Phylogenetic analysis of a biofilm bacterial population in a water pipeline in the Gulf of Mexico

The aim of this study was to assess the bacterial diversity associated with a corrosive biofilm in a steel pipeline from the Gulf of Mexico used to inject marine water into the oil reservoir. Several aerobic and heterotrophic bacteria were isolated and identified by 16S rRNA gene sequence analysis. Metagenomic DNA was also extracted to perform a denaturing gradient gel electrophoresis analysis of ribosomal genes and to construct a 16S rRNA gene metagenomic library. Denaturing gradient gel electrophoresis profiles and ribosomal libraries exhibited a limited bacterial diversity. Most of the species detected in the ribosomal library or isolated from the pipeline were assigned to Proteobacteria (Halomonas spp., Idiomarina spp., Marinobacter aquaeolei, Thalassospira sp., Silicibacter sp. and Chromohalobacter sp.) and Bacilli (Bacillus spp. and Exiguobacterium spp.). This is the first report that associates some of these bacteria with a corrosive biofilm. It is relevant that no sulfate-reducing bacteria were isolated or detected by a PCR-based method. The diversity and relative abundance of bacteria from water pipeline biofilms may contribute to an understanding of the complexity and mechanisms of metal corrosion during marine water injection in oil secondary recovery.     

非培养方法解析北京地区甘薯叶际细菌的群落结构

利用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术和磷脂脂肪酸(PLFA)分析方法,比较了北京通州、顺义、昌平、延庆地区甘薯叶际细菌的多样性和生物量,并调查了通州地区甘薯叶际细菌群落在不同生长季节的变化情况。PLFA分析结果发现所有检测样品中,革兰氏阳性细菌生物量均高于革兰氏阴性细菌生物量。PCR-DGGE方法与PLFA方法聚类分析结果较一致,甘薯叶际细菌群落结构受到时空因素、甘薯生理特性等的影响,不同地点、不同生长季节甘薯叶际细菌群落结构有较大差异,DGGE条带测序分析表明,Pseudomonas sp.在不同地点的甘薯叶际均为保守菌群,Bacillus sp.,Acinetobacter sp.,     

造纸废水活性污泥的微生物群落结构及多样性分析

为探究造纸废水活性污泥中微生物群落结构多样性以及对造纸废水处理效果的影响，利用Illumina MiSeq 高通量测序方法，分析在处理造纸废水过程中，同一运行阶段两个并联氧化沟内活性污泥的微生物群落与多样性组成。结果表明，系统中处理造纸废水的活性污泥在同一废水条件下微生物群落结构总体稳定，优势细菌为绿弯菌门(Chloroflexi)、拟杆菌门（Bacteroidota）、变形菌门（Proteobacteria）、Myxococcota、放线菌门(Actinobacteria）、厚壁菌门(Firmicutes）等。最重要的优势细菌类群为Chloroflexi，相对丰度占比为47.67%~48.22%，远远高于其他废水中Chloroflexi的占比，其中厌氧绳菌纲（Anaerolineae）是其主要成员，占比84.39%~88.34%，可针对性地去除造纸废水中的污染物。造纸废水活性污泥样品中存在大量特殊功能菌群，其在废水中污染物尤其是木质素的去除中发挥着重要作用。     

Electrochemical and microbiological characterization of paper mill biofilms

Biofilm samples collected from inside and outside the press and former sections of paper machines in a Northwestern Ontario paper mill for a period of 2 years were characterized microbiologically and electrochemically. Bacterial community profiling was done using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and selected bacterial isolates were identified using 16S rDNA analysis. The bacterial community showed the presence of Proteobacteria, Firmicutes, and Actinobacteria. Sphingomonas sp. was found to be the most common bacterial species, which showed the highest production of extracellular polymeric substances. Bacteria isolated from biofilms showed better adhesion properties than those from water samples. Cyclic voltammetry and electrochemical impedance spectroscopy studies showed that bacteria isolated from biofilms and feed water collected from inside the machine were more easily oxidized than those from outside, suggesting the need for a more rigorous biofilm abatement strategy for inside paper machines.     

太湖浮游细菌与春末浮游藻类群落结构演替的相关分析

为研究浮游细菌与浮游藻类群落演替的相关性,2005年4月至6月在太湖5个观测点采集浮游细菌及浮游藻类样本。分别采用聚合酶链式反应-变性梯度凝胶电泳（PCR—DGGE）和显微观察的方法分析浮游细菌及浮游藻类群落组成。结果表明,春末夏初,浮游细菌与藻类均呈现较高的多样性,浮游细菌DGGE图谱具有43种不同条带,浮游藻类的常见种有29种。浮游细菌群落聚类分析显示,丝藻（Ulothrix sp．）和微囊藻（Microcystis spp．）占优势时,浮游细菌群落基因组成存在明显差异。以藻类种群Shannon—Wiener多样性指数（Hp）,浮游藻类总细胞数（N）以及Microcystis spp．（M）百分含量为变量,典型对应分析（CCA）结果显示浮游细菌与浮游藻类群落结构变化的相关系数为30．9％,表明春末夏初太湖浮游细菌与浮游藻类群落演替具有较高的相关性。     

Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis

The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions.     

Microbial biofilms on facial prostheses

The composition of microbial biofilms on silicone rubber facial prostheses was investigated and compared with the microbial flora on healthy and prosthesis-covered skin. Scanning electron microscopy showed the presence of mixed bacterial and yeast biofilms on and deterioration of the surface of the prostheses. Microbial culturing confirmed the presence of yeasts and bacteria. Microbial colonization was significantly increased on prosthesis-covered skin compared to healthy skin. Candida spp. were exclusively isolated from prosthesis-covered skin and from prostheses. Biofilms from prostheses showed the least diverse band-profile in denaturing gradient gel electrophoresis (DGGE) whereas prosthesis-covered skin showed the most diverse band-profile. Bacterial diversity exceeded yeast diversity in all samples. It is concluded that occlusion of the skin by prostheses creates a favorable niche for opportunistic pathogens such as Candida spp. and Staphylococcus aureus. Biofilms on healthy skin, skin underneath the prosthesis and on the prosthesis had a comparable composition, but the numbers present differed according to the microorganism.     

The Influence of Iron Availability on Human Salivary Microbial Community Composition

It is a well-recognized fact that the composition of human salivary microbial community is greatly affected by its nutritional environment. However, most studies are currently focused on major carbon or nitrogen sources with limited attention to trace elements like essential mineral ions. In this study, we examined the effect of iron availability on the bacterial profiles of an in vitro human salivary microbial community as iron is an essential trace element for the survival and proliferation of virtually all microorganisms. Analysis via a combination of PCR with denaturing gradient gel electrophoresis demonstrated a drastic change in species composition of an in vitro human salivary microbiota when iron was scavenged from the culture medium by addition of the iron chelator 2,2'-bipyridyl. This shift in community profile was prevented by the presence of excessive ferrous iron (Fe(2+)). Most interestingly, under iron deficiency, the in vitro grown salivary microbial community became dominated by several hemolytic bacterial species, including Streptococcus spp., Gemella spp., and Granulicatella spp. all of which have been implicated in infective endocarditis. These data provide evidence that iron availability can modulate host-associated oral microbial communities, resulting in a microbiota with potential clinical impact.     

16S ribosomal DNA-based analysis of bacterial diversity in purified water used in pharmaceutical manufacturing processes by PCR and denaturing gradient gel electrophoresis

The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.     

溶微囊藻细菌的富集筛选及其菌群结构特征

利用铜绿微囊藻(Microcystis aeruginosa)作为溶藻对象富集、筛选, 获得一个稳定的溶藻菌群。采用叶绿素、PCR和变性梯度凝胶电泳(DGGE)方法研究溶藻过程及其细菌种群结构的变化。结果显示, 富集的溶藻菌经1×10-5稀释后仍有显著溶藻效果。Rubritepida菌C1、假单胞菌C2和鞘氨醇单胞菌C3是存在于铜绿微囊藻中的3种伴生细菌。加入富集的溶藻菌群后, 菌群结构发生明显的变化, Rubritepida菌C1、假单胞菌C2消失, 混合菌群包含未培养黄杆菌A2、鞘氨醇单胞菌C3和噬氢     

Microbial communities in wetlands of the Athabasca oil sands: genetic and metabolic characterization

Naphthenic acids are a complex family of naturally occurring cyclic and acyclic carboxylic acids that are present in the acidic fraction of petroleum. Naphthenic acids are acutely toxic to aquatic organisms. Previous studies showed that wetland sediments exposed to oil sands process water containing naphthenic acids had higher rates of naphthenic acid degradation in vitro compared with unexposed wetlands. In this study we compare the microbial community structures in sediments from wetlands exposed to different amounts of oil sands process water using BIOLOG, phospholipid fatty acid analysis and denaturing gradient gel electrophoresis of total bacterial DNA. Community profiles were compared using cluster analysis. BIOLOG profiles were primarily influenced by seasonal trends rather than naphthenic acids content. In contrast, phospholipid fatty acid analysis comparisons clustered communities that had higher levels of residual oil, although this association was not strong. In contrast, cluster diagrams produced from the denaturing gradient gel electrophoresis data clearly separated bacterial communities according to naphthenic acids concentrations, indicating that naphthenic acids content was a major influence on the composition of the bacterial community. In addition, denaturing gradient gel electrophoresis profiles indicated that naphthenic acids-exposed bacterial communities were homogeneous on a scale of meters, whereas unexposed (off-site) wetlands were less homogeneous.     

Characterization of the bacterial consortium associated with black band disease in coral using molecular microbiological techniques

The bacterial community associated with black band disease (BBD) of the scleractinian corals Diploria strigosa, Montastrea annularis and Colpophyllia natans was examined using culture-independent techniques. Two complementary molecular screening techniques of 16S rDNA genes [amplified 16S ribosomal DNA restriction analysis (ARDRA) of clone libraries and denaturing gradient gel electrophoresis (DGGE)] were used to give a comprehensive characterization of the community. Findings support previous studies indicating low bacterial abundance and diversity associated with healthy corals. A single cyanobacterial ribotype was present in all the diseased samples, but this was not the same as that identified from Phormidium corallyticum culture isolated from BBD. The study confirms the presence of Desulfovibrio spp. and sulphate-reducing bacteria that have previously been associated with the BBD consortium. However, the species varied between diseased coral samples. We found no evidence of bacteria from terrestrial, freshwater or human sources in any of the samples. We report the presence of previously unrecognized potential pathogens [a Cytophaga sp. and an alpha-proteobacterium identified as the aetiological agent of juvenile oyster disease (JOD)] that were consistently present in all the diseased coral samples. The molecular biological approach described here gives an increasingly comprehensive and more precise picture of the bacterial population associated with BBD. To understand the pathogenesis of BBD, our attention should be focused on the pervasive ribotypes identified in this study (the Cyanobacterium sp., the Cytophaga sp. and the JOD pathogen).     

Influence of crude oil on changes of bacterial communities in Arctic sea-ice

The danger of a petroleum hydrocarbon spillage in the polar, ice-covered regions is increasing due to oil exploration in Arctic offshore areas and a growing interest in using the Northern Sea Route (NSR) as an alternative transportation route for Arctic oil and gas. However, little is known about the potential impact of accidental oil spills on this environment. We investigated the impact of crude oil on microbial community composition in six different Arctic sea-ice samples incubated with crude oil at 1 degrees C in microcosms for one year. Alterations in the composition of bacterial communities were analyzed with the culture-independent molecular methods DGGE (denaturing gradient gel electrophoresis) and FISH (fluorescence in situ hybridization). DGGE, FISH and cultivation methods revealed a strong shift in community composition toward the gamma-proteobacteria in sea-ice and melt pool samples incubated with crude oil. Marinobacter spp., Shewanella spp. and Pseudomonas spp. were the predominant phylotypes in the oil-treated microcosms. The ability of indigenous sea-ice bacteria to degrade hydrocarbons at low temperature (1 degrees C) was tested using four representative strains cultivated from sea-ice enriched with crude oil. [14C]Hexadecane was degraded by the sea-ice isolates at 20-50% capacity of the mesophilic type strain Marinobacter hydrocarbonoclasticus, a known hydrocarbon degrader, incubated at 22 degrees C.     

Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico

Microbial communities associated to biofilms promote corrosion of oil pipelines. The community structure of bacteria in the biofilm formed in oil pipelines is the basic knowledge to understand the complexity and mechanisms of metal corrosion. To assess bacterial diversity, biofilm samples were obtained from X52 steel coupons corroded after 40 days of exposure to normal operation and flow conditions. The biofilm samples were directly used to extract metagenomic DNA, which was used as template to amplify 16S ribosomal gene by PCR. The PCR products of 16S ribosomal gene were also employed as template for sulfate-reducing bacteria (SRB) specific nested-PCR and both PCR products were utilized for the construction of gene libraries. The V3 region of the 16S rRNA gene was also amplified to analyse the bacterial diversity by analysis of denaturing gradient gel electrophoresis (DGGE). Ribosomal library and DGGE profiles exhibited limited bacterial diversity, basically including Citrobacter spp., Enterobacter spp. and Halanaerobium spp. while Desulfovibrio alaskensis and a novel clade within the genus Desulfonatronovibrio were detected from the nested PCR library. The biofilm samples were also taken for the isolation of SRB. Desulfovibrio alaskensis and Desulfovibrio capillatus, as well as some strains related to Citrobacter were isolated. SRB consists in a very small proportion of the community and Desulfovibrio spp. were the relatively abundant groups among the SRB. This is the first study directly exploring bacterial diversity in corrosive biofilms associated to steel pipelines subjected to normal operation conditions.     

Microbial biofilms on facial prostheses

The composition of microbial biofilms on silicone rubber facial prostheses was investigated and compared with the microbial flora on healthy and prosthesis-covered skin. Scanning electron microscopy showed the presence of mixed bacterial and yeast biofilms on and deterioration of the surface of the prostheses. Microbial culturing confirmed the presence of yeasts and bacteria. Microbial colonization was significantly increased on prosthesis-covered skin compared to healthy skin. Candida spp. were exclusively isolated from prosthesis-covered skin and from prostheses. Biofilms from prostheses showed the least diverse band-profile in denaturing gradient gel electrophoresis (DGGE) whereas prosthesis-covered skin showed the most diverse band-profile. Bacterial diversity exceeded yeast diversity in all samples. It is concluded that occlusion of the skin by prostheses creates a favorable niche for opportunistic pathogens such as Candida spp. and Staphylococcus aureus. Biofilms on healthy skin, skin underneath the prosthesis and on the prosthesis had a comparable composition, but the numbers present differed according to the microorganism.     

16S Ribosomal DNA-Based Analysis of Bacterial Diversity in Purified Water Used in Pharmaceutical Manufacturing Processes by PCR and Denaturing Gradient Gel Electrophoresis

The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.     

Relationship between bacterioplankton and phytoplankton community dynamics during late spring and early summer in Lake Taihu, China

The relationship between bacterioplankton and phytoplankton community compositions was investigated from April to June, 2005 with samples taken at five different stations in Lake Taihu, China. Bacterioplankton and phytoplankton community compositions as determined by denaturing gradient gel electrophoresis of polymerase chain reaction amplified bacterial 16S rRNA gene fragments (PCR-DGGE) and microscopic examination, respectively, revealed a high degree of diversity. In total, 43 unique bands were identified amongst the DGGE profiles, and 29 dominant phytoplankton populations were observed. The DGGE profiles were clustered into two distinct groups. These two clusters were closely associated with two dominant phytoplanktons, Ulothrix sp. and Microcystis spp., as observed on April 19, May 27 and June 27, 2005, respectively. The relationship between bacterioplankton and phytoplankton community compositions was investigated using Canonical Correspondence Analysis (CCA) with the Shannon-Wiener diversity index of phytoplankton community (Hp), the total cell abundance of phytoplankton (N) and the relative abundance of Microcystis spp. (M) as the variables. The CCA results indicated a 30.9 % correlation coefficient, suggesting a high degree of correlation between bacterioplankton and phytoplankton community compositions.     

Relationship between bacterioplankton and phytoplankton community dynamics during late spring and early summer in Lake Taihu, China

The relationship between bacterioplankton and phytoplankton community compositions was investigated from April to June, 2005 with samples taken at five different stations in Lake Taihu, China. Bacterioplankton and phytoplankton community compositions as determined by denaturing gradient gel electrophoresis of polymerase chain reaction amplified bacterial 16S rRNA gene fragments (PCR-DGGE) and microscopic examination, respectively, revealed a high degree of diversity. In total, 43 unique bands were identified amongst the DGGE profiles, and 29 dominant phytoplankton populations were observed. The DGGE profiles were clustered into two distinct groups. These two clusters were closely associated with two dominant phytoplanktons, Ulothrix sp. and Microcystis spp., as observed on April 19, May 27 and June 27, 2005, respectively. The relationship between bacterioplankton and phytoplankton community compositions was investigated using Canonical Correspondence Analysis (CCA) with the Shannon-Wiener diversity index of phytoplankton community (Hp), the total cell abundance of phytoplankton (N) and the relative abundance of Microcystis spp. (M) as the variables. The CCA results indicated a 30.9 % correlation coefficient, suggesting a high degree of correlation between bacterioplankton and phytoplankton community compositions.     

Denaturing gradient gel electrophoresis analysis of bacterial community profiles in the rhizosphere of <Emphasis Type="Italic">cry1AC-carrying Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis>

The effect of genetically modified (GM) Brassica rapa subsp. pekinensis (Chinese cabbage) expressing Bt toxin gene (cry1AC) to the rhizosphere bacterial community was examined using the denaturing gradient gel electrophoresis (DGGE) fingerprinting method. From the visual comparison of the DGGE profiles, there were no significant differences between the profiles of Bt and control rhizosphere in both Suwon and Yesan samples. From the sequence analysis of the individual bands, Sphingomonas sp. of Alphaproteobacteria and several actinobacterial members were identified as the main bacterial taxa in both Suwon and Yesan samples. In the multiple correspondence analysis, no clear separation between Bt and control rhizosphere was seen in both Suwon and Yesan datasets. The profiles of bulk soils were separated from those of rhizosphere. The DGGE fingerprinting analyses indicated that Bt crops did not significantly alter the genetic composition of rhizosphere bacterial communities.     

Bacterial communities in a crude oil gathering and transferring system (China)

Bacterial communities in crude oil and oil field production water samples from an oil gathering and transferring system in Changqing Oil field in China were investigated by 16S rRNA denaturing gradient gel electrophoresis (DGGE) analysis followed by gene cloning and sequencing. DGGE profiles showed that bacterial communities are far more rich in the water samples than that in the crude oil samples, and that bacteria related to Ochrobactrum sp. and Stenotrophomonas sp. were detected in all crude oil and oil field water samples. Bacteria related to Burkholderia sp., Brevundimonas sp., and Propionibacterium sp. were detected in the crude oil samples but not in water samples. Bacteria related to Hippea sp., Acidovorax sp., Arcobacter sp., Pseudomonas sp., Thiomicrospira sp., Brevibacterium sp., Tissierella sp. and Peptostreptococcus sp. were detected in the water samples but not in crude oil samples. Using an archaea-specific primer set, methanogens related to Methanomicrobials and Methanosarcinales were found in water samples but not in crude oil samples. The comparability of the microbial communities in the water and crude oil phase during the period of oil gathering and transferring process was 83.3% and 88.2%, respectively, indicating a stable structure of the microbial communities.