Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

Enrichment of the hydrogen-producing microbial community from marine intertidal sludge by different pretreatment methods

To determine the effects of pretreatment on hydrogen production and the hydrogen-producing microbial community, we treated the sludge from the intertidal zone of a bathing beach in Tianjin with four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing. The results showed that acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. The efficiency of the hydrogen production of the acid-pretreated sludge was 0.86 +/- 0.07 mol H-2/mol glucose (mean +/- S.E.), whereas that of the sludge treated with heat-shock, freezing and thawing, base method and control was 0.41 +/- 0.03 mol H-2/mol glucose, 0.17 +/- 0.01 mol H-2/mol glucose, 0.11 +/- 0.01 mol H-2/mol glucose and 0.20 +/- 0.04 mol H-2/mol glucose, respectively. The result of denaturing gradient gel electrophoresis (DGGE) showed that pretreatment methods altered the composition of the microbial community that accounts for hydrogen production. Acid and heat pretreatments were favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp., Enterococcus sp. and Bacillus sp., However, besides hydrogen-producing bacteria, much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Therefore, based on our results, we concluded that, among the four pretreatment methods using acid, heat-shock, base or freezing and thawing, acid pretreatment was the most effective method for promoting hydrogen production of microbial community. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

Vertical distribution of bacterial and archaeal communities along discrete layers of a deep-sea cold sediment sample at the East Pacific Rise (similar to 13 degrees N)

The community structure and vertical distribution of prokaryotes in a deep-sea (ca. 3,191 m) cold sediment sample (ca. 43 cm long) collected at the East Pacific Rise (EPR) similar to 13 degrees N were studied with 16SrDNA-based molecular analyses. Total community DNA was extracted from each of four discrete layers EPRDS-1, -2, -3 and -4 (from top to bottom) and 16S rDNA were amplified by PCR. Cluster analysis of DGGE profiles revealed that the bacterial communities shifted sharply between EPRDS-1 and EPRDS-2 in similarity coefficient at merely 49%. Twenty-three sequences retrieved from DGGE bands fell into 11 groups based on BLAST and bootstrap analysis. The dominant groups in the bacterial communities were Chloroflexi, Gamma proteobacteria, Actinobacterium and unidentified bacteria, with their corresponding percentages varying along discrete layers. Pairwise Fst (F-statistics) values between the archaeal clone libraries indicated that the archaeal communities changed distinctly between EPRDS-2 and EPRDS-3. Sequences from the archaeal libraries were divided to eight groups. Crenarchaea Marine Group I (MGI) was prevalent in EPRDS-1 at 83%, while Uncultured Crenarchaea group II B (UCII B) abounded in EPRDS-4 at 61%. Our results revealed that the vertically stratified distribution of prokaryotic communities might be in response to the geochemical settings and suggested that the sampling area was influenced by hydrothermalism. The copresence of members related to hydrothermalism and cold deep-sea environments in the microbial community indicated that the area might be a transitional region from hydrothermal vents to cold deep-sea sediments.

海藻表面附着弧菌多样性分析及方法研究

对四种大型海藻：江篱(Gracilaria textorii)、孔石莼（Ulva pertusa）、海带苗（Laminaria japonica）和多管藻（Polysiphonia urceolata）表面附着弧菌的多样性进行了分析，同时对多样性分析中采用的不同方法进行了比较。 利用TCBS培养基从四种海藻表面总共分离到12株细菌：G1、G2分离自江篱，U3分离自孔石莼，L4、L5、L6分离自海带苗，P7、P8、P9、P10、P11、P12分离自多管藻。菌株G1，G2属于盐单胞菌(Halomonas)，其余10株细菌都属于弧菌(Vibrio)。 对12株细菌的酶学活性、抗生素抗性进行了研究。发现除G1、G2外，其余菌株都可产生淀粉酶和明胶酶。菌株G1、G2和U3对氨苄青霉素、链霉素和四环素有很强抗性。 采用多种分子生物学方法（16S rDNA、gyrB、RFLP、DGGE ）分析了上述12株菌的系统发育关系，对不同方法获得的结果进行了比较。 12株菌的16S rDNA系统发生树可分为4个明显分支。G1、G2与细菌H. meridiana构成一个分支。U3与V. lentus构成一个分支。L4、L5、L6、P9、P10、P11、P12与V. tasmaniesis形成一个分支。P7、P8与V. splendidus形成一个分支。 以gyrB基因序列为基础构建的系统发生树将G1、G2以外的10株弧菌分成4个较大分支，L4、P9属于同一分支，L6、P8、P10、P11和P12属于一个大的分支， U3、L5各自构成一个分支。菌株G1、G2没有得到gyrB基因序列扩增带。表明试验设计的引物是弧菌特异性引物。相对16S rDNA，不同菌株间gyrB基因序列差异性更大。 RFLP分析结果显示，12株细菌HinfI酶切后得到6种带型，SmaI酶切后得到4种带型。结合16S rDNA、gyrB基因的分析结果可知，RFLP可以在种的水平上有效进行细菌多样性分析。 12株细菌的DGGE分析结果显示，G1、G2与其它菌株电泳图谱有明显不同。菌株U3独自构成一种带型，L5、L6构成一种带型。表明DGGE技术在属的水平上分析细菌多样性效果较好，在种的水平上分析细菌多样性有一定的局限性。

胶州湾和东太平洋海隆(~13°N)沉积物微生物多样性研究

海洋是一个巨大的生态系统，多样的微生物是构成海洋生态系统的基本元素。海洋微生物的群落结构及演变深刻的反映着海洋生态系统的变迁。本文采用分子生物学技术，研究了近海沉积物生态系统——胶州湾沉积物中细菌的多样性、群落结构的时空演替规律以及远洋深海沉积物生态系统——东太平洋海隆北纬13o附近深海沉积物中细菌和古细菌群落结构沿沉积物断层的分布情况，结果表明在两处沉积物中，微生物群落的结构都与环境因子有显著的相关性，是反映海洋沉积物环境特征的重要（分子）标志物，并且可能在这些环境中参与生物地球化学循环等重要过程。 1.从胶州湾不同区域的8个代表性站点采集4个季度的沉积物样品。提取总基因组DNA，利用16S rDNA作为分子标记，采用克隆文库对胶州湾沉积物中细菌群落的组成、空间分布和季节演替规律进行了研究。结果显示沉积物中的细菌具有高度多样性，来自于13个细菌门，同时还有28%的未鉴定克隆，表明胶州湾沉积物中蕴藏着巨大的微生物资源。其中已鉴定的优势种群是α-、β-、γ-、δ-变形细菌、绿弯菌、厚壁菌、蓝细菌和放线菌。同时还包括酸杆菌、拟杆菌、浮霉菌、疣微菌、芽单胞菌、绿菌、梭杆菌、异常球菌-栖热菌等类群的存在。将各克隆库的组成与温度、总碳、总氮等环境因子结合分析，结果显示细菌群落结构更替的主要驱动力是季节变化所带来的温度等环境因子的演变。对数据库中与本研究所获得序列具有最近亲缘关系序列的来源环境进行分析表明，胶州湾中细菌群落受航运活动、水产养殖、重金属污染等人类活动的明显影响，同时这些活动表现出显著的空间特异性，比如C4和D6等站点明显受到航运活动的影响，而A3和Y1等站点则容易受到沿岸径流所带来的淡水和油污染的影响。 2.分别利用PCR-DGGE和克隆文库技术对东太平洋海隆北纬13o附近深海柱状沉积物样品中细菌和古菌群体进行研究，结果显示这些微生物群落沿四个分别代表不同沉积年代断层明显的成层分布，与环境因子结合分析表明这种成层分布与氧化还原性质等地球化学特征的成层分布相吻合，提示我们该生态系统中的微生物受到环境因子的巨大作用，同时也表明这些微生物可能参与该生态系统中硫、金属元素代谢等过程。通过系统发育分析，四个断层中的微生物群落中呈现出很多与热液活动相关的个体（其中34.7%的细菌序列和31%的古菌序列与来源于各种热液环境的序列具有最近的亲缘关系）。但总体群落结构分析表明该区域可能属于热液活动影响区域的边缘，处于从热液活动环境到普通的低温沉积物环境的过渡区域。 3.将在胶州湾和东太平洋海隆北纬13o附近海洋沉积物生态系统中都存在的优势细菌类群（α-、β-、γ-、δ-变形细菌和放线菌、绿弯菌、厚壁菌、酸杆菌、浮霉菌）进行系统发育分析和背景比较分析，结果显示两处沉积物中的细菌优势种群虽然在大类群上很多是相同的，但是可能由于两处沉积物中不同物理化学等环境因子的选择作用（如胶州湾的近海特征和人为活动，东太平洋深海特点和热液活动），而导致优势种群在系统发育关系上距离比较远。这表明独特的微生物群落结构，特别是优势种群的群落结构信息是描述特定环境生态系统的重要方面。本研究表明在全球环境变迁中,自然环境因子和人类活动都在深刻改变着微生物群落的结构和功能。本文阐述了在环境变迁特定时期两处沉积物生态系统中的微生物群落结构及时空差异，为研究大范围生态系统的演变提供了依据，同时也为在两处沉积物环境中进行微生物参与的生物地球化学研究奠定了基础。

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。

Análise molecular de fungos micorrízicos arbusculares em raízes de milho.

A comunidade nativa de fungos micorrízicos arbusculares (FMA), presente em amostras de raiz e de solo rizosférico de 15 genótipos de milho contrastantes para eficiência no uso de fósforo, foi avaliada pela técnica de eletroforese em gel de gradiente desnaturante (DGGE). Foram amplificados fragmentos do DNA ribossomal com primers específicos para as famílias Acaulosporaceae e Glomeraceae. Os primers para a família Glomeraceae foram eficientes em diferenciar a estrutura da população de fungos micorrízicos, indicando grande diversidade da comunidade entre os genótipos. No DGGE específico para Glomeraceae, foram observadas bandas exclusivas nas linhagens eficientes L228-3 e L3, ambas cultivadas sob baixo teor de fósforo, indicando uma associação preferencial entre os genótipos e os simbiontes, que pode resultar em melhor eficiência na aquisição de fósforo. Além disso, a presença de Glomus clarum nestas duas linhagens eficientes, cultivadas sob baixo P, indica uma possível relação dessa espécie à tolerância ao estresse de P nesse solo. Com relação à família Acaulosporaceae, a técnica de DGGE detectou pouca variação entre os genótipos cultivados em baixo P, além de menor diversidade de fungos micorrízicos dessa família colonizando as raízes de milho.

Spatio-temporal variability in ammonia oxidation and ammonia oxidising bacteria and archaea in coastal sediments of the Western English Channel

The abundance of ammonia-oxidising bacterial (AOB) and ammonia-oxidising archaeal (AOA) (amoA) genes and ammonia oxidation rates were compared bimonthly from July 2008 to May 2011 in 4 contrasting coastal sediments in the western English Channel. Despite a higher abundance of AOA amoA genes within all sediments and at all time-points, rates of ammonia oxidation correlated with AOB and not AOA amoA gene abundance. Sediment type was a major factor in determining both AOB amoA gene abundance and AOB community structure, possibly due to deeper oxygen penetration into the sandier sediments, increasing the area available for ammonia oxidation. Decreases in AOB amoA gene abundance were evident during summer and autumn, with maximum abundance and ammonia oxidation rates occurring in winter and early spring. PCR-DGGE of AOB amoA genes indicated that no seasonal changes to community composition occurred; however, a gradual movement in community composition occurred at 3 of the sites studied. The lack of correlation between AOA amoA gene abundance and ammonium oxidation rates, or any other environmental variable measured, may be related to the higher spatial variation amongst measurements, obscuring temporal trends, or the bimonthly sampling, which may have been too infrequent to capture temporal variability in the deposition of fresh organic matter. Alternatively, AOA may respond to changing substrate concentrations by an increase or decrease in transcript rather than gene abundance.

Disturbance to conserved bacterial communities in the cold water gorgonian coral Eunicella verrucosa

The bacterial communities associated with healthy and diseased colonies of the cold-water gorgonian coral Eunicella verrucosa at three sites off the south-west coast of England were compared using denaturing gradient gel electrophoresis (DGGE) and clone libraries. Significant differences in community structure between healthy and diseased samples were discovered, as were differences in the level of disturbance to these communities at each site; this correlated with depth and sediment load. The majority of cloned sequences from healthy coral tissue affiliated with the Gammaproteobacteria. The stability of the bacterial community and dominance of specific genera found across visibly healthy colonies suggest the presence of a specific microbial community. Affiliations included a high proportion of Endozoicomonas sequences, which were most similar to sequences found in tropical corals. This genus has been found in a number of invertebrates and is suggested to have a role in coral health and in the metabolisation of dimethylsulfoniopropionate (DMSP) produced by zooxanthellae. However, screening of colonies for the presence of zooxanthellae produced a negative result. Diseased colonies showed a decrease in affiliated clones and an increase in clones related to potentially harmful/transient microorganisms but no increase in a particular pathogen. This study demonstrates that a better understanding of these bacterial communities, the factors that affect them and their role in coral health and disease will be of critical importance in predicting future threats to temperate gorgonian communities.

Microbial analysis of soil and groundwater from a gasworks site and comparison to a sequenced biological reactive barrier remediation process (SEREBAR)

Aims: To investigate the distribution of a polymicrobial community of biodegradative bacteria in (i) soil and groundwater at a former manufactured gas plant (FMGP) site and (ii) in a novel SEquential REactive BARrier (SEREBAR) bioremediation process designed to bioremediate the contaminated groundwater. Methods and Results: Culture-dependent and culture-independent analyses using denaturing gradient gel electrophoresis (DGGE) and polymerase chain reaction (PCR) for the detection of 16S ribosomal RNA gene and naphthalene dioxygenase (NDO) genes of free-living (planktonic groundwater) and attached (soil biofilm) samples from across the site and from the SEREBAR process was applied. Naphthalene arising from groundwater was effectively degraded early in the process and the microbiological analysis indicated a dominant role for Pseudomonas and Comamonas in its degradation. The microbial communities appeared highly complex and diverse across both the sites and in the SEREBAR process. An increased population of naphthalene degraders was associated with naphthalene removal. Conclusion: The distribution of micro-organisms in general and naphthalene degraders across the site was highly heterogeneous. Comparisons made between areas contaminated with polycyclic aromatic hydrocarbons (PAH) and those not contaminated, revealed differences in the microbial community profile. The likelihood of noncultured bacteria being dominant in mediating naphthalene removal was evident. Significance and Impact of the Study: This work further emphasizes the importance of both traditional and molecular-based tools in determining the microbial ecology of contaminated sites and highlights the role of noncultured bacteria in the process.

Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms

Rhizosphere microorganisms play an important role in soil carbon flow, through turnover of root exudates, but there is little information on which organisms are actively involved or on the influence of environmental conditions on active communities. In this study, a (CO2)-C-13 pulse labelling field experiment was performed in an upland grassland soil, followed by RNA-stable isotope probing (SIP) analysis, to determine the effect of liming on the structure of the rhizosphere microbial community metabolizing root exudates. The lower limit of detection for SIP was determined in soil samples inoculated with a range of concentrations of C-13-labelled Pseudomonas fluorescens and was found to lie between 10(5) and 10(6) cells per gram of soil. The technique was capable of detecting microbial communities actively assimilating root exudates derived from recent photo-assimilate in the field. Denaturing gradient gel electrophoresis (DGGE) profiles of bacteria, archaea and fungi derived from fractions obtained from caesium trifluoroacetate (CsTFA) density gradient ultracentrifugation indicated that active communities in limed soils were more complex than those in unlimed soils and were more active in utilization of recently exuded C-13 compounds. In limed soils, the majority of the community detected by standard RNA-DGGE analysis appeared to be utilizing root exudates. In unlimed soils, DGGE profiles from C-12 and C-13 RNA fractions differed, suggesting that a proportion of the active community was utilizing other sources of organic carbon. These differences may reflect differences in the amount of root exudation under the different conditions.

Identifying trophic variation in a marine suspension feeder:DNA- and stable isotope-based dietary analysis in Mytilus spp.

Accurate field data on trophic interactions for suspension feeders are lacking, and new approaches to dietary analysis are necessary. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was integrated with stable isotope analysis to examine dietary patterns in suspension-feeding Mytilus spp. from seven spatially discrete locations within a semi-enclosed marine bay (Strangford Lough, Northern Ireland) during June 2009. Results of the two methods were highly correlated, reflecting dietary variation in a similar manner. Variation in PCR-DGGE data was more strongly correlated with the principal environmental gradient (distance from the opening to the Irish Sea), while values of dC and dN became progressively enriched, suggesting a greater dependence on animal tissue and benthic microalgae. Diatoms and crustaceans were the most frequently observed phylotypes identified by sequencing, but specific DNA results provided little support for the trophic trends observed in the stable isotope data. This combined approach offers an increased level of trophic insight for suspension feeders and could be applied to other organisms. © 2012 Springer-Verlag Berlin Heidelberg.

Balancing Inflammatory, Lipid, and Xenobiotic Signaling Pathways by VSL#3, a Biotherapeutic Agent, in the Treatment of Inflammatory Bowel Disease

Background: The interleukin 10 knockout mouse (IL10-KO) is a model of human inflammatory bowel disease (IBD) used to Study host microbial interactions and the action of potential therapeutics. Using Affymetrix data analysis, important signaling pathways and transcription factors relevant to gut inflammation and antiinflammatory probiotics were identified.

Methods: Affymetrix microarray analysis on both wildtype (WT) and IL10-KO mice orally administered with and without the probiotic VSL#3 was performed and the results validated by real-time polymerase chain reaction (PCR), immunocytochemistry, proteomics, and histopathology. Changes in metabolically active bacteria were assessed with denaturing gradient gel electrophoresis (DGGE).

Results: Inflammation in IL10-KO mice was characterized by differential regulation of inflammatory, nuclear receptor, lipid, and xenobiotic signaling pathways. Probiotic intervention resulted in downregulation of CXCL9 (fold change [FC] = -3.98, false discovery rate [FDR] = 0.019), CXCL10 (FC = -4.83, FDR = 0.0008), CCL5 (FC -3.47 FDR = 0.017), T-cell activation (Itgal [FC = -4.72, FDR = 0.00009], Itgae [FC = -2.54 FDR = 0.0044]) and the autophagy gene IRGM (FC = -1.94, FDR = 0.01), a recently identified susceptibility gene in human IBD. Consistent with a marked reduction in integrins, probiotic treatment decreased the number of CCL5+ CD3+ double-positive T Cells and upregulated galectin2, which triggers apoptosis of activated T cells. Importantly, genes associated with lipid and PPAR signaling (PPAR alpha [FC = 2.36, FDR = 0.043], PPARGC1 alpha [FC 2.58, FDR = 0.016], Nrld2 [FC = 3.11, FDR = 0.0067]) were also upregulated. Altered microbial diversity was noted in probiotic-treated mice.

Conclusions: Bioinformatics analysis revealed important immune response. phagocytic and inflammatory pathways dominated by elevation of T-helper cell 1 type (TH1) transcription factors in IL10-KO mice. Probiotic intervention resulted in a site-specific reduction of these pathways but importantly upregulated PPAR, xenobiotic, and lipid signaling genes. potential antagonists of NF-kappa B inflammatory pathways.

Microbial Communities Associated with Healthy and White Syndrome-Affected Echinopora lamellosa in Aquaria and Experimental Treatment with the Antibiotic Ampicillin

Prokaryotic and ciliate communities of healthy and aquarium White Syndrome (WS)-affected coral fragments were screened using denaturing gradient gel electrophoresis (DGGE). A significant difference (R = 0.907, p < 0.001) in 16S rRNA prokaryotic diversity was found between healthy (H), sloughed tissue (ST), WS-affected (WSU) and antibiotic treated (WST) samples. Although 3 Vibrio spp were found inWS-affected samples, two of these species were eliminated following ampicillin treatment, yet lesions continued to advance, suggesting they play a minor or secondary role in the pathogenesis. The third Vibrio sp increased slightly in relative abundance in diseased samples and was abundant in non-diseased samples. Interestingly, a Tenacibaculum sp showed the greatest increase in relative abundance between healthy and WS-affected samples, demonstrating consistently high abundance across all WS-affected and treated samples, suggesting Tenacibaculum sp could be a more likely candidate for pathogenesis in this instance. In contrast to previous studies bacterial abundance did not vary significantly (ANOVA, F2, 6 = 1.000, p = 0.422) between H, ST, WSU or WST. Antimicrobial activity (assessed on Vibrio harveyi cultures) was limited in both H and WSU samples (8.1% ±8.2 and 8.0% ±2.5, respectively) and did not differ significantly (Kruskal-Wallis, χ2 (2) = 3.842, p = 0.146). A Philaster sp, a Cohnilembus sp and a Pseudokeronopsis sp. were present in all WS-affected samples, but not in healthy samples. The exact role of ciliates in WS is yet to be determined, but it is proposed that they are at least responsible for the neat lesion boundary observed in the disease.

Macrofaunal assemblages from mud volcanoes in the Gulf of Cadiz

Desde a descoberta em 1999 do primeiro vulcão de lama no Golfo de Cádis, cerca de 40 locais, de profundidade variável entre os 200 e os 3900 m, com diferentes graus de emissão de hidrocarbonetos foram localizados e amostrados dentro do programa IOC-UNESCO “Training Through Research (TTR) “ e mais recentemente dentro do projecto europeu HERMES. Neste estudo investigamos as comunidades da macrofauna dos vulcões de lama do Golfo de Cádis utilizando uma diversidade de equipamento de amostragem quantitativo e não quantitativo. Mais de 14550 espécimes foram examinados e incluídos nos diferentes grupos taxonómicos, sendo fornecida uma lista taxonómica detalhada com o menor nível taxonómico possível. A biodiversidade, distribuição dos principais taxa, as espécies quimiossintéticas e a biodiversidade regional e substituição de espécies são apresentados e discutidos. Dentro da macrofauna, os bivalves (nomeadamente super-familia Thyasiroidea, espécies quimisimbióticos e comunidade de bivalves) e os ofiurideos são estudados em pormenor. Os Thyasiroidea colhidos nos vulcões de lama do Golfo de Cádis são revistos. Das sete espécies identificadas, apenas uma Thyasira vulcolutre. sp. nov se encontra associada a um ambiente quimiossintético. Esta espécie é restrita a locais activos, mas não se verificam padrões de distribuição para as outras espécies. Os bivalves quimiosimbióticos amostrados são revistos. Das 10 espécies fortemente associadas a ambientes quimiossintéticos duas Solemyidae, Petrasma elarraichensis sp. nov. e Acharax gadirae sp. nov., uma Lucinidae, Lucinoma asapheus sp. nov., e uma Vesicomyidae, Isorropodon megadesmus sp. nov. são descritas e comparadas com similares das respectivas famílias. As comunidades de bivalves foram analisadas em detalhe e do estudo de 759 espécimes (49 espécies em 21 familias) descreve-se a diversidade e padrões de distribuição. Os Ophiuroidea amostrados nos vulcões de lama e ambientes batiais adjacentes são revistos. Treze espécies são incluídas em 4 famílias, Ophiacanthidae, Ophiactidae, Amphiuridae e Ophiuridae e são identificadas, tendo sido descrita uma nova espécie Ophiopristis cadiza sp. nov. Rácios isotópicos (δ13C, δ15N, δ34S) foram determinados em várias espécies no intuito de investigar a ecologia trófica das comunidades bênticas dos vulcões do Golfo de Cádis. Os valores de δ13C para os bivalves Solemyidae, Lucinidae e Thyasiridae estão de acordo com os valores para outros bivalves conhecidos por possuírem simbiontes tiotróficos. Por outro lado os valores de δ13C e δ34S para Bathymodiolus mauritanicus sugerem a ocorrência de metanotrofia. A análise da fauna heterotrófica indica igualmente que as espécies habitantes da cratera dos vulcões de lama derivam a sua nutrição de fontes quimiossintéticas. A indicação pela análise isotópica que as bactérias autotróficas contribuem substancialmente para a nutrição dos bivalves hospedeiros, levou-nos a investigar os endossimbiontes e as suas relações filogenéticas relativamente a outros bivalves através da análise comparativa de análises de sequências de 16S ribossomal RNS. Análises moleculares PCR-DGGE (Denaturing Gradient Gel Electrophoresis) e clonagem de genes de bacterias 16S rRNA confirmaram a presença de simbiontes oxidantes de enxofre e colocam a possibilidade de uma simbiose dupla para o B. mauritanicus. A diversidade microbiana dentro dos Frenulata foi igualmente estudada recorrendo a métodos moleculares e revelou a não existência de padrão entre espécies, vulcões, profundidade e idade do animal sugerindo assim a não procura de simbiontes específicos.