Numbers,diversity, and morphological characteristics of aerobic,chemoheterotrophic bacteria in deep subsurface sediments from a site in South Carolina

The aerobic, chemoheterotrophic bacteria indigenous to deep aquifers and other subsurface sediments (depths to 265 m) at a site in South Carolina were characterized by direct microscopy, enumeration of viable cells, analysis of colony morphologies on plates, and analysis of cell morphologies of isolated strains. Substantial numbers of viable bacteria (10^5‐10⁸/g) were present in all transmissive, aquifer sediments, and their numbers did not decrease with depth. Fewer bacteria (<10³/g) were detected in nontransmissive, confining layers. The highest viable counts were obtained on dilute media, but 10–50% of the bacteria in most aquifer sediments also grew rapidly on concentrated, nutrient‐rich media (indicating a high degree of metabolic flexibility). Most of the bacteria were mesophilic; relatively few psychrophiles or thermophiles were detected (<10³/g; in many cases, none). The bacterial flora was diverse (11–62 distinct colony types on enumeration plates of most aquifer sediments). Diversity did not decrease with depth, but the composition of the microflora (based on colony analysis) varied extensively from one geological formation to another. Almost 95% of the platable colonies that grew on enumeration plates contained nonstreptomycete bacteria, more than 80% of which were gram‐negative rods. Light microscopy of films released from aquifer sediments by flotation revealed the presence of dividing cells and microcolonies, thus implying that the in situ deep aquifer microflora was more metabolically active than that seen previously in shallow aquifers.     

Metal resistance among aerobic chemoheterotrophic bacteria from the deep terrestrial subsurface

The metal resistance of 350 subsurface bacterial strains from two U.S. Department of Energy facilities, the Savannah River Site (SRS), South Carolina, and the Hanford site, Washington, was determined to assess the effect of metal toxicity on microorganisms in the deep terrestrial subsurface. Resistance was measured by growth inhibition around discs containing optimized amounts of Hg(II), Pb(II), and Cr(VI). A broad range of resistance levels was observed, with some strains of Arthrobacter spp. demonstrating exceptional tolerance. A higher level of resistance to Hg(II) and Pb(II) (P < 0.05) and a higher occurrence of multiple resistances suggested that metals more effectively influenced microbial evolution in subsurface sediments of the SRS than in those of the Hanford site. Common resistance to heavy metals suggests that toxic metals are unlikely to inhibit bioremediation in deep subsurface environments that are contaminated with mixed wastes.     

Grain size and depth constraints on microbial variability in coastal plain subsurface sediments

We have examined the effects of sediment grain size and depth on the abundance and activity of aerobic bacteria at two coastal plain sites in Virginia. Samples were collected at centimeter intervals as well as meter intervals because fine‐scale sampling can be essential to assess microbial variability. At the Oyster site, grain size varied from 0.12 to 0.25 mm below 1.5 m depth and did not correlate with either bacterial abundance or activity. Perhaps due to the fairly uniform grain size at this site, variations in bacterial numbers were less than fivefold between replicate samples of 0,1 to 100 g and generally less than 15‐fold among closely spaced intervals (～5 cm). At the Abbott Pit site, grain size was about threefold greater (0.50 ± 0.17mm) in an interval of 4.35 to 5.0m below land surface than grain size in the surrounding sediments. In the same interval, bacterial abundance increased by 11‐fold and activity increased by 217‐fold relative to the surrounding sediments. Overall, grain size correlated significantly with bacterial abundance and activity below the soil zone at the Abbott Pit site. This suggests that changes in grain size, even at the centimeter scale, could have a predominant effect on microbial variability in sandy aquifers of the coastal plain. Besides grain size, depth correlated significantly with total organic carbon and bacterial abundance and activity at both sites, suggesting that depth is also an important factor controlling microbial variability in the subsurface environments.     

Distribution of aerobic bacteria,protozoa, algae,and fungi in deep subsurface sediments

The distribution of microorganisms in deep subsurface profiles was determined at three sites at the Savannah River Plant, Aiken, South Carolina. Acridine orange direct counts (AODC) of bacteria were highest in surface soil samples and declined to the 10⁶ to 10⁷ per gram range in the subsurface, but then did not decline further with depth. In the subsurface, AODC values varied from layer to layer, the highest being found in samples from sandy aquifer formations and the lowest in clayey interbed layers. Sandy aquifer sediments also contained the highest numbers of viable bacteria as determined by aerobic spread plate counts (CFU) on a dilute heterotrophic medium. In some of these samples bacterial CFU values approached 100% of the AODC values. Viable protozoa (amoebae and flagellates, but no ciliates) were found in samples with high bacterial CFU values. A variety of green algae, phytoflagellates, diatoms, and a few cyanobacteria were found at low population densities in samples from two of the three boreholes. Low numbers of fungi were evenly distributed throughout the profiles at all three sites. Microbial population density estimates correlated positively with sand content and pore‐water pH, and negatively with clay content and pore‐water metal concentration. A large diversity of prokaryotic and eukaryotic microorganisms was found in samples with high population densities. A survey of bacterial strains isolated from subsurface samples revealed associations of gram‐positive bacteria with high clay sediments and gram‐negative bacteria with sandy sediments. The ability to deposit lipophilic storage material (presumably poly‐ß‐hydroxybutyrate) was found in a high proportion of isolates from sandy sediments, but only rarely in isolates from high clay sediments.     

丢糟窖窖泥细菌多样性评价

目的 对丢糟窖窖泥细菌多样性进行评价。方法 选取3个丢糟窖窖池为研究对象,每个窖池分别从上、中和下部取样,采用MiSeq高通量测序技术以16S rRNA为靶点,从分类操作单元（operational taxonomic units,OTU）分布、菌群分类学地位、核心OTU、菌群α和β多样性等维度对其细菌多样性进行研究。结果 在门水平上,Firmicutes和Actinobacteria的平均含量分别为93.89%和4.73%。在属水平上,Lactobacillus、Clostridium、Bacillus、Paenibacillus和Thermoactinomyces的平均相对含量分别为59.67%、8.28%、5.73%、4.97%和1.29%。发现7个平均相对含量大于1.0%的核心OTU,累计相对含量为63.30%。结论 丢糟窖窖泥中的细菌主要由隶属于Firmicutes的5个属构成,不同样品间共有大量的核心菌群。     

泥浸汁对太湖沉积物中的好氧可培养细菌多样性的影响

【目的】研究添加泥浸汁与否对太湖沉积物中可培养细菌的影响。【方法】采用R2A培养基和添加泥浸汁R2A培养基对沉积物中细菌进行分离培养,16S r RNA基因系统发育分析比较种群结构。【结果】培养基中添加泥浸汁,可使可培养细菌的种类数量增加到1.6倍。16S r RNA基因序列分析表明,培养的优势细菌类群存在明显差别。R2A培养基上生长的细菌主要为厚壁菌门(52%)、放线菌门(24%)、变形菌门(20%)和拟杆菌门(4%),其中大部分细菌与芽孢杆菌属、假单胞菌属、节杆菌属等关系密切;而添加泥浸汁的R2A培养基上生长的细菌则主要为变形菌门(40%)、放线菌门(35%)、厚壁菌门(22.5%)和拟杆菌门(2.5%),与鞘脂单胞菌属、芽孢杆菌属、副球菌属、节杆菌属等关系密切。【结论】添加泥浸汁原始营养因子可提高沉积物中可培养细菌的多样性,提高菌种可培养效率。     

Microbial diversity in coastal subsurface sediments: a cultivation approach using various electron acceptors and substrate gradients

Microbial communities in coastal subsurface sediments are scarcely investigated and have escaped attention so far. But since they are likely to play an important role in biogeochemical cycles, knowledge of their composition and ecological adaptations is important. Microbial communities in tidal sediments were investigated along the geochemical gradients from the surface down to a depth of 5.5 m. Most-probable-number (MPN) series were prepared with a variety of different carbon substrates, each at a low concentration, in combination with different electron acceptors such as iron and manganese oxides. These achieved remarkably high cultivation efficiencies (up to 23% of the total cell counts) along the upper 200 cm. In the deeper sediment layers, MPN counts dropped significantly. Parallel to the liquid enrichment cultures in the MPN series, gradient cultures with embedded sediment subcores were prepared as an additional enrichment approach. In total, 112 pure cultures were isolated; they could be grouped into 53 different operational taxonomic units (OTU). The isolates belonged to the Proteobacteria, "Bacteroidetes," "Fusobacteria," Actinobacteria, and "Firmicutes." Each cultivation approach yielded a specific set of isolates that in general were restricted to this single isolation procedure. Analysis of the enrichment cultures by PCR and denaturing gradient gel electrophoresis revealed an even higher diversity in the primary enrichments that was only partially reflected by the culture collection. The majority of the isolates grew well under anoxic conditions, by fermentation, or by anaerobic respiration with nitrate, sulfate, ferrihydrite, or manganese oxides as electron acceptors.     

Microbial transformation of heterocyclic molecules in deep subsurface sediments

Recently attempts have been made to establish the presence and to determine the metabolic versatility of microorganisms in the terrestrial deep subsurface at the Savannah River Plant, Aiken, SC, USA. Sediment samples obtained at 20 different depths of up to 526 m were examined to determine carbon mineralization under aerobic, sulfate-reducing, and methanogenic conditions. The evolution of¹⁴CO₂ from radiolabelled glucose was observed under aerobic conditions in all sediments, whereas pyridine was transformed in 50% of the 20 sediments and indole was metabolized in 85% of the sediments. Glucose mineralization in certain sediments was comparable to that in the surface environment. Sulfate was reduced in only five sediments, and two were carbon limited. Methane production was detected in ten sediments amended with formate only after long-term incubations. The transformation of indole and pyridine was only rarely observed under sulfate-reducing conditions and was never detected in methanogenic incubations. This study provides information concerning the metabolic capability of both aerobic and anaerobic microorganisms in the deep subsurface and may prove useful in determining the feasibility of microbial decontamination of such environments.     

Phylogenetic and enzymatic diversity of deep subseafloor aerobic microorganisms in organics- and methane-rich sediments off Shimokita Peninsula

"A meta-enzyme approach" is proposed as an ecological enzymatic method to explore the potential functions of microbial communities in extreme environments such as the deep marine subsurface. We evaluated a variety of extra-cellular enzyme activities of sediment slurries and isolates from a deep subseafloor sediment core. Using the new deep-sea drilling vessel "Chikyu", we obtained 365 m of core sediments that contained approximately 2% organic matter and considerable amounts of methane from offshore the Shimokita Peninsula in Japan at a water depth of 1,180 m. In the extra-sediment fraction of the slurry samples, phosphatase, esterase, and catalase activities were detected consistently throughout the core sediments down to the deepest slurry sample from 342.5 m below seafloor (mbsf). Detectable enzyme activities predicted the existence of a sizable population of viable aerobic microorganisms even in deep subseafloor habitats. The subsequent quantitative cultivation using solid media represented remarkably high numbers of aerobic, heterotrophic microbial populations (e.g., maximally 4.4 x 10(7) cells cm(-3) at 342.5 mbsf). Analysis of 16S rRNA gene sequences revealed that the predominant cultivated microbial components were affiliated with the genera Bacillus, Shewanella, Pseudoalteromonas, Halomonas, Pseudomonas, Paracoccus, Rhodococcus, Microbacterium, and Flexibacteracea. Many of the predominant and scarce isolates produced a variety of extra-cellular enzymes such as proteases, amylases, lipases, chitinases, phosphatases, and deoxyribonucleases. Our results indicate that microbes in the deep subseafloor environment off Shimokita are metabolically active and that the cultivable populations may have a great potential in biotechnology.     

Bacterial diversity in deep Mediterranean sediments: relationship with the active bacterial fraction and substrate availability

We investigated vertical distribution and depth-related patterns (from 670 to 2,570 metres) of bacterial diversity in sediment samples collected along a transect in the warm deep Mediterranean sea. Analyses of bacterial diversity were compared with the abundance of benthic bacteria, their metabolically active fraction and the substrates potentially available for their growth. The number of active bacteria was dependent upon the availability of organic substrate in the sediment deriving from phytopigment inputs from the photic layer. The T-RFLP analysis revealed that the surface layers of all sediments analysed were dominated by the same ribotypes, but clear shifts in bacterial community structure were observed in deeper sediment layers. High values of bacterial diversity (expressed as D, H') and evenness (as J) were observed at all stations (a total of 61 ribotypes was identified), and as a result of the large fraction of rare ribotypes (c. 35%), the overall bacterial diversity in the deep sea region investigated was among the highest reported so far in literature. Biodiversity parameters did not display any relationship with water depth, but ribotype richness was related with the number and percentage of active bacteria, suggesting a coupling between organic inputs stimulating bacterial growth and deep-sea bacterial diversity.     

Aperiodic variations of the turbidity maxima of two German coastal plain estuaries

Suspended particulate matter dynamics in estuaries can be split into more or less regular and periodic phenomena dominated by the tide and aperiodic events like river spates which have lasting effects on the suspended matter distribution. The catchment areas of the estuaries of both the River Elbe and R. Weser (Germany) are subject to almost the same meteorological conditions. But the mean freshwater runoff of R. Elbe is about twice the mean R. Weser runoff. In the turbidity maxima of both estuaries, suspended matter dynamics are dominated by the tide most of the year. The turbidity maxima are associated with the low salinity regions, and the locations of both the mixing zones and turbidity maxima depend on runoff. In both estuaries, mixing zone and turbidity maximum react almost immediately and simultaneously on strong increases of runoff. During river floods no turbidity maxima can be observed in the inner estuaries. With decreasing runoff after a river flood the re-establishment of the turbidity maximum lags behind the return of the mixing zone. The restoration of the turbidity maximum to its normal magnitude lasts for months. For the 7 river floods presented here the restoration period varied from 1 to 6 months for the Weser and from 5 to 7 months for the Elbe estuary.     

Aerobic and anaerobic microbial activity in deep subsurface sediments from the savannah river plant

Methanogenesis, sulfate reduction, and rates of carbon mineralization were determined for samples derived at different depths from four cores drilled at the Savannah River Plant, Aiken South Carolina. Three‐gram subsamples of the sediments were dispensed to 10‐mL serum bottles under 5% H₂/95% N₂ and amended with 0.5 mL degassed distilled water with or without the following solutes: formate plus acetate, bicarbonate, lactate, and radiolabeled sulfate, glucose, and Índole. After incubating 1 to 5 days, the sediments were assayed for methane, H₂, ³⁵S, and ^I4CO₂. No methanogenesis was detected at any depth in any core and sulfate was rarely reduced. Evolution of ¹⁴CO₂ from glucose and indole was detected in sediments as deep as 262 and 259 m, respectively. At some depths the ¹⁴CO₂ evolution rate was comparable to that of surface soils; however, at other depths no ¹⁴CO₂ evolution could be detected. Injection of sterile air into anaerobic incubations increased rates of carbon mineralization at all depths that had demonstrated anaerobic activity and stimulated mineralization activity in sediments that were inactive anaerobically, suggesting a predominance of aerobic metabolism. Increasing the concentration of added glucose and indole often increased the resulting rates of ¹⁴CO₂ evolved from these substrates. Our data indicate that both aerobic and anaerobic microorganisms are present and metabolically active in samples from deep subsurface environments.     

海岸带沉积物中氮循环功能微生物多样性

海岸带生境类型多样,环境梯度明显,是研究微生物多样性、群落结构与功能关系及调控机制的天然实验场.沉积物是海岸带环境中营养盐再生与转化发生的重要场所,其中多种微生物类群在氮素循环过程中扮演重要角色.本文重点介绍海岸带沉积物中固氮菌、氨氧化菌、厌氧氨氧化菌、反硝化与硝酸盐铵化微生物的基于16SrRNA基因的物种多样性和基于关键酶基因nifH、amoA、narG、nirS、nirK、nosZ、nrfA、hzo、hzs等的功能多样性;总结了在海岸带特有生境(如河口、潮间带、海草藻床、红树林、盐沼、珊瑚礁、浅海等)及污染胁迫、生物扰动等条件下各功能类群的群落组成特征及时空变化规律,并提出今后需要重点关注新的培养技术和方法的开发,以进一步提高微生物的可培养性,将单细胞基因组测序与分析技术、DNA和RNA结合起来研究,以全面了解氮循环微生物多样性、参与介导硝酸盐铵化过程的微生物多样性等方面.     

High virus-to-cell ratios indicate ongoing production of viruses in deep subsurface sediments

Marine sediments cover two-thirds of our planet and harbor huge numbers of living prokaryotes. Long-term survival of indigenous microorganisms within the deep subsurface is still enigmatic, as sources of organic carbon are vanishingly small. To better understand controlling factors of microbial life, we have analyzed viral abundance within a comprehensive set of globally distributed subsurface sediments. Phages were detected by electron microscopy in deep (320 m below seafloor), ancient (∼14 Ma old) and the most oligotrophic subsurface sediments of the world''s oceans (South Pacific Gyre (SPG)). The numbers of viruses (10⁴–10⁹ cm⁻³, counted by epifluorescence microscopy) generally decreased with sediment depth, but always exceeded the total cell counts. The enormous numbers of viruses indicate their impact as a controlling factor for prokaryotic mortality in the marine deep biosphere. The virus-to-cell ratios increased in deeper and more oligotrophic layers, exhibiting values of up to 225 in the deep subsurface of the SPG. High numbers of phages might be due to absorption onto the sediment matrix and a diminished degradation by exoenzymes. However, even in the oldest sediments, microbial communities are capable of maintaining viral populations, indicating an ongoing viral production and thus, viruses provide an independent indicator for microbial life in the marine deep biosphere.     

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。     

双台子河口沉积物中细菌多样性分析

【目的】掌握双台子河口沉积物中细菌多样性及其群落结构的季节变化特征。【方法】于2009年4月、7月、10月和12月共4个航次在内陆河流入海口处进行四季样品的采集, 采用PCR-DGGE技术对沉积物中细菌多样性进行分析。【结果】通过序列比对发现, 该处沉积物中的细菌主要归属于5个细菌类群, 分别为变形菌门(52.6%)、放线菌门(15.8%)、拟杆菌门(10.5%)、酸杆菌门(5.3%)以及绿弯菌门(5.3%), 此外还有一部分分类地位尚不明确的细菌(10.5%)。在四季样品中变形菌门(52.6%)为优势菌群, 而在变形菌门中, δ亚群又占绝对优势地位。实验结果还显示四季沉积物中细菌Shannon-Wiener多样性指数范围为1.84?2.79, 且春夏两季沉积物中的Shannon-Wiener多样性指数比秋冬两季沉积物中Shannon-Wiener多样性指数大。【结论】双台子河口沉积物中的细菌多样性符合典型河口沉积物中细菌多样性的特征; 低温能导致沉积物中细菌多样性的减少。本研究为初步掌握双台子河口沉积物中细菌种类和组成状况提供了一定的参考, 同时也为该处海洋环境的监测及生物资源的保护提供了科学依据。     

Investigation of bacterial diversity in Brazilian tropical estuarine sediments reveals high actinobacterial diversity

Phylogenetic and statistical analyses of 16S rRNA gene libraries were used for the investigation of actinobacterial communities present in two tropical estuarine sediments (Santos-São Vicente estuary, Brazil). The libraries were constructed from samples collected at the brackish end of the estuary, highly hydrocarbon-contaminated, and at the marine end, uncontaminated. Clones from the marine end of the estuary were all related to sequences from non-cultured Actinobacteria and unidentified bacteria recovered from a wide range of environmental samples, whereas clones from the brackish end were mainly related to sequences from cultured Actinobacteria. Statistical analyses showed that the community recovered from the hydrocarbon-contaminated sediment sample, at the brackish end, was less diverse than the uncontaminated one, at the marine end, and that the communities from the two libraries were differently structured, suggesting that these may have not originated from the same community. The recognition of the spatial pattern of actinobacterial distribution in a natural environment is a first step towards understanding the way these communities are organized, providing valuable data for further investigations of their taxonomic and functional diversity.     

Acidobacteria phylum sequences in uranium-contaminated subsurface sediments greatly expand the known diversity within the phylum

The abundance and composition of bacteria of the phylum Acidobacteria were surveyed in subsurface sediments from uranium-contaminated sites using amplification of 16S rRNA genes followed by clone/sequence analysis. Analysis of sequences from this study and public databases produced a revised and greatly expanded phylogeny of the Acidobacteria phylum consisting of 26 subgroups.