近海沉积物与水体中天然大分子聚合物降解菌的原位富集与多样性分析

本研究选取5种海洋动植物大分子高聚物或其天然组织，包括虾壳、鱼鳞、海带叶片、几丁质和壳聚糖，分别在海水表层和沉积物环境中进行富集，定期取样，通过高通量测序分析菌群多样性。结果发现，不同有机物原位富集的细菌多样性存在较大的差异，而且同种底物在海水表层与沉积物中的降解菌菌群差异较大。从物种多样性看，在海水表层环境中富集的鱼鳞样品种群最丰富，而沉积物环境中富集的海带叶片样品菌群多样性最低，除其优势菌群为热袍菌门外，其他所有富集物中优势菌均为变形菌门。其中脱硫杆菌科、黄杆菌科、脱硫弧菌科和弧菌科占有较大比例，脱硫杆菌科在所有样品中优势较大，黄杆菌科在海水表层环境样品中为优势菌群，弧菌科在沉积物样品几丁质和壳聚糖样品中占比较高。本研究通过对同种海洋环境中不同富集样品之间以及同种底物不同环境中富集菌群之间的比较，分析结果得到了原位条件下参与大分子聚合物降解的菌群种类，但有待于在更多不同的海域富集物中进行验证。     

胶州湾沉积物中石油降解菌的降解能力及多样性研究

2013年青岛输油管道爆炸，大量石油污染了附近海岸。课题组采集了污染的沉积物样品，以原油为唯一碳源和能源，富集了四个石油降解菌群。生物多样性和群落分析表明，Luteibacter、Parvibaculum 和属于食烷菌科的一个属是降解菌群的主要优势菌，都属于变形菌门。从石油降解菌群中分离筛选，获得了9株具有不同16S rRNA基因序列的降解菌，分别属于8个属。重量法测定降解菌的石油降解率，其中5株的石油降解率大于30%。GC-MS分析结果表明，石油降解菌多倾向于降解烷烃，对多环芳烃的降解能力较差，其中5株细菌的烷烃降解率较大，仅1株菌D2对多环芳烃的降解率较大，其降解率在34.9%到77.5%。通过对高通量数据的分析，表明食烷菌属是菌群A和菌群E的主要降解菌群，其中筛选获得的菌株E4可能是菌群E的一株优势降解菌。本研究所筛选菌株证明了其石油降解潜力，为油污染海滩生物修复提供了菌株资源。     

深海来源芳烃类降解菌筛选与盐单胞菌芘降解能力测定

海洋来源的菌株在高盐污水类修复处理中有重要的应用前景,而烃类物质是污水中的重要组成部分,其中含有多个苯环的芳香烃族化合物,对人类和环境具有较大的毒性,而目前关于多环芳烃(PAHs)厌氧代谢的相关报道相对较少。本研究以PAHs(萘、菲和芘等)为唯一碳源和能源,通过厌氧富集对太平洋深海沉积物中的PAHs降解菌展开研究。富集物细菌菌群分析结果表明,盐单胞菌属(Halomonas)、海旋菌属(Thalassospira)、海杆菌属(Marinobacter)、海洋杆菌属(Oceanobacter)和食烷菌属(Alcanivorax)等是主要的功能类群;其中盐单胞菌属是最主要的功能类群。通过筛选分离获得一高效烃降解盐单胞菌株,鉴定并命名为泰坦尼克盐单胞菌(Halomonas titanicae) PA16-9,该菌与模式菌株Halomonas titanicae BH1^T的16S rRNA相似性为99.52%。基于16S rRNA基因序列比对搜寻NCBI数据库,发现Halomonas titanicae广泛存在于废水、活性污泥、油田、湿地等有机质复杂区域,生态位分布极广,可利用丰富多样的底物。通过生长实验表明,菌株PA16-9能在厌氧条件下以硝酸盐为主要电子受体,利用芘、苯并芘和十六烷等烃类物质为唯一碳源进行生长,在培养45 d后芘的降解率达到61.9%,期间亚硝酸盐逐渐积累,浓度达到约0.24 mmol/L。盐单胞菌广泛存在于厌氧烃降解富集菌群中,暗示了盐单胞菌属在厌氧烃代谢中可能发挥了重要作用,在实际应用上具有很大潜力。     

青岛潮间带沉积物可培养厌氧细菌多样性的研究

为了验证设计的简易厌氧培养方法,作者以青岛潮间带沉积物为研究对象,采用5种培养基,从青岛潮间带沉积物共分离获得138株厌氧细菌。16S r DNA序列分析表明,这些细菌分属3个门15个属32个种,其中γ-变形菌纲64株18个种,在种类上处于优势地位;此外还包括δ-变形菌纲(δ-Proteobacteria)16株2个种,ε-变形菌纲4株1个种,拟杆菌门(Bacteroidetes)29株8个种,梭杆菌门(Fusobacteria)25株3个种。在属水平上,弧菌属(Vibrio)、Marinifilum属、泥杆菌属(Ilyobacter)、脱硫弧菌属(Desulfovibrio)、希瓦氏菌属(Shewanella)在数量上占优势。此外,有26株8种细菌(占总菌株数的18.84%)与已知菌的同源性介于89.38%~94.22%,为潜在的海洋细菌新科或新属,表明此简易厌氧菌培养方法在获得新菌方面具有较大优势。另外,研究结果还表明,不同培养基对特定的类群具有选择性:2216E培养基对γ-变形菌纲分离培养效率较高;SPG培养基在获得新菌方面具有优势(占新菌数62.5%),且这些新菌大多属于拟杆菌门和梭杆菌门,其中SPG-1培养基对于分离硫酸盐还原菌和难培养的ε-变形菌纲细菌具有优势,SPG-4培养基对分离硝酸盐还原菌具有优势。     

南沙深海沉积物中石油降解菌的分离鉴定和多样性分析

为了解我国南沙海域中石油降解菌的多样性,认识其在环境自净中作用并获得用于石油污染生物修复的菌种资源,以不同链长烷烃及支链烷烃对南沙深海沉积物样品进行富集与烃降解菌的分离鉴定,并结合16S rRNA基因文库和DGGE技术对降解菌群的结构进行了分析.结果表明：通过16S rRNA基因文库构建及PCR-DGGE分析发现,不同链长烷烃富集后的菌群中,食烷菌（Alcanivorax）在各降解菌群中都是绝对优势菌;另外,除烃海杆菌（Marinobacter hydrocarbonoclasticus）、海绵假单胞菌（Pseudomonas pachastrellae）,以及Idiomarina与Kangiella属的细菌也是菌群中较重要的优势菌.此外,通过2种平板培养基从烷烃富集的降解菌群中分离获得了8个不同属的细菌.本研究还首次发现Kangiella属（食烷菌科）的细菌可能参与了烷烃降解.南沙沉积物烷烃降解菌多为γ-Proteobacteria;其中,食烷菌科（Alcanivoraceae）（Alcanivorax与Kangiella属）的细菌在降解菌群中是绝对优势菌,假单胞菌与海杆菌在烷烃降解过程中也起着重要作用.     

深海沉积物中龙须菜降解菌群的筛选与鉴定及多样性分析

以龙须菜为唯一的营养源对深海沉积物进行富集和筛选,获得一个可降解龙须菜(Gracilaria lemaneiformis)、紫菜(Porphyra umbilicalis)和海带(Laminaria japonica)产生还原性寡糖的菌群.通过16S rRNA序列及16S rRNA-RFLP分析对该菌群中的细菌多样性进行了研究.结果表明,降解菌群中的细菌组成主要为弧菌属(Vibrio,8株)、火色杆菌属(Flammeovirga,7株)和希瓦氏菌属(Shewanella,5株),其中希瓦氏菌属和火色杆菌属的细菌在菌群中的丰度较高.采用龙须菜为唯一营养源的筛选培养基对菌群中的细菌进行分离,获得4株具有琼胶酶活力的细菌,包括2株火色杆菌属和2株希瓦氏菌属细菌.培养和未培养的结果均表明火色杆菌和希瓦氏菌这2个属为所研究的深海沉积物主要的龙须菜降解菌.对原始降解菌群和所分离的关键菌株进行了龙须菜酶解产糖能力的比较,结果发现菌群中的弧菌属菌株虽然自身没有酶解龙须菜的能力,但可能可以协助关键菌株,提高菌群对龙须菜的降解效率.因此本研究中所获得的菌群和菌株有望在琼胶寡糖的绿色生产中得到广泛应用.     

白令海和楚科奇海表层沉积物中多环芳烃降解微生物多样性

为了研究白令海(海盆及陆架)至楚科奇海陆架表层沉积物中多环芳烃(PAHs)降解菌的多样性,并获得新的PAHs降解菌资源。在GC-MS分析沉积物中PAHs种类和含量的基础上,以萘、菲和芘的混合物为唯一碳源和能源对表层沉积物样品进行富集,通过平板分离鉴定可培养菌株,并验证其降解能力;同时利用变性梯度凝胶电泳(DGGE)和Illumina高通量测序技术分析降解菌群结构。GC-MS测定结果表明,14个表层沉积物中PAHs总干质量介于32.99~276.97ng/g。富集菌群中共分离获得51株可培养细菌,平板纯培养、PCR-DGGE及Illumina测序结果均表明,菌群中优势的降解菌是γ-proteobacteria的Marinobacter,Pseudoalteromonas,Pseudomonas和Actinobacteria门的Dietzia菌。此外,Illumina测序结果还表明14个降解菌群在菌群结构组成上,可分为海盆区和陆架区两种类群;同时检测到一些低丰度的海洋专属PAHs降解菌,如Cycloclasticus,Alteromonas和Neptunomonas等。本文结果将加深对白令海及楚科奇海表层沉积物中PAHs降解菌资源与生物多样性的认识。     

不同碳源富集的石油烃降解菌群结构的分析

以原油和正十六烷为唯一碳源,从长期受石油污染的环境中富集和分离具有不同功能的石油烃降解菌,并利用平板法和PCR-DGGE法时不同碳源富集到的菌群结构进行分析.结果表明,利用2216E平板从以原油和正十六烷为碳源的富集液中各得到两株菌,分另11为TJ-1、TJ-2和TJL-1和TJL-2,分子生物学方法鉴定结果表明,这4株菌分别为短小芽孢杆菌(Bacillus pumilus)、威尼斯不动杆菌(Acinetobacter venetianus)、Oceanobacillus picturaeP和短小芽孢杆菌(Bacillus pumilus).PCR-DGGE分析结果表明,以原油和正十六烷为碳源的富集液中优势菌分别有5种和2种,且2种富集液中的优势菌明显不同.对比PCR-DGGE和平板法分析结果,可以看出PCR-DGGE法能够提供更全面的菌群结构信息.     

异养细菌与虾病关系的研究

对对虾养殖池海水、底质、虾池进水及其邻近水域海水中的异养细菌和弧菌进行了调查研究.结果表明,在对虾养殖环境中,异养细菌菌群结构和各类群属细菌数量变化异常.弧菌成为优势菌属,并显示了喜欢在营养丰富的环境中生长的特点.弧菌可作为有机污染的指示菌.弧菌的数量与对虾发病有一定关系.     

南极低温降解菌的筛选、鉴定与低温降解适应性分析

以南极海洋细菌为研究对象,采用2216E和MMC液体培养基结合的方法,从菌种库里筛选南极低温降解菌,进行石油烃降解率测定、表型特征确定和16SrRNA分子鉴定,构建系统发育树,并对南极低温降解菌NJ49的低温降解适应性进行了初步研究。结果表明,3株南极海洋细菌NJ41、NJ49和NJ289可以在以柴油为惟一碳源和能源的培养基中生长,被视为南极低温降解菌;16SrRNA分子鉴定结果表明,南极低温降解菌NJ41、NJ49和NJ289分别属于Planococcus、Shewanella和Pseudoalteromonas属;NJ49通过改变脂肪酸组分的膜修饰方式适应低温降解。     

曹妃甸近岸海域石油烃污染物的来源与空间分布研究

以曹妃甸邻近海域为研究区,利用生物标志化合物地球化学参数分析了表层沉积物中石油烃类的主要组成和分布特征,探讨了石油烃污染物的来源。结果表明,曹妃甸邻近海域表层沉积物中正构烷烃为混源,且陆源贡献小于海洋源。地质构型藿烷、甾烷在甾萜类化合物中为优势组分,且萜甾烷参数显示了有机质成熟度较高。饱和烃的多种地球化学参数显示了该区域表层沉积物可能受到成熟度较高的石油烃输入及其后期微生物降解的影响。该研究将为曹妃甸近岸沉积物输移研究提供基础资料,进而为曹妃甸围填海工程的科学决策提供相关科学数据。     

Evaluation method and application of the relative contribution of marine hydrocarbon source rocks in the Tarim basin: A case study from the Tazhong area

In total, 2.37 million tons of marine crude oil originating from mixed source rocks has been discovered in the Tarim basin. Geological and geochemical analyses have confirmed that these mixed hydrocarbons are mainly from two sets of source rocks, including the Cambrian – Lower Ordovician and Middle-Upper Ordovician hydrocarbon source rocks. In this study, we determined the set of source rocks primarily responsible for the mixed hydrocarbons and the next location to be explored. Differences in n-alkane carbon isotopes in end-member oils from Cambrian–Lower Ordovician and Middle-Upper Ordovician source rocks were examined. A material balance model and simulation methods were used to evaluate the relative amounts contributed by each source. The results from known reserves in the Tazhong area show that the mixing ratio or contribution is up to 65% from Cambrian–Lower Ordovician source rocks and is generally higher than that from Middle-Upper Ordovician source rocks. The discovery of deep hydrocarbons has caused the total oil contribution from the Cambrian–Lower Ordovician to increase. The mixing ratio of Cambrian–Lower Ordovician oil varies depending on the well, formation, and block. It increases from west to east horizontally and from top to bottom vertically. Hydrocarbons from Cambrian–Lower Ordovician source rocks migrate upward along faults, and the mixing ratio decreases as the distance from the oil source fault increases. Favorable areas for Cambrian–Lower Ordovician hydrocarbon exploration are deep layers and areas near the fault zone that are connected to deep layers. The material balance model for carbon isotopes and evaluation methods for relative contributions considered differences in relative concentration and carbon isotope structure of n-alkanes. Herein, new methods for the identification and evaluation of hydrocarbons in the petroleum system of this superimposed basin are presented.     

Petroleum hydrocarbons in sediments of Southampton water estuary

The sediments of Southampton Water were analysed for petroleum hydrocarbons to determine the fate of petrochemical refinery waste in the estuary. Much of the petroleum hydrocarbons appears to be removed by adsorption onto estuarine sediments close to source. Surface sediment concentrations range from 3·1mg/g dry weight near a refinery to 0·5 mg/g dry weight at locations distant from the refinery.The absence of any change with depth in total hydrocarbon concentrations from some sediment cores and the presence of distinct ‘oil’ horizons in others suggests that mixing of sediments in the area is very random. Sedimentation rates in areas where a distinct oil horizon was observed ranged from 1 to 3cm a year.     

Biota: sediment partitioning of aluminium smelter related PAHs and pulp mill related diterpenes by intertidal clams at Kitimat, British Columbia

The question of polycyclic aromatic hydrocarbon (PAH) bioavailability and its relationship to specific PAH sources with different PAH binding characteristics is an important one, because bioavailability drives PAH accumulation in biota and ultimately the biochemical responses to the PAH contaminants. The industrial harbour at Kitimat (British Columbia, Canada) provides an ideal location to study the bioavailability and bioaccumulation of sediment hydrocarbons to low trophic level biota. Samples of soft shell clams (Mya arenaria) and intertidal sediment collected from multiple sites over six years at various distances from an aluminium smelter and a pulp and paper mill were analysed for 106 PAHs, plant diterpenes and other aromatic fraction hydrocarbons. Interpretation using PAH source ratios and multivariate data analysis reveals six principal hydrocarbon sources: PAHs in coke, pitch and emissions from anode combustion from the aluminium smelter, vascular plant terpenes and aromatised terpenes from the pulp and paper mill, petroleum PAHs from shipping and other anthropogenic activities and PAHs from natural plant detritus. Harbour sediments predominantly contain either pitch or pyrogenic PAHs from the smelter, while clams predominantly contain plant derived PAHs and diterpenes from the adjacent pulp mill. PAHs from the smelter have low bioavailability to clams (Biota-Sediment Accumulation Factors; BSAFs <1 for pitch and coke; <10 for anode combustion, decreasing to ∼0.1 for the mass 300 and 302 PAHs), possibly due to binding to pitch or soot carbon matrices. Decreases in PAH isomer ratios between sediments and clams likely reflect a combination of variation in uptake kinetics of petroleum PAHs and compound specific metabolism, with the importance of petroleum PAHs decreasing with increasing molecular weight. Plant derived compounds exhibit little natural bioaccumulation at reference sites, but unsaturated and aromatised diterpenes released from resins by industrial pulping processes are readily accumulated by the clams (BSAFs >500). Thus while most of the smelter associated PAHs in sediments may not be bioavailable to benthic organisms, the plant terpenes (including retene, totarol, ferruginol, manool, dehydroabietane and other plant terpenes that form the chemical defence mechanism of conifers) released by pulp mills are bioavailable and possess demonstrated toxic properties. The large scale release of plant terpenes by some of the many pulp mills located in British Columbia and elsewhere represents a largely undocumented risk to aquatic biota.     

Hydrocarbons in the sediments of Port Valdez,Alaska: Consequences of five years' permitted discharge

Hydrocarbons have been investigated in the sediments of Port Valdez, Alaska after three to five years of oil terminal operation with a routine daily discharge of 170 kg of petroleum residue in an otherwise undeveloped area. Surficial benthic sediments (0–5 cm) and core segments down to 30 cm have been analyzed. Information about total hydrocarbons, unresolved complex mixture, normal alkanes, pristane, phytane, hopanes and polycyclic aromatic hydrocarbons indicates that petroleum, biogenic hydrocarbons, and combustion-derived aromatic hydrocarbons are present in sediments around the terminal at concentrations up to 218 μg g^?1 dry sediment. The vertical, horizontal and temporal distributions of anthropogenic hydrocarbons indicate that most of the sedimentary hydrocarbon accumulation has occurred within 1 km of the terminal. A simple calculation suggests that less than 3% of the total oil discharged during routine operations of the terminal has entered the sediments of Port Valdez. For comparison sediment hydrocarbon accumulation associated with a nearby small boat harbor was also examined.     

A STUDY ON MICROBIAL DEGRADATION FOR OIL POLLUTANTS IN MARINE ENVIRONMENT

This paper presents the degradation of 10 strains of hydrocarbon-degrading bacteria isolated from Xiamen harbor for 8 kinds of hydrocarbon and their mixed hydrocarbons incubated for 7 days at 25℃. The results indicate that the degradation rates of different genera of bacteria, which are measured with gas chromatography, are different for 8 hydrocarbons, thus showing the differences among genera; that the degradation rates of most strains for alkane hydrocarbons are higher than those for aromatic hydrocarbons; that the mixed strains favour the degratation for mixed hydrocarbons; and that the concentration of hydrocarbon is an important factor affecting degradation rates.     

Use of the Comet assay to assess DNA damage in hemocytes and digestive gland cells of mussels and clams exposed to water contaminated with petroleum hydrocarbons

Oil spills can result in the deposition of large quantities of petroleum hydrocarbons into intertidal and shallow waters seriously impacting bivalve populations. Petroleum hydrocarbons are enriched in polycyclic aromatic hydrocarbons (PAH) and PAH analogs many of which may have potential to damage DNA. The Comet assay is useful for assessing DNA damage and has been used to a limited degree with aquatic organisms, but mostly with studies in vitro. We have carried out studies with the Comet assay to assess the DNA damaging potential of complex mixtures of petroleum hydrocarbons for bivalves. Experiments were carried out with mussels (Mytilus edulis) and clams (Mya arenaria) with dispersions and water soluble fractions of an Arabian crude oil which was also chemically characterized in detail by GC-MS. Pilot studies were first conducted to evaluate test performance and reproducibility. An interindividual coefficient of variation ranging from 17 to 30% was established for the assay with hemocytes and digestive gland cells of both species. Exposure to hydrocarbon fractions had no significant impact on clams. However, an increase in DNA damage was observed at P < 0.1 with digestive gland cells of mussels exposed to aqueous fractions of a light crude oil. These studies have demonstrated a potential for DNA damage in bivalves exposed to oil spills in inshore waters as well as potential for interspecies sensitivity.     

浙江镇海潮间带沉积物中可培养微生物初步研究

针对浙江镇海潮间带沉积物样 品,采用纯培养法分离培养海洋微生物,并基于16S rRNA基因序列,开展系统发育学研究,分析沉积物细菌群落结构及多样性。分离获得细菌39株,16S rRNA基因序列分析表明,这些菌株分别属于厚壁菌门Firmicutes(51. 3%)、变形杆菌门Proteobacteria(30.8%)、拟杆菌门Bacteroidetes(15. 4%)和放线菌门Actinobacteria(2. 6%)4个类群。厚壁菌门和变形杆菌门的菌株主要归属于芽孢杆菌纲Bacilli和a-变形杆菌纲Alphaproteobacteria,它们可能在近海海洋生态系统的元素地球化学循环中发挥了重要作用。此外,部分菌株16S rRNA基因序列与已报道物种的相似性较低,可能代表了新的分类单元。研究丰富了浙江近海微生物种质资源,并拓展了对浙江近海可培养细菌多样性的认识,为今后开发利用海洋微生物资源积累了资料。     

Hydrocarbons in bivalve mollusks of Port Valdez, Alaska: Consequences of five years' permitted discharge

Petroleum hydrocarbons were investigated in Mytilus edulis and Macoma balthica at six intertidal sites in Port Valdez, Alaska after three to five years of oil terminal operation. Information about the concentrations of total hydrocarbons, unresolved complex mixture, normal alkanes, isoprenoids, olefins, hopanes and polycyclic aromatic hyrocarbons shows that petroleum accumulation in these bivalve mollusks is greatest at the Valdez municipal boat harbor and a crude oil terminal which has been permitted to discharge 170 kg oil day⁻¹. Accumulation two- to ten-fold less were observed at 3 km from the terminal and boat harbor. The temporal trends in petroleum hydrocarbon concentrations in M. edulis and M. balthica reflect the effects of selective retention and depuration of different hydrocarbon classes as well as variation in hydrocarbon sources. It is not clear whether the petroleum concentrations in Port Valdez bivalves had reached steady state by 1982, the final sampling time reported here.     

大亚湾大鹏澳油污染状况和自净能力的研究 --污染有机物可降解性研究

将已知浓度的污染物石油烃加入到取自大亚湾大鹏澳天然海水中，进行污染物自净能力实验室模拟试验，探讨了各种条件下微生物对有机物降解的影响，用有机物浓度、ATP、DO、BOD5、COD描述了有机物的降解过程。根据实验结果，得出BOD5和ATP可用来指示降解过程中微生物量的变化；此外，在一定条件下，石油烃的生物降解一级反应方程可描述为OIL=17.442e^-0.1949d。