Substrate Response Mechanism Of Antibiotic Resistance Genes Transfer In Livestock Manure And Residual Sludge During Anaerbic Digestion

Anaerobic digestion(AD)is an important treatment method for livestock manure and residual sludge.It can reduce antibiotic resistance genes(ARGs)and help reduce environmental pollution and ecological risks caused by it.At present,there are different opinions on the outcome of antibiotic resistance genes in the anaerobic digestion process.There have been many studies focusing on the influence of reactor configuration,operating conditions,and temperature.However,the properties of substrates such as differences in substrate types and their composition,the influence of substrate microorganisms and the selection of inoculum mud are unclear.Therefore,it is worth further study.There are two main forms of resistance genes: intracellular resistance genes(iARGs)in microbial cells and extracellular resistance genes(eARGs)exposed in the environment.Horizontal gene transfer(HGT)involving eARGs and iARGs plays an important role in ARGs diffusion.However,differences between eARGs and iARGs in the outcome of anaerobic digestion remain unclear.Therefore,this study selected three typical ARGs sources in different environments: pig manure(PM),chicken manure(CM)and urban surplus sludge(SS)for anaerobic fermentation batch test,so as to explore the variation of substrate type difference and its composition on ARGs.The influence of substrate microorganisms on ARGs during anaerobic digestion was investigated by pretreatment of three substrates with autoclastic steam sterilization(121℃,30min)(i.e.,sterilized pig feces(TPM),sterilized chicken feces(TCM)and sterilized urban residual sludge(TSS).The trend of intracellular resistance genes(iARGs)and extracellular resistance genes(eARGs)during anaerobic digestion was studied by extracting total DNA and intracellular DNA(iDNA)and extracellular DNA(eDNA)from samples.The experimental results showed that the pretreatment substrate of autoclave did not significantly change the methane production of AD in the three environmental samples.However,the gas production of TPM and TCM was greater than that of PM and CM in the early anaerobic digestion phase,while that of sludge was the opposite.In addition,the inhibition of gas production in the chicken feces group was consistent with the previous studies.QPCR quantitative test showed that the total AMOUNT of iDNA in the three substrates was greater than the total amount of eDNA,and iARGs in PM and CM were higher than eARGs,and eARGs in SS was twice higher than iARGs.In addition,eARGs in SS and TSS groups accounted for 5.47% and 5.18% of the total ARGs.ARGs in eDNA in residual sludge is a potential and not to be ignored in the environment.The relative abundance of int I1 was highest on the 6th day of PM anaerobic digestion and the 55 th day of SS anaerobic digestion,thus promoting the diffusion of ARGs by horizontal transfer.By analyzing the phyla level and genera level of the microbial community in the process of AD,the dominant genera of PM and CM were similar.In addition,there are anaerobic fermentation hydrogen-producing bacteria in SS.Mantel test and Procrustes analysis showed that the change of microbial community played an important role in ARGs transformation,and the intracellular microbial community showed a significant correlation with iARGs.Procrustes analysis showed no significant correlation between the extracellular microbial community and eARGs.The differences of different substrate types,their composition and substrate microbial communities not only have significant differences in methane gas production in AD but also influence the change of AGRs.The conversion of eARGs in anaerobic digestion plays an important role in ARGs control in different substrates.EARGs degradation is an important way to achieveARGs reduction.This study provides a reference for the variation of ARGs in the process of AD for different substrate types.