Microbial Community Composition in Acid Mine Drainage Lake of Xiang Mountain Sulfide Mine in Anhui Province,China

Acid mine drainage (AMD) lake of Xiang Mountain in Anhui Province, China, was characterized by acidic waters (pH around 2.8) containing high concentrations of soluble metals and sulfate. To investigate the function and dynamics of this extreme ecosystem, four water samples were collected from the lake in the fall of 2010. The acidophilic community structure was analyzed by molecular approaches, and bacterial and archaeal clone libraries of 16S rRNA genes were constructed. In contrast to dominance of chemolithotrophic acidophiles in typical AMD environments, autotrophic iron/sulfur-oxidizing bacteria were detected in only one sample with low abundance. Unexpectedly, the Cyanobacteria group was the predominant in all four samples (54.9%?77%). Chemoheterotrophs Acidiphilium and Acidisphaera were also abundant. These two heterotrophic groups contain bacteriochlorophyll that can perform photosynthesis, an advantage to grow and survive in such oligotrophic acidic environments. Only two clone sequences related to Legionella (2.8% of the total clones) were recovered from one sample in sharp contrast to its higher abundance (12.7%) in the summer of 2009. All archaeal sequences were affiliated to the phylum Crenarchaeota. The results of statistical analysis suggested that the water chemistry of the AMD lake controlled microbial composition of the AMD ecosystem.     

封闭环境下酸性矿坑水中微生物生态多样性的研究

铜绿山铜矿是世界开采时间最长的矿井之一,在开采过程中有许多矿井被废弃,许多废弃的矿井内产生了大量的对环境有害的酸性矿坑水.酸性矿坑水取自铜绿山铜矿某废弃矿井,利用限制性酶切片断多样性分析(RFLP分析)对酸性矿坑水中的微生物生态多样性进行了研究.研究表明,酸性矿坑水呈酸性,相对于其他极端与非极端生态环境,酸性矿坑水中的细菌与古菌的群落多样性较低.RFLP分析与系统发育分析表明,酸性矿坑水中细菌主要由A.fcrrooxidans(属于gamma-Proteobacteria)和L.ferrooxidans(属于Nitospira)成;古菌主要由Thermoplasma相关古菌组成.在这种封闭环境的酸性矿坑水中首次发现了类似于产甲烷古菌的克隆片断,其占古菌种群的四分之一左右.本研究将促进对酸性矿坑水中细菌及古菌群落组成及其对酸性矿坑水产生的作用的研究.     

云南东川黄铜矿酸性浸矿废水中微生物群落分析

利用限制性片段长度多态性和16s rRNA PCR扩增及其序列分析来研究中国云南东川黄铜矿酸性浸矿废水(AMD)中微生物群落结构。基于建立16s rRNA克隆文库的分子方法常常被用来研究AMD中微生物生态。一小部分的rRNA被PCR扩增,克隆,然后用限制性片段长度多态性来筛选出其不同种类。在三个样地中,共挑选了357个克隆子,经限制性酶切分析后聚为100个可操作分类单元(OTU)。一个OTU代表各个样地中出现的同一种酶切图谱。有58个OTU被测序分析,结果在基因数据库中比对分析,其系统发育分析表明它们聚类为六大类:γ-proteobacteria,Acidobacteria,Clostridia,Actinobacteria,Nitrospira,α-pro- teobacteria。其中,γ-proteobacteria为最大的群落,在三个样地DCK-Ⅰ,DCN-Ⅰand DC-Ⅱ中分别占36%,57%,62%的比例。但与浸矿相关的微生物如At.ferrooxidans;L ferrooxidans等却发现的很少,仅在两个样地中各发现四个相关的OTUs。在这个AMD环境中浸矿相关的微生物都不是优势种群。     

Culturable and molecular phylogenetic diversity of microorganisms in an open-dumped,extremely acidic Pb/Zn mine tailings

A combination of cultivation-based and molecular-based approaches was used to reveal the culturable and molecular diversity of the microbes inhabiting an open-dumped Pb/Zn mine tailings that was undergoing intensive acid generation (pH 1.9). Culturable bacteria found in the extremely acidic mine tailings were Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum, Sulfobacillus thermotolerans and Acidiphilium cryptum, where the number of acidophilic heterotrophs was ten times higher than that of the iron- and sulfur-oxidizing bacteria. Cloning and phylogenetic analysis revealed that, in contrast to the adjacent AMD, the mine tailings possessed a low microbial diversity with archaeal sequence types dominating the 16S rRNA gene library. Of the 141 clones examined, 132 were represented by two sequence types phylogenetically affiliated with the iron-oxidizing archaea Ferroplasma acidiphilum and three belonged to two tentative groups within the Thermoplasma lineage so far represented by only a few environmental sequences. Six clones in the library were represented by the only bacterial sequence type and were closely related to the well-described iron-oxidizer L. ferriphilum. The significant differences in the prokaryotic community structures of the extremely acidic mine tailings and the AMD associated with it highlights the importance of studying the microbial communities that are more directly involved in the iron and sulfur cycles of mine tailings.     

Diversity of Prokaryotic Communities Indigenous to Acid Mine Drainage and Related Rocks from Baiyin Open-Pit Copper Mine Stope,China

Metagenomic approach permits us to obtain the latent resources from culturable and unculturable microorganisms in ecosystem. In this study, high-throughput sequencing was practiced to comprehensively probe prokaryotic community within extreme acidic environment of Baiyin open-pit mine stope, which varied in pH and other physicochemical parameters. Bioinformatics analysis was further accomplished to process millions of Illumina reads and analyzed alpha and beta diversities, and prokaryotic community profile in different samples obtained from the acidic mine stope. Diversity indices such as ACE, Chao, Shannon, and Simpson were varied among samples. Both taxon richness and evenness were significantly higher in the solid samples than that of the water samples. Taxonomic diversity was unexpectedly higher within confined pit ecosystem. Most of the sequences were assigned to phyla Proteobacteria, Firmicutes, and Acidobacteria. In archaea, Euryarchaeota and Thaumarchaeota were major phyla reported, however, archaea occupied very little share in the metagenome. At class level, variation in community structure was higher within samples. Among iron- and sulfur-related acidophiles, 30.8% of the sequences were unidentified at genera level, while the remaining were dominated by sulfur and/or iron oxidizing Acidithiobacillus and heterotrophic Acidiphilum related groups. The community profile of solid and water groups was different and metagenomic biomarkers were higher in solid, while acidophiles and archaea were reported only in water group by using LEfSe. Among samples, community structure and abundance was varied in terms of OTUs abundance, which clearly indicates spatial variation and proposed the influence of physicochemical and geochemical properties on phylogenetic diversity. This study offers numerous treasured datasets for better understanding the community composition under the influence of geochemical and physicochemical factors and possible novelty in terms of taxonomic/phylogenetic diversity in acidic ecosystem.     

Sulfate Reduction Based Bioprocesses for the Treatment of Acid Mine Drainage and the Recovery of Metals

Biological sulfate reduction is increasingly replacing chemical unit processes in mining biotechnology. Sulfate reducing bacteria (SRB) can be used for treating ground‐ and surface waters contaminated with acid mine drainage (AMD), and for recovering metals from wastewater and process streams. Biologically produced H₂S precipitates metals as metal sulfides, while biogenic bicarbonate alkalinity neutralizes acidic waters. This paper reviews various passive and active SRB‐based alternatives as well as some process design aspects, such as reactor types, process configurations, and choices of substrates for sulfate reduction. The latest developments of using various low‐cost substrates together with new bioprocess designs are increasing the uses and applications of SRB‐based bioreactors in AMD control and selective metal recovery.     

Phylogenetic analysis of the archaeal community in an alkaline-saline soil of the former lake Texcoco (Mexico)

The soil of the former lake Texcoco is an extreme environment localized in the valley of Mexico City, Mexico. It is highly saline and alkaline, where Na⁺, Cl⁻, HCO₃⁻ and CO₃²⁻ are the predominant ions, with a pH ranging from 9.8 to 11.7 and electrolytic conductivities in saturation extracts from 22 to 150 dS m⁻¹. Metagenomic DNA from the archaeal community was extracted directly from soil and used as template to amplify 16S ribosomal gene by PCR. PCR products were used to construct gene libraries. The ribosomal library showed that the archaeal diversity included Natronococcus sp., Natronolimnobius sp., Natronobacterium sp., Natrinema sp., Natronomonas sp., Halovivax sp., “Halalkalicoccus jeotgali” and novel clades within the family of Halobacteriaceae. Four clones could not be classified. It was found that the archaeal diversity in an alkaline-saline soil of the former lake Texcoco, Mexico, was low, but showed yet uncharacterized and unclassified species. César Valenzuela-Encinas and Isabel Neria-González contributed equally to this publication.     

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.     

Molecular analysis of microbial communities in mobile deltaic muds of Southeastern Papua New Guinea

A PCR-based approach combined with microbiological cultivation methods was employed to determine the occurrence of sulfate-reducing bacteria (SRB) in colon biopsy samples from ulcerative colitis patients and from non-colitic controls. The detection of mucosa-associated SRB was carried out by digoxigenin-dUTP-labelled PCR amplification, in liquid Postgate medium B and in a new liquid medium, termed VM medium I. Using Postgate medium B, the growth of SRB was confirmed in 92% of the colitic specimens and in 52% of non-colitic samples. However, PCR analysis and incubation in VM medium I detected SRB in 100% of biopsy material indicating ubiquitous presence of SRB in human colon mucosa.     

健康妇女及3例细菌性阴道病患者阴道菌群的非培养分析

根据Amsel标准及Nugent标准, 确诊筛选健康妇女及细菌性阴道病(bacterial vaginosis, BV)患者各3例。提取其阴道分泌物样本的总DNA, 构建16S rRNA基因克隆文库, 并对阳性克隆进行ARDRA和测序分析。结果表明, 健康妇女样本的基因文库中, 分别以卷曲乳酸杆菌(L. crispatus)和惰性乳酸杆菌(L. iners)的克隆子占较大比例, 另外存在少量的阴道乳酸杆菌(L. vaginalis)或詹氏乳酸杆菌(L. jensenii)的克隆子。BV患者样本的基因文库中, 克隆子所代表的菌种类型明显增多, 但均以阴道加德纳氏菌(Gardnerella vaginalis)和阴道阿托波氏菌(Atopobium vaginae)的克隆子占较大比例, 且无乳酸杆菌克隆子。说明健康妇女阴道菌群的种类单一, 以乳酸杆菌占优势, L. iners为优势菌种之一; BV患者阴道菌群的种类复杂多样, 但均以Gardnerella vaginalis及Atopobium vaginae共同占优势。     

健康妇女及3例细菌性阴道病患者阴道菌群的非培养分析

根据Amsel标准及Nugent标准,确诊筛选健康妇女及细菌性阴道病(bacterial vaginosis,BV)患者各3例.提取其阴道分泌物样本的总DNA,构建16S rRNA基因克隆文库,并对阳性克隆进行ARDRA和测序分析.结果表明,健康妇女样本的基因文库中,分别以卷曲乳酸杆菌(L.crispatus)和惰性乳酸杆菌(L. iners)的克隆子占较大比例,另外存在少量的阴道乳酸杆菌(L.vaginalis)或詹氏乳酸杆菌(L.jensenii)的克隆子.BV患者样本的基因文库中,克隆子所代表的菌种类型明显增多,但均以阴道加德纳氏菌(Gardnerella vaginalis)和阴道阿托波氏菌(Atopobium vaginae)的克隆子占较大比例,且无乳酸杆菌克隆子.说明健康妇女阴道菌群的种类单一,以乳酸杆菌占优势,L. iners为优势菌种之一;BV患者阴道菌群的种类复杂多样,但均以Gardnerella vaginalis及Atopobium vaginae共同占优势.     

Macrofilamentous microbial communities in the metal-rich and acidic River Tinto, Spain

A novel type of macroscopic microbial community consisting of large dendritic filaments (up to 1.5 m) in a pH 2.0 dam of the River Tinto (South-western Spain) is described. The combined use of 16S rRNA-gene surveys and fluorescent in situ hybridisation (FISH) suggested that gamma-proteobacteria and a relative large diversity of alpha-proteobacteria dominated these structures. beta-Proteobacteria, Actinobacteria and Firmicutes were also detected. Whereas acidophilic bacteria of the genera Acidithiobacillus, Leptospirillum and Acidiphilium, and archaea belonging to the Thermoplasmatales dominate mine acid drainage waters and streamers (riverbed filamentous biofilms), none of the lineages identified in this study affiliate to typical acid mine drainage acidophilic bacteria. Bacteria of the Tinto macrofilaments might be heterotrophic, and could be feeding on the organic matter entrapped in the filamentous structure.     

Tetraether-linked membrane monolayers in <Emphasis Type="Italic">Ferroplasma</Emphasis> spp: a key to survival in acid

Ferroplasma acidarmanus thrives in hot, extremely low pH, metal-rich solutions associated with dissolving metal sulfide ore deposits. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thin layer chromatography analyses of F. acidarmanus membranes indicate that tetraether lipids predominate, with at least three core lipid structures. NMR measurements indicate that the cytoplasmic pH of F. acidarmanus is ~5.6. The optimal growth pH is ~1.2, and the lowest growth pH is ~0.0. Thus, these organisms maintain pH gradients across their membranes that approach 5 pH units. Tetraether lipids were originally thought to be specifically associated with thermophiles but are now known to be widely distributed within the archaeal domain. Our data, in combination with recently published results for thermophilic and mesothermophilic acidophilic archaea, indicate that there may be a stronger association between tetraether lipids and tolerance to acid and/or large metal ion gradients.     

Microbial sulfate reduction in acidic (pH 3) strip-mine lakes

Abstract ³⁵SO₄ reduction was detected in slurries of sediments obtained from Reservoir 29 (pH 3.8) and Lake B (pH 6.2), two acid strip-mine lakes in Indiana. The rates varied seasonally and were higher in summer and fall than in the spring. The optimal pH for sulfate reduction in Reservoir 29 sediments was 5, but samples had increased activity at pH 7 within 24 h after adjusting the pH to this value. In Lake B, the optimal pH for sulfate reduction was the in situ pH (6.2). Sulfate reduction in both lakes was stimulated 2–3-fold by increasing p _H2. High concentrations (5 mM) of organic acids inhibited sulfate reduction at pH 3.8, but stimulation was observed at concentrations of 0.1 mM. Acid-volatile sulfides accounted for about 70% of the products of ³⁵SO₄ reduction.     

Culturable Diversity and Community Fatty Acid Profiling of Sulfate-Reducing Fluidized-Bed Reactors Treating Acidic,Metal-Containing Wastewater

Cultivable strains were identified from sulfate-reducing fluidized-bed reactors (FBR) treating acidic metal-containing wastewater. The FBR-communities were further characterized using culture-independent phenotypic markers, phospholipid fatty acid (PLFA) profiling. After morphological screening of 128 bacterial strains and partial sequencing of 55 strains, 17 distinct phylogenetic types were identified and characterized further. A total of 14 and 6 different bacterial strains were isolated from ethanol- and lactate-fed FBRs, respectively. Sequencing of the 16S rRNA gene showed that these strains were affiliated with members of the δ-Proteobacteria, Firmicutes and Bacteroidetes The strains were affiliated with members of the genera Desulfovibrio, Desulfotomaculum, Desulfobulbus, Desulfitobacterium, Clostridium, Caloramator, Oxobacter, and Bacteroides. Many of the strains were only distantly related to previously described species and, thus, may represent novel species or genera. A number of the strains were not detected in previously employed molecular analyses of the FBR communities, and the major component of each FBR as identified in the molecular analyses were not retrieved as cultures in this study. Most of the SRB, and two of the non-SRB utilized ethanol and lactate as a source of carbon and energy, but none of the isolates grew on acetate, an intermediate in the oxidation of ethanol and lactate. PLFA analysis revealed that the FBR community members contained large amounts of saturated fatty acids. Although the PLFA analysis showed some signatures consistent with sulfate-reducing communities, it did not show any substantial difference in the microbial communities between the reactors, an outcome that was quite contrary to the culture-independent phylogenetic analyses.     

Archaeaplankton in the Columbia River, its estuary and the adjacent coastal ocean, USA

PCR-amplified 16S rRNA genes from particle-attached and free-living Archaea in the Columbia River estuary, particle-attached Archaea in the river, and Archaea in the adjacent coastal ocean were cloned, and 43 partial sequences were determined. There was a high diversity of Archaea in the estuary, especially among the particle-attached Archaea, with representatives from four major phylogenetic clusters. Eighteen of 21 estuarine clones were closely related to clones from the river and the coastal ocean or to clusters of marine and soil clones identified in other studies. This contrasts with a similar study of the estuarine bacterial community that found 62% of bacterial 16S rRNA clones to be unique to the estuary. Archaea in the estuary were primarily allochthonous, and therefore, unlike the bacteria, probably do not form a native estuarine community.     

Laboratory Scale Bioremediation of Acid Mine Water Drainage from a Disused Tin Mine

Real acidic mine-water drainage was seeded with Acidithiobacillus ferrooxidans to catalyse the removal of iron contained therein. The addition of At. ferrooxidans increased metal precipitation kinetics and decreased the water iron content by ~70%. Supplementing non-sterile mine water with a bacterial growth medium accelerated metal removal by indigenous micro-organisms both at the 500 ml shake-flask and 5 l bioreactor scale.     

Cultivation-dependent and cultivation-independent characterization of the microbial community in acid mine drainage associated with acidic Pb/Zn mine tailings at Lechang, Guangdong, China

Cultivation-based and molecular approaches were used to characterize the phylogenetic composition and structure of the microbial community in an extremely acidic (pH 2.0) acid mine drainage (AMD) associated with Pb/Zn mine tailings that were undergoing vigorous acid generation. Acidophilic bacteria were isolated and enumerated on solid media, and were found to be restricted to isolates related to Acidithiobacillus ferrooxidans and Acidiphilium cryptum. By contrast, cloning and phylogenetic analysis of 16S rRNA genes revealed that, although low in total taxonomically distinct groups, the tailings AMD ecosystem harbored a wide range of phylogenetically diverse microbes. Of the 141 clones examined, 104 were phylogenetically affiliated with the recently discovered, iron-oxidizing Leptospirillum group III within the Nitrospira. It thus appears that iron serves as the major electron donor in this habitat. Thirty clones were affiliated with the Proteobacteria, half of which belonged to organisms related to Alphaproteobacteria species capable of ferric iron reduction. Other clones were grouped with Betaproteobacteria and Gammaproteobacteria (six clones each), and even with Deltaproteobacteria (three clones), a subdivision with anaerobic sulfate or metal (iron) reduction as the predominant physiological trait of its members. Finally, four clones were clustered within the Firmicutes and the Acidobacteria. Approximately half of the sequence types representing the majority of the total clones fell into lineages that are poorly represented by cultured organisms or have thus far been represented by only a few environmental sequences. Thus, the present study extends our knowledge of the biodiversity of microorganisms populating highly acidic AMD environments.     

Analysis of the microbial community in moderately acidic drainage from the Yanahara pyrite mine in Japan

Acid rock drainage (ARD) originating from the Yasumi-ishi tunnel near the main tunnel of the Yanahara mine in Japan was characterized to be moderately acidic (pH 4.1) and contained iron at a low concentration (51?mg/L). The composition of the microbial community was determined by sequence analysis of 16S rRNA genes using PCR and denaturing gradient gel electrophoresis. The analysis of the obtained sequences showed their similarity to clones recently detected in other moderately acidic mine drainages. Uncultured bacteria related to Ferrovum- and Gallionella-like clones were dominant in the microbial community. Analyses using specific primers for acidophilic iron- or sulfur-oxidizing bacteria, Acidithiobacillus ferrooxidans, Leptospirillum spp., Acidithiobacillus caldus, Acidithiobacillus thiooxidans, and Sulfobacillus spp. revealed the absence of these bacteria in the microbial community in ARD from the Yasumi-ishi tunnel. Clones affiliated with a member of the order Thermoplasmatales were detected as the dominant archaea in the ARD microbial population.