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题名: 猪粪厌氧消化过程中四环素类抗生素和铜的影响及其抗性基因归趋研究
作者: 王瑞
学位类别: 博士
答辩日期: 2015-05
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 魏源送
关键词: 猪粪中温厌氧消化,金霉素,铜,复合污染,swine manure mesophilic anaerobic digestion, chlortetracycline, copper, combined pollution
其他题名: Effect and fate of tetracyclines, Cu and their resistant genes on swine manure anaerobic digestion
学位专业: 环境工程
中文摘要:     我国集约化畜禽养殖业产生了大量的畜禽粪便。残留在畜禽粪便中的高浓度兽用抗生素和重金属不仅会带来严重的环境污染,而且会诱发诸如耐药细菌、抗性基因的产生和传播,对人类健康造成巨大的潜在危害。厌氧消化是畜禽粪便资源化、减量化和无害化的主要处理方法。然而,目前人们对兽用抗生素与重金属的复合污染在畜禽粪便厌氧消化中的影响及其抗性基因的归趋缺乏深入的认识。因此,针对畜禽粪便中残留四环素类抗生素和重金属铜的复合污染特征及其控制需求,本文选取猪粪为研究对象,考察了四环素类抗生素、铜及其复合污染对猪粪中温厌氧消化的影响及其归趋;研究了四环素和铜的抗性基因、功能菌群在猪粪中温厌氧消化过程中的变化,以期为我国畜禽粪便的资源化和安全利用提供科技支撑。
    首先以猪场厌氧消化污泥为接种污泥,考察了不同浓度金霉素、铜及其复合污染对猪粪中温厌氧消化产甲烷潜势的影响(500 mL反应器)。试验结果表明,单独添加金霉素(150, 500, 1000 mg/kg DW)和单独添加低浓度的铜(2000 mg/kg DW)对累积产甲烷总量有一定的促进作用,而单独添加高浓度的铜(5000 mg/kg DW,10000 mg/kg DW)却对累积产甲烷总量有一定的抑制作用,但影响均不显著(P > 0.05),可能是接种物中的微生物已经适应了高浓度的四环素类抗生素或铜。然而,复合污染条件下(同时添加金霉素和铜),累积产甲烷总量显著降低(P < 0.05)。经过猪粪中温厌氧消化后,金霉素(包括差向异构金霉素)的去除率高达85%左右;四环素、差向异构四环素、土霉素和差向异构土霉素只有在铜添加组才有比较明显的去除效果,这说明铜可能促进了四环素、差向异构四环素、土霉素和差向异构土霉素在猪粪中温厌氧消化过程中的降解。
    为了进一步明确金霉素和铜对猪粪中温厌氧消化过程的影响,以北京某污水处理厂厌氧消化污泥为接种污泥,考察了一定浓度的金霉素(500 mg/kg DW)和铜(5000 mg/kg DW)及其复合污染对猪粪中温厌氧消化过程的影响(2 L反应器)。结果表明,单独添加金霉素或铜可以极大地增加VFA浓度,其中金霉素添加组、铜添加组、空白组VFA的最大值分别为3307、5745、1909 mg/L,进而提高了累积产甲烷总量,例如,金霉素和铜添加组分别提高了21%和15%。金霉素和铜复合污染条件下,猪粪厌氧消化上清液中的VFA浓度(1394 mg/L)明显低于空白组(1909 mg/L),说明同时添加这两种物质会抑制水解酸化过程,导致累积产甲烷总量明显减少(-30%)。
    采用定量PCR,考察了猪粪中温厌氧消化过程中四环素和铜的抗性基因变化特征。研究结果表明,经过中温厌氧消化后,tetO、tetX、tetL、tetW和pcoA、pcoC、pcoD的绝对丰度减少了一个log左右,分别是tetO:1.45,tetX:1.51,tetL:1.12,tetW:0.77 log copies /g TS和pcoA:0.83,pcoC:1.37,pcoD:0.87 log copies /g TS;而tetA和intI1的绝对丰度却有所增加。在金霉素或铜单独添加组,tet基因和pco基因的相对丰度(金霉素组tet:8.5%,pco:0.006%;铜添加组tet:9.3%,pco:0.006%)均高于空白组(tet:6.2%,pco:0.005%),这可能是抗生素和重金属协同抗性的表现。
    采用高通量测序,考察了金霉素、铜及其复合污染对猪粪中温厌氧消化过程中微生物群落结构的变化及其影响。结果表明,复合污染对第一阶段(1-13天)的功能菌群(主要以可降解大分子有机物的微生物菌群占优势,如普雷沃氏菌科、紫单胞菌科和瘤胃球菌科等)有抑制作用。金霉素或铜单独添加能够促进这一阶段微生物菌群的生长,但是铜的添加会延迟厌氧消化进入第二阶段(13-27天)。
    采用Pearson双变量方法分析了猪粪中温厌氧消化过程中抗性基因与微生物群落的相互关系,结果表明,抗性基因tetO、tetX、tetL、tetW与普雷沃氏菌属、拟杆菌属和韦荣氏球菌属微生物显著正相关。这三种微生物很可能是这几种抗性基因的优势宿主菌群。
英文摘要:     A large amount of animal manure has been produced with the development of confined animal feeding operations (CAFOs) in China. The high level residual veterinary antibiotics and trace heavy metals in animal manure not only cause serious environmental pollution, it will also induce antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARG), and cause a potential damage to human health. Anaerobic digestion is one of methods widely used in resource recovery, reduction and sanitation of animal manure. However, there is still information gap regarding the combined effect of veterinary antibiotics such as tetracyclines and heavy metals on swine manure anaerobic digestion. Thus the purpose of this study was to investigate effects of tetracyclines, copper and their combined pollution on batch swine manure mesophilic anaerobic digestion, in order to better estimate and control risk from antibiotics and heavy metals to anaerobic digestion. Meanwhile resistant genes of tetracycline and Cu, and microbial community evolution under CTC, Cu and their combined pollution conditions were investigated during swine manure mesophilic anaerobic digestion.
    The results of anaerobic digestion biochemical methane potential (500 mL BMP tests) assays showed that spiked chlortetracycline (CTC) (150, 500, 1000 mg/kg DW) or Cu (2000, 5000, 10000 mg/kg DW), respectively, had no significant impacts on the total methane yield (P > 0.05), and the possible reason was that the inoculum collected from an anaerobic digestion tank in this study had already acclimated to high level of TCs and Cu. The combined effect of CTC and Cu had a significant influence on the total methane yield (P < 0.05). The removal rate of CTC (including ECTC) was up to 85% during swine manure anaerobic digestion. Tetracycline, epi-tetracycline, oxytetracycline and epi-oxytetracycline had an obviously removal rate except that in copper spiked assays.
    In order to further investigate the effects of CTC and Cu on swine manure anaerobic digestion, the inoculum was collected as anaerobic digestion sludge from a municipal wastewater treatment plant in Beijing. The certain concentrations of CTC (500 mg/kg DW), Cu (5000 mg/kg DW) and their combination were spiked on mesophilic anaerobic digestion (2 L) to investigate their combined effect on anaerobic digestion process. The results showed that concentrations of VFA were much higher in spiked CTC (3307 mg/L) or Cu (5745 mg/L) assays.Although daily methane production flow rate was inhibited, the final methane yield was increased by 21% and 15% in the CTC or the Cu spiked assays. VFA concentration in the combined pollution assay (1394 mg/L) was much lower than that in the control assay (1909 mg/L). This demonstrated that spiking CTC and Cu together would inhibit hydrolysis-acidification process, and thus result in low methane yield by -30%.
    The results from tetracycline and Cu resistant genes showed that absolute abundance of tetO, tetX, tetL, tetW, pcoA, pcoC and pcoD had reduced by approximately one order of magnitude (tetO:1.45, tetX:1.51, tetL:1.12, tetW: 0.77, pcoA: 0.83, pcoC:1.37, pcoD: 0.87 log copies /g TS) after mesophilic anaerobic digestion; absolute abundance of tetA and intI1 had increased after mesophilic anaerobic digestion. In addition, in CTC or Cu spiked assays, relative abundance of tet genes (8.5%, 9.3%) and pco genes (0.006%, 0.006%) were higher than the control assay (tet:6.2%, pco:0.005%). It may be co-selection resistance mechanisms of antibiotic and heavy metals.
    Evolution of microbial community structure of mesophilic anaerobic digestion was investigated using high throughput sequencing method. The results demonstrated that during the three stages of the swine manure mesophilic anaerobic digestion, the combined effect of CTC and Cu would inhibit functional flora of the first stage (1-13 days), including microorganisms such as Prevotellaceae, Porphyromonadaceae, Ruminococcaceae which could degrade macromolecular organic matter. Spiked CTC or Cu separately could promote growth of functional flora in the first stage. However, spiked Cu alone would prolong anaerobic digestion to step into the second stage (13-27 days).
    The Pearson correlation analysis showed that there was a positive correlation among tetO、tetX、tetL、tetW, pcoA、pcoC、pcoD genes and several microorganisms, such as Veillonella, Prevotella and Bacteriodes. It implied that these kinds of microorganisms might be the host bacteria of these resistant genes.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34444
Appears in Collections:水污染控制技术研究室_学位论文

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Recommended Citation:
王瑞. 猪粪厌氧消化过程中四环素类抗生素和铜的影响及其抗性基因归趋研究[D]. 北京. 中国科学院研究生院. 2015.
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