Microbial diversity in tropical marine sediments assessed using culture-dependent and culture-independent techniques

The microbial communities associated with marine sediments are critical for ecosystem function yet remain poorly characterized. While culture-independent (CI) techniques capture the broadest perspective on community composition, culture-dependent (CD) methods can select for low abundance taxa that are missed using CI approaches. This study aimed to assess microbial diversity in tropical marine sediments at five shallow-water sites in Belize using both CD and CI techniques. The CD methods captured approximately 3% of the >800 genera detected across all sites using the CI approach. Additionally, 39 genera were only detected in culture, revealing rare taxa that were missed with the CI approach. Significantly different communities were detected across sites, with rare taxa playing an important role in distinguishing among communities. This study provides important baseline data describing shallow-water sediment microbial communities, evidence that standard cultivation techniques may be more effective than previously recognized, and the first steps towards identifying new taxa that are amenable to agar plate cultivation.     

南海局部海洋沉积物中真菌多样性及产酶活性

【目的】从11份南海海洋沉积物中分离耐盐真菌,并对其物种多样性及产酶活性进行研究。【方法】利用平板涂布法分离耐盐真菌,基于形态学和ITS序列的系统进化研究耐盐真菌多样性;利用6种筛选培养基对耐盐真菌进行产酶活性筛选。【结果】分离得到1689株耐盐真菌,共41个形态种。形态学和ITS序列分析表明,这些真菌归于15个属,其中曲霉属(Aspergillus)和青霉属(Penicillium)为优势菌群。对已测序的41株耐盐真菌的产酶活性研究表明,8株产纤维素酶,9株产淀粉酶,5株产复合酶,16株产蛋白酶,3株产脂肪酶,未发现产壳聚糖酶的菌株,其中Acrodontium sp.8m和Aspergillus sp.86b产复合酶的活性相对较高,而Penicillium sp.41m产蛋白酶的活性相对较高。【结论】南海局部海洋沉积物中耐盐真菌丰富,多数菌株具有产酶活性。     

南海西沙海槽表层沉积物微生物多样性

利用非培养的分子技术研究了西沙海槽表层沉积物中的微生物群落.沉积物中扩增的古菌16S rDNA 序列分属两个大类:泉古生菌(Crenarchaeota)和广古生菌(Euryarchaeota).以Marine Crenarchaeotic GroupⅠ (古菌16S rDNA文库的49.2%)和Terrestrial Miscellaneous Euryarchaeotal Group (16.9%)为主要类群;其余为Marine Benthic Group B (9.7%)、 Marine Benthic Group A (4%)、 Marine Benthic Group D (1.6%)、Novel Euryarchaeotic Group (0.8%)和 C3(0.8%).细菌克隆子多样性明显高于古菌,16S rDNA序列分别来自变形杆菌(Proteobacteria)(细菌16S rDNA文库的30.5%)、浮霉菌(Planctomycetes)(20.3%)、放线菌(Actinobacteria)(14.4%)、厚壁菌(Firmicutes)(15.3%)、屈桡杆菌(Chloroflexi)(8.5%)、酸杆菌(Acidobacteria)(3.4%)、candidate division OP8 (2.5%)、拟杆菌/绿菌(Bacterioidetes/Chlorobi)(1.7%)和疣微菌(Verrucomicrobia)(1.7%).变形杆菌为优势类群(包括Alpha-和Delta-Proteobacteria亚群).多数克隆子为未培养细菌和古菌.结果表明南海表层沉积物中蕴含大量未知的微生物资源.     

Microbial diversity in surface sediments of the Xisha Trough, the South China Sea

Microbial communities were obtained from the surface sediments of the Xisha Trough using the culture-independent technique. The characteristics of the 16S rDNA gene amplified from the sediments indicated that archaeal clones could be grouped into Euryarchaeota and Crenarchaeota, respectively. Two archaeal groups, Marine Crenarchaeotic GroupI and Terrestrial Miscellaneous Euryarchaeotal Group, were the most dominant archaeal 16S rDNA gene components in the sediments. The remaining components were related to the members of Marine Benthic Group B, Marine Benthic Group A, Marine Benthic Group D, Novel Euryarchaeotic Group and C3. The bacterial clones exhibited greater diversity than the archaeal clones with the 16S rDNA gene sequences from the members of Proteobacteria, Planctomycetes, Actinobacteria, Firmicutes, Chloroflexi, Acidobacteria, candidate division OP8, Bacterioidetes/Chlorobi and Verrucomicrobia. Most of these lineages represented uncultured microorganisms. The result suggests that a vast amount of microbial resource in the surface sediments of the South China Sea has not been known.     

Seasonal and spatial diversity of microbial communities in marine sediments of the South China Sea

This study was conducted to characterize the diversity of microbial communities in marine sediments of the South China Sea by means of 16S rRNA gene clone libraries. The results revealed that the sediment samples collected in summer harboured a more diverse microbial community than that collected in winter, Deltaproteobacteria dominated 16S rRNA gene clone libraries from both seasons, followed by Gammaproteobacteria, Acidobacteria, Nitrospirae, Planctomycetes, Firmicutes. Archaea phylotypes were also found. The majority of clone sequences shared greatest similarity to uncultured organisms, mainly from hydrothermal sediments and cold seep sediments. In addition, the sedimentary microbial communities in the coastal sea appears to be much more diverse than that of the open sea. A spatial pattern in the sediment samples was observed that the sediment samples collected from the coastal sea and the open sea clustered separately, a novel microbial community dominated the open sea. The data indicate that changes in environmental conditions are accompanied by significant variations in diversity of microbial communities at the South China Sea.     

Bacterial diversity in marine hydrocarbon seep sediments

Marine seeps introduce significant amounts of hydrocarbons into oceans and create unusual habitats for microfauna and -flora. In the vicinity of chronic seeps, microbes likely exert control on carbon quality entering the marine food chain and, in turn, hydrocarbons could influence microbial community composition and diversity. To determine the effects of seep oil on marine sediment bacterial communities, we collected sediment piston cores within an active marine hydrocarbon seep zone in the Coal Oil Point Seep Field, at a depth of 22 m in the Santa Barbara Channel, California. Cores were taken adjacent to an active seep vent in a hydrocarbon volcano, on the edge of the volcano, and at the periphery of the area of active seepage. Bacterial community profiles were determined by terminal restriction fragment length polymorphisms (TRFLPs) of 16S ribosomal genes that were polymerase chain reaction (PCR)-amplified with eubacterial primers. Sediment carbon content and C/N ratio increased with oil content. Terminal restriction fragment length polymorphisms suggested that bacterial communities varied both with depth into sediments and with oil concentration. Whereas the apparent abundance of several peaks correlated positively with hydrocarbon content, overall bacterial diversity and richness decreased with increasing sediment hydrocarbon content. Sequence analysis of a clone library generated from sediments collected at the periphery of the seep suggested that oil-sensitive species belong to the gamma Proteobacteria and Holophaga groups. These sequences were closely related to sequences previously recovered from uncontaminated marine sediments. Our results suggest that seep hydrocarbons exert a strong selective pressure on bacterial communities in marine sediments. This selective pressure could, in turn, control the effects of oil on other biota in the vicinity of marine hydrocarbon seeps.     

Prokaryotic diversity in Zostera noltii-colonized marine sediments

The diversity of microorganisms present in a sediment colonized by the phanerogam Zostera noltii has been analyzed. Microbial DNA was extracted and used for constructing two 16S rDNA clone libraries for Bacteria and Archaea. Bacterial diversity was very high in these samples, since 57 different sequences were found among the 60 clones analyzed. Eight major lineages of the Domain Bacteria were represented in the library. The most frequently retrieved bacterial group (36% of the clones) was delta-Proteobacteria related to sulfate-reducing bacteria. The second most abundant group (27%) was gamma-Proteobacteria, including five clones closely related to S-oxidizing endosymbionts. The archaeal clone library included members of Crenarchaeota and Euryarchaeota, with nine different sequences among the 15 analyzed clones, indicating less diversity when compared to the Bacteria organisms. None of these sequences was closely related to cultured Archaea organisms.     

Microbial diversity in lake sediments detected by PCR-DGGE

In this study,PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu,Lake Mochou in Nanjing and Lake Taihu in Wuxi.Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted.The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g),while that of sediments in Lake Taihu was relatively low.After DNA purification,the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE.The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments.The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of micro-organisms were identified in the sediment samples of both lakes.These results suggest that the sediment samples of these two city lakes (Xuanwu,Mochou) have similar microbial communities.However,the DGGE profiles of sediment samples in Lake Taihu were significantly differ-ent from these two lakes.Furthermore,the DGGE pro-files of sediment samples in different locations in Lake Taihu were also different,suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou.The differences in microbial diversity may be caused by the different environmental conditions,such as redox potential,pH,and the concentrations of organic matters.Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced.The results of sequencing analysis indicate that five sequences shared 99%-100% homology with known sequences (Bacillus and Brevibacillus,uncultured bacteria),while the other two sequences shared 93%-96% homology with known sequences (Acinetobacter,and Bacillus).The study shows that the PCR-DGGE tech-nique combined with sequence analysis is a feasible and efficient method for the determination of microbial com-munities in sediment samples.     

Microbial diversity in lake sediments detected by PCR-DGGE

In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu, Lake Mochou in Nanjing and Lake Taihu in Wuxi. Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted. The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g), while that of sediments in Lake Taihu was relatively low. After DNA purification, the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE. The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments. The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of microorganisms were identified in the sediment samples of both lakes. These results suggest that the sediment samples of these two city lakes (Xuanwu, Mochou) have similar microbial communities. However, the DGGE profiles of sediment samples in Lake Taihu were significantly different from these two lakes. Furthermore, the DGGE profiles of sediment samples in different locations in Lake Taihu were also different, suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou. The differences in microbial diversity may be caused by the different environmental conditions, such as redox potential, pH, and the concentrations of organic matters. Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced. The results of sequencing analysis indicate that five sequences shared 99%–100% homology with known sequences (Bacillus and Brevibacillus, uncultured bacteria), while the other two sequences shared 93%–96% homology with known sequences (Acinetobacter, and Bacillus). The study shows that the PCR-DGGE technique combined with sequence analysis is a feasible and efficient method for the determination of microbial communities in sediment samples. __________ Translated from Acta Ecologica Sinica, 2006, 26(11): 3610–3616 [译自: 生态学报]     

南海北部陆坡神狐海域HS-PC500 岩心微生物多样性

[目的]本文研究南海北部陆坡神狐HS-PC500重力活塞岩心沉积物中微生物多样性.[方法]使用吖啶橙染色法计数沉积物中微生物丰度;提取沉积物微生物总DNA,使用特异性引物扩增古菌及细菌16SrRNA基因序列;对克隆文库进行系统发育分析.[结果]系统发育分析显示表层PC500-l(0-5 cm below sea floor,bsf)古菌以C3为主要类群,占该层总序列的25.6%;中层PC500-6(350-355 cm bsf)和底层PC500-11(790-795 cm bsf)古菌以Marine Benthic Group(MBG)-B为主要类群,分别占该层总序列的48.1%和38.9%.另有部分克隆序列属于MBG-A、Miscellaneous Crenarchaeotic Group(MCG)、Thermoprotei、NGC、Halobacteriales、MBG-E、South African Gold Mine Euryarchaeotic Group(SAGMEG).表层细菌以变形菌(Proteobacteria)为主要类群,占该层文库的38.3%.中层和底层细菌以绿弯菌(Choloflexi)和JS1为主要类群,分别占该层文库的28.1%、29.2%和39%、24.7%.另有部分克隆序列属于硝化螺旋菌(Nitrospirae)、放线菌(Actinobacteria)、酸杆菌(Acidobacteria)、OP8、螺旋体菌(Spirochaetes)、TM6、脱铁杆菌(Deferribacteres)、浮霉菌(Plantomycete).[结论]结果显示,HS-PC500岩心微生物丰度与甲烷浓度变化相吻合;微生物丰度较低可能与较低的总有机碳量有关;微生物多样性较高,并且随深度的增加群落结构变化明显;岩心中有关硫酸盐还原的微生物类群占优势,说明微生物的硫代谢在该海区沉积物的物质循环过程中占有重要地位.     

北极太平洋扇区海洋沉积物细菌多样性的系统发育分析

对北极太平洋扇区3个不同深度的海洋沉积物样品,采用PCR结合变性梯度凝胶电泳(DGGE)技术进行细菌16S rRNA基因V3区序列的系统发育分析。结果表明,同一个沉积物样品不同层次的DGGE电泳图谱不完全相同。从3个沉积物样品中共获得50条序列,大部分序列与从海洋环境尤其海洋沉积物获得的细菌16S rDNA序列相似性较高(88%~100%),归属于变形细菌(Proteobacteria)的gamma亚群、alpha亚群、beta亚群、epsilon亚群、delta亚群,Cytophaga_Flavobacterium_Bacteroides(CFB)群细菌和高G C含量的革兰氏阳性细菌等系统分类群,其中变形细菌(Proteobacteria)的gamma亚群为沉积物中的优势细菌类群。     

Actinobacterial diversity from marine sediments collected in Mexico

Seventeen different media known to support the growth and isolation of members of the class Actinobacteria were evaluated as selective isolation media for the recovery of this microbial group from marine sediments samples collected in the Gulf of California and the Gulf of Mexico. A general selective isolation procedure was employed for six sediments and nearly 300 actinomycetes were recovered from the selective isolation plates. Full 16S rRNA gene sequencing revealed that the isolates belonged to several actinobacterial taxa, notably to the genera Actinomadura, Dietzia, Gordonia, Micromonospora, Nonomuraea, Rhodococcus, Saccharomonospora, Saccharopolyspora, Salinispora, Streptomyces, “Solwaraspora” and Verrucosispora. Previous works on marine sediments have been restricted to the isolation of members of the genera Micromonospora, Rhodococcus and Streptomyces. This study provides further evidence that Actinobacteria present in marine habitats are not restricted to the Micromonospora-Rhodococcus-Streptomyces grouping. Indeed, this first systematic study shows the extent of actinobacterial diversity that can be found in marine sediments collected in Mexico and probably, worldwide. The 16S rRNA gene sequences of marine isolates A1, AA2, AA6, AB1, AB2, AG1, AI2, AK1, AL2, AO1, AO3, AR1, AW1, B1, BB1, BC1, C5, R1, R2, R3, AV1, AE1, AI1, AN1 and AP1 determined in this study have been deposited under GenBank accession numbers EU714241–EU714258 and FJ462359–FJ462365, respectively.     

Microbial consumption of dimethyl sulfide and methanethiol in coastal marine sediments

Abstract: Samples were taken from oxic and anoxic zones of three ecosystems: a cyanobacterial mat, a diatom film and a carbonate sediment. Dimethylsulfide (DMS) concentrations were determined by headspace analysis of sediment slurries; maximal amounts were in the upper 5–10 mm of the sediments of 20 μM (cyanobacterial mat), 8 μM (diatom film) and < 1 μM in the carbonate sediment. Dissolved DMS in the cyanobacterial mat, determined by centrifugation and cryogenic trapping, was about two orders of magnitude lower than from slurry estimations but its variation with depth was similar. CH₃SH concentrations in slurried samples, determined after treatment with tributylphosphine, ranged from 2 to 7 μM in the diatom mat and was below the limit of detection (< 0.1 μM) in the carbonate sediment. MPN counts of bacteria that grew on DMS under oxic and anoxic (nitrate added) conditions were determined at all three sites. Aerobic DMS utilizers peaked in the surface and decreased with depth, while the population of anaerobic DMS utilizers was relatively constant in the top 20 mm. Populations of DMS utilizers were highest in the cyanobacterial mat and lowest in the carbonate sediment. MPN's of thiosulfate utilizers, aerobic and anaerobic (nitrate added) were determined in the cyanobacterial mat. Populations of aerobic and anaerobic S₂O₃²⁻ utilizers were similar throughout the top 20 mm and comparable to those of DMS utilizers in the top 5 mm, but higher by about 100-fold below that zone. DMS and CH₃SH consumption rates were measured in slurries of sediments and aerobic rates were similar or only slightly higher than anaerobic rates; the latter were stimulated by nitrate.     

Phylogenetic diversity of gram-positive bacteria cultured from marine sediments

Major advances in our understanding of marine bacterial diversity have been gained through studies of bacterioplankton, the vast majority of which appear to be gram negative. Less effort has been devoted to studies of bacteria inhabiting marine sediments, yet there is evidence to suggest that gram-positive bacteria comprise a relatively large proportion of these communities. To further expand our understanding of the aerobic gram-positive bacteria present in tropical marine sediments, a culture-dependent approach was applied to sediments collected in the Republic of Palau from the intertidal zone to depths of 500 m. This investigation resulted in the isolation of 1,624 diverse gram-positive bacteria spanning 22 families, including many that appear to represent new taxa. Phylogenetic analysis of 189 representative isolates, based on 16S rRNA gene sequence data, indicated that 124 (65.6%) belonged to the class Actinobacteria while the remaining 65 (34.4%) were members of the class Bacilli. Using a sequence identity value of >/=98%, the 189 isolates grouped into 78 operational taxonomic units, of which 29 (37.2%) are likely to represent new taxa. The high degree of phylogenetic novelty observed during this study highlights the fact that a great deal remains to be learned about the diversity of gram-positive bacteria in marine sediments.     

黄海海域海洋沉积物细菌多样性分析

【背景】海洋独特的环境造就了海洋生物的多样性,海洋沉积物中细菌对海洋环境具有至关重要的作用。【目的】研究陆地土壤和海洋沉积物间细菌群落相似性和差异性,以便更好地认识海洋细菌多样性,深入了解沉积物细菌在海洋环境中的潜在作用。【方法】从中国黄海海域及大连市大黑山脚下分别采集样品,以陆地土壤为对照,采用16SrRNA基因高通量测序技术分析海洋沉积物的细菌群落结构。【结果】海洋沉积物样品中芽孢杆菌纲(Bacilli)、鞘氨醇单胞菌属(Sphingomonas)和芽孢杆菌属(Bacillus)丰度高于陆地土壤样品;海洋沉积物中亚硝化单胞菌(unculturedbacterium f. Nitrosomonadaceae)和厌氧绳菌(uncultured bacterium f. Anaerolineaceae)丰度虽低于陆地土壤,但丰度值也均高于1%;样品分类学统计显示酸杆菌门(Acidobacteria)在海洋沉积物和陆地土壤样品中的序列丰度比例都较大,鞘氨醇单胞菌属(Sphingomonas)在海洋沉积物样品中的序列丰度大于陆地土壤样品。【结论】海洋沉积物细菌多样性可作为海洋环境恢复情况的重...     

High bacterial diversity in permanently cold marine sediments.

A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. Screening 353 clones by dot blot hybridization with group-specific oligonucleotide probes suggested a predominance of sequences related to bacteria of the sulfur cycle (43.4% potential sulfate reducers). Within this fraction, the major cluster (19.0%) was affiliated with Desulfotalea sp. and other closely related psychrophilic sulfate reducers isolated from the same habitat. The cloned sequences showed between 93 and 100% similarity to these bacteria. Two additional groups were frequently encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated with Myxobacteria spp. and Bdellovibrio spp. Many clones (18.1%) belonged to the gamma subclass of the class Proteobacteria and were closest to symbiotic or free-living sulfur oxidizers. Probe target groups were further characterized by amplified rDNA restriction analysis to determine diversity within the groups and within the clone library. Rarefaction analysis suggested that the total diversity assessed by 16S rDNA analysis was very high in these permanently cold sediments and was only partially revealed by screening of 353 clones.     

Statistical approaches for estimating actinobacterial diversity in marine sediments

Bacterial diversity in a deep-sea sediment was investigated by constructing actinobacterium-specific 16S ribosomal DNA (rDNA) clone libraries from sediment sections taken 5 to 12, 15 to 18, and 43 to 46 cm below the sea floor at a depth of 3,814 m. Clones were placed into operational taxonomic unit (OTU) groups with >/= 99% 16S rDNA sequence similarity; the cutoff value for an OTU was derived by comparing 16S rRNA homology with DNA-DNA reassociation values for members of the class Actinobacteria. Diversity statistics were used to determine how the level of dominance, species richness, and genetic diversity varied with sediment depth. The reciprocal of Simpson's index (1/D) indicated that the pattern of diversity shifted toward dominance from uniformity with increasing sediment depth. Nonparametric estimation of the species richness in the 5- to 12-, 15- to 18-, and 43- to 46-cm sediment sections revealed a trend of decreasing species number with depth, 1,406, 308, and 212 OTUs, respectively. Application of the LIBSHUFF program indicated that the 5- to 12-cm clone library was composed of OTUs significantly (P = 0.001) different from those of the 15- to 18- and 43- to 46-cm libraries. F(ST) and phylogenetic grouping of taxa (P tests) were both significant (P < 0.00001 and P < 0.001, respectively), indicating that genetic diversity decreased with sediment depth and that each sediment community harbored unique phylogenetic lineages. It was also shown that even nonconservative OTU definitions result in severe underestimation of species richness; unique phylogenetic clades detected in one OTU group suggest that OTUs do not correspond to real ecological groups sensu Palys (T. Palys, L. K. Nakamura, and F. M. Cohan, Int. J. Syst. Bacteriol. 47:1145-1156, 1997). Mechanisms responsible for diversity and their implications are discussed.     

Bacterial diversity in surface sediments from the Pacific Arctic Ocean

In order to assess bacterial diversity within four surface sediment samples (0–5 cm) collected from the Pacific Arctic Ocean, 16S ribosomal DNA clone library analysis was performed. Near full length 16S rDNA sequences were obtained for 463 clones from four libraries and 13 distinct major lineages of Bacteria were identified (α, β, γ, δ and ε-Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Actinobacteria, Firmicutes, Planctomycetes, Spirochetes, and Verrucomicrobia). α, γ, and δ-Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria were common phylogenetic groups from all the sediments. The γ-Proteobacteria were the dominant bacterial lineage, representing near or over 50% of the clones. Over 35% of γ-Proteobacteria clones of four clone library were closely related to cultured bacterial isolates with similarity values ranging from 94 to 100%. The community composition was different among sampling sites, which potentially was related to geochemical differences.