Dynamics of bacterial community exposed to hydrocarbons and oleophilic fertilizer in high-Arctic intertidal beach

Exposure of pristine microbial environments to hydrocarbon contamination stimulates growth of the initially small fraction of indigenous hydrocarbon-degrading bacteria. Custom-made oleophilic fertilizers have been demonstrated to promote oil bioremediation by boosting this proliferation. In the present study, the temporal dynamics of the bacterial community structure and the individual influences of hydrocarbons and an oleophilic fertilizer in shaping the community structure was explored during a 78 days bioremediation experiment in a high-Arctic intertidal beach environment. A combination of cultivation-independent 16S rRNA gene length-heterogeneity polymerase chain reaction (LH-PCR) profiling and identification of hydrocarbon-degrading isolates based on partial 16S rRNA gene sequences was employed. LH-PCR community profiles in the fertilizer alone and fertilized kerosene plots were largely indistinguishable throughout the experimental period, while kerosene alone plots showed a markedly different composition of dominant groups. This pointed to the fertilizer as the more decisive factor in shaping the community structure. Most prominent LH-PCR fragments which emerged after kerosene or fertilizer addition could be provisionally assigned to bacterial taxa through coinciding LH-PCR fragment lengths with hydrocarbon-degrading isolates obtained from the same type of experimental units. However, a few quantitatively significant LH-PCR groups had no counterparts among the cultivated bacteria. One of these was affiliated to a hitherto unspeciated subgroup within the Alkanindiges/Acinetobacter clade of Moraxellaceae by a 16S rRNA gene cloning approach.     

Plant and soil properties determine microbial community structure of shared Pinus-Vaccinium rhizospheres in petroleum hydrocarbon contaminated forest soils

Rhizosphere communities are critical to plant and ecosystem function, yet our understanding of the role of disturbance in structuring these communities is limited. We tested the hypothesis that soil contamination with petroleum hydrocarbons (PHCs) alters spatial patterns of ecto- (ECM) and ericoid (ERM) mycorrhizal fungal and root-associated bacterial community structure in the shared rhizosphere of pine (Pinus contorta var. latifolia) and lingonberry (Vaccinium vitis-idaea) in reconstructed sub-boreal forest soils. Pine seeds and lingonberry cuttings were planted into containers with an organic (mor humus, FH or coarse woody debris, CWD) layer overlying sandy mineral horizons (Ae and Bf) of forest soils collected from field sites in central British Columbia, Canada. After 4 months, 219 mg cm^-2 crude oil was applied to the soil surface of half of the systems; systems were sampled 1 or 16 weeks later. Composition, relative abundance and vertical distribution of pine ECMs were assessed using light microscopy; community profiles were generated using LH-PCR of ribosomal DNA. Multivariate analysis revealed that plant and soil factors were more important determinants of community composition than was crude oil treatment. Fungal communities differed between pine and lingonberry roots; ECM communities were structured by soil layer whereas ERM communities varied between FH and CWD soil systems. Bacterial communities varied between plants and soil layers, indicating properties of ECM and ERM rhizospheres and the soil environment influence bacterial niche differentiation. This integration of mycorrhizal and bacterial community analysis contributes to a greater understanding of forest soil sustainability in forest ecosystems potentially contaminated with PHCs.     

Early succession of bacterial biofilms in paper machines

Formation of biofilms causes severe problems in paper machines, and hence financial costs. It would be preferable to prevent attachment of the primary-colonizing bacteria than to control the growth of secondary communities, which are sheltered by exopolysaccharide slime layers. We have therefore investigated the early succession of paper-machine biofilms by incubating stainless-steel test coupons in the process water-flow lines in two paper machines operating in slightly alkaline conditions in temperatures (45 and 49°C) supporting thermophilic microbes. Microbial succession was profiled using length heterogeneity analysis of PCR-amplified 16S rRNA genes (LH-PCR) and linking the sequence data of the created 16S rRNA gene libraries to the dominant LH-PCR peaks. Although the bacterial fingerprints obtained from the attached surface communities varied slightly in different samples, the biomarker signals of the dominating primary-colonizing bacterial groups remained high over time in each paper machine. Most of the 16S rRNA gene copies in the early biofilms were assigned to the genera Rhodobacter, Tepidimonas, and Cloacibacterium. The dominance of these sequence types decreased in the developing biofilms. Finally, as phylogenetically identical primary-colonizers were detected in the two different paper mills, the machines evidently had similar environmental conditions for bacterial growth and potentially a common source of contamination.     

Use of length heterogeneity PCR and fatty acid methyl ester profiles to characterize microbial communities in soil

In length heterogeneity PCR (LH-PCR) a fluorescently labeled primer is used to determine the relative amounts of amplified sequences originating from different microorganisms. Labeled fragments are separated by gel electrophoresis and detected by laser-induced fluorescence with an automated gene sequencer. We used LH-PCR to evaluate the composition of the soil microbial community. Four soils, which differed in terms of soil type and/or crop management practice, were studied. Previous data for microbial biomass, nitrogen and carbon contents, and nitrogen mineralization rates suggested that the microbial characteristics of these soils were different. One site received two different treatments: no-till and conventional till perennial ryegrass. The other sites were no-till continuous grass plots at separate locations with different soil types. Community composition was characterized by assessing the natural length heterogeneity in eubacterial sequences amplified from the 5' domain of the 16S rRNA gene and by determining fatty acid methyl ester (FAME) profiles. We found that LH-PCR results were reproducible. Both methods distinguished the three sites. The most abundant bacterial community members, based on cloned LH-PCR products, were members of the beta subclass of the class Proteobacteria, the Cytophaga-Flexibacter-Bacteriodes group, and the high-G+C-content gram-positive bacterial group. Strong correlations were found between LH-PCR results and FAME results. We found that the LH-PCR method is an efficient, reliable, and highly reproducible method that should be a useful tool in future assessments of microbial community composition.     

应用LH-PCR研究党参、麻黄和独一味内生细菌多样性

通过直接提取药用植物样品的总DNA,采用长度多态片段PCR (length heterogeneity PCR, LH-PCR)技术研究四川省甘孜藏族自治州的党参、麻黄和独一味3种药用植物内生细菌多样性.结果表明： 同种植物根、茎、叶的LH-PCR图谱相似度很高,条带丰富度差别不大;但不同植物样品之间的差异较大.党参的植物样品条带丰富度最大,麻黄次之,独一味最低.3种药用植物中474 bp长度的细菌是绝对的优势菌群.植物内生细菌多样性与土壤速效磷呈负相关,而与土壤pH值呈正相关.海拔和土壤总氮是影响植物样品内生细菌多样性分布的两个重要环境因子.LH-PCR所得的种群信息能较直观地反映不同植物样品间细菌多样性的差异.因此LH-PCR适用于分析药用植物内生菌多样性.     

Decreasing prevalence of rhizosphere IAA producing and seedling root growth promoting bacteria with barley development irrespective of protozoan grazing regime

Barley was grown in soil with either bacteria and a mixed protozoan community (Mixed protozoa) or bacteria and a single vahlkampfiid amoebal species (Single amoeba). We assessed the influence of plant age (day 29, 43 and 57 after sowing) on two aspects of rhizosphere bacterial functioning: (1) the proportion of indole-3-acetic acid (IAA) producing bacteria and (2) the effect of mixed rhizosphere bacterial assemblages on barley seedling root growth in an agar based assay. The proportion of IAA producers was significantly lower at day 57 than at day 29 and 43, and mixed bacterial assemblages extracted from rhizospheres of 29 days old plants were significantly less harmful to seedling growth than bacterial assemblages from older plants. Hence both assays indicated that bacterial communities from rhizospheres of older plants were less beneficial for root growth than bacterial communities from younger plants. Genetic fingerprinting of rhizosphere bacterial communities was compared by use of length heterogeneity polymerase chain reaction (LH-PCR). This analysis showed a clear succession from the inoculum bacterial community with a rather low diversity to a community with much higher diversity at day 29. However, diversity did not change after day 29, and no relationship between protozoan treatment nor plant age and genetic fingerprinting was found. Responsible Editor: Petra Marschner     

Black Band Disease Microbial Community Variation on Corals in Three Regions of the Wider Caribbean

Black band disease (BBD) is a pathogenic consortium of microorganisms that primarily affects massive framework-building scleractinian corals on reefs worldwide. There has been considerable debate concerning the microbial community composition of BBD. The aim of this study was to utilize microbial profiling to assess overall patterns of variation in the BBD bacterial community with respect to geographic location, host coral species, time, and nutrient regime. Length heterogeneity polymerase chain reaction (LH-PCR) was employed to differentiate BBD communities based on the natural variation in the sequence lengths within hypervariable domains of the 16S rRNA gene. Analysis of LH-PCR profiles of 97 BBD samples using multivariate ordination methods and analysis of similarity revealed significant clustering with respect to geographic region when comparing BBD sampled from reefs near Lee Stocking Island in the Bahamas’ Exuma Chain, the Northern Florida Keys (NFK), and St. John in the US Virgin Islands. There was much variability in BBD community composition on a regional basis, between sites in the NFK, and in terms of coral host species. The observed differences among BBD microbial community profiles were driven primarily by variation in relative abundance of 313–316-bp amplicons, which correspond to cyanobacteria and α-proteobacteria. The results obtained in this study support previous reports of intrinsic variability and complexity of the BBD microbial community but also suggest that this variability has biogeographic patterns.     

Links between Plant and Rhizoplane Bacterial Communities in Grassland Soils, Characterized Using Molecular Techniques

Molecular analysis of grassland rhizosphere soil has demonstrated complex and diverse bacterial communities, with resultant difficulties in detecting links between plant and bacterial communities. These studies have, however, analyzed “bulk” rhizosphere soil, rather than rhizoplane communities, which interact most closely with plants through utilization of root exudates. The aim of this study was to test the hypothesis that plant species was a major driver for bacterial rhizoplane community composition on individual plant roots. DNA extracted from individual roots was used to determine plant identity, by analysis of the plastid tRNA leucine (trnL) UAA gene intron, and plant-related bacterial communities. Bacterial communities were characterized by analysis of PCR-amplified 16S rRNA genes using two fingerprinting methods: terminal restriction fragment length polymorphisms (T-RFLP) and denaturing gradient gel electrophoresis (DGGE). Links between plant and bacterial rhizoplane communities could not be detected by visual examination of T-RFLP patterns or DGGE banding profiles. Statistical analysis of fingerprint patterns did not reveal a relationship between bacterial community composition and plant species but did demonstrate an influence of plant community composition. The data also indicated that topography and other, uncharacterized, environmental factors are important in driving bacterial community composition in grassland soils. T-RFLP had greater potential resolving power than DGGE, but findings from the two methods were not significantly different.     

Links between plant and rhizoplane bacterial communities in grassland soils, characterized using molecular techniques

Molecular analysis of grassland rhizosphere soil has demonstrated complex and diverse bacterial communities, with resultant difficulties in detecting links between plant and bacterial communities. These studies have, however, analyzed "bulk" rhizosphere soil, rather than rhizoplane communities, which interact most closely with plants through utilization of root exudates. The aim of this study was to test the hypothesis that plant species was a major driver for bacterial rhizoplane community composition on individual plant roots. DNA extracted from individual roots was used to determine plant identity, by analysis of the plastid tRNA leucine (trnL) UAA gene intron, and plant-related bacterial communities. Bacterial communities were characterized by analysis of PCR-amplified 16S rRNA genes using two fingerprinting methods: terminal restriction fragment length polymorphisms (T-RFLP) and denaturing gradient gel electrophoresis (DGGE). Links between plant and bacterial rhizoplane communities could not be detected by visual examination of T-RFLP patterns or DGGE banding profiles. Statistical analysis of fingerprint patterns did not reveal a relationship between bacterial community composition and plant species but did demonstrate an influence of plant community composition. The data also indicated that topography and other, uncharacterized, environmental factors are important in driving bacterial community composition in grassland soils. T-RFLP had greater potential resolving power than DGGE, but findings from the two methods were not significantly different.     

Diversity of ndo genes in mangrove sediments exposed to different sources of polycyclic aromatic hydrocarbon pollution

Polycyclic aromatic hydrocarbon (PAH) pollutants originating from oil spills and wood and fuel combustion are pollutants which are among the major threats to mangrove ecosystems. In this study, the composition and relative abundance in the sediment bacterial communities of naphthalene dioxygenase (ndo) genes which are important for bacterial adaptation to environmental PAH contamination were investigated. Three urban mangrove sites which had characteristic compositions and levels of PAH compounds in the sediments were selected. The diversity and relative abundance of ndo genes in total community DNA were assessed by a newly developed ndo denaturing gradient gel electrophoresis (DGGE) approach and by PCR amplification with primers targeting ndo genes with subsequent Southern blot hybridization analyses. Bacterial populations inhabiting sediments of urban mangroves under the impact of different sources of PAH contamination harbor distinct ndo genotypes. Sequencing of cloned ndo amplicons comigrating with dominant DGGE bands revealed new ndo genotypes. PCR-Southern blot analysis and ndo DGGE showed that the frequently studied nah and phn genotypes were not detected as dominant ndo types in the mangrove sediments. However, ndo genotypes related to nagAc-like genes were detected, but only in oil-contaminated mangrove sediments. The long-term impact of PAH contamination, together with the specific environmental conditions at each site, may have affected the abundance and diversity of ndo genes in sediments of urban mangroves.     

Response of Antarctic Soil Bacterial Assemblages to Contamination by Diesel Fuel and Crude Oil

Abstract The effects of diesel fuel and ``Arabian light' crude oil addition on Antarctic bacterial assemblages were studied in four contaminated soils during 1 year in the Terre Adelie land area. Monthly sampling allowed a regular survey of the bacterial changes occurring in the contaminated soils. All samples were analyzed for total bacteria, heterotrophic culturable microbiota, and hydrocarbon-utilizing microbiota. Crude oil contamination induced an initial increase of all bacterial parameters in all contaminated soils. Diesel oil contamination had a more complex effect. Hydrocarbon degrading bacterial abundance increases occurred after diesel oil addition. In contrast, general heterotrophic bacterial abundance could significantly decrease in the same conditions. In all cases the stimulatory effects of oil addition disappeared after several months of contamination. Received: 13 April 1999; Accepted: 24 February 2000; Online Publication: 29 May 2000     

Molecular assessment of microbiota structure and dynamics along mixed olive oil and winery wastewaters biotreatment

The major parcel of the degradation occurring along wastewater biotreatments is performed either by the native microbiota or by added microbial inocula. The main aim of this study was to apply two fingerprinting methods, temperature gradient gel electrophoresis (TGGE) and length heterogeneity-PCR (LH-PCR) analysis of 16S rRNA gene fragments, in order to assess the microbiota structure and dynamics during mixed olive oil and winery wastewaters aerobic biotreatment performed in a jet-loop reactor (JLR). Sequence homology analysis showed the presence of bacterial genera Gluconacetobacter, Klebsiella, Lactobacillus, Novosphingobium, Pseudomonas, Prevotella, Ralstonia, Sphingobium and Sphingomonas affiliated with five main phylogenetic groups: alpha-, beta- and gamma-Proteobacteria, Firmicutes and Bacteroidetes. LH-PCR analysis distinguished eight predominant DNA fragments correlated with the samples showing highest performance (COD removal rates of 67 up to 75%). Cluster analysis of both TGGE and LH-PCR fingerprinting profiles established five main clusters, with similarity coefficients higher than 79% (TGGE) and 62% (LH-PCR), and related with hydraulic retention time, indicating that this was the main factor responsible for the shifts in the microbiota structure. Canonical correspondence analysis revealed that changes observed on temperature and O₂ level were also responsible for shifts in microbiota composition. Community level metabolic profile analysis was used to test metabolic activities in samples. Integrated data revealed that the microbiota structure corresponds to bacterial groups with high degradative potential and good suitability for this type of effluents biotreatments.     

An assessment of soil bacterial community structure and physicochemistry in two microtopographic locations of a palustrine forested wetland

We studied redoximorphic features, field indicators and bacterial communities of soils in hummocks and hollows of a palustrine forested wetland in Virginia. We hypothesized that presence of hydric soils, soil physicochemistry and soil bacterial community structure would differ between hummocks and hollows. We fingerprinted soils collected from different microtopographic locations using Length Heterogeneity Polymerase Chain Reaction (LH-PCR) to study their bacterial community structures. Two hummocks had silty/sandy loam soils with mean chroma values of > 4, showing no indication of ‘hydric soils’ (i.e., wetland soils). Two hollows, however, had clay loam soils with mean chroma values of 2 with gleying and redox concentrations observed, indicative of seasonally inundated wetlands. The soils of hollows also had higher organic matter content and soil moisture compared to the soils of hummocks (P < 0.05). Multidimensional scaling (MDS) and Analysis of similarity (ANOSIM) of the fingerprints revealed differences in soil microbial community structures between hummocks and hollows (Global R = 0.30, P < 0.01). The diversity measures of the fingerprints (Shannon’s H′) were also different by microtopography with higher diversity in hollows relative to hummocks (P < 0.05). LH-PCR proves to be a useful tool in examining bacterial community composition of wetland soils in this study. However, cloning and sequencing of specific community LH-PCR profiles of interest is necessary to fully characterize the community down to genus/species level. With species identities we should be able to not only better explain differences observed in the community profiles, but study their relations to hydrologic and/or physicochemical conditions of wetlands.     

火电厂周边不同生物结皮细菌群落特征差异及其影响因素

为探究大气降尘重金属污染对矿区周边不同类型生物结皮细菌群落结构的影响,利用高通量测序技术分析位于宁东能源化工基地典型火电厂周边的3类生物结皮(藻结皮ZB、混生结皮HB、苔藓结皮TB)和对照(CK,裸土)的细菌丰度和群落结构,并探讨了影响细菌群落结构的环境因子。结果表明: 不同类型生物结皮的理化性质和重金属含量存在差异,且由于生物结皮对大气降尘重金属的富集作用造成各类结皮均达重度污染级别。在相对丰度排名前10的优势细菌门中,芽单胞菌门、蓝细菌门在不同类型生物结皮之间差异显著。细菌群落α多样性由高到低排序依次为CK>TB>HB>ZB。非度量多维排序(NMDS)结果显示,裸土细菌群落与其他3种生物结皮存在明显差异。相关性分析表明,生物结皮演替对细菌群落组成具有显著影响,细菌多样性和组成与pH、养分、重金属含量等密切相关。放线菌门、绿弯菌门相对丰度与pH值呈显著正相关关系,而与全氮(TN)、全磷(TP)、Pb、Zn、Cd均呈显著负相关关系;冗余分析结果表明,TN、pH、TP、有机碳(SOC)是影响3种生物结皮细菌群落α多样性以及一些优势菌群相对丰度的主要土壤环境因子,而重金属Pb、Zn、Cd是影响细菌群落结构的主要重金属元素,对细菌群落数量和多样性有抑制或刺激作用。说明pH、重金属和养分是影响结皮细菌群落组成的关键因子。总体而言,长期的重金属富集作用会对生物结皮的细菌多样性和群落组成产生影响。     

Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment

The bacterial diversity of a chronically oil-polluted retention basin sediment located in the Berre lagoon (Etang-de-Berre, France) was investigated. This study combines chemical and molecular approaches in order to define how the in situ petroleum hydrocarbon contamination level affects the bacterial community structure of a subsurface sediment. Hydrocarbon content analysis clearly revealed a gradient of hydrocarbon contamination in both the water and the sediment following the basin periphery from the pollution input to the lagoon water. The nC17 and pristane concentrations suggested alkane biodegradation in the sediments. These results, combined with those of terminal-restriction fragment length polymorphism analysis of the 16S rRNA genes, indicated that bacterial community structure was obviously associated with the gradient of oil contamination. The analysis of bacterial community composition revealed dominance of bacteria related to the Proteobacteria phylum (Gamma-, Delta-, Alpha-, Epsilon- and Betaproteobacteria), Bacteroidetes and Verrucomicrobium groups and Spirochaetes, Actinobacteria and Cyanobacteria phyla. The adaptation of the bacterial community to oil contamination was not characterized by dominance of known oil-degrading bacteria, because a predominance of populations associated to the sulphur cycle was observed. The input station presented particular bacterial community composition associated with a low oil concentration in the sediment, indicating the adaptation of this community to the oil contamination.     

The application of amplicon length heterogeneity PCR (LH-PCR) for monitoring the dynamics of soil microbial communities associated with cadaver decomposition

The placement of cadavers in shallow, clandestine graves may alter the microbial and geochemical composition of the underlying and adjacent soils. Using amplicon length heterogeneity-PCR (LH-PCR) the microbial community changes in these soils can be assessed. In this investigation, nine different grave sites were examined over a period of 16 weeks. The results indicated that measurable changes occurred in the soil bacterial community during the decomposition process. In this study, amplicons corresponding to anaerobic bacteria, not indigenous to the soil, were shown to produce differences between grave sites and control soils. Among the bacteria linked to these amplicons are those that are most often part of the commensal flora of the intestines, mouth and skin. In addition, over the 16 week sampling interval, the level of indicator organisms (i.e., nitrogen fixing bacteria) dropped as the body decomposed and after four weeks of environmental exposure they began to increase again; thus differences in the abundance of nitrogen fixing bacteria were also found to contribute to the variation between controls and grave soils. These results were verified using primers that specifically targeted the nifH gene coding for nitrogenase reductase. LH-PCR provides a fast, robust and reproducible method to measure microbial changes in soil and could be used to determine potential cadaveric contact in a given area. The results obtained with this method could ultimately provide leads to investigators in criminal or missing person scenarios and allow for further analysis using human specific DNA assays to establish the identity of the buried body.     

Bacterial succession in a petroleum land treatment unit

Bacterial community dynamics were investigated in a land treatment unit (LTU) established at a site contaminated with highly weathered petroleum hydrocarbons in the C(10) to C(32) range. The treatment plot, 3,000 cubic yards of soil, was supplemented with nutrients and monitored weekly for total petroleum hydrocarbons (TPH), soil water content, nutrient levels, and aerobic heterotrophic bacterial counts. Weekly soil samples were analyzed with 16S rRNA gene terminal restriction fragment (TRF) analysis to monitor bacterial community structure and dynamics during bioremediation. TPH degradation was rapid during the first 3 weeks and slowed for the remainder of the 24-week project. A sharp increase in plate counts was reported during the first 3 weeks, indicating an increase in biomass associated with petroleum degradation. Principal components analysis of TRF patterns revealed a series of sample clusters describing bacterial succession during the study. The largest shifts in bacterial community structure began as the TPH degradation rate slowed and the bacterial cell counts decreased. For the purpose of analyzing bacterial dynamics, phylotypes were generated by associating TRFs from three enzyme digests with 16S rRNA gene clones. Two phylotypes associated with Flavobacterium and Pseudomonas were dominant in TRF patterns from samples during rapid TPH degradation. After the TPH degradation rate slowed, four other phylotypes gained dominance in the community while Flavobacterium and Pseudomonas phylotypes decreased in abundance. These data suggest that specific phylotypes of bacteria were associated with the different phases of petroleum degradation in the LTU.     

Effectiveness of bioremediation for oil-polluted Antarctic seawater

The effectiveness of a specific fertiliser (INIPOL EAP 22) addition on bioremediation of oil-contaminated Antarctic coastal seawater was determined in the “Terre Adelie” area. Mesocosm studies were conducted to evaluate the effects of “Arabian light” crude oil contamination on coastal bacterioplanktonic communities. After oil addition, regular surveys of the bacterial changes of the oil-contaminated seawater were performed during 5-week periods during the austral summer of 1992/1993 and 1993/1994. All results (total, saprophytic and hydrocarbon-utilising bacterial abundance) clearly revealed a significant response of Antarctic bacterial communities to hydrocarbon contamination. A 1 order of magnitude increase of bacterial microflora occurred in seawater after crude oil contamination. A concomitant enrichment in oil-degrading bacteria was generally observed, from less than 0.001% of the community in uncontaminated samples to up to 50% after 3 weeks of contamination. Addition of fertiliser (INIPOL EAP 22) induced clear enhancement of both saprophytic and hydrocarbon-utilising microflora. Chemical analysis of the residual hydrocarbon fractions confirmed that fertiliser application increased the rate of oil biodegradation. Received: 27 March 1997 / Accepted: 20 October 1997