34.24%) and Syner-01 (12.31%) at the last stage. This work provides a fundamental basis for applying and regulating SDAD with multiple electron acceptors for the remediation of nitrogen- and sulfide- laden wastewaters.

A novel strategy for anammox consortia preservation: Transformation into anoxic sulfide oxidation consortia.

The preservation of anaerobic ammonia oxidation (anammox) consortia is crucial for the rapid start-up and the process stability of the anammox based bioreactor. This work proposed and evaluated the feasibility of an anammox consortia preservation strategy, in which the anammox sludge was transformed into intermediate anoxic sulfide oxidation (ASO) functional microorganisms. Initially, the ASO process was successfully started up by inoculating anammox sludge and the overall sulfide and nitrate removal rates stabilized at 57.5 ± 0.22 and 10.0 ± 0.18 kg m-3 day-1, respectively. Then, the bioreactor function was reversely transformed into anammox, whose nitrogen removal rate reached 1.68 kg m-3 day-1. Granule characteristics analysis revealed that both biomass and extracellular polymeric substance content returned to their original states after the reverse start-up. Although the population of Candidatus_Kuenenia was greatly declined during ASO process, its richness was successfully recovered after the reverse start-up of the anammox process. The inferred metagenomes analysis demonstrated that the shifts in functional microorganisms were related to variation in the main metabolic pathways. The specific activities of anammox and ASO both are regarded as key indicators for the successful start-up of bioreactor. This work revealed a novel technique for the preservation of anammox consortia and might be a potential strategy for overcoming the drawback of long start-up time.

Deciphering the microbial community and functional genes response of anammox sludge to sulfide stress.

Sulfide has attracted increasing attention due to its odor nuisance, toxicity and corrosion. Although variations in the nitrogen removal performance of anammox under sulfide stress have been reported previously, understanding the microorganisms at the molecular level is of greater significance. This study first deciphered the microbial community and functional gene response of anammox sludge to sulfide stress. Results showed that 20 mg L-1 sulfide could reduce specific anammox activity by 61.7%. The protein-like substances within extracellular polymeric substances were quenched at the end of the experiment. Moreover, the relative abundance of Candidatus Kuenenia significantly decreased from 28.7% to 6.4% while Thiobacillus increased from 0 to 7.2% due to sulfide stress. Furthermore, the abundances of functional genes (hzsA, hdh, nirK and nirS) significantly decreased when the sulfide concentration reached 20 mg L-1. These findings provide a further theoretical basis for the anammox process for nitrogen removal from wastewater containing sulfide.

Whether glycine betaine improves the thermotolerance of mesophilic anammox consortia.

While the application of mesophilic anammox process is currently the state of the art, the feasibility of a thermophilic anammox bioprocess is still unclear. In this study, we investigate whether glycine betaine (GB) addition can enhance the thermotolerance of mesophilic anammox biomass in the upflow anaerobic sludge blanket (UASB) reactors fed with synthetic wastewater at a nitrogen loading of approximately 4 kg N m-3 d-1. The results showed that during a long-term operation at 45°C with GB (0, 0.1, 1, 2 mM) addition, anammox performance became worse with the final effluent concentrations of NO2 -N of 145 ± 11.6 mg L-1 and nitrogen removal efficiency decreased from 92.3-6.9%. Specific anammox activity decreased from 392.1 ± 12.1-6.0 ± 0.8 mg N g-1 VSS d-1, which were not significantly higher than those in the control reactor. The content of heme c showed a stronger downward trend in T1 (with GB addition) than in the control reactor T0. The qPCR results showed that the relative abundance of Candidatus Kuenenia decreased in both the experimental (from 53.5-28.8%) and control reactors (from 54.1-35.1%). Overall, continuous addition of exogenous GB did not improve the thermotolerance of mesophilic anammox consortia at 45°C.

Conversion of municipal wastewater-derived waste to an adsorbent for phosphorus recovery from secondary effluent.

The sustainable management and recirculation of phosphorus resources are essential to our human lives. In this work, phosphorus removal and recovery from secondary effluent were achieved using municipal wastewater-derived materials as adsorbents. Through modification with 0.5 M NaOH for 30 min, iron containing sludge that originated from the coagulation pretreatment of municipal wastewater was successfully converted to phosphorus adsorbent. The maximal adsorption capacity of the prepared adsorbent was estimated to be 22 mg-P/g, and the adsorption performance remained stable in the pH range of 5-8. FeO(OH) was identified as the key adsorption site, and the ligand exchange mediated chemical adsorption was the main mechanism for phosphorus removal by the prepared material. Moreover, a laboratory-scale continuous-flow adsorption column experiment showed that the surplus phosphorus in secondary effluent could be readily reduced to <0.1 mg/L. By pyrolysis of P-laden alkali-treated iron sludge under oxygen limited conditions, the phosphorus was recovered and successfully applied to support wheat growth. This work provides valuable information for both the sustainable management of phosphorus streams in wastewater and cyclic utilization of waste sludge.

Microbial community evolution and fate of antibiotic resistance genes in anammox process under oxytetracycline and sulfamethoxazole stresses.

The microbial community characteristics, functional and antibiotic resistance genes (ARGs), anammox performance under individual and combined oxytetracycline (OTC) and sulfamethoxazole (SMX) were tested under environmentally relevant levels. The results showed that anammox performance was inhibited when the OTC or SMX concentration increased from 0.5 to 1.0 mg L-1. The absolute abundance of tetX in OTC (3.03 × 106 copies mg-1), SMX (2.80 × 106 copies mg-1) and OTC + SMX (2.03 × 106 copies mg-1) was the highest and one more order of magnitude higher than that of tetG, tetM, intI1, or sul2. The anammox performance in the presence of OTC or SMX was lower than that sum of their independent effects. The enrichment of sludge resistomes with prolonged exposure time and increasing OTC and SMX doses might be due to succession of bacterial hosts and potential elevation of ARGs by horizontal transfer.

Adaption and restoration of anammox biomass to Cd(II) stress: Performance, extracellular polymeric substance and microbial community.

Cadmium (Cd) can cause the deterioration of biological systems through inhibiting the enzymes activity and disturbing the microbial metabolism. Although the influence of Cd on conventional wastewater treatment process has been studied, the response of anammox to Cd exposure still remains unclear. This study firstly investigated the adaption and restoration of anammox biomass to Cd(II) stress. Results showed that long-term exposure of anammox bacteria to 2 mg L-1 Cd(II) was beneficial for the reactor performance, while 5 mg L-1 Cd(II) would cause the decline of SAA, extracellular polymeric substance content and relative abundance of Candidatus kuenenia by 40%, 25% and 31%, respectively. Furthermore, these indexes could approximately recover to the initial status after withdrawing Cd(II) from the influent. Overall, the anammox biomass exhibited a certain adaption and restoration ability to the suppression of Cd(II). This study may provide key valuable information for the biological treatment of wastewater containing Cd(II).

The performance and microbial community in response to MnO2 nanoparticles in anammox granular sludge.

The response of anaerobic ammonium oxidation (anammox) process and granular sludge in the upflow anaerobic sludge blanket reactor was analyzed under long-term exposure to MnO2 nanoparticles (NPs) (1-200 mg L-1). The addition of 200 mg L-1 MnO2 NPs had a significantly positive effect on nitrogen removal and this system exhibited excellent performance, with a total nitrogen removal efficiency of 93.1%. Moreover, the specific anammox activity enhanced with increasing concentrations of MnO2 NPs up to the maximum value of 657.3 ±â€¯9.3 mg TN g-1 VSS d-1 under MnO2 NPs concentration of 200 mg L-1. This value was approximately 1.6-fold higher than that of the reactor in the absence of MnO2 NPs. The extracellular polymeric substances and settling velocity were both increased with MnO2 NPs addition. Meanwhile, the high-throughput sequencing results revealed that MnO2 NPs increased the relative abundance of dominant bacteria (Candidatus Kuenenia) from 17.3% at the absence of MnO2 NPs to 23.9% at 200 mg L-1 MnO2 NPs, which resulted in a higher efficiency of biological nitrogen removal on the anammox system. These results indicated that MnO2 NPs enhanced nitrogen removal performance of anammox process.

Merely inoculating anammox sludge to achieve the start-up of anammox and autotrophic desulfurization-denitrification process.

Anammox and autotrophic desulfurization-denitrification (AADD) process is feasible for the nitrogen and sulfide removal in the same reactor, and the influence of excess nitrate produced by anammox could also be alleviated simultaneously. This study firstly proposed a novel strategy with inoculating single anammox sludge to start up the AADD process. Results demonstrated that the 90% nitrogen removal efficiency (NRE), 2.55kgm-3 d-1 nitrogen removal rate (NRR), and 95% sulfide removal efficiency (SRE) were obtained at the influent total nitrogen of 280mgL-1 and sulfide of 221.5mgL-1, and the final effluent nitrate concentration was as low as 8mgL-1 under the appropriate operation conditions. Tryptophan-like and protein-like substances were characterized as the main components in bound EPS. Thiobacillus (35.68%) and Pseudoxanthomonas (11.61%) were identified as the predominant genera. This study will pave a potential avenue to promote the treatment of high concentration nitrogen and sulfide in wastewater in the future.

The revolution of performance, sludge characteristics and microbial community of anammox biogranules under long-term NiO NPs exposure.

Given the increasing applications of NiO nanoparticles (NPs) in battery products, the potential effects of NiO NPs on anaerobic ammonium oxidation (anammox) systems were studied for the first time. The results showed that the anammox system performance obviously differed under the stresses of different NiO NPs concentrations. After the withdrawal of NiO NPs, the nitrogen removal performance of the anammox reactor returned to nearly that of the initial phase within 35 days. Compared with 0 mg L-1 NiO NPs, the specific anammox activity first increased and then decreased to the minimum value of 116.8 ±â€¯13.8 mg TN g-1 VSS d-1 at 60 mg L-1 NiO NPs. The variations in the heme c contents and extracellular polymeric substance amounts were similar to the variations in the specific anammox activity throughout the whole experiment. Additionally, the relative abundance of the dominant bacteria (Candidatus kuenenia) increased from 20.44% at 60 mg L-1 NiO NPs to 23.14% at the end of the last phase. Thus, the potential effects of NiO NPs on anammox systems should be a cause for great concern.

Anammox granule as new inoculum for start-up of anaerobic sulfide oxidation (ASO) process and its reverse start-up.

The feasibility of implementing anaerobic ammonium oxidation (anammox) granules to start up high-loading anaerobic sulfide oxidation (ASO) in an upflow anaerobic sludge bed (UASB) reactor was investigated. An innovation method of the reverse start-up of anammox was also validated. Firstly, the reactor was operated to treat sulfide-rich wastewaters into which nitrite was introduced as an electron acceptor. An high-rate performance with sulfide and nitrate removal rates of 105.5 ±â€¯0.11 kg S m-3 d-1 and 28.45 ±â€¯3.40 kg N m-3 d-1, respectively, was accomplished. Sulfurovum were enriched with the increase of the substrate load and then conquered Candidatus Kuenenia to be the predominant bacteria. Excitation-emission matrix (EEM) spectroscopy showed that the intensities of fluorescence decreased and protein-like substrates were the main components associated with the process of start-up. FT-IR analysis found that the main functional groups indicator were O-H groups. Secondly, the reverse start-up of anammox (achieving 90% TN removal) was achieved immediately when the substrate changed. 16S rRNA analysis indicated the successfully enrichment of anammox bacteria (Candidatus Kuenenia). These results suggest that anammox granules can act as inoculum of high-loading ASO process and the reverse start-up provides a new perspective for the fast initiation of anammox process.

Insight into the short- and long-term effects of quinoline on anammox granules: Inhibition and acclimatization.

The short- and long-term influence of quinoline on the properties of anaerobic ammonium oxidation (anammox) biogranules was evaluated. During batch tests, the bioactivity of anammox granules in the presence of different quinoline concentrations was monitored, and the IC50 of quinoline was calculated to be 13.1 mg L-1 using a non-competitive inhibition model. The response of anammox granules to pre-exposure to quinoline was dependent on metabolic status, and the presence of both quinoline and NO2--N had a rapid detrimental effect, resulting in a 64.5% decrease within 12 h. During continuous-flow experiments, the nitrogen removal rate (NRR) of the reactor decreased sharply within 3 days in the presence of 10 mg L-1 quinoline and then was restored to 2.6 kg N m-3 d-1. In the presence of quinoline-induced stress, the specific anammox activity and levels of extracellular polymeric substance and heme c were decreased, while settling velocity persistently increased. After cultivation and acclimation obtained by adding a medium level of quinoline to the influent, the anammox granule sludge was able to tolerate 10 mg L-1 quinoline in 178 days.

Discrepant effects of metal and metal oxide nanoparticles on anammox sludge properties: A comparison between Cu and CuO nanoparticles.

Metal and metal oxide nanoparticles (NPs) show differences in antimicrobial activity due to different chemical and physical properties. Using copper as a representative example, this study compared the NP effects on anaerobic ammonium oxidation (anammox) bacteria in wastewater treatment. Long-term exposure to 5 mgCu L-1 CuNPs reduced the physiological activity and abundance of anammox bacteria, thereby causing deterioration of reactor performance. However, anammox granules exhibited stronger resistance and resilience to perturbation by 1-160 mgCu L-1 CuONPs, and no adverse effects on performance were observed. Moreover, the level of Cu(II) released from NPs in the influent exhibited good correlations with variations of the community structure and sludge properties. Therefore, the effects of Cu-based NPs on anammox sludge properties are dependent on their forms and levels, and their discrepant effects are partially attributed to their ability to release ionic copper.

Increased salinity improves the thermotolerance of mesophilic anammox consortia.

While the application of anammox-based process for mesophilic sidestream treatment is at present the state of the art and mainstream treatment at ambient temperature is also in development, the feasibility of thermophilic anammox process is still unclear. This study investigated the effects of salinity on the thermotolerance of mesophilic anammox sludge. In batch activity tests, 45 °C seems to be the critical temperature for the tolerance of mesophilic anammox consortia without acclimatization or amendments. The optimal anammox activity at 40, 42.5, and 45 °C can be achieved with the amendment of salt at 5-8, 8-10, and ~12 g NaCl L-1, respectively. However, this improvement effect was limited at 50 °C or when the shock duration was longer than 24 h even at 45 °C. In continuous-flow bioreactors, mesophilic anammox consortia could gradually adapt to 40-50 °C under a transition of 2.5 °C, and the performance was enhanced by an increase in salinity, which may be associated with the increase in extracellular polymeric substances. A nitrogen removal rate of 0.53 kgN m-3 d-1 was finally obtained at 50 °C. Overall, these interesting results facilitate further opportunities for thermophilic anammox process.

Evaluating the effects of metal oxide nanoparticles (TiO2, Al2O3, SiO2 and CeO2) on anammox process: Performance, microflora and sludge properties.

The increasing use of engineered metal oxide nanoparticles (MONPs) in consumer products raises great concerns about their environmental impacts, but their potential impacts on anaerobic ammonium oxidation (anammox) process in wastewater treatment remain unclear. In this study, the presence of MONPs (1, 50, 200 mg L-1) exhibited no visible effects on the nitrogen removal performance of anammox reactors, but high levels (200 mg L-1) of SiO2NPs, Al2O3NPs and CeO2NPs had a distinct effect on shaping the anammox community. Long-term exposure of MONPs caused different responses in the relative abundance of Ca. Kuenenia, the level of functional gene HzsA and the activities of three key enzymes involved in anammox metabolism, but no significant inhibition effects on specific anammox activity were detected. Overall, the effects of MONPs on anammox community structure and sludge properties depended on their types and levels and followed the order SiO2 > CeO2 > Al2O3 > TiO2.

Roles of MnO2 on performance, sludge characteristics and microbial community in anammox system.

The long-term impacts of MnO2 on performance, sludge characteristics and microbial community of biogranule-based anaerobic ammonium oxidation (anammox) process were evaluated in an up-flow anaerobic sludge blanket reactor. It was found that the total nitrogen removal efficiency of reactor was fluctuated between 90%-93% at 1-200mgL-1 MnO2. Notably, the specific anammox activity was increased to maximum value of 657.3±10.6mgTNg-1VSSd-1 at 50mgL-1 MnO2 and then slightly decreased, but still higher than that achieved at 0-15mgL-1 MnO2, which had similar variation trends to the content of heme c and extracellular polymeric substances in anammox granules. High throughput sequencing indicated that MnO2 could improve the microbial richness and diversity of anammox granules and Candidatus Kuenenia was always the dominant species, and its abundance continued to increase to 21.3% at the end of operational experiment. Therefore, MnO2 could be applied to enhance the anammox process and the optimal influent MnO2 concentration was lower than 50mgL-1 in view of the reactor performance and cost issues.

Anammox granules show strong resistance to engineered silver nanoparticles during long-term exposure.

The increasing use of engineered Ag nanoparticles (NPs) in consumer products raises great concerns about their environmental impacts, but their potential impact on anaerobic ammonium oxidation (anammox) bacteria in wastewater treatment remains unclear. In this study, the response of anammox consortia inhabiting in granules to long-term exposure of AgNPs was investigated. The gradually increased stress of AgNPs (1, 10, 50 mg L-1) showed no adverse effects on the nitrogen removal performance and on the relative abundance of Ca. Kuenenia inhabiting in granules. Notably, in response to the escalating burden of AgNPs, anammox consortia tended to secrete more protein but less polysaccharide, and meanwhile increase the relative abundances of Ag+ efflux protein SilA and regulatory protein SilR. Overall, these results suggested that the strong resistance of anammox granules to AgNPs might be mainly attributed to the defense role of extracellular polymeric substances and gene-based regulation of ion transport.

Effect of chromium on granule-based anammox processes.

In this study, the feasibility of application of anaerobic ammonium oxidation (anammox) to teat wastewater containing chromium was assessed. Anammox granule activity decreased with increasing Cr(VI) concentration and pre-exposure time in batch tests, and the 50% inhibition concentration of Cr(VI) on anammox biomass was 296.5 mg L-1. Approximately 93.9% chromium was absorbed by loosely bound-extracellular polymeric substances and tightly bound-extracellular polymeric substances when less than 60 mg L-1 Cr(VI) was added. During long-term operation in up-flow anaerobic sludge blanket reactor, significant inhibitory effects anammox performance were observed for Cr(VI) concentrations up to 2 mg L-1. The nitrogen removal rate (NRR) rapidly decreased to 1.49 ±â€¯0.89 kg N m-3 d-1, whereas the NRR was 11.37 ±â€¯1.30 kg N m-3 d-1 in a control reactor. Compared with initial levels, specific anammox granule activity was 22%. The tolerance of the anammox process to Cr(VI) can be enhanced after a long-term adaptive phase.

Transient disturbance of engineered ZnO nanoparticles enhances the resistance and resilience of anammox process in wastewater treatment.

The increasing use of engineered nanoparticles (NPs) in consumer and industrial products raises concerns about their environmental impacts, but their potential influence on anaerobic ammonium oxidation (anammox) process in wastewater treatment remains unknown. In this study, the response of granule-based anammox reactor to different loads of ZnONPs was investigated. The introduction of 1-5mgL-1 ZnONPs did not affect reactor performance, but 90% of the nitrogen removal capacity was deprived by a shock of 10mgL-1 ZnONPs within 3days. Anammox activity was significantly inhibited, but no significant stimulation of intracellular reactive oxygen species (ROS) production or extracellular lactate dehydrogenase (LDH) activity was observed. The inhibition was thus mainly due to the accumulation of toxic Zn(II) ions in anammox biomass. However, the resistance and resilience of this anammox reactor to ZnONPs were enhanced by intermittent perturbations in the mode of "shock-recovery". The up-regulated abundance of Zn(II)-exporter ZntA might contribute to the enhanced resistance. In addition, these repeated transient disturbances improved the functional specificity of the anammox community despite the reduction of its diversity. Overall, these results may provide useful references for evaluating and controlling the risk of NPs to anammox process.

Enhanced effects of maghemite nanoparticles on the flocculent sludge wasted from a high-rate anammox reactor: Performance, microbial community and sludge characteristics.

Magnetic nanoparticles (NPs) have been widely applied in environmental remediation, biomass immobilization and wastewater treatment, but their potential impact on anaerobic ammonium oxidation (anammox) biomass remains unknown. In this study, the short-term and long-term impacts of maghemite NPs (MHNPs) on the flocculent sludge wasted from a high-rate anammox reactor were investigated. Batch assays showed that the presence of MHNPs up to 200 mg L-1 did not affect anammox activity, reactive oxygen species production, or cell membrane integrity. Moreover, long-term addition of 1-200 mg L-1 MHNPs had no adverse effects on reactor performance. Notably, the specific anammox activity, the abundance of hydrazine synthase structural genes and the content of extracellular polymeric substance were increased with elevated MHNP concentrations. Meanwhile, the community structure was shifted to higher abundance of Candidatus Kuenenia indicated by high-throughput sequencing. Therefore, MHNPs could be applied to enhance anammox flocculent sludge due to their favorable biocompatibility.