External redox couple enhanced anammox sludge activity at low temperature: Insight into intracellular resource synthesis.

Anaerobic ammonium oxidation (anammox), an energy-efficient deamination biotechnology, faces operational challenges in low-temperature environments. Enhancing the metabolic activity of anammox bacteria (AnAOB) is pivotal for advancing its application in mainstream municipal wastewater treatment. Inspired by the metabolic adaptability of AnAOB and based on our previous findings, this work investigated the enhancement of intracellular ATP and NADH synthesis through the exogenous supply of reduced humic acid (HAred) and H2O2 redox couple, aiming to augment AnAOB activity under low-temperature conditions. Our experimental setup involved continuous dosing of 0.0067 µmol g-1 volatile suspended solid of H2O2 and 10 mg g-1 volatile suspended solid of HAred into a mainstream anammox reactor operated at 15 °C with an influent TN content of 60 mg/L. The results showed that HAred / H2O2 couple succeeded in maintaining the effluent TN at 10.72 ± 0.91 mg l-1. The specific anammox activity, ATP and NADH synthesis levels of sludge increased by 1.34, 2.33 and 6.50 folds, respectively, over the control setup devoid of the redox couple. High-throughput sequencing analysis revealed that the relative abundance of Candidatus Kuenenia after adding HAred / H2O2 couple reached 3.65 % at the end of operation, which was 5.14 folds higher than that of the control group. Further metabolomics analysis underscored an activation in the metabolism of amino acids, nucleotides, and phospholipids, which collectively enhanced the availability of ATP and NADH for the respiratory processes. These findings may provide guidance on strategy development for improving the electron transfer efficiency of AnAOB and underscore the potential of using redox couples to promote the mainstream application of anammox technology.

A new substrate equalization method for optimizing the influent conditions and fluid flow patterns of a multifed upflow anaerobic sludge blanket reactor with mature anammox granules.

To improve nitrogen removal efficiency (NRE) and achieve homogenous distribution of anammox sludge and substrate, a new substrate equalization theory and a cumulative overload index was proposed for multifed upflow anaerobic sludge bed (MUASB) reactors with mature anammox granules. The performance and flow patterns of MUASB reactors were investigated under various influent conditions. The results showed that the nitrogen removal performance and stability of MUASB reactors could be optimized by minimizing the cumulative load. The NRE gradually increased from 83.3 ± 2.2 %, 86.8 ± 4.2 % to 89.3 ± 4.1 % and 89.7 ± 1.6 % in feeding flow tests and feeding port tests, respectively. Furthermore, the flow patterns were compared based on residence time distribution and computational fluid dynamics, indicating that a better equilibrium distribution of microorganisms and substrates could be achieved in the MUASB reactors under the lowest cumulative load. Therefore, substrate equalization theory can be used to optimize the nitrogen removal performance of MUASB reactors with low-carbon footprints.

Microbial response and recovery strategy of the anammox process under ciprofloxacin stress from pure strain and consortia perspectives.

Ciprofloxacin (CIP) poses a high risk of resistance development in water environments. Therefore, comprehensive effects and recovery strategies of CIP in anaerobic ammonia oxidation (anammox) process were systematically elucidated from consortia and pure strains perspectives. The anammox consortia was not significantly affected by the stress of 10 mg L-1 CIP, while the higher concentration (20 mg L-1) of CIP caused a dramatic reduction in the nitrogen removal performance of anammox system. Simultaneously, the abundances of dominant functional bacteria and corresponding genes also significantly decreased. Such inhibition could not be mitigated by the recovery strategy of adding hydrazine and hydroxylamine. Reducing nitrogen load rate from 5.1 to 1.4 kg N m-3 d-1 promoted the restoration of three reactors. In addition, the robustness and recovery of anammox systems was evaluated using starvation and shock strategies. Simultaneously, antibiotic resistance genes and key metabolic pathways of anammox consortia were upregulated, such as carbohydrate and energy metabolisms. In addition, 11 pure stains were isolated from the anammox system and identified through phylogenetic analysis, 40 % of which showed multidrug resistance, especially Pseudomonas. These findings provide deep insights into the responding mechanism of anammox consortia to CIP stress and promote the application of anammox process for treating wastewater containing antibiotics.

Feasibility of anaerobic ammonium oxidation process for treatment of pretreated printed circuit board wastewater.

The treatment of pretreated printed circuit board (PCB) wastewater by anaerobic ammonium oxidation (anammox) process has been rarely reported. This study sought to investigate the performance of the anammox process during various phases of pretreated PCB wastewater treatment. The nitrogen removal efficiency (NRE) reached 90 ± 10% at a Cu2+ concentration of 2.5 mg·L-1, but declined to 22 ± 11% as the Cu2+ level increased to 10.3  mg·L-1. During phase III, there was a 38% increase in the relative abundance of Candidatus Kuenenia compared to phase I. By adjusting the substrate concentration and introducing synthetic wastewater into the reactor, the anammox performance was nearly restored to that of phase I. These findings underscore the potential of the anammox process for treating pretreated PCB wastewater and expanding its practical applications to industrial wastewater treatment.

Light-Driven Electron Uptake from Nonfermentative Organic Matter to Expedite Nitrogen Dissimilation by Chemolithotrophic Anammox Consortia.

Nonphotosynthetic microorganisms are typically unable to directly utilize light energy, but light might change the metabolic pathway of these bacteria indirectly by forming intermediates such as reactive oxygen species (ROS). This work investigated the role of light on nitrogen conversion by anaerobic ammonium oxidation (anammox) consortia. The results showed that high intensity light (>20000 lx) caused ca. 50% inhibition of anammox activity, and total ROS reached 167% at 60,000 lx. Surprisingly, 200 lx light was found to induce unexpected promotion of the nitrogen conversion rate, and ultraviolet light (<420 nm) was identified as the main contributor. Metagenomic and metatranscriptomic analyses revealed that the gene encoding cytochrome c peroxidase was highly expressed only under 200 lx light. 15N isotope tracing, gene abundance quantification, and external H2O2 addition experiments showed that photoinduced trace H2O2 triggered cytochrome c peroxidase expression to take up electrons from extracellular nonfermentative organics to synthesize NADH and ATP, thereby expediting nitrogen dissimulation of anammox consortia. External supplying reduced humic acid into a low-intensity light exposure system would result in a maximal 1.7-fold increase in the nitrogen conversion rate. These interesting findings may provide insight into the niche differentiation and widespread nature of anammox bacteria in natural ecotopes.

Research advances in application of mainstream anammox processes: Roles of quorum sensing and microbial metabolism.

Anaerobic ammonium oxidation (anammox) is a low-carbon biological nitrogen removal process, that has been widely applied to treat high-strength wastewater. However, the practical application of mainstream anammox treatment is limited due to the slow growth rate of anammox bacteria (AnAOB). Therefore, it is important to provide a comprehensive summary of the potential impacts and regulatory strategies for system stability. This article systematically reviewed the effects of environmental fluctuations on anammox systems, summarizing the bacterial metabolisms and the relationship between metabolite and microbial functional effects. To address the shortcoming of mainstream anammox process, molecular strategies based on quorum sensing (QS) were proposed. Sludge granulation, gel encapsulation and carrier-based biofilm technologies were adopted to enhance the QS function in microbial aggregation and reduction of biomass loss. Furthermore, this article discussed the application and progress of anammox-coupled processes. Valuable insights were provided for the stable operation and development of mainstream anammox process from the perspectives of QS and microbial metabolism.

Deciphering endogenous and exogenous regulations of anammox consortia in responding to lincomycin by multiomics: quorum sensing and CRISPR system.

The widespread use of antibiotics has created an antibiotic resistance genes (ARGs)-enriched environment, which causes high risks on human and animal health. Although antibiotics can be partially adsorbed and degraded in wastewater treatment processes, striving for a complete understanding of the microbial adaptive mechanism to antibiotic stress remains urgent. Combined with metagenomics and metabolomics, this study revealed that anammox consortia could adapt to lincomycin by spontaneously changing the preference for metabolite utilization and establishing interactions with eukaryotes, such as Ascomycota and Basidiomycota. Specifically, quorum sensing (QS) based microbial regulation and the ARGs transfer mediated by clustered regularly interspaced short palindromic repeats (CRISPR) system and global regulatory genes were the principal adaptive strategies. Western blotting results validated that Cas9 and TrfA were mainly responsible for the alteration of ARGs transfer pathway. These findings highlight the potential adaptative mechanism of microbes to antibiotic stress and fill gaps in horizontal gene transfer pathways in the anammox process, further facilitating the ARGs control through molecular and synthetic biology techniques.

Evaluation the role of soluble microbial products for denitrification sludge characteristic under starvation stress.

Physiological changes with the assist role of soluble microbial products (SMP) of preserved denitrifying sludge (DS) undergoing long-term stress of starvation under different storage temperature is extremely important. In this study, SMP extracted from DS were added into DS in starvation condition under room temperature (15-20 °C), 4 °C and -20 °C with three different bio-augmentation phases of 10, 15 and 30 days. Experimental results showed that added SMP in room temperature was optimal for preservation of DS under starvation stress with optimized dosage of 2.0 mL mL-1 sludge and bio-augmentation phase of 10 d. SMP was more effective in maintaining the specific denitrification activity of DS, and it was nearly boosted to 94.1 % of control one due to assist of 2 times SMP addition with 10 days interval of each. Under assist of SMP, extracellular polymeric substances (EPS) secretion was enhanced as the defense layer to withstand starvation stress, and the protein may be utilized as an alternative substrate to gain energy, accelerate electron transport and transfer during denitrification process. This investigation revealed the feasibility of SMP as an economical and robust strategy for preservation of DS.

Role of quorum sensing-based regulation in development of anaerobic ammonium oxidation process.

Shortage of anaerobic ammonium oxidation (anammox) sludge greatly limits the extensive full-scale application of anammox-based processes. Although numerous start-up strategies have been proposed, the interaction among microbial consortia and corresponding mechanism during the process development remain unknown. In this study, three reactors were established based on different seed sludges. After 27 days, the anammox process inoculated with anammox granules and activated sludge (1:5) was firstly achieved, and the highest nitrogen removal rate was 1.17 kg N m-3 d-1. Correspondingly, the anammox activity and abundances of related functional genes increased. Notably, the dominant anammox bacteria shifted from Candidatus Kuenenia to Candidatus Brocadia. Metagenomic analysis indicated that quorum sensing-based regulation mainly contributed to the proliferation and accumulation of anammox bacteria. This work provides an insight into the quorum sensing (QS)-regulated microbial interactions in the anammox and activated sludge consortia during the process development.

Decoding the response of complete autotrophic nitrogen removal over nitrite (CANON) performance and microbial succession to hydrazine and hydroxylamine: Linking performance to mechanism.

The influence of hydrazine (N2H4) and hydroxylamine (NH2OH) on performance and microbial community structures of complete autotrophic nitrogen removal over nitrite (CANON) process were assessed in this study. Experimental results demonstrated that CANON process was successfully started up and got total nitrogen removal efficiency (TNRE) of 53.6 % ± 9.8 % and 56.4 % ± 6.5 % under 1.0 and 0.5 mg L-1 N2H4 and NH2OH, respectively. N2H4 and NH2OH promoted activity of ammonia-oxidizing bacteria (AOB) and anaerobic ammonium oxidation bacteria (AnAOB), and inhibited activity of nitrite-oxidizing bacteria (NOB). Meanwhile, the stable operation of CANON process could be maintained without N2H4 auxiliary. While, performance assisted by NH2OH was fluctuated without NH2OH addition, suggesting that both N2H4 and NH2OH had a persistent and reversible inhibition on NOB. This study reveals new insights into influence of N2H4 and NH2OH on CANON performance.

A review of anammox-based nitrogen removal technology: From microbial diversity to engineering applications.

The anaerobic ammonium oxidation (anammox) process has the advantages of high efficiency and low energy consumption, so it has broad application prospects in biological denitrification of wastewater. However, the application of anammox technology to existing wastewater treatment is still challenging. The main problems are the insufficient supply of nitrite and the susceptibility of anammox bacteria to environmental factors. In this paper, from the perspective of the diversity of anammox bacteria, the habitats and characteristics of anammox bacteria of different genera were compared. At the same time, laboratory research and engineering applications of anammox technology in treating wastewater from different sources were reviewed, and the progress of and obstacles to the practical application of anammox technology were clarified. Finally, a focus for future research was proposed to intensively study the water quality barrier factors of anammox and its regulation strategies. Meanwhile, a combined process was developed and optimized on this basis.

Inorganic quantum dots - anammox consortia hybrid for stable nitrogen elimination under high-intensity solar-simulated irradiation.

External stimulus such as light irradiation is able to deteriorate intracellular redox homeostasis and induce photooxidative damage to non-photogenic bacteria. Exploiting effective strategies to help bacteria resisting infaust stress is meaningful for achieving a stable operation of biological treatment system. In this work, selenium-doped carbon quantum dots (Se-CQDs) were blended into anaerobic ammonia oxidation (anammox) bacteria and an inorganic nanoparticle-microbe hybrid was successfully fabricated to evaluate its nitrogen removal performance under solar-simulated irradiation. It was found that the specific anammox activity decreased by 29.7 ± 5.2% and reactive oxygen species (ROS) content increased by 134.8 ± 4.1% under 50,000 lux light. Sludge activity could be completely recovered under the optimum dosage of 0.42 mL·(g volatile suspended solid) -1 Se-CQDs. Hydroxyl radical (·OH) and superoxide anion radical (·O2-) were identified as the leading ROS inducing lipid peroxidation and antioxidase function detriment. Also, the structure of ladderane lipids located on anammoxosome was destroyed by ROS and functional genes abundances declined accordingly. Although cell surface coated Se-CQDs could absorb ultraviolet light and partially mitigated the photoinhibition, the direct scavenging of ROS by intracellular Se-CQDs primarily contributed to the cellular redox homeostasis, antioxidase activity recovery and sludge activity improvement. The findings of this work provide in-depth understanding the metabolic response mechanism of anammox consortia to light irradiation and might be valuable for a more stable and sustainable nitrogen removal technology, i.e., algal-bacterial symbiotic system, development.

Mitigating the detrimental effects of salt stress on anammox process: A comparison between glycine betaine and mannitol.

The use of seawater to alleviate water shortages causes an increase of salinity in municipal pipe networks, posing challenges for biological wastewater treatment. The impacts of two compatible solutes on the anammox process under salt stress (20 g L-1) were compared here at the genetic and microbial levels. The findings revealed that both 0.3 mM glycine betaine (GB) and mannitol (MA) could alleviate the salt stress on anammox process, with GB exhibiting a better effect. Specifically, the addition of GB recovered the nitrogen removal efficiency (NRE) from 40 % to >80 % within 13 days. The addition of MA caused the reduction of the absolute abundance of hdh and hzsA, implying that 0.6 mM was not the optimal concentration. Moreover, salt stress induced an increase in the absolute abundance of nitrification functional genes and a decrease in the abundance of denitrification functional genes. Notably, compared with the initial level, the abundance of Candidatus Kuenenia increased by 7.1 % and 4.3 % after adding GB and MA, respectively. According to the network analysis, two compatible solutes promoted the bacterial interactions in anammox systems, which promoted the nitrogen circulation and further the nitrogen removal performance. This work provides a feasible strategy to relieve the salt stress on anammox process and then facilitates its application for treating saline wastewater.

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.

Removal of extracellular deoxyribonucleic acid increases the permeability and mass transfer of anammox granular sludge with different sizes.

As a key component of extracellular polymeric substances (EPS), extracellular deoxyribonucleic acid (eDNA) acts as a bridge in maintaining the structural stability of granular sludge. However, its ability of carrying antibiotic resistance genes (ARGs) promotes the gene horizontal transfer, raising a high risk for human health. In this study, a series of batch tests were performed to elucidate the response of anammox granular sludge (AnGS) with different sizes (S-AnGS with diameters lower than 0.9 mm and L-AnGS with diameters of 0.9-2 mm) to the removal of eDNA and corresponding mechanism. The results showed that the highest bioactivity of S-AnGS and L-AnGS was achieved by adding DNase I, and the absolute abundance of hzsA in the systems also increased. The dominant microorganism in each sludge was Candidatus Kuenenia, which maintained a higher relative abundance of 24% in S-AnGS. Settling experiments demonstrated that the permeability of AnGS was positively correlated with the addition of DNase I. The permeability index of granular sludge, Г, rose by 58.54% in S-AnGS and 11.79% in L-AnGS. The absence of eDNA is conducive to the increase in the permeability and porosity of AnGS. Similarity in the functional genes and microbial communities of intracellular and extracellular DNA implied the occurrence of gene transmembrane transfer. The findings enrich our knowledge of eDNA in anammox granules and provide a guidance for the specific control of gene transfer through reducing eDNA.

The response of anaerobic ammonium oxidation process to bisphenol-A: Linking reactor performance to microbial community and functional gene.

As a typical endocrine disruptor, bisphenol A (BPA) has been widely detected in various water bodies. Although the influence of BPA on traditional biological treatment system has been investigated, it is not clear whether it has potential impact on anaerobic ammonium oxidation (anammox) process. The short- and long-term influences of BPA on reactor operational performance, sludge characteristics and microbial community were investigated in this study. Results revealed that 1 and 3 mg L-1 BPA exhibited a limited adverse impact on granular sludge reactor performance. However, exposure of sludge under 10 mg L-1 BPA would cause an obvious inhibition on nitrogen removal rate from 10.3 ± 0.2 to 7.6 ± 0.4 kg N m-3 d-1. BPA would affect granular sludge metabolic substance excretion and lead to effluent dissolved organic content increase. Both the microbial community and redundancy analysis showed that BPA exhibited a negative influence on Ca. Kuenenia but a positive correlation with SBR1031. Low BPA concentration appeared a limited impact on functional genes while 10 mg L-1 BPA would cause decline of hzsA and hdh abundances. The results of this work might be valuable for in-depth understanding the potential influence of endocrine disruptor on anammox sludge.

Intracellular and extracellular protective mechanisms of the anammox consortia against exogenous sulfadimidine.

Antibiotics and antibiotic resistance genes (ARGs) have been recognized as emerging high-risk pollutants for human and animal health. This study systematically investigated the comprehensive effects of a typical antibiotic (sulfadimidine, SDM) in livestock and poultry breeding wastewater on the anammox process, with the aim of elucidating the intracellular and extracellular protective mechanisms of the anammox consortia to the antibiotic stress. Results revealed that the high-concentration SDM significantly reduced the specific anammox activity (SAA) by 37.8%. Changes in the abundance of Candidatus Kuenenia showed a similar trend with that of SAA, while other nitrogen-related microorganisms (e.g., Nitrosomonas and Nitrospira) contributed to the nitrogen removal especially during the inhibitory period. Resistance of the anammox consortia to SDM mainly depended on the protection of ARGs and EPS. Network analysis revealed the host range of eARGs was relatively larger than that of iARGs, and intI1 was closely associated with representative denitrifiers. In addition, metaproteomic analysis and molecular docking results indicated that abundant proteins in EPS could detain SDM in the extracellular matrix through forming complex via hydrogen bond. These findings provide a guidance for the stable operation of anammox process and ARGs transfer controlling.

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.

Anammox sludge preservation: Preservative agents, temperature and substrate.

The difficulties of enrichment and preservation of anaerobic ammonium oxidation bacteria (AnAOB) greatly limit their application in practice. Herein, traditional and emerging preservative agents (e.g., EPS + N2H4, betaine, glycerol and trehalose) were evaluated for their preservation of AnAOB-dominant sludge at different temperatures (e.g., 4 °C and room temperature). In addition, the effects of substrates on preservation were also considered. The results showed that adding betaine or glycerol at 4 °C was the optimal strategy for preserving anammox granular sludge. The relative anammox activities (rAA) increased by 145.26% and 158.30% at the recovery phase, respectively. Moreover, the absolute abundances of functional gene hzsA increased by 339% and 46%, respectively. Although the granular properties and microbial community structures changed during the preservation, the general performance of anammox granules could effectively restored. Collectively, this study provides the optimal strategies for anammox sludge preservation at low temperatures.

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.