Archaeal and bacterial communities of heavy metal contaminated acidic waters from zinc mine residues in Sepetiba Bay

Mining of metallic sulfide ore produces acidic water with high metal concentrations that have harmful consequences for aquatic life. To understand the composition and structure of microbial communities in acid mine drainage (AMD) waters associated with Zn mine tailings, molecular diversity of 16S genes was examined using a PCR, cloning, and sequencing approach. A total of 78 operational taxonomic units (OTUs) were obtained from samples collected at five different sites in and around mining residues in Sepetiba Bay, Brazil. We analyzed metal concentration, physical, chemical, and microbiological parameters related to prokaryotic diversity in low metal impacted compared to highly polluted environments with Zn at level of gram per liter and Cd–Pb at level of microgram per liter. Application of molecular methods for community structure analyses showed that Archaea and Bacteria groups present a phylogenetic relationship with uncultured environmental organisms. Phylogenetic analysis revealed that bacteria present at the five sites fell into seven known divisions, α-Proteobacteria (13.4%), β-Proteobacteria (16.3%), γ-Proteobacteria (4.3%), Sphingobacteriales (4.3%), Actinobacteria (3.2%) Acidobacteria (2.1%), Cyanobacteria (11.9%), and unclassified bacteria (44.5%). Almost all archaeal clones were related to uncultivated Crenarchaeota species, which were shared between high impacted and low impacted waters. Rarefaction curves showed that bacterial groups are more diverse than archaeal groups while the overall prokaryotic biodiversity is lower in high metal impacted environments than in less polluted habitats. Knowledge of this microbial community structure will help in understanding prokaryotic diversity, biogeography, and the role of microorganisms in zinc smelting AMD generation and perhaps it may be exploited for environmental remediation procedures in this area.     

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.     

Microalgal adaptation to a stressful environment (acidic,metal-rich mine waters) could be due to selection of pre-selective mutants originating in non-extreme environments

At least six species of eukaryotic microalgae inhabit the acidic (pH 2.4–2.7), metal-rich mine waters from ponds in the copper mine district of Mynydd Parys (N Wales, UK). Consequently, these ponds constitute interesting natural laboratories for analysis of adaptation by microalgae to extremely stressful conditions. To distinguish between the pre-selective and post-selective origin of adaptation processes that allow the existence of microalgae in these ponds, a Luria-Delbrück fluctuation analysis was performed with the chlorophycean Dictyosphaerium chlorelloides isolated from non-acidic waters. In this analysis, natural Mynydd Parys pond water (MPW) was used as selective factor. Pre-selective, resistant D. chlorelloides cells appeared with a frequency of 1.6 × 10^?6 per cell per generation. MPW-resistant mutants, with a diminished Malthusian fitness, are maintained in non-extreme waters as the result of a balance between new MPW-resistant cells arising by mutation and MPW-resistant mutants eliminated by natural selection (equilibrium at ca. 19 MPW-resistant per 10⁷ wild-type cells). We propose that the microalgae inhabiting these stressful ponds could be the descendents of chance mutants that arrived in the past or are even arriving at the present.     

Microbial communities and geochemical dynamics in an extremely acidic, metal-rich stream at an abandoned sulfide mine (Huelva, Spain) underpinned by two functional primary production systems

An extremely acidic (pH 2.5-2.75) metal-rich stream draining an abandoned mine in the Iberian Pyrite Belt, Spain, was ramified with stratified macroscopic gelatinous microbial growths ('acid streamers' or 'mats'). Microbial communities of streamer/mat growths sampled at different depths, as well as those present in the stream water itself, were analysed using a combined biomolecular and cultivation-based approach. The oxygen-depleted mine water was dominated by the chemolithotrophic facultative anaerobe Acidithiobacillus ferrooxidans, while the streamer communities were found to be highly heterogeneous and very different to superficially similar growths reported in other extremely acidic environments. Microalgae accounted for a significant proportion of surface streamer biomass, while subsurface layers were dominated by heterotrophic acidophilic bacteria (Acidobacteriacae and Acidiphilium spp.). Sulfidogenic bacteria were isolated from the lowest depth streamer growths, where there was also evidence for selective biomineralization of copper sulfide. Archaeal clones (exclusively Euryarchaeota) were recovered from streamer samples, as well as the mine stream water. Both sunlight and reduced inorganic chemicals (predominantly ferrous iron) served as energy sources for primary producers in this ecosystem, promoting complex microbial interactions involving transfer of electron donors and acceptors and of organic carbon, between microorganisms in the stream water and the gelatinous streamer growths. Microbial transformations were shown to impact the biogeochemical cycling of iron and sulfur in the acidic stream, severely restricting the net oxidation of ferrous iron even when the initially anoxic waters were oxygenated by indigenous acidophilic algae. A model accounting for the biogeochemistry of iron and sulfur in the mine waters is described, and the significance of the acidophilic communities in regulating the geochemistry of acidic, metal-rich waters is described.     

Selective removal of transition metals from acidic mine waters by novel consortia of acidophilic sulfidogenic bacteria

Two continuous‐flow bench‐scale bioreactor systems populated by mixed communities of acidophilic sulfate‐reducing bacteria were constructed and tested for their abilities to promote the selective precipitation of transition metals (as sulfides) present in synthetic mine waters, using glycerol as electron donor. The objective with the first system (selective precipitation of copper from acidic mine water containing a variety of soluble metals) was achieved by maintaining a bioreactor pH of ～2.2–2.5. The second system was fed with acidic (pH 2.5) synthetic mine water containing 3 mM of both zinc and ferrous iron, and varying concentrations (0.5–30 mM) of aluminium. Selective precipitation of zinc sulfide was possible by operating the bioreactor at pH 4.0 and supplementing the synthetic mine water with 4 mM glycerol. Analysis of the microbial populations in the bioreactors showed that they changed with varying operational parameters, and novel acidophilic bacteria (including one sulfidogen) were isolated from the bioreactors. The acidophilic sulfidogenic bioreactors provided ‘proof of principle’ that segregation of metals present in mine waters is possible using simple online systems within which controlled pH conditions are maintained. The modular units are versatile and robust, and involve minimum engineering complexity.     

Decay of sewage-associated bacterial communities in fresh and marine environmental waters and sediment

Applied Microbiology and Biotechnology - Understanding the microbial quality of recreational waters is critical to effectively managing human health risks. In recent years, the development of new...     

Effect of dispersed oil on heterotrophic bacterial communities in cold marine waters

Mesocosm studies were conducted to evaluate the effect of dispersed oil on total and heterotrophic bacterial communities of under-ice seawater from the St. Lawrence Estuary. A regular survey of bacterial changes in the oil-contaminated seawater was performed during a two week period. The bacterial community structure was investigated by carrying out 27 morphological and biochemical tests on 168 isolated strains. The results show a detectable but transient response of the bacterial community to crude oil addition. While total bacterial counts were approximately constant during the experiment, dispersed oil induced an increase in heterotrophic bacterial microflora (from 10⁴ to 10⁵ bacteria ml^-1 after two weeks of contamination). The dispersed oil appeared to have an inhibitory effect on some components of the bacterial community. A decrease of most probable number values was observed just after addition of crude oil in the most polluted tanks and one day later in the less polluted tank. However, except for the most polluted tank, this adverse effect disappeared rapidly. While the dispersed oil induced a total disappearance of some components of the bacterial community in the most polluted tank, the structure of the bacterial community in the less polluted tank appeared relatively unchanged after 14 days of contamination.Offprint requests to: Daniel Delille     

Attached and unattached bacterial communities in a 120-meter corehole in an acidic, crystalline rock aquifer

The bacteria colonizing geologic core sections (attached) were contrasted with those found suspended in the groundwater (unattached) by examining the microbiology of 16 depth-paired core and groundwater samples using a suite of culture-independent and culture-dependent analyses. One hundred twenty-two meters was continuously cored from a buried chalcopyrite ore hosted in a biotite-quartz-monzonite porphyry at the Mineral Park Mine near Kingman, Ariz. Every fourth 1.5-m core was acquired using microbiologically defensible methods, and these core sections were aseptically processed for characterization of the attached bacteria. Groundwater samples containing unattached bacteria were collected from the uncased corehole at depth intervals corresponding to the individual cores using an inflatable straddle packer sampler. The groundwater was acidic (pH 2.8 to 5.0), with low levels of dissolved oxygen and high concentrations of sulfate and metals, including ferrous iron. Total numbers of attached cells were less than 10(5) cells g of core material(-1) while unattached cells numbered about 10(5) cells ml of groundwater(-1). Attached and unattached acidophilic heterotrophs were observed throughout the depth profile. In contrast, acidophilic chemolithotrophs were not found attached to the rock but were commonly observed in the groundwater. Attached communities were composed of low numbers (<40 CFU g(-1)) of neutrophilic heterotrophs that exhibited a high degree of morphologic diversity, while unattached communities contained higher numbers (ca. 10(3) CFU ml(-1)) of neutrophilic heterotrophs of limited diversity. Sulfate-reducing bacteria were restricted to the deepest samples of both core and groundwater. 16S ribosomal DNA sequence analysis of attached, acidophilic isolates indicated that organisms closely related to heterotrophic, acidophilic mesophiles such as Acidiphilium organovorum and, surprisingly, to the moderately thermophilic Alicyclobacillus acidocaldarius were present. The results indicate that viable (but possibly inactive) microorganisms were present in the buried ore and that there was substantial distinction in biomass and physiological capabilities between attached and unattached populations.     

Seasonal changes in nitrogen-cycle gene abundances and in bacterial communities in acidic forest soils

The abundance of genes related to the nitrogen biogeochemical cycle and the microbial community in forest soils (bacteria, archaea, fungi) were quantitatively analyzed via real-time PCR using 11 sets of specific primers amplifying nifH, bacterial amoA, archaeal amoA, narG, nirS, nirK, norB, nosZ, bacterial 16S rRNA gene, archaeal 16S rRNA gene, and the ITS sequence of fungi. Soils were sampled from Bukhan Mountain from September of 2010 to July of 2011 (7 times). Bacteria were the predominant microbial community in all samples. However, the abundance of archaeal amoA was greater than bacterial amoA throughout the year. The abundances of nifH, nirS, nirK, and norB genes changed in a similar pattern, while narG and nosZ appeared in sensitive to the environmental changes. Clone libraries of bacterial 16S rRNA genes were constructed from summer and winter soil samples and these revealed that Acidobacteria was the most predominant phylum in acidic forest soil environments in both samples. Although a specific correlation of environmental factor and gene abundance was not verified by principle component analysis, our data suggested that the combination of biological, physical, and chemical characteristics of forest soils created distinct conditions favoring the nitrogen biogeochemical cycle and that bacterial communities in undisturbed acidic forest soils were quite stable during seasonal change.     

Characterisation and differentiation of oligotrophic waters by culturable particle-attached and free-living bacterial communities

This study reports the development of a tool to characterise and differentiate northern Adriatic waters, particularly oligotrophic, high-salinity waters, based on the cellular fatty acids of culturable heterotrophic bacterioplankton. The growth abilities and population dominance were observed for particle-attached and free-living bacteria cultured in three types of media: Marine Broth, diluted Marine Broth (1:10) and R2 broth. Three groups of water layers were distinguished by hierarchical clustering analysis: eutrophic, oligotrophic and oligotrophic nutrient-selected. Significant differences between the resulting groups were tested by two-way ANOVA (with replication). Eutrophic layers were characterised by readily culturable particle-attached and free-living fractions of the bacterial community in all three media, all dominated by fast-growing γ-Proteobacteria. In contrast, oligotrophic water layers with low productivity had a much weaker culturability and a different population dominance for the free-living community, as compared to their attached or growth-arrested counterparts, for all media. The free-living bacteria from strictly oligotrophic environments demonstrated minimum culturability in Marine Broth, while those from selective oligotrophic environments were culturable and were dominated either by Cytophaga–Flavobacter complex, α-Proteobacteria or γ-Proteobacteria. The conclusive evidence regarding the selective and refractory nature of organic compounds in these waters demonstrates the dominant culturability of the Cytophaga–Flavobacter complex and α-Proteobacteria in free-living communities in all growth-media. The response of fatty acid dominance ratios depends significantly on the trophic state and fraction (p < 0.05), although the effect of the trophic state is completely different in attached and free fractions. Both fractions were tested separately, demonstrating a significant influence of the trophic state (p < 0.05), while the effect of the media on the fatty acid response was not significant (p > 0.05). An interaction between media and trophic status was present in the attached fraction (p < 0.05), yet this was not observed in the free fraction (p > 0.05), indicating that any systematic difference between trophic states was the same for each media tested. Accordingly, the free-living fraction of bacterioplankton is a more informative attribute and can be used solely as an indicator of the water layer trophic condition.     

Effects of enrichment on protist abundances and bacterial composition in simple microbial communities

We experimentally investigated effects of nutrient enrichment and trophic structure in a microbial food web consisting of mixed bacteria, two bacterivorous ciliates ( Tetrahymena sp. and Colpidium sp.) and an omnivorous ciliate ( Blepharisma sp.) feeding on both trophic levels. We assembled all possible food webs including one or more of the ciliate species and cross-classified them with four levels of enrichment of the bacterial medium. The qualitative outcome of food web interactions was independent of enrichment and always the same: Tetrahymena strongly depressed or excluded Colpidium , and Blepharisma strongly depressed or excluded both bacterivores. Consequently, in all sub-webs only the dominant ciliate species responded positively to enrichment. The total density of bacteria increased with enrichment irrespective of food web composition. In contrast, the response of single-celled bacteria to enrichment depended on food web composition and was only weakly positive in most food webs with the omnivore. Enrichment had a positive effect on the relative success of (presumably more defended) bacterial aggregates. The outcome of interspecific interactions among ciliates could not be predicted from monoculture experiments and deviated from earlier experiments in which each bacterivore coexisted separately with the omnivore. As a potential explanation we suggest that changes in experimental protocol reduced spatial heterogeneity and increased attack rates. A simple, dynamical model shows that increased attack rates can indeed greatly decrease the upper limit and range of enrichment over which intermediate consumers can coexist with omnivores.     

A reference system for continental running waters: plant communities as bioindicators of increasing eutrophication in alkaline and acidic waters in north-east France

Two bioindication scales of the degree of eutrophication based on aquatic macrophyte communities were established in two types of running waters free of organic matter, the one in acidic soft waters (pH 5.5–7.0, conductivity 40–110 S.cm^–1), the other in alkaline hard waters (pH 7–8, conductivity 500–900 S.cm^–1). We show that the main determining factor of the macrophyte distribution is the nutrient level (trophy), especially the level of phosphate and ammonia. The acidic scale, with increasing pH, includes four stages ranging from oligotrophic to eutrophic level (traces to 300 g.l^–1 N-NHinf4^p+ and P-PO₄ ^3–), while the alkaline scale at constant pH comprises six stages of a trophic gradient. For the most part, the floristic composition found in the two sequences is different and depends on conductivity and alkalinity variation. However, some species occur in the two scales and may reflect differences in the trophic level, depending on whether the waters are alkaline or acidic. This change of trophic level for these species is discussed.     

广东北部山区若干森林群落

应用植被样方调查法,对地处粤北山区的连州田心、乐昌八宝山、曲江罗坑、仁化范子山、始兴刘张家山、平远龙文-黄田等自然保护区的森林植被优势种进行比较分析,并用极点排序法对上述6地的森林群落进行二维排序。结果表明:各地共有优势种仅有红楠和枫香2种,且优势度差异明显。各地群落的相似性系数以田心与龙文-黄田之间的65.49%为最大;八宝山与其它各地的群落相似性程度均小于50%。群落相似性系数与极点排序结果均提示森林群落的相似性程度受土壤基质、海拔高度的影响要大于经纬度和地理距离的影响。各地森林群落间接地反映了它们的生境条件,对保护区森林群落的比较研究,可为今后广东自然保护区的网络建设提供参考。     

Effects of abiotic factors on the phylogenetic diversity of bacterial communities in acidic thermal springs

Acidic thermal springs offer ideal environments for studying processes underlying extremophile microbial diversity. We used a carefully designed comparative analysis of acidic thermal springs in Yellowstone National Park to determine how abiotic factors (chemistry and temperature) shape acidophile microbial communities. Small-subunit rRNA gene sequences were PCR amplified, cloned, and sequenced, by using evolutionarily conserved bacterium-specific primers, directly from environmental DNA extracted from Amphitheater Springs and Roaring Mountain sediment samples. Energy-dispersive X-ray spectroscopy, X-ray diffraction, and colorimetric assays were used to analyze sediment chemistry, while an optical emission spectrometer was used to evaluate water chemistry and electronic probes were used to measure the pH, temperature, and E(h) of the spring waters. Phylogenetic-statistical analyses found exceptionally strong correlations between bacterial community composition and sediment mineral chemistry, followed by weaker but significant correlations with temperature gradients. For example, sulfur-rich sediment samples contained a high diversity of uncultured organisms related to Hydrogenobaculum spp., while iron-rich sediments were dominated by uncultured organisms related to a diverse array of gram-positive iron oxidizers. A detailed analysis of redox chemistry indicated that the available energy sources and electron acceptors were sufficient to support the metabolic potential of Hydrogenobaculum spp. and iron oxidizers, respectively. Principal-component analysis found that two factors explained 95% of the genetic diversity, with most of the variance attributable to mineral chemistry and a smaller fraction attributable to temperature.     

Isolation and phylogenetic characterization of acidophilic microorganisms indigenous to acidic drainage waters at an abandoned Norwegian copper mine

The biodiversity of culturable acidophilic microbes in three acidic (pH 2.7–3.7), metal-rich waters at an abandoned subarctic copper mine in central Norway was assessed. Acidophilic bacteria were isolated by plating on selective solid media, and dominant isolates were identified from their physiological characteristics and 16S rRNA gene sequences. The dominant iron-oxidizing acidophile in all three waters was an Acidithiobacillus ferrooxidans -like eubacterium, which shared 98% 16S rDNA identity with the type strain. A strain of Leptospirillum ferrooxidans was obtained from one of the waters after enrichment in pyrite medium, but this iron oxidizer was below detectable levels in the acidic waters themselves. In two sites, there were up to six distinct heterotrophic acidophiles, present at 10³ ml⁻¹. These included Acidiphilium -like isolates (one closely related to Acidiphilium rubrum , a second to Acidiphilium cryptum and a third apparently novel isolate), an Acidocella -like isolate (96% 16S rDNA identity to Acidocella facilis ) and a bacterium that shared 94.5% 16S rDNA identity to Acidisphaera rubrifaciens. The other numerically significant heterotrophic isolate was not apparently related to any known acidophile, with the closest match (96% 16S rDNA sequence identity) to an acetogen, Frateuria aurantia . The results indicated that the biodiversity of acidophilic bacteria, especially heterotrophs, in acidic mine waters may be much greater than previously recognized.     

Diversity and functional analysis of bacterial communities associated with natural hydrocarbon seeps in acidic soils at Rainbow Springs, Yellowstone National Park

In this paper we describe the bacterial communities associated with natural hydrocarbon seeps in nonthermal soils at Rainbow Springs, Yellowstone National Park. Soil chemical analysis revealed high sulfate concentrations and low pH values (pH 2.8 to 3.8), which are characteristic of acid-sulfate geothermal activity. The hydrocarbon composition of the seep soils consisted almost entirely of saturated, acyclic alkanes (e.g., n-alkanes with chain lengths of C15 to C30, as well as branched alkanes, predominately pristane and phytane). Bacterial populations present in the seep soils were phylogenetically characterized by 16S rRNA gene clone library analysis. The majority of the sequences recovered (>75%) were related to sequences of heterotrophic acidophilic bacteria, including Acidisphaera spp. and Acidiphilium spp. of the alpha-Proteobacteria. Clones related to the iron- and sulfur-oxidizing chemolithotroph Acidithiobacillus spp. were also recovered from one of the seep soils. Hydrocarbon-amended soil-sand mixtures were established to examine [14C]hexadecane mineralization and corresponding changes in the bacterial populations using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Approximately 50% of the [14C]hexadecane added was recovered as 14CO2 during an 80-day incubation, and this was accompanied by detection of heterotrophic acidophile-related sequences as dominant DGGE bands. An alkane-degrading isolate was cultivated, whose 16S rRNA gene sequence was identical to the sequence of a dominant DGGE band in the soil-sand mixture, as well as the clone sequence recovered most frequently from the original soil. This and the presence of an alkB gene homolog in this isolate confirmed the alkane degradation capability of one population indigenous to acidic hydrocarbon seep soils.     

Microbial communities,processes and functions in acid mine drainage ecosystems

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十八河铜尾矿库坝面细菌群落时空动态及其驱动力

群落多样性的维持机制是群落生态学研究的热点问题.尾矿库作为人工原生裸地,土壤重金属含量较高,随着恢复年限的延长,土壤理化性质发生变化,是研究土壤微生物群落多样性驱动机制的理想场地.本研究在调查十八河尾矿库土壤因子、植物群落多样性和土壤细菌群落多样性的基础上,探究了局域小尺度下细菌群落结构和多样性的驱动机制.结果表明: 尾矿库土壤养分含量随恢复年限的延长显著提高,并具有一定的季节变化,不同恢复年限土壤养分含量的季节动态存在差异.细菌群落的Shannon多样性和丰富度指数随恢复年限呈显著增加趋势,群落稳定性逐步提高;而其季节变化受到植物群落的影响,在不同恢复年限下具有一定的差异.RDA分析显示,环境因子对土壤细菌群落多样性有决定作用.结构方程模型分析表明,土壤养分(TC、TN、NO₃^--N、NO₂^--N)含量、植物群落多样性和土壤酶活性共同驱动该铜尾矿库土壤细菌群落的构建过程.     

In-depth characterization of bacterial and archaeal communities present in the abandoned Kettara pyrrhotite mine tailings (Morocco)

Extremophiles - In Morocco, pollution caused by closed mines continues to be a serious threat to the environment, like the generation of acid mine drainage. Mine drainage is produced by...