Molecular analysis of the bacterial community in a continental high-temperature and water-flooded petroleum reservoir

Water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China was analysed for its bacterial community and diversity. The bacteria were characterized by their 16S rRNA genes. A 16S rRNA gene clone library was constructed from the community DNA, and using restriction fragment length polymorphism analysis, 337 randomly selected clones were clustered with 74 operational taxonomic units. Sequencing and phylogenetic analyses showed that the screened clones were affiliated with Gammaproteobacteria (85.7%), Thermotogales (6.8%), Epsilonproteobacteria (2.4%), low-G+C Gram-positive (2.1%), high-G+C Gram-positive, Betaproteobacteria and Nitrospira (each <1.0%). Thermopilic bacteria were found in the high-temperature water from the flooded petroleum reservoir, as well as mesophilic bacteria such as Pseudomonas-like clones. The mesophilic bacteria were probably introduced into the reservoir as it was being exploited. This work provides significant information on the structure of bacterial communities in high-temperature, long-term water-flooded petroleum reservoirs.     

Succession of bacterial community composition over two consecutive years in two aquatic systems: a natural lake and a lake-reservoir

The succession in bacterial community composition was studied over two years in the epilimnion and hypolimnion of two freshwater systems: a natural lake (Pavin Lake) and a lake-reservoir (Sep Reservoir). The bacterial community composition was determined by cloning-sequencing of 16S rRNA and by terminal restriction fragment length polymorphism. Despite large hydrogeological differences, in the Sep Reservoir and Pavin Lake the dominant bacteria were from the same taxonomic divisions, particularly Actinobacteria and Betaproteobacteria. In both ecosystems, these major bacterial divisions showed temporal fluctuations that were much less marked than those occurring at a finer phylogenetic scale. Nutrient availability and mortality factors, the nature of which differed from one lake to another, covaried with the temporal variations in the bacterial community composition at all sampling depths, whereas factors related to seasonal forces (temperature and outflow for Sep Reservoir) seemed to account only for the variation of the hypolimnion bacterial community composition. No seasonal reproducibility in temporal evolution of bacterial community from one year to the next was observed.     

有机污染物对水体真细菌群落结构的影响

为了揭示有机污染物对环境真细菌组成和多样性的影响,应用末端限制性片段长度多态性(tRFLP)和16S rDNA文库技术并结合水质分析方法,比较分析了松花江流域内受不同程度有机污染的4个水体及其沉积物中真细菌的群落结构。tRFLP分析表明各水体及底泥均呈现较为复杂的群落结构模式,不同底泥群落形成的末端限制性片段(TRF)图谱具有很高的相似性,但随着污染程度的加强,部分类群明显富集,而且在水样组和泥样组内,群落结构的相似性同水质相似性是一致的,主成分分析(PCA)显示水样和泥样中的真细菌TRF形成不同的群。16SrDNA文库分析表明松花江哈尔滨段底泥中真细菌分布于10个门,Proteobacteria门占优势,达群落总数的21.92%(β-Proteobacteria亚门占10.96%),而有机染污物严重超标的生活污水排污道底泥中的微生物多样性较低,分布于7个门,Proteobacteria门为优势群,占群落的47.37%(α-Proteobacteria亚门占21.05%,δ/ε-Proteobacteria亚门占15.79%)。该研究表明向水体中长期排放高浓度有机物能使系统中微生物群落多样性降低,与污染物降解相关的功能微生物类群明显富集。     

Characterization of the bacterial community associated with the surface and mucus layer of whiting (Merlangius merlangus)

The bacterial community inhabiting the mucus layer and surface of whiting was examined to determine whether the bacteria present are a reflection of the surrounding water or an indigenous bacterial flora is present. The outer mucus, mouth mucus and gut of four whiting harvested from a site in the Irish Sea and the surrounding water were examined by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA gene and clone library construction. The water community was the most diverse, with only a small number of shared water-mucus phylotypes present. The bacterial flora associated with the outer mucus layer were more diverse than that of the mouth mucus and gut. All three mucus layers were characterized by the presence of a dominant phylotype, identified as clone wom-1, highly similar to Photobacterium iliopiscarium. In addition to other Photobacterium phylotypes, members of the CFB and Clostridia groups were also detected. Subsequently, whiting from 11 different sites along the east and south coast of Ireland were compared by tRFLP analysis. Strikingly, the mucus layer of whiting at all sites was characterized by the presence and dominance of a TRF corresponding to the clone wom-1 which was virtually absent from the water column.     

长春花内生细菌多样性与柑橘黄龙病菌的相关性

【目的】分析感柑橘黄龙病长春花植株与健康长春花植株不同部位内生细菌菌群结构变化,为柑橘黄龙病菌与长春花内生细菌的相关性研究提供理论基础。【方法】本研究利用兼性厌氧可培养技术、16S rDNA限制性片段长度多态性分析(Restriction fragment length polymorphism,RFLP)以及16S rDNA序列分析相结合的方法。【结果】分别从感病和健康长春花叶、茎、根的组织中分离获得67株内生细菌,与GenBank中29种细菌的相似性达到97%-100%。其中短小杆菌属(Curtobacterium sp.)、欧文氏菌属(Erwinia sp.)、蜡样芽胞杆菌(Bacillus cereus)为感病长春花内生细菌的优势菌群,鞘胺醇单胞菌属(Brevundimonas sp.)、芽胞杆菌属(Bacillus sp.)为健康长春花内生细菌的优势菌群;马胃葡萄球菌(Staphylococcus equorum)为两者的共同优势菌群。通过RFLP方法分析,感病株得到16个、健株得到23个操作分类单元(Operational TaxonomicUnits,OTUs),感病植株中除柑橘黄龙病菌Candidatus Liberibacter asiaticus外,还有丰富的CandidatusLiberibacter sp.存在。【结论】感病与健康长春花植株中均含有丰富的内生细菌,黄龙病菌的存在改变了长春花原有内生细菌的菌群结构,且菌群多样性下降。可见长春花内生细菌在一定程度上受到柑橘黄龙病菌的抑制。     

16S rDNA clone library-based bacterial phylogenetic diversity associated with three South China Sea sponges

Culture-independent molecular techniques, 16S rDNA clone library alongside RFLP and phylogenetic analysis, were applied to investigate the bacterial diversity associated with three South China Sea sponges, Stelletta tenui, Halichondria rugosa and Dysidea avara. A wide bacterial diversity was detected according to total genomic DNA-based 16S rDNA clone library, abundant clones with low identify with sequences retrieved from database were found as well as uncultured sponge symbionts. The phylogenetic analysis shows that the bacterial community structure of Stelletta tenui is similar to that of Halichondria rugosa comprising gamma-Proteobacteria and Firmicutes. Whereas, alpha-Proteobacteria, gamma-Protebacteria, Bacteroidetes and uncultured sponge symbionts were found in sponge Dysidea avara, suggesting that Dysidea avara has the highest bacteria diversity among these sponges. A specific sponge–microbe association is suggested based on the difference of bacterial diversity among these three sponges from the same geography location and the observed sponge species-specific bacteria.     

Name that microbe: rapid identification of taxa responsible for individual fragments in fingerprints of microbial community structure

Polymerase chain reaction (PCR)‐based ‘fingerprinting’ methods, such as Terminal restriction fragment length polymorphism, Length Heterogeneity‐Polymerase Chain Reaction (LH‐PCR) and Automated Ribosomal Intergenic Spacer Analysis (ARISA) make possible quantitative studies of microbial community structure and dynamics. Here we outline a strategy for the rapid and cost‐effective isolation of 16S clones corresponding to particular fragment sizes in a fingerprint, based on applying the fingerprinting method to pools of colonies from a clone library. This allows the definitive identification of taxa responsible for the most important bands in the community fingerprint from a full 16S sequence. It offers significant advantages over random selection of clones and removes a significant barrier to the use of these methods.     

水葫芦根际细菌群落结构多样性分析

【目的】了解水葫芦根际细菌群落结构。【方法】运用末端限制性片段长度多态性(Terminal restriction fragment length polymorphism,T-RFLP)技术分析富营养化水体中水葫芦根际和水葫芦近、远水样的细菌群落特征及多样性,结合克隆文库技术和培养法分析根际的细菌种群类型。【结果】同一时期水葫芦根际细菌多样性(Shannon-Weiner指数H′或Simpson指数D)更高,水葫芦近水样次之,远水样最小。10月份的细菌多样性高于5月份的。通过水葫芦根际细菌的克隆文库可知变形杆菌门(Proteobacteria)是水葫芦根际细菌的主要类群,占总群体的65.1%,包括噬菌弧菌(Bacteriovorax sp.)、Dechloromonas sp.、Leptothrix sp.、红螺菌科(Rhodospirillaceae)、Rhodoferax sp.和红环菌科(Rhodocyclaceae)等。T-RFLP图谱显示159 bp为最大优势菌,247 bp为第二大优势菌,对照克隆文库及培养结果分析247 bp属于γ-Proteobacteria,159 bp为不动杆菌(Acinetobacter sp.)。【结论】水葫芦根际细菌的群落结构丰富,不同时段水葫芦根际细菌的丰度略有变化,主要类群为变形杆菌门。     

Analysis of bacterial community structure in the natural circulation system wastewater bioreactor by using a 16S rRNA gene clone library

A variety of physical and chemical parameters are routinely monitored during operation of the Natural Circulation System, a wastewater purification bioreactor in which only natural materials and no synthetic chemicals are used. However, the microbial community structures existing in the Natural Circulation System have not been well characterized. Thus, bacterial community structure and composition in this system were studied using clone library analysis of 16S ribosomal RNA genes amplified using PCR with universal bacterial primer sets. The PCR products were then subcloned into the pGEM-T vector. Each unique restriction fragment length polymorphism pattern, created by using two pairs of restriction endonucleases, was designated as an operational taxonomic unit (OTU). The Natural Circulation System comprises five tanks, the second and third of which play a major role in the bioreactor. Clone library pro-files and principal component analysis revealed differences in the bacterial community structures of the second (anaerobic chamber) and the third (aerobic chamber) tanks. However, the beta-proteobacteria, Bacteroidetes/ Chlorobi and gamma-proteobacteria groups were dominant in both tanks. Bacterial composition was more complex in the second tank (107 OTUs) than in the third tank (68 OTUs). Of a total of 154 OTUs in the clone libraries, only 21 were common to the two tanks. The results obtained in this study should provide important information for future research into and management of the Natural Circulation System wastewater bioreactor.     

克拉玛依油田石油污染土壤细菌群落结构与环境因子的关系

【目的】以16S rRNA为分子标记,探讨克拉玛依油田石油污染土壤中细菌群落多样性和系统发育,并分析环境因子对群落分布的影响,为生物降解石油污染物提供理论基础。【方法】在克拉玛依油田分别采集深度为5、20、50 cm的石油污染土壤样品,测定环境参数;提取石油污染土壤细菌群落基因组DNA,分别构建3个土层细菌16S rRNA基因文库,利用限制性片段长度多态性分析(Restriction fragment length polymorphisms RFLP)技术初步分群,确定各文库中的代表菌株并测定16S rRNA基因序列;利用软件Biodap计算各群落多样性和丰富度指数,以Neighbor-Joining法构建3个土层细菌的系统发育树;运用软件CANOCO 4.5结合不同样品环境因子的差异进行典型对应分析(CCA),并探讨了环境因子对细菌多样性的影响。【结果】环境参数结果表明20 cm土层总磷(TP)、总氮(TN)含量最低,50 cm含量最高;5 cm土层中有机碳(TOC)含量最高,50 cm含量最低。基于16S rRNA序列的生物多样性和物种丰富度指数表明20 cm土层生物多样性和丰富度指数较高,而50 cm土层各项指数均较低。各土层供试序列RFLP聚类分析表明,克拉玛依油田石油污染土壤细菌种群具有丰富的多样性。Neighbor-Joining构建的系统发育分析表明,石油污染土壤被分为5个类群(I–V),分别为变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、厚壁菌门(Firmicute)、拟杆菌门(Bacteroidetes)、浮霉状菌门(Planctomycetes),其中群Ⅰ占78.57%,广泛分布于不同的生态环境;其中来自5 cm土层代表菌的69.23%分布于群Ⅰ。CCA分析结果显示TN、TP和TOC对大部分细菌影响较大;TOC含量对Pseudomonas影响明显。【结论】克拉玛依油田石油污染土壤细菌群落具有丰富的多样性;环境因子是影响石油污染土壤细菌群落空间分布的重要因素。     

Identification of dominant bacterial phylotypes in a cadmium-treated forest soil

The presence of heavy metals in soils can lead to changes in microbial community structure, characterized by the dominance of groups that are able to tolerate contamination. Such groups may provide good microbial indicators of heavy-metal pollution in soil. Through terminal restriction fragment length polymorphism (T-RFLP) profiling, changes in the bacterial community structure of an acidic forest soil that had been incubated with cadmium (Cd) for 30 days were investigated. T-RFLP revealed, in particular, three operational taxonomic units (OTUs) strongly dominating in relative abundance in the contaminated soil. By cloning of the amplified 16S rRNA genes and partial sequencing of 25 clones, these three dominant OTUs were phylogenetically characterized. One dominant OTU in the cadmium-contaminated soil was derived from Betaproteobacteria, genus Burkholderia, and the other two were from uncultured members of the class Actinobacteria, closely related to the genus Streptomyces. To confirm T-RFLP data, four primers were designed on the basis of this study's dominant sequences, targeting the OTUs corresponding to Burkholderia or Actinobacteria. Real-time PCR showed that Burkholderia target sequences were more abundant in cadmium-treated soil (7.8 x 10(7)+/- 3.0 x 10(7) targets g(-1) soil) than in untreated soil (4.0 x 10(6)+/- 8.9 x 10(5) targets g(-1) soil). It was concluded that the genus Burkholderia includes species that may be particularly dominant under cadmium contamination.     

Microbial community analysis of a biogas-producing completely stirred tank reactor fed continuously with fodder beet silage as mono-substrate

The bioconversion of renewable raw material to biogas by anaerobic microbial fermentation processes in completely stirred tank reactors (CSTR) is a valuable alternative resource of energy especially for rural areas. However, knowledge about the microorganisms involved in the degradation of plant biomass is still poor. In this study, a first analysis of the biogas-forming process within a CSTR fed continuously with fodder beet silage as mono-substrate is presented in the context of molecular data on the microbial community composition. As indicated by the conventional process parameters like pH value, content of volatile fatty acids, N:P ratio and the biogas yield, the biogas-forming process within the CSTR occurred with a stable and efficient performance. The average biogas yield based on volatile solids was 0.87m(3)kg(-1) at an organic loading rate of 1.2-2.3kgm(-3)d(-1). This amounts to 94% of the theoretical maximum. In order to identify microorganisms within the CSTR, a 16S rDNA clone library was constructed by PCR amplification applying a prokaryote-specific primer set. One hundred and forty seven clones were obtained and subsequently characterized by amplified rDNA restriction analysis (ARDRA). The sequences of 60 unique ARDRA patterns were estimated in a length of approximately 800-900bp each. Four of them were assigned to the domain Archaea and 56 to the domain Bacteria. Within the domain Archaea, all clones showed a close relationship to methanogenic species. Major bacterial groups represented in the clone library were the class Clostridia of the phylum Firmicutes (22% of all 16S rDNA clones), the class Deltaproteobacteria of the phylum Proteobacteria (24%), the class Bacilli of the phylum Firmicutes (22%) and members of the phylum Bacteroidetes (21%). Within these major groups, the highest biodiversity was found within the class Clostridia (35% of all operational taxonomic units). Members of the phyla Actinobacteria and Spirochaetes were represented only by 5 and 2 clonal sequences, respectively.     

Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants

The aim of this study was to examine whether the terminal restriction fragment length polymorphism (T-RFLP) analysis represents an appropriate technique for monitoring highly diverse soil bacterial communities, i.e. to assess spatial and/or temporal effects on bacterial community structure. The T-RFLP method, a recently described fingerprinting technique, is based on terminal restriction fragment length polymorphisms between distinct small-subunit rRNA gene sequence types. This technique permits an automated quantification of the fluorescence signal intensities of the individual terminal restriction fragments (T-RFs) in a given community fingerprint pattern. The indigenous bacterial communities of three soil plots located within an agricultural field of 110 m(2) were compared. The first site was planted with non-transgenic potato plants, while the other two were planted with transgenic GUS and Barnase/Barstar potato plants, respectively. Once prior to planting and three times after planting, seven parallel samples were taken from each of the three soil plots. The T-RFLP analysis resulted in very complex but highly reproducible community fingerprint patterns. The percentage abundance values of defined T-RFs were calculated for the seven parallel samples of the respective soil plot. A multivariate analysis of variance was used to test T-RFLP data sets for significant differences. The statistical treatments clearly revealed spatial and temporal effects, as well as spacextime interaction effects, on the structural composition of the bacterial communities. T-RFs which showed the highest correlations to the discriminant factors were not those T-RFs which showed the largest single variations between the seven-sample means of individual plots. In summary, the T-RFLP technique, although a polymerase chain reaction-based method, proved to be a suitable technique for monitoring highly diverse soil microbial communities for changes over space and/or time.     

Molecular bacterial diversity of a forest soil under residue management regimes in subtropical Australia

A major operational change in exotic pine plantations of subtropical Australia has been the decision to retain postharvest residues on site. A long-term field experiment was established in February 1996 to examine the impacts of residue management regimes [i.e. the postharvest residues removed (G0R), natural amount of residues retained (G1R) and residue quantity doubled and retained (G2R)] on tree growth (F1 hybrid pine) and sustainable soil management. Twelve soil samples, which included the above three residue regimes with four replicates, were collected at plantation age 6.4 years. A 16S rRNA gene clone library was established following soil community DNA extraction, polymerase chain reaction amplification and cloning. A total of 324 clones, including 27 from each sample, were randomly selected and sequenced to represent the bacterial composition and diversity of the clone library and thus the soil bacterial community under the residue management regimes. Phylogenetic analyses indicated that Acidobacteria (37.6%) and Proteobacteria (35.6%) were the dominant components of the soil bacterial community, followed by Actinobacteria (14.7%), Chlamydiae/Verrucomicrobia (7.3%), Unclassified Bacteria (3.8%) and Gemmatimonadetes (1.0%). Analysis of molecular variance revealed that there was no significant difference in bacterial composition and diversity among the residue management regimes or their replicated samples.     

Molecular analysis of the bacterial communities in the live Pacific oyster (Crassostrea gigas) and the influence of postharvest temperature on its structure

Aims: To evaluate the effect of postharvest temperature on bacterial communities in live Pacific oysters (Crassostrea gigas) using nonculture‐based methods. Methods and Results: Live oysters were compared before and after storage at 4, 6, 15, 20 and 30°C using terminal restriction fragment length polymorphism (T‐RFLP). Bacterial communities in freshly harvested (control) vs stored oysters were significantly different. Changes in bacterial communities at 4, 15 and 30°C observed by T‐RFLP were further investigated by clone library analysis. Members of the Proteobacteria predominated (43·0–57·0% of clones) in control oysters, while storage altered the bacterial profile. At 4°C, Psychrilyobacter spp. (phylum Fusobacteria) predominated (43·8% of clones), while at 15 and 30°C, members of the phylum Bacteroidetes represented 63·0 and 60·2% of clones, respectively. High microbial diversity in oysters was observed, with at least 73 different genera‐related clones among all samples. Conclusions: Changes in the overall bacterial community of Pacific oysters were influenced by storage temperature and would likely not be detected by standard culture‐based methods currently used to assess oyster quality. Certain dominant genera, such as Psychrilyobacter, Polynucleobacter and a bacterial group related to Alkaliflexus, should be further studied as possible indicators for postharvest temperature control. Significance and Impact of the Study: This work is the first report describing the effect of different storage temperatures on bacterial diversity in postharvest live Pacific oysters using molecular‐based methods.     

新疆艾比湖湿地博乐河入口处土壤细菌多样性分析

【目的】了解新疆艾比湖湿地国家级自然保护区非培养土壤细菌群落组成及多样性。【方法】采用非培养法直接从湿地土壤提取总DNA进行16S r RNA基因扩增,构建细菌16S r RNA基因克隆文库。使用MspⅠ和AfaⅠ限制性内切酶对阳性克隆进行16S r RNA基因扩增片段的限制性酶切分析(Amplified r DNA restriction analysis,ARDRA),挑取具有不同双酶切图谱的克隆进行测序,序列比对并构建16S r RNA基因系统发育树。【结果】从土壤细菌的16S r RNA基因文库中随机挑取75个不同谱型的克隆子,共得到58个OTUs,系统发育归类为8个细菌类群:绿弯菌门(Chloroflexi)、蓝藻门(Cyanobacteria)、变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrob)和芽单胞菌门(Gemmatimonadetes)。其中,变形菌门为第一优势菌群,拟杆菌门为第二优势菌群,两者约占总克隆的65%。【结论】艾比湖湿地博乐河入口处土壤细菌多样性丰富,且存在一定数量的潜在微生物新种。