基因工程菌在厌氧膜生物反应器中对偶氮染料废水的脱色

印染和染料废水色度大,COD高,可生化性差.采用基因工程菌Escherichia coli JM109(pGEX-AZR)在厌氧膜生物反应器中,对模拟偶氮染料废水进行脱色研究.结果表明,系统对酸性红B有很好的脱色能力,且启动期短,脱色效率高,脱色率稳定在95%以上,对COD的去除率能达到68%.系统运行过程中,反应器内生物量稳定在0.4 g/L.EPS与生物量变化趋势一致,先升高后降低;pH值在运行初期由6.5降至6.2,后维持在6.6左右.系统运行27 d后进行第一次膜清洗,膜通量恢复为初始值的92%,系统运行周期约为26 d.     

七星河湿地氨氧化古菌多样性和丰度及其与环境因子的相关性分析

以典型东北寒温带沼泽湿地——七星河湿地中芦苇和小叶樟植被下不同深度土壤中氨氧化古菌(Ammonia-Oxidizing Archaea,AOA)为研究对象,以编码氨氧化作用关键酶——氨单加氧酶(AMO)的amoA基因为分子标记,通过构建克隆文库和生物信息学方法分析数据,考察了不同植被环境中AOA多样性和丰度的垂直分布规律,及其与环境因子的相关性.研究结果表明,小叶樟植被土壤中AOA amoA基因多样性显著高于芦苇植被土壤中amoA基因多样性,芦苇植被土壤中AOA amoA基因多样性在深度为40~60 cm土层中最高, AOA 多样性与NH4+-N、NO3--N含量呈正相关;小叶樟植被土壤中AOA amoA基因多样性在深度为20~40 cm土层中最高,AOA多样性与NH4+-N、NO3--N含量呈正相关.芦苇植被土壤中AOA丰度最低值出现于0~20 cm土层中,到 20~40 cm土层中出现最高值,随着深度增加AOA丰度下降,AOA丰度则与NO2--N含量呈正相关.小叶樟植被土壤中AOA丰度变化趋势与芦苇不同,最高值出现于0~20 cm土层中,随着土层深度增加AOA丰度下降,到40~60 cm出现最低值,AOA丰度与NO2--N、TOC含量呈正相关.两种植被土壤中AOA丰度均在60~90 cm处有升高趋势.芦苇植被土壤中AOA丰度约是小叶樟植被土壤中AOA丰度的1.49倍.以上结果为湿地AOA生态功能及分布模式的研究提供基础数据,对全球氮循环的研究提供补充.     

宁波三江口水域原核生物群落结构分析

首次应用16S rRNA基因-Illumina Miseq高通量测序技术对甬江流域宁波三江口区域的表层水体原核生物群落进行了分析,共获得215 504条高质量序列.多样性指数分析表明,该流域的水体原核生物群落具有较高的遗传多样性和丰富度.菌群分类分析发现,β-变形菌纲(β-Proteobacterium)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)是主要的优势菌群,其相对丰度占总群落的78.88%.对比不同站位的原核生物群落,发现水文环境可能是影响三江口水域原核生物群落组成和结构的重要因素之一.与以往报道对比分析表明,甬江流域分布有多种污水和粪便污染指示菌,其中余姚江污染指示菌的丰度最高,说明其受污染的风险可能较高.病原菌BLASTN分析表明,在种和亚种水平上,分别检出76和18种潜在病原菌,占序列总量的2.19%和0.40%.本研究为系统认识甬江流域原核生物群落结构及生态功能提供了重要基础数据.     

Monitoring aromatic hydrocarbon biodegradation by functional marker genes

The development of biological treatment technologies for contaminated environments requires tools for obtaining direct information about the biodegradation of specific contaminants. The potential of functional gene array analysis to monitor changes in the amount of functional marker genes as indicators of contaminant biodegradation was investigated. A prototype functional gene array was developed for targeting key functions in the biodegradation of naphthalene, toluene and xylenes. Internal standard probe based normalization was introduced to facilitate comparison across multiple samples. Coupled with one-colour hybridization, the signal normalization improved the consistency among replicate hybridizations resulting in better discrimination for the differences in the amount of target DNA. During the naphthalene biodegradation in a PAH-contaminated soil slurry microcosm, the normalized hybridization signals in naphthalene catabolic gene probes were in good agreement with the amount of naphthalene-degradation genes and the production of 14CO2. Gene arrays provide efficient means for monitoring of contaminant biodegradation in the environment.     

磺胺甲恶唑胁迫对2种微塑料上细菌群落和抗性基因影响的初步研究

微塑料异质性生物膜成为抗生素抗性基因(antibiotic resistance genes, ARGs)传播扩散的潜在汇。抗生素胁迫浓度以及微塑料种类对其生物膜上ARGs时间动态演化特征的影响目前尚不明确。本研究以聚乙烯和聚苯乙烯作为代表性的微塑料,研究低(10 μg·L^-1)、中(100 μg·L^-1)、高浓度(1 000 μg·L^-1)磺胺甲恶唑(sulfamethoxazole, SMX)胁迫下,微塑料生物膜上磺胺类抗性基因(sul1和sul2)、Ⅰ类整合子整合酶基因(intI1)和细菌群落的动态演化特征及其关联性。结果表明,2种不同微塑料上ARGs相对丰度无显著差异,但SMX胁迫作用明显提高ARGs丰度并促进ARGs的传播扩散,尤其是高浓度SMX具有最显著的胁迫作用;高浓度SMX胁迫下ARGs具有明显的时间序列演化特征,中浓度胁迫下ARGs无明显变化,低浓度胁迫仅在第60天对ARGs具有明显的促进作用;2种微塑料生物膜上细菌群落组成结构具有差异性特征,然而与ARGs强关联性的细菌在门水平无显著性差异。尽管2种微塑料上细菌属与ARGs共现模式存在差异,但未对不同微塑料上ARGs的相对丰度产生差异性影响。微塑料生物膜上intI1与ARGs具有明显的相关性,但2种微塑料上intI1无明显差异,未引起不同种类微塑料上ARGs差异性分布。     

Use of reporter-gene based bacteria to quantify phenanthrene biodegradation and toxicity in soil

A phenanthrene-degrading bacterium, Sphingomonas paucimobilis EPA505 was used to construct two fluorescence-based reporter strains. Strain D harboring gfp gene was constructed to generate green fluorescence when the strain started to biodegrade phenanthrene. Strain S possessing gef gene was designed to die once phenanthrene biodegradation was initiated and thus to lose green fluorescence when visualized by a live/dead cell staining. Confocal laser scanning microscopic observation followed by image analysis demonstrates that the fluorescence intensity generated by strain D increased and the intensity by strain S decreased linearly at the phenanthrene concentration of up to 200 mg/L. Such quantitative increase and decrease of fluorescence intensity in strain D (i.e., from 1 to 11.90 ± 0.72) and strain S (from 1 to 0.40 ± 0.07) were also evident in the presence of Ottawa sand spiked with the phenanthrene up to 1000 mg/kg. The potential use of the reporter strains in quantitatively determining biodegradable or toxic phenanthrene was discussed.     

Bioanalytical and instrumental analysis of thyroid hormone disrupting compounds in water sources along the Yangtze River

Thyroid hormone (TH) agonist and antagonist activities of water sources along the Yangtze River in China were surveyed by a green monkey kidney fibroblast (CV-1) cell-based TH reporter gene assay. Instrumental analysis was conducted to identify the responsible thyroid-active compounds. Instrumentally derived l-3,5,3′-triiodothyronine (T₃) equivalents (T₃-EQs) and thyroid receptor (TR) antagonist activity equivalents referring to dibutyl phthalate (DBP-EQs) were calculated from the concentrations of individual congeners. The reporter gene assay demonstrated that three out of eleven water sources contained TR agonist activity equivalents (TR-EQs), ranging from 286 to 293 ng T₃/L. Anti-thyroid hormone activities were found in all water sources with the TR antagonist activity equivalents referring to DBP (Ant-TR-EQs), ranging from 51.5 to 555.3 μg/L. Comparisons of the equivalents from instrumental and biological assays suggested that high concentrations of DBP and di-2-ethylhexyl phthalate (DEHP) were responsible for the observed TR antagonist activities at some locations along the Yangtze River.     

利用16S rRNA高通量测序技术考察温度对生物脱氮硝化过程中亚硝酸盐氧化菌代谢功能的影响

为探究温度对生物脱氮硝化过程微生物代谢及功能基因的影响,采用16S rRNA高通量测序技术,以富集的亚硝酸盐氧化菌(NOB)为研究对象,考察了活性污泥系统硝化过程中菌属与比亚硝酸盐氧化速率(SNiOR)的相关性。通过LEfSe分析筛选出3种温度(25、35和40℃)条件下的生物标记物,并利用KEGG数据库解析了微生物的代谢功能、氮代谢相关酶类型及相对丰度。结果表明,在3种温度条件下,NOB富集系统内的Nitrospira是SNiOR的唯一影响因子,且两者呈现显著的正相关性,Spearman相关系数(ρ)为0.95。此外,在温度条件为25、35和40℃时,分别筛选出14种、6种和8种生物标记物,显著优势菌分别是Xanthomonadales目、Sphingomonadales目和Gammaproteobacteria纲。以上结果说明：温度对微生物的代谢功能具有一定程度的影响,低温有利于碳水化合物代谢、能量代谢;较高温度会抑制能量代谢,NOB丰度降低;随着温度的升高,亚硝酸盐氧化还原酶(nxr)类基因(nxrA和nxrB)的数量基本保持不变。     

生物强化系统微生物分子诊断技术的应用及新发展

现代分子生物技术的飞速发展及其在环境研究领域的应用，为生物强化技术的研究和发展提供了新方法和新思路。本文从生物强化系统特异微生物检测及定量化技术、生物强化系统微生物群落结构组成及动态演替规律研究、生物强化作用机制的分子生物学解析、生物强化菌的基因工程构建、生物强化系统微生物的安全释放及控制技术几方面，对生物强化系统微生物分子诊断技术的应用和发展作了较为全面的综述。