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题名: 不同抗生素压力下中、高温厌氧消化对剩余污泥中抗性基因的控制研究
作者: 田哲
学位类别: 博士
答辩日期: 2015-04
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 杨敏 ; 张昱
关键词: 抗生素 ; 抗性基因 ; 剩余污泥 ; 中温厌氧消化 ; 高温厌氧消化 ; 控制,Antibiotics ; Antibiotic resistance genes ; Excess sludge ; Mesophilic anaerobic digestion ; Thermophilic anaerobic digestion ; Control
其他题名: Control of antibiotic resistance genes in excess sludge by mesophilic and thermophilic anaerobic digestion under different antibiotic stresses
学位专业: 环境工程
中文摘要:     抗生素抗性基因作为一种新型的环境污染物,引起了国际社会的广泛关注。我们的前期研究表明抗生素生产废水处理系统的剩余污泥与市政污泥相比含有高浓度的抗生素残留和高水平抗性基因,需进行有效处理。厌氧消化(Anaerobic digestion, AD)作为一种广泛采用的污泥处理工艺,最近被发现对市政污泥中的抗性基因也具有一定的消减效果,并且高温系统效果优于中温AD。然而至今为止,关于中、高温厌氧消化去除污泥中抗性基因的机制及高抗生素压力下厌氧消化系统中抗性基因的响应和控制未见系统报道。
    本论文系统研究了中、高温AD 对于污泥中抗性基因的控制效果,取得以下主要成果:
(1)本研究首先考察了直接升温策略从稳定运行的中温AD 系统启动高温AD 的有效性。发现采用直接升温的方式,可以在20 天左右时间内从稳定运行的中温AD 系统启动高温AD 系统。利用定量PCR 和454 焦磷酸测序分析了直接升温后60 天内消化污泥中微生物群落的演替情况。发现随着乙酸型产甲烷古菌Methanosarcina 和两种氢型产甲烷古菌( Methanothermobacter 和Methanoculleus)的快速增殖,嗜热产甲烷古菌群落至少在升温后11 内已经建立。升温后消化污泥中细菌群落结构也发生了显著地变化, 其中嗜热菌Fervidobacterium 在升温后18 天内相对丰度从0 快速升高至28.52%,从26 天开始出现其它潜在嗜热菌属丰度的增加,形成了稳定而多样化的嗜热细菌群落。上述结果表明,直接将消化温度升至嗜热产甲烷群落的最适生长温度(55℃),可以从一开始就促进其在消化系统中的快速增殖,从而促进高温AD 的快速启动。
(2)为探索中、高温AD 对市政污泥中抗性基因消减效果差异的机制,本研究针对上述高温厌氧消化启动过程,同时追踪了三大类共24 种抗性基因、3种水平转移元件和抗性基因细菌宿主(垂直转移)的动态变化。发现高温AD(抗性基因相对丰度去除率:64.99%)比中温厌氧(38.82%)表现出了更有效的抗性基因总量消减,其中四环素类和大环内酯类抗性基因在高温AD 中得到了更好的消减,而氨基糖苷类抗性基因在中温系统中得到了更好的消减。经中、高温AD 处理后,I 型整合子(intI1)丰度分别下降了47.51%和71.90%。intI1 与三种四环素抗性基因(tet(A)、tet(G)和tet(Q))表现出强的正相关性(P < 0.05),表明高温AD 可能通过更有效地阻断其水平转移途径而促进了三种基因的消减。另一方面,升温后消化污泥中抗性基因宿主的总丰度由16.16%降为7.77%。同时,转化过程中tet(X)、strB、tet(A)、aacA4 和ermF 等基因与其宿主表现出强相关性(tet(X)P < 0.01,strB P < 0.01,tet(A) P < 0.1,aacA4 P < 0.1,ermF P < 0.1),而其中大部分细菌宿主的丰度在升温后逐渐降低。上述结果表明,高温AD 可能同时更好地阻断了污泥中抗性基因的水平和垂直转移途径,从而保证了其更优的抗性基因消减能力。
(3)为探索抗生素压力下,中、高温AD 对剩余污泥中抗性基因的控制效果,考察了不同土霉素压力水平下(0,40,200,1000 mg/L)中、高温系统的运行效果及其中抗性基因的响应情况。中、高温系统在0~200mg/L 土霉素压力下运行效果稳定。然而,在1000mg/L 土霉素压力下,中温系统停止产气,而高温AD 的产气效果出现波动但经过一段时间适应后,效果恢复。中、高温系统对于污泥中吸附态土霉素的消减能力相似,但是高温系统相比中温系统能够有效去除污泥中溶解态土霉素含量。随着污泥中抗生素浓度提高,中、高温污泥中四环
素抗性基因的丰度均逐渐升高,而氨基糖苷类和大环内酯类抗性基因丰度同样随着污泥中土霉素浓度提高丰度逐渐提高,表明高抗生素压力下抗性基因共选择作用的存在。在不同抗生素压力下上述基因在高温污泥中的丰度仍是明显低于中温污泥。同时,3 种水平转移元件(intI1、Tn916/1545 和ISCR1)也随污泥中抗生素压力提高而增加,并且3 种转移元件分别与其能够转移的共线性抗性基因表现出强的正相关性,表明高抗生素压力下水平转移作用在抗性基因的扩增中发挥着重要作用。
英文摘要:     Numerous studies have demonstrated that sewage sludge from municipal wastewater treatment plant is an important reservoir for antibiotic resistance genes (ARGs), which could shade potential detriment to human health. It is thus important to handle the excess sewage sludge properly to prevent the release of ARGs to the environment. Anaerobic digestion (AD) is a widely applied technology for the disposal of excess sewage sludge. Recently, AD was found to be also effective in mitigating the quantities of ARGs in sewage sludge, and thermophilic AD always showed higher capacity in reducing ARGs than mesophilic system. In addition, excess sludge from antibiotic production wastewater treatment plant usually contains high levels of antibiotic resistance genes as well as high concentration of antibiotics, which might adversely affect the operation performance of digester and decrease the effectiveness of AD in the reduction of ARGs. However, previous studies were mainly carried out during the stable operation of the digester to compare the performance of different processes on the control of ARGs, which is hard to illustrate the mechanism that drives the difference in ARGs reduction by meso- and thermo- AD systems. On the other hand, the response of ARGs during AD under high concentration of antibiotics also requires in-depth studies. Meanwhile, for the thermophilic AD itself, whose microbial community composition is significantly different with the conventional mesophilic one, it’s currently unclear for the method and microbial ecology mechanism of its quick startup.
    In this study, we systematically studied the control of ARGs in excess sludge by thermophilic and mesophilic AD, and the main results are as follows:
(1) Stable thermophilic AD was achieved within 20 days from a mesophilic digester treating sewage sludge by adopting the one-step startup strategy. The succession of archaeal and bacterial populations over a period of 60 days after the temperature increment was followed by using 454-pyrosequencing and quantitative PCR. After the increase of temperature, thermophilic methanogenic community was established within 11 days, which was characterized by the fast colonization of Methanosarcina thermophila and two hydrogenotrophic methanogens (Methanothermobacter spp. and Methanoculleus spp.). At the same time, the bacterial community was dominated by Fervidobacterium, whose relative abundance rapidly increased from 0 to 28.52 % in 18 days, followed by other potential thermophilic genera, such as Clostridium, Coprothermobacter, Anaerobaculum and EM3. The above result demonstrated that the one-step startup strategy could allow the rapid
establishment of the thermophilic anaerobic microbial community.
(2) Though thermophilic AD was reported to be more effective than mesophilic AD in reducing ARGs in excess sewage sludge, the reason was not well understood. In this study, we examined the changes in the abundances of twenty-four ARGs conferring resistance to tetracyclines, macrolides and aminoglycosides, three mobile elements and the reported hosts of the targeted ARGs in sewage and digested sludge using quantitative PCR and 454 pyrosequencing during the transformation of an anaerobic digester from meshophilic to thermophilic. Tetracycline and macrolide resistance genes were more effectively reduced by thermophilic AD, while aminoglycoside resistance genes were more effectively attenuated by mesophilic AD. However, thermophilic AD still exhibited a significantly higher reduction (64.99 %) of the total quantity of the twenty-four ARGs than mesophilic one (38.82 %).
Decreased by 47.51 % and 71.90 % under mesophilic and thermophilic conditions, respectively, class 1 integron (intI1) exhibited significant correlations with three tetracycline resistance genes (tet(A), tet(G) and tet(Q)) (P < 0.05), suggesting that the blockage of the horizontal gene transfer pathway might be responsible for the reduction of these three ARGs. On the other hand, the relative abundance of the reported hosts of the twenty-four ARGs decreased from 16.16 % to 7.77 % after temperature increase. Meanwhile, tet(X), strB, tet(A), aacA4 and ermF genes showed significant or marginally significant positive correlation with the occurrences of their reported hosts (tet(X) P < 0.01, strB P < 0.01, tet(A) P < 0.1, aacA4 P < 0.1, ermF P <0.1) during the transition period. The findings demonstrated that thermophilic AD might be more effective than mesophilic one in blocking both the horizontal and vertical gene transfer pathways for the reduction of ARGs in sewage sludge.
(3) We established the lab-scale AD systems and used oxytetracycline (OTC) as the representative of antibiotics to explore the control of ARGs in excess sludge by mesophilic and thermophilic AD under high antibiotic pressure. Thermophilic AD showed a better tolerance of OTC: although its gas production fluctuated when the concentration of OTC in feed sludge reached 1000mg/L, but after a period of adaption its performance recovered. While the mesophilic digester was upset under the above OTC pressure, which required stoping dosing of OTC to recover the sigestion system. Mesophilic and thermophilic AD had a similar capability in the mitigation of adsorpted OTC, while soluble OTC was significantly lower in the thermophilic sludge than that in mesophilic one. As for the control of ARGs, together with the increase of OTC concentration, the relative abundances of nine tetracycline resistance genes increased, but their quantities in the thermophilic sludge were still lower than that in the mesophilic one. Aminoglycoside and macrolide resistance genes in digested sludge exhibited a similar trend with that of tetracycline resistance genes, proving the important roles of coselection for their dissemination during AD. Meanwhile, three mobile elements (intI1, Tn916/1545 and ISCR1) also increased followed the increase of antibiotic pressure, and they displayed significantly positive correlation with their corresponding ARGs during the experiment, indicating that during AD with a high antibiotic pressure horizontal gene transfer also played an important role in the amplification of ARGs.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34377
Appears in Collections:环境水质学国家重点实验室_学位论文

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田哲. 不同抗生素压力下中、高温厌氧消化对剩余污泥中抗性基因的控制研究[D]. 北京. 中国科学院研究生院. 2015.
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