Phenacetin promoted the rapid start-up and stable maintenance of partial nitrification: Responses of nitrifiers and antibiotic resistance genes.

Phenacetin (PNCT) belongs to one of the earliest synthetic antipyretics. However, impact of PNCT on nitrifying microorganisms in wastewater treatment plants and its potential microbial mechanism was still unclear. In this study, PN could be initiated within six days by PNCT anaerobic soaking treatment (8 mg/L). In order to improve the stable performance of PN, 21 times of PNCT aerobic soaking treatment every three days was conducted and PN was stabilized for 191 days. After PN was damaged, ten times of PNCT aerobic soaking treatment every three days was conducted and PN was recovered after once soaking, maintained over 88 days. Ammonia oxidizing bacteria might change the dominant oligotype to gradually adjust to PNCT, and the increase of abundance and activity of Nitrosomonas promoted the initiation of PN. For nitrite-oxidizing bacteria (NOB), the increase of Candidatus Nitrotoga and Nitrospira destroyed PN, but PN could be recovered after once aerobic soaking illustrating NOB was not resistant to PNCT. KEGG and COG analysis suggested PNCT might disrupt rTCA cycle of Nitrospira, resulting in the decrease of relative abundance of Nitrospira. Moreover, PNCT did not lead to the sharp increase of absolute abundances of antibiotic resistance genes (ARGs), and the risk of ARGs transmission was negligible.

The spread of different resistance genes fractions in nitrification system under chronic exposure to varying alkyl chain length benzalkyl dimethylammonium compounds.

Benzalkyl dimethylammonium compounds (BACs) are generally applied as surfactants and disinfectants. In this study, the nitrification systems were exposed to different alkyl chain lengths (C12-C16) and different levels of BACs (0-5 mg/L), respectively, totally 120 days and to explore the chronic effect of BACs on resistance genes (RGs). RGs were classified into four fractions based on activated sludge properties. Ammonia oxidation performance were not significantly affected by BACs, whereas BACs increased the absolute abundance of most intracellular RGs in sludge (si-RGs). Under the exposure of BACs, extracellular RGs in water (we-RGs) showed a decrease trend and si-RGs tended to be converted to we-RGs. Tightly bound-Tyrosine side chain was significantly correlated with most we-RGs, and we-intI1 might contribute to the propagation of RGs. Therefore, the risk of transmission of different fractions of RGs in the nitrification system under the stress of BACs should be taken seriously.

Metatranscriptome Revealed the Efficacy and Safety of a Prospective Approach for Agricultural Wastewater Reuse: Achieving Ammonia Retention during Biological Treatment by a Novel Natural Inhibitor Epsilon-Poly-l-Lysine.

Appropriate inhibitors might play important roles in achieving ammonia retention in biological wastewater treatment and its reuse in agriculture. In this study, the feasibility of epsilon-poly-l-lysine (ε-PL) as a novel natural ammonia oxidation inhibitor was investigated. Significant inhibition (ammonia oxidation inhibition rate was up to 96.83%) was achieved by treating the sludge with ε-PL (400 mg/L, 12 h soaking) only once and maintaining for six cycles. Meanwhile, the organic matter and nitrite removal was not affected. This method was effective under the common environmental conditions of biological wastewater treatment. Metatranscriptome uncovered the possible action mechanisms of ε-PL. The ammonia oxidation inhibition was due to the co-decrease of Nitrosomonas abundance, ammonia oxidation genes, and the cellular responses of Nitrosomonas. Thauera and Dechloromonas could adapt to ε-PL by stimulating stress responses, which maintained the organic matter and nitrite removal. Importantly, ε-PL did not cause the enhancement of antibiotic resistance genes and virulent factors. Therefore, ε-PL showed a great potential of ammonia retention, which could be applied in the biological treatment of wastewater for agricultural reuse.

Different acesulfame potassium fate and antibiotic resistance propagation pattern in nitrifying and denitrifying sludge systems.

Acesulfame potassium (ACE-K) is a widely utilized sugar substitute with increasing demand, which is frequently detected in various environmental matrix due to recalcitrance. However, a general consensus on the contribution of nitrifying and denitrifying process to ACE-K removal is lacking. Therefore, ACE-K removal, its effects on antibiotic resistant genes (ARGs) propagation and microbial community in nitrifying sequencing batch reactor (N-SBR) and denitrifying sequencing batch reactor (D-SBR) inoculated with the identical activated sludge were investigated. In this study, ACE-K can be eliminated in N-SBR with satisfying removal efficiency (96.76 ± 8.33 %) after 13 d acclimation, while it remained persistent (average ACE-K removal efficiency of 2.24 ± 1.86 %) in D-SBR during 84 d exposure. Moreover, ACE-K hardly affected the performances of these two types of reactors and had little impact on nitrifying and denitrifying functional genes. However, initial contact with ACE-K would increase ARGs abundance, network analysis showed functional bacteria in each reactor were possible ARGs hosts. Potential ACE-K degrading genera Chelatococcus, Bosea and Aquamicrobium were found in both reactors. LefSe analysis showed that Phyllobacteriaceae containing Aquamicrobium genus was a differentially enriched family in N-SBR. This research might provide a perspective for better understanding factor affecting ACE-K fate in wastewater treatment process and its ecological risks.

The fate and behavior mechanism of antibiotic resistance genes and microbial communities in flocs, aerobic granular and biofilm sludge under chloroxylenol pressure.

Chloroxylenol (PCMX), an antibacterial agent, has been widely detected in water environment and has toxic effects on biology and ecology. During 270 d, the influence of PCMX on the performance of three nitrification systems was investigated, including floc-based sequencing batch reactor (FSBR), aerobic granule-based SBR (AGSBR) and biofilm SBR (BSBR). The nitrification capability of three systems was inhibited by PCMX, but recovered after domestication, and PCMX made three systems realize partial nitrification for 10, 100 and 35 days, respectively. The extracellular polymeric substances of three systems increased first and then decreased with the increment of PCMX. The granular structure of AGSBR may be conducive to the enrichment of antibiotic resistance genes (ARGs), and almost all ARGs of BSBR were reduced during the addition of 5.0 mg/L PCMX. The microbial community results showed that Rhodococcus as potential degrading bacteria was continuously enriched in three systems. Piscinibacter was regarded as the potential antibiotic resistant bacteria, which was positively associated with multiple ARGs in three systems. Additionally, quaternary ammonium compounds resistance genes had a variety of positive correlations with bacteria in three systems. This study provided a new perspective for the usage and treatment of PCMX.

Feeding low-level benzethonium chloride can promote the start-up, fast recovery and long-term stable maintenance of partial nitrification for low-ammonium wastewater.

The establishment of stable partial nitrification (PN) is beneficial to promote the application of anaerobic ammonium oxidation, especially for low-ammonium wastewater. This study demonstrated an innovative approach for achieving PN through feeding low-level benzethonium chloride (BZC). PN was started and maintained for 125 days after the sequential feeding of 0.2 and 1 mg/L BZC for 50 days. The damaged PN recovered rapidly within eight days by feeding 2 mg/L BZC, and it thrived for more than 172 days, indicating that nitrite-oxidizing bacteria did not adapt to BZC. The removal of BZC changed from adsorption to degradation gradually. Increased extracellular polymeric substances secretion and altered protein secondary structures explained sludge granulation during BZC feeding, which may be closely related to long-term stable maintenance of PN. PICRUSt2 revealed the underlying microbial mechanisms in depth. Overall, this research proposed a novel scheme to achieve robust PN treating low-ammonium wastewater through feeding low-level BZC.

Enhancement of antibiotic resistance dissemination by artificial sweetener acesulfame potassium: Insights from cell membrane, enzyme, energy supply and transcriptomics.

The abuse of antibiotics on animals could induce the development of antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB), and acesulfame potassium (ACE) is the widely used artificial sweetener in animal feed. Generally speaking, ACE and ARB often coexist in livestock wastewater, however, the impact of the co-occurrence of ACE and ARB on the transmission of ARGs is still unknown. In this study, the effects of ACE on vertical gene transfer (VGT) and horizontal gene transfer (HGT) were both evaluated. For VGT, ACE may hinder the spread of sul gene in Pseudomonas HLS-6 by blocking ARB growth. As for HGT (from Escherichia coli DH5α to Pseudomonas HLS-6), environmentally relevant ACE concentration could facilitate the conjugative transfer. The underlying mechanisms of HGT were characterized by enhanced cell membrane permeability, reactive oxygen species overproduction, SOS response, energy supply, which were all further verified by the changes in transcription levels of related genes. Interestingly, intracellular Mg2+ in donor strain was found for the first time as an indicator for the conjugation occurrence in ACE treated mating system. This study may provide new insights into the role of ACE on ARGs proliferation and highlight its potential environmental impacts.

Metagenomics insights into the selective inhibition of NOB and comammox by phenacetin: Transcriptional activity, nitrogen metabolism and mechanistic understanding.

Phenacetin (PNCT), a common antipyretic and analgesic drug, is often used to treat fever and headache. However, the effect of PNCT on nitrifiers in wastewater treatment processes remains unclear. The practicability of attaining partial nitrification (PN) through inhibitor-PNCT was investigated in this study. The optimal treatment conditions of soaking once for 18 h with 2.50 × 10-3 g PNCT/(g MLSS) were applied to the PN stability experiment. The results showed that ammonia oxidation activity recovered quickly after 3 cycles of operation, while nitrite oxidation activity was suppressed steadily. In addition, average ammonium removal efficiency and nitrite accumulation ratio during 138 cycles could reach 94.94% and 85.38%, respectively. Complimentary DNA high-throughput sequencing and oligotyping analysis showed that the activity of Nitrosomonas would gradually surpass Nitrospira after PNCT treatment only once. The decrease of Nitrospira activity was accompanied by the simplification of oligotypes after PNCT treatment, while Nitrosomonas could adapt to PNCT stress by reducing the differences between oligotypes. Metagenomics revealed that the decrease in the number of NXR in the nitrogen metabolism pathways was the key reason for achieving PN. The potential mechanisms might be that the dominant nitrite-oxidizing bacteria and complete ammonia oxidizers were bio-killed by PNCT.

Chloroxylenol at environmental concentrations can promote conjugative transfer of antibiotic resistance genes by multiple mechanisms.

The intergeneric conjugative transfer of antibiotic resistance genes (ARGs) is recognized as an important way to the dissemination of antibiotic resistance. However, it is unknown whether the extensive use of chloroxylenol (para-chloro-meta-xylenol, PCMX) in many pharmaceutical personal care products will lead to the spread of ARGs. In this study, the abilities and mechanisms of PCMX to accelerate the intergeneric conjugative transfer were investigated. Results showed that exposure of bacteria to environmental concentrations of PCMX (0.20-1.00 mg/L) can significantly stimulate the increase of conjugative transfer by 8.45-9.51 fold. The phenotypic experiments and genome-wide RNA sequencing revealed that 0.02-5.00 mg/L PCMX exposure could increase the content of alkaline phosphatase and malondialdehyde, which are characteristic products of cell wall and membrane damage. In addition, PCMX could lead to excessive production of reactive oxygen species (ROS) by 1.26-2.00 times, the superoxide dismutase and catalase produced by bacteria in response to oxidative stress were not enough to neutralize the damage of ROS, thus promoting the conjugative transfer. Gene Ontology enrichment analysis indicated that cell membrane permeability, pili, some chemical compounds transport and energy metabolism affected conjugative transfer. This study deepened the understanding of PCMX in promoting propagation of ARGs, and provided new perspectives for use and treatment of personal care products.

Achieving stable and long-term partial nitrification of domestic wastewater by side-stream sludge treatment using a novel nitrite oxidation inhibitor chloroxylenol.

Using inhibitors to selectively suppress the activity of nitrite-oxidizing bacteria (NOB) was an emerging way to rapidly achieve partial nitrification (PN). This study explored the feasibility of inactivating NOB by a novel inhibitor chloroxylenol (PCMX) in real domestic wastewater. Different frequencies (periodic strategy and concentrative time strategy) of PCMX side-stream sludge treatment were used to achieve and maintain PN during 250 days. PN was realized by PCMX treatment once a day about 20 days, due to the inhibition of Nitrospira. PN was completely destroyed after 212 days by periodic strategy, caused by the increase of Candidatus Nitrotoga. PN maintained without PCMX in following 201 days by concentrative time strategy. The risks of PCMX were assessed and almost no PCMX was detected in the effluent of mainstream sequencing batch reactors. These results meant PN realized by PCMX side-stream sludge treatment was feasible and concentrative time strategy was a better operating strategy.

Chicoric acid attenuates hyperglycemia-induced endothelial dysfunction through AMPK-dependent inhibition of oxidative/nitrative stresses.

BACKGROUND: Endothelial dysfunction is a driving force during the development and progression of cardiovascular complications in diabetes. Targeting endothelial injury may be an attractive avenue for the management of diabetic vascular disorders. Chicoric acid is reported to confer antioxidant and anti-inflammatory properties in various diseases including diabetes. However, the role and mechanism of chicoric acid in hyperglycemia-induced endothelial damage are not well understood. METHODS: In the present study, human umbilical vein endothelial cells (HUVECs) were incubated with high glucose/high fat (HG + HF) to induce endothelial cell injury. RESULTS: We found that exposure of HUVECs to HG + HF medium promoted the release of cytochrome c (cytc) from mitochondrion into the cytoplasm, stimulated the cleavage of caspase-3 and poly ADP-ribose-polymerase (PARP), then inducing cell apoptosis, the effects that were prevented by administration of chicoric acid. Besides, we found that chicoric acid diminished HG + HF-induced phosphorylation and degradation of IκBα, and subsequent p65 NFκB nuclear translocation, thereby contributing to its anti-inflammatory effects in HUVECs. We also confirmed that chicoric acid mitigated oxidative/nitrative stresses under HG + HF conditions. Studies aimed at exploring the underlying mechanisms found that chicoric acid activated the AMP-activated protein kinase (AMPK) signaling pathway to attenuate HG + HF-triggered injury in HUVECs as AMPK inhibitor Compound C or silencing of AMPKα1 abolished the beneficial effects of chicoric acid in HUVECs. CONCLUSION: Collectively, chicoric acid is likely protected against diabetes-induced endothelial dysfunction by activation of the AMPK signaling pathway. Chicoric acid could be a novel candidate for the treatment of the diabetes-associated vascular endothelial injury.