石油污染土壤自然衰减过程多环芳烃降解基因动态特征

新疆石油污染土壤中微生物多环芳烃(polycyclic aromatic hydrocarbons,PAHs)降解功能基因研究甚少,且环境因子和功能基因之间相关性仍不清楚。【目的】揭示新疆石油污染砂质土壤自然衰减过程中多环芳烃降解关键基因结构和变化规律。【方法】以新疆准东油田为研究区,分析同一采油区不同石油污染年限土壤理化因子和多环芳烃含量变化,采用扩增子测序研究石油自然衰减过程中多环芳烃降解酶基因结构变化规律,利用Mental检验探讨其环境驱动因子。【结果】石油污染时间1年和3年的土壤中有多项理化指标与背景土存在显著性差异,而污染5年土壤与背景土之间仅2项指标具有显著性差异,随石油自然衰减逐渐恢复至正常。石油污染1年的土壤中16种多环芳烃除苊烯和?以外,其余14种多环芳烃均高于石油污染3年和5年土壤,多环芳烃总量和含油率污染1年土壤均显著高于污染3年和5年的土壤,多环芳烃会在污染后短时间内迅速被降解。扩增子测序结果显示,萘双加氧酶基因分类操作单元(operational taxonomic units,OTUs)序列随污染年限延长逐渐增多;芳环羟化双加氧酶基因OTUs序列BLAST(...     

温州市境内城市河流底泥氨氧化菌富集培养物微生物群落结构分析

【背景】城市河流底泥含有丰富的微生物资源,底泥表面更是硝化作用的主要位点之一,其表面微生物在河流生态系统氮的转化过程中发挥着重要作用。【目的】以温州市境内的城市河流水系温瑞塘河茶山段舜岙河和横江河的4条河道作为采样点,比较分析4种不同环境下城市河流表层底泥氨氧化菌富集培养物的微生物群落结构。【方法】通过野外采样及室内培养对底泥中氨氧化功能菌进行富集培养,采用高通量测序技术分析微生物群落的组成、丰度和多样性。【结果】富集培养后主要优势类群为变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)。4个样品共涉及氨氧化细菌3个属,分别为亚硝化单胞菌属(Nitrosomonas)、亚硝化螺菌属(Nitrosospira)、亚硝化球菌属(Nitrosococcus),涉及氨氧化古菌1个属为Nitrososphaera,其中所有样品均以Nitrosomonas为主。不同底泥富集样品氨氧化微生物可操作分类单元(Operational taxonomic unit,OTU)组成存在明显差异,栽种有水生植物的河道底泥样品DA2具有最高的氨氧化细菌OTU数量和相对丰度,而存在生活餐饮污染的河道底泥样品DA4具有最高的氨氧化古菌OTU数量和相对丰度;相较于滞留水体,采自相对流动水体的富集样品DA2、DA4具有更高的氨氧化微生物OTU数量和相对丰度。【结论】阐述了4种不同环境下城市河流底泥氨氧化菌富集培养物微生物群落结构的多样性,确定了富集培养之后的优势类群,为氨氧化微生物培养源的选择提供了参考,也为城市河流底泥中氨氧化菌进一步的筛选分离及其生理生态特征的研究提供了科学依据。     

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.     

Responses of Microbial Communities in Arctic Sea Ice After Contamination by Crude Petroleum Oil

Microbial communities associated with Arctic fjord ice polluted with petroleum oils were investigated in this study. A winter field experiment was conducted in the Van Mijen Fjord (Svalbard) from February to June 2004, in which the ice was contaminated with a North Sea paraffinic oil. Holes were drilled in the ice and oil samples frozen into the ice at the start of the experiment. Samples, including cores of both oil-contaminated and clean ice, were collected from the field site 33, 74, and 112 days after oil application. The sampled cores were separated into three sections and processed for microbiological and chemical analyses. In the oil-contaminated cores, enumerations of total prokaryotic cells by fluorescence microscopy and colony-forming units (CFU) counts of heterotrophic prokaryotes both showed stimulation of microbial growth, while concentrations of oil-degrading prokaryotes remained at similar levels in contaminated and clean ice. Analysis of polymerase chain reaction (PCR)-amplified bacterial 16S rRNA gene fragments by denaturing gradient gel electrophoresis (DGGE) revealed that bacterial communities in oil-contaminated ice generated fewer bands than communities in clean ice, although banding patterns changed both in contaminated and clean ice during the experimental period. Microbial communities in unpolluted ice and in cores contaminated with the paraffinic oil were examined by cloning and sequence analysis. In the contaminated cores, the communities became predominated by Gammaproteobacteria related to the genera Colwellia, Marinomonas, and Glaciecola, while clean ice included more heterogeneous populations. Chemical analysis of the oil-contaminated ice cores with determinations of n-C17/Pristane and naphthalene/phenanthrene ratios indicated slow oil biodegradation in the ice, primarily in the deeper parts of the ice with low hydrocarbon concentrations.     

Phytoremediation of petroleum hydrocarbons by using a freshwater fern species Azolla filiculoides Lam

In this study, the phytoremediation capacity of Azolla filiculoides Lam. for the water resources contaminated with petroleum hydrocarbons was investigated. The plants were grown in nitrogen-free Hoagland nutrient solution containing 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% crude oil under greenhouse conditions for 15 days. Although the growth rate of the plants were not negatively influenced by the presence of crude oil in the media for the concentration of 0.005% and 0.01% v/v, a gradual impeding effect of crude oil in the growth media has been observed at concentrations 0.05–0.1%. More than 0.1% crude oil in the growth medium ostensibly retarded the growth. For example, 0.2% oil in the media reduced growth approximately 50% relative to the control, and the presence of crude oil at concentrations 0.3% or more were lethal. The data about the percentage of plant growth, fresh weight increase and root growth clearly indicated that the tolerance level of A. filiculoides plants to crude oil ranges between 0.1% and 0.2%. In comparison to control samples, the biodegradation rate of total aliphatic and aromatic (phenathrene) hydrocarbons at 0.05–0.2% oil concentrations, was 94–73% and 81–77%, respectively. On the other hand, in case of further increases in oil concentration in media, i.e.; 0.3–0.5%, the biodegradation rate was still higher in the experimental samples, respectively 71–63% and 75–71%. The high biodegradation rates of petroleum hydrocarbons in the experimental samples suggested that A. filiculoides plants could be a promising candidate to be used for the phytoremediation of low crude oil contaminated precious freshwater resources.     

Relationships between root growth of Zinnia hybrid “profusion orange” flowers and phytoremediation of oil-contaminated soil

Relationships exist between plant root growth and the phytoremediation of oil-contaminated soils. In a previous study, we demonstrated that zinnia flowers are well suited for the remediation of oil-contaminated soil. In this study, our goal was to quantify the relationship between zinnia root growth and purification of oil-contaminated soils. Three treatments were used: (1) cultivation of zinnia in oil-contaminated soil (contaminated pots), (2) cultivation in non-contaminated soil (non-contaminated pots), and (3) contaminated soil with no cultivation and only irrigation (irrigated pots). Growth of the Zinnia plants, including their roots, was significantly reduced in the contaminated pots compared with the noncontaminated pots. The soil dehydrogenase activity increased between 45 and 90?days after planting in all parts of the contaminated pots, especially the upper parts. The soil total petroleum hydrocarbon (TPH) concentrations in the contaminated pots decreased throughout the study period. Interestingly, the soil dehydrogenase activity increased, and the soil TPH concentration decreased even in lower parts of the pots where there was very little root growth. Therefore, the cultivation of plants can have a remediative effect on oil-contaminated soil even below the depth reached by the plant roots.     

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.     

Sulphur‐oxidizing and sulphate‐reducing communities in Brazilian mangrove sediments

Mangrove soils are anaerobic environments rich in sulphate and organic matter. Although the sulphur cycle is one of the major actors in this ecosystem, little is known regarding the sulphur bacteria communities in mangrove soils. We investigated the abundance, composition and diversity of sulphur‐oxidizing (SOB) and sulphate‐reducing (SRB) bacteria in sediments from three Brazilian mangrove communities: two contaminated, one with oil (OilMgv) and one with urban waste and sludge (AntMgv), and one pristine (PrsMgv). The community structures were assessed using quantitative real‐time polymerase chain reaction (qPCR), polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) and clone libraries, using genes for the enzymes adenosine‐5′‐phosphosulphate reductase (aprA) and sulphite reductase (Dsr) (dsrB). The abundance for qPCR showed the ratio dsrB/aprA to be variable among mangroves and higher according to the gradient observed for oil contamination in the OilMgv. The PCR‐DGGE patterns analysed by Nonmetric Multidimensional Scaling revealed differences among the structures of the three mangrove communities. The clone libraries showed that Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria were the most abundant groups associated with sulphur cycling in mangrove sediments. We conclude that the microbial SOB and SRB communities in mangrove soils are different in each mangrove forest and that such microbial communities could possibly be used as a proxy for contamination in mangrove forests.     

Diversity and identification of methanogenic archaea and sulphate-reducing bacteria in sediments from a pristine tropical mangrove

Mangrove sediments are anaerobic ecosystems rich in organic matter. This environment is optimal for anaerobic microorganisms, such as sulphate-reducing bacteria and methanogenic archaea, which are responsible for nutrient cycling. In this study, the diversity of these two functional guilds was evaluated in a pristine mangrove forest using denaturing gradient gel electrophoresis (DGGE) and clone library sequencing in a 50 cm vertical profile sampled every 5.0 cm. DGGE profiles indicated that both groups presented higher richness in shallow samples (0–30 cm) with a steep decrease in richness beyond that depth. According to redundancy analysis, this alteration significantly correlated with a decrease in the amount of organic matter. Clone library sequencing indicated that depth had a strong effect on the selection of dissimilatory sulphate reductase (dsrB) operational taxonomic units (OTUs), as indicated by the small number of shared OTUs found in shallow (0.0 cm) and deep (40.0 cm) libraries. On the other hand, methyl coenzyme-M reductase (mcrA) libraries indicated that most of the OTUs found in the shallow library were present in the deep library. These results show that these two guilds co-exist in these mangrove sediments and indicate important roles for these organisms in nutrient cycling within this ecosystem.     

Reproductive cycle of the mangrove oyster Crassostrea rhizophorae (Bivalvia: Ostreidae) in Camamu Bay, Bahia, Brasil

The mangrove oyster Crassostrea rhizophorae is important fishery resource along the entire Brasilian coast with excellent potential for marine culture. The purpose of this paper was to examine the reproductive characteristics of the oyster of the Maraú river estuary in Camamu Bay, Bahia, Brasil. The samples were collected monthly, from September 2006 to August 2007, at two points (I and II) in Camamu Bay. At each site 20 oysters were collected for histological analysis, fixed in Davidson's solution, embedded in paraffin, dehydrated in an ethanol series, sectioned (7 microm thick) and stained with Harris hematoxylin and Eosin (HE). Additionally, 30 oysters were sampled, at each point, for a condition index analysis. The water temperature ranged from 23.5 degrees C to 30 degrees C and the salinity from 15 to 25 ups at Point I (Maraú) and from 25 to 35 at Point II (Tanque Island). The oyster's height ranged from 30 to 92 mm at Point I and from 27 to 102 mm at Point II, with an average of 49.0 mm +/- 9.1 (n = 230) and 49.9 mm +/- 9.9 (n = 237), respectively. Among the sampled oysters at Point I, 59.1% were females, 31.3% males, 1.3% hermaphrodites and 8.2% of the oysters of undetermined sex. At Point II, 66.2% were females, 30.4% males, 0.8% hermaphrodites and 2.5% (n = 237) of undetermined sex. The gonadic stage analysis indicated that the reproduction period of the C. rhizophorae in the Maraú Peninsula was continuous all year, without any regressive phase. The condition index (R) ranged from 8.0% to 17.7%. The peak periods of R coincided with the expressive oyster's percentage in the maturation and liberation gametic stages. The results of these findings will contribute information for the oyster spat collection and to the process installation of the oyster culture in Camamu Bay.     

Bioremediation of oil by marine microbial mats

Cyanobacterial mats developing in oil-contaminated sabkhas along the African coasts of the Gulf of Suez and in the pristine Solar Lake, Sinai, were collected for laboratory studies. Samples of both mats showed efficient degradation of crude oil in the light, followed by development of an intense bloom of Phormidium spp. and Oscillatoria spp. Isolated cyanobacterial strains, however, did not degrade crude oil in axenic cultures. Strains of sulfate-reducing bacteria and aerobic heterotrophs were capable of degrading model compounds of aliphatic and aromatic hydrocarbons. Results indicate that degradation of oil was done primarily by aerobic heterotrophic bacteria. The oxygenic photosynthesis of oil-insensitive cyanobacteria supplied the molecular oxygen for the efficient aerobic metabolism of organisms, such as Marinobacter sp. The diurnal shifts in environmental conditions at the mat surface, from highly oxic conditions in the light to anaerobic sulfide-rich habitat in the dark, may allow the combined aerobic and anaerobic degradation of crude oil at the mat surface. Hence, coastal cyanobacterial mats may be used for the degradation of coastline oil spills. Oxygen microelectrodes detected a significant inhibition of photosynthetic activity subsequent to oil addition. This prevailed for a few hours and then rapidly recovered. In addition, shifts in bacterial community structure following exposure to oil were determined by denaturing gradient gel electrophoresis of PCR-amplified fractions of 16S rRNA from eubacteria, cyanobacteria and sulfate-reducing bacteria. Since the mats used for the present study were obtained from oil-contaminated environments, they were believed to be preequilibrated for petroleum remediation. The mesocosm system at Eilat provided a unique opportunity to study petroleum degradation by mats formed under different salinities (up to 21%). These mats, dominated by cyanobacteria, can serve as close analogues to the sabkhas contaminated during the Gulf War in Kuwait and Saudi Arabia. Electronic Publication     

Crude Oil Biodegradation by a Soil Indigenous Bacillus sp. Isolated from Lavan Island

The application of microorganisms for removing crude oil pollution from contaminated sites as a type of bioremediation has long been a matter of study in scientific communities. In this study, 35 morphologically different spore-forming Bacilli were isolated from an oil-contaminated soil in Lavan Island. The objective of this study was to investigate the oil-biodegrading ability of these indigenous bacilli. Therefore, their biosurfactant production, using Du Neuy ring, and the crude oil aliphatic and aromatic content alteration after bacterial treatment, respectively, using gas chromatography and high-performance liquid chromatography, were studied. An isolate with high endurance of a wide range of temperature and pH and optimized growth at 30°C and pH 6.8 that could reduce the surface tension from 60 to 40 mN/m and cause the most alteration in aliphatic and aromatic content of crude oil was selected. Using biochemical and molecular analyses of 16SrRNA, this suitable bacterium for oil biodegradation was characterized as Bacillus cereus sp. 4.     

PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites

tmoA and related genes encode the alpha-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of tmoA-like gene sequences in environmental samples using a newly designed moderately degenerate primer set suitable for that purpose. In 35 BTEX-degrading bacterial strains isolated from a hydrocarbon polluted aquifer, tmoA-like genes were only detected in two o-xylene degraders and were identical to the touA gene of Pseudomonas stutzeri OX1. The diversity of tmoA-like genes was examined in DNA extracts from contaminated and non-contaminated subsurface samples at a site containing a BTEX-contaminated groundwater plume. Differences in DGGE patterns were observed between strongly contaminated, less contaminated and non-contaminated samples and between different depths, suggesting that the diversity of tmoA-like genes was determined by environmental conditions including the contamination level. Phylogenetic analysis of the protein sequences deduced from the amplified amplicons showed that the diversity of TmoA-analogues in the environment is larger than suggested from described TmoA-analogues from cultured isolates, which was translated in the DGGE patterns. Although different positions on the DGGE gel can correspond to closely related TmoA-proteins, relationships could be noticed between the position of tmoA-like amplicons in the DGGE profile and the phylogenetic position of the deduced protein sequence.     

Spatial distribution and abundances of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in mangrove sediments

We investigated the diversity, spatial distribution, and abundances of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in sediment samples of different depths collected from a transect with different distances to mangrove forest in the territories of Hong Kong. Both the archaeal and bacterial amoA genes (encoding ammonia monooxygenase subunit A) from all samples supported distinct phylogenetic groups, indicating the presences of niche-specific AOA and AOB in mangrove sediments. The higher AOB abundances than AOA in mangrove sediments, especially in the vicinity of the mangrove trees, might indicate the more important role of AOB on nitrification. The spatial distribution showed that AOA had higher diversity and abundance in the surface layer sediments near the mangrove trees (0 and 10 m) but lower away from the mangrove trees (1,000 m), and communities of AOA could be clustered into surface and bottom sediment layer groups. In contrast, AOB showed a reverse distributed pattern, and its communities were grouped by the distances between sites and mangrove trees, indicating mangrove trees might have different influences on AOA and AOB community structures. Furthermore, the strong correlations among archaeal and bacterial amoA gene abundances and their ratio with NH₄⁺, salinity, and pH of sediments indicated that these environmental factors have strong influences on AOA and AOB distributions in mangrove sediments. In addition, AOA diversity and abundances were significantly correlated with hzo gene abundances, which encodes the key enzyme for transformation of hydrazine into N₂ in anaerobic ammonium-oxidizing (anammox) bacteria, indicating AOA and anammox bacteria may interact with each other or they are influenced by the same controlling factors, such as NH₄⁺. The results provide a better understanding on using mangrove wetlands as biological treatment systems for removal of nutrients.     

18S rDNA Sequences from Microeukaryotes Reveal Oil Indicators in Mangrove Sediment

Background

Microeukaryotes are an effective indicator of the presence of environmental contaminants. However, the characterisation of these organisms by conventional tools is often inefficient, and recent molecular studies have revealed a great diversity of microeukaryotes. The full extent of this diversity is unknown, and therefore, the distribution, ecological role and responses to anthropogenic effects of microeukaryotes are rather obscure. The majority of oil from oceanic oil spills (e.g., the May 2010 accident in the Gulf of Mexico) converges on coastal ecosystems such as mangroves, which are threatened with worldwide disappearance, highlighting the need for efficient tools to indicate the presence of oil in these environments. However, no studies have used molecular methods to assess the effects of oil contamination in mangrove sediment on microeukaryotes as a group.

Methodology/Principal Findings

We evaluated the population dynamics and the prevailing 18S rDNA phylotypes of microeukaryotes in mangrove sediment microcosms with and without oil contamination, using PCR/DGGE and clone libraries. We found that microeukaryotes are useful for monitoring oil contamination in mangroves. Our clone library analysis revealed a decrease in both diversity and species richness after contamination. The phylogenetic group that showed the greatest sensitivity to oil was the Nematoda. After contamination, a large increase in the abundance of the groups Bacillariophyta (diatoms) and Biosoecida was detected. The oil-contaminated samples were almost entirely dominated by organisms related to Bacillariophyta sp. and Cafeteria minima, which indicates that these groups are possible targets for biomonitoring oil in mangroves. The DGGE fingerprints also indicated shifts in microeukaryote profiles; specific band sequencing indicated the appearance of Bacillariophyta sp. only in contaminated samples and Nematoda only in non-contaminated sediment.

Conclusions/Significance

We believe that the microeukaryotic targets indicated by our work will be of great applicability in biomonitoring hydrocarbons in mangroves under oil contamination risk or during recovery strategies.     

Screening of plants for phytoremediation of oil-contaminated soil

Several species of ornamental flowering plants were evaluated regarding their phytoremediation ability for the cleanup of oil-contaminated soil in Japanese environmental conditions. Thirty-three species of plants were grown in oil-contaminated soil, and Mimosa, Zinnia, Gazania, and cypress vine were selected for further assessment on the basis of their favorable initial growth. No significant difference was observed in the above-ground and under-ground dry matter weight of Gazania 180 days after sowing between contaminated and non-contaminated plots. However, the other 3 species of plants died by the 180^th day, indicating that Gazania has an especially strong tolerance for oil-contaminated soil. The total petroleum hydrocarbon concentration of the soils in which the 4 species of plants were grown decreased by 45–49% by the 180^th day. Compared to an irrigated plot, the dehydrogenase activity of the contaminated soil also increased significantly, indicating a phytoremediation effect by the 4 tested plants. Mimosa, Zinnia, and cypress vine all died by the 180^th day after seeding, but the roots themselves became a source of nutrients for the soil microorganisms, which led to a phytoremediation effect by increase in the oil degradation activity. It has been indicated that Gazania is most appropriate for phytoremediation of oil-contaminated soil.     

海绵中石油降解微生物的分离筛选及降解特性研究

从大连湾原油污染海域生长的海绵中分离筛选到一株原油降解菌OA58,根据其生长形态、培养特征、16S rRNA序列相似性比对分析和生化指标检测,初步鉴定为脱叶链霉菌（Streptomyces exfoliatus）。同时,考察了该链霉菌对原油的降解效果,OA58能以原油为唯一碳源生长,在人工海水培养基中,14 d内对原油（初始浓度为1 g/L）的平均降解率为83%,是一株具有开发潜力的原油降解放线菌。     

Identification of a bacterium that specifically catalyzes the reductive dechlorination of polychlorinated biphenyls with doubly flanked chlorines

A microorganism whose growth is linked to the dechlorination of polychlorinated biphenyls (PCBs) with doubly flanked chlorines was identified. Identification was made by reductive analysis of community 16S ribosomal DNA (rDNA) sequences from a culture enriched in the presence of 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB), which was dechlorinated at the para position. Denaturing gradient gel electrophoresis (DGGE) analysis of total 16S rDNA extracted from the culture led to identification of three operational taxonomic units (OTUs 1, 2, and 3). OTU 1 was always detected when 2,3,4,5-CB or other congeners with doubly flanked chlorines were present and dechlorinated. Only OTUs 2 and 3 were detected in the absence of PCBs and when other PCBs (i.e., PCBs lacking doubly flanked chlorines) were not dechlorinated. Partial sequences of OTUs 2 and 3 exhibited 98.2% similarity to the sequence of "Desulfovibrio caledoniensis" (accession no. DCU53465). A sulfate-reducing vibrio isolated from the culture generated OTUs 2 and 3. This organism could not dechlorinate 2,3,4,5-CB. From these results we concluded that OTU 1 represents the dechlorinating bacterium growing in a coculture with a Desulfovibrio sp. The 16S rDNA sequence of OTU 1 is most similar to the 16S rDNA sequence of bacterium o-17 (89% similarity), an ortho-PCB-dechlorinating bacterium. The PCB dechlorinator, designated bacterium DF-1, reductively dechlorinates congeners with doubly flanked chlorines when it is supplied with formate or H(2)-CO(2) (80:20).     

Phylogenetic diversity of bacterial communities associated with bioremediation of crude oil in microcosms

Bioremediation, mainly by indigenous bacteria, has been regarded as an effective way to clean up oil pollution after an oil spill. In order to obtain a systematic understanding of the succession of bacterial communities associated with oil bioremediation, sediments collected from the Penglai 19-3 oil platform were co-incubated with crude oil. Oil biodegradation was assessed on the basis of changes in oil composition monitored by GC–MS. Changes in the bacterial community structure were detected by two 16S rRNA gene based culture-independent methods, denaturing gradient gel electrophoresis (DGGE) and clone library. The results suggested that crude oil was rapidly degraded during the 30-day bioremediation period. Bacteria affiliated with the genus Pseudomonas dominated all three clone libraries. But dramatic changes were also detected in the process of biodegradation of crude oil. The “professional hydrocarbonocastic bacteria” (e.g., Alcanivorax) became abundant in the two samples during the bioremediation period. Meanwhile, δ-proteobacteria was only detected in the two samples. Information on the bacterial community revealed in this study will be useful in developing strategies for bioremediation of crude oil dispersed in the marine ecosystem.