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题名: 养猪废弃物中多种抗生素同时分析及抗性基因在厌氧处理中的转归研究
作者: 王瑞1
学位类别: 博士后
答辩日期: 2017-07
授予单位: 中国科学院大学
授予地点: 北京
导师: 魏东斌 ; 魏源送
关键词: 抗生素筛查,调研,厌氧消化,抗性基因,微生物群落 ; Antibiotics screening, investigation, anaerobic digestion, ARGs, microbial community
学位专业: 生物学
中文摘要: 近年来,饲料中添加抗生素以提高畜禽养殖行业经济效益的行为所带来的潜在环境和生态危害日益受到关注,持续、过量和滥用抗生素不仅诱导抗性基因、耐药菌的传播,更会扰乱环境微生物的自然选择、演替和进化。然而,当前对于我国畜禽养殖行业中所使用的抗生素的种类、以及这些抗生素在环境中的降解转化规律的系统全面研究还比较欠缺,这已成为制约畜禽养殖行业用抗生素环境危害评估及其科学削减方案实施的瓶颈问题。本文以生猪养殖废水和粪便为研究对象,分析了其中的抗生素残留种类、含量及其不同季节分布情况,考察了不同生猪废水和粪便处理工艺对抗生素残留和抗性基因的去除效果,可为有效削减抗生素残留及抗性基因提供基础数据和科学依据。主要结果如下: 开发了适于生猪养殖废水与粪便的残留抗生素快速高通量筛查方法,建立了猪场废水中6大类11种抗生素的高效液相色谱-三重四极杆质谱分析定量方法,分别是四环素类(TC,OTC,CTC),磺胺类(SDMD),大环内酯类(CLA,TILM)氟喹诺酮类(CIP,OFL,ENR),β-内酰胺类(PCN-G)和林可胺类(LIM)。猪粪中抗生素分析方法学的优化结果表明,选取甲醇和Na2EDTA-McIlvaine缓冲溶液的混合液为提取液,以超声方式进行提取、固相萃取方法进行净化,各个目标物的回收率在30%-160%范围内,检出限为0.010-2.885 μg/L,定量限为0.034-9.615 μg/L,线性范围均在5-500 μg/L。 以北京地区两个规模化养猪场为对象,明确了春、夏、冬三季生猪养殖废水和粪便中的抗生素残留特征。结果表明,四环素类抗生素(TCs)是这两个猪场使用的主要抗生素,平均占比58%,其次是大环内酯类的替米考星(TILM),平均占比33%。1#猪场的磺胺类抗生素磺胺二甲嘧啶(SDMD)的残留量也相对较高,平均占比10%,但2#猪场未检测到SDMD。冬季和春季抗生素的残留量均高于夏季,抗生素总量在粪便中的平均比例为98.5%,在废水中的平均比例只有1.5%,说明抗生素主要被吸附在固体上。磺胺类抗生素在废水中的比例相对较高,平均达到5.29%。经过厌氧和好氧工艺后约80%的抗生素均可被去除,但磺胺类抗生素的去除效果较差,导致SDMD在出水中占比较高。 厌氧消化能有效削减猪粪中的抗生素和抗性基因。不同含固率(TS:B=4%,常规厌氧消化;C=8%,D=11%,E=14%,高含固厌氧消化)猪粪中温厌氧消化的试验结果表明,抗生素总量分别下降了35%、46%、39%、14%,其中四环素类、大环内酯类TILM和氟喹诺酮类均下降55%左右,但磺胺类总量均有明显升高。经厌氧消化后,抗性基因的相对丰度总和在B、C、D、E组分别下降了17%、20%、20%、7%,其中ermB下降幅度最大,分别下降了16%、23%、23%、28%,说明厌氧消化对猪粪中优势ARGs有明显削减作用,但大环内酯类mefA基因、磺胺类sulI和sulII基因、四环素类tetG基因却在厌氧消化后明显增长。整合子IntI1在常规厌氧消化中呈下降趋势,而在高含固厌氧消化过程中呈升高趋势,原因可能是高含固厌氧消化使得水平转移的几率相对更大。 基于抗性基因的分析结果表明,厚壁菌门在整个厌氧消化过程中始终是丰度最高的细菌,平均丰度分别为70%,其次是拟杆菌门和变形菌门,这三类细菌的总丰度为90%。高含固与常规厌氧消化微生物群落结构演替的途径有明显差异。常规厌氧消化过程中Sedimentibacter、Ercella、Acinetobacter和Comamonas丰度明显升高;高含固试验中Corynebacterium、Thermacetogenium、Wohlfahrtiimonas、Tepidimicrobium、Paenalcaligenes、Thiopseudomonas六种细菌丰度明显升高,且这些细菌与VFAs和TAN具有一定的相关性。猪粪厌氧消化过程中呈明显下降趋势的抗性基因ermB、blaTEM、tetX、pcoA与Anaerococcus、Peptoniphilus、Streptococcus、Lactobacillus、Methanosphaera、Olsenella、Syntrophococcus、Peptostreptococcus、Bacteroides、Anaerosphaera、Methanothrix存在一定的相关性,说明细菌群落结构的演替对ARGs的变化起到了至关重要的作用。
英文摘要: In recent years, the potential hazard on environment and ecology that adding antibiotics as feed additive in order to improve the economic benefits caught more and more attention. Antibiotics are not only induced antibiotics resistance genes, antibiotics resistance bacteria proliferation and spread; it would disrupt the nature selection and evolution of the microbial community. In this paper, swine wastes were chosen as the research object, residual antibiotics screening trial were carried out using high-resolution MS with full scan mode, in order to determine the species of residual antibiotics in the swine farm wastewaters and build these antibiotics quantitative analysis method of wastewater and solid wastes. Investigations about antibiotics residues in the swine wastewater and solid wastes in different waste treatment process were carried out during spring, summer and winter in two typical swine farm in north of China, in order to determine the antibiotics residues situation and removal effect. Antibiotics, antibiotics resistance genes (ARGs) and horizontal transfer elements (HTE) were detected during the swine manure anaerobic digestion with different solid loading, in order to clarify the effect of solid loading on the removal of antibiotics and ARGs during anaerobic digestion. High-throughput sequence method was used to detect the evolution of microbial community with different solid loading during the swine manure anaerobic digestion, in order to illuminate the effect of the functional bateria and routine parameters on the change of ARGs and to provide scientific evidence for reducing the risk of antibiotics resistance by anaerobic digestion (AD). The results of antibiotics screening shown that there were 11 antibiotics, including TC,OTC,CTC,SDMD,CLA,TILM,CIP,OFL,ENR,PCN-G and LIM existed in the swine wastewater. The 11 antibiotics quantitative analysis methods were build by optimizing solid phase extraction (SPE), the condition of HPLC and MS/MS. Methanol and Na2EDTA-McIlvaine buffer were used as extracted solvents, the method of extracting antibiotics from the solid swine manure was established by ultrasonic extraction. The recoveries of every compound were over 50% during SPE process, and the ranges of recoveries were in 30%-160% during SPE + ultrasonic extraction. Limit of determination and quantitation were 0.010-2.885 μg/L and 0.034-9.615 μg/L, and the linear range was in 5-500 μg/L. Two intensive swine farms were selected; antibiotics residues in the swine wastewater and solid wastes were investigated during the spring, summer and winter. The results shown that tetracyclines (TCs) were the most abundant antibiotics residues in these two swine farm, which the average percentage was 58%. The second abundant antibiotic was macrolides TILM, which the average percentage was 33%. Sulfonamides SDMD was detect in the swine farm 1 and accounted for 10%, and it was not detected in the swine farm 2. Antibiotics residues in the winter and spring were higher than that in the summer, since infectious disease is prevail during winter and spring. Additionally, most of antibiotics was concentrated in the solid phase, which accounted for 98.5% averagely, only 1.5% antibiotics in the liquid phase. Otherwise, the percentage of SDMD in the liquid phase was relatively high, which was 5.29% averagely. Approximately 80% antibiotics were removed after anarobic digestion and aerobic process. The removal effect of SDMD was bad, and SDMD occupied relatively higher proportion in the effluent. Fresh swine manure was collected and brought back from the swine farm 1. Different solid loading (TS:B=4%,the convertional AD;C=8%,D=11%,E=14%,high solid loading AD) were carried out. The results shown that the total amount of antibiotics decreased by 35%, 46%, 39%, 14%, respectively, after the swine manure AD, e.g., TCs, TILM and Fluoroquinolones (FQs) decreased by around 55% and SDMD increased evidently. These results were accordance with the investigation results. The total relative abundance of ARGs decreased by 17%, 20%, 20%, 7%, respectively in B, C, D, E assays after the swine manure AD. ermB was the dominant ARGs in the swine manure and decreased largely by 16%, 23%, 23%, 28%, respectively. This result indicated that AD could eliminate the dominant ARGs in the swine manure. It should pay more attention to mefA, sulI, sulII, tetG, which increased obviously after AD. The difference was significant on the change of IntI1 during the conventional AD and high solid loading AD, IntI1 increased dramatically on the 63th, 77th in the high solid loading AD, which was much higher than that in the conventional AD. The high solid loading AD might cause horizontal transfer rate relatively higher than the conventional AD. Firmicutes was the most abundance bacteria during the whole AD, the average abundance was 70%, the second and the third abundance bacteria were Bacteroidetes and Proteobacteria. The sum of these three bacteria accounted for 90%. There were significantly differences on microbial community evolution between the conventional AD and the high solid loading AD, e.g., the abundance of Sedimentibacter, Ercella, Acinetobacter and Comamonas were increased during the conventional AD but the high solid loading AD; Corynebacterium, Thermacetogenium, Wohlfahrtiimonas, Tepidimicrobium, Paenalcaligenes, Thiopseudomonas were increased during the high solid loading AD but the conventional AD, which these bacteria shown the obviously correlation with VFAs (Volatile fatty acids) and TAN (Total NH3-N). ermB、blaTEM、tetX、pcoA shown a significantly correlation with Anaerococcus, Peptoniphilus, Streptococcus, Lactobacillus, Methanosphaera, Olsenella, Syntrophococcus, Peptostreptococcus, Bacteroides, Anaerosphaera, Methanothrix,which took on decreasing trend during the AD. These demonstrated that the evolution of microbial community played an important role on the change of ARGs
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/38702
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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作者单位: 1.中国科学院生态环境研究中心

Recommended Citation:
王瑞. 养猪废弃物中多种抗生素同时分析及抗性基因在厌氧处理中的转归研究[D]. 北京. 中国科学院大学. 2017.
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