Influence Of Antibiotics On The Occurrence Characteristics Of Antibiotic Resistance Genes In Sludge Anaerobic Digestion System

Antibiotic resistance genes（ARGs）are one of the important challenges facing human health in today’s society.At present,high abundance of ARGs,antibiotic-resistant bacteria and even multi-drug-resistant bacteria are commonly detected in the environment.Sludge from WWTPs is considered as an important reservoir off ARGs and antibiotic-resistant bacteria,as well as a potential hotspot for horizontal gene transfer between bacteria.It is particularly important to strengthen the control of ARGs during sludge treatment and disposal.Anaerobic digestion,as one of the main technologies for sludge treatment in the world,can realize the reduction,resource utilization,and innocuity treatment of sludge,and convert organic matters in sludge into valuable energy substances through biochemical reactions.The produced methane can be burned as a clean energy.It is necessary to carry out in-depth research on the change law and enhanced control of ARGs in the process of sludge anaerobic digestion.In addition,antibiotics in wastewater are often adsorbed in sludge.Whether antibiotic pressure will promote the development of sludge ARGs during anaerobic digestion has not been systematically studied.Moreover,integron is an important mobile genetic element that promotes the development of multiple antibiotic resistance,however the occurrence characteristics of integron carried ARGs has not been in previous studies on ARGs in sludge anaerobic digestion system.In this study,the occurrence characteristics of ARGs in the sludge of several different types of wastewater treatment plants were first investigated.On the basis of understanding the distribution characteristics of ARGs in the sludge,sludge anaerobic digestion reactors were built.Typical antibiotics were selected to investigate the occurrence characteristics of ARGs during volatile fatty acids（VFAs）production and methane production in sludge anaerobic digestion system under the selection pressure of single antibiotic or mixed antibiotics.the influences of antibiotics on occurrence characteristics of ARGs in sludge anaerobic digestion system were therefore revealed.Based on the above works,the following main conclusions are drawn:（1）The types of ARGs in sludge of wastewater treatment plants are mainly aminoglycosides,β-lactams,multi-drug resistance,macrolide-lincosamide-streptomycin B（MLSB）and tetracycline resistance genes.Sulfonamide ARGs are widely present in all samples with an abundance of 1.18×10⁹-9.86×10⁹copies/g sludge,which was the most abundant among the different ARGs classes.qac Edelta1,qac H,and erm F are several ARGs with high abundances that are widely detected in industrial wastewater treatment systems.Aminoglycoside ARGs are the most abundant genes detected in class1 integron,among which aad A,aad A2 and aad A5 are typical representatives of ARGs.Multidrug-resistant and chloramphenicol ARGs are also detected in high abundance in class I integron.The abundance of qac H was relatively high in most samples;bla OXA10,ere A,cat B3 and cat B8were several ARGs that widely exist in the class1 integron of sludge from municipal wastewater treatment plants with high abundances.The types of ARGs in both the total DNA and class I integron were municipal wastewater treatment plants>industrial wastewater treatment systems.However,the abundance of ARGs in total DNA was industrial wastewater treatment systems>municipal wastewater treatment plants,while the abundance of ARGs in class I integron was municipal wastewater treatment plants>industrial wastewater treatment Systems.The Microbial community is an important factor that causes the differences in the occurrence characteristics of ARGs in wastewater treatment systems.（2）The addition of levofloxacin（OFL）,erythromycin（ERY）,and the combined OFL and ERY all inhibited the sludge anaerobic fermentation of VFAs production,and the inhibition intensity was as follows:OFL&ERY>OFL>ERY.The inhibition of OFL and OFL&ERY on the VFAs production was long-term,and the performance did not tend to resile within 28 days of the experiment.The inhibitory effect of ERY was short,and the VFAs production tended to recover after the 21 days operation.The anaerobic VFAs production process reduced the absolute abundance of ARGs,mainly of aminoglycosides,sulfonamides and MLSB resistance genes.Among all antibiotic-added groups,ERY had the strongest promoting effect on the increase of ARGs abundance.Clostridia is one of the most critical microorganisms in the process of VFAs production.The high proportion of Clostridia is an important reason for the increase of VFAs production in the later stage of the ERY added group,while OFL and OFL&ERY decreased the abundance of Clostridia,thereby inhibited the production of VFA.The significant increase in the abundance of various ARGs in the ERY added group might be caused by the increase in the abundance of their potential host,Firmicutes,during anaerobic VFAs production.（3）The addition of OFL,ERY,and the combined OFL and ERY can inhibit the production of CH₄in anaerobic digestion,in which OFL&ERY has the strongest inhibition.OFL and OFL&ERY seriously inhibited the activity of microorganisms in the anaerobic digestion system,and the anaerobic digestion system almost stopped biogas production at last.Under long-term ERY stress,the microorganisms in anaerobic digestion showed a adaptation and ultimately increase the methanogenesis rate of the system.Antibiotics addition increased the overall abundances of ARGs in total DNA,however,different antibiotics had different effects on the abundance of different types of ARGs.OFL&ERY has the most significant effect on promoting the increase of ARGs abundance,mainly due to the increase ofβ-lactam,multidrug resistance,and MLSB resistance genes.ERY and OFL&ERY significantly increased the abundance of int I1,and promoted the development of long gene cassette arrays in class I integrons.Oppositely,OFLinhibited the development of long gene cassette array carried by class1 integron,and the increase of int I1 abundance.ERY improved the diversity of bacteria and archaea,as well as the absolute abundance of archaea,and increased the proportion of Bacteroides,Clostridia,and Methanosarcina,which might be the key reason that promoted methane production in the late stage of ERY added anaerobic digestion reactor.However,OFL and OFL&ERY reduced the diversity of bacteria,decreased the proportions of key hydrolytic fermentation bacteria,such as Thermotogae,Bacteroides,Clostridia,which resulted in the long-term inhibition of OFL and OFL&ERY on methane production.