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题名: A2/O工艺污泥膨胀时期微生物群落及PAEs生物降解菌的研究
作者: 金德才
学位类别: 博士
答辩日期: 2012
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 庄国强
关键词: 膨胀污泥 ; sludge bulking ; 微生物多样性 ; microbial diversity ; 邻苯二甲酸酯 ; PAEs ; 多相分类 ; polyphasic approach ; 生物降解 ; biodegradation
其他题名: Analysis of bacteria communities of bulking sludge and characterization of PAEs-degrading strains in a wastewater treatment plant with A2/O process
中文摘要:       随着工业化和城镇化的发展,污水处理厂应运而生,在脱氮除磷,有机污染物降解方面发挥着重要作用。然而,污泥膨胀已成为污水处理厂实际运行过程中普遍存在的问题,对于污水处理的效果造成了严重的影响,目前,关于污泥膨胀时期的微生物群落结构的研究鲜见报道,以及该时期邻苯二甲酸酯类环境激素相关降解菌的研究非常缺乏,本论文首先采用可培养和不可培养技术分析了污泥膨胀时期的细菌多样性,获得了比较详尽的细菌群落组成。其次,通过富集培养,分离筛选到多株具备邻苯二甲酸酯(PAEs)降解能力的菌株,并在富集液中分离到一株细菌新种。研究结果概括如下: 1. 利用6种培养基对膨胀时期的活性污泥的细菌进行了分离鉴定,结果表明63株可培养微生物可划分为28个种,属于6个大的系统发育类群(γ-Proteobacteria,β-Proteobacteria,α-Proteobacteria,Firmicutes,Actinobacteria,和Bacteriodetes),其中γ-Proteobacteria为最优势类群。利用rep-PCR技术对其中的优势菌株进一步区分,结果表明,利用REP 和BOX该技术可以明显区别同属之间的差异。16Sr RNA基因克隆文库结果表明,在膨胀污泥中β-Proteobacteria 是优势类群,Candidatus Accumulibacter phosphatis是优势菌株,占据了14.7%。以上结论揭示了采用不同的研究手段,可以得到更加丰富的微生物多样性信息,从而对于膨胀污泥的调控提供更多的参考。 2. 从邻苯二甲酸酯类化合物的富集液中,分离到了10株细菌。其中4株细菌从以邻苯二甲酸酯化合物为唯一碳源的无机盐培养基上分离得到;其余6株为LB琼脂培养基所分离。这10株细菌包括4株Gordonia细菌,2株Delftia细菌,2株Pseudomonas细菌,1株Diaphorobacter细菌,1株Terrimonas属潜在新种。 3. 首次报道了一株DBP降解新属,并对其降解特性进行了详细研究,结果表明该菌对DBP降解的最佳条件为pH7.0-8.0,温度30-35℃,转速150–225 r/min;在最适条件下,该菌能把500 mg/L的DBP 在12 h内降解完全。此外,考查了重金属(Cr6+和Cu2+)存在下对该菌对DBP降解的影响,结果显示在5-30 mg/L范围内,均能显著抑制该菌的降解能力。 4. 对分离到的潜在新种QHT进行多相分类学的鉴定。结果:菌株QHT为革兰氏阴性,无运动性,不产孢子,严格好氧的杆菌。细胞大小为1.0-3.0 μm长和0.3-0.5 μm宽。在R2A培养基上培养2 d后,形成直径为1.0 mm左右,乳白色,表面光滑整齐。菌株生长温度是10-37℃(最适30℃),pH范围是5.0-8.0 (最适pH7.0),NaCl浓度范围是 0-1%(w/v,最适0%)。不产色素,具有氧化酶和过氧化氢酶活性。16S rRNA序列分析显示,菌株QHT归类于Terrimonas属,最近缘种是Terrimonas lutea BCRC 17944T,其序列同源性是96.3%,基因组DNA的(G+C)mol%含量是41.0%。菌株QHT主要包含的脂肪酸类型是iso-C15:0,iso-C15:0 G和summed feature 3(由C16:1 ω6c 和或C16:1 ω7c 组成),主要的醌型是MK-7,主要的磷脂成分为磷脂酰乙醇胺(PE)。基于表型特征和系统发育分析,菌株QHT是Terrimonas属内的一个新种,命名为Terrimonas pekingensis。作为典型菌株保藏编号是CICC 10452T和NCCB 100397T)。 5. 首次报道了一株可以利用多种PAEs和中间代谢产物(PA)的Gordonia细菌,代号为QH-11。QH-11的DBP最佳降解条件为PH7.0,温度30℃。在最适条件下,考查了不同初始浓度下(100、200、300、500和750 mg/L)该菌对DBP的降解能力,结果显示此浓度范围内该菌的DBP降解过程可用Gompertz曲线来描述,相关系数均为0.98以上。此外,在菌株QH-11中检测到邻苯二甲酸双加氧酶的基因序列,以葡萄糖为唯一碳源做对照,RT-qPCR结果显示,该基因在DBP和PA诱导下,表达水平可以显著上升,这是首次报道定量Gordonia菌株中邻苯二甲酸双加氧酶的差异表达。
英文摘要:
      With the development of industrialization and urbanization, sewage treatment plants came into play an important role in nitrogen and phosphorus removal and degradation of organic contaminants. Sludge bulking, however, has become a common problem affecting plant operation of the sewage treatment plant, and it also has a serious impact on performance of wastewater treatment plant. Up to now, there is relatively little attention paid on the microbial community structure and function during the process of sludge bulking, and phthalate esters-degrading bacteria are rarely reported in A2/O process. In this study, the detailed information about bacterial community composition was obtained based on culture-dependent and-independent method. Moreover, several bacteria strains were isolated from PAEs-degrading consortium, including new specie in genus Terrimonas. The results are summarized as follows: 1. Six different culture media were used to reveal the bacterial composition during this stage. A neighbor-joining tree of the partial 16S rRNA gene sequences showed that 63 isolates were phylogenetically clustered into 6 major groups and 28 distinct lineages or species, γ-Proteobacteria was most dominant group. Moreover, these dominant strains (number ≥5) were further analyzed by rep-pcr technology (REP-pcr and BOX-pcr). The results showed that rep-pcr produced highly discriminatory banding patterns among the same genus using REP and BOX primer sets. While the culture-independent assessment revealed that β-Proteobacteria was the dominant group in the bulking sample. The sequences of an operational taxonomic unit were 98% identical to that of Candidatus Accumulibacter phosphatis, which is used to remove phosphorous from wastewater, representing the highest proportion (14.7%) of the total clones. These results indicated that combining different approaches can produce complementary information, thus generating a more accurate view of microbial community in bulking sludge. 2. Ten bacteria strains were isolated from an enriched PAEs-degrading microbial consortium. Of four strains were cultured on minimal salts agar plates supplemented with PAEs, six strains were cultured on LB agar plate. Ten bacteria strains including four Gordonia strains, two Delftia strains, two Pseudomonas strains, one Diaphorobacter strain, and one possible new species of genus Terrimonas. 3. For the first time, we studied the biodegradation ability of PAEs by genus Diaphorobacter. The HPLC analysis revealed that the optimum conditions for DBP degradation were pH 7.0–8.0, temperature 30–35°C, and agitation rate 150–225 r/min. Under these conditions, 500 mg/L of DBP could be completely degraded within 12 h. We investigated the effects of heavy metals (Cr6+ and Cu2+) on the DBP degradation. The results demonstrated that the heavy metals at a wide concentration range of 5-30 mg/L can restrain the DBP degradation. 4. Strain QH was characterized using a polyphasic approach. The results were as follows: Cells are Gram-staining-negative, strictly aerobic, non-motile, and rod-shaped. Poly-β-hydroxybutyrate granules are not accumulated and flagella are not observed. After 72 h incubation at 30°C, the mean cell size is approximately 0.3-0.5 μm in diameter and 1.0-3.0 μm in length. Colonies are milky white, viscous, smooth, circular with entire edges, approximately 1.0 mm in diameter on R2A agar after 72 h incubation at 30°C. Growth occurs at 10-37°C (optimum, 30°C), pH5.0-8.0 (optimum, pH7.0) and with 0-1% NaCl (optimum, 0%). Flexirubin-type pigments are not present. Positive for oxidase and catalase activities. Analysis of the 16S rRNA gene sequence of strain QH revealed that it is a member of the genus Terrimonas. Terrimonas lutea BCRC 17944T was the nearest relative (96.3% 16S rRNA gene sequence similarity). The G+C content of the genomic DNA was 41.0 mol%. The major fatty acids were iso-C15: 0, summed feature 3 (comprising C16: 1ω7c and/or C16: 1ω6c), and iso-C15: 1 G. The predominant isoprenoid quinone was MK-7. The predominant polar lipid of strain QHT was phosphatidylethanolamine. On the basis of phenotypic properties and phylogenetic inference, strain QH represents a novel species of the genus Terrimonas, for which the name Terrimonas pekingensis is proposed. The type strain is QHT (=CICC 10452T = NCCB 100397T). 5. A bacterial strain designated as QH-11 was isolated and found to be able to degrade various PAEs and intermediate product phthalic acid (PA). The optimal conditions for DBP degradation was pH 7.0 and 30°C, and kinetics studies of DBP degradation by the strain QH-11 revealed that DBP depletion curves fit with the modified Gompertz model (R2>0.98). Moreover, a gene encoding the large subunit of the phthalate dioxygenase, which is responsible for PA degradation, was successfully detected in strain QH-11. Furthermore, the results of reverse transcription quantitative PCR demonstrate that mRNA expression level of phthalate dioxygenase increased significantly after strain QH-11 was induced by DBP and PA.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/35093
Appears in Collections:中科院环境生物技术重点实验室_学位论文

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Recommended Citation:
金德才. A2/O工艺污泥膨胀时期微生物群落及PAEs生物降解菌的研究[D]. 北京. 中国科学院研究生院. 2012.
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