石油污染对土壤微生物群落多样性的影响

土壤中的微生物主要有细菌、放线菌、真菌三大类群,微生物在石油污染的土壤中发挥着维持生态平衡和生物降解的功能。文中以四川省遂宁市射洪县某废弃油井周围不同程度石油污染土壤为供试土壤,首先对各组供试土壤的基本理化性质进行测定分析;然后采用平板菌落计数法测定了供试土壤中三大类微生物数量的变化,结果表明:相比未被污染的对照土壤,石油污染的土壤中细菌、放线菌、真菌数量均减少,并且土壤中可培养微生物的数量与土壤含水量呈正相关;再采用454焦磷酸测序技术对土壤中的细菌群落多样性及变化进行16S rRNA基因分析。在所有供试的4个土壤样品中,共鉴定出不少于23 982个有效读取序列和6 123种微生物,相比于未被污染的对照土壤,石油污染土壤中细菌的种类更加丰富,主要优势门类为酸杆菌门、放线菌门、拟杆菌门、绿弯菌门、浮霉菌门和变形菌门。但不同土壤样品中优势菌群的群落结构有所差异,石油污染的土壤中,酸杆菌门、放线菌门和变形菌门的数量最多,未被石油污染的土壤中,放线菌门、拟杆菌门和变形菌门的数量最多。     

分子生物学在石油微生物多样性研究中的应用

该文较系统地概述了目前以16SrRNA为基础的核酸探针检测技术、利用引物的PCR技术、DNA序列分析技术和电泳分离及显示技术在油藏微生物群落多样性和石油环境污染研究中的研究进展,并探讨了未来分子生物学技术在石油微生物研究中的发展方向。     

溴代阻燃剂污染底泥与未污染底泥中细菌群落结构之比较分析

【目的】通过免培养的分子生物学方法,比较受溴代阻燃剂污染严重的河流底泥和与未污染水库底泥中细菌多样性差异,解析二者间细菌群落结构,为污染河流的治理与生物修复提供相关的理论依据。【方法】从中国贵屿溴代阻燃剂污染区练江底泥样品和未污染水库底泥样品中分别提取微生物总DNA,用细菌通用引物27F和1500R扩增16S rRNA基因,构建16S rRNA基因文库。用HhaⅠ和HinfⅠ2种限制性内切酶对克隆子进行扩增产物rDNA的限制性酶切分析(ARDRA),挑取不同的操作分类单元OUT中的克隆进行测序并构建系统发育树,比较代表克隆子的基本分类类群和生物多样性构成。【结果】未污染水库底泥中细菌群落组成主要包括变形细菌门(Proteobacteria)的α、β、γ和δ4个亚门、浮霉菌门(Planctomycetes)、酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、绿弯菌门(Chloroflexi)、疣微菌门(Verrucomicrobia)和厚壁菌门(Firmicutes)。优势菌是酸杆菌,占文库克隆的30.2%。污染河流底泥中细菌群落组成包括变形细菌门(Proteobacteria)的α、β、δ和ε4个亚门、浮霉菌门(Planctomycetes)、绿菌门或拟杆菌门(Chlorobi orBacteroidetes)、酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、绿弯菌门(Chloroflexi)、待定菌群candidatedivision OP01、candidate division OP08和candidate division WS3的相似菌。优势菌是ε-变形细菌和绿弯菌,占文库克隆的44.9%。【结论】受溴代阻燃剂污染河流底泥中的细菌群落具有较高水平的多样性,与未污染底泥有显著区别,主要体现在ε-变形细菌和绿弯菌在细菌群落中具有优势地位。这种优势种群的改变可能与污染物的过度富集具有一定的相关性,对于我们进一步探索和了解溴代阻燃剂的微生物修复具有一定的指导意义。     

蜜蜂成虫工蜂肠道可培养细菌群落结构分析

【目的】研究2种蜜蜂(健康意大利蜜蜂和健康中华蜜蜂)成虫工蜂肠道可培养细菌的群落结构组成。【方法】利用16S r RNA基因的聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)分析技术,结合菌落形态观察和生理生化特征鉴定细菌种类。【结果】从2种蜜蜂成虫工蜂肠道200株可培养细菌得到18种不同细菌遗传型,分属于肠杆菌科(Enterobacteriaceae)、弧菌科(Vibrionaceae)和肠球菌科(Enterococcaceae)3个科。其中肠杆菌科是肠道可培养细菌最优势的细菌种类。同样以序列相似性大于97%的菌株归为相同细菌种类为标准,找到了2种蜜蜂可培养细菌的共有菌种,结合菌落形态观察和生理生化特征鉴定,确定肠道可培养细菌为肠杆菌属8株,克雷伯氏菌属1株,肠球菌属2株,以及气单胞菌属1株。【结论】通过研究健康意大利蜜蜂和中华蜜蜂成虫工蜂肠道可培养细菌群落结构组成,可为开展蜜蜂的微生态研究提供基础性资料。     

浓香型白酒两个产区窖泥微生物群落结构分析

【目的】探索浓香型白酒两个典型产区窖泥微生物群落结构和多样性,分析窖泥微生物群落地域特征及对白酒风格形成的影响。【方法】分别提取四川和安徽两个产区窖泥样品总DNA,应用PCR-ARDRA和16S rRNA基因克隆测序技术对两个产区窖泥细菌和古菌进行研究。【结果】两个浓香型白酒产区窖泥细菌丰富,包括:厚壁菌门(Firmicute)、拟杆菌门(Bacteroidetes)、绿弯菌门(Chloroflexi)、互养菌门(Synergistetes)、Armatimonadetes类群和未分类细菌(Unclassified bacteria)。两个产区窖泥绝对优势种群均为厚壁菌门中梭菌纲(Clostridia)细菌,在四川产区窖泥中检出较多的互营单胞菌属(Synthrophomonas)和紫单胞菌属(Petrimonas)。古菌的群落组成较为简单,主要是甲烷囊菌属(Methanoculleus)、甲烷八叠球菌属(Methanosarcina)、甲烷鬃菌属(Methanosaeta)和甲烷杆菌属(Methanobacterium)4个产甲烷古菌类群,四川产区窖泥优势古菌为甲烷囊菌和甲烷八叠球菌,安徽产区则为甲烷八叠球菌属和甲烷鬃菌。【结论】四川和安徽两个产区窖泥微生物的16S rRNA基因克隆文库系统地反映了两者微生物群落的相似性和差异性,对揭示浓香型白酒两个产区的酒体风格差异形成有一定的参考价值。     

舟山群岛不同功能区划海域细菌群落结构分析

浮游细菌在海洋生态系统中不可或缺,在海洋生物地球化学循环过程中起着关键性作用。【目的】为了解舟山群岛不同功能区划海域细菌群落结构及丰度变化,探索海洋生态因子对细菌群落结构的影响。【方法】于2016年夏季(8月)在舟山群岛不同功能区划海域共设置8个典型站位采集表层海水,基于细菌16S rRNA基因进行高通量测序;利用流式细胞术揭示各海域细菌丰度;利用典范对应分析(Canonical correspondence analysis,CCA)探讨海洋生态因子与细菌多样性之间的关系。【结果】共获取到305487条原始序列,基于97%相似性水平进行OTU(Operational Taxonomic Units)聚类分析,共得到1088个OTUs,包括29个门、62个纲、138个目、239个科、416个属。细菌群落组成在各个站位之间不尽相同,但都主要包括Flavobacteria、Alphaproteobacteria、Gammaproteobacteria三大优势菌纲。CCA结果表明细菌群落结构和多样性情况与站位分布和所在站位的环境因子息息相关,Cyanobacteria受硝酸盐影响显著,Parcubacteria受温度影响最大,而磷酸盐对本实验海域菌群影响甚微。对海洋菌群潜在功能进行预测的结果显示,各海域菌群在氨基酸代谢、碳水化合物代谢、膜运输等方面功能较为突出,为今后舟山海洋微生物研究提供了新的方向。【结论】高通量测序分析可以更精确地揭示海洋菌群的群落结构信息。该研究为细菌群落结构与环境因素的关联提供参考。本研究所取得的大量数据既可以作为对舟山市海洋功能区划施行情况的响应,又将为舟山及其邻近海域浮游细菌群落结构的进一步研究奠定基础。     

乌梁素海浮游细菌群落结构及其对富营养化因子的响应

【目的】揭示乌梁素海浮游细菌的群落结构及其对富营养化环境因子的响应,认识乌梁素海浮游细菌多样性,以丰富微生物与富营养化关系的理论。【方法】提取水体浮游细菌总DNA,利用细菌通用引物对63F/1387R进行PCR扩增,构建小口、沙尖北和红圪卜3个湖区16S rRNA基因克隆文库;以典型对应分析(CCA)方法解析浮游细菌群落结构对环境因子的响应。【结果】各湖区中富营养化程度最重的红圪卜位点细菌多样性、丰富度及均匀度最高,而富营养化程度最轻的小口位点细菌多样性、丰富度及均匀度最低。Proteobacteria、Bacteroidetes和Actinobacteria为水体中优势类群。α-、γ-、δ-Proteobacteria,Actinobacteria及Bacteroidetes等菌群丰度随着水体的富营养化程度改变而有显著的变化。各湖区皆存在许多可能具有污染物降解及驱动生源要素循环能力的细菌类群。CCA分析表明TN、NH4+、NO3-和COD等水体指标对浮游细菌群落结构组成的影响较大。此外,乌梁素海水体内未知细菌类群很多;且不同于一般淡水生态系统,乌梁素海中存在9.6%的轻度嗜盐碱性细菌。【结论】乌梁素海水体中浮游细菌多样性较高,细菌群落结构复杂、其类群组成与富营养化因子紧密相关。     

云冈石窟石质文物表面及周边岩石样品中微生物群落分析

【目的】通过对云冈石窟石质文物表面及云冈石窟周边岩石样品中微生物的研究,建立可用于快速检测石质文物中微生物的方法。【方法】选取云冈石窟石质样品和云冈石窟周边岩石样品作为研究对象,应用PCR-DGGE技术对样品中的微生物群落结构进行了分析研究。【结果】根据系统发育树聚类分析,可以得出云冈石窟中检测出的微生物主要分为四大类群:γ-变形菌纲、鞘脂杆菌门、α-变形菌纲和放线菌纲;根据GenBank数据库中的序列比对结果,可以知道云冈石窟周边类似岩石样品中的微生物主要属于γ-变形菌纲、厚壁菌门和α-变形菌纲等。【结论】本实验成功检测出云冈石窟石质样品表面及云冈石窟周边岩石样品中的微生物类群,为云冈石窟的保护工作提供了有力依据,同时也证明了DEEG和分子克隆技术相结合的方法是检测石质文物中微生物群落结构的一种可操作性强、快速、准确的检测手段。     

不同日粮饲养黄牛的胃部微生物群落多样性比较分析

为分析不同饲料喂养对黄牛胃中微生物群落结构的影响,分别用芒草单一喂养和农家混合饲料喂养两年的黄牛为实验组和对照组。以瘤胃、蜂巢胃、重瓣胃和皱胃四个胃中的微生物为研究对象,采用原位包埋裂解法和脉冲场电泳（pulsed field gel electrophoresis,PFGE）提取微生物总 DNA,脉冲场电泳（pulsed field gel elec-trophoresis,PFGE）。使用细菌16S rRNA 引物341F ／534R 及真菌18S rDNA 引物 NS1／GCFungi 进行降落 PCR 扩增。变性梯度凝胶电泳（denaturing gradient gel electrophoresis,DGGE）对扩增产物进行区分,使用硝酸银染色。电泳扫描结果通过 Quantity one 软件进行分析,SPSS 软件进行数据统计。针对实验组和对照组瘤胃样品共性条带和特性条带测序并比对。结果表明：实验组与对照组细菌群落结构变化较大,UPGAM聚类图上被分为两支,相似性只有0．35。且香农多样性指数及条带丰度都明显少于对照组。真菌 DGGE 图谱条带差别不大,聚类图上显示实验组四个样品与对照组四个样品相似性在0．43～0．68之间。多样性及条带丰度在实验组与对照组之间差异0．0027～0．5999。测序结果,细菌与数据库中未培养细菌种类较为接近,而真菌中部分与已知菌种接近。芒草单一饲养对牛胃中的微生物群落结构具有极大的影响,对细菌的影响尤为明显。     

巴丹吉林沙漠盐湖微生物多样性

【目的】研究内蒙古巴丹吉林沙漠碱性盐湖中的原核生物多样性及其与环境因子之间的关系。【方法】利用分子生物学方法构建16S rRNA基因克隆文库,对盐湖中的嗜盐碱微生物进行系统发育分析;利用R语言绘图,对不同盐湖的微生物群落结构进行对比研究。【结果】该区域盐湖含盐量很高,矿化度达165g/L-397 g/L。同时,水体呈强碱性,p H均在10以上。三个湖的盐度和p H值等理化参数有梯度变化,因此微生物多样性和群落结构存在明显差异。整体而言,细菌的多样性大于古菌。样品中含有的主要的细菌门类为γ-变形菌亚门(Gammaproteobacteria)、拟杆菌门(Bacteroidetes)、α-变形菌亚门(Alphaproteobacteria)、厚壁菌门(Firmicutes)、疣微菌门(Verrucomicrobia),古菌则全部属于广古菌门(Euryarchaeota)中的盐杆菌科(Halobacteriaceae)。【结论】盐度是决定细菌群落结构的主要因素,古菌群落结构则由多种环境因素综合影响。一些已知的嗜盐碱菌,如Roseinatronobacter spp.、Halohasta spp.等,可以生活在比其盐度和碱度生长范围更高的极端盐碱环境中。此外,样品中还含有大量未培养的嗜盐碱细菌和古菌,对进一步开发极端盐碱环境中的微生物资源有重要意义。     

Bacterial communities in a crude oil gathering and transferring system (China)

Bacterial communities in crude oil and oil field production water samples from an oil gathering and transferring system in Changqing Oil field in China were investigated by 16S rRNA denaturing gradient gel electrophoresis (DGGE) analysis followed by gene cloning and sequencing. DGGE profiles showed that bacterial communities are far more rich in the water samples than that in the crude oil samples, and that bacteria related to Ochrobactrum sp. and Stenotrophomonas sp. were detected in all crude oil and oil field water samples. Bacteria related to Burkholderia sp., Brevundimonas sp., and Propionibacterium sp. were detected in the crude oil samples but not in water samples. Bacteria related to Hippea sp., Acidovorax sp., Arcobacter sp., Pseudomonas sp., Thiomicrospira sp., Brevibacterium sp., Tissierella sp. and Peptostreptococcus sp. were detected in the water samples but not in crude oil samples. Using an archaea-specific primer set, methanogens related to Methanomicrobials and Methanosarcinales were found in water samples but not in crude oil samples. The comparability of the microbial communities in the water and crude oil phase during the period of oil gathering and transferring process was 83.3% and 88.2%, respectively, indicating a stable structure of the microbial communities.     

Microbial Community Composition in Crude Oils and Asphalts from the Kurdistan Region of Iraq

Abstract

To identify hydrocarbon-degrading microorganisms contributing to the formation of heavy oil we investigated the microbial community composition in different types of crude oils from oil-production facilities and in crude oil and asphalt from different natural seeps from the Kurdistan Region of Iraq (KRI). Crude oils from five out of six production facilities did not contain microorganisms detectable by 16S rRNA gene PCR amplicon sequencing likely reflecting a low microbial abundance in these samples. Crude oil and asphalt from the natural seeps hosted diverse microbial communities. The same phylotypes of uncultivated Deferribacteres and Thermodesulfobacteraceae were predominant community members across crude oils and asphalts from separate geographical locations. Soils surrounding seeps did not contain these phylotypes suggesting that they originate from the subsurface and although they seem commonly detected in hydrocarbon-rich environments their role in hydrocarbon-degradation is unknown. GC-MS analyses showed that mainly aromatic hydrocarbons were present in the crude oil and asphalt and that they were undergoing biodegradation - likely with sulfate and nitrate as terminal oxidants. In agreement, only bssA gene, but not assA gene-carrying microorganisms were detectable in the analyzed sampled. Overall our study identified several abundant uncultivated taxa with likely roles in transformation of nitrate, sulfate and hydrocarbons.     

Bacterial Communities Inside and Surrounding Soil Iron-Manganese Nodules

Bacterial community structures of a Fe-Mn nodule sample and its surrounding soil were investigated using PCR, amplified ribosomal DNA restriction analysis, cloning and sequencing methods. Result showed that phylogenetically diverse bacteria were present in the nodule and soil samples, and Acidobacteria- and Proteobacteria-affiliated bacteria dominated in both samples. However, Firmicutes were only found in the nodules, while the soil had much more Acidobacteria and Verrucomicrobia than the nodules. Many clones retrieved in this study closely resembled the clones previously obtained from environments with high metal contents. These findings may shed light on the biological formation of Mn oxides in soil environment.     

PCR‐DGGE Fingerprinting Analysis of Plankton Communities and Its Relationship to Lake Trophic Status

Plankton communities in eight lakes of different trophic status near Yangtze, China were charac‐terized by using denatured gradient gel electrophoresis (DGGE). Various water quality parameters were also measured at each collection site. Following extraction of DNA from plankton communi‐ties, 16S rRNA and 18S rRNA genes were amplified with specific primers for prokaryotes and eu‐karyotes, respectively; DNA profiles were developed by DGGE. The plankton community of each lake had its own distinct DNA profile. The total number of bands identified at 34 sampling stations ranged from 37 to 111. Both prokaryotes and eukaryotes displayed complex fingerprints composed of a large number of bands: 16 to 59 bands were obtained with the prokaryotic primer set; 21 to 52 bands for the eukaryotic primer set. The DGGE‐patterns were analyzed in relation to water quality parameters by canonical correspondence analysis (CCA). Temperature, pH, alkalinity, and the con‐centration of COD, TP and TN were strongly correlated with the DGGE patterns. The parameters that demonstrated a strong correlation to the DGGE fingerprints of the plankton community differed among lakes, suggesting that differences in the DGGE fingerprints were due mainly to lake trophic status. Results of the present study suggest that PCR‐DGGE fingerprinting is an effective and precise method of identifying changes to plankton community composition, and therefore could be a useful ecological tool for monitoring the response of aquatic ecosystems to environmental perturbations. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Impacts of Bioremediation Schemes on Bacterial Population in Naphthalene-Contaminated Marine Sediments

Microcosm experiments were conduced in which the surface of marine sediment was contaminated with naphthalene and subjected to either of three different bioremediation schemes, i.e., biostimulation (BS) by supplementing with slow-release nitrogen and phosphorus fertilizers, bioaugmentation (BA) by inoculating with Cycloclasticus sp. E2, an aromatics-degrading bacterium identified to play an important role for aromatic-hydrocarbon degradation in marine environments and combination (CB) of BS and BA. These three schemes were found to be similarly effective for removing naphthalene, while naphthalene disappearance in sediment without any treatment (WT) was slower than those in the treated sediments. Shifts in bacterial populations during and after bioremediation were analyzed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. It was found that the Cycloclasticus rRNA type occurred as the strongest bands in the course of naphthalene degradation. Clustering analysis of DGGE profiles showed that bacterial populations in the WT, BS and CB sediments differed consistently from those in the uncontaminated control, while the profile for the BA sediment was finally included in the cluster for uncontaminated control sediments after a 150-day treatment. The results suggest that bioaugmentation with ecologically competent pollutant-degrading bacteria is an ecologically promising bioremediation scheme.     

Bacterial Communities in Soil Under Moss and Lichen-Moss Crusts

Biological soil crusts are symbiotic microbial communities formed by green algae, mosses, fungi, lichens, cyanobacteria and bacteria in different proportions. Crusts contribute to soil fertility and favour water retention and infiltration. However, little is known about the bacterial community structure in soil under the crusts. Soil was sampled under a moss crust (considered the MOSS group), lichen plus moss (considered the LICHEN group) and bare soil (considered the BARE group) and the microbial communities determined using nearly full 16S rRNA gene libraries. Bacteria belonging to seven different phyla were found and the Acidobacteria and Alphaproteobacteria were the dominant in each group. The crusts affected negatively the abundance of the Burkholderiales. The phylogenetic diversity and bacterial community membership were different in the LICHEN group compared to the BARE and MOSS groups, but not species richness and community structure. The beta diversity analysis also revealed a different bacterial community structure beneath the LICHEN and MOSS crusts, suggesting species-specific influence. This is a first insight into the effect of a biological soil crust on the bacterial community structure in an organic matter rich soil of a high altitude mountain forest.     

健康妇女及3例细菌性阴道病患者阴道菌群的非培养分析

根据Amsel标准及Nugent标准, 确诊筛选健康妇女及细菌性阴道病(bacterial vaginosis, BV)患者各3例。提取其阴道分泌物样本的总DNA, 构建16S rRNA基因克隆文库, 并对阳性克隆进行ARDRA和测序分析。结果表明, 健康妇女样本的基因文库中, 分别以卷曲乳酸杆菌(L. crispatus)和惰性乳酸杆菌(L. iners)的克隆子占较大比例, 另外存在少量的阴道乳酸杆菌(L. vaginalis)或詹氏乳酸杆菌(L. jensenii)的克隆子。BV患者样本的基因文库中, 克隆子所代表的菌种类型明显增多, 但均以阴道加德纳氏菌(Gardnerella vaginalis)和阴道阿托波氏菌(Atopobium vaginae)的克隆子占较大比例, 且无乳酸杆菌克隆子。说明健康妇女阴道菌群的种类单一, 以乳酸杆菌占优势, L. iners为优势菌种之一; BV患者阴道菌群的种类复杂多样, 但均以Gardnerella vaginalis及Atopobium vaginae共同占优势。     

健康妇女及3例细菌性阴道病患者阴道菌群的非培养分析

根据Amsel标准及Nugent标准,确诊筛选健康妇女及细菌性阴道病(bacterial vaginosis,BV)患者各3例.提取其阴道分泌物样本的总DNA,构建16S rRNA基因克隆文库,并对阳性克隆进行ARDRA和测序分析.结果表明,健康妇女样本的基因文库中,分别以卷曲乳酸杆菌(L.crispatus)和惰性乳酸杆菌(L. iners)的克隆子占较大比例,另外存在少量的阴道乳酸杆菌(L.vaginalis)或詹氏乳酸杆菌(L.jensenii)的克隆子.BV患者样本的基因文库中,克隆子所代表的菌种类型明显增多,但均以阴道加德纳氏菌(Gardnerella vaginalis)和阴道阿托波氏菌(Atopobium vaginae)的克隆子占较大比例,且无乳酸杆菌克隆子.说明健康妇女阴道菌群的种类单一,以乳酸杆菌占优势,L. iners为优势菌种之一;BV患者阴道菌群的种类复杂多样,但均以Gardnerella vaginalis及Atopobium vaginae共同占优势.     

油藏水样细菌群落16S rRNA基因的RFLP分析

通过16S rRNA基因的限制性酶切片段长度多态性分析(RFLP)方法,考察了油藏水样中细菌群落及多样性。从水样中分离纯化微生物总DNA,选择性扩增细菌16S rRNA基因,并构建16S rDNA克隆文库。337个16S rDNA克隆片段分别用限制性内切酶HinfⅠ和HaeⅢ酶切分析,得到74个操作分类单元(OTUs),其中数量最多的4个OTUs共占克隆子总数的73.6%,另外70个OTUs的丰度均处于较低水平,有57个OTUs仅含有1个克隆子。结果表明,运用RFLP方法分析16S rDNA克隆片段能够有效评估油藏水样中的细菌群落和多样性。     

Firmicutes is an Important Component of Microbial Communities in Water-Injected and Pristine Oil Reservoirs,Western Siberia,Russia

The dominant microbial components of fluids from wells in pristine and water-injected, high-temperature, Western Siberian oil fields, were analyzed by PCR-DGGE. Particular emphasis was placed on sulphate-reducing organisms, due to their ecological and industrial importance. Bacterial phylotypes obtained from the non-water-injected Stolbovoye oil field were more diverse than those from the Samotlor field, which is subject to secondary oil recovery by reinjection of recycled production water. The majority of phylotypes from both sites were related to Firmicutes. The low similarity to their closest relatives indicates unique bacterial communities in deep underground production waters and crude oil. Archaeal phylotypes detected only in the Samotlor samples were represented by Methanosarcinales and Methanobacteriales.