大连湾石油污染沉积物中细菌群落结构分析

大连新港"7.16"输油管道爆炸溢油事故发生后,为探究石油污染与细菌群落结构变化之间的关系及在石油生物降解过程中起重要作用的细菌菌群,本研究对大连湾表层沉积物中石油烃含量和细菌宏基因组16SrDNA V3区进行分析。结果表明:溢油初期2010年8月DLW01站位表层沉积物石油烃含量高达1 492mg/kg,符合第三类沉积物质量标准,随着时间推移,2011年4月、2011年7月、2011年12月航次各站位沉积物中石油烃含量基本呈下降趋势,且均符合第一类沉积物质量标准;16S rDNA PCR-DGGE方法分析表明,石油烃含量高的区域优势细菌种类少,反之则较丰富;海洋环境中同一地点的细菌群落能保持一定稳定性;大连湾石油污染沉积物中变形菌门γ-变形菌纲和拟杆菌门一直保持较高的优势度,是在石油生物降解过程中起重要作用的细菌菌群,而厚壁菌门只在石油烃含量低的区域出现;此外,出现的对污染物敏感的嗜冷杆菌可作为石油污染指示生物进行深入研究。     

海洋石油降解菌群的固定化及石油降解特性

为克服岸滩溢油生物修复过程中海浪冲刷等不利环境对石油降解菌(群)岸滩定植的影响,本文利用聚乙烯醇(PVA)和海藻酸钠作为载体对石油降解菌群DC10进行固定化,通过研究细菌固定化微球的机械性能、传质性能及石油降解特性等参数,确定石油降解菌群的最优固定化条件。实验结果表明:6%PVA,2%海藻酸钠及0.5%活性炭制备的凝胶可以通过蠕动泵方便快捷制备细菌固定化微球,其粘度小、易成型、机械强度高。海洋石油降解分析表明,与游离菌体(FB)相比,固定化菌群12d石油降解率提高了近7%;GC-MS分析显示,石油烷烃和芳烃降解效果显著。实验证明,石油降解菌群DC10经过固定化处理,其石油降解活性提高,连续降解能力增强,该研究为溢油岸滩的生物修复提供新的技术方法。     

Bacterial communities fluctuate in abundance and diversity under simulated oil-contaminated seawater conditions

Marine bacteria have recently been identified as a potent solution for petroleum hydrocarbon degradation in response to hazardous oceanic oil spills. In this study, a mesocosm experiment simulating a petroleum spill event was performed to investigate changes in the abundance, structure, and productivity of bacterial communities in response to oil pollution. Cultured heterotrophic bacteria and total bacteria showed a consistent trend involving an immediate decrease in abundance, followed by a slight increase, and a steady low-level thereafter. However, the changing trend of bacterial productivity based on bacterial biomass and bacterial volume showed the opposite trend. In addition, the density of oil-degrading bacteria increased initially, then subsequently declined. The change in the bacterial community structure at day 0 and day 28 were also analyzed by amplified ribosomal DNA restriction analysis (ARDRA), which indicated that the species diversity of the bacterial community changed greatly after oil pollution. Alphaproteobacteria (40.98%) replaced Epsilonproteobacteria (51.10%) as the most abundant class, and Gammaproteobacteria (38.80%) became the second most dominant class in the whole bacterial community. The bacterial communities in oil-contaminated seawater (32 genera) became much more complex than those found in the natural seawater sample (16 genera). The proportion of petroleum-degrading bacteria in the oil-contaminated seawater also increased. In this study, culture-dependent and culture-independent approaches were combined to elucidate changes in both bacterial productivity and community structure. These findings will contribute to a better understanding of the role that bacteria play in material cycling and degradation in response to oil pollution.     

Does historical exposure to hydrocarbon contamination alter the response of benthic communities to diesel contamination?

A microcosm experiment was used to compare the influence of diesel contamination on two benthic salt-marsh communities, one chronically exposed to petroleum hydrocarbons for decades (Louisiana [LA]) and the other relatively uncontaminated (Mississippi [MS]). Initial meiofaunal community composition of the two sites was similar. Higher organic content of MS sediments should have reduced bioavailability, and thus the toxicity of hydrocarbons relative to the LA site. Nevertheless, although responses to diesel contamination at the two sites were generally qualitatively similar, a species-specific and several community-response variables were influenced to a much greater degree in the MS community. In particular, the abundance of total nauplii, ostracods, and copepods were negatively impacted to a greater extent in MS than in LA, as was grazing by ostracods on benthic microalgae. Nematode:copepod ratios in contaminated sediments were much higher in MS than in LA sediments. Pseudostenhelia wellsi (a benthic copepod) nauplii suffered greater adverse effects of diesel in MS than in LA. We conclude that the MS community was more sensitive to diesel contamination than was the LA community. The differential sensitivity is presumably a manifestation of different tolerances to hydrocarbon contaminants, mediated by a higher proportion of more tolerant species and/or increased tolerance among individual species in LA. Although the MS site was more sensitive to diesel contamination, qualitative response of the LA and MS communities were similar, and comparable to previous studies of diesel contamination. The spatial and temporal consistency of diesel impacts on salt-marsh communities suggests that hydrocarbon contamination results in predictable community responses. Specifically, crustacean (e.g. copepods, ostracods, and nauplii) benthos are most sensitive to hydrocarbons. Reductions in abundance and grazing activity of crustaceans leads to enhanced algal biomass, reduced copepod diversity, and alters competitive interactions among meiofauna.     

渤海海域浮游病毒和浮游细菌的丰度变化与环境因子之间的耦联关系

Uncovering the role of environmental factors and finding critical factors which harbor significant fractions in governing microbial communities remain key questions in coastal marine systems. To detect the interactions between environmental factors and distributions of virio-and bacterioplankton in trophic coastal areas, we used flow cytometry to investigate the abundance of virio-and bacterioplankton covering 31 stations in the Bohai Sea of China. Our results suggested that the average abundance of total virus(TV) in winter(～2.29×10~8 particles/mL)was slightly lower than in summer(～3.83×10~8 particles/mL). The mean total bacterial abundance(TB) was much lower in winter(～2.54×107 particles/mL) than in summer(～5.43×10~7 particles/mL). Correlation analysis via redundancy analysis(RDA) and network analysis among virioplankton, bacterioplankton and environmental factors revealed that the abundances of viral and bacterial subpopulations depend on environmental factors. In winter, only temperature significantly influenced the abundances of virio-and bacterioplankton. In summer, in addition to temperature, both salinity and nutrient(SiO_2) had a remarkable impact on the distribution of virioand bacterioplankton. Our results showed a clear seasonal and trophic pattern throughout the whole water system, which revealed that temperature and eutrophication may play crucial roles in microbial distribution pattern.     

渤海湾表层海水中浮游细菌群落随离岸距离的分布特征及其影响因素

为研究渤海湾近岸污染对远近岸海域微生态的影响,利用高通量测序技术,对渤海湾不同离岸距离的6个站位采集表层海水,进行浮游细菌群落结构分析,结合环境、空间因素探究影响其变化的主要因素。结果表明:研究区域存在环境因子的梯度变化,如氮营养盐在近岸高于远岸;细菌α-多样性在不同站位间差异不显著,但仍显示在近岸相对较高;细菌群落结构随离岸距离变化显著,γ-变形菌和拟杆菌在近岸显著富集,且与氮营养盐的含量有关;蓝细菌在远岸显著富集,且与氨氮、透明度、电导率有关;邻体矩阵主坐标单独解释部分对群落结构变异的贡献率最大(38.1%),说明可能存在尚未测量但具有空间结构的环境变量影响群落空间分布;结合功能预测的结果推测近岸区域的富营养与烃类污染等可能影响群落变化。本文从环境和空间影响两方面探讨了渤海湾不同离岸距离的海域浮游细菌群落结构变化,为研究渤海湾海洋生态及环境保护提供一定的参考。     

Heterotrophic bacteria of the Dapeng Bay in the South China Sea: community structure, abundance, and the relationships of culturablity with environmental factors

The community composition,abundance and culturablity of heterotrophic bacterioplankton in the Dapeng Bay of the South China Sea were investigated by morphological method,bacterial cultivation and biochemical methods over an annual cycle.Aeromonas and Pseudomonas were the predominant genera among the Gram-negative bacteria.The bacterioplankton abundance fluctuated from 2.07×10 8 to 2.44×10 9 cells/dm 3 ,and culturable bacteria abundance ranged between 0.1×10 6 and 16.07×10 6 CFU/dm 3 .The Culturablity of heterotrophic bacteria ranged between 0.01% and 1.31%.The relationship between bacterial culturablity and the environmental factors was also studied in different sites of the Dapeng Bay.Throughout the bay,a significantly positive relationship was evidenced between bacterial culturability and temperature,meanswhile bacterial culturability was significantly inversely related with salinity.The positive relationship was shown at the central bay while a negative relationship was observed at the bay mouth and there was no significant correlation at top bay between bacterial culturablity and chlorophyll a of sea water.A positive relationship was found between culturability and DIN all the bay,but DIP showed a significantly negative relationship with bacterial culturability only at the mouth of the bay.     

Bacterioplankton in the northeastern part of the Black Sea during the summer and autumn of 2005

On the basis of luminescent microscopy using a DAPI dye, the abundance, morphometric characteristics, and biomass of bacteria in the coastal waters off the northeastern part of the Black Sea in 2005 were considered. The material was collected in June, September, and October along the cross section from Golubaya Bay towards the open part of the sea. An analysis of the microorganism development level showed that the bacteria achieved their maximal abundance in September, amounting, in the closest near-shore area, to 2435 × 10³ cells/ml, or 276 mg/m³ of wet weight; the lowest values were characteristic of October. During all the survey periods, coccal forms were prevailing among the microorganisms; regardless of the season, their fraction was maximal at the coastal stations. The greatest prevalence of coccal forms was characteristic of October (83%). The average sizes of the bacterial cells in 2005 varied within 0.073–0.257 μm³. The highest values of the bacterial abundance and biomass, almost in all the cases, were found at coastal stations; with the distance from the coast, a decrease in the bacterioplankton abundance was observed. A comparison of methodically similar data on the concentration of bacteria (dying with acridine orange and DAPI) from 1985 till now shows a trend of the annual growth in the bacterial abundance.     

Molecular analyses of bacterioplankton communities with highly abundant Vibrio clades: a case study in Bohai Sea coastal waters

We investigated the bacterioplankton abundance, community composition and the associated Vibrio clades of natural seawater in Bohai Sea coastal waters. Seawater samples (10 L in triplicate) were collected at 0.5, 3, and 5 m depths near the coastal aquaculture zone of the Bohai Sea on May 12, 2016. Real-time PCR and 16S rRNA gene amplicon high-throughput sequencing methods were employed by which 485 operational taxonomic units (OTUs) at a 97% sequence similarity level were generated. Gammaproteobacteria, Alphaproteobacteria, and Bacteroidetes were the most abundant groups, accounting for 49.5%, 23.5%, and 18.9% of the total assemblage, respectively. Obvious variations in Pseudoalteromonas, Vibrio, and Octadecabacter , which were the most abundant genera, could be observed among diff erent samples. Notably, the results of Vibrio -specifi c real-time PCR indicated that Vibrio had extremely high 16S rRNA gene copy numbers. The 16S rRNA gene sequencing results across all the samples also indicated that they occupied a large proportion of the total assemblage. Both the alpha diversity and major bacterioplankton group Pseudoalteromonas had significant correlations with the concentration of PO4^3-. Overall, studies on bacterioplankton communities with highly abundant Vibrio clades can provide interesting insight into the microbial function and health assessment of the Bohai Sea coastal ecosystem.     

Field observations on the variability of crude oil impact on indigenous hydrocarbon-degrading bacteria from sub-Antarctic intertidal sediments

Oil pollution of the oceans has been a problem ever since man began to use fossil fuels. Biodegradation by naturally occurring populations of micro-organisms is a major mechanism for the removal of petroleum from the environment. To examine the effects of crude oil pollution on intertidal bacteria, we repeated the same contamination experiments on nine different sub-Antarctic intertidal beaches using specifically built enclosures (PVC pipe, 15 cm in inner diameter and 30 cm in height). Despite the pristine environmental conditions, significant numbers of indigenous hydrocarbon-degrading bacteria were observed in all the studied beaches. Introduction of oil into these previously oil-free environments resulted in several orders of magnitude of increase in hydrocarbon-degrading micro-organisms within a few days in some of the studied sites but has no obvious effects on two others. The physical environment of the bacterial assemblage seems to play a major role in the biodegradation capacities. After 3 months of contamination, both remaining oil concentrations and biodegradation indexes differ strongly between the different stations. Thus, chemical and biological parameters reveal a strong heterogeneity of biodegradation capacities between the different sites.     

Macrobenthos monitoring in the Gulf of Suez, Egypt

The biogeography of macrobenthic communities with large-scale differences in species distribution and abundance was studied along the eastern and western coastal regions of the Gulf of Suez to indicate biodiversity of benthos in relation to the prevailing environmental conditions. In general, benthos have a wide variety of species of mainly polychaet worms, crustaceans, bivalves, and other bottom invertebrates living in burrows in/or on the sediment surface. Coastal development clearly affects the ecosystem after the construction of coastal projects. The highest values of benthic diversity, evenness, richness and dominance are found at the Gulf of Suez （WG 1, WG 3）, Ras Gharib （WG 7, WG 8） and Ras Shukier （WG 9a） by contrast to others. The abundance of macrobenthic organsims along the coasts of Gulf of Suez can be arranged in the following sequence： other groups （34%） greater than crustaceans （25%） greater than polychaetes （22%） greater than molluscs （17%） greater than echinoderms （2%）. The biomass of benthic assemblages at different sampling sites can also be ranked as follows： echinoderms （52%） greater than molluscs （27%） greater than crustaceans （16%） greater than polychaetes （2%） greater than other groups （3%）. Some benthic groups or species indicated a distinctive correlation to pollution sources in the marine ecosystem which obviously increased in the coastal areas due to the extension of the coastal development. These activities needed regular information to establish database for the biology and distribution of marine benthic communities. The density and diversity of benthos are significantly affected by eutrophication at Ain Sukhna, whereas the benthos in the open Gulf of Suez do not seem to be adversely affected by pollution.     

Microbial pollution indicators and culturable heterotrophic bacteria in a Mediterranean area (Southern Adriatic Sea Italian coasts)

In the present study we evaluated the degree of microbial water pollution along the coast line between Brindisi and Santa Maria di Leuca (Southern Adriatic Sea) as well as the culturable heterotrophic bacteria abundances and biodiversity in relation to the microbiological quality of the water. A total of 3773 colonies were isolated, subcultured and identified by several morphological, cultural and biochemical methods including the standardized API 20 E and API 20 NE tests. Along the examined coastal tract the microbial pollution indicators were always below the tolerance limits for bathing waters defined by the CEE directive, suggesting a good sanitary quality. Concerning culturable heterotrophic bacteria, different temporal density trends were observed in the four sites in relation to their geographical position. A positive relationship between the bacterial abundances and the temperature was observed in S. Cataldo and Otranto. The culturable bacterial community was mainly composed of the genera Aeromonas, Pseudomonas, Photobacterium and Flavobacterium. The Enterobacteriaceae family represented a conspicuous component of the bacterial community too. Bacilli were predominant among the Gram-positive bacteria. Of interest is the isolation of yeasts (2% at the surface and 1% at the bottom) taking into account their capability of biodegradation of various materials. Because of the low level of microbial pollution recorded, our results are indicative of the natural variation and diversity of the culturable bacterial community in such an oligotrophic ecosystem and could represent a good point of comparison with other ecosystems as well as a baseline for long term studies aimed to evaluate the effects of environmental fluctuations and human impacts on this aspect of biodiversity in coastal areas.     

河北黄骅近岸石油污染对海洋生物体内石油烃含量的影响

对河北黄骅沿海1995年2次调查所捕获的鱼、虾、贝类等18种样品的测定结果表明,受测海洋生物均检出石油烃,双壳类生物体内石油烃含量高于甲壳类和鱼类.该海域海洋生物体内石油烃含量水平远低于国内外污染严重的水域.从分析结果看,该海域目前石油污染特点主要以慢性油污染为主.     

青岛多种天气下生物气溶胶中细菌群落特征

为探究雾、霾、沙尘等多种天气下生物气溶胶中细菌群落结构特征,于2017年12月—2018年4月采集了青岛晴天和多种特殊天气下的大气生物气溶胶样品,运用16S rRNA高通量测序方法分析了样品中细菌群落结构。结果表明,在雾、霾、沙尘天均检测到对人体健康存在潜在危害的条件致病菌,其中变形菌门(Proteobacteria)均是优势细菌门,不受天气条件变化影响;霾天时其相对丰度最大。除此以外,沙尘和霾发生时,细菌群落结构显著变化,丰富度和多样性高于晴天,但随霾污染程度的加剧,群落丰富度和多样性下降;雾天多样性下降,丰富度变化不显著。在霾天,不同气团来源中细菌群落结构也存在显著差异。属水平上,霾天不动杆菌(Acinetobacter)和鞘氨醇单胞菌(Sphingomonas)占主导地位;雾天微球菌(Micrococcus)、副球菌(Paracoccus)和假单胞菌(Pseudomonas)占主导地位;沙尘天的优势菌为芽孢杆菌(Bacillus)。RDA分析显示O₃是影响菌群的主要环境因子。     

Responses of macrobenthic communities to patchy distributions of heavy metals and petroleum hydrocarbons in sediments:A study in China's Zhoushan Archipelago

This study conducted four cruises during 2014–2017 to investigate relationships between macrobenthic communities and sediment contaminations in sea area around the Zhoushan Archipelago. Fourteen sites were categorized into three groups: high total heavy metal contamination content(HHMC), high total petroleum hydrocarbon content(HTPH), and low content ratio of heavy metal contamination content to total petroleum hydrocarbon content(HMC/TPH) areas. Four main taxa of macrofauna(polychaetes, bivalves, gastropods, and crustaceans) were determined to respond to environmental factors differently. While tolerant polychaetes being the minimal impact by environmental factors, bivalves were threated by heavy metal pollutions in sediment.Additionally, body size distribution frequency demonstrated that macrofauna in the low HMC/TPH areas were less disturbed by contamination than those in the HHMC and HTPH areas. The result represented the presentation of sensitive species while tolerant species are usually considered as small size organisms. Overall,this study confirmed the hypothesis that the contamination levels of small-scale patches is indicated by the condition of macrobenthic communities.     

Variation in microbial biomass and community structure in sediments of peter the great bay (sea of japan/east sea), as estimated from fatty acid biomarkers

Variation in the microbial biomass and community structure found in sediment of heavily polluted bays and the adjacent unpolluted areas were examined using phospholipid fatty acid analysis. Total microbial biomass and microbial community structure were responding to environmental determinants, sediment grain size, depth of sediment, and pollution due to petroleum hydrocarbons. The marker fatty acids of microeukaryotes and prokaryotes - aerobic, anaerobic, and sulfate-reducing bacteria -were detected in sediments of the areas studied. Analysis of the fatty acid profiles revealed wide variations in the community structure in sediments, depending on the extent of pollution, sediment depth, and sediment grain size. The abundance of specific bacterial fatty acids points to the dominance of prokaryotic organisms, whose composition differed among the stations. Fatty acid distributions in sediments suggest the high contribution of aerobic bacteria. Sediments of polluted sites were significantly enriched with anaerobic bacteria in comparison with clean areas. The contribution of this bacterial group increased with the depth of sediments. Anaerobic bacteria were predominantly present in muddy sediments, as evidenced from the fatty acid profiles. Relatively high concentrations of marker fatty acids of sulfate-reducing bacteria were associated with organic pollution in this site. Specific fatty acids of microeukaryotes were more abundant in surface sediments than in deeper sediment layers. Among the microeukaryotes, diatoms were an important component. Significant amounts of bacterial biomass, the predominance of bacterial biomarker fatty acids with abundance of anaerobic and sulfate-reducing bacteria are indicative of a prokaryotic consortium responsive to organic pollution.     

Sampling and GC-FID,GC/MS analysis of petroleum hydrocarbons in the ocean surface microlayer off Richards Bay,South Africa

In view of the demonstrated concentration of hydrophobic pollutants at the air/water interface and their expected deleterious effects on the associated neuston, surface microlayer sampling with a Teflon disc was introduced as part of a coastal oil pollution survey. Differentiation of the petroleum assemblages comprising the samples is demonstrated, using gas chromatography with flame-ionization detection and GC/MS with computer-reconstructed mass fragmentograms. The former technique provides parameters such as the carbon-number preference index, which distinguishes biogenic from petrogenic n-alkanes, whilst the relative abundances of ions characteristic of steranes and triterpanes indicate similarities among samples, and give further evidence of the petrogenic origin of the hydrocarbon assemblages found. Levels of petroleum hydrocarbons found are uniformly higher (expressed as μg/m²) than those reported previously, where sampling techniques differed from the present one in taking a finite volume of surface water for analysis.     

通过焦磷酸测序揭示北极海水的微生物群落结构

This study aimed to determine the microbial community structure of seawater in(ICE-1) and out(FUBIAO) of the pack ice zone in the Arctic region.Approximate 10 L seawater was filtrated by 0.2 μm Whatman nuclepore filters and the environmental genomic DNA was extracted.We conducted a detailed census of microbial communities by pyrosequencing.Analysis of the microbial community structures indicated that these two samples had high bacterial,archaeal and eukaryotic diversity.Proteobacteria and Bacteroidetes were the two dominant members of the bacterioplankton community in both samples,and their relative abundance were 51.29% and 35.39%,72.95%and 23.21%,respectively.Euryarchaeota was the most abundant archaeal phylum,and the relative abundance was nearly up to 100% in FUBIAO and 60% in ICE-1.As for the eukaryotes,no_rank_Eukaryota,Arthropoda and no_rank_Metazoa were the most abundant groups in Sample FUBIAO,accounting for 85.29% of the total reads.The relative abundance of the most abundant phylum in Sample ICE-1,no_rank_Eukaryota and no_rank_Metazoa,was up to 90.69% of the total reads.Alphaproteobacteria,Flavobacteria and Gammaproteobacteria were the top three abundant classes in the two samples at the bacterial class level.There were also differences in the top ten abundant bacterial,archaeal and eukaryotic OTUs at the level of 97% similarity between the two samples.     

仿生人工贻贝在筼筜湖海水石油烃污染监测上的应用

仿生人工贻贝（Ｓｅｍｉ－Ｐｅｒｍｅａｂｌｅ Ｍｅｍｂｒａｎｅ Ｄｅｖｉｃｅｓ）是近年来海洋污染监测中新技术,其制作简单,吊挂取样方便,在筼筜湖纳潮进水渠和排污出水口进行夏、冬应用实验,结果显示筼筜湖内石油烃污染仍然较严重,其每ｇ甘油三油酸酯中石油烃浓度夏季可达２．１～７．８ｍｇ。退潮排污出水口人工贻贝石油烃浓度为纳潮进水口的２倍,冬季人工贻贝石油烃浓度又为夏季的１．５倍。同时也说明仿生人工贻贝（ＳＰＭＤ）可克服天然贻贝因性别、生长期、健壮程度和海区条件引起的差异,应用前景良好。