Bacterial diversity in marine hydrocarbon seep sediments

Marine seeps introduce significant amounts of hydrocarbons into oceans and create unusual habitats for microfauna and -flora. In the vicinity of chronic seeps, microbes likely exert control on carbon quality entering the marine food chain and, in turn, hydrocarbons could influence microbial community composition and diversity. To determine the effects of seep oil on marine sediment bacterial communities, we collected sediment piston cores within an active marine hydrocarbon seep zone in the Coal Oil Point Seep Field, at a depth of 22 m in the Santa Barbara Channel, California. Cores were taken adjacent to an active seep vent in a hydrocarbon volcano, on the edge of the volcano, and at the periphery of the area of active seepage. Bacterial community profiles were determined by terminal restriction fragment length polymorphisms (TRFLPs) of 16S ribosomal genes that were polymerase chain reaction (PCR)-amplified with eubacterial primers. Sediment carbon content and C/N ratio increased with oil content. Terminal restriction fragment length polymorphisms suggested that bacterial communities varied both with depth into sediments and with oil concentration. Whereas the apparent abundance of several peaks correlated positively with hydrocarbon content, overall bacterial diversity and richness decreased with increasing sediment hydrocarbon content. Sequence analysis of a clone library generated from sediments collected at the periphery of the seep suggested that oil-sensitive species belong to the gamma Proteobacteria and Holophaga groups. These sequences were closely related to sequences previously recovered from uncontaminated marine sediments. Our results suggest that seep hydrocarbons exert a strong selective pressure on bacterial communities in marine sediments. This selective pressure could, in turn, control the effects of oil on other biota in the vicinity of marine hydrocarbon seeps.     

Yeasts from Marine and Estuarine Waters with Different Levels of Pollution in the State of Rio de Janeiro, Brazil

Yeast counts were made at 24 marine and estuarine sites in the vicinity of Rio de Janeiro, Brazil. Mean salinities of estuarine sites ranged from 14.2 to 27.4‰, and mean temperatures ranged from 25 to 28°C. Total coliform counts varied from 80% above 100,000 colony-forming units (CFU)/100 ml at heavily polluted sites to 100% below 100 CFU/100 ml at unpolluted sites. Total yeast counts above 100 CFU/100 ml were typical of heavily and moderately polluted water but atypical of lightly polluted and unpolluted water. Mean total yeast counts were 2,880 CFU/100 ml for heavily polluted sites, 202 CFU/100 ml for moderately polluted sites, and 3 CFU/100 ml for lightly polluted and unpolluted sites. Total yeast counts had a positive response to increased pollution levels, and Candida krusei and phenotypically similar yeasts as a group were prevalent in polluted estuarine water but rare in unpolluted seawater. The 549 strains of yeasts and yeast-like organisms isolated were grouped into 67 species, of which the 21 most prevalent made up 86% of the total yeast population. The prevalent genera in the polluted estuary were Candida, Rhodotorula, Torulopsis, Hanseniaspora, Debaryomyces, and Trichosporon.     

Biodegradation of petroleum hydrocarbons in estuarine sediments: metal influence

In this work, the potential effect of metals, such as Cd, Cu and Pb, on the biodegradation of petroleum hydrocarbons in estuarine sediments was investigated under laboratory conditions. Sandy and muddy non-vegetated sediments were collected in the Lima River estuary (NW Portugal) and spiked with crude oil and each of the metals. Spiked sediments were left in the dark under constant shaking for 15 days, after which crude oil biodegradation was evaluated. To estimate microbial abundance, total cell counts were obtained by DAPI staining and microbial community structure was characterized by ARISA. Culturable hydrocarbon degraders were determined using a modified most probable number protocol. Total petroleum hydrocarbons concentrations were analysed by Fourier Transform Infrared Spectroscopy after their extraction by sonication, and metal contents were determined by atomic absorption spectrometry. The results obtained showed that microbial communities had the potential to degrade petroleum hydrocarbons, with a maximum of 32 % degradation obtained for sandy sediments. Both crude oil and metals changed the microbial community structure, being the higher effect observed for Cu. Also, among the studied metals, only Cu displayed measurable deleterious effect on the hydrocarbons degradation process, as shown by a decrease in the hydrocarbon degrading microorganisms abundance and in the hydrocarbon degradation rates. Both degradation potential and metal influence varied with sediment characteristics probably due to differences in contaminant bioavailability, a feature that should be taken into account in developing bioremediation strategies for co-contaminated estuarine sites.     

Contaminated marine wounds--the risk of acquiring acute bacterial infection from marine recreational beaches.

An animal model was used to determine the potential for causing wound infections of bacteria isolated from marine recreational beaches in Hong Kong. Water samples were characterized physically, chemically and bacteriologically and used to inoculate artificially-induced wounds in rats. Morbidity and mortality correlated significantly (P < 0.01) with MacConkey plate counts and faecal coliform counts (membrane filtration) and inversely with salinity of the water. The majority of deaths were due to infection caused by marine and estuarine bacteria rather then enteric organisms. A total of 318 bacterial strains was isolated from the wounds and blood of animals inoculated with seawater, of which 242 were marine/estuarine (predominantly Vibrio spp., Aeromonas hydrophila and Pseudomonas putrefaciens) and 40 were enterobacteria. The virulence of the animal strains were comparable with those from clinical sources.     

Contaminated marine wounds—the risk of acquiring acute bacterial infection from marine recreational beaches

An animal model was used to determine the potential for causing wound infections of bacteria isolated from marine recreational beaches in Hong Kong. Water samples were characterized physically, chemically and bacteriologically and used to inoculate artificially-induced wounds in rats. Morbidity and mortality correlated significantly ( P < 0.01) with MacConkey plate counts and faecal coliform counts (membrane filtration) and inversely with salinity of the water. The majority of deaths were due to infection caused by marine and estuarine bacteria rather then enteric organisms. A total of 318 bacterial strains was isolated from the wounds and blood of animals inoculated with seawater, of which 242 were marine/estuarine (predominantly Vibrio spp., Aeromonas hydrophila and Pseudomonas putrefaciens ) and 40 were enterobacteria. The virulence of the animal strains were comparable with those from clinical sources.     

Bacterial Community Response to Petroleum Hydrocarbon Amendments in Freshwater,Marine, and Hypersaline Water-Containing Microcosms

Hydrocarbon-degrading bacterial communities from freshwater, marine, and hypersaline Brazilian aquatic ecosystems (with water salinities corresponding to 0.2%, 4%, and 5%, respectively) were enriched with different hydrocarbons (heptadecane, naphthalene, or crude oil). Changes within the different microcosms of bacterial communities were analyzed using cultivation approaches and molecular methods (DNA and RNA extraction, followed by genetic fingerprinting and analyses of clone libraries based on the 16S rRNA-coding gene). A redundancy analysis (RDA) of the genetic fingerprint data and a principal component analysis (PCA) of the clone libraries revealed hydrocarbon-enriched bacterial communities specific for each ecosystem studied. However, within the same ecosystem, different bacterial communities were selected according to the petroleum hydrocarbon used. In general, the results demonstrated that Acinetobacter and Cloacibacterium were the dominant genera in freshwater microcosms; the Oceanospirillales order and the Marinobacter, Pseudomonas, and Cycloclasticus genera predominated in marine microcosms; and the Oceanospirillales order and the Marinobacter genus were selected in the different hydrocarbon-containing microcosms in hypersaline water. Determination of total petroleum hydrocarbons (TPHs) in all microcosms after 32 days of incubation showed a decrease in the hydrocarbon concentration compared to that for the controls. A total of 50 (41.3%) isolates from the different hydrocarbon-contaminated microcosms were associated with the dominant operational taxonomic units (OTUs) obtained from the clone libraries, and their growth in the hydrocarbon contaminating the microcosm from which they were isolated as the sole carbon source was observed. These data provide insight into the general response of bacterial communities from freshwater, marine, and hypersaline aquatic ecosystems to petroleum hydrocarbon contamination.     

Predominant role of hydrocarbon solubilization in the microbial uptake of hydrocarbons

Using EDTA and proteolytic enzymes to suppress hydrocarbon solubilization, direct evidence is presented in support of the mechanism of liquid hydrocarbon uptake by microbial cells predominantly from the solubilized or accommodated substrate. EDTA (2-5mM) strongly inhibited growth of three yeast species and one bacterial species on n-hexadecane and the inhibition was removed by surfactant-emulsified and surfactant-solubilized alkane and also by excess addition of Ca(2+). EDTA had no inhibitory effect on the growth of the organisms on soluble substrates such as sodium acetate and nutrient broth or on n-pentane, a volatile alkane which was primarily transported by diffusion from gas phase. EDTA was shown to have no significant effect on the adsorption of cells on alkane drops. EDTA inhibition of growth was considered to be due to suppression of alkane solubilization, brought about by the solubilizing factor(s) produced by cells. It was shown that this chelating agent did not inhibit the growth of yeast on solubilized alkane but strongly inhibited its growth on alkane drops. It was demonstrated that adherent capacity of microbial cell to oil phase was closely related to the state of hydrocarbon emulsification and had no relationship to the ability of organisms to grow on hydrocarbon. Certain proteolytic enzymes inhibited the growth of yeast on alkane, presumably by digesting the alkane solubilizing protein, but not on glucose, and the inhibition was removed by a supply of surfactant-emulsified and surfactant-solubilized alkane. Specific solubilization of various hydrocarbon types during growth of the prokaryotic bacterial strain was demonstrated. The specific solubilization of hydrocarbon was strongly inhibited strain was demonstrated. The specific solubilization of hydrocarbon was strongly inhibited by EDTA, and the inhibition was removed by excess Ca(2+). It was concluded that specific solubilization of hydrocarbons is an important mechanism in the microbial uptake of hydrocarbons.     

Composition of humic acid-degrading estuarine and marine bacterial communities

We examined the bacterial decomposition of humic acids (HA) in two flow-through culture experiments, one inoculated by marine and one by estuarine bacterial communities. In both experiments, the cultures were fed with HA media of salinities of 28 and 14, close to their ambient and a distinctly different, foreign salinity. HA were decomposed to >?60% of the initial concentration within 70?days, and the foreign salinity yielded the highest decomposition. A detrended correspondence analysis of denaturing gradient gel electrophoresis (DGGE) banding patterns showed that during incubation, the bacterial community composition underwent distinct changes. A phylogenetic analysis of DGGE bands excised and bacteria isolated at the end on HA as the sole carbon source showed that Alphaproteobacteria and Gammaproteobacteria largely dominated the communities in the marine flow-through cultures, whereas Gammaproteobacteria, Actinobacteria and Alphaproteobacteria dominated the estuarine communities. Eleven of 13 isolates obtained from both experiments were able to grow on HA as the sole carbon source, seven on phenol and three, affiliated to the Roseobacter clade, on various aromatic acids. The bacteria retrieved from the flow-through cultures were closely (96-99%) affiliated to organisms capable of degrading humic matter, aromatic and aliphatic compounds and also to other bacteria reported previously from the Wadden Sea and Weser estuary.     

Metabolic and phylogenetic analysis of microbial communities during phytoremediation of soil contaminated with weathered hydrocarbons and heavy metals

In the current study, the microbial ecology of weathered hydrocarbon and heavy metal contaminated soil undergoing phytoremediation was studied. The relationship of functional diversity, measured as carbon source utilisation in Biolog plates and extracellular enzymatic activities, and genetic diversity of bacteria was evaluated. Denaturing gradient gel electrophoresis was used for community analyses at the species level. Bulk soil and rhizosphere soil from pine and poplar plantations were analysed separately to determine if the plant rhizosphere impacted hydrocarbon degradation. Prevailing microbial communities in the field site were both genetically and metabolically diverse. Furthermore, both tree rhizosphere and fertilisation affected the compositions of these communities and increased activities of extracellular aminopeptidases. In addition, the abundance of alkane hydroxylase and naphthalene dioxygenase genes in the communities was low, but the prevalence of these genes was increased by the addition of bioavailable hydrocarbons. Tree rhizosphere communities had greater hydrocarbon degradation potential than those of bulk soil. Hydrocarbon utilising communities were dominated generally by the species Ralstonia eutropha and bacteria belonging to the genus Burkholderia. Despite the presence of viable hydrocarbon-degrading microbiota, decomposition of hydrocarbons from weathered hydrocarbon contaminated soil over four years, regardless of the presence of vegetation, was low in unfertilised soil. Compost addition enhanced the removal of hydrocarbons.     

Influence of spatial and temporal variations on organic pollutant biodegradation rates in an estuarine environment.

The influence of spatial and temporal environmental variations on rates of organic pollutant biodegradation were assessed by using heterotrophic uptake kinetics. These studies were conducted at three sites, representing the gradient from freshwater to estuarine to marine systems. Of the compounds tested, total uptake Vmax rates decreased in the order of nitrilotriacetic acid, m-cresol, chlorobenzene, and 1,2,4-trichlorobenzene. In general, the freshwater site exhibited the highest uptake rates, with somewhat lower rates at the estuarine site. Rates at the marine site were much lower than at the other sites, except during the winter. Metabolic rates at both the freshwater and estuarine areas were significantly decreased during periods of low water temperature. Rates at the marine site were relatively uniform throughout the year. Linear regression analysis was used to compare m-cresol biodegradation rates to characteristics of the microbial community, which included direct microscopic counts, CFU counts, and cellular incorporation of amino acids. The observed rates did not consistently correlate well with any of the measured characteristics of the microbial community.     

Isolation of Non-O1 Vibrio cholerae Serovars from Oregon Coastal Environments

Water, sediment, and shellfish from three Oregon estuaries were cultured for pathogenic Vibrio species. Non-O1 serovars of V. cholerae were the most common pathogenic Vibrio species recovered. Non-O1 V. cholerae were isolated from all three estuaries sampled, covering an area of about 170 miles along the Oregon coast. Non-O1 V. cholerae were isolated from water and sediment, but not shellfish, at temperatures ranging from 11 to 19°C and salinities of 2.3 to 26‰. Sixteen isolates representing 12 different non-O1 serovars were identified, while four non-O1 V. cholerae isolates failed to react with any of the 54 antisera tested. These results indicate that non-O1 V. cholerae serovars can be found over a large geographic area and under a variety of environmental conditions. These organisms are apparently an autochthonous component of these estuarine microbial communities.     

Microbiological Survey of Adirondack Lakes with Various pH Values

Nine high-altitude oligotrophic Adirondack lakes in upstate New York having water of pH 4.3 to 7.0 were surveyed for total bacterial numbers and possible adaptation of the microbial communities to environmental pH. The number of heterotrophic bacteria from water samples recoverable on standard plate count agar were low (10¹ to 10³ per ml) for most of the lakes. Acridine orange direct counts were approximately two orders of magnitude higher than plate counts for each lake. Sediment aerobic heterotrophs recovered on standard plate count agar ranged from 1.4 × 10⁴ to 1.3 × 10⁶ per g of sediment. Direct epifluorescence counts of bacteria in sediment samples ranged from 3.0 × 10⁶ to 1.4 × 10⁷ per g. Low density values were consistent with the oligotrophic nature of all the lakes surveyed. There were no apparent differences in numbers of bacteria originally isolated at pH 5.0 and pH 7.0 between circumneutral lakes (pH > 6.0) and acidic lakes (pH < 5.0). Approximately 1,200 isolates were recultured over a range of pH from 3.0 to 7.0. Regardless of the original isolation pH (pH 5.0 or pH 7.0), less than 10% of the isolates grew at pH < 5.0. Those originally isolated at pH 5.0 also grew at pH 6.0 and 7.0. Those originally isolated at pH 7.0 preferred pH 7.0, with 98% able to grow at pH 6.0 and 44% able to grow at pH 5.0. A chi-square contingency test clearly showed (P < 0.005) that two distinct heterotrophic populations had been originally isolated at pH 5.0 and pH 7.0, although there is undoubtedly some overlap between the two populations.     

Bionomy of Macrobenthic Molluscs in Evros Delta (North Aegean Sea)

The composition and distribution of the macrobenthic molluscs in Evros Delta (North Aegean Sea) were investigated by quantitative sampling. The molluscan distribution shows a zonation mainly related to the degree of “confinement”. Based on similarity in distribution among stations, 2 groups of species were distinguished. One group includes marine species (Tellina tenuis, Bittium reticulatum) found only in coastal stations and the second group is typical of estuarine species (Hydrobia acuta, Abra ovata and Cerastoderma glaucum) distributed in almost all stations. Spatial fluctuations of the 3 estuarine species reflect their preferences in the various parameters (“confinement”, salinity, grain size and organic matter of sediment). The most isolated stations support the highest values of total mollusc abundance and biomass.     

Distribution of thermophilic endospores in a temperate estuary indicate that dispersal history structures sediment microbial communities

Endospores of thermophilic bacteria are found in cold and temperate sediments where they persist in a dormant state. As inactive endospores that cannot grow at the low ambient temperatures, they are akin to tracer particles in cold sediments, unaffected by factors normally governing microbial biogeography (e.g., selection, drift, mutation). This makes thermophilic endospores ideal model organisms for studying microbial biogeography since their spatial distribution can be directly related to their dispersal history. To assess dispersal histories of estuarine bacteria, thermophilic endospores were enriched from sediments along a freshwater‐to‐marine transect of the River Tyne in high temperature incubations (50°C). Dispersal histories for 75 different taxa indicated that the majority of estuarine endospores were of terrestrial origin; most closely related to bacteria from warm habitats associated with industrial activity. A subset of the taxa detected were marine derived, with close relatives from hot deep marine biosphere habitats. These patterns are consistent with the sources of sediment in the River Tyne being predominantly terrestrial in origin. The results point to microbial communities in estuarine and marine sediments being structured by bi‐directional currents, terrestrial run‐off and industrial effluent as vectors of passive dispersal and immigration.     

Variability of some microbial parameters in relation to the hydrological features of the water column in the Eolian Basin (Southern Tyrrhenian Sea).

During the EOCUMM '94 cruise, 15 stations located in the Eolian Islands area (Southern Tyrrhenian Sea) were sampled to analyse the distributions of the total bacterioplankton densities and the heterotrophic viable bacteria counts on Marine Agar 2216. According to the TS (temperature-salinity) diagrams, obtained by processing the CTD (conductivity-temperature-depth) vertical profiles, the sampled stations were grouped in "hydrological clusters". The bacteriological variables, together with the chlorophyll a and the particulate organic carbon measures obtained during the same cruise were used to compare the stations of the same and different clusters. The results indicated that variabilities of the analysed microbial parameters were not obviously related to the hydrographic features of the sampling stations. This work is an attempt to verify the possibility of using microbial parameters to characterize the structure of the water column.     

Comparison of microbial numbers in soils by using various culture media and temperatures

The influence of different media and incubation temperatures on the quantification of microbial populations in sorghum, eucalyptus and forest soils was evaluated. Microbial growth was compared by using complex (tryptone soybean agar, TSA, casein-starch, CS, and Martin) and saline (Thorton, M3, Czapeck) media and incubation temperatures of 25 and 30 degrees C. Higher numbers of total bacterial and fungal colony-forming units (CFU) were observed in sorghum soils, and of spore-forming and Gram-negative bacteria in forest soils than other soils. Actinomycetes counts were highest in forest soil when using CS medium at 30 degrees C and in sorghum soil at 25 degrees C in M3 medium. Microorganism counts were dependent on the media and incubation temperatures. The counts at temperatures of 30 degrees C were significantly higher than at 25 degrees C. Microbial quantification was best when using TSA medium for total and spore-forming bacteria, Thorton for Gram-negative bacteria, M3 for actinomycetes, and Martin for fungi.     

Culturability as an Indicator of Succession in Microbial Communities

Successional theory predicts that opportunistic species with high investment of energy in reproduction and wide niche width will be replaced by equilibrium species with relatively higher investment of energy in maintenance and narrower niche width as communities develop. Since the ability to rapidly grow into a detectable colony on nonselective agar medium could be considered as characteristic of opportunistic types of bacteria, the percentage of culturable cells may be an indicator of successional state in microbial communities. The ratios of culturable cells (colony forming units on R2A agar) to total cells (acridine orange direct microscopic counts) and culturable cells to active cells (reduction of 5-cyano-2,3-ditolyl tetrazolium chloride) were measured over time in two types of laboratory microcosms (the rhizosphere of hydroponically grown wheat and aerobic, continuously stirred tank reactors containing plant biomass) to determine the effectiveness of culturabilty as an index of successional state. The culturable cell:total cell ratio in the rhizosphere decreased from approximately 0.25 to less than 0.05 during the first 30-50 days of plant growth, and from 0.65 to 0.14 during the first 7 days of operation of the bioreactor. The culturable cell:active cell ratio followed similar trends, but the values were consistently greater than the culturable cell:total cell ratio, and even exceeded I in early samples. Follow-up studies used a cultivation-independent method, terminal restriction fragment length polymorphisms (TRFLP) from whole community DNA, to assess community structure. The number of TRFLP peaks increased with time, while the number of culturable types did not, indicating that the general decrease in culturability is associated with a shift in community structure. The ratio of respired to assimilated C-14-labeled amino acids increased with the age of rhizosphere communities, supporting the hypothesis that a shift in resource allocation from growth to maintenance occurs with time. Results from this work indicate that the percentage of culturable cells may be a useful method for assessing the successional state of microbial communities.     

Occurrence and Growth of Yeasts in Yogurts

Yogurts purchased from retail outlets were examined for the presence of yeasts by being plated onto oxytetracycline malt extract agar. Of the 128 samples examined, 45% exhibited yeast counts above 10³ cells per g. A total of 73 yeast strains were isolated and identified as belonging to the genera Torulopsis, Kluyveromyces, Saccharomyces, Candida, Rhodotorula, Pichia, Debaryomyces, and Sporobolomyces. Torulopsis candida and Kluyveromyces fragilis were the most frequently isolated species, followed by Saccharomyces cerevisiae, Rhodotorula rubra, Kluyveromyces lactis, and Torulopsis versatilis. The growth of yeasts in yogurts was related to the ability of the yeasts to grow at refrigeration temperatures, to ferment lactose and sucrose, and to hydrolyze milk casein. Most yeast isolates grew in the presence of 100 μg of sorbate and benzoate preservatives per ml. Higher yeast counts from yogurts were obtained when the yogurts were plated onto oxytetracycline malt extract agar than when they were plated onto acidified malt extract agar.     

微生物降解石油烃的功能基因研究进展

微生物对石油烃的降解在自然衰减去除土壤和地下水石油烃污染的过程中发挥了重要作用。微生物通过其产生的一系列酶来利用和降解这类有机污染物,其中,编码关键降解酶的基因称为功能基因。功能基因可作为生物标志物用于分析环境中石油烃降解基因的多样性。因此,研究石油降解功能基因是分析土著微生物群落多样性、评价自然衰减潜力与构建基因工程菌的重要基础。本文主要介绍了烷烃和芳香烃在有氧和无氧条件下的微生物降解途径,重点总结了烷烃和芳香烃降解的主要功能基因及其作用,包括参与羟化作用的单加氧酶和双加氧酶基因、延胡索酸加成反应的琥珀酸合酶基因以及中心中间产物的降解酶基因等。     

长江口中华鲟自然保护区潮间带鱼类群落结构

为深入了解鱼类对潮间带水域的利用,2010年5月、8月、11月在长江口中华鲟自然保护区潮间带水域进行了插网调查。结果表明:调查期间共出现28种鱼类,隶属于10目16科,鲤形目种类最多,其次为鲈形目;群落优势种为中国花鲈(Lateolabrax maculates)、鮻(Liza haematocheila)和窄体舌鳎(Cynoglossus gracilis);长江口中华鲟自然保护区潮间带鱼类群落可以分为河口定居鱼类、海洋偶见鱼类、降海洄游鱼类、溯河洄游鱼类、海洋洄游鱼类和淡水鱼类6种生态类群,其中河口定居鱼类和海洋洄游鱼类占据明显优势。对调查水域鱼类群落进行聚类分析,结果表明秋季和春夏季差异显著,鮻、中国花鲈、斑尾刺虾虎鱼(Acanthogobius ommaturus)是对季节差异贡献最大的3种鱼类。主成分分析结果表明,夏季鱼类群落主要受到海洋洄游鱼类的影响,而秋季主要受到河口定居鱼类的影响,这2种鱼类的幼鱼在不同季节利用潮间带水域索饵肥育可能是造成季节差异的主要原因。