| Clarithromycin(CLA)is a representative second-generation macrolide antibiotic containing a 14-membered lactone ring,it has been widely used in the prevention and treatment of human,livestock and poultry diseases since it was listed in the world in the 1990s.The mass production and use of CLA caused the release of CLA into the environment inevitably.According to literature reports,the concentration of CLA detected in the natural environment has reached the ppb or even ppm level,posing potential risks to human health and the ecological environment.Sewage treatment plants is a line of defense before CLA enters the natural environment.However,the current traditional biological treatment process is difficult to effectively degrade CLA in sewage.Most CLA will be concentrated and adsorbed by waste activated sludge(WAS)or remain in the sewage,which will bring adverse effects to the subsequent treatment and disposal process.Anaerobic fermentation or digestion is a technology favored by many engineering practices and scholars to realize the stabilization and resource utilization of WAS.However,when CLA exist as pollutant in WAS,its influence behavior,mechanism and its own metabolic transformation behavior on WAS anaerobic fermentation or digestion process have not been explored systematically.Therefore,this paper comprehensively studied the effect of CLA on the production of short-chain fatty acids(SCFAs),H2 and CH4 by WAS anaerobic fermentation/digestion,explored its potential mechanism,and analyzed its degradation behavior in WAS anaerobic system.Meanwhile,the methods of strengthening CLA degradation with calcium peroxide and sulfate reducing bacteria(SRB)sludge were proposed,and the feasibility of these methods was preliminarily evaluated.Firstly,the effect of CLA at different concentrations on the production of short-chain fatty acid(SCFAs)and H2 during WAS thermophilic fermentation(60?)was investigated through batch experiments,and the potential mechanism of CLA on acidification and H2 consumption process was studied in depth.Results showed that CLA temporarily inhibited the production of SCFAs(48 hours),and the inhibitory effect on acetic acid was more obvious.However,CLA significantly promoted the maximum cumulative H2 yield and the maximum H2 production rate,and prolonged the lag phase time of H2production.With the increase of CLA concentration from 0 to 60 mg/kg TSS(total suspended solid),the maximum cumulative H2 yield increased from 25.5±1.3 to 36.1±1.1 m L/g VSS.When CLA concentration further increased to 300?2000 mg/kg TSS,H2 yield slightly increased to 37.3±1.2?39.7±1.3 m L/g VSS.The mechanism exploration showed that the temporary inhibition of CLA on acidification process was the main reason why CLA inhibited SCFAs production in the first 48 hours and prolonged the lag phase time of H2 production.While the inhibition of CLA on homo-acetogens was the important reason for CLA to the increase of the maximum H2 production rate and the maximum cumulative H2 yield.Secondly,the effect and mechanism of CLA on CH4production from WAS anaerobic digestion were comprehensively analyzed.Results showed that CLA inhibited the maximum CH4 production rate but did not affect the final cumulative CH4 yield.When the CLA concentration increased from 0 to 1000 mg/kg TSS,the maximum CH4 production rate decreased from 20.0±0.4 to 13.7±0.3 m L/(g VSS·d),further increasing the CLA concentration to 2000 mg/kg TSS,the maximum CH4 production rate did not further decrease significantly(p>0.05).CLA promoted the solubilization of polysaccharide and protein,but inhibited hydrolysis,acidogenesis,acetogenesis,homo-acetogenesis,methanogenesis and sulfate reduction process to varying degrees.The longer-time and more-severely inhibitory effect of CLA on acetogenesis and methanogenesis than other processes were the main reason for the lower CH4 production rate.The results of microbial analysis showed that the increased abundance of typical syntrophic acetogens,such as Syntrophomonas and Syntrophorhabdus,and the methanogens,such as Methanosaeta and the hydrogenotrophic methanogens in CLA-contained reactor contributed to the recovery of CH4 production in the later period of anaerobic digestion.Based on the above research,the degradation behavior and mechanism of CLA in WAS anaerobic fermentation and digestion system were analyzed,and the potential effect of CLA's typical metabolite on WAS anaerobic fermentation and digestion process were revealed.Results showed that the degradation efficiency of CLA(60 mg/kg TSS)was 5±2%after 3 days in WAS thermophilic fermentation system,the main metabolites were De(cladinosyl)-CLA(De-CLA,accounting for 97%)and Lactone ring hydrolyzed-CLA(3%).The degradation efficiency of CLA was 62.9±3.2%after 36 days in WAS mesophilic anaerobic digestion system,the lactone ring hydrolysis pathway was the main degradation pathway of CLA,and the metabolites were Lactone ring hydrolyzed-CLA(account for 32.6%)and Rearranged-CLA(28.0%).Followed by the cladinose hydrolysis pathway,the metabolite was De-CLA(37.4%),and the third was the phosphorylation pathway,the metabolite was Phosphorylated-CLA(2%).The 23S r RNA methylation mediated by erm F gene was the main mechanism of anaerobic microorganisms to develop drug resistance to CLA,and esterase and phosphotransferase synthesis mediated by ere gene and mph gene,respectively,were beneficial to the biodegradation of CLA.De-CLA had no effect on the solubilization,hydrolysis and homo-acetogenesis,had slight inhibition on the acidogenesis,acetogenesis,methanogenesis and sulfate reduction,but the inhibition was far less than CLA.Finally,the feasibility of using calcium peroxide and anaerobic SRB sludge to strengthen the degradation of CLA was investigated.The results showed that calcium peroxide accelerated the degradation of CLA in the sludge,and the removal efficiency of 60 mg/kg TSS CLA reached85.6%after 2 days of treatment.The effective degradation of CLA reduced the risk of drug resistance caused by CLA in the subsequent anaerobic digestion process.The long-term domesticated SRB sludge can effectively degrade CLA,the removal efficiency of CLA in the0.01,1 and 10 mg/L stable operated reactors were 91.5±3.6%,61.3±2.2%and 49.1±1.2%,respectively,after 3 days of treatment.The removal of CLA in SRB sludge was mainly attributed to biodegradation,sorption also contributed to the removal of CLA.The biological metabolites of CLA in SRB sludge were De-CLA(92%),Lactone ring hydrolysis-CLA(6%)and Rearranged-CLA(2%).Microbial community analysis showed that CLA could induce the enrichment of potentially drug-resistant microorganisms,such as Escherichia Shigella,Trichococcus and Lactobacillus.The efflux pump,23S r RNA methylation,esterase hydrolysis and exogenous ARGs capture were important mechanisms for the expression and spread of drug resistance of SRB sludge microorganisms.This study comprehensively explored the behavior and mechanism of CLA affecting the anaerobic fermentation and digestion process of WAS,proposed methods to enhance the degradation of CLA by using calcium peroxide and SRB sludge for the first time,filling a fundamental gap between CLA and WAS anaerobic digestion,and providing a theoretical basis references for the development of antibiotics degradation technology in the future. |
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