Response of the mainstream anammox process to the biodegradable carbon sources in the granule-based systems: The difference in self-stratification of the microbial community.

The inevitable introduction of biodegradable carbon sources (such as monosaccharides and volatile fatty acids) originating from pretreatment units might affect the performance of the mainstream anaerobic ammonium oxidation (anammox) process. Two model carbon sources (glucose and acetate) were selected to investigate their effects on granule-based anammox systems under mainstream conditions (70 mg total nitrogen (TN) L-1, 15 °C). At a nitrogen loading rate of 2.87 ± 0.80 kg N m-3 d-1, a satisfactory effluent quality (TN < 10 mg L-1) was achieved in the presence of glucose or acetate at a chemical oxygen demand (COD/N) ratio of 0.5. The contribution of anammox to nitrogen removal decreased with increasing COD/N ratio to 1.0 because the expression of anammox functional genes was inhibited, whereas the expression of denitrifying functional genes was promoted. However, the nitrogen removal efficiency of the two considered reactors was maintained above 80 %. Self-stratification of the microbial community along the reactor height facilitated a functional balance through the retention of anammox bacteria in granules but resulted in washout of denitrifying bacteria in flocs under a high-flow pattern. These findings highlighted the advantages of granule-based systems in the mainstream anammox process due to their inherent biomass self-segregation property and the need for the development of targeted biomass retention strategies.

Molecular spectroscopy and docking simulation revealed the binding mechanism of phenol onto anammox sludge extracellular polymeric substances.

The rapid development of chemical industry has induced to the large amount of phenolic wastewater production. When the promising anaerobic ammonium oxidation (anammox) was employed to treat the industrial wastewater, phenolic compounds would possibly inhibit the microbial performance. Extracellular polymeric substances (EPSs) play an essential role in protecting cells from being intoxicated by phenolic compound while the distinct mechanism remains elusive. In this work, the interaction of phenol with anammox sludge EPSs and transmembrane ammonium transport (Amt) domain was explored at molecular level by using spectral method and molecular docking simulation. It was found that phenol statically quenched the fluorescent components of EPSs and the protein component dominated the interaction between EPSs and phenol. The overall interaction was an entropy-driven process with hydrophobic interaction as the main driving force, and the CO vibration responded preferentially. As phenol continued to penetrate into the cell surface, there were hydrogen bond, hydrophobic interaction force and π-π base-stacking forces between the Amt domain and phenol. The interaction between phenol and amino acid residues of the Amt domain would interfere the NH4+ transport and further affect the activity of anammox sludge. This work is beneficial for in-depth understanding the role of EPSs in protecting anammox sludge from inhibiting by phenolic pollutants.

A review of heavy metals inhibitory effects in the process of anaerobic ammonium oxidation.

Anaerobic ammonium oxidation (anammox) is a promising biological technology for treating ammonium-rich wastewaters. However, due to the high sensitivity of anammox bacteria, many external factors have inhibitory effects on this process. As one of the commonly found toxic substances in wastewater, heavy metals (HMs) are possible to cause inhibition on anammox sludge, which then results in a declined treatment performance. Getting insights into the response mechanism of anammox sludge to HMs is meaningful for its application in treating this kind of wastewater. This review summarized the effect of different HMs on treatment performance of anammox bioreactor. In addition, the mechanism of toxication raised by HMs was discussed. Also, the potential mitigation strategies were summarized and the future prospects were outlooked. This review might provide useful information for both scientific research on and engineering application of anammox process for treating HMs containing wastewater.

Comprehensive evaluation of the long-term effect of Cu2+ on denitrifying granular sludge and feasibility of in situ recovery by phosphate.

With increased industrial development, vast heavy metals are inevitably discharged into wastewater. Cu2+ is one of the most hazardous heavy metals in biotreatment. However, the potential effect of Cu2+ on denitrifying granular sludge is still unknown. This work assesses the response of denitrifying granular sludge to Cu2+ stress from multiple aspects. The denitrifying granular sludge could tolerate 5 mg L-1 Cu2+, while the nitrogen removal efficiency decreased to 48.5% under 10 mg L-1 Cu2+. Enzyme activity and carbohydrate metabolism were inhibited, and the denitrifying bacteria were washed out under Cu2+ stress. The resulting deteriorated state was reversed by phosphate. The nitrogen removal efficiency recovered to 99% after 10 days, and the enzyme activity also recovered to the original level. Membrane transport, transcription and cellular processes were promoted. Overall, the results of this work provide a feasible strategy to rapidly restore the metabolic activity of denitrifying granular sludge under Cu2+ stress.

Molecular Insight into the Binding Property and Mechanism of Sulfamethoxazole to Extracellular Proteins of Anammox Sludge.

Antibiotics are widely found in nitrogen-containing wastewater, which may affect the operation stability of anaerobic ammonium oxidation (anammox)-based biological treatment systems. Extracellular polymeric substances (EPSs) of anammox sludge play a pivotal role in combining with antibiotics; however, the exact role and how the structure of the leading component of EPSs (i.e., extracellular proteins) changes under antibiotic stress remain to be elucidated. Here, the interaction between sulfamethoxazole and the extracellular proteins of anammox sludge was investigated via multiple spectra and molecular simulation. Results showed that sulfamethoxazole statically quenched the fluorescent components of EPSs, and the quenching constant of the aromatic proteins was the largest, with a value of 1.73 × 104 M-1. The overall binding was an enthalpy-driven process, with ΔH = -75.15 kJ mol-1, ΔS = -0.175 kJ mol-1 K-1, and ΔG = -21.10 kJ mol-1 at 35 °C. The O-P-O and C═O groups responded first under the disturbance of sulfamethoxazole. Excessive sulfamethoxazole (20 mg L-1) would decrease the ratio of α-helix/(ß-sheet + random coil) of extracellular proteins, resulting in a loose structure. Molecular docking and dynamic simulation revealed that extracellular proteins would provide abundant sites to bind with sulfamethoxazole, through hydrogen bond and Pi-Akyl hydrophobic interaction forces. Once sulfamethoxazole penetrates into the cell surface and combines with the transmembrane ammonium transport domain, it may inhibit the NH4+ transport. Our findings enhance the understanding on the interaction of extracellular proteins and sulfamethoxazole, which may be valuable for deciphering the response property of anammox sludge under the antibiotic stress.

Extracellular polymeric substances excreted by anammox sludge act as a barrier for As(III) invasion: Binding property and interaction mechanism.

The arsenic in livestock wastewater would induce adverse impact on the biological treatment technology such as anaerobic ammonium oxidation (anammox) process. Extracellular polymeric substances (EPS) play an important role in resisting such toxicity. Unfortunately, the role of EPS in protecting anammox from As(III) and the mechanisms underlying the protection still remains unclear. This work comprehensively evaluated the acute toxicity of arsenic on anammox sludge and investigated the binding property and interaction mechanism. The results revealed that the half maximal inhibitory concentration (IC50) of As(III) on anammox sludge was estimated to be 408 mg L-1, which decreased to 41.97 mg L-1 when EPS was exfoliated. Complexation and hydrophobic interactions were the leading forces in preventing arsenic invasion. Protein was the main component that complexes with As(III), and O-H, -NH, -CO were binding sites. The response sequence of organic component in EPS to As(III) was ordered as hydrocarbons-proteins-polysaccharides-aliphatic amines.

Linear anionic surfactant (SDBS) destabilized anammox process through sludge disaggregation and metabolic inhibition.

The increase of emerging contaminants, such as surfactants, is one of the major challenges to biological wastewater treatment. However, the potential impact of linear alkylbenzene sulphonates (LAS), a major class of anionic surfactants, on anammox process is unclear. The long-term effects of sodium dodecyl benzene sulfonate (SDBS, as a model LAS) on reactor performance, microbial community and sludge properties were investigated in this study. The presence of 5 mg L-1 SDBS promoted the release of extracellular microbial products from anammox granules and the wash-out of anammox population via effluent. Despite sludge disaggregation, the reactor performance was robust to the exposure of 5 mg L-1 SDBS due to functional redundancy. With the further increase of SDBS to 10 mg L-1, the metabolic activity of anammox biomass and the transcription and post-translation of hydrazine dehydrogenase were significantly decreased. The potential mechanism might be associated with the damage on cell membrane that induced the leakage of intracellular matrix. These results highlight the need to consider the potential risk of LAS to operation of anammox process in biological wastewater treatment plant.

Anammox Granules Acclimatized to Mainstream Conditions Can Achieve a Volumetric Nitrogen Removal Rate Comparable to Sidestream Systems.

The implementation of mainstream anammox has gained increasing attention. In this study, the feasibility of using sidestream anammox granules to start up mainstream reactors was investigated by comparing two switching strategies. A maximum nitrogen removal potential of 3.6 ± 0.2 kg N m-3 d-1 was obtained for the reactor after direct switching to mainstream conditions (70 mg TN L-1, 15 °C). Nevertheless, the reactor preacclimatized to 25 °C (Ma) exhibited a higher nitrogen removal potential of 7.0 ± 0.3 kg N m-3 d-1 at 15 °C, which is the highest volumetric nitrogen removal rate of mainstream anammox reactors to date. Candidatus Kuenenia stuttgartiensis was identified as the dominant anammox bacterium, and its relative abundance in two reactors remained stable throughout the whole operation (200 days). Moreover, with the aid of acclimatization, the activation energy was reduced and the specific growth rate became higher. These results indicated that the physiological evolution of the dominant anammox bacterium instead of interspecies selection was the main reason for the high potential during the switch to mainstream conditions. Therefore, using sidestream anammox granules as seed sludge to start up mainstream reactors was demonstrated to be feasible, and a switching strategy of acclimatization at 25 °C was recommended.

Long-term effects of Fe3O4 NPs on the granule-based anaerobic ammonium oxidation process: Performance, sludge characteristics and microbial community.

The performance of anaerobic ammonium oxidation (anammox) granules were studied under long-term exposure to Fe3O4 NPs. The Fe3O4 NPs had no negative impacts on nitrogen removal performance with the addition of 2-200 mg L-1. The specific anammox activity (SAA) slightly decreased from 287.0 ± 13.2 to -253.0 ± 9.2 mg TN g-1VSS d-1 with the increase in Fe3O4 NPs level from 2 to 60 mg L-1, and then significantly enhanced to 381.8 ± 15.7 mg TN g-1VSS d-1 at 200 mg L-1 Fe3O4 NPs. And the change trends of the heme c content, extracellular polymeric substance amount and settling velocity were consistent with that of SAA. The Candidatus_Kuenenia was the dominant species during the entire experiment and its relative abundance was up to 33.4 % at the end the experiment. The results provide some useful information for comprehending the impact of Fe3O4 NPs on the performance of wastewater biological treatment systems.

Resistance of anammox granular sludge to copper nanoparticles and oxytetracycline and restoration of performance.

Nanoparticles and antibiotics, the two most frequently detected emerging pollutants from different wastewater sources, are eventually discharged into wastewater treatment plants. In this study, the widely used materials CuNPs and oxytetracycline (OTC) were selected as target pollutants to investigate their joint effects on anaerobic ammonium oxidation (anammox). The results indicated that the environmental concentration slightly inhibited the performance of the reactors, while the performance rapidly deteriorated within a week under high-level combined shocks (5.0 mg L-1 CuNPs and 2.0 mg L-1 OTC). After the second shock (2.5 mg L-1 CuNPs and 2.0 mg L-1 OTC), the resistance of anammox bacteria was enhanced, with an elevated relative abundance of Candidatus Kuenenia and absolute abundance of hzsA, nirS, and hdh. Moreover, the extracellular polymeric substance (EPS) content and specific anammox activity (SAA) showed corresponding changes. Improved sludge resistance was observed with increasing CuNP and OTC doses, which accelerated the recovery of performance.

Determination of the response characteristics of anaerobic ammonium oxidation bioreactor disturbed by temperature change with the spectral fingerprint.

Anaerobic ammonium oxidation (anammox) bacteria are sensitive and susceptible to operating condition fluctuations that can lead to the instability of a bioreactor. Through multivariate spectral analysis, the dynamic changes of intracellular and extracellular metabolites of anammox sludge under the declined temperature stress were characterized. It was found that effluent fluorescence components were positively related to the bacterial activity, and the response of the protein-like substances to the temperature change was more sensitive than that of humic substances. Under the transient disturbance during temperature change from 35 to 15 °C, anammox system tended to considerably excrete extracellular polymeric substances to resist the low temperature inhibition. However, the long-term exposure of the sludge at 10 °C resulted in the considerably inhibition of sludge activity, granular disintegration and heterotrophic denitrification bacteria increase. The two-dimensional correlation analysis further revealed that the humic acid in extracellular polymeric substances was preferentially responded to the temperature change than protein. Anammox bacteria tended to increase the intracellular protein and electron transfer-related reactive substance excretion to counteract the low temperature inhibition. Herein, both the intra- and extra-cellular response characteristics of anammox sludge to temperature variation were successfully resolved via the combined spectra. This work provides a comprehensive understanding on the mechanism of anammox sludge to temperature variation and may be valuable for the development of bioreactor monitoring techniques.

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.

Long-term effects of copper nanoparticles on granule-based denitrification systems: Performance, microbial communities, functional genes and sludge properties.

The widespread use of copper nanoparticles (CuNPs) has attracted increasing concern because of their potential effects on biological wastewater treatment. However, their effect on granule-based denitrification systems is unclear. Hence, the effects of CuNPs on denitrifying granules were investigated during long-term operation. The results showed that 51.9% of nitrogen removal capacity was lost after exposure to 5 mg L-1 CuNPs, with the amount of Cu(II) gradually increasing with elevating CuNP levels. Moreover, the relative abundance of denitrifying bacteria (Castellaniella) and denitrifying functional genes (nirK, napA, narG and nosZ) obviously decreased. Meanwhile, the specific denitrification activity, the content of extracellular polymeric substances and dehydrogenase activity decreased by 44.0%, 15.2% and 99.9%, respectively, compared to their values in the initial sludge. Considering the downtrend in the abundance of copper resistance genes, it was deduced that the toxicity of CuNPs was mainly or at least partially due to the release of Cu(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.

Effects of ZnO nanoparticles on high-rate denitrifying granular sludge and the role of phosphate in toxicity attenuation.

The increasing release of engineered nanoparticles (NPs) from consumer products has raised great concerns about their impacts on biological wastewater treatment. In this study, the widely-used ZnO NP was selected as a model NP to investigate its impact on high-rate denitrifying granular sludge in terms of sludge properties and community structure. A hormesis effect was observed during short-term exposure, in which the specific denitrification activity (SDA) was stimulated by 10% at 1 mg L-1 ZnO NPs, but inhibited by 23% at 5.0 mg L-1 ZnO NPs. When continuously exposed to 2.5 mg L-1 ZnO NPs, the nitrogen removal capacity of the denitrification reactor was nearly deprived within 15 days, and the relative abundance of the dominant denitrifying bacterium (Castellaniella) was decreased from 51.0 to 8.0%. Meanwhile, the dehydrogenase activity (DHA) and the content of extracellular polymeric substance (EPS) significantly decreased to 22.3 and 61.1%, respectively. Nevertheless, the presence of phosphate substantially weakened the adverse effects of ZnO NPs on the SDA, EPS, DHA and the relative abundance of functional genes even exposed to 6.25 mg L-1 ZnO NPs, which was associated with the fact that the level of Zn(II) released from ZnO NPs was significantly reduced in the presence of phosphate. Therefore, the toxicity of ZnO NPs may be mainly attributed to the release of toxic Zn(II) and could be attenuated in the presence of phosphate. Overall, this study provided further reference and meaningful insights into the impact of engineered NPs on biological wastewater treatment.

Evaluating the effects of Zn(II) on high-rate biogranule-based denitrification: Performance, microbial community and sludge characteristics.

High-rate denitrification is a popular and efficient process for treatment of nitrate-rich wastewater. Knowing the effect of heavy metals on denitrification is essential for industrial development. In the present study, the long-term impacts of Zn(II) on denitrifying granular sludge were investigated. The suppression threshold of Zn(II) on denitrifying bacteria was 10 mg L-1 for long-term exposure. The nitrogen removal rate was decreased by long-term addition of 10 mg L-1 Zn(II). Castellaniella and Klebsiella were the two dominant genera under Zn(II) stress. The relative abundance of Klebsiella sharply decreased to 4.64% after the addition of 10 mg L-1 Zn(II), whereas Castellaniella was susceptible to 2 mg L-1 Zn(II), revealing that Castellaniella mainly was devoted to denitrification under no or low Zn(II) stress conditions, whereas Klebsiella was effective under high Zn(II) stress.

Deciphering the evolution characteristics of extracellular microbial products from autotrophic and mixotrophic anammox consortia in response to nitrogen loading variations.

Extracellular microbial products (EMP) in biological wastewater treatment systems vary with operational conditions and in turn indicate the metabolic status of functional bacteria. In this study, the response of EMP from autotrophic and mixotrophic anammox consortia (AAC and MAC) to the variation of total nitrogen loading rates (TNLR) were investigated as well as their correlations with the community evolution. The variation of TNLR showed a significantly negative correlation with the production of bound microbial products (BMP) but a significantly positive correlation with the production of soluble microbial products (SMP). The presence of organic matters with COD/TN ratio of 0.15 limited the abundance of anammox bacteria in MAC at the full-load phase and suppressed their proliferation at the restart phase. Due to the improved abundance of carbohydrate metabolism genes, MAC with lower abundance of anammox bacteria produced lower soluble polysaccharides than AMC at the full-load phase. Furthermore, four components (C1-4) were identified on the excitation-emission matrix fluorescence spectra of SMP using parallel factor analysis. C1 exhibited a relative higher proportion at the full-load phase, whereas C4 was generated only at the light-load phase or empty-load phase. At the restart phase, C2 and C3 appeared simultaneously and accounted for a high proportion. The information of four components also suggested the metabolic status of AC as revealed by the specific anammox activity, which therefore provided a novel complementary but direct approach for monitoring the operation status of anammox bioreactors.

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.

Achieving completely anaerobic ammonium removal over nitrite (CAARON) in one single UASB reactor: Synchronous and asynchronous feeding regimes of organic carbon make a difference.

At least 11% of total nitrogen (TN) remains in the anammox effluent, making it difficult to meet increasingly stringent discharge standards. To overcome this bottleneck, an innovative process to achieve completely anaerobic ammonium removal over nitrite (CAARON) in one single up-flow anaerobic sludge blanket reactor was proposed in this study. The synchronous feeding of acetate at a C/N (nitrite) ratio of 0.6 significantly reduced the nitrogen removal capacity of anammox reactor by limiting the abundance and metabolism of anammox bacteria. In contrast, the asynchronous feeding of acetate optimized the partition of the reactor column into two specific compartments: the lower half favoring anammox and the upper half dominated by DEAMOX (DEnitrifying AMmonium Oxidation). A high TN removal efficiency of 96.2±0.4% and a low effluent TN concentration of 9.3±0.9mgL-1 were obtained under a high TN loading rate of 9.0kgNm-3d-1. The dominant functional microbes in the CAARON process were identified as Candidatus Kuenenia and Thauera, which were responsible for the anammox and denitratation reactions, respectively. Overall, the results in this study provide valuable insight into the coupling of anammox with denitratation, which is a cost-efficient approach for treating ammonium-rich wastewaters.