| Lincomycin(LCM),a representative of Macrolides-LincosamidesStreptogramins(LMS)antibiotics,is the second most consumed antibiotic in China.Large quantities of antibiotic production and use lead to the inevitable release of LCM into the environment,and the concentration detected in antibiotic production wastewater and livestock and poultry wastewater can even reach thousands of ppm,posing enormous threat to the environment waters and human health.Sewage treatment plant is an effective place to reduce residual LCM entering the environment.Anaerobic digestion technology is a common sewage treatment process used by engineering practitioners and researchers,which can efficiently treat high organic wastewater and generate clean energy.The effect of LCM on anaerobic digestion system treating wastewater contains high concentration of LCM,as well as its influence mechanism and its own metabolic pathway have not been systematically and comprehensively analyzed.Therefore,this study comprehensively and systematically investigated the effects of lincomycin on the short-term and long-term operation of anaerobic digestion and explored the strengthening method of alleviating lincomycin stress and analyzed the degradation path of lincomycin according the evolution of microbial community structure and specific metabolic pathway of organic matter under different environmental factors.It provides theoretical basis and technical support for the control strategy of methanogenesis process of high organic wastewater containing antibiotics.It was first found out through short-term batch experiments that anaerobic granular sludge was more tolerant to LCM than anaerobic suspended sludge,and that1000 mg/L LCM increased methane production in the anaerobic granular sludge reactor by 20.8%.LCM significantly stimulated the secretion of the outermost adhesive extracellular polymer instantly,which can provide immediate protection to microorganisms and also provided more available substrate for methanogenesis.During the anaerobic acidification phase,LCM increased the activity of acetate kinase and ferric pyruvate oxidoreductase,which is directly related to acetate production and glucose metabolizing to acetate and hydrogen respectively.LCM significantly inhibited the uptake and utilization rate of propionate and butyrate,but facilitate the hydrogenconsuming methanogenesis process by promoting potential hydrogen-mediated interspecies electron transfer.Anaerobic sludge mainly carried LCM resistance genes erm F and erm B with ribosomal protection mechanisms.Their abundance increased with increasing LCM concentrations may be related to the fact that LCM achieves antimicrobial effects by inhibiting protein synthesis.Then,the long-term effects of LCM on mesophilic and thermophilic anaerobic reactors with different substrates(carbohydrate-rich substrate and protein-rich substrate)were investigated by semi-continuous operation.After the addition of 1000 mg/L LCM,the average daily methane production mesophilic reactor with carbohydrate-rich substrate increased by 31%.The methanogenic kinetics showed that the maximum methane production rate in two mesophilic reactors increased by 24.8%-37.7%,while the maximum methane production and maximum methane production rate for reactors under thermophilic condition decreased by 9.2%-25.5% and 28.5%-43.2%,respectively.Metagenomic analysis showed that the relative abundance of hydrogen-consuming methanogenic archaea dominated in mesophilic reactors after the addition of LCM.Through organic matter metabolic pathway analysis with metagenomic functional gene annotation,LCM also promoted the gene abundance of enzymes with significant changes associated in the hydrogen-consuming methanogenic pathway in the mesophilic reactors.The abundance of bacteria responsible for hydrolytic acidification in reactor R1 also increased,which helped to promote efficient operation of the anaerobic system.It was found that the addition of LCM would decrease the methane production ability from organic acids,particularly for thermophilic reactor using carbohydrate-rich substrates,hydrogentrophic methanogenesis was also significantly inhibited.According gene abundances of enzyme with significant changes,all organic acids generation pathways have been promoted in thermophilic conditions,causing organic acid accumulation which decreased the methane production sharply.The protein-rich substrate can significantly enrich Firmicutes and Methanosarcina which can utilize a variety of organic compounds,reducing the inhibition effect of LCM on thermophilic reactor.Further analysis of LCM resistance gene distribution and corresponding microbial hosts revealed that the highest expressed ribosomal protection resistance genes increased by 12.2%-237.7% among the reactors.The resistance genes showed both horizontal and vertical transfer in the mesophilic reactor,while exhibited vertical transfer in the thermophilic reactor.Finally,the regulatory strategy with bioaugmentation and exogenous mediation to LCM-stressed anaerobic digestion systems were examined.Through a stepwise load enhancing method,propionic enriched consortia(PEC)which can sustain an organic loading of 2.0 g SCOD/L/d and a tolerance of 9 g SCOD/L propionic acid and a hydrogen enriched consortia(HEC)contains a relative abundance of 89.64% of hydrogen consuming methanogenic archaea were obtained.The semi-continuous biaugmentation experiments revealed that,daily addition of 2% VS bioaugmented consortia was more effective in the lincomycin stress system than the addition of 10%VS bioaugmented consortia every 5 days,which can enable the reactor to adapt to higher organic load compared with the control group(10000 mg/L)and the maximum methane production increased by 73.3%-99.8% compared with the control group.The addition of 2 g/L micron-sized zero-valent iron to the anaerobic reactor with LCM increased the maximum methane yield by 77.2%,while the highest LCM removal rate(40.0%)was obtained using 5 g/L micron-sized zero-valent iron.It was analyzed by liquid-phase mass spectrometry that least types of LCM degradation products was observed in anaerobic system strengthened by nano zero-valent iron and hydroxy LCM derivatives can be obtained via the hydroxylation pathway,together with other intermediate products by direct desulfurization,oxidation,and opening of pyranose rings. |
