【作者】 张鑫；

【导师】 张社奇； 谷洁；

【作者基本信息】 西北农林科技大学 ， 生物物理学， 2019， 博士

【摘要】 随着养殖业的迅速发展,抗生素被用作兽药和饲料添加剂促进动物的生长和疾病防治,致使畜禽粪便中含有高浓度的兽用抗生素残留。厌氧发酵技术可以有效的实现畜禽粪便的资源化利用,并且实现能源转换生成沼气。然而畜禽粪便中残留的抗生素会影响发酵系统的性能从而影响沼气生成。且会诱导富集抗生素抗性基因（Antibiotic Resistance Genes,ARGs）,ARGs通过沼液沼渣施肥转移至土壤农田,具有一定的生态风险。也可通过水平基因转移使致病菌获得抗性,降低了抗生素效用,威胁到人类健康。本文以新鲜牛粪为原材料进行厌氧发酵,通过添加不同种类、浓度的抗生素,研究抗生素对牛粪厌氧发酵过程中发酵参数（产气速率,pH值,VFAs,COD）、产气途径关键酶基因、微生物群落多样性的影响和ARGs的变化,分析了ARGs、MGEs、关键酶基因和微生物群落的关系,以揭示微生物影响ARGs变化的机制,为抗生素污染畜禽粪便无害化处理和提高发酵效率提供技术支持和理论依据。得到如下主要结果:（1）泰乐菌素（TYL）（50mg/kg）、环丙沙星（CIP）（30mg/kg）和磺胺二甲嘧啶（SM₂）（20mg/kg）使厌氧发酵总甲烷产量比未添加抗生素处理分别减少了7.5%、21.9%和16.0%。抗生素主要是通过影响乙酸盐,丙酸盐和丁酸盐在发酵初期的降解,引起VFAs积累降低了发酵系统pH值,从而使发酵系统产甲烷微生物活性降低。不同抗生素作用于发酵系统不同种类微生物,如泰乐菌素降低了Spirochaeta和Fibrobacteres的丰度,增加了Syntrophomonas spp丰度,环丙沙星降低Syntrophomonas spp丰度,SM₂处理中ST-12K33显著增加,这些属于发酵酸化和产氢产乙酸阶段微生物。TYL和SM₂分别使乙酸产甲烷途径微生物（Methanosarcina）丰度提高了29.04%和52.79%,CIP降低了Methanobrevibacter和Methanosarcina丰度。同时CIP对mcrA基因有较强的抑制作用,其丰度较对照组降低49%。冗余分析表明,Spirochaeta,Fibrobacteres和Methanosarcina与CH₄、mcrA呈正相关关系。值得注意的是CIP增加了人类致病菌Treponema的丰度。（2）10 mg/kg泰乐菌素使甲烷产量增加了19.6%,100 mg/kg泰乐菌素使甲烷产量减少了9.5%。低浓度的泰乐菌素通过促进乙酸产甲烷途径提高了甲烷产量,表现在提高了发酵后期Methanosarcina和Methanobacterium的丰度,且ACAS绝度丰度较对照组增加了1.3 logs,乙酸产甲烷途径基因丰度增加。高浓度泰乐菌素在发酵初期对系统中微生物形成选择压力,破坏了发酵过程酸化和产酸阶段的动态平衡,抑制系统产气性能。在泰乐菌素影响下厌氧发酵提高了ermQ、sul1、sul2、aac（6’）-ib-cr、intI2和Tn916/1545的相对丰度,可能会促进这些ARGs在环境中的传播和扩散。Network结果表明,ARGs的潜在宿主菌主要属于Firmicutes和Actinobacteria,MGEs拥有与其相似的宿主菌。微生物群落演替是厌氧发酵过程ARGs变化的主要原因。产甲烷途径关键酶基因表现出与sul1、sul2、aac（6’）-ib-cr、tetC的共现性,一些关键功能微生物被鉴定为ARGs的潜在宿主,这使发酵中ARGs的变化值得被关注。（3）10mg/kg的环丙沙星使发酵系统CH₄的累积量增加了9.9%,50mg/kg的环丙沙星引起发酵系统中VFAs的大量积累致使发酵系统pH值降低到6.4,抑制了系统产甲烷微生物的生长破坏发酵系统稳定性。乙酸产甲烷途径被证明是发酵系统中甲烷贡献率最高的产甲烷途径,10mg/kg环丙沙星通过提高发酵初期VFAs浓度,提高Methanosarcina在发酵系统中对乙酸盐的利用率增加了系统沼气产量。高浓度（50mg/kg）环丙沙星在发酵后期使mcrA和ACAS的相对丰度降低,抑制了降解H₂产甲烷的途径,使氢营养型产甲烷微生物活性降低。在环丙沙星在影响下厌氧发酵过程中tetC、sul1、ermF、intI1的相对丰度显著升高,特别是在10mg/kg的浓度影响下,这可能是由于环丙沙星对微生物的选择性压力使整合子基因和ARGs丰度增加。冗余分析结果表明ARGs的变化同时受到MGEs和细菌/古菌群落的影响。Network结果展示,ARGs的大部分宿主菌属于Proteobacteria和Firmicutes,产甲烷过程的关键酶基因与4种ARGs和整合子基因共存,导致厌氧发酵稳定性越强,ARGs在发酵过程更易得到富集。（4）10、50mg/kg的磺胺二甲嘧啶均使发酵系统CH₄产量降低,并随着磺胺二甲嘧啶浓度增加抑制作用增强。在磺胺二甲嘧啶作用下关键酶基因（mcrA、ACAS、FTHFS）和乙酸型产甲烷微生物Methanosarcina在发酵初期丰度下降,是磺胺二甲嘧啶影响产气量的主要原因。磺胺二甲嘧啶诱导增加了发酵产物中sul1和sul2基因的相对丰度,且高浓度具有更强和更持久的作用。冗余分析得到ARGs变化主要受微生物群落演替、VFAs和COD的影响。ARGs和整合子的潜在宿主大都属于Actinobacteria和Firmicutes门类微生物,intI1是造成添加磺胺二甲嘧啶牛粪厌氧发酵过程中ARGs的水平转移起的主要原因。（5）20g/kg的沸石提高了被复合抗生素污染的牛粪厌氧发酵系统的甲烷产量,甲烷增加了23.9%,主要是通过加强对乙酸盐和丙酸盐的降解,增加mcrA和ACAS基因在发酵过程中的丰度,从而提高发酵系统产气性能。沸石显著改变了发酵系统细菌和古菌的群落结构,通过提高了Methanocorpusculum、Methanosarcina的丰度,使产甲烷微生物成为优势菌属,降低了复合抗生素的抑制作用。与复合抗生素处理比较,沸石削减了发酵产物中tetX、tetW、sul1、ermX和总ARGs的相对丰度,ARGs潜在宿主菌主要为Firmicutes。Network分析表明,ARGs的变化与微生物群落组成的变化密切相关,沸石通过影响微生物群落和基因遗传元件的丰度从而影响ARGs。综上所述,抗生素通过改变发酵系统中的产甲烷途径来影响沼气的生成,其中Methanosarcina可以作为发酵系统产甲烷能力的生物学标志。抗生素的存在会诱导提高发酵产物中ARGs丰度,且随浓度增加诱导作用增强。沸石有利于降低被复合抗生素污染畜禽粪便的发酵产物中的ARGs丰度。更多还原

【Abstract】 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.更多还原