Comparative analyses of viable bacterial counts in foods and seawater under microplate based liquid- and conventional agar plate cultivation: increased culturability of marine bacteria under liquid cultivation

Bacterial counts under liquid cultivation using 96-well microplates were performed. The counts under liquid and under solid cultivation were equivalent in foods, although the counts under liquid cultivation exceeded those under solid cultivation in seawater, suggesting that some bacteria in seawater were viable but did not form detectable colonies. Phylogenetic analysis of bacteria obtained under liquid cultivation was also performed.     

Diversity and structure of bacterial communities in Fildes Peninsula, King George Island

To examine the bacterial community structure in the Fildes Peninsula, King George Island, Antarctica, we examined the bacterial diversity and community composition of samples collected from lacustrine sediment, marine sediment, penguin ornithogenic sediments, and soils using culture-dependent and culture-independent methods. The 70 strains fell into five groups: Actinobacteria, Bacteroidetes, Firmicutes, Gammaproteobacteria, and Betaproteobacteria. Bacterial diversity at the phylum level detected in Denaturing Gradient Gel Electrophoresis (DGGE) profiles comprised Proteobacteria (including the subphyla Alpha-, Beta-, Gamma-, Deltaproteobacteria), Bacteroidetes, Firmicutes, Chlorobi, and Deinococcus-Thermus. Gammaproteobacteria was identified to be the dominant bacterial subphylum by cultivation and DGGE method. By cluster analysis, the overall structure and composition of bacterial communities in the soil and lacustrine sediment were similar to one another but significantly different from bacterial communities in penguin ornithogenic sediment and marine sediment, which were similar to one another. The majority of 16S rDNA sequences from cultured bacteria were closely related to sequences found in cold environments. In contrast, a minority of 16S rDNA sequences from the DGGE approach were closely related to sequences found in cold environments.     

Multiplex bacterial growth monitoring in 24-well microplates using a dual optical sensor for dissolved oxygen and pH

Non-invasive, simultaneous optical monitoring of oxygen and pH during bacterial cultivation in 24-well microplates is presented using an integrated dual sensor for dissolved oxygen and pH values. The dual sensor is based on oxygen-sensitive organosilica microparticles and pH-sensitive microbeads from a polymethacrylate derivative embedded into a polyurethane hydrogel. The readout is based on a phase-domain fluorescence lifetime-based method referred to as modified frequency domain dual lifetime referencing using a commercially available detector system for 24-well microplates. The sensor was used for monitoring the growth of Pseudomonas putida bacterial cultures. The method is suitable for parallelized, miniaturized bioprocessing, and cell-based high-throughput screening applications.     

Measuring beta-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays.

We describe a high-throughput procedure for measuring beta-galactosidase activity in bacteria. This procedure is unique because all manipulations, including bacterial growth and cell permeabilization, are performed in a 96-well format. Cells are permeabilized by chloroform/SDS treatment directly in the 96-well blocks and then transferred to 96-well microplates for standard colorimetric assay of beta-galactosidase activity as described by Miller [J. H. Miller (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Absorbance data are collected with a microplate reader and analyzed using a Microsoft Excel spreadsheet. The beta-galactosidase specific activity values obtained with the high-throughput procedure are identical to those obtained by the traditional single-tube method of Miller. Thus, values obtained with this procedure may be expressed as Miller units and compared directly to Miller units reported in the literature. The 96-well format for permeabilization and assay of enzyme specific activity together with the use of 12-channel and repeater pipettors enables efficient processing of hundreds of samples in an 8-h day.     

Predominant growth of Alcanivorax during experiments on "oil spill bioremediation" in mesocosms

Mesocosm experiments were performed to study the changes on bacterial community composition following oil spill in marine environment. The analysis of 16S crDNA revealed a shift in the structure of initial bacterial population that was drastically different from that one measured after 15 days. The results showed that, after 15 days, bacteria closely related to the genus Alcanivorax became the dominant group of bacterial community in petroleum-contaminated sea water nitrogen and phosphorus amended. This suggested that these bacteria played the most important role in the process of bioremediation of oil-contaminated marine environments.     

Viability, diversity and composition of the bacterial community in a high Arctic permafrost soil from Spitsbergen, Northern Norway

The viable and non-viable fractions of the bacterial community in a 2347-year-old permafrost soil from Spitsbergen were subjected to a comprehensive investigation using culture-independent and culture-dependent methods. LIVE/DEAD BacLight staining revealed that 26% of the total number of bacterial cells were viable. Quantitatively, aerobic microcolonies, aerobic colony-forming units and culturable anaerobic bacteria comprised a minor fraction of the total number of viable bacteria, which underlines the necessity for alternative cultivation approaches in bacterial cryobiology. Sulfate reduction was detected at temperatures between -2 degrees C and 29 degrees C while methanogenesis was not detected. Bacterial diversity was high with 162 operational taxonomic units observed from 800 16S rDNA clone sequences. The 158 pure cultures isolated from the permafrost soil affiliated with 29 different bacterial genera, the majority of which have not previously been isolated from permafrost habitats. Most of the strains isolated were affiliated to the genera Cellulomonas and Arthrobacter and several of the pure cultures were closely related to bacteria reported from other cryohabitats. Characterization of viable bacterial communities in permafrost soils is important as it will enable identification of functionally important groups together with the as yet undescribed adaptations that bacteria have evolved for surviving subzero temperatures for millennia.     

Community level physiological study of algicidal bacteria in the phycospheres of Skeletonema costatum and Scrippsiella trochoidea

Bacteria in the phycosphere have a unique ecological relationship with host algae due to their utilization of algal extracellular products as nutrients. Some bacteria control the growth of algal cells and even lyse them. The diversity of bacteria and their community dynamics in the phycosphere of microalgae are still relatively little understood, especially of those associated with red tide-causing algae. In this study, scanning electron microscope (SEM) images of algal cell morphology revealed that the phycosphere bacteria of the red tide-causing algae, Skeletonema costatum and Scrippsiella trochoidea, could lyse them within 72 h. The community level physiology of the algicidal bacteria was studied using Biolog ECO microplates, a common method for the ecological study of microbial communities. The average well color development (AWCD) values of bacteria in the phycospheres of both species were low, indicating that the bacteria had low metabolic activity overall. The diversity indices were both lower than the bacterial diversity from natural environments. However, the bacteria associated with S. trochoidea demonstrated a higher AWCD value and diversity than those in the phycosphere of S. costatum. The utilization of carbon sources significantly changed at different lytic times, reflecting that the bacterial community structure changed during the algae-lysing process. These results revealed that the bacterial communities in phycospheres had a simple structure and low diversity. When the balance between algae and bacteria broke down, the total bacterial density increased while the algicidal bacteria accumulated and became the dominant species, changing the bacterial community structure in this micro-ecosystem.     

利用PCR-DGGE未培养技术对中国白酒高温和中温大曲细菌群落结构的分析

采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术,研究了5种高温和中温白酒大曲细菌群落结构,通过优势条带切胶鉴定确定了大曲中优势细菌种属信息。结果表明,Weissella cibaria、Lactobacillus helveticus、L.fermentum、L.panis等乳酸菌普遍存在于5种大曲中,Ther-moactinomyces sanguinis仅存在于高温曲酱曲中,同时DGGE检测到了传统方法未能分离鉴定的Staphylococcus xylosus、Klebsiella oxytoca。不同工艺大曲细菌群落结构存在明显差异,随着制曲温度的升高,大曲细菌多样性指数有下降趋势。PCR-DGGE技术是一种能够快速有效地研究白酒大曲细菌群落结构的技术。     

The Hoopoe's Uropygial Gland Hosts a Bacterial Community Influenced by the Living Conditions of the Bird

Molecular methods have revealed that symbiotic systems involving bacteria are mostly based on whole bacterial communities. Bacterial diversity in hoopoe uropygial gland secretion is known to be mainly composed of certain strains of enterococci, but this conclusion is based solely on culture-dependent techniques. This study, by using culture-independent techniques (based on the 16S rDNA and the ribosomal intergenic spacer region) shows that the bacterial community in the uropygial gland secretion is more complex than previously thought and its composition is affected by the living conditions of the bird. Besides the known enterococci, the uropygial gland hosts other facultative anaerobic species and several obligated anaerobic species (mostly clostridia). The bacterial assemblage of this community was largely invariable among study individuals, although differences were detected between captive and wild female hoopoes, with some strains showing significantly higher prevalence in wild birds. These results alter previous views on the hoopoe-bacteria symbiosis and open a new window to further explore this system, delving into the possible sources of symbiotic bacteria (e.g. nest environments, digestive tract, winter quarters) or the possible functions of different bacterial groups in different contexts of parasitism or predation of their hoopoe host.     

舟山群岛不同功能区划海域细菌群落结构分析

浮游细菌在海洋生态系统中不可或缺,在海洋生物地球化学循环过程中起着关键性作用。【目的】为了解舟山群岛不同功能区划海域细菌群落结构及丰度变化,探索海洋生态因子对细菌群落结构的影响。【方法】于2016年夏季(8月)在舟山群岛不同功能区划海域共设置8个典型站位采集表层海水,基于细菌16S rRNA基因进行高通量测序;利用流式细胞术揭示各海域细菌丰度;利用典范对应分析(Canonical correspondence analysis,CCA)探讨海洋生态因子与细菌多样性之间的关系。【结果】共获取到305487条原始序列,基于97%相似性水平进行OTU(Operational Taxonomic Units)聚类分析,共得到1088个OTUs,包括29个门、62个纲、138个目、239个科、416个属。细菌群落组成在各个站位之间不尽相同,但都主要包括Flavobacteria、Alphaproteobacteria、Gammaproteobacteria三大优势菌纲。CCA结果表明细菌群落结构和多样性情况与站位分布和所在站位的环境因子息息相关,Cyanobacteria受硝酸盐影响显著,Parcubacteria受温度影响最大,而磷酸盐对本实验海域菌群影响甚微。对海洋菌群潜在功能进行预测的结果显示,各海域菌群在氨基酸代谢、碳水化合物代谢、膜运输等方面功能较为突出,为今后舟山海洋微生物研究提供了新的方向。【结论】高通量测序分析可以更精确地揭示海洋菌群的群落结构信息。该研究为细菌群落结构与环境因素的关联提供参考。本研究所取得的大量数据既可以作为对舟山市海洋功能区划施行情况的响应,又将为舟山及其邻近海域浮游细菌群落结构的进一步研究奠定基础。     

Monitoring the bacterial community during fermentation of sunki, an unsalted, fermented vegetable traditional to the Kiso area of Japan

Aims:  To investigate the microbial community in sunki , an indigenous, unsalted, fermented vegetable, made from the leaves of red beet.
Methods and Results:  Fermenting samples were collected at 1- to 2-day intervals from four houses and investigated by culture-dependent and culture-independent techniques. PCR-Denaturing-Gradient-Gel-Electrophoresis profiles indicated that the bacterial community was stable and Lactobacillus delbrueckii , Lact. fermentum and Lact. plantarum were dominant during the fermentation. This result agreed well with that obtained by the culturing technique. Moulds, yeasts or bacteria other than lactic acid bacteria (LAB) were not detected.
Conclusions:  The bacterial community was stable throughout the fermentation, and Lact. delbrueckii , Lact. fermentum and Lact. plantarum were dominant. The acidic pH and lactic acid produced by LAB probably preserve the sunki from spoilage.
Significance and Impact of the Study:  This is the first report on the use of both culture-dependent and culture-independent techniques to study the bacterial community in sunki . A combination of culture-dependent and culture-independent techniques is necessary for the analysis of complex microbial communities.     

马里亚纳海沟共培养细菌多样性动态变化及潜在微生物互作关系

马里亚纳海沟是世界已知最深的海沟,其寡营养、高压、低温、低氧等极端的深海环境孕育出独特的细菌群落结构及多样性特征。选取寡营养培养基对马里亚纳海沟海水及表层沉积物分别进行液体共培养,并在不同培养阶段取样进行高通量测序,分析细菌群落结构组成及其多样性的动态变化,探讨微生物之间可能的互作关系。研究结果表明：液体共培养样品中一共检测到19个门、34个纲、76个目、131个科、227个属的细菌,其中变形菌门（Proteobacteria）和拟杆菌门（Bacteroidetes）为优势菌群,其次为厚壁菌门（Firmicutes）;与其他样品相比,1000米海水样品中细菌群落的多样性最高,并且蓝细菌门（Cyanobacteria）具有更高的相对丰度。共培养样品中细菌丰富度、多样性、群落结构均随培养时间而改变,其中共培养中期样品的细菌多样性较高;表层沉积物样本中,盐单胞菌属（Halomonas）可能由于较强的竞争能力在共培养后期占据优势地位。基因功能预测与代谢通路富集结果显示,随着共培养时间的增加,微生物生长相关的代谢通路丰度明显下降,而与互作相关的代谢通路丰度明显增加。共培养样品检测到的细菌多样性远高于单独分离培养的多样性,仅有少量菌属为单独分离培养与共培养样品均检测到的共有属。综上所述,马里亚纳海沟细菌群落中存在竞争、互利共生的相互作用,共培养法有利于揭示细菌间的互作关系。研究为深渊及深海等极端环境下微生物生态系统组成及维持奠定了理论基础,也为进一步研究极端微生物的生存策略提供了科学指导。     

Bacterial diversity in organically-enriched fish farm sediments

The bacterial diversity and community structure within both organically enriched and adjacent, unimpacted, near-shore marine sediments at two fish farms in southern Tasmania, Australia, was examined using 16S rRNA gene clone library construction and analysis. Sediments at both caged and reference sites at both farms showed a very high level of microbial diversity. Over 900 clones were analysed and grouped into 631 unique phylotypes. Reference sites were dominated by Delta- and Gammaproteobacteria and the Cytophaga-Flavobacteria-Bacteroides group. Cage site sediments were also dominated by these phylotypes, as well as members of the Alpha- and Epsilonproteobacteria. Diversity and coverage indices indicated that the actual diversity of the sediments was much greater than that detected, despite a large sampling effort. All libraries were shown to be statistically different from one another (P < 0.05). Many phylotypes did not group with cultured bacteria, but grouped with other environmental clones from a wide array of marine benthic environments. Diversity and evenness indices suggested that although both parameters changed after farming, diverse communities were present in all sediments. The response of the microbial community to organic load suggested that random, rather than predictable, succession events determine community composition and diversity, and that sediment type may influence bacterial community and sediment response to organic perturbation.     

Biodiversity of polycyclic aromatic hydrocarbon-degrading bacteria from deep sea sediments of the Middle Atlantic Ridge

The bacteria involved in the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in deep sea subsurface environments are largely unknown. In order to reveal their biodiversity, sediments from 2.2 m under the bottom surface at a water depth of 3542 m were sampled on the Middle Atlantic Ridge with a gravity column sampler. The sediments were promptly enriched with either crude oil or a mixture of PAHs (naphthalene, phenanthrene and pyrene) as the sole carbon source, and further enriched with the PAH mixture mentioned above in the lab. The resulting consortia were named C2CO and C2PPN respectively. Their bacterial composition was analysed with plate cultivation, PCR-DGGE and 16S rDNA library analysis. On plates, isolates belonging to Pseudoalteromonas , Halomonas , Marinobacter , Thalassospira and Tistrella dominated the culturable populations. With PCR-DGGE, five major bands closely related to Cycloclasticus , Alteromonas , Thalassospira , Alcanivorax and Rhodospirillaceae were detected in consortium C2CO, while only one major band of Cycloclasticus was detected in consortium C2PPN. In addition, the dynamics of community structure in response to aromatic substrate alterations were examined. As a result, three ribotypes of Cycloclasticus were detected by 16S rDNA library analysis, one which played a key role in phenanthrene degradation; two Alteromonas bacteria dominated the naphthalene reselected consortium. Although bacteria of the two genera grew as the main members of the communities, none of them were isolated, probably owing to their poor cultivability. These results confirm that bacteria of Cycloclasticus are important obligate PAH degraders in marine environments, and coexist with other degrading bacteria that inhabit the deep subsurface sediment of the Atlantic. This supports the view that PAH accumulation and bioattenuation occur in remote areas consistently and continuously.     

Seawater Inundation from the 2011 Tohoku Tsunami Continues to Strongly Affect Soil Bacterial Communities 1 Year Later

The effects of inundation caused by the 2011 Tohoku tsunami on soil bacterial communities in agricultural fields were evaluated. Bacterial communities were compared across three different types of soil, unflooded field (UF) soil, soil flooded for 2 weeks (short term (ST)), and soil flooded for 2 months (long term (LT)), using polymerase chain reaction-pyrosequencing of 16S rRNA genes. Acidobacteria were dominant in UF, with a relative abundance of approximately 35 %, and Proteobacteria dominated flooded soils (30–67 %). Hierarchical cluster analysis indicated that the community structure of soil bacteria in flooded soils (ST and LT) clearly differed from that in UF. Differences between LT and ST fields were rarely observed in terms of chemical properties and microbial community structure at the phylum level. However, sulfur-oxidizing bacteria (SOB) and nitrite-oxidizing bacteria (NOB) in LT tended to occur at high and low abundances, respectively. Halothiobacillus, a halotolerant SOB, was detected in all LT fields. Unexpectedly, a zeta-Proteobacteria, which had previously only been detected in marine environments, was detected in LT fields only. Our results demonstrate that the effects of the 2011 Tohoku tsunami on soil bacterial communities in agricultural fields may have lasted at least 1 year. Furthermore, SOB, NOB, and zeta-Proteobacteria may serve as indicators of the effects of seawater inundation on microorganisms.     

Heterogeneity of surface attached microbial communities from Sydney Harbour, Australia

There is limited information on bacterial communities attached to marine surfaces. These surface attached bacterial communities can vary at a micro scale and these differences may be due to surface characteristics in marine environments. The current study investigates the heterogeneity of bacterial communities on five different marine invertebrates (Heliocidaris erythrogramma, Austrocochlea concamerata, Crassostrea gigas, Dendrilla rosea, and Actinia tenebrosa), the alga, Lobophora variegata and marine gravel from a 20 m × 20 m quadrant in Camp Cove, Sydney Harbour, Australia. Terminal restriction fragment length polymorphism (TRFLP) of 16S sequences showed that each surface contained unique combinations of TRFLP fragment lengths. Phylogenetic analysis of random clones picked from clone libraries constructed from the amplified 16S sequences revealed that 16S sequences from the communities on different surfaces clustered into distinct clades. None of the bacteria identified by 16S sequencing of the whole (uncultured) microbial communities was detected after cultivation. Overall, the study shows surface type plays a major role in shaping microbial communities in marine environments.     

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.     

Diversity of the bacterial community in the rice rhizosphere managed under conventional and no-tillage practices

Bacterial diversity in the rice rhizosphere at different rice growth stages, managed under conventional and no-tillage practices, was explored using a culture-based approach. Actinobacteria are among the bacterial phyla abundant in the rice rhizosphere. Their diversity was further examined by constructing metagenomic libraries based on the 16S rRNA gene, using actinobacterial- and streptomycete-specific polymerase chain reaction (PCR) primers. The study included 132 culturable strains and 125 clones from the 16S rRNA gene libraries. In conventional tillage, there were 38% Proteobacteria, 22% Actinobacteria, 33% Firmicutes, 5% Bacteroidetes, and 2% Acidobacteria, whereas with no-tillage management there were 63% Proteobacteria, 24% Actinobacteria, 6% Firmicutes, and 8% Bacteroidetes as estimated using the culture-dependent method during the four stages of rice cultivation. Principal coordinates analysis was used to cluster the bacterial communities along axes of maximal variance. The different growth stages of rice appeared to influence the rhizosphere bacterial profile for both cultivation practices. Novel clones with low similarities (89–97%) to Actinobacteria and Streptomyces were retrieved from both rice fields by screening the 16S rRNA gene libraries using actinobacterial- and streptomycete-specific primers. By comparing the actinobacterial community retrieved by culture-dependent and molecular methods, it was clear that a more comprehensive assessment of microbial diversity in the rice rhizosphere can be obtained using a combination of both techniques than by using either method alone. We also succeeded in culturing a number of bacteria that were previously described as unculturable. These were in a phylogenetically deep lineage when compared with related cultivable genera.     

A marine bacterial adhesion microplate test using the DAPI fluorescent dye: a new method to screen antifouling agents

AIMS: To develop a method to screen antifouling agents against marine bacterial adhesion as a sensitive, rapid and quantitative microplate fluorescent test. METHODS AND RESULTS: Our experimental method is based on a natural biofilm formed by mono-incubation of the marine bacterium Pseudoalteromonas sp. D41 in sterile natural sea water in a 96-well polystyrene microplate. The 4'6-diamidino-2-phenylindole dye was used to quantify adhered bacteria in each well. The total measured fluorescence in the wells was correlated with the amount of bacteria showing a detection limit of one bacterium per 5 microm(2) and quantifying 2 x 10(7) to 2 x 10(8) bacteria adhered per cm(2). The antifouling properties of three commercial surface-active agents and chlorine were tested by this method in the prevention of adhesion and also in the detachment of already adhered bacteria. The marine bacterial adhesion inhibition rate depending on the agent concentration showed a sigmoid shaped dose-response curve. CONCLUSIONS: This test is well adapted for a rapid and quantitative first screening of antifouling agents directly in seawater in the early steps of marine biofilm formation. Significance AND IMPACT OF THE STUDY: In contrast to the usual screenings of antifouling products which detect a bactericidal activity, this test is more appropriate to screen antifouling agents for bacterial adhesion removal or bacterial adhesion inhibition activities. This screening test focuses on the antifouling properties of the products, especially the initial steps of marine biofilm formation.     

Quantitative aspects of anin situ hybridization procedure for detecting mRNAs in cells using 96-well microplates

The universal quantitation of the DNA hybridization reaction has been a goal sought by many researchers. Part of this search has been the need to develop a rapid, sensitive, easy-to-perform, and quantitative method to measure the abundance of specific mRNAs directly within cells. Conventionally mRNA detection can be done by advanced quantitativein situ hybridization (ISH) using either image analysis or fluorescencein situ hybridization (FISH), or indirectly by extraction of mRNA from cells or tissue and using Northern blot or quantitative polymerase chain reaction (PCR). We examined the quantitative nature of probe binding to intracellular mRNA in a sensitive and easy-to-use nonisotopic method of ISH previously developed in our laboratories. The method is applicable to isolated primary cells or cells in culture. The procedural details are very simple, with cells being centrifuged into 96-well microplates, fixed with formalin, and pretreated with Triton X-100 and Nonidet P-40 before photobiotin-labeled cDNA probes are applied. Biotin from the hybridization of probe to target is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and visualized by thep-nitrophenyl phosphate conversion method. The quantitative parameters of the ISH procedure were determined by measuring the levels of expression of erythropoietin (EPO) mRNA and its translated protein in transfected COS-7 cells. There is a log-linear relationship between the levels of signal obtained in the ISH reaction in 96-well microplates and the EPO protein levels measured by enzyme-linked immunosorbent assay (ELISA). This demonstrated relationship is important in the standardization and use of these procedures to measure quantitatively mRNAs within cells.