Abundance Of ARGs On Different Types Of DNA During Sludge Anaerobic Digestion Process

Along with human activities,antibiotics have entered the environment in large quantities,promoting the development of antibiotic resistance genes（ARGs）in the environment.Wastewater treatment plants are important“sinks”and“sources”of ARGs.Up to 10¹²copies/g abundance of ARGs are often detected in the excess sludge.Direct discharge will inevitably cause huge ecological pressure on the environment.Therefore,it is necessary to strengthen the control of ARGs during the treatment of excess sludge.As anaerobic digestion（AD）is a widely used sludge treatment technology in the world,studying the changes of ARGs in the process of sludge AD,and enhancing the control of ARG in sludge AD are of great significance.However,the conclusion of ARGs removal under high temperature are not consistent.Besides,the current research on ARGs in the sludge AD is mainly carried out by extracting total DNA for analysis.It does not distinguish between ARGs carried by different DNA types such as dead bacteria,live bacteria,plasmids,and cell-free DNA,and the mechanism and risk of ARGs transmission by different DNA types are obviously different.Studies based on extracting total DNA for ARGs research is not conducive to accurately analyzing the control efficiency of AD on ARGs in sludge.Therefore,this study focused on the abundance characteristics of ARGs carried on different types of DNA in the entire process of sludge pretreatment and AD,and focused on comparing the abundance differences of ARGs carried on different types of DNA in AD systems under different temperatures.In addition,the correlations of ARGs abundance with microbial communities were analyzed.This study is in order to provide theoretical support for enhancing the control of sludge ARGs by AD process.First,the effects of different sludge pretreatment methods,i.e.thermal hydrolysis,thermo-alkaline,ultrasound,microwave,on the removal of ARGs were studied.The results show that the thermal hydrolysis and thermo-alkaline pretreatment have a significantly better removal effect on sludge ARGs than ultrasonic and microwave pretreatment,and show higher removal characteristics on ARGs.However,the abundance of ARGs carried by dead cells is higher in thermo-alkaline treated sludge than that in thermal hydrolysis treated sludge.The abundance of ARGs carried by the cell-free DNA in the sludge after all the pretreatments was low.Proteobacteria is the most dominant phylum in sludge.As the thermal hydrolysis pretreatment time continues to prolong,the relative abundance of Firmicutes in the live bacterial community greatly increased.sul I has a high correlation with tet C,and has a significant positive correlation with microorganisms in multiple genera such as Anaerolineaceae and Rhodocyclaceae.It is speculated that the main hosts of the two ARGs are the same and the host species are diverse.In general,thermal hydrolysis pretreatment achieves the best removal effect of sludge ARGs,but when thermal hydrolysis time is insufficient,it is easy to leave high abundance of dead bacteria ARGs in the sludge.Therefore,proper extension of thermal hydrolysis pretreatment time can effectively reduce ARGs in sludge.Subsequently,a study was conducted on the abundances of ARGs carried by various types of DNA in AD systems under different temperature.The results show that,from the perspective of intracellular DNA carried ARGs,the abundance of ARGs such as tet C,tet O,tet W,and sul I in the normal temperature AD system showed an increasing trend,and tet A and sul II decreased slightly;the abundance of tet C,tet O,and sul I in moderate temperature AD gradually increased slightly,and tet A,tet W,and sul II decreased;each ARGs in high temperature AD decreased significantly.The high-temperature AD showed better removal characteristics of intracellular ARGs than normal-temperature AD and medium-temperature AD.From the perspective of the plasmid carried ARGs,the abundance of tet C,tet W,sul I,and sul II is relatively high,and the abundance of tet A and tet C is relatively low.In normal temperature AD and medium temperature AD,the abundance of each ARGs is higher than that of high temperature AD,and tet A,tet O,tet W,sul I showed a gradual upward trend;high temperature AD has lower abundances of plasmid ARGs.From the perspective of cell-free DNA,cell-free ARGs in the AD systems under three temperatures were relatively low,and the difference was not significant,indicating that AD can effectively maintain cell-free ARGs in a low abundance.Correlation analysis showed that intracellular tet A,tet W,sul I and int I1 had significant correlations and there was the possibility of multiple resistances carried by integron I.Plasmids tet A,tet W and int I1showed significant correlation and had the possibility of coexistence in plasmid integron.In general,high temperature AD has a better ARGs reduction effect than normal temperature and moderate temperature AD.Finally,the microbial community in the AD system of sludge under different temperatures and its correlation with intracellular and plasmid ARGs were studied.The results show that in different temperature sludge AD systems,the bacterial and archaeal communities in the normal temperature and intermediate temperature AD systems were relatively similar,but the bacterial and archaeal communities in the high temperature AD system showed significant differences from the other two temperature AD systems.Bacteroidetes and Firmicutes were the main bacterial phylum in normal temperature and moderate temperature AD systems,while Thermotogae is the main bacterial phylum in the high temperature AD systems.Methanomicrobia was the archaeal phylum that was dominant in the high-temperature AD system,but there were also high-abundance archaea such as Thermoplasmata and Thermococci in the normal temperature and medium temperature AD systems.Intracellular ARGs showed significant positive correlation with many bacteria and archaeal microorganisms,and there are multiple co-correlations with int I1,indicating that the potential ARGs host strains in the sludge AD system might be rich,with type I integron multi-resistance induced by plasmids.Plasmid ARGs were less relevant to bacteria and archaea communities,which might be due to the ease of plasmid conjugation and transformation by microorganisms,and thus achieving the inter-species transfer of ARGs and making the dynamically change of the host of the plasmid ARGs.Studies have shown that the application of thermal hydrolysis pretreatment and high temperature anaerobic digestion treatment has a good removal effect on sludge ARGs.