Effects Of Antibiotics On Functional Microbial Communities And Resistance Genes During Cattle Manure Anaerobic Digestion

With the rapid development of livestock farming,antibiotics have been used as veterinary drugs and feed additives to promote animal growth and disease prevention,resulting in high concentration of veterinary antibiotics residues in livestock manure.Anaerobic digestion can effectively utilize the livestock manure to produce biogas through the energy conversion.However,the residues of antibiotics in livestock manure can affect the performance of digestion system and biogas production.The presence of antibiotics can induce the generation of antibiotic resistance genes?ARGs?.ARGs transfer to soil and farmland through spread manure,which has a certain ecological risk.They can also lead to resistance of human pathogenic bacteria by horizontal transfer,affecting the therapeutic effect of antibiotics and threatening human health.In this paper,the effects of antibiotics on digestion parameters?gas production rate,pH value,VFAs,COD?,key enzymes genes of gas production pathway,diversity of microbial communities and ARGs changes during anaerobic digestion of fresh cow dung are investigated by adding antibiotics of different kinds and concentrations.The relationship between key enzymes genes of methanogenesis and microbial communities was analyzed.The relationships among ARGs,MGEs,key enzyme genes and microbial communities are analyzed in order to reveal the microbiological mechanism of ARGs changes and provide technical support and theoretical basis for innocuous treatment of livestock and poultry manure containing antibiotics and improvement of digestion efficiency.?1?Tylosin?50mg/kg?,ciprofloxacin?30mg/kg?and sulfamethazine?20mg/kg?reduced the total methane production by 7.5%,21.9%and 16.0%respectively,compared with the treatment without antibiotics.Antibiotics mainly affect the degradation of acetate,propionate and butyrate in the early stage of digestion,resulting in the accumulation of VFAs,which reduces the pH value of the digestion system,thus reducing the activity of methane-producing microorganisms in the digestion system.Different antibiotics acted on different kinds of microorganisms in digestion system,such as TYL,which reduces the abundance of Spirochaeta and Fibrobacteres and increases the abundance of Syntrophomonas spp,CIP decreases the abundance of Syntrophomonas spp,and ST-12K33increases significantly in SM₂ treatment.These microorganisms belong to the stage of digestion acidification and hydrogen production acetic acid.TYL and SM₂ increase the abundance of Methanosarcina by 29.04%and 52.79%,respectively.While CIP decreases the abundance of Methanobrevibacter and Methanosarcina,and it has a strong inhibitory effect on mcrA gene,with its abundance was 49%lower than that of the control group.Redundancy analysis shows that Spirochaeta,Fibrobacters and Methanosarcina are positively correlated with CH₄ and mcrA.?2?10 mg/kg tylosin increases methane production by 19.6%and 100 mg/kg tylosin reduces methane production by 9.5%.Low concentration of Tylosin increases methane production by promoting acetic acid methanogenesis pathway,which can be shown that the abundance of Methanosarcina and Methanobacterium increases in late digestion period,and the absolute abundance of ACAS increases by 1.3 logs compared with the control group,and the gene abundance of acetic acid methanogenesis pathway increases.In the early stage of digestion,high concentration of tylosin formed selective pressure on microorganisms in the system,which destroys the dynamic balance of acidification and acidification in the digestion process,and inhibits the gas production performance of the system.Under the influence of tylosin,anaerobic digestion increases the relative abundance of ermQ,sul1,sul2,aac?6'?-ib-cr,intI2 and Tn916/1545,which may promote the spread and diffusion of these ARGs in the environment.Network analysis shows that the potential host bacteria of ARGs and MGEs mainly belong to Firmicutes and Actinobacteria,and microbial community succession is the main reason for the change of ARGs during anaerobic digestion.The key enzymes genes of methanogenesis pathway show co-occurrence with sul1,sul2,aac?6'?-ib-cr and tetC.Some key functional microorganisms have been identified as potential hosts of ARGs,which makes the changes of ARGs in digestion worthy of attention.?3?10 mg/kg of ciprofloxacin increases the accumulation of CH₄ in digestion system by 9.9%.50 mg/kg of ciprofloxacin causes the accumulation of VFAs in digestion system,which reduces the pH of digestion system to 6.4,inhibits the growth of methanogenic microorganisms and destroys the stability of digestion system.Methanosarcina has been proved to be the most methane-producing microorganism in the digestion system.10 mg/kg ciprofloxacin increases methane production by increasing acetic acid methane production.High concentration?50mg/kg?of ciprofloxacin reduces the relative abundance of mcrA and ACAS,inhibits the degradation of H₂ to methane,and reduces the activity of hydrogen-trophic methanogens.The relative abundance of tetC,sul1,ermF and intI1increased significantly under the influence of ciprofloxacin during anaerobic digestion,especially under the influence of 10 mg/kg concentration.This may be due to the selective pressure of ciprofloxacin on microorganisms,which increases the abundance of integron genes and ARGs.Redundancy analysis shows that the changes of ARGs are affected by both MGEs and bacterial/archaeological communities.Network analysis shows that most of the host bacteria of ARGs belong to Firmicutes and Proteobacteria,the coexistence of four ARGs and integron genes may lead to the increase of ARGs abundance under better anaerobic dogestion performance.?4?Both 10 mg/kg and 50 mg/kg sulfamethazine reduce the gas production of digestion system,and the inhibition increases with the increase of sulfamethazine concentration.Sulfamethazine reduces the abundance of key enzymes?mcrA,ACAS,FTHFS?and acetic acid methanogen Methanosarcina in the early stage of digestion,which is the main reason for the effect of sulfamethazine on gas production.The inducement of Sulfamethazine increases the relative abundance of sul1 and sul2 genes in digestion products,and high concentration has stronger and more lasting effects.The changes of ARGs map were mainly influenced by microbial community succession,VFAs and COD.Most potential hosts of ARGs and integrons belong to Firmicutes and Actinobacteria.IntI1plays an important role in the horizontal transfer of ARGs during anaerobic digestion of cow dung supplemented with sulfamethazine.?5?20g/kg zeolite increases methane production by 23.9%in the anaerobic digestion system of cow dung contaminated by compound antibiotics.The degradation of acetate and propionate is enhanced and the abundance of mcrA and ACAS genes in the digestion process is increased,thus improving the gas production performance of the digestion system.Zeolite significantly changed the community structure of bacteria and archaea in digestion system.By increasing the abundance of Methanocorpusculum and Methanosarcina,methanogenic microorganisms becomes dominant bacteria and reduces to the inhibitory effect of compound antibiotics.Compared with compound antibiotic treatment,the addition of zeolite reduces the relative abundance of tetX,tetW,sul1,ermX and total ARGs in digestion products.The potential host bacteria of ARGs is mainly Firmicutes.Network analysis shows that the changes of ARGs are closely related to the changes of microbial community composition,and ARGs are affected by Zeolite through influencing the abundance of microbial community and genetic elements.In conclusion,antibiotics affect methane production by changing the methane production pathway in digestion system,and Methanosarcina can be used as a biological marker of methane production capacity in digestion system.The presence of antibiotics could induce the increase of ARGs abundance in digestion products,and the induction effect increases with the increase of concentration.Zeolite can contribute to reduce the ARGs abundance in digestion products of livestock manure contaminated by compound antibiotics.