【作者】 毛跃建；

【导师】 赵立平；

【作者基本信息】 上海交通大学 ， 微生物学， 2009， 博士

【摘要】 在废水生物处理系统中,污染物的去除主要是由其中的微生物群落完成的,微生物群落的结构和功能往往决定了整个装置的性能。Thauera属细菌就是一类广泛存在于各种类型的废水处理装置中并具有多种芳香族污染物降解能力的重要功能类群,深入研究其种群组成与降解功能,具有重要的理论意义和应用价值。由于分离比较困难、缺少专一性的分子检测方法,人们对于废水处理系统中Thauera属种群的结构与功能的了解还非常有限。为此,本论文建立了Thauera特异性、嵌套式PCR-DGGE (denaturing gradient gel electrophoresis)的方法用于环境样品中Thauera种群结构的研究。对一个来自废水处理系统的生物膜样品以Thauera特异性PCR扩增产物建立了克隆文库,所有测序的克隆(91个)都属于Thauera属,说明该PCR方法具有很高特异性。为了快速简便的比较不同样品中Thauera的结构,以特异性PCR扩增产物为模板进行16S rRNA基因V3区嵌套式扩增后用于DGGE分析,建立了Thauera特异性PCR-DGGE方法。13个带有不同V3序列的克隆在DGGE上形成了10条条带,说明该方法具有较高的分辨率,可以用于检测样品中的Thauera属的组成。在一个大型焦化废水处理装置由于回流泵机械故障而导致化学需氧量(COD)去除率逐渐降低时,我们使用建立的特异性PCR-DGGE方法对系统中Thauera的结构变化进行了追踪监测。对DGGE指纹图谱进行主成份分析(PCA)后,发现当系统COD的去除率从前4周的84.1±2.7%降到第5和第6周的低于75%时,Thauera种群结构呈现出相应的时间演替轨迹,表明Thauera种群结构的变化与系统COD去除功能的波动密切相关。为了分离焦化废水处理系统中常规方法难以分离的Thauera细菌,我们设计了一套以Thauera特异性16S rRNA基因为靶向的分离方法。根据已知Thauera的代谢特性及目标菌的生活环境,设计了6种培养基,并使用Thauera特异性PCR-DGGE方法对焦化废水处理装置反硝化池样品在不同培养条件下获得的Thauera多样性进行了分析。选取多样性较高的培养基1/10 NB与MMQ在好氧条件下进行分离培养。以Thauera特异性PCR方法筛选阳性菌落,并用DGGE方法检验菌落的纯度。将含有Thauera的混合菌落在不同的选择性培养基上多次划线,使用特异性PCR及DGGE方法追踪含有Thauera的菌落并分析其纯度,最终从反硝化池样品中纯化获得了3株Thauera菌株(Q4、3-35和Q20-C)。这种以特异性分子标记为靶向分离培养细菌的方法,提高了细菌筛选的灵敏度,可协助分离常规方法难以分离的细菌。随后,对这3株Thauera菌株的基因及其污染物降解能力进行了比较分析。测序结果表明这3株Thauera细菌具有相同的16S rRNA基因,但是它们的ERIC-PCR基因组指纹图谱却存在显著差异(相似性<65%)。使用气相色谱-质谱(GC/MS)联用技术对这3株菌的污染物降解能力进行了测定。好氧条件下,它们能降解焦化废水中除喹啉外的所有主要有机污染物(苯酚、甲酚、二甲酚及吲哚等),但苯酚降解速度却各不相同(Q4>3-35> Q20-C)。在已知的8个Thauera种中,只有T. phenylacetia能在好氧条件下降解其中的一种污染物(苯酚)。虽然Q20-C含有亚硝酸盐还原酶基因(nirS),但这3株菌都没有显现出反硝化能力,而已知的Thauera细菌则都是反硝化菌。以上结果表明,这3个从焦化废水处理装置中分离到的细菌可能代表了一种新的Thauera类型,具有广泛的芳香族污染物降解能力。为了了解Thauera属细菌在废水处理脱氮过程中的作用,我们研究了8个不同来源的Thauera菌株(T. aminoaromatica、T. linaloolentis、T. phenylacetica、T. terpenica、Thauera sp. DNT-1、Thauera sp. 27、Thauera sp. 28及Thauera sp. 63)的反硝化功能及基因。反硝化过程中,所有Thauera均只产生少量的NO(<50 nmol/flask;< 32.9 nM in liquid)。除T. phenylacetica外,其余Thauera菌株都可将硝态氮彻底转化为氮气。PCR分析后发现T. phenylacetica缺失了N2O还原酶(nos)基因,使它的反硝化终产物为N2O。对T. aminoaromatica在不同pH、O2及NOx条件下的反硝化过程进行分析后发现,在pH7-9范围内,随着pH的升高,积累的中间产物(NO和N2O)会减少。相比于NO3-,以NO2-作为电子受体,不仅使得积累的N2O量显著升高(6-40倍),还使其开始反硝化的O2浓度从<0.8μM提高到了>4μM。这说明NO2-能诱导T. aminoaromatica反硝化基因的表达。在反硝化进行时,只有N2O还原酶的活性会被O2立刻抑制,而其他反硝化酶的活性基本不受的影响。这进一步阐明了氧气调控反硝化过程的机制。本论文建立了废水处理系统中重要功能类群Thauera属种群结构的专一性检测方法,并在该方法的协助下从废水处理系统中分离到了常规方法难以分离的Thauera菌株;揭示了Thauera属细菌在废水处理系统的有毒有机污染物降解及脱氮过程中的作用,并阐述了不同环境条件对Thauera反硝化功能的影响,为维持及强化废水处理装置的功能提供了菌种资源和理论依据。更多还原

【Abstract】 It has been well recognized that the performance of a wastewater treatment plant (WWTP) is mainly determined by the structure and activity of its microbial community. Thauera genus has been known as one of the functionally important groups, which has been widely found in WWTPs and mostly shown high versatile organic substrate degrading capacity. However, our understanding of the structure and function of Thauera genus in WWTPs was still limited, due to its resistance to isolation and lack of group-specific analysis method.Therefore, a Thauera-specific nested-PCR denaturing gradient gel electrophoresis (DGGE) method was firstly developed, and its usefulness was demonstrated by monitoring the structural shifts of Thauera spp. in an anaerobic-anoxic-oxic fixed-biofilm coking wastewater treatment plant (WWTP) responding to operational perturbations. The specificity of the PCR method was demonstrated by the fact that all 16S rRNA gene sequences, which were cloned from the amplicons of a biofilm sample, belonged to Thauera genus. 16S rRNA gene V3 region was then amplified from the first round Thauera-specific PCR product and applied for DGGE analysis. Different amplified fragments of Thauera clones, with 13 different V3 regions, migrated into 10 positions on DGGE gel, which demonstrated the high resolution of this DGGE method.When the WWTP experienced a gradual deterioration in chemical oxygen demand (COD) removal function due to a mechanical failure of the recirculation pump, biofilm samples were collected from the reactor and analyzed by this method. Principal component analysis (PCA) of the DGGE fingerprinting data showed that the composition of Thauera group exhibited a time related trajectory when the plant’s COD removal rate decreased from 84.1±2.7% in the first 4 weeks to less than 75% at week 5 and 6, suggesting the structural shift of Thuaera genus was closely related with the system’s COD removal function.A new isolation method that was guided by Thauera-specific PCR and DGGE fingerprinting was developed to get purified cultures of the Thauera spp. from the WWTP. According to the physiological characteristics of known Thauera strains and the living environment of the target bacteria, six types of media were designed. The biofilm from the denitrifying bioreactor of the coking WWTP was inoculated to these media and cultured under both aerobic and anaerobic conditions, the diversity of Thauera spp. that grew under different conditions were analyzed by Thauera-specific PCR-DGGE. Two media (1/10 NB and MMQ) which recovered higher diversity of Thauera spp. and lower number of colonies were used to isolate Thauera sp. under aerobic condition. The colonies were screened by Thauera-specific PCR. The homogeneity of colonies showing positive PCR signals was then checked by DGGE. The colonies with multiple species were further purified by being streaked on different selective media. The positive colonies were tracked by Thauera-specific PCR, and their homogeneity analyzed by DGGE. Finally, three Thauera strains (Q4, Q20-C and 3-35) were isolated to pure cultures. Guided with group-specific PCR and DGGE method, the efficiency and sensitivity of bacterial isolation can thus be significantly improved.The functional genes and pollutants-degrading capacity of these Thauera isolates were then characterized. Although sequencing analysis showed they had identical 16S rRNA genes, their ERIC-PCR fingerprinting patterns were significantly different (similarity <65%), indicating wide variation of genomic structures. The degradation of organic pollutants in coking wastewater by these strains was studied with gas chromatography-mass spectrometry (GC/MS). All the main organic pollutants (phenol, methylphenol, quinoline and indole) in the coking wastewater, except quinoline, were degraded by them under aerobic condition. Their phenol degradation rates were different (Q4> 3-35> Q20-C). However, within all the eight Thauera species, only T. phenylacetia has ability to degrade one of these aromatic compounds (phenol) under aerobic condition. Nitrite reductase gene (nirS) was detected in Q20-C, but none of these strains showed denitrification capacity. However, all the known Thauera species were denitrifiers. These results suggested the Thauera strains that isolated from coking WWTP may represent a new Thauera species, and have high versatile aromatic compounds degrading capacity.To understand the roles of Thauera spp. played in nitrogen removal in WWTPs, the denitrification genes and functions of 8 Thauera strains (T. aminoaromatica, T. linaloolentis, T. phenylacetica, T. terpenica, Thauera sp. DNT-1, Thauera sp. 27, Thauera sp. 28 and Thauera sp. 63) that originated from different environments were studied. All the strains emitted little NO (<50 nmol per flask; < 32.9 nM in liquid) during the denitrification process. All of them were able to transform nitrate to N2, except T. phenylacetica, which produced N2O as the final product and was found to be lack of N2O reductase (nos) gene. Analysis of the denitrification of T. aminoaromatica under different pH、O2 and NOx condition, indicated that in its activity pH range (pH 7-9), less intermediates (NO and N2O) were produced at higher pH level. In comparison to nitrate, the N2O production was significantly increased (6-40 times) under nitrite condition; the O2 level that denitrification started was also increased from < 0.8μM (in nitrate) to > 4μM (in nitrite), showed the induction effect of nitrite on denitrification of T. aminoaromatica. The activity of N2O reductase was inhibited by O2, but the other denitrification enzymes were not affected. It improved our knowledge on the regulation mechanism of O2 on denitrification.In conclusion, a Thauera specific PCR-DGGE method was developed for analyzing the structure of this functionally important population in WWTPs. The Thauera strains in the coking WWTP, which were known as difficult to be isolated by conventional methods, were isolated under the guidance of this group-specific method. This study demonstrated the important roles of Thauera spp. in degradation of toxic organic pollutants and nitrogen removal in WWTPs, illustrated the impacts of different conditions on the denitrification of Thauera, and provided new insights, strains and methods for optimizing the function of WWTPs.更多还原