Anaerobic Digestion Efficiency And Resistance Genes Reduction Of Swine Manure Under Selective Pressure Of Antibiotics And Heavy Metals

As an emerging environmental pollutant,antibiotic resistance genes（ARGs）can cause resistance to pathogenic bacteria through horizontal gene transfer（HGT）,which leads to the effectiveness of the antibiotics,thereby pose great threat on public health.In recent years,in order to prevent livestock diseases and promote animal growth,antibiotics and heavy metals have been widely used as feed additives in livestock farming,resulting in livestock manure becoming an important reservoir of antibiotics,heavy metals and ARGs.Anaerobic digestion is one of the main methods for recycling animal manure.At present,the migration and transformation mechanism of ARGs during anaerobic digestion of manure polluted by antibiotics and heavy metals are still unclear.Therefore,this study systematically studied the variation of ARGs during anaerobic digestion of swine manure,revealing the biological mechanism of antibiotics and heavy metals affecting the fate of ARGs in anaerobic digestion of pig manure.In order to solve the problems such as poor passivation effect of heavy metals,unstable digestive system and diffusion of ARGs during the anaerobic digestion of pig manure,the straw biochar（BC）and KH₂PO₄-modified biochar（BP）were used to solved these problems.The main purpose of this paper is to strengthen the effect of anaerobic digestion of pig manure and further reduce the ecological risks caused by the spread of heavy metals and resistance genes.In this study,the fate of ARGs were investigated during anaerobic digestion of swine manure when norfloxacin（NOR）and sulfamethoxazole（SMX）were present.By adding NOR and SMZ separately or in combination to the anaerobic digestion,the reduction of antibiotics,cumulative methane production,microbial community composition and resistance gene abundance were investigated.The mechanism and key factors of selective antibiotic pressure on the transformation of ARGs during anaerobic digestion of pig manure were analyzed.The degradation of SMZ and NOR was found to be significantly different in the sludge and water phases.The degradation rate of SMZ in anaerobic digestion was significantly higher than that of NOR,while the degradation rate of antibiotics in the coexistence of NOR and SMZ was faster than the control.The introduction of NOR and SMZ has a negative impact on the anaerobic digestion process for methane production.NOR inhibits the anaerobic methanogenesis process and increases the delay time of methane production.SMZ had less effect on the anaerobic methanogenesis process,but significantly increased the delay time of methane production.Both NOR and SMZ exert selective pressure on microorganisms during anaerobic digestion,increasing the risk of horizontal transfer of resistance genes and the abundance of some ARGs（such as aac（6’）-Ib-cr,sul1 and sul2）.The addition of antibiotics decreased the microbial diversity in anaerobic digestion system,but SMZ promoted the growth of Methanospirillum,Methanobacterium and Methanosarcina.In the presence of antibiotics,the results indicated that the factors including the microbial community structure,environment conditions,int I1 and MRGs accounted for 84%of variations in ARGs,among which microbial community contributed the most（43.4%）to the evolution of ARGs followed by int I1（14.4%）,environment conditions（7.30%）and MRGs（4.80%）.In this work,the fate of ARGs were investigated during anaerobic digestion of swine manure with presence of Cu and Zn.The effects of heavy metal residues on the anaerobic digestion of pig manure and ARGs were investigated by adding Cu and Zn separately or in combination to the anaerobic digestion.The mechanism and key factors of selective heavy metal pressure on ARGs transformation during anaerobic digestion of pig manure were analyzed.It is found that Cu and Zn improve the methanogenic activity of the system and shorten the lag time for methanogenic activity.The digestion process had a good passivation effect on zinc,and the proportion of unstable zinc in the system was significantly reduced.Anaerobic digestion process can reduce the abundance of most resistance genes in the system,and the removal efficiency of ARGs is about 20%-90%.However,the addition of heavy metals inhibited the removal of ARGs during anaerobic digestion,and the abundance of total resistance genes,ARGs and mobile genetic elements（MGEs）increased significantly.The addition of heavy metal decreased the microbial diversity in anaerobic digestion system,but the heavy metals did not significantly change the community structure of anaerobic bacteria.The results showed that the bio-available heavy metals might also contribute to the proliferation of ARGs,considering the still high bio-available fractions after digestion,the presence of heavy metals contribute more to the distribution of ARG during anaerobic digestion.Beyond the critical role of int I1 in the adaptation of microbial communities against heavy metal obstructions,the co-selection effects driven by heavy metals might also contributed to ARG proliferation.The results indicated that int I1 had significantly positive influences on the variation of ARGs and it had the most standardized direct effects on ARGs abundance（R=0.85,p<0.01）.On the basis of the previous research results,the impacts of two types of biochar（BC and BP）on the changes of ARGs during the anaerobic digestion were studied.The resulted indicated that biochar showed great potential in improving AD performance and shortened the methane production lag time.The addition of biochar served as an electronic conductor to achieve long-distance electron transfer,thus reducing the content of cyt-c in the system.Biochar addition alleviated the need of cytochrome-c as interspecies electron connection components and enriched the microbes involved in direct interspecies electron transfer（DIET）.Methanothrix might participate the electric syntrophy similar to DIET with Defluviitoga,Thermovirga and Cloacibacillus.BC and BP addition decreased DTPA-extractable Cr,Cu,Pb and Zn amount,transformed these HMs to more stable state,and decreased the ecological risks of these heavy metals by 2grades accordingly.The results suggested that the addition of BP might release PO₄^3-that further participated in the formation of metal phosphate precipitation through complexation reaction,thus enhancing the passivation effect of heavy metals.However,PO₄^3-could compete with As for sorption sites on biochar,leading to subsequent As（Ⅲ）release,resulting in high mobility of As.The addition of biochar was put forward to remove the ARGs in anaerobic digestion.Correlation analysis revealed that the reductions in intracellular 16S r RNA contributed to intracellular ARGs removal,while the variations of other ARGs related to MGEs abundance.Additionally,the reduction in potential host bacteria also resulted in the intracellular ARGs removal.BP with larger specific surface area was propitious to decrease the absolute copy number of extracellular resistance genes.The addition of biochar induced the oxidative stress response of microorganisms and enhances the antioxidant capacity of bacteria.And due to the negative feedback,biochar negatively affected SOS response by inducing antioxidant system.The elevated antioxidant levels implied cell membrane damage and further weakened the energy generation process.These changes ultimately lead to the suppression of horizontal transfer of ARGs.