Unraveling potential mechanism of different metal ions effect on anammox through big data analysis, molecular docking and molecular dynamics simulation.

Heavy metals (HMs) have been widely reported to pose an adverse effect on anaerobic ammonia oxidation (anammox) bacteria, yet the underlying mechanisms remain unclear. This study provides new insights into the potential mechanisms of interaction between HMs and functional enzymes through big date analysis, molecular docking and molecular dynamics simulation. The statistical analysis indicated that 10 mg/L Cu(II) and Cd(II) reduced nitrogen removal rate (NRR) by 85% and 43%, while 5 mg/L Fe(II) enhanced NRR by 29%. Additionally, the results of molecular simulations provided a microscopic interpretation for these macroscopic data. Molecular docking revealed that Hg(II) formed a distinctive binding site on ferritin, while other HMs resided at iron oxidation sites. Furthermore, HMs exhibited distinct binding sites on hydrazine dehydrogenase. Concurrently, the molecular dynamics simulation results further substantiated their capacity to form complexes. Cu(II) displayed the strongest binding affinity with ferritin for -1576 ± 79 kJ/mol in binding free energy calculation. Moreover, Cd(II) bound to ferritin and HDH for -1052.67 ± 58.49 kJ/mol, -290.02 ± 49.68 kJ/mol, respectively. This research addressed a crucial knowledge gap, shedding light on potential applications for remediating heavy metal-laden industrial wastewater.

Spatiotemporal Assembly and Immigration of Heterotrophic and Anammox Bacteria Allow a Robust Synergy for High-Rate Nitrogen Removal.

The novel partial denitrification-driven anammox (PD/A) is an energy-efficient method for nitrogen removal from wastewater. However, its stability and efficiency are impeded by the competition between heterotrophic denitrifying bacteria and relatively slow-growing anammox bacteria. In this study, a PD/A granular sludge system was developed, which achieved a nitrogen removal efficiency of 94% with 98% anammox contribution, even as the temperature dropped to 9.6 °C. Analysis of bacterial activity in aggregates of different sizes revealed that the largest granules (>2.0 mm) exhibited the highest anammox activity, 2.8 times that of flocs (<0.2 mm), while the flocs showed significantly higher nitrite production rates of PD, more than six times that of the largest granules. Interestingly, fluorescent in situ hybridization (FISH) combined with confocal laser scanning microscopy (CLSM) revealed a nest-shaped structure of PD/A granules. The Thauera genus, a key contributor to PD, was highly enriched at the outer edge, providing substrate nitrite for anammox bacteria inside the granules. As temperature decreased, the flocs transformed into small granules to efficiently retain anammox bacteria. This study provides multidimensional insights into the spatiotemporal assembly and immigration of heterotrophic and autotrophic bacteria for stable and high-rate nitrogen removal.

Beyond an Applicable Rate in Low-Strength Wastewater Treatment by Anammox: Motivated Labor at an Extremely Short Hydraulic Retention Time.

The application of anammox technology in low-strength wastewater treatment is still challenging due to unstable nitrite (NO2--N) generation. Partial denitrification (PD) of nitrate (NO3--N) reduction ending with NO2--N provides a promising solution. However, little is known about the feasibility of accelerating nitrogen removal toward the practical application of anammox combined with heterotrophic denitrification. In this work, an ultrafast, highly stable, and impressive nitrogen removal performance was demonstrated in the PD coupling with an anammox (PD/A) system. With a low-strength influent [50 mg/L each of ammonia (NH4+-N) and NO3--N] at a low chemical oxygen demand/NO3--N ratio of 2.2, the hydraulic retention time could be shortened from 16.0 to 1.0 h. Remarkable nitrogen removal rates of 1.28 kg N/(m3 d) and excellent total nitrogen removal efficiency of 94.1% were achieved, far exceeding the applicable capacity for mainstream treatment. Stimulated enzymatic reaction activity of anammox was obtained due to the fast NO2--N jump followed by a famine condition with limited organic carbon utilization. This high-rate PD/A system exhibited efficient renewal of bacteria with a short sludge retention time. The 16S rRNA sequencing unraveled the rapid growth of the genus Thauera, possibly responsible for the incomplete reduction of NO3--N to NO2--N and a decreasing abundance of anammox bacteria. This provides new insights into the practical application of the PD/A process in the energy-efficient treatment of low-strength wastewater with less land occupancy and desirable effluent quality.

Flexible Nitrite Supply Alternative for Mainstream Anammox: Advances in Enhancing Process Stability.

Anaerobic ammonium oxidation (anammox) has attracted extensive attention as a potentially sustainable and economical municipal wastewater treatment process. However, its large-scale application is limited by unstable nitrite (NO2--N) production and associated excessive nitrate (NO3--N) residue. Thus, our study sought to evaluate an efficient alternative to the current nitritation-based anammox process substituting NO2--N supply by partial-denitrification (PD; NO3--N → NO2--N) under mainstream conditions. Ammonia (NH4+-N) was partly oxidized to NO3--N and removed via a PD coupled anammox (PD/A) process by mixing the nitrifying effluents with raw wastewater (NH4+-N of 57.87 mg L-1, COD of 176.02 mg L-1). Excellent effluent quality was obtained with< 5 mg L-1 of total nitrogen (TN) despite frequent temperature fluctuations (25.7-16.3 °C). The genus Thauera (responsible for PD) was the dominant denitrifiers (36.4%-37.4%) and coexisted with Candidatus Brocadia (anammox bacteria; 0.33%-0.46%). The efficient PD/A allowed up to 50% reduction in aeration energy consumption, 80% decrease in organic resource demand, and lower nitrous oxide (N2O) production compared to conventional nitrification/denitrification process. Our study demonstrates that coupling anammox with flexible NO2--N supply has great potential as a stable and efficient mainstream wastewater treatment.

Primary sludge as solid carbon source for biological denitrification: System optimization at micro-level.

Commercial carbon source (e.g. methanol) has been frequently used to enhance heterotrophic denitrification for nitrate removal. However, this is not sustainable due to the high cost of chemical purchasing and excessive sludge production. To address these issues, this study reports an integrated denitrification system using primary sludge as solid carbon source. Complete denitrification without any nitrite accumulation achieved at the primary sludge dosage of 6.0 g VSS/g N with the maximum specific nitrate reduction rate of 6.4 mg N/g VSS/h, which was comparable with the reported soluble carbon source. More importantly, as a solid "waste" in municipal wastewater treatment plants (WWTPs), the primary sludge was simultaneously reduced by 65.3%-85.1%, and this avoids the intensive denitrification biomass generation that generally occurs in using the commercial carbon source. Ammonium, phosphate, and recalcitrant organic matter were released meantime. Interestingly, the concentration of ammonium and phosphate declined during the denitrification process. The refractory dissolved organics mainly composed of aromatic protein and microbial by-products. The detailed cycle study suggests that an appropriate denitrification cycle/duration time would largely lower the effluent organics concentration, which can be achieved by monitoring the pH turning point. This study clearly demonstrates that primary sludge is a promising alternative carbon source for biological denitrification with great economic benefits and environmental sustainability.

Correction to: Mechanisms and microbial structure of partial denitrification with high nitrite accumulation.

In the published version of the above article, the affiliation of one of the co-authors (Baikun Li) is incorrect.

Correction to: High-throughput profiling of microbial community structures in an ANAMMOX-UASB reactor treating high-strength wastewater.

The published online version contains mistake in the affiliation ID of the author Baikun Li. The correct presentation is given above.

New direction in biological nitrogen removal from industrial nitrate wastewater via anammox.

Anaerobic ammonium oxidation (anammox) is an important scientific discovery in the field of wastewater treatment. This process is a sustainable option in nitrogen removal due to its energy-efficient and cost-effective advantage. Great effort has been made recently to remove ammonium from industrial and municipal wastewater via the anammox process with a preceding partial nitrification (PN) converting part of NH4+ to NO2-. Anammox process is seldom involved in the nitrate removal. Nitrate (NO3-), one of the main nitrogen compounds produced from various industries, is typically converted to nitrogen gas via denitrification process where a large amount of carbon source is consumed. Within this context, we reviewed the current technologies for high-strength nitrate wastewater treatment. It is found that nitrite accumulation often occurs during nitrate reduction, and its accumulating level would be increased at certain conditions (i.e., low C/N ratio and high pH). Hence, this provides a great opportunity to employ the anammox process to further convert nitrite in a more sustainable way. In this review, we highlight a new approach for industrial nitrate wastewater treatment via partial denitrification coupled with anammox process (PD-A). We also discuss the conditions to achieve successful PD-A process, economic and environmental benefits, and potential challenges as well as the future perspectives in practical application.

High-throughput profiling of microbial community structures in an ANAMMOX-UASB reactor treating high-strength wastewater.

In this study, the microbial community structure was assessed in an anaerobic ammonium oxidation-upflow anaerobic sludge blanket (ANAMMOX-UASB) reactor treating high-strength wastewater (approximately 700 mg N L(-1) in total nitrogen) by employing Illumina high-throughput sequencing analysis. The reactor was started up and reached a steady state in 26 days by seeding mature ANAMMOX granules, and a high nitrogen removal rate (NRR) of 2.96 kg N m(-3) day(-1) was obtained at 13.2∼17.6 °C. Results revealed that the abundance of ANAMMOX bacteria increased during the operation, though it occupied a low proportion in the system. The phylum Planctomycetes was only 8.39 % on day 148 and Candidatus Brocadia was identified as the dominant ANAMMOX species with a percentage of 2.70 %. The phylum of Chloroflexi, Bacteroidetes, and Proteobacteria constituted a percentage up to 70 % in the community, of which the Chloroflexi and Bacteroidetes were likely to be related to the sludge granulation. In addition, it was found that heterotrophic denitrifying bacteria of Denitratisoma belonging to Proteobacteria phylum occupied a large proportion (22.1∼23.58 %), which was likely caused by the bacteria lysis and decay with the internal carbon source production. The SEM images also showed that plenty of other microorganisms existed in the ANAMMOX-UASB reactor.

Mechanisms and microbial structure of partial denitrification with high nitrite accumulation.

Nitrite (NO2 (-)-N) accumulation in denitrification can provide the substrate for anammox, an efficient and cost-saving process for nitrogen removal from wastewater. This batch-mode study aimed at achieving high NO2 (-)-N accumulation over long-term operation with the acetate as sole organic carbon source and elucidating the mechanisms of NO2 (-)-N accumulation. The results showed that the specific nitrate (NO3 (-)-N) reduction rate (59.61 mg N VSS(-1) h(-1) at NO3 (-)-N of 20 mg/L) was much higher than specific NO2 (-)-N reduction rate (7.30 mg N VSS(-1) h(-1) at NO3 (-)-N of 20 mg/L), and the NO2 (-)-N accumulation proceeded well at the NO3 (-)-N to NO2 (-)-N transformation ratio (NTR) as high as 90 %. NO2 (-)-N accumulation was barely affected by the ratio of chemical oxygen demand (COD) to NO3 (-)-N concentration (C/N). With the addition of NO3 (-)-N, NO2 (-)-N accumulation occurred and the specific NO2 (-)-N reduction rate declined to a much lower level compared with the value in the absence of NO3 (-)-N. This indicated that the denitrifying bacteria in the system preferred to use NO3 (-)-N as electron acceptor rather than use NO2 (-)-N. In addition, the Illumina high-throughput sequencing analysis revealed that the genus of Thauera bacteria was dominant in the denitrifying community with high NO2 (-)-N accumulation and account for 67.25 % of total microorganism. This bacterium might be functional for high NO2 (-)-N accumulation in the presence of NO3 (-)-N.

Reactivation performance and sludge transformation after long-term storage of Partial denitrification/Anammox (PD/A) process.

This study explores the startup of innovative Partial denitrification/Anammox (PD/A) process using long-term stored sludge (>2 years at 4 °C). Results indicate a swift recovery performance, characterized by a progressive increase in the activity of functional microorganisms with improved nitrogen volumetric loading rate during operation. Stable nitrogen removal efficiency of 99.6 % was attained at 14.2 °C under influent nitrate and ammonium of 120 and 100 mg/L, respectively. A distinctive transformation was observed as the initially black seeding sludge transitioned to brownish-red, accompanied by rapid sludge granulation with size notably increased from 263.1 µm (day 4) to 1255.0 µm (day 128), significantly contributing to the rapid PD/A performance recovery. Microbial community analysis revealed substantial increases in functional bacteria, Thauera (0.09 %-10.4 %) and Candidatus Brocadia (0.003 %-1.98 %), coinciding with enhanced nitrogen removal performance. Overall, this study underscores the viability of long-term stored PD/A sludge as a seed for rapid reactor startup, offering useful technical support to advance practical PD/A process implementation.

Selective genes expression and metabolites transformation drive a robust nitrite accumulation during nitrate reduction under alternating feast-famine condition.

Nitrite production via denitrification has been regarded as a key approach for survival of anaerobic ammonium oxidation (anammox) bacteria. Despite the important carbon substrate, little is known about the role of differential genes expression and extracellular metabolite regulation among diverse microbial communities. In this study, a novel alternating feast-famine strategy was proposed and demonstrated to efficiently accumulate nitrite in a low-nitrogen loading rate (NLR) (0.2∼0.8 kg N/m3/d) denitrification system. Highly selective expression of denitrifying genes was revealed as key regulators. Interestingly, in absence of carbon source (ACS) condition, the expression of narG and narI/V genes responsible for reduction of nitrate to nitrite jumped to 2.5 and 5.1 times higher than that in presence of carbon source (PCS) condition with carbon to nitrate ratio of 3.0. This fortunately facilitated a rapid nitrite accumulation once acetate was added, despite a significantly down-regulated narG and narI/narV and up-regulated nirS/nirK. This strategy selected Thauera as the most dominant denitrifier (50.2 %) with the highest contribution to narG and narI/narV genes, responsible for the high nitrite accumulation. Additionally, extracellular xylose, pyruvate, and glucose jointly promoted carbon-central metabolic pathway of key denitrifiers in ACS stage, playing an important role in the process of self-growth and selective enrichment of functional bacteria. The relatively rapid establishment and robust performance obtained in this study shows an engineering-feasible and economically-favorable solution for the regulation of partial denitrification in practical application.

Stable continuous flow CANDAN process transitioning from anammox UASB reactor by facilitating indigenous nitrite-producing denitrification community.

The emerging nitrogen removal process known as CANDAN (Complete Ammonium and Nitrate removal via Denitratation-Anammox over Nitrite) has been developed in Sequencing Batch Reactors (SBRs). Yet, starting up and maintaining stability in continuous-flow reactors remain challenging. This study explores the feasibility of transitioning the CANDAN process from an anammox-dominated process by introducing appropriate external organics to facilitate indigenous nitrite-producing denitrification community in an Upflow Anaerobic Sludge Blanket (UASB) reactor. 150-day operation results indicate that under feeding rates of domestic wastewater at 0.54 L/h and nitrate-containing wastewater at 1.08 L/h, excellent N removal was achieved, with effluent TN below 10.0 mg N/L. Adding external sodium acetate at a COD/NO3--N = 2.0 triggered denitratation, ex-situ denitrification activity tests showed increased nitrite production rates, maintaining the nitrate-to-nitrite transformation ratio (NTR) above 90 %. Consequently, anammox activity was consistently maintained, dominating Total Nitrogen (TN) removal with a contribution as high as 78.3 ± 8.0 %. Anammox functional bacteria, Brocadia and Kuenenia were identified and showed no decrease throughout the operation, indicating the robustness of the anammox process. Notably, the troublesome of sludge flotation, did not occur, also contributing to sustained outstanding performance. In conclusion, this study advances our understanding of the synergistic interplay between anammox and denitrifying bacteria in the Anammox-UASB system, offering technical insights for establishing a stable continuous-flow CANDAN process for simultaneous ammonium and nitrate removal.

Deciphering retention effect of extracellular polymeric substances to typical heavy metals and their interaction with key inner enzymes of Candidatus Kuenenia.

This study employed spectroscopy, metagenomics, and molecular simulation to investigate the inhibitory effects of Cd(II) and Cu(II) on the anammox system, examining both intracellular and extracellular effects. At concentrations of 5 mg/L, Cd(II) and Cu(II) significantly reduced nitrogen removal efficiency by 41.46 % and 62.03 %, respectively. Additionally, elevated metal concentrations were correlated with decreased extracellular polymeric substances (EPS), thereby reducing their capacity to absorb heavy metals, particularly Cu(II), which decreased from 76.47 % to 14.67 %. Spectral analysis revealed alterations in the secondary structures of EPS induced by Cd(II) and Cu(II), decreasing the ratio of extracellular protein α-helix to (ß-sheet + random coil), which resulted in looser extracellular protein configurations. The results of the metagenomics study showed that the abundance of Candidatus Kuenenia and its genes encoding nitrogen removal-related enzymes was reduced. The abundance of hzs-γ was reduced by 35.09 % at a concentration of 5 mg/L Cu(II). Conversely, genes associated with metal efflux enzymes, like czcR, increased by 54.86 % at 2 mg/L Cd(II). Molecular docking revealed robust bindings of Cd(II) to HZS-α (-342.299 ± 218.165 kJ/mol) and Cu(II) to HZS-γ (-880.934 ± 55.526 kJ/mol). This study elucidated the inhibitory mechanisms of Cd(II) and Cu(II) on the anammox system, providing insights into the resistance of anammox bacteria to heavy metals.

Redox mediator chlorophyll accelerates low-temperature biological denitrification with responses of extracellular polymers and changes in microbial community composition.

Low temperatures limit the denitrification wastewater in activated sludge systems, but this can be mitigated by addition of redox mediators (RMs). Here, the effects of chlorophyll (Chl), 1,2-naphthoquinone-4-sulfonic acid (NQS), humic acid (HA), and riboflavin (RF), each tested at three concentrations, were compared for denitrification performance at low temperature, by monitoring the produced extracellular polymeric substances (EPS), and characterizing microbial communities and their metabolic potential. Chl increased the denitrification rate most, namely 4.12-fold compared to the control, followed by NQS (2.62-fold increase) and HA (1.35-fold increase), but RF had an inhibitory effect. Chl promoted the secretion of tryptophan-like and tyrosine-like proteins in the EPS and aided the conversion of protein from tightly bound EPS into loosely bound EPS, which improved the material transfer efficiency. NQS, HA, and RF also altered the EPS components. The four RMs affected the microbial community structure, whereby both conditionally abundant taxa (CAT) and conditionally rare or abundant taxa (CRAT) were key taxa. Among them, CRAT members interacted most with the other taxa. Chl promoted Flavobacterium enrichment in low-temperature activated sludge systems. In addition, Chl promoted the abundance of nitrate reduction genes narGHI and napAB and of nitrite reduction genes nirKS, norBC, and nosZ. Moreover, Chl increased abundance of genes involved in acetate metabolism and in the TCA cycle, thereby improving carbon source utilization. This study increases our understanding of the enhancement of low-temperature activated sludge by RMs, and demonstrates positive effects, in particular by Chl.

Microecology of aerobic denitrification system construction driven by cyclic stress of sulfamethoxazole.

The construction of aerobic denitrification (AD) systems in an antibiotic-stressed environment is a serious challenge. This study investigated strategy of cyclic stress with concentration gradient (5-30 mg/L) of sulfamethoxazole (SMX) in a sequencing batch reactor (SBR), to achieve operation of AD. Total nitrogen removal efficiency of system increased from about 10 % to 95 %. Original response of abundant-rare genera to antibiotics was changed by SMX stress, particularly conditionally rare or abundant taxa (CRAT). AD process depends on synergistic effect of heterotrophic nitrifying aerobic denitrification bacteria (Paracoccus, Thauera, Hypomicrobium, etc). AmoABC, napA, and nirK were functionally co-expressed with multiple antibiotic resistance genes (ARGs) (acrR, ereAB, and mdtO), facilitating AD process. ARGs and TCA cycling synergistically enhance the antioxidant and electron transport capacities of AD process. Antibiotic efflux pump mechanism played an important role in operation of AD. The study provides strong support for regulating activated sludge to achieve in situ AD function.

Potential causes of partial-denitrification (PD) granular sludge breakdown under high nitrate loading rates.

The granule instability has been frequently reported during the operation of high loading rates. While, there no research was performed on the recently developed anoxic partial-denitrification (PD) granules, a novel pathway in producing nitrite from nitrate for anammox process. Herein, this work, for the first time, investigated the influence of nitrate loading rates on the instability of PD granules and identified the key causes. Two lab-scale sequencing batch reactors (SBRs) were operated with nitrate loading rates (NLR) increased from 0.48 to 3.84 kg N/m3/d (R1, 8 cycles/d), and 0.96 to 7.68 kg N/m3/d (R2, 16 cycles/d) by gradually elevating the influent nitrate concentration. Results showed that nitrite production rates increased with the NLRs, with a maximal value of 5.26 kg N/m3/d obtained. However, the compact regular PD granules were not stable and broke down when NLR was above 3.84 kg N/m3/d, which resulted in serious sludge washing out from SBR. The high NLRs led to the extracellular polymeric substances (EPS) transformation in terms of its composition and structure, which the protein content in the EPS and the tightly bound EPS (T-EPS) fraction was significantly decreased, this was supposed to be the major reason causing the breakdown of PD granules. Besides, it was found the PD granule in R2 was more deteriorated than that in R1 under the same high NLR, suggesting the short starvation (idle) times in SBR cycle was likely another reason impairing the stability of PD granules. Overall, this research provides useful information in development of granule-based PD systems and sheds light on achieving high-rate nitrite production in SBR with great stability.