Variation Mechanism Of Antibiotic Resistance Genes During Anaerobic Digestion With Livestock Manure

As an emerging environmental pollutant,ARGs can transfer into pathogens via HGT,thereby causing the antibiotics lose their effectiveness,thus pose great threat on public health.Animal manure has become an important reservoir of antibiotics,heavy metals and ARGs because they are largely used as feed additive in livestock farming.Anaerobic digestion is one of the main methods for recycling animal manure.The agriculture use of digestion product is an important way for spreading ARGs from manure to environment.By using lab scale experimental simulation,we studied the effects of residual antibiotic and heavy metal in animal manure,and digestion conditions such as temperature,total solid content and biochar on ARGs during the anaerobic digestion.The re lationship between ARGs and MGEs and microbial community was analyzed to reveal the underlying microbial mechanism.The main results and conclusions are:(1)We found that 8/10 detected ARGs declined and 5/10 decreased more than 1.0 log during thermophilic digestion,whereas only four and five ARGs decreased during moderate and mesophilic digestion,respectively.The changes in ARGs and bacterial communities were similar under the moderate and mesophilic treatments,but distinct from those in the thermophilic system.Potential pathogens such as Bacteroidetes,Proteobacteria,and Corynebacterium were removed by thermophilic digestion but not by moderate and mesophilic digestion.The bacterial community succession was the dominant mechanism that influenced the variation in ARGs and integrons during anaerobic digestion.Thermophilic digestion decreased the amount of mesophilic bacteria(Bacteroidetes and Proteobacteria)carrying ARGs.Anaerobic digestion generally decreased the abundance of integrons by eliminating the aerobic hosts of integrons(Actinomycetales and Bacilli).Thermophilic anaerobic digestion is recommended for the treatment and reuse of animal manure.(2)Compared to the liquid anaerobic digestion(LAD),the solid anaerobic digestion(SAD)performed better at reducing ARGs and MGEs.Specially,the SAD at mesophilic temperature significantly declined 5/10 ARGs and 4 MGEs by 0.47–0.86 log and 0.16–1.03 logs,respectively.Lower abundance of Clostridium XII(significantly positively correlated with ARGs)and higher abundance of Sphaerochaeta and Petrimonas(significantly negatively correlated with ARGs)were responsible for lower ARGs in mesophilic SAD.Unlike LAD,thermophilic temperature did not show higher deductions on ARGs and MGEs than mesophilic temperature under SAD.Integrase genes and ISCR1 played an important role in the HGT of ARGs during SAD.62.5% of the potential hosts for ARGs and MGEs belonged to Firmicutes,and the variations of microbial community structure were the main reasons for differences of ARGs among treatments.(3)The addition of Enrofloxacin significantly increased the abundance of sul1,aac(6')-ib-cr and int I2 during anaerobic digestion.int I1 was improved 0.86–0.90 log in the digestive product of Enrofloxacin addition treatments,and this may because the Enrofloxacin exert selective pressure on microbe,thereby improved abundances of integrons and ARGs.The microbial community was altered by Enrofloxacin from day 15,and specifically,the Methasarsina having positive relation with ARGs were improved significantly.The alteration caused by Enrofloxacin disappeared at the end of digestion.The variation trend of ARGs profiles were mainly affected by microbial community evolution,changes of p H and NO3--N.The primary potential host bacteria of ARGs and integrons were Firmicutes,Actinobacteria and Euryarchaeota,and int I1 played important roles in the ARGs transfer during anaerobic digestion with Enrofloxacin.(4)High concentrations of residual arsanilic acid occur in pig manure due to its use in feed to promote growth and control diseases.This study compared the effects of arsanilic acid at three concentrations(0,325,and 650 mg/kg dry pig manure)on the abundance of antibiotic resistance genes(ARGs)and the microbial community during anaerobic digestion.Addition of 650 mg/kg arsanilic acid enhanced the absolute abundances of tet C,sul2,erm B,and gyrA more than twofold in the digestion product.Redundancy analysis indicated that the change in the microbial community structure was the main driver of variation in the ARGs profile.Network analysis showed that the potential hosts of ARGs were mainly Firmicutes and Proteobacteria,where the As resistance gene arsC co-occurred with four ARGs and int I1,possibly causing the increase in ARGs under pressure by arsanilic acid.High arsanilic acid concentrations can increase the risk of ARGs occurring in anaerobic digestion products.The amount of arsanilic acid used as a feed additive should be controlled.(5)Addition with 20 g/L biochar significantly decreased the abundances of tet W,tet X,sul2,qnrA,qnrS,aac(6')-ib-cr,erm X,ISCR1 and total ARGs.Addition of 5 g/L biochar declined 5/13 ARGs in the digestion product.The microbial communities were affec ted by the addition of biochar,and 20 g/L treatment showed greater influence than 5 g/L treatment.Firmicutes were the primary potential host bacteria of ARGs,and biochar addition can influence Firmicutes and Proteobacteria thus changing ARGs profiles.MGEs were the secondary influence factor responsible for changes of ARGs profiles,and int I2 and ISCR1 player important parts for the spread of ARGs during anaerobic digestion with biochar.In conclusion,thermophilic anaerobic digestion reduced ARGs abundances through removing mesophilic host bacteria.High total solid digestion was conductive to lower the abundances of MGEs.Adding 20 g/L biochar was beneficial for reducing tetracyclines and quinolones resistance genes in digestion product.Enrofloxacin and As could enhance ARGs abundance by generating selective pressure,therefore,the amount of enrofloxacin and arsanilic acid used as feed additives should be controlled.