2010年夏季白令海浮游细菌的多样性和群落组成分析

海洋浮游细菌在海洋生态系统的运行中起着关键性的生物地球化学作用。利用2010年夏季第4次北极考察获得的白令海不同深度水样,运用变性梯度凝胶电泳技术以及克隆建库等方法,来了解白令海中浮游细菌的多样性和群落组成等信息。结果表明:在白令海海盆区B07站位的50 m处,其浮游细菌的香农多样性指数是最高的,为2.61;香农多样性指数最低的是B07站位的3 m处,为1.99。白令海海盆区的细菌多样性变化比陆架区要大,有可能与海洋环境的复杂变化有关。通过克隆测序,鉴定出的浮游细菌类群分为4大类：α-变形杆菌、β-变形杆菌、γ-变形杆菌和拟杆菌。其中γ-变形杆菌所占的比例最大,为53%,是白令海中的优势种群;拟杆菌其次,为37%。这些浮游细菌在白令海中的分布情况为：γ-变形杆菌和拟杆菌存在于3个位点的所有水层中,α-变形杆菌只存在于B07站位的50m和100 m水层中,β-变形杆菌除B13站位的0 m处外,存在于其他站位的所有水层中。白令海B断面3个站位的温度随着深度的增加而降低,盐度随着深度的增加而升高。大体上白令海海盆区（B07站位）的硝酸盐、磷酸盐和硅酸盐浓度比陆架区（B15和B13站位）高,陆架区的铵盐浓度较海盆区高。     

Molecular identification of Prorocentrum （Dinophyceae） species

A fragment of a large sub-unit ribosomal DNA (LrDNA) of 12 strains of Prorocentrum species was amplified by polymerase chain reaction (PCR). The PCR products were digested by 3 restriction endonucleases (Cfo Ⅰ, Hae Ⅲ, and RSA Ⅰ) and then resolved in agarose gels. Results show that different species had different RFLP patterns, except for P arcuatum (ME 131), which had the same pattern to P. micans (ME 160 and 04).The same fragment of 19 strains of the genus was also amplified and subjected to denaturing gradient gel electrophoresis (DGGE). 11 different patterns were resolved. Different cultures of a same species had the same pattern. The results of RFLP and DGGE analyses showed that eight newly isolated epibenthic Prorocentrum species were different from each other, and also from other cultured ones examined in this study. P arcuatum(MEI32) could not be differentiated from P. micans (MEI60 and 04), it was probably mis-identified, since they are quite different morphologically. P. redfieldii (MEI38) could also not be distinguished form P. triestinium(MEI32), it should be regarded as a synonym ofP. triestinium. Unexpectedly, a restriction site was found in P.micans, compared with previous sequence data.     

贵州赫章土法炼锌导致的土壤重金属污染特征及微生物生态效应

贵州省赫章县妈姑镇新关寨附近的土法炼锌不仅导致植被的破坏，而且使附近土壤中重金属有不同程度的积累，Zn达到162.23-877.88mg/kg,Pb为37.24-305.56mg/kg,Cd为0.50-16.43mg/kg，大大超过了当地的土壤背景值；而且重金属含量（特别是Pb和Zn含量）与土壤中的Fe2O3和Al2O3有极显著的正相关关系，显示土壤中铁氧化物和粘土矿物对重金属的固定作用。化学形态研究表明，Pb和Zn在土壤中主要表现为铁锰氧化物结合态与残渣态，而Cd的情况与Pb和Zn完全相反，其形态以可交换态为主。本区土壤微生物生物量（用含碳量表示）较低，仅为57.00-388.00μg/g，而且与土壤重金属间有显著的负相关关系，特别是与Zn的相关系数高达-0.7801，反映出重金属对微生物的毒害作用。Biolog测试结果表明，土壤微生物群落结构间没有明显的差别；土壤微生物DNA经PCR（聚合酶链式反应）和DGGE（变性梯度胶电泳）反应后的基因片段非常一致，反映出不同程度的重金属污染并没有导致土壤微生物群落结构的改变和基因损伤，另一方面也表明，土壤本身有强烈的固定重金属能力，抵御了重金属对土壤生物的进一步毒害。     

Analysis of community structure of a microbial consortium capable of degrading benzo(a)pyrene by DGGE

A microbial consortium was obtained by enrichment culture of sea water samples collected from Botan oil port in Xiamen, China, using the persistent high concentration of a mixture of polycyclic aromatic hydrocarbons enrichment strategy. Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial composition and community dynamic changes based on PCR amplification of 16S rRNA genes during batch culture enrichment. Using the spray-plate method, three bacteria, designated as BL01, BL02 and BL03, which corresponded to the dominant bands in the DGGE profiles, were isolated from the consortium. Sequence analysis showed that BL01, BL02 and BL03 were phylogenetically close to Ochrobactrum sp., Stenotrophomonas maltophilia and Pseudomonas fluorescens, respectively. The degradation of benzo(a)pyrene (BaP), a model high-molecular-weight polycyclic aromatic hydrocarbon (HMW PAH) compound was investigated using individual isolates, a mixture of the three isolates, and the microbial consortium (BL) originally isolated from the oil port sea water. Results showed that the order of degradative ability was BL > the mixture of the three isolates > individual isolates. BL degraded 44.07% of the 10 ppm BaP after 14 days incubation, which showed the highest capability for HMW PAH compound degradation.Our results revealed that this high selective pressure strategy was feasible and effective in enriching the HMW PAH-degraders from the original sea water samples.     

Darstellung der mikrobiellen Besiedlungsstruktur verschiedener Grundwasserhabitate durch Anwendung molekularbiologischer Methoden

Community Structures of Different Groundwater Habitats Investigated Using Methods of Molecular Biology The degradation of pollutants in groundwater and aquifers depends on microbiological and hydrogeochemical processes. To understand the transport and fate of anthropogenic compounds during bank filtration and artificial recharge of groundwater it is necessary to gain more information about the structure of microbial populations in these systems. The population structure of aerobic, anaerobic groundwater habitats and of water samples during artificial groundwater recharge was examined by 16S rDNA based analysis. Water and sediment samples were collected from a groundwater catchment area with artificial groundwater recharge near the river Ruhr in NW-Germany. 16S rRNA genes of mixed bacterial DNA from different samples were amplified by PCR (polymerase chain reaction) with eubacterial primer sequences. To reveal eubacterial population structure amplified PCR-products were separated by DGGE (denaturing gradient gel electrophoresis) on the basis of melting domain structure and nucleotide composition. DGGE patterns of groundwater enrichment cultures and groundwater samples were compared to demonstrate differences between the use of cultivation dependent and molecularbiological approaches. The DGGE pattern of groundwater is very complex and differs significantly from DGGE patterns of groundwater enrichment cultures characterized by a small number of distinct bands. This shows the small quantity of culturable microorganisms in groundwater eco-systems. Aerobic and anaerobic groundwater and sediment samples differ markedly in their DGGE profiles. Different hydrogeochemical zones of this groundwater catchment area are mirrowed by distinct DGGE patterns indicating changes in microbial community structure.Analysis of bacterial population structure in the course of artificial groundwater recharge shows identical DGGE patterns comparing surface water samples to samples taken be-fore gravel prefiltration and before sand filtration. In contrast the DGGE pattern of artificial recharged groundwater differs markedly, indicating significant changes in microbial population during underground passage.     

应用DGGE技术分析北黄海和青岛近海藻类DNA病毒遗传多样性

采用DGGE技术对北黄海和青岛近海藻类DNA病毒群落多样性进行调查。结果表明,在北黄海A604站、B107站和C803站分别得到10个、8个、8个OTUs,在青岛近海ZQ站得到6个OTUs,说明不同海域藻类DNA病毒多样性不同,在北黄海较丰富,青岛近海较低。北黄海3个站间相比,OTUs数量和位置均存在差异,表明同一海域不同站位藻类DNA病毒多样性也存在差异。在所得OTUs中,有2个OTUs在4个站中均存在,说明在北黄海和青岛近海存在着一些共同的藻类DNA病毒。4个站点藻类DNA病毒优势种均不相同,在A604站和ZQ仅有一个优势种,而在B107和C803各有两个优势种。     

海洋微微型真核生物分子多样性的研究方法

微微型真核生物在海洋中广泛存在,是初级生产力的重要贡献者,也是微食物环的重要组成部分.由于微微型真核生物形体微小且不同种类缺乏明显形态学特点,故而难以利用电镜等传统方法精确研究其系统发生关系和种类组成.运用分子生物学技术直接对微微型真核生物种类组成进行研究已成为微微型真核生物多样性研究的重要方法.本文概述了目前世界范围内的微微型真核生物分子多样性研究进展以及我国在该领域中的研究状况.综述了微微型真核生物分子多样性研究中常用的靶标基因,包括18S rDNA和ITS序列等真核生物保守基因及psbA和rbcL等功能基因;常用的分子生物学方法,如克隆文库构建、DGGE/TGGE、PCR-RFLP/T-RFLP、FISH、宏基因组等;以及目前研究方法中存在的一些问题.     

Study on diversity and temporal-spatial characteristics of eukaryotic microorganisms on Glacier No.1 at the Urumqi River Head, Tianshan

Surface snow samples of different altitudes and snow pit samples were collected from Glacier No.1 at the Urumqi River Head, Tianshan. Denaturing gradient gel electrophoresis (DGGE) was used to examine the diversity and temporal-spatial characteristics of eukaryotic microorganisms with different altitudes and depths. Results show that the eukaryotic microorganisms belong to four kingdoms—Viridiplantae, Fungi, Amoebozoa, and Alveolata. Among them, algae (especially Chlamydomonadales) were the dominant group. The diversity of eukaryotic microorganisms was negatively correlated with altitude and accumulation time, but positively correlated with δ¹⁸O values. These results indicate that temperature is the main factor for the temporal- spatial change of eukaryotic microorganisms, and the diversity of eukaryotic microorganisms could be an index for climate and environmental change.     

应用PCR-DGGE指纹技术分析高温季节仿刺参养殖水环境中菌群多样性

采用16S rDNA的PCR-DGGE基因指纹技术,于6~9月间,对辽宁普兰店湾2个相邻的刺参养殖池塘进行了养殖水环境中菌群多样性的分析,首先用2216E固体培养基对仿刺参养殖水环境中的细菌总数进行统计,再采用PCR-DGGE指纹技术分析菌群多样性。结果表明,池塘水环境中较高水平的细菌数,且9月为6月的3倍;高温季节刺参养殖水环境中细菌种群较为丰富,其中大多数属于变形菌门;6~9月份样品具有一致的优势菌群,分别为Pseudomonas sp.,Uncul-tureddelta bacterium,Ostreococcus sp.以及Unculturedalpha proteobacterium。同时发现,同一时间采集的样品,有一定的菌群相似性,并在聚类分析图中分别成簇,检测样品的细菌多样性指数在1.7至2.5之间。     

Deep-sea bacterial communities in sediments and guts of deposit-feeding holothurians in Portuguese canyons (NE Atlantic)

Deposit-feeding holothurians often dominate the megafauna in bathyal deep-sea settings, in terms of both abundance and biomass. Molpadia musculus is particularly abundant at about 3400 m depth in the Nazaré Canyon on the NE Atlantic Continental Margin. However, these high abundances are unusual for burrowing species at this depth. The objective of this research was to understand the reasons of the massive occurrence of these molpadiid holothurians in the Nazaré Canyon. To address this question we investigated possible trophic interactions with bacteria at sites where the organic content of the sediment was different (Setúbal and Cascais Canyons, NE Atlantic Continental Margin). The molecular fingerprinting technique of Denaturing Gradient Gel Electrophoresis (DGGE) with band sequencing, combined with non-metric multi-dimensional scaling and statistical analyses, was used to compare the bacterial community diversity in canyon sediments and holothurian gut contents. Our results suggest that M. musculus does not need to develop a specialised gut bacterial community to aid digestion where the sediment is rich in organic matter (Nazaré Canyon); in contrast, such a community may be developed where the sediment is poorer in organic matter (Cascais Canyon).