Heterogeneous aerobic benzene-degrading communities in oxygen-depleted groundwaters

A sandstone aquifer beneath a petrochemicals plant (SIReN site, UK) is heterogeneously contaminated with benzene and oxygen-depleted. Despite low redox potentials in three of the most contaminated groundwaters (benzene concentrations from 17.8 to 294 mg L(-1)), we observed aerobic benzene degradation in microcosms, indicating the presence in situ of a latent community of obligate aerobic microorganisms or an active community of facultative aerobes responding rapidly to oxygen ingress. Moreover, benzene degradation occurred at the ambient pH of 8.9 and 9.4, considerably more alkaline conditions than previously reported. 16S rRNA analyses showed that the groundwater microcosm communities were distinct from each other, despite sharing the function of aerobic benzene degradation. From DNA fingerprinting, one consortium was dominated by Acidovorax spp., another by Pseudomonas spp.; these benzene-degrading consortia were similar to the in situ communities, perhaps indicating that these organisms are active in situ and degrading benzene microaerophilically or by denitrification. Conversely, in the third sample, benzene degradation occurred only after the community changed from a Rhodoferax-dominated community to a mix of Rhodococcus and Hydrogenophaga spp. Four of the main benzene-degrading strains were brought into culture: Hydrogenophaga and Pseudomonas spp., and two strains of Rhodococcus erythropolis, a ubiquitous and metabolically versatile organism.     

Novel T-RFLP method to investigate six main groups of 2,4-diacetylphloroglucinol-producing pseudomonads in environmental samples

Strains of fluorescent pseudomonads producing 2,4-diacetylphloroglucinol (DAPG) are involved in the protection of plant roots against soil-borne plant pathogens. Recently, a multilocus sequence analysis of a world wide collection of DAPG-producers led to the identification of six main groups (A-F). In this study a T-RFLP method based on the phlD gene was developed to efficiently identify the members of these six groups in environmental samples. A combination of six restriction enzymes was identified which leads to group specific terminal fragments (T-RF). The detection limit of the phlD-T-RFLP method was determined for the two P. fluorescens strains F113 (group B) and CHA0 (group F) in rhizosphere samples and was found to be 5 × 10³ CFU/g and 5 × 10⁴ CFU/g respectively. PhlD-T-RFLP and phlD-DGGE analysis of wheat and maize root samples from greenhouse and field revealed similarly the presence of multilocus groups A, B and D. However, they were more frequently detected with phlD-T-RFLP. Additionally, groups C and F were detected in greenhouse samples but only by phlD-T-RFLP and not by phlD-DGGE. In conclusion, the new phlD-T-RFLP method proved to be a fast and reliable method to detect strains of the six main groups of DAPG-producers in environmental samples with an improved detection limit compared to phlD-DGGE.     

同一生境下白及与黄花白及内生菌群差异的初步研究

为揭示白及与黄花白及内生菌群落特征异同的内在机制,该文运用末端限制性酶切片段长度多态性分析技术对白及和黄花白及叶、茎、块茎、根等组织中内生细菌16S rDNA、内生真菌ITS区进行检测,分析内生菌丰度及多样性指数,运用主成分分析、聚类分析和相关性分析对比白及与黄花白及内生菌差异。结果表明:(1)白及和黄花白及各组织内生细菌H指数为1.77～2.51,内生真菌H指数为1.79～3.18。(2)内生细菌表现为茎、块茎和根中相似,叶中差异较大; 内生真菌则表现出明显的特异性。(3)白及药用部位多糖含量与内生细菌中的7个T-RFs、内生真菌中的2个T-RFs呈相关性,黄花白及药用部位多糖含量与内生细菌中的3个T-RFs、内生真菌中的6个T-RFs呈相关性。综上结果表明,同一生境下,白及和黄花白及内生细菌除叶组织外,其他组织相似,内生真菌则差异显著,部分内生菌和药用部位多糖含量存在相关性。     

Function-specific response to depletion of microbial diversity

Recent meta-analyses suggest that ecosystem functioning increases with biodiversity, but contradictory results have been presented for some microbial functions. Moreover, observations of only one function underestimate the functional role of diversity because of species-specific trade-offs in the ability to carry out different functions. We examined multiple functions in batch cultures of natural freshwater bacterial communities with different richness, achieved by a dilution-to-extinction approach. Community composition was assessed by molecular fingerprinting of 16S rRNA and chitinase genes, representing the total community and a trait characteristic for a functional group, respectively. Richness was positively related to abundance and biomass, negatively correlated to cell volumes and unrelated to maximum intrinsic growth rate. The response of chitin and cellulose degradation rates depended on the presence of a single phylotype. We suggest that species identity and community composition rather than richness matters for specific microbial processes.     

Relative diversity and community structure analysis of rumen protozoa according to T-RFLP and microscopic methods

Protozoa are common inhabitants of the rumen where they play roles in host nutrition and methanogenesis. Knowledge of how changes in the composition of protozoa communities affect these processes is limited in part due to a lack of efficient methods for protozoa community analysis. In this study, a terminal-restriction fragment length polymorphism (T-RFLP) assay targeting the 18S rRNA gene was developed for comparative analysis of rumen protozoa communities. Comparison of diversity and structure of protozoa communities from hay-fed versus silage/grain-fed cattle via T-RFLP analysis yielded similar overall results to microscopy analysis. According to both methods, Entodinium spp. were more abundant in the silage/grain-fed cattle and protozoa diversity (as calculated using the Shannon index) was higher for the hay-fed cattle due to greater species evenness. Type B protozoa were more prevalent in the hay-fed cattle, whereas Type A protozoa were more prevalent in the silage/grain-fed cattle. Analysis of similarity (ANOSIM) indicated that the protozoa communities from hay-fed and silage/grain-fed cattle were different, and multivariate analysis indicated that pen mates (i.e., cattle fed the same diet and housed together) tended to have similar protozoa communities types. In summary, we present a T-RFLP method for analyzing rumen protozoa communities which complements traditional microscopy approaches but has the advantage of being amenable to high-throughput.     

Carbon Mineralization in Peatlands: Does the Soil Microbial Community Composition Matter?

Peat from four geographically separated peatlands (up to 1,500 km apart) with distinct vegetation across North America was sterilized and inoculated with microbial consortia from either the home site or from the other sites. This reciprocal inoculation microcosm experiment examined how different microbial communities adapted to various peat substrates and how this in turn influenced C-mineralization patterns. The experimental approach allows distinctions to be made as to whether microbial community structure, peat properties, or imposed environmental conditions are primary drivers of peat C mineralization. Two additional inocula collected from other freshwater environments (industrially polluted harbor and lake sediments) were also added to each peat type to investigate the response of clearly disparate microbial communities. We hypothesized that the peat properties, such as substrate quality and physical structure, would dictate microbial community composition and activity, thus inoculations from different sites into the same peat soil would lead to the establishment of very similar microbial communities both phylogenetically and functionally. Post-incubation, the bacterial communities in each site converged towards a similar community regardless of the inoculum source, with the exception of peat inoculated with polluted harbor sediment. Inoculum type had no effect on C mineralization rates compared with controls, except for the two disparate inocula, which had lower rates in all peat types. Variation in microbial community structure measured as nonmetric multidimensional scaling axes scores or richness did not correlate significantly with microbial activity. Overall, these findings suggest that abiotic variables (e.g., pH, aeration, moisture content, and temperature) are the dominant control on peatland microbial activity and community composition, and in natural peatlands the microbial community can quickly adapt to future environmental change.     

Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies.     

Differentiation of Staphylococcus spp. by terminal-restriction fragment length polymorphism analysis of glyceraldehyde-3-phosphate dehydrogenase-encoding gene

Classical phenotypic and biochemical testing do not lead to correct identification of the distinct Staphylococcus species. Therefore, the aim of our study was to develop a method for the reliable and accurate determination of distinct Staphylococcus species.

In the present study, the 931–934-bp partial sequences of the glyceraldehyde-3-phosphate dehydrogenase-encoding (gap) gene of 28 validly described Staphylococcus species were amplified and sequenced. By using the respective sequence information we performed a terminal-restriction fragment length polymorphism (T-RFLP) analysis. For T-RFLP the partial gap gene was amplified with double-fluorescently labelled primers and digested with the restriction enzymes DdeI, BspHI and TaqI. Distinctive T-RFLP patterns were rendered by the use of capillary electrophoresis with laser-induced fluorescence detection. This molecular method allowed us to identify all 28 Staphylococcus species with high specificity. This was validated by analysis of 34 Staphylococcus epidermidis and 28 Staphylococcus haemolyticus isolates.

These results demonstrate the feasibility and applicability of the T-RFLP method based on the partial gap gene sequences for rapid and accurate species identification.     

Host effects on ectomycorrhizal fungal communities: insight from eight host species in mixed conifer-broadleaf forests

To advance our understanding of host effects on the community structure of ectomycorrhizal fungi (EMF), EMF communities were compared among different host species, genera and families in two mixed conifer-broadleaf forests in Japan. Using molecular identification methods we examined EMF root tips of eight coexisting species belonging to six genera (three families): Abies and Tsuga (Pinaceae), Betula and Carpinus (Betulaceae) and Fagus and Quercus (Fagaceae). In total, 205 EMF species were detected, and the total richness was estimated to exceed 300 species using major estimators. Of the 55 EMF species occurring three or more times, eight showed significantly biased host preference. A Mantel test showed a significantly negative correlation between EMF community similarity and host taxonomic distance. Detrended correspondence analysis separated EMF communities mainly by host taxonomic and successional status. Thus, EMF communities are similar on hosts with similar taxonomic and successional status. A significant proportion of EMF exhibited host specificity, which may contribute to the extremely diverse EMF community in conifer-broadleaf forests.     

T-RFLP技术分析油藏微生物多样性

应用T-RFLP(末端限制性片段长度多态性)技术分析和比较了胜利油田单12区块的一口注水井(S12-zhu)和三口采油井(S12-4、S12-5和S12-19)的油藏微生物多样性。基于T-RFLP图谱的多样性指数表明注入水样品具有更丰富的细菌和古菌多样性。相似性指数表明,样品间细菌群落结构的相似性介于22.4%～30.8%之间,古菌群落结构的相似性介于20.8%～34.5%之间。查询RDP数据库推测这4个油藏样品所共有的优势微生物可能为Pseudomonas属,Marinobacter属和产甲烷微生物。T-RFLP技术能方便快捷的分析微生物多样性,其在油藏微生物多样性研究上的应用可以为MEOR提供有用的信息。