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.

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.

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.

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.

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.

How anammox process resists the multi-antibiotic stress: Resistance gene accumulation and microbial community evolution.

The effects of multiple antibiotics on the anaerobic ammonia oxidation (anammox) process were investigated. The resistance of the anammox system to high-concentration antibiotics was also demonstrated through gradual acclimation experiments. Inhibition of the anammox process (R1) occurred when the concentrations of erythromycin (ERY), sulfamethoxazole (SMX) and tetracycline (TC) were 0.1, 5.0 and 0.1 mg L-1, respectively. The nitrogen removal efficiency (NRE) of R1 was reduced from 97.2% to 60.7% within 12 days and then recovered to 88.9 ± 9.5% when the nitrogen loading declined from 4.52 ± 0.69 to 2.11 ± 0.58 kg N m-3 d-1. Even when the concentrations of ERY, SMX and TC were as high as 1.0, 15.0 and 1.0 mg L-1, respectively, R1 maintained stable operation. The increases in the abundance of antibiotic resistance genes (ARGs) and in extracellular polymeric substances (EPS) content showed that the anammox process alleviated stress from multiple antibiotics mainly by producing ARGs and secreting EPS. The molecular docking simulation results illustrated the potential binding sites between ammonium transporter and different antibiotics. The upregulation of functional gene expression and the stable abundance of Candidatus Kuenenia in R1 compared with that in the control suggested that the R1 reactor generally maintained more stable long-term operation. This work provides a new understanding of the application of the anammox process to treat wastewater containing multiple antibiotics.

Inhibition of wastewater pollutants on the anammox process: A review.

The anaerobic ammonium oxidation (anammox) process has been recognized as an efficient nitrogen removal technology. However, anammox bacteria are susceptible to surrounding environments and different pollutants, which limits the extensive application of the anammox process worldwide. Numerous researchers investigate the effects of various pollutants on the anammox process or bacteria, and related findings have also been reviewed with the focused on their inhibitory effects on process performance and microbial community. This review systemically summarized the recent advances in the inhibition, mechanism and recovery process of traditional and emerging pollutants on the anammox process over a decade, such as organics, metals, antibiotics, nanoparticles, etc. Generally, low-concentration pollutants exhibited a promotion on the anammox activity, while high-concentration pollutants showed inhibitory effects. The inhibitory threshold concentration of different pollutants varied. The combined effects of multipollutant also attracts more attentions, including synergistic, antagonistic and independent effects. Additionally, remaining problems and research needs are further proposed. This review provides a foundation for future research on the inhibition in anammox process, and promotes the proper operation of anammox processes treating different types of wastewaters.

Resistance genes and extracellular proteins relieve antibiotic stress on the anammox process.

The anaerobic ammonium oxidation (anammox) process is regarded as a promising approach to treat antibiotic-containing wastewater. Therefore, it is urgent to elucidate the effects of various antibiotics on the anammox process. Moreover, the mechanism of extracellular polymeric substance (EPS) as protective barriers to relieve antibiotic stress remain unclear. Therefore, the single and combined effects of erythromycin (ETC) and sulfamethoxazole (SMZ), and interactions between EPS and antibiotics were investigated in this study. Based on a 228-day continuous flow experiment, high concentrations of ETC and SMZ had significant inhibitory effects on the nitrogen removal performance of the anammox process, with the abundances of corresponding antibiotic resistance genes (ARGs) increasing. In addition, the combined inhibitory effect of the two antibiotics on the anammox process was more significant and longer-lasting than that of the single. However, the anammox process was able to quickly recover from deterioration. The tolerance of anammox granules to the stress of low-concentration antibiotics was probably attributed to the increase in ARGs and secretion of EPS. Molecular docking simulation results showed that proteins in EPS could directly bind with SMZ and ETC at the sites of GLU-307, HYS-191, ASP-318 and THR-32, respectively. These findings improved our understanding of various antibiotic effects on the anammox process and the interaction mechanism between antibiotics and proteins in EPS.

Comparison of the dynamic responses of different anammox granules to copper nanoparticle stress: Antibiotic exposure history made a difference.

Two types of anaerobic ammonium oxidation (anammox) seed sludge were selected to evaluate their responses to copper nanoparticles (CuNPs) exposure. Antibiotic-exposed anammox granules (R1) were more likely to be inhibited by 5.0 mg L-1 CuNPs than the normal anammox granules (C1). The nitrogen removal efficiency (NRE) of C1 decreased by 9.00% after two weeks of exposure to CuNPs, whereas that of R1 decreased by 20.32%. Simultaneously, the abundance of Candidatus. Kuenenia decreased by 27.65% and 36.02% in C1 and R1 under CuNPs stress conditions, respectively. Generally, R1 was more susceptible to CuNPs than C1. The correlation analysis indicated that the horizontal transfer of antibiotic resistance genes and copA triggered by intI1 facilitated the generation of multiresistance in the anammox process. Moreover, the potential multiresistance mechanism of anammox bacteria was hypothesized based on previous results. The results will generate new ideas for the treatment of complex wastewater using the anammox process.