基因芯片技术在环境微生物群落研究中的应用

基因芯片技术作为一种快速、敏感、高通量的检测技术,近几年来在环境微生物群落研究中的应用越来越广泛并且得到充分的发展.它不仅可以研究环境微生物群落的微生物分布、种类、功能、动力学变化,还能分析环境污染等环境因素改变对其微生物生态的影响.本文按照基因芯片探针的设计方法,将环境样品群落研究基因芯片分为系统寡核苷酸芯片、功能基因芯片、群落基因组芯片、宏基因组芯片,并简要综述了该技术在活性污泥、土壤、水等环境样品微生物群落研究上的应用,最后,本文展望了该技术的研究方向和在寻找不同环境微生物群落之间差异微生物、差异基因或差异表达基因研究中的应用前景.     

基因芯片及其在环境微生物研究中的应用

基因芯片因其具有高密度、高灵敏度、快速 (实时 )检测、经济、自动化和低背景水平等特点 ,而广泛应用于不同的研究领域。目前 ,应用于环境微生物研究的基因芯片主要有功能基因芯片 (FGAs)、系统发育的寡核苷酸芯片 (POAs)和群落基因组芯片 (CGAs)。综述了基因芯片在环境微生物研究中的应用 ,包括自然环境中微生物的基因表达分析、比较基因组分析和混合微生物群落的分析等。讨论了基因芯片面临的挑战和前景展望     

土层厚度对种植屋面土壤微生物群落功能的影响

采用控制性模拟试验方法,设置40、70、100 cm 3个土层厚度,研究了种植屋面土壤微生物群落功能和土层厚度之间的关系,尝试从土壤生物学角度揭示土层厚度在提高种植屋面整体生态功能方面所起的重要作用。结果表明,种植屋面土壤微生物群落代谢功能整体活性AWCD值、对碳源CI、AI、MI的代谢作用和McIntosh U指数,100 cm土层均显著大于其他土层(P<0.05),而70和40 cm土层之间大多差异不显著(P>0.05)。主成分分析(PCA)排序图显示,100 cm土层代表的微生物群落碳源代谢能力最强,而70和40 cm土层对多数碳源的代谢能力相当。土层厚度显著影响了高杆女贞胸径年生长率(AGR),从40到70 cm,AGR增加了11%;从70到100 cm,AGR增加了15%,增幅明显加大。种植屋面土层厚度对土壤微生物群落功能的影响和植物的生长率有着较好的一致性。合理的土层厚度能为种植屋面的生物提供适宜的生长环境,促进其生态效益的最大化。     

兰州市空气微生物群落的碳代谢特征及功能多样性研究

应用Biolog ECO技术和RDA分析研究了兰州市不同功能区,即风景区(Scenic Area,SA)、交通干线(Main Traffic Line,MTL)、公共服务区(Public Service Area,PSA)、文教区(Culture and Education Area,CEA)空气微生物群落代谢功能多样性及其与环境因子的相关性。结果表明,不同功能区的AWCD值在培养192 h后进入稳定期,对碳源总体利用能力呈现显著的区域性差异,SAPSACEA≈MTL。受环境因素影响,Mc Intosh指数差异明显,SA和PSA物种均一度较高。兰州市空气微生物相对利用率较高的碳源类型是酯类、氨基酸类及醇类,与主成分1显著相关的碳源有10种,其中氨基酸类是导致兰州市空气微生物群落代谢功能区域性差异的主要分异类型。环境因子与空气微生物群落代谢功能密切相关,温度、风速是不同功能区存在差异的主导因子。     

非培养生物方法在堆肥微生物生态中的应用展望

微生物是堆肥化处理研究中被重点关注的因素,但大部分堆肥微生物都处于存活不可培养状态,使得了解堆肥过程中微生物实际群落结构比较困难。非培养生物方法的快速发展,为解决这一难题提供了技术依据。分别就生物化学,分子生物学,生理学中不依赖于传统培养技术的代表方法PLFA谱图分析法、FISH技术、Biolog微量板分析法进行了介绍,综述了3类方法的组合应用以及在堆肥微生物生态中的应用展望。     

稻草覆盖与间作三叶草对丘陵茶园土壤微生物群落功能的影响

不同的土壤管理方式对土壤的质量有重要影响,而土壤微生物群落功能多样性是反映土壤质量变化的重要指标之一。本研究旨在利用Biolog微孔板鉴定系统研究不同培肥措施下茶园土壤微生物群落功能多样性的变化。比较6种处理的结果发现,与NPK肥＋清耕（CK）相比,平均吸光值（average well color development,AWCD）的变化速度（斜率）和最大值顺序为：饼肥＋PK＋间作三叶草〉NPK肥＋稻草覆盖〉饼肥＋PK＋稻草覆盖〉饼肥＋PK＋清耕〉NPK肥＋间作三叶草〉CK。可见间作三叶草与稻草覆盖不同程度地提高了微生物整体活性和丰富度,其中饼肥＋PK＋间作三叶草处理处理效果最好。分析多样性指数发现,虽然稻草覆盖与间作三叶草对土壤常见微生物种类影响并不大,但微生物群落均匀度有所降低。对碳源利用主成分起分异作用的主要是糖类和羧酸类物质。     

MAR-FISH技术及其在环境微生物群落与功能研究中的应用

对复杂环境中微生物群落结构和功能的研究是微生物生态学的重要任务。尽管现代分子生物学技术已经成功地用于解析环境中微生物的群落结构, 但是这些方法并不能提供微生物的原位生理学信息。而一种新的方法, 微观放射自显影和荧光原位杂交集成技术(MAR-FISH)则能够同时在单细胞水平上, 检测复杂环境中微生物的系统发育信息及其生理特性。本文总结了MAR-FISH方法的原理, 实验步骤及其在环境微生物群落与功能研究中的应用。     

环境微生物群落功能研究的新方法和新策略

微生物群落在驱动生物地球化学循环中扮演着重要角色,传统的研究方法可对微生物群落进行遗传结构的解析,但不能有效地与功能研究耦联.概述了近年发展起来的基于核酸和蛋白质水平的分子生物学新方法--环境mRNA 和 rRNA同时荧光原位杂交(FISH)、寡核苷酸微阵列技术(Oligonucleotide Microarray)、 稳定性同位素联合宏基因组学(SIP-enabled Metagenomics)和环境蛋白质组学(Metaproteomics)在环境微生物群落功能研究中的应用,并且对其发展趋势进行了分析和展望.     

可用于微生物群落分子生态学研究的活性污泥总DNA提取方法研究

活性污泥样品经液氮速冻、沸水浴融化、溶菌酶处理和 SDS裂解后 ,99%以上细胞裂解。所提取的 DNA经琼脂糖凝胶电泳检测和荧光法浓度测定 ,其片断大小在 2 0 kb左右 ,产量可达 1 .75 6± 0 .1 mg/g MLSS。样品 ABS2 6 0 nm/ABS2 80 nm的比值为 1 .96± 0 .2。以提取的总 DNA为模板 ,进行细菌核糖体小亚基 1 6Sr DNA基因 V3区和多组分苯酚羟化酶大亚基基因 (Lm PHs)的 PCR扩增 ,均获得成功 ,为活性污泥中微生物群落的分子生态学研究提供了一种简便、可靠的 DNA提取方法。     

科尔沁沙地小叶锦鸡儿固沙群落土壤微生物功能多样性

为探讨科尔沁沙地流动沙丘上建立小叶锦鸡儿固沙植被后土壤微生物群落代谢功能的变化,采用Biolog-ECO微平板检测法,研究了9、16和26年生人工群落以及流动沙丘和天然小叶锦鸡儿群落土壤微生物碳源代谢的多样性变化特征.结果表明:在培养的264 h内,流动沙丘土壤微生物平均颜色变化率,以及对羧酸类、糖类、多聚物类、氨基酸类、胺类和芳香类化合物类的代谢能力明显高于人工群落;随着人工群落年龄的增加,土壤微生物代谢碳源种类增加,26年生人工群落土壤微生物的碳源代谢模式已接近天然群落.流动沙丘建立小叶锦鸡儿植被后,土壤微生物群落碳源代谢功能多样性和均匀度指数下降;随着植物群落发育年龄的增长,微生物功能多样性逐渐提高.     

Assessment of Pollution-Induced Microbial Community Tolerance to Heavy Metals in Soil Using Ammonia-Oxidizing Bacteria and Biolog Assay

This study attempted to investigate if the tolerance of soil bacterial communities in general, and autotrophic ammonia-oxidizing bacteria (AOB) in particular, evolved as a result of prolonged exposure to metals, and could be used as an indigenous bioindicator for soil metal pollution. A soil contaminated with copper, chromium, and arsenic (CCA) was mixed with an uncontaminated garden soil (GS3) to make five test soils with different metal concentrations. A modified potential ammonium oxidation assay was used to determine the metal tolerance of the AOB community. Tolerance to Cr, Cu, and As was tested at the beginning and after up to 13 months of incubation. Compared with the reference GS3 soil, the five CCA soils showed significantly higher tolerance to Cr no matter which form of Cr (Cr³⁺, CrO₄ ^2?, or Cr₂O₇ ^2?) was tested, and the Cr tolerance correlated with the total soil Cr concentration. However, the tolerance to Cu²⁺, As³⁺, and As⁵⁺ did not differ significantly between the GS3 soil and the five CCA soils. Community level physiological profiles using Biolog microtiter plates were also used to examine the chromate tolerance of the bacterial communities extracted after six months of exposure. Our results showed that the bacterial community tolerance was altered and increased as the soil Cr concentration was increased, indicating that the culturable microbial community and the AOB community responded in a similar manner.     

现代生物技术在根际微生物群落研究中的应用

根际微生物的定义为植物的根表及受根系直接影响的土壤区域中的微生物群落.根际微生物与植物根系相互作用,是植物生态系统物质交换的主要媒介.根际微生物群落的研究对于微生物生态学与植物生态学均具有重要意义.近年来现代生物学技术发展迅速,对根际自然群落中不可培养微生物的研究取得了突破.综述了目前已取得的研究进展,对现代生物技术在根际微生物群落研究中的应用做了讨论.     

Microbial Community Composition in a Rehabilitated Bauxite Residue Disposal Area: A Case Study for Improving Microbial Community Composition

The chemical composition of rehabilitated Bauxite Residue Disposal Areas (BRDA) remains the primary indicator of rehabilitation success, with little consideration of microbial community development. We investigated links between the chemical and microbial components of rehabilitated residue at the Aughinish Alumina BRDA (Ireland). Rehabilitated was compared to unamended residue and to an analogy reference soil from unmanaged grassland within the refinery boundary. Bauxite residue comprised of areas with 1, 11, and 12 years following rehabilitation establishment, and gypsum applied at 45 and 90 t/ha. The unamended residue was typical of bauxite residues with high pH (10), sodicity (exchangeable sodium percentage [ESP]‐79), exchangeable sodium (19 cmol/kg), salinity (electrical conductivity [EC] 2.6 mS/cm), and low/negligible nutrient content, microbial biomass (71 µg‐C/g), and fungal phospholipid fatty acid (PLF). Microbial biomass increased 10‐fold with only 1 year of rehabilitation. Gypsum application rate had no effect on microbial biomass. Phospholipid fatty acid analysis (PLFA) demonstrated the emergence of distinct microbial community dependent on rehabilitation time and gypsum application rates. Changes of PLFA profiles were correlated (multiple regressions analysis) to shifts in residue chemical properties (sodicity, organic C, total C, total N, salinity, Mg). An increase of the arbuscular mycorrhizal fungi fatty acid (16:1ω5) with reducing pH has implications on rehabilitation practices. The microbial characteristics of the rehabilitated residue were approaching that of a soil from an unmanaged reference site adjacent to the working site. Gypsum affected PLFA properties, and thereby application has implications for rehabilitation success. For successful ecosystem reconstruction, it is critical that rehabilitation practices consider microbial development.     

PCR-DGGE技术分析染整废水微生物群落多样性

旨在揭示水解酸化-生物接触氧化工艺处理染整废水过程中的微生物多样性.取初级沉淀池,水解酸化池,生物接触氧化池和二沉池的活性污泥,通过细胞裂解直接提取基因组DNA,以细菌通用引物进行16S rRNA基因V3区域PCR扩增,将PCR产物进行变性梯度凝胶电泳,获得微生物群落的DNA特征指纹图谱,并对条带进行统计分析和切胶测序,进行了同源性分析并建立了系统发育树.研究表明,整个水处理过程中含有丰富的微生物群落,其中初级沉淀池、水解酸化池、二沉池和生物接触氧化池的污泥样品分别测出36条带、42条带、30条带和29条带.不同区段微生物群落间相似度最高达68％,最低达42.4％,说明群落间演替明显,不同工艺区段既存在共同的微生物种属也存在特异微生物种属.     

Biolog微生物鉴定系统中接种液的替代

目的：采用市售试剂自行配制Biolog菌种鉴定接种液,代替Biolog公司的原装接种液,用以降低菌种鉴定成本。方法：用实验室自行配制的接种液和Biolog公司的接种液,对120株细菌和50株丝状真菌进行Biolog微生物系统鉴定,对比鉴定结果。结果：用2种接种液进行菌种鉴定时,鉴定板上部分孔的颜色反应不同,但鉴定结果一致。结论：自行配制的接种液可以满足菌株鉴定的要求,具有推广意义。     

Shifts in Microbial Community Composition Following Surface Application of Dredged River Sediments

Sediment input to the Illinois River has drastically decreased river depth and reduced habitats for aquatic organisms. Dredging is being used to remove sediment from the Illinois River, and the dredged sediment is being applied to the surface of a brownfield site in Chicago with the goal of revegetating the site. In order to determine the effects of this drastic habitat change on sediment microbial communities, we examined sediment physical, chemical, and microbial characteristics at the time of sediment application to the soil surface as well as 1 and 2 years after application. Microbial community biomass was determined by measurement of lipid phosphate. Microbial community composition was assessed using phospholipid fatty acid (PLFA) analysis, terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes, and clone library sequencing of 16S rRNA genes. Results indicated that the moisture content, organic carbon, and total nitrogen content of the sediment all decreased over time. Total microbial biomass did not change over the course of the study, but there were significant changes in the composition of the microbial communities. PLFA analysis revealed relative increases in fungi, actinomycetes, and Gram positive bacteria. T-RFLP analysis indicated a significant shift in bacterial community composition within 1 year of application, and clone library analysis revealed relative increases in Proteobacteria, Gemmatimonadetes, and Bacteriodetes and relative decreases in Acidobacteria, Spirochaetes, and Planctomycetes. These results provide insight into microbial community shifts following land application of dredged sediment.     

CRISPR-Cas系统在微生物研究中的应用进展

来源于细菌防御系统的CRISPR-Cas系统因其功能强大、多样性受到广泛关注,也在动物、植物、微生物等各个领域得到了应用并迅速发展。概述了CRISPR-Cas系统的作用机理和分类,并对CRISPR-Cas系统在微生物基因编辑和病毒核酸检测方面的研究及应用进展进行了综述,以期为微生物研究工作提供参考。     

Microbial Interactions within a Cheese Microbial Community

The interactions that occur during the ripening of smear cheeses are not well understood. Yeast-yeast interactions and yeast-bacterium interactions were investigated within a microbial community composed of three yeasts and six bacteria found in cheese. The growth dynamics of this community was precisely described during the ripening of a model cheese, and the Lotka-Volterra model was used to evaluate species interactions. Subsequently, the effects on ecosystem functioning of yeast omissions in the microbial community were evaluated. It was found both in the Lotka-Volterra model and in the omission study that negative interactions occurred between yeasts. Yarrowia lipolytica inhibited mycelial expansion of Geotrichum candidum, whereas Y. lipolytica and G. candidum inhibited Debaryomyces hansenii cell viability during the stationary phase. However, the mechanisms involved in these interactions remain unclear. It was also shown that yeast-bacterium interactions played a significant role in the establishment of this multispecies ecosystem on the cheese surface. Yeasts were key species in bacterial development, but their influences on the bacteria differed. It appeared that the growth of Arthrobacter arilaitensis or Hafnia alvei relied less on a specific yeast function because these species dominated the bacterial flora, regardless of which yeasts were present in the ecosystem. For other bacteria, such as Leucobacter sp. or Brevibacterium aurantiacum, growth relied on a specific yeast, i.e., G. candidum. Furthermore, B. aurantiacum, Corynebacterium casei, and Staphylococcus xylosus showed reduced colonization capacities in comparison with the other bacteria in this model cheese. Bacterium-bacterium interactions could not be clearly identified.