Microbial coadaptation drives the dynamic stability of microecology in mainstream and sidestream anammox systems under exposure of progesterone.

Microbial cooperation determines the efficacy of wastewater biological treatment, and the adaptability of microorganisms to environmental stresses varies. Recently, extensive use of hormones results in their inevitable discharge into aquatic environment. Therefore, mainstream and sidestream anammox reactors were constructed in this study to evaluate their removal performance of progesterone and nitrogen simultaneously, the adaptability of anammox consortia to progesterone stress and the corresponding regulation mechanism. Both anammox processes had the resilience to progesterone stress, with the average nitrogen removal efficiency exceeding 90 %. At the same time, progesterone removal efficiency also exceeded 70 %. In contrast, microbial community in the mainstream reactors was more susceptible to progesterone interference. The adaptation of anammox consortia mainly depended on microbial cooperation and molecular regulation. Initially, bacteria secreted more extracellular polymeric substances to detain progesterone. Biodegradation also contributed to mitigating the side effect of progesterone, which was demonstrated by the proliferation of potential degrading bacteria such as Bacillus salacetis, Bacillus wiedmannii and Rhodococcus erythropolis. In addition, the enhancement of microbial interaction intensity drove their cooperation to enhance adaptability and maintain stable performance. Combined with metagenomic and metatranscriptomic analyses, such microbial adaptability was enhanced through molecular regulations, including the energy redistribution for amino acid synthesis and alteration of key metabolic pathways. Related functional gene expressions and microbial interactions were, in turn, regulated by quorum sensing. This work verifies the feasibility of anammox process in hormone-containing wastewater treatment and provides a holistic understanding of molecular mechanism of microbial interaction and coadaptation to stress.

Polystyrene nanoparticles regulate microbial stress response and cold adaptation in mainstream anammox process at low temperature.

Nanoplastic pollution has become one of the most pressing environmental issues, and its bioaccumulation in aquatic environment also causes a great difficulty in treatment. Therefore, this work investigated the microbial dynamics of mainstream anaerobic ammonia oxidizing (anammox) process to treat the wastewater containing typical nanoplastics, as well as the fate and regulation mechanism of polystyrene nanoparticles (PS-NPs) with different concentrations. The results showed that 0.1-0.5 mg L-1 of PS-NPs had no significant effect on the nitrogen removal efficiency (NRE). When the concentration of PS-NPs increased from 0.5 mg L-1 to 2 mg L-1, the NRE of R1 with PS-NPs decreased from 94.9 ± 2.3 % to 77.0 ± 1.6 %, while the control reactor R0 maintained a stable NRE. Notably, the relative abundance of Ca. Kuenenia decreased from 17.4 % to 14.8 %, and that of Ca. Brocadia slightly decreased from 5.9 % to 5.0 % in R1. In addition, PS-NPs induced oxidative stress in anammox consortia, leading to the significant increase in reactive oxygen species (ROS) and lactate dehydrogenase (LDH) as well as cell membrane damage. PS-NPs also downregulated the content of heme c and further inhibited anammox activity. Based on the molecular docking simulation and western blotting, cold shock proteins (CSPs) could bind to PS-NPs and reduce the performance of anammox processes at low temperatures.

Response of anammox to organics with different degradation characteristics and exposure time: Performance, sludge characteristics and bacterial community.

The effects of typical organic compounds including easily degradable organic matters sodium acetate, yeast and methanol, and refractory organic matter (ROM) humic acid on anaerobic ammonium oxidation (anammox) systems in short-term and medium-term exposure time were studied. During short-term experiments, nitrogen removal activity (NRA) was inhibited at sodium acetate level of 150 mg L-1 total organic carbon (TOC) and methanol level of 30-150 mg L-1 TOC, but humic acid and yeast (≤150 mg L-1 TOC) enhanced nitrogen removal in anammox systems. The greatest NRA of 30.10 mg TN g-1 VSS h-1 was recorded at yeast level of 90 mg L-1 TOC. In medium-term experiments, organics significantly inhibited the nitrogen removal ability. As a ROM, humic acid enhanced sludge aggregation and biological diversity, but decreased the bioactivity and extracellular polymeric substances levels. Due to the endogenous denitrification, the relative abundance of anammox bacteria (AnAOB) was decreased. Candidatus Kuenenia is still dominant in sludge with methanol and humid acid, but AnAOB are not dominant due to the addition of sodium acetate and yeast. This research would be beneficial for the full-scale application of the anammox process in treating real wastewater with organics and ammonia.

Beta-blocker drives the conjugative transfer of multidrug resistance genes in pure and complex biological systems.

Drug resistance poses a high risk to human health. Extensive use of non-antibiotic drugs contributes to antibiotic resistance genes (ARGs) transfer. However, how they affect the spread of broad-host plasmids in complex biological systems remains unknown. This study investigated the effect of metoprolol on the transfer frequency and host range of ARGs in both intrageneric and intergeneric pure culture systems, as well as in anammox microbiome. The results showed that environmental concentrations of metoprolol significantly promoted the intrageneric and intergeneric conjugative transfer. Initially, metoprolol induced excessive oxidative stress, resulting in high cell membrane permeability and bacterial SOS response. Meanwhile, more pili formation increased the adhesion and contact between bacteria, and the abundance of conjugation-related genes also increased significantly. Activation of the electron transport chain provided more ATP for this energy-consuming process. The underlying mechanism was further verified in the complex anammox conjugative system. Metoprolol induced the enrichment of ARGs and mobile genetic elements. The enhanced bacterial interaction and energy generation facilitated the high conjugative transfer frequency of ARGs. In addition, plasmid-borne ARGs tended to transfer to opportunistic pathogens. This work raises public concerns about the health and ecological risks of non-antibiotic drugs.

Mechanistic insight into microbial interaction and metabolic pattern of anammox consortia on surface-modified biofilm carrier with extracellular polymeric substances.

The extremely slow growth rate of anaerobic ammonia oxidation (anammox) bacteria limits full-scale application of anammox process worldwide. In this study, extracellular polymeric substances (EPS)-coated polypropylene (PP) carriers were prepared for biofilm formation. The biomass adhesion rate of EPS-PP carrier was 12 times that of PP carrier, and EPS-PP achieved significant enrichment of E. coli BY63. The 120-day continuous flow experiment showed that the EPS-PP carrier accelerated the formation of anammox biofilm, and the nitrogen removal efficiency increased by 10.5 %. In addition, the abundance of Candidatus Kuenenia in EPS-PP biofilm was 27.1%. Simultaneously, amino acids with high synthesis cost and the metabolites of glycerophospholipids related to biofilm formation on EPS-PP biofilm were significantly up-regulated. Therefore, EPS-PP carriers facilitated the rapid formation of anammox biofilm and promoted the metabolic activity of functional bacteria, which further contributed to the environmental and economic sustainability of anammox process.

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.

Quorum sensing mediated response mechanism of anammox consortia to anionic surfactant: Molecular simulation and molecular evidence.

The widespread use of surfactants raise challenges to biological wastewater treatment. Anaerobic ammonium oxidation (anammox) process has the potential to treat wastewater containing anionic surfactants, but the response of anammox consortia at the molecular level under long-term exposure is unclear. Using high-throughput sequencing and gene quantification, combined with molecular docking, the effect of sodium dodecyl sulfonate (SDS) on anammox consortia were investigated. Levels of reactive oxygen species (ROS) might be lower than the threshold of oxidative damage, while the increase of lactate dehydrogenase (LDH) represented the cell membrane damage. Decreased abundance of functional genes (hdh, hzsA and nirS) indicated the decrease of the anammox bacterial abundance. Trace amounts of N-acyl homoserine lactone (AHL, C6-HSL, C8-HSL and C12-HSL) contained in influent could induce endogenous quorum sensing (QS), which could regulate the correlation between functional bacteria to optimize the microbial community and strengthen the resistance of anammox consortia to SDS. In addition, the proliferation of disinfectant resistance genes might increase the environmental pathogenicity of sewage discharge. This work highlights the potential response mechanism of anammox consortium to surfactants and provides a universal microbial-friendly bioenhancement strategy based on QS.

Reason and control strategy for denitrification and anammox sludge flotation in nitrogen removal process: Mechanisms, strategies and perspectives.

Anaerobic biological treatment technology, especially denitrification and anaerobic ammonia oxidation (anammox) technology as mainstream process, played dominant role in the field of biological wastewater treatment. However, the above process was prone to sludge floating during high load operation and thereby affecting the efficient and stable operation of the system. Excessive production of extracellular polymeric substance (EPS) was considered to be the main reason for anaerobic granular sludge flotation, but the summaries in this area were not comprehensive enough. In this review, the potential mechanisms of denitrification and anammox sludge floatation were discussed from the perspective of granular sludge structural characteristics, nutrient transfer, and microbial flora change respectively, and the corresponding control strategies were also summarized. Finally, this paper indicated that future research on sludge flotation should focus on reducing the negative effects of EPS in sludge particles.

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.

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.

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.

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.