Molecular Phylogenetic Analysis of Archaea and Bacteria in Wind Cave,South Dakota

The diversity of bacteria and archaea was characterized from sediments collected from Wind Cave located in Wind Cave National Park in the Black Hills of South Dakota. Wind Cave is a limestone dissolution cave with strata that started forming over 300 million years ago, making it one of the oldest in the world. Previous work suggested that the cave was largely a detritus based system ultimately dependent upon allochthonous energy and carbon from photosynthesis of the overlying vegetation, and algae growing near lights along the tour routes. In this work, we used a molecular phylogenetic approach to characterize the microbial structure and infer a corresponding ecosystem function where appropriate. Four bacterial divisions and subdivisions were found in the culture collection, which represented 14 phylotypes, whereas 12 divisions and subdivisions were identified in the clonal analysis comprising 49 phylotypes. The predominant groups were the γ-Proteobacteria and Acidobacteria. Although a few of the clones resembled sequences from other cave and subterranean systems, no cave-specific bacterial community was evident in this work. Archaeal phylotypes (20 Crenarchaeota and 2 Euryarchaeota) were detected, with a large proportion of the Crenarchaeota resembling sequences from a South African gold mine. One archaeal cluster in particular appears to be specific to the subterranean environment. Most of the microbial sequences were not related to known chemolithoautotrophs, therefore we conclude that this particular community is likely detritus based where allochthonous energy and carbon are transported into the cave by infiltrating waters.     

16S rDNA克隆文库方法分析太岁样品中细菌的多样性

通过构建16S rDNA克隆文库的方法,分析太岁样品中细菌的群落结构及多样性。太岁样品中的细菌归属于4个门9个目,优势类群依次是芽胞杆菌目(Bacillales,33.01%)、柄杆菌目(Caulobacterales,32.04%)和伯克霍尔德氏菌目(Burkholderiales,12.62%);优势属为短波单胞菌属(Brevundimonas,30.10%)、葡萄球菌属(Staphylococcus,29.13%)和食酸菌属(Acidovorax,7.77%)。并且其中的5个目中含有未培养的细菌,红杆菌目(Rhodobacterales)、伯克霍尔德氏菌目和红环菌目(Rhodocyclales)的11个克隆子的细菌16S rDNA序列同源性低于97%。研究表明太岁样品中细菌多样性较丰富,且蕴藏着许多未知的微生物资源。     

Bacterial Communities Inside and Surrounding Soil Iron-Manganese Nodules

Bacterial community structures of a Fe-Mn nodule sample and its surrounding soil were investigated using PCR, amplified ribosomal DNA restriction analysis, cloning and sequencing methods. Result showed that phylogenetically diverse bacteria were present in the nodule and soil samples, and Acidobacteria- and Proteobacteria-affiliated bacteria dominated in both samples. However, Firmicutes were only found in the nodules, while the soil had much more Acidobacteria and Verrucomicrobia than the nodules. Many clones retrieved in this study closely resembled the clones previously obtained from environments with high metal contents. These findings may shed light on the biological formation of Mn oxides in soil environment.     

Microbial leaching of zinc concentrate in fresh‐water microcosms: Comparison between aerobic and oxygen‐limited conditions

Microbially mediated leaching and solubilization of zinc ore concentrate by native aquatic microbial communities incubated under aerobic and oxygen‐limited conditions were examined in static microcosms consisting of stream sediment and water. Sterile controls provided information on abiotic sulfide oxidation and leaching of zinc, lead, cadmium, and copper. The flux of these heavy metals from the sediments to the water column was greatest under biotic oxygen‐limited conditions. When calculated as the percentage of total metal available in zinc concentrate‐amended microcosms, the order of metal solubilization under oxygen‐limited conditions was lead, copper, zinc, and cadmium. Under biotic aerobic conditions, the order of solubilization was zinc, lead, cadmium, and copper. This study indicates that aquatic heterotrophs are capable of leaching and solubilizing metallic sulfides under conditions of neutral to slightly acidic pH and are effective in releasing heavy metals to the water column under oxygen‐limited conditions.     

Microbial Characteristics of a Submerged Karst Cave System in Northern Florida

Karst aquifer systems contain submerged caves that act as conduits for subterranean water flow and are subject to rapid surface recharge at points such as sink holes and submerging streams. We examined the microbial communities in six conduits of the Northern Florida Wakulla Springs cave system and in several hydrologically connected surface sinks. Culturable bacteria were assessed using both oligotrophic and copiotrophic media, and specific media for Enterococcus and Escherichia coli. Culture independent methods included using 16S rRNA PCR amplified DNA for T-RFLP analysis and development of clone libraries for sequencing. Pronounced seasonality was found in all microbiological parameters suggesting responsiveness to surface conditions from recharge of the groundwater despite near constant groundwater temperature. Other differences may reflect the character of the drainage areas feeding different conduits and flow rates and flow reversals that affected residence time in cave conduits. In a region of groundwater flow divide, elevated numbers of a plate counts, flocculent material, and a sulfide smell were reflected in T-RFLP pattern differences. Sequence data from five selected sampling locations revealed the presence of Enterobacter and Klebsiella sequences in surface waters, but not in conduits. One conduit contained a high percentage of sequences with close homology to the archaeal species Thermococcalles archaeon. Other cave-specific sequences were found that do not match previously characterized bacteria. Overall, the data suggest both temporal and spatial differences in the microbial communities within the extensive cave system conduits feeding the spring vent, reflecting both drainage area influences and undocumented subterranean microbial diversity.     

河北承德地区两个温泉中细菌的多样性分析

通过构建16S rDNA克隆文库,对承德地区两温泉中的细菌多样性水平及系统发育关系进行了初步研究。研究表明:68°C的A11文库中阳性克隆的16S rDNA序列分属5个细菌类群,分别为Firmicutes(6.25%)、Deinococcus-Thermus(25.0%)、Gammaproteobacteria(12.5%)、Betaproteobacteria(50.0%)、Alphaproteobacteria(6.25%);而74.5°C的A12文库仅属于一个细菌类群:厚壁菌门(Firmicutes)。两温泉中细菌多样性的差异表明,温度是影响温泉中细菌多样性水平的重要因素。此外,A11文库中克隆的16S rDNA序列与许多已知的可产色素的好氧菌相似性很高,而A12文库中的细菌多数为专性厌氧或兼性厌氧型,其中厌氧芽孢杆菌属(Anoxybacillus)中的Anoxybacillus flavithermus可以作为研究泉华形成的理想材料。     

太湖地区典型菜地土壤微生物16S rDNA的PCR-RFLP分析

土壤微生物多样性是土壤生态功能的基础,但长期以来缺乏对高强度土地利用条件下的土壤微生物多样性的认识.作者采用间接法提取了江苏省太湖地区典型菜地土壤微生物的总DNA,以细菌的通用引物27F和1492R扩增16S rDNA片段,将扩增产物与T-载体酶连,转化大肠杆菌,建立土壤微生物16S rDNA克隆文库.PCR扩增基因文库中插入的16S rDNA外源片段,用两种限制性内切酶Hha I和Rsa I分别酶切,获得该土壤173个克隆的酶切指纹图谱.结果表明,Hha I和Rsa I联合酶切产生了63个基因分型,文库的覆盖度达76.30%,单一酶切产生的基因分型少,但文库的覆盖度高;克隆文库中存在两种优势类群,分别占总克隆的16%和12%.16S rDNA测序结果表明,太湖地区菜地土壤细菌在分类方面主要属于α-和γ-变形杆菌亚门.以上结果为进一步研究太湖地区菜地土壤微生物生态功能提供了基础资料.     

Bacterial Community Structure in a Semi-Arid Haloalkaline Soil Using Culture Independent Method

Bacterial community structures in two physicochemically different soils from the coastal region of Gujarat, India were investigated using PCR, 16S rRNA gene clone libraries and sequencing methods. The aim of the study was to determine the diversity of bacterial communities inhabiting haloalkaline soil from a semi-arid coastal region. The phylogenetic diversity of bacteria in a haloalkaline soil (EC 20 dS/m; pH 9.5) was compared with a normal soil (EC 0.93 dS/m; pH 7.2). Clones representing phyla Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Actinobacteria, Acidobacteria and Planctomycetes were found in both soils. Cyanobacteria, Verrucomicrobia, OP10 and Bacteria incertae sedis were detected in normal soil whereas Nitrospira was found only in haloalkaline soil. The dominant phylum in the haloalkaline soil was Bacteroidetes followed by Proteobacteria whereas normal soil was dominated by Proteobacteria and Actinobacteria. About 82% of the sequences from the haloalkaline library were related to those previously retrieved from various saline, alkaline and oil-natural gas field ecosystems whereas 50% of the sequences from normal soil resembled sequences of bacteria retrieved from agriculture-related habitats viz. agriculture fields, rhizosphere and grasslands. One third of the total sequences from both soil samples showed low BLAST identities (<95%) suggesting that these soils may harbor unique, novel taxa. Further, the correlation analysis revealed negative correlations of Shannon diversity indices and species evenness with salinity (EC) and pH but positive correlations with total carbon and total nitrogen contents of the soil samples. The haloalkaline soil exhibited less bacterial diversity and communities were significantly different from those of normal soil. In this study, the haloalkaline soil from a semi-arid region supports oligotrophic microbes.

Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

16S rDNA克隆文库法探索转基因香石竹对土壤细菌群落的影响

【目的】通过研究转基因香石竹对土壤细菌群落的影响,为转基因香石竹的环境安全性评价提供依据。【方法】通过构建16S rDNA克隆文库,分析种植转基因和非转基因香石竹的土壤中细菌的群落结构组成。【结果】转基因和非转基因香石竹土壤中,共有的菌群有变形菌门(Proteobacteria)、浮霉菌门(Planctomycetes)、酸杆菌门(Acidobacteria),其中α-变形菌门、β-变形菌门、浮霉菌门为优势菌群;而在放线菌门(Actinobacteria)、疣微菌门(Verrucomicrobia)及未培养菌(Uncultured bacterium clone)等菌群存在部分差异。【结论】通过16S rDNA克隆文库方法揭示了转基因香石竹的土壤中细菌多样性十分丰富,其栽培对土壤细菌群落结构影响有限。     

Bacterial community composition of a shallow hypertrophic freshwater lake in China, revealed by 16S rRNA gene sequences

The phylogenetic composition of a bacterial community from a hypertrophic freshwater lake in China was investigated by sequencing cloned 16S rRNA genes. Three hundred and thirty-six bacterial clones from four clone libraries in different months (March, May, July and September in 2004) were classified into 142 operational taxonomic units, most of which were affiliated with bacterial divisions commonly found in freshwater ecosystem, e.g. Alpha-, Beta-, Gamma- and Deltaproteobacteria, Bacteriodetes and Actinobacteria. The results showed that the composition of bacterial community in the July library was the most diverse one. Actinobacteria was the most significant lineage in Lake Taihu, with dominant numbers of operational taxonomic units in the May, July and September libraries. Phylogenetic analysis suggested that 53 sequences were grouped into six novel clusters which may represent specific populations indigenous to the environment. Coverage analyses indicated that the clone libraries could provide a fine inventory of bacterial diversity in the lake.     

Bacterial Diversity in Linglong Gold Mine,China

Bacteria have been actively regulating cycles of various elements in the environment. To explore the potential bacterial role in gold biogeochemical cycling, this study analyzed the bacterial diversity of mine rock (MR) and surface soil (SS) samples from Linglong gold mine using 16S rRNA gene clone library analysis and cultivation method. From MR, 24 operational taxonomic units (OTUs) were identified from MR, covering 3 phyla and 18 genera. Meanwhile, 24 OTUs were identified from SS, including 4 phyla and 18 genera. Compared with 16S rRNA gene clone library analysis, 28 aerobic and 34 anaerobic isolates were obtained, whereas 26 aerobic and 71 anaerobic strains were isolated from SS. The cultivable bacteria were affiliated with Firmicutes, Proteobacteria and Actinobacteria phyla, and dominated by Firmicutes. These results underscore the high level of bacterial diversity in the gold mine. Our study provides information on the microbial diversity in Linglong gold mine and sheds light on the existence and potential function of bacteria in the gold biogeochemical cycling.     

塔河天然胡杨林地可培养细菌的生态分布研究

目的：研究塔里木河天然胡杨林部分地区可培养细菌的生态分布。方法：通过塔里木河胡杨林采样,可培养菌分离及16S rDNA序列鉴定。结果：从3种不同样品（水样、土样和胡杨树杆分泌物）中分离筛选了22株细菌,其中17株菌为革兰氏阳性菌,5株为革兰氏阴性菌。根据生理生化特征与16S rDNA序列分析结果表明,其中15株菌属于芽孢杆菌属,4株属于不动杆菌属、假单胞菌属、动性球菌属和Agrococcus属各含有1个分离株。结论：塔里木河胡杨林可培养微生物中芽孢杆菌比较丰富,其中有3个可能的新种。     

Five-Year Monitoring of Bacterial Communities in Dripping Water from the Heshang Cave in Central China: Implication for Paleoclimate Reconstruction and Ecological Functions

Microbial lipids in stalagmites are increasingly used to reconstruct the paleoclimate change, necessitating the investigations on microbial communities in dripping waters. A pilot molecular survey was conducted on bacterial communities of dripping waters at two sites (1D and 3D) in Heshang Cave of Hubei Province in central China for a period of 2008 to 2013. The samples were subjected to genome DNA extraction, 16S rRNA gene amplification, clone library construction and phylogenetic analysis to explore the seasonal variation of bacterial communities and their association with environmental factors including regional air temperature, precipitation, cave temperature, pH, conductivity and dripping rate of the water samples. Seasonal variations were clearly observed in components and diversities of bacterial communities at both sites. Bacterial community was dominated by Gammaproteobacteria in autumn and winter, whereas Betaproteobacteria became dominant in samples collected in summer and spring. Among the environmental factors investigated, regional air temperature was found to have a strong impact on bacterial communities indicated by cluster and redundancy analysis. Moreover the bacterial biodiversity was observed to increase with the temperature rising. Bacteria identified in dripping water were either oligotrophs or able to acquire nutrients from minerals under oligotrophic conditions. They may also be able to induce calcite precipitation in cave systems. Our data shed light on the potential of microbes used as a tool for the reconstruction of paleo-temperature as well as on the ecological functions of bacterial communities in oligotrophic caves.     

应用16S rDNA克隆文库解析人工快速渗滤系统细菌种群多样性

本文通过构建16S rDNA克隆文库开展了人工快速渗滤(CRI)系统表层(10 cm深)细菌种群多样性研究, 结果表明, 在CRI系统表层填料中细菌多样性十分丰富, 存在7个主要类群, 其中不可培养的酸杆菌纲(uncultured Acidobacteria)和其它不可培养细菌(uncultured bacteria)在整个克隆文库中比例最大, 比例为53.72%, 其次是浮霉菌纲(uncultured planctomycete)和β-变形菌纲(β-proteobacterium), 分别占文库比例的13.89%和8.33%; 克隆文库中反硝化菌的比例高于亚硝化单胞菌属细菌(0.925%), 没有出现Nitrospira属的克隆子。本文通过16S rDNA克隆文库揭示了在生物膜上存在的优势菌为不可培养菌, 而假单胞菌(Pseudomonas aeruginosa)和紫色色杆菌(Chromobacterium sp.)等在平板分离培养中出现的频率较高, 两种方法之间的结果存在差异。本研究采用16S rDNA克隆文库方法揭示的结果, 将为CRI系统生物降解的进一步提高提供依据。     

应用16S rDNA克隆文库解析人工快速渗滤系统细菌种群多样性

本文出不穷通过构建16S rDNA克隆文库开展了人工快速渗滤(CRI)系统表层(10cm深)细菌种群多样性研究,结果表明,在CRI系统表层填料中细菌多样性十分丰富,存在7个主要类群,其中不可培养的酸杆菌纲(uncultured Acidobacteria)和其它不可培养细菌(uncultured bacteria)在整个克隆文库中比例最大,比例为53.72%,其次是浮霉菌纲(uncultured planctomycete)和β-变形菌纲(β-proteobacterium),分别占文库比例的13.89%和8.33%;克隆文库中反硝化菌的比例高于亚硝化单胞菌属细菌(O.925%),没有出现Nitrospira属的克隆子.本文通过16S rDNA克隆文库揭示了在生物膜上存在的优势茵为不可培养菌,而假单胞菌(Pseudomonas aeruginosa)和紫色色杆菌(Chromobacterium sp.)等在平板分离培养中出现的频率较高,两种方法之间的结果存在差异.本研究采用16S rDNA克隆文库方法揭示的结果,将为CRI系统生物降解的进一步提高提供依据.     

Bacterial diversity in biofilms formed on condenser tube surfaces in a nuclear power plant

To elucidate the bacterial diversity in biofilms formed on a condenser tube from a nuclear power plant, 16S rRNA gene sequences were examined using a PCR-cloning-sequencing approach. Twelve operational taxonomic units were retrieved in the clone library, and the estimated species richness was low (13.2). Most of the clones (94.7%) were affiliated with α-Proteobacteria; Planctomycetes and γ-Proteobacteria were much rarer. Interestingly, except for one clone belonging to Pseudoalteromonas, most of the sequences displayed sequence similarities <97% of those of the closest type strains. Based on 16S rRNA phylogenetic analysis, most bacteria were assigned to novel taxa above the species level. The low species richness and unusual bacterial composition may be attributable to selective pressure from chlorine in the cooling water. To prevent or control bacterial biofilms in cooling circuits, additional studies of the physiology and ecology of these species will be essential.     

新疆艾比湖湿地博乐河入口处土壤细菌多样性分析

【目的】了解新疆艾比湖湿地国家级自然保护区非培养土壤细菌群落组成及多样性。【方法】采用非培养法直接从湿地土壤提取总DNA进行16S r RNA基因扩增,构建细菌16S r RNA基因克隆文库。使用MspⅠ和AfaⅠ限制性内切酶对阳性克隆进行16S r RNA基因扩增片段的限制性酶切分析(Amplified r DNA restriction analysis,ARDRA),挑取具有不同双酶切图谱的克隆进行测序,序列比对并构建16S r RNA基因系统发育树。【结果】从土壤细菌的16S r RNA基因文库中随机挑取75个不同谱型的克隆子,共得到58个OTUs,系统发育归类为8个细菌类群:绿弯菌门(Chloroflexi)、蓝藻门(Cyanobacteria)、变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrob)和芽单胞菌门(Gemmatimonadetes)。其中,变形菌门为第一优势菌群,拟杆菌门为第二优势菌群,两者约占总克隆的65%。【结论】艾比湖湿地博乐河入口处土壤细菌多样性丰富,且存在一定数量的潜在微生物新种。     

Molecular phylogenetic diversity of bacteria associated with the leachate of a closed municipal solid waste landfill

A 16S rDNA-based molecular study was performed to determine the nature of the bacterial constituents of the leachate from a closed municipal solid waste landfill. Total community DNA was extracted and bacterial 16S rRNA genes were subsequently amplified and cloned. Recombinant rDNA clones in the library were randomly selected, and they were sequenced for a single run and then grouped. A total of 76 sequence types representing 138 randomly selected nonchimeric clones were identified. Full-length sequencing and phylogenetic analysis of the sequence types revealed that more than 90% of the screened clones were affiliated with low-G+C gram-positive bacteria (38.4%), Proteobacteria (35.5%), the Cytophaga Flexibacter Bacteroides group (11.6%), and Spirochaetes (5.1%). Minor portions were affiliated with Verrucomicrobia (2.9%), candidate division OP11 (2.2%), and the green nonsulfur bacteria, Cyanobacteria and the Deinococcus Thermus group (each <1.0%). Although some rDNA sequences clustered with genera or taxa that were classically identified within anaerobic treatment systems and expected with known functions, a substantial fraction of the clone sequences showed relatively low levels of similarity with any other reported rDNA sequences and thus were derived from unknown taxa. These results suggest that bacterial communities in landfill environment are far more complex than previously expected and remain largely unexplored.     

污水处理活性污泥微生物群落多样性研究

为研究污水处理活性污泥微生物多样性,提取了活性污泥宏基因组DNA,并采用细菌通用引物27F和1492R扩增了上海污泥厂活性污泥细菌16S rDNA片段,构建了细菌16S rDNA克隆文库,并对该文库中的微生物群落进行了分析。共获得200条高质量序列并建立系统发育树,结果显示活性污泥主要的细菌类群为变形菌门(Proteobacteria)(91.9%)、厚壁菌门(Firmicures)(4.6%)、拟杆菌门(Bacteroidetes)(2%)、绿弯菌门(Chloroflexi)(0.5%)、硝化螺菌门(Nitrospirae)(1%)。其中,明显的优势菌群为Alcaligenes feacalis(55%)、Pseudomonas aeruginosa(12.8%)和Stenotrophomonas(12.8%),优势菌的产酶能力在活性污泥中显示生态修复功能菌的作用。