| Antibiotics and heavy metals are used widely to treat disease and promote growth in intensive livestock farming,thereby leading to livestock manure being the repository of antibiotics,heavy metals,and antibiotic resistance genes(ARGs).Antibiotic resistance has become a major global public helth issure.ARGs can transfer into pathogens via horizontal transfer,thereby causing the antibiotics lose their effectiveness,thus pose great threat on public health.Anaerobic digestion is one of the main methods for recycling animal manure.Organic fertilizer is obtained as the product of anaerobic digestion and its use in agriculture can increase the risk of ARGs entering the environment.This study systematically investigated the contamination characteristics of ARGs in the anaerobic digestion process and products based on laboratory simulations with antibiotics,as well as with single heavy metals comprising zinc and copper.The relationships were analyzed between ARGs and environmental factors(antibiotics,heavy metals,and environmental quality parameters),mobile genetic elements(MGEs),heavy metal resistance genes(HMRGs),and microbial communities.The aims of this study were to understand the behavior of ARGs during the anaerobic digestion of swine manure contaminated with heavy metals.Laboratory scale experimental simulations of anaerobic digestion with a specific concentration of residual copper and added graphene oxide were also conducted to determine their effect on reducing the abundances of ARGs and the main factors responsible.The results obtained in this study provide a theoretical basis for evaluating the ecological risk of ARGs and reducing their abundances during anaerobic digestion with heavy metals.The main conclusions of this study are as follows.(1)The anaerobic digestion of swine manure with a residual level of sulfachloropyridazine hydrazine sodium(SCPS)increased the accumulation of methane by 1.7 times.However,the amounts of methane that accumulated in the reactors contaminated with zinc and SCPS at different concentrations were significantly lower,where SL and SH reduced the amounts by 22% and 65%,respectively.Gas production was unstable and stagnant in an anaerobic digestion system with high residual concentrations of zinc and SCPS.When zinc and SCPS were present in the anaerobic digestion system at the same time,a higher residual concentration of zinc increased the degradation half-life of SCPS.The absolute abundances of sul1 and sul2 were increased in the anaerobic digestion products in the presence of combined contamination with zinc and SCPS at different concentrations.According to redundancy analysis,the total environmental parameters explained 67.1% of the variations in ARGs,intI1,and intI2.The main factor related to the dynamic changes in ARGs was the pH.The concentration of SCPS was significantly correlated with ARGs(sul1,sul2,sul3,and drfA7).The abundances of sul1,sul2,sul3 and drfA7 were closely related to the changes in the concentration of SCPS.(2)Zinc increased the daily methane production by the anaerobic digestion system,where the amounts of biogas accumulated in the reactors with zinc concentrations of 125 mg/L and 1250 mg/L were 51.2% and 56.0% higher,respectively,compared with the control.However,the abundances of 12/14 ARGs were significantly higher in the final anaerobic digestion products from the two reactors with added zinc compared with the control treatment,and the abundance of intI2 increased by 6.5 times.Correlation analysis showed that bio-Zn was significantly correlated with the relative abundances of 9/14 ARGs as well as with the absolute abundances of 6/14 ARGs and intI2.Therefore,bio-Zn was the most important factor that determined the ARG profiles.(3)The addition of Cu 75 and 227 mg/L significantly increased the abundances of most ARGs during anaerobic digestion.Different concentrations of copper also increased the total abundances of ARGs in the anaerobic digestion products,where the abundances of 12 category 5 ARGs were significantly higher than that of the control.The abundances of ARGs were significantly higher in the digestion products obtained from the treatment with a high concentration of copper compared with a low concentration of copper.The dynamic changes in ARGs in the anaerobic digestion process depended on the succession of the bacterial community.Redundancy analysis showed that the pH was the main factor responsible for the changes in the bacterial community structure,followed by sCOD,VFAs,and bio-Cu.pH,sCOD,and VFAs were all strongly correlated with bio-Cu,thereby indicating that the changes in the bacterial community were influenced by bio-Cu in the anaerobic digestion system.Network analysis based on the ARGs,HMRGs,MGEs,and bacterial communities in three digestion systems showed that the selective pressure imposed by copper increased the co-occurrence of ARGs,HMRGs,and MGEs in the same bacteria.Network analysis based on ARGs,HMRGs,and MGEs showed that the co-occurrence of MGEs and ARGs increased under the pressure due to copper,whereas the co-occurrence of HMRGs and ARGs did not change significantly.In general,copper pollution can accelerate the transfer of ARGs via MGEs and increase the risk of ARGs being transmitted in the environment.(4)Graphene oxide effectively reduced the abundances of ARGs,MGEs,and HMRGs in an anaerobic digestion system containing a high concentration of copper(227 mg/L).The abundances of MRGs,ARGs,and MGEs in the GO1 reactor decreased by 51.8%,78.2%,and 83.8%,respectively.The abundances of MRGs,ARGs,and MGEs in the GO2 reactor decreased by 6.9%,71.8%,and 68.4%,respectively.Redundancy analysis showed that MGEs explained most of the changes in ARGs,i.e.,37.53%,thereby indicating that the abundances of ARGs were reduced by graphene oxide mainly by controlling the horizontal transfer of ARGs by MGEs.The bacterial community explained 33.94% of the variations in ARGs,and thus it was the second most important factor after MGEs.The pH,sCOD,VFAs,and bio-Cu were the main environmental factors related to the changes in the bacterial community.In particular,bio-Cu had negative correlations with the pH,sCOD,and VFAs.(5)Methanobrevibacter,Methanocorpusculum,Methanosarcina,and Methanobacterium were the major bacilli detected during the anaerobic digestion process.Different concentrations of graphene oxide had diverse effects on the major methanogens in various digestion stages.In particular,graphene oxide inhibited Methanocorpusculum.In addition,a low concentration of graphene oxide significantly enhanced the abundance of Methanosarcina whereas it was inhibited by a high concentration.By contrast,a high concentration of graphene oxide increased the abundance of Methanobacterium.Redundancy analysis showed that sCOD,pH,and VFAs were the main factors that affected the methanogenic flora.Moreover,the fluctuations in sCOD,pH,and VFA were greater in the digestion system with a high concentration of graphene oxide,and they also affected the succession of the methanogenic bacterial community.Thus,the application of a low concentration of graphene oxide was safer and more reliable in terms of the stable structure of methanogens during anaerobic digestion. |
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