石油降解菌的分离鉴定及石油污染土壤的细菌多样性

从石油污染的土壤中分离筛选到28株石油降解菌,经鉴定分别为短杆菌属、假单胞菌属、邻单胞菌属和微球菌属;对4个石油不同程度污染的土壤样品中嗜油微生物分布状况进行分析,发现污染严重的土壤样品中嗜油菌的数量相对较多;用聚合酶链式反应(PCR)、变性梯度凝胶电泳(DGGE)和切胶测序相结合的方法对4个土壤样品中的细菌多样性进行分析,结果显示在受污染的土壤中,My cobacterium和B acillus在污染程度较低的样品中分布的较为集中,F lavobacterium和A zosp ira在污染程度较高的样品中丰度较高。属于B eta p roteobacterium类群的细菌在受污染的土壤中占有优势,同时还有一些不可培养的菌群存在。气质联用(GC-M S)分析结果表明石油污染程度及污染物中芳香烃类的含量对细菌多样性有着显著影响。在石油污染程度高,芳香烃类含量高的样品中细菌的多样性相对较低。     

不同石油污染区微生物修复技术研究

采集辽河油田不同石油污染地区的土样 ,经富集、驯化、分离、筛选 ,得到优势石油降解菌。它们分别是假单胞菌属、黄单胞菌属、黄杆菌属、节杆菌属、动胶杆菌属 ,这些菌既有普遍性 ,同时又不乏地域特色。经系统测定其生理特性及表面活性物质对其利用多环芳烃物质的影响 ,结果表明不同石油污染区土壤的理化特性与优势细菌的生理特性相关 ;表面活性剂Tween 80可使优势细菌降解菲的速率及程度均有提高。     

石油污染盐碱土壤翅碱蓬根围的细菌多样性及耐盐石油烃降解菌筛选

为了更好地了解石油污染盐碱土壤翅碱蓬根围的细菌多样性,采用16S rRNA基因克隆文库方法对其进行分析,在此基础上采用富集培养方法从该生境中分离筛选耐盐石油烃降解菌.16S rRNA基因克隆文库分析结果表明,海杆菌属(Marinobacter)、食烷菌属(Alcanivorax)和假单胞菌属(Pseudomonas)是该生境中的优势菌.他们可能在石油污染盐碱土壤翅碱蓬植物修复过程中起重要作用.进一步采用富集培养方法,从该生境中分离得到8株耐盐石油烃降解菌,可以耐受6％-10％浓度的NaCl,石油烃降解率在32.3％-57.0％之间.经16S rRNA基因序列分析,8株菌隶属于戈登氏菌属(Gordonia)、无色杆菌属(Achromobacter)、迪茨菌属(Dietzia)、芽胞杆菌属(Bacillus)和假单胞菌属(Pseudomonas).他们可能参与石油污染盐碱土壤翅碱蓬植物修复过程中的石油烃降解.     

基于高通量测序的辐射污染区细菌群落特征分析

【目的】为了更加全面地揭示辐射污染区细菌种群多样性,了解辐射污染对辐射区土壤中细菌群落结构的影响。【方法】运用高通量测序方法,分别进行了土样细菌16S r RNA基因的V3可变区测序,进而对无辐射污染对照和不同辐射污染程度的土样中细菌群落组成和多样性进行分析。【结果】研究共获得110 348条有效序列,17 604个OTUs,共涉及细菌域的19个门和6个潜在菌门和其它未分类菌群的726个属。多样性分析表明,辐射污染会引起土壤样品中微生物群落的分布显著差异化,显著提高细菌群落种群多样性和微生物丰度。微生物群落组成分析发现,在辐射污染胁迫下,辐射污染区样品中变形杆菌门分布比例显著下降;随着辐射污染程度的提高,放线菌门所占比例逐步提高,未分类菌门、厚壁菌门和酸杆菌门也有明显的提高。同时,研究发现辐射污染区中存在着大量未分类菌属。【结论】研究揭示了辐射污染区极为丰富的细菌多样性,大量微生物新物种资源有待发掘。     

3种假单胞菌CaCl2法感受态细胞制备及转化条件

假单胞菌因其生境和代谢类型的多样性,在污染环境修复、生物转化、生物防治等领域具有广阔的应用潜力;外源基因的导入是假单胞菌遗传改造的重要环节,而感受态细胞的制备和转化方法的建立是导入外源基因的重要方法学基础.本研究以从石油污染土壤中分离筛选的假单胞菌属的3个菌株Pseudomonas putida TS11、P.stutzeri DNB、P.mendocina JJ12为对象,通过3因素4水平正交实验设计,研究了不同CaCl2浓度、热激时间及复苏时间对不同假单胞菌感受态细胞制备及转化效率的影响.结果表明:CaCl2浓度是影响假单胞菌转化效率的最主要因素(P＜0.05),且在制备感受态细胞之前用无菌蒸馏水多次洗涤菌体细胞,转化率明显提高.3种假单胞菌的CaCl2转化优化条件分别为:100 mmol·L-1 CaCl2,热激3 min,复苏1.5 h;50 mmol·L-1 CaCl2,热激6 min,复苏1.5 h;75 mmol·L-1 CaCl2,热激4.5 min,复苏0.5h.在上述转化条件下,3种假单胞菌的外源质粒转化效率均达到105个转化子·μg-1DNA水平.     

石油污染土壤强化修复前后细菌多样性变化研究

采用高通量测序技术,对石油污染土壤及石油降解菌强化修复土壤的细菌群落多样性进行了分析。发现污染前后各组间在门水平和属水平上变化显著,污染前细菌多样性丰富,包括34门675属,主要优势菌群依次为变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、酸杆菌门(Acidobacteria)、拟杆菌门(Bacteroidetes)、芽单胞菌门(Gemmatimonadetes)等。优势菌属依次为芽单胞菌属(Gemmatimonas)、鞘氨醇单胞菌属(Sphingomonas)、节杆菌属(Arthrobacter)等。石油污染110 d后土壤细菌类群多样性降低,分布在29门507属,细菌优势门变化不显著等,优势菌属依次为鞘氨醇单胞菌属、假单胞菌属(Pseudomonas)、GP6、芽单胞菌属、GP4、微小杆菌属(Exiguobacterium)、寡养单胞菌(Stenotrophomonas)和类诺卡氏菌属(Nocardioides)。添加铜绿假单胞菌1217、红平红球菌KB1和混合菌剂的三个强化修复组细菌分别分布在31门471属、32门474属和29门473属,在细菌组成上差异不显著,在丰度上差异显著。鞘氨醇单胞菌属、假单胞菌属、芽单胞菌属和类诺卡氏菌属细菌是主要的石油污染物降解菌。     

台州市路桥区重金属污染对土壤动物群落结构的影响

通过对台州市路桥区峰江再生园区及周围的8个样地采样调查,分析了重金属污染对土壤动物多样性的影响。对研究区域进行大量取样调查并测定土样中Cu、Zn、Pb、Cd 的含量。计算出重金属综合污染指数,运用地理信息系统(GIS)技术中的插值法根据重金属综合污染指数制作重金属综合污染趋势图。结合重金属综合污染趋势图与实地勘察,共设置8个采样地。研究方法为大型土壤动物采用野外分层手捡计数,中小型土壤动物用取土器分层取土,带回室内分别用干湿漏斗分离并镜检。结果表明:(1)8个样地共获得土壤动物844只,分属于4门9纲20个类群,其中优势类群为蜱螨目、弹尾目和线虫纲,其他为常见类群和稀有类群。(2)8个样地中土壤动物的个体数量存在极显著性差异(P<0.01),类群数存在极显著性差异(P<0.01)。在严重污染区,土壤动物的数量和类群数稀少,而轻度污染区土壤动物的密度大、群落多样性高,稀有类群大量出现。表明重金属污染能对土壤动物多样性构成严重影响,土壤动物群落的类群和个体数量随污染程度的加重而减少(3)用群落多样性指标DG衡量重金属污染对土壤动物多样性的影响,结果为随着综合污染指数的减小,DG指数逐渐增大。(4)用简单相关性分析表明土壤动物数量变化与综合污染指数具有极显著负相关性(相关系数-0.86),与有机质含量有一定相关性(相关系数0.42)。在轻度污染的样地中,土壤动物的数量随有机质含量的升高而上升,表明在严重的污染区,重金属的毒性对土壤动物的影响是主要的,而在轻度污染区,有机质含量可能成为影响土壤动物多样性的主要因素。(5)从弹尾目的等节跳科和球角跳科在8个样地中的分布特点可知,本地区的等节跳科对重金属污染的耐受力最强。球角跳科数量大、密度高,对重金属污染较敏感,可作为重金属污染的指示生物。     

软腐白菜细菌群落结构多样性与生长环境的相关性

[目的]通过对比分析不同生境白菜软腐病变组织与根系土壤相关细菌的群落结构,探讨白菜软腐细菌种群的多样性,以及与生境土壤细菌种群的相关性.[方法]样品采自河南省2个不同生态型白菜田,以成熟白菜软腐组织及病株根系土壤为目标,利用模拟原环境的培养基成分和条件,对样品中的细菌进行高通量分离培养和细胞16S rRNA基因序列比对分析,获得各样品细菌种群结构及其丰度,进而对各样品的优势菌群进行对比分析.[结果]两种不同生境白菜软腐组织细菌总量M05T为4.0×l0s cell/g、Q2T为1.2×1011 cell/g,分别获得纯菌56株和85株.M05T优势菌为萎蔫短小杆菌萎蔫亚种(Curtobacterium flaccunfaciens pv.Flaccumfaciens);Q2T优势菌为假单胞菌(Pseudomonas spp.)(柄木槿假单胞菌(P.hibiscicola)、台湾假单胞菌(P.taiwanensis)、托木尔假单胞菌(P.tuomuerensis)、莫塞尔假单胞菌(P.mosselii)).根系土壤细菌总量M05S为2.7 × 105 cell/g、Q2S为6.2 × 107 cell/g,分别获得纯菌36株和70株.M05S优势菌为巨大芽胞杆菌(Bacillus megatherium);Q2S优势菌为假单胞菌(Pseudomonas spp.)(香鱼假单胞菌(P.plecoglossicida)、栖木槿假单胞菌(P.hibiscicola)、类黄色假单胞菌(P.parafulva)、蒙氏假单胞菌(P.monteilii)、膝形假单胞菌(P.geniculata)).[结论]依据不同生境的白菜软腐组织和根系土壤细菌群落结构对比分析,认为白菜软腐菌可能具有多样性和多种致病来源,本研究为软腐病多种防治措施的制定提供基础研究和菌种资源.     

3种假单胞菌CaCl2法感受态细胞制备及转化条件

假单胞菌因其生境和代谢类型的多样性,在污染环境修复、生物转化、生物防治等领域具有广阔的应用潜力;外源基因的导入是假单胞菌遗传改造的重要环节,而感受态细胞的制备和转化方法的建立是导入外源基因的重要方法学基础.本研究以从石油污染土壤中分离筛选的假单胞菌属的3个菌株Pseudomonas putida TS11、P. stutzeri DNB、P. mendocina JJ12为对象,通过3因素4水平正交实验设计,研究了不同CaCl₂浓度、热激时间及复苏时间对不同假单胞菌感受态细胞制备及转化效率的影响.结果表明： CaCl₂浓度是影响假单胞菌转化效率的最主要因素(P<0.05),且在制备感受态细胞之前用无菌蒸馏水多次洗涤菌体细胞,转化率明显提高.3种假单胞菌的CaCl₂转化优化条件分别为：100 mmol·L^-1 CaCl₂,热激3 min,复苏1.5 h;50 mmol·L^-1 CaCl₂,热激6 min,复苏1.5 h;75 mmol·L^-1 CaCl₂,热激4.5 min,复苏0.5 h.在上述转化条件下,3种假单胞菌的外源质粒转化效率均达到10.5个转化子·μg^-1 DNA水平.     

湘江流域工业污染源对农田生态系统土壤动物群落影响的研究

对湘江流域工业污染区的土壤动物群落研究表明,接近污染源和污染物质富集的农田,土壤动物的种类和数量减少;工业污染区土壤的污染主要是重金属有毒物质的过量累积。调查共获得29类土壤动物,隶属于4门10纲,动物的种类与数量随着重金属污染程度的增加而递减,土壤动物密度与重金属元素Hg、Cd,zn、Cu、As、Pb的浓度密切相关,这在污染模拟实验中也得到证明。     

Diversity of Arbuscular Mycorrhizal Fungus Populations in Heavy-Metal-Contaminated Soils

High concentrations of heavy metals have been shown to adversely affect the size, diversity, and activity of microbial populations in soil. The aim of this work was to determine how the diversity of arbuscular mycorrhizal (AM) fungi is affected by the addition of sewage-amended sludge containing heavy metals in a long-term experiment. Due to the reduced number of indigenous AM fungal (AMF) propagules in the experimental soils, several host plants with different life cycles were used to multiply indigenous fungi. Six AMF ecotypes were found in the experimental soils, showing consistent differences with regard to their tolerance to the presence of heavy metals. AMF ecotypes ranged from very sensitive to the presence of metals to relatively tolerant to high rates of heavy metals in soil. Total AMF spore numbers decreased with increasing amounts of heavy metals in the soil. However, species richness and diversity as measured by the Shannon-Wiener index increased in soils receiving intermediate rates of sludge contamination but decreased in soils receiving the highest rate of heavy-metal-contaminated sludge. Relative densities of most AMF species were also significantly influenced by soil treatments. Host plant species exerted a selective influence on AMF population size and diversity. We conclude based on the results of this study that size and diversity of AMF populations were modified in metal-polluted soils, even in those with metal concentrations that were below the upper limits accepted by the European Union for agricultural soils.     

Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils

Abstract: A total of 132 different fluorescent Pseudomonas strains were isolated from several agricultural and industrial soils. The bacteria from the two different soil environments were compared for species and biotype variation, antibiotic and heavy metal resistance profiles, ability to degrade polyaromatic hydrocarbons, and plasmid incidence. Irrespective of the soil type, the isolates belonged to Pseudomonas fluorescens biotypes I–VI and Pseudomonas putida biotype B. Except for a streptomycin resistant isolate from one of the industrial soils, all the strains had the same antibiotic resistance profile. However, there was a higher incidence of heavy metal resistance and polyaromatic hydrocarbon degradation phenotypes in the isolates from industrial soils than from the agricultural soils. Only 2 out of 68 strains from agricultural soil were found to carry plasmids, while 28 out of 64 strains from industrial soil had plasmids. A majority of the plasmids (56%) were estimated to be larger than 50 kb, indicating that they could encode transfer functions. However, transferability as indicated by the ability to mobilize an IncQ plasmid (tra⁻, mob⁺), was observed with only one plasmid. None of the plasmid(s) containing isolates hybridized to a ³²P-labelled repP probe suggesting that none of the indigenous plasmids in the soil fluorescent Pseudomonas strains was related to the IncP group of conjugative plasmids commonly associated with resistance and catabolic genes.     

Decreased abundance and diversity of culturable Pseudomonas spp. populations with increasing copper exposure in the sugar beet rhizosphere

Recent studies have indicated that culturable bacteria constitute highly sensitive bioindicators of metal-induced stress in soil. We report the impact of different copper exposure levels characteristic of contaminated agricultural soils on culturable Pseudomonas spp. in the rhizosphere of sugar beet. We observed that the abundance of Pseudomonas spp. was much more severely affected than that of the general population of culturable heterotrophic bacteria by copper. For diversity assessment, Pseudomonas isolates were divided into operational taxonomic units based on amplified ribosomal DNA restriction analysis and genomic PCR fingerprinting by universally primed PCR. Copper significantly decreased the diversity of Pseudomonas spp. in the rhizosphere and significantly increased the frequency of copper-resistant isolates. Concomitant chemical and biological analysis of copper in the rhizosphere and in bulk soil extracts indicated no rhizosphere effect and a relatively low copper bioavailability in the studied soil, suggesting that the observed effects of copper may occur at lower total concentrations in other soils. We conclude that culturable Pseudomonas sensu stricto constitutes a highly sensitive and relevant bioindicator group for the impact of copper in the rhizosphere habitat, and suggest that continued application of copper to agricultural soils poses a significant risk to successful rhizosphere colonization by Pseudomonas spp.     

Genetic diversity and biological control activity of novel species of closely related pseudomonads isolated from wheat field soils in South Australia

Rhizobacteria closely related to two recently described species of pseudomonads, Pseudomonas brassicacearum and Pseudomonas thivervalensis, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either Pseudomonas corrugata or P. brassicacearum-P. thivervalensis type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen Gaeumannomyces graminis var. tritici, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that P. brassicacearum and P. thivervalensis are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.     

Source apportionment and health risk assessment of heavy metals in agricultural soils in Xinglonggang,Northeastern China

Heavy metals in soil can affect human health through the exposure pathways of oral ingestion, dermal contact, and inhalation. In this study, to assess the health risk of heavy metals in the agricultural area of Xinglonggang, 52 soil samples were collected and tested to obtain the concentrations of As, Cd, Cr, Cu, Ni, Pb, V, and Zn in the soil. The enrichment factor indicated that the heavy metals of the agricultural soils were enriched, but the degree of enrichment was mild for all of the heavy metals. Coefficient analysis and principal component analysis indicated that V, Cr, Ni, and Pb were mainly from natural sources, As was from irrigation, Cu and Cd tended to be from chemical fertilizers and pesticides, and Zn was from mixed sources including irrigation, chemical fertilizers, and pesticides. A human-health risk assessment indicated that the residents in the study area face high risk from carcinogens and low risk from noncarcinogens; As and Cr are the major heavy metals affecting human health. This study provides a reference and a basis for formulating effective measures to prevent and control heavy metal enrichment in agricultural soils.     

Comparison of ectomycorrhizas of Quercus garryana (Fagaceae) on serpentine and non-serpentine soils in southwestern Oregon

The diversity of ectomycorrhizal communities associated with Quercus garryana on and off serpentine soils was compared and related to landscape-level diversity. Serpentine soils are high in magnesium, iron, and heavy metals and low in fertility. In plant communities on serpentine soils, a high proportion of flowering plant species are endemic. At three sites with paired serpentine and nonserpentine soils in southwestern Oregon, we sampled Q. garryana roots and categorized ectomycorrhizas by morphotyping and by restriction fragment length patterns. Ectomycorrhizas were abundant at all sites; no single fungal species dominated in the ectomycorrhizas. Of 74 fungal species characterized by morphotype and pattern of restriction fragment length polymorphisms, 46 occurred on serpentine soils, and 32 were unique to serpentine soil. These species are potentially endemic to serpentine soil. Similarities in species composition between paired serpentine and nonserpentine soils were not significantly lower than among three serpentine sites or among three nonserpentine sites. We conclude that mycorrhizal communities associated with oaks on serpentine soil do not differ in species richness or species evenness from those on neighboring nonserpentine soil.     

接种泡囊假单胞菌对土壤生物性质及A. corsicum吸收Ni的影响

采用室内模拟试验方法,研究了在水稻土、元江土和墨江土中添加泡囊假单胞菌（Pseulormanas vesicularis）后土壤中微生物种群数量、土壤酶活性和镍超积累植物Alyssum corsicum对土壤镍的富集效果.土壤接种泡囊假单胞菌70d后,水稻土中DTPA提取态镍较对照土中的明显减少、元江土和墨江土中的有所减少;土壤中细菌、真菌和放线菌数量增加,5种土壤酶活性提高.试验结果表明,水稻土、元江土、墨江土添加泡囊假单菌后植物地上部生物量较对照分别增加了29%、309%和43%,进而提高了A.corsicum自土壤中富集镍的效率：水稻土中增加54%,元江土中增加306%,墨江土中增加32%.泡囊假单胞菌这一新用途的发现,可为植物修复微生物制剂和基因工程菌的开发提供本土的微生物的菌种资源.     

Cultivation-independent analysis of Pseudomonas species in soil and in the rhizosphere of field-grown Verticillium dahliae host plants

Despite their importance for rhizosphere functioning, rhizobacterial Pseudomonas spp. have been mainly studied in a cultivation-based manner. In this study a cultivation-independent method was used to determine to what extent the factors plant species, sampling site and year-to-year variation influence Pseudomonas community structure in bulk soil and in the rhizosphere of two Verticillium dahliae host plants, oilseed rape and strawberry. Community DNA was extracted from bulk and rhizosphere soil samples of flowering plants collected at three different sites in Germany in two consecutive years. Pseudomonas community structure and diversity were assessed using a polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) system to fingerprint Pseudomonas-specific 16S rRNA gene fragments amplified from community DNA. Dominant and differentiating DGGE bands were excised from the gels, cloned and sequenced. The factors sampling site, plant species and year-to-year variation were shown to significantly influence the community structure of Pseudomonas in rhizosphere soils. The composition of Pseudomonas 16S rRNA gene fragments in the rhizosphere differed from that in the adjacent bulk soil and the rhizosphere effect tended to be plant-specific. The clone sequences of most dominant bands analysed belonged to the Pseudomonas fluorescens lineage and showed closest similarity to culturable Pseudomonas known for displaying antifungal properties. This report provides a better understanding of how different factors drive Pseudomonas community structure and diversity in bulk and rhizosphere soils.     

IN-SITU SELECTION OF SUITABLE PLANTS FOR THE PHYTOREMEDIATION OF MULTI-METALS–CONTAMINATED SITES IN CENTRAL TAIWAN

Soils contaminated with metals are a serious problem in central Taiwan; 70% of the metals-contaminated soils in Taiwan are distributed there. We used soil turnover and dilution methods to reduce the total concentration of metals in soil, but this technique may be not suitable for other sites because of their shallow soil depths, which were less than 60 cm. Central Taiwan has the largest flower market in Taiwan and we propose that using local flower species to clean up the metals-contaminated soils is a feasible solution. A 1.3-ha area contaminated by multiple metals (As, Cr, Ni, Cu, and Zn) located in central Taiwan was selected for this large-area phytoremediation experiment. According to the Taiwan Environmental Protection Administration project contract, in-situ selection experiments were conducted to select 12 potential species from 33 tested species for further large area experiment. After in-situ planting of 33 species of plants in the contaminated soil for 33 d, bougainvillea and cockscomb showed yellow-colored leaves and withered as the result of the toxicity of metals. Herbaceous plants can accumulate higher concentration of metals and have higher bioconcentration factor in relative to woody plants. Three weighting models of growth condition and the metal-accumulated concentration of plants growing in the site were evaluated and compared. Six woody plants and six herbaceous plants were selected as high potential metal accumulators for a further large-area experiment.     

农用化学品污染对土壤微生物群落DNA序列多样性影响研究

采用ＲＡＰＤ分子遗传标记技术研究了农用化学品不同使用环境下的４种土壤微生物群落ＤＮＡ序列多样性的变化。结果表明,４种土壤微生物群落ＤＮＡ序列在其丰富度、多样性指数、均匀度等方面均存在差异;农用化学品的使用会对土壤微生物群落在ＤＮＡ分子水平上的多样性产生影响;而冰同的农用化学品对土壤微生物群落ＤＮＡ序列多样性影响各不相同：化肥污染会引起某些土壤微生物的富集和一些微生物物种的丧失;农药杂会引起土壤微生