Seasonal variations in microbial populations and environmental conditions in an extreme acid mine drainage environment.

Microbial populations, their distributions, and their aquatic environments were studied over a year (1997) at an acid mine drainage (AMD) site at Iron Mountain, Calif. Populations were quantified by fluorescence in situ hybridizations with group-specific probes. Probes were used for the domains Eucarya, Bacteria, and Archaea and the two species most widely studied and implicated for their role in AMD production, Thiobacillus ferrooxidans and Leptospirillum ferrooxidans. Results show that microbial populations, in relative proportions and absolute numbers, vary spatially and seasonally and correlate with geochemical and physical conditions (pH, temperature, conductivity, and rainfall). Bacterial populations were in the highest proportion (>95%) in January. Conversely, archaeal populations were in the highest proportion in July and September ( approximately 50%) and were virtually absent in the winter. Bacterial and archaeal populations correlated with conductivity and rainfall. High concentrations of dissolved solids, as reflected by high conductivity values (up to 125 mS/cm), occurred in the summer and correlated with high archaeal populations and proportionally lower bacterial populations. Eukaryotes were not detected in January, when total microbial cell numbers were lowest (<10(5) cells/ml), but eukaryotes increased at low-pH sites ( approximately 0.5) during the remainder of the year. This correlated with decreasing water temperatures (50 to 30 degrees C; January to November) and increasing numbers of prokaryotes (10(8) to 10(9) cells/ml). T. ferrooxidans was in highest abundance (>30%) at moderate pHs and temperatures ( approximately 2.5 and 20 degrees C) in sites that were peripheral to primary acid-generating sites and lowest (0 to 5%) at low-pH sites (pH approximately 0.5) that were in contact with the ore body. L. ferrooxidans was more widely distributed with respect to geochemical conditions (pH = 0 to 3; 20 to 50 degrees C) but was more abundant at higher temperatures and lower pHs ( approximately 40 degrees C; pH approximately 0.5) than T. ferrooxidans.     

Quantitative proteomic analyses of the response of acidophilic microbial communities to different pH conditions

Extensive genomic characterization of multi-species acid mine drainage microbial consortia combined with laboratory cultivation has enabled the application of quantitative proteomic analyses at the community level. In this study, quantitative proteomic comparisons were used to functionally characterize laboratory-cultivated acidophilic communities sustained in pH 1.45 or 0.85 conditions. The distributions of all proteins identified for individual organisms indicated biases for either high or low pH, and suggests pH-specific niche partitioning for low abundance bacteria and archaea. Although the proteome of the dominant bacterium, Leptospirillum group II, was largely unaffected by pH treatments, analysis of functional categories indicated proteins involved in amino acid and nucleotide metabolism, as well as cell membrane/envelope biogenesis were overrepresented at high pH. Comparison of specific protein abundances indicates higher pH conditions favor Leptospirillum group III, whereas low pH conditions promote the growth of certain archaea. Thus, quantitative proteomic comparisons revealed distinct differences in community composition and metabolic function of individual organisms during different pH treatments. Proteomic analysis revealed other aspects of community function. Different numbers of phage proteins were identified across biological replicates, indicating stochastic spatial heterogeneity of phage outbreaks. Additionally, proteomic data were used to identify a previously unknown genotypic variant of Leptospirillum group II, an indication of selection for a specific Leptospirillum group II population in laboratory communities. Our results confirm the importance of pH and related geochemical factors in fine-tuning acidophilic microbial community structure and function at the species and strain level, and demonstrate the broad utility of proteomics in laboratory community studies.     

Pyruvate Inhibition of Ferrobacillus ferrooxidans Reversed by Rhodotorula Yeast

Ferrobacillus ferrooxidans was isolated in this laboratory from uranium ores mined at Jaduguda, Bihar. Investigations on a laboratory scale indicated that this organism could leach uranium from ores. Pyruvic acid (10^-4 M) was found to inhibit growth of F. ferrooxidans as well as leaching of uranium ore by F. ferrooxidans . However, in the presence of a red pigmented Rhodotorula yeast isolated from the same ores, the pyruvate inhibition was found to be reversed.     

Chemolithotrophic Bacteria in Copper Ores Leached at High Sulfuric Acid Concentration

Extensive bacterial growth was observed when copper sulfide ores were leached with 0.6 N sulfuric acid. The bacterial population developed in this condition was examined by characterization of the spacer regions between the 16S and 23S rRNA genetic loci obtained after PCR amplification of the DNA extracted from the leached ore. The spacers observed had the sizes found in strains of "Leptospirillum ferrooxidans" and Thiobacillus thiooxidans, except for a larger one, approximately 560 bp long, that was not observed in any of the strains examined, including those of Thiobacillus ferrooxidans. The bacteria with this last spacer were selected after culturing in mineral and elemental sulfur media containing 0.7 N sulfuric acid. The spacer and the 16S ribosomal DNA of this isolate were sequenced and compared with those in species commonly found in bioleaching processes. Though the nucleotide sequence of the spacer showed an extensive heterologous region with T. thiooxidans, the sequence of its 16S rDNA gene indicated a close relationship (99.85%) with this species. These results indicate that a population comprised of bacterial strains closely related to T. thiooxidans and of another strain, possibly related to "L. ferrooxidans," can develop during leaching at high sulfuric acid concentration. Iron oxidation in this condition is attributable to "L. ferrooxidans" and not T. ferrooxidans, based on the presence of spacers with the "L. ferrooxidans" size range and the absence of spacers characteristic of T. ferrooxidans.     

Comparison of Acid Mine Drainage Microbial Communities in Physically and Geochemically Distinct Ecosystems

This study presents population analyses of microbial communities inhabiting a site of extreme acid mine drainage (AMD) production. The site is the inactive underground Richmond mine at Iron Mountain, Calif., where the weathering of a massive sulfide ore body (mostly pyrite) produces solutions with pHs of ~0.5 to ~1.0. Here we used a suite of oligonucleotide probes, designed from molecular data recently acquired from the site, to analyze a number of microbial environments by fluorescent in situ hybridization. Microbial-community analyses were correlated with geochemical and mineralogical data from those environments. The environments investigated were within the ore body and thus at the site of pyrite dissolution, as opposed to environments that occur downstream of the dissolution. Few organism types, as defined by the specificities of the oligonucleotide probes, dominated the microbial communities. The majority of the dominant organisms detected were newly discovered or organisms only recently associated with acid-leaching environments. “Ferroplasma” spp. were detected in many of the communities and were particularly dominant in environments of lowest pH and highest ionic strength. Leptospirillum spp. were also detected in many slime and pyrite-dominated environments. In samples of an unusual subaerial slime, a new uncultured Leptospirillum sp. dominated. Sulfobacillus spp. were detected as a prominent inhabitant in warmer (~43°C) environments. The information gathered here is critical for determining organisms important to AMD production at Iron Mountain and for directing future studies of this process. The findings presented here also have relevance to the microbiology of industrial bioleaching and to the understanding of geochemical iron and sulfur cycles.     

Comparison of acid mine drainage microbial communities in physically and geochemically distinct ecosystems

This study presents population analyses of microbial communities inhabiting a site of extreme acid mine drainage (AMD) production. The site is the inactive underground Richmond mine at Iron Mountain, Calif., where the weathering of a massive sulfide ore body (mostly pyrite) produces solutions with pHs of approximately 0.5 to approximately 1.0. Here we used a suite of oligonucleotide probes, designed from molecular data recently acquired from the site, to analyze a number of microbial environments by fluorescent in situ hybridization. Microbial-community analyses were correlated with geochemical and mineralogical data from those environments. The environments investigated were within the ore body and thus at the site of pyrite dissolution, as opposed to environments that occur downstream of the dissolution. Few organism types, as defined by the specificities of the oligonucleotide probes, dominated the microbial communities. The majority of the dominant organisms detected were newly discovered or organisms only recently associated with acid-leaching environments. "Ferroplasma" spp. were detected in many of the communities and were particularly dominant in environments of lowest pH and highest ionic strength. Leptospirillum spp. were also detected in many slime and pyrite-dominated environments. In samples of an unusual subaerial slime, a new uncultured Leptospirillum sp. dominated. Sulfobacillus spp. were detected as a prominent inhabitant in warmer ( approximately 43 degrees C) environments. The information gathered here is critical for determining organisms important to AMD production at Iron Mountain and for directing future studies of this process. The findings presented here also have relevance to the microbiology of industrial bioleaching and to the understanding of geochemical iron and sulfur cycles.     

Heterotrophic bacteria from cultures of autotrophic Thiobacillus ferrooxidans: relationships as studied by means of deoxyribonucleic acid homology.

From several presumably pure cultures of Thiobacillus ferrooxidans, we isolated a pair of stable phenotypes. One was a strict autotroph utilizing sulfur or ferrous iron as the energy source and unable to utilize glucose; the other phenotype was an acidophilic obligate heterotroph capable of utilizing glucose but not sulfur or ferrous iron. The acidophilic obligate heterotroph not only was encountered in cultures of T. ferrooxidans, but also was isolated with glucose-mineral salts medium, pH 2.0, directly from coal refuse. By means of deoxyribonucleic acid homology, we have demonstrated that the acidophilic heterotrophs are of a different genotype from T. ferrooxidans, not closely related to this species; we have shown also that the acidophilic obligate heterotrophs, regardless of their source of isolation, are related to each other. Therefore, cultures of T. ferrooxidans reported capable of utilizing organic compounds should be carefully examined for contamination. The acidophilic heterotrophs isolated by us are different from T. acidophilis, which is also associated with T. ferrooxidans but is facultative, utilizing both glucose and elemental sulfur as energy sources. Since they are so common and tenacious in T. ferrooxidans cultures, the heterotrophs must be associated with T. ferrooxidans in the natural habitat.     

Seasonal Variations in Microbial Populations and Environmental Conditions in an Extreme Acid Mine Drainage Environment

Microbial populations, their distributions, and their aquatic environments were studied over a year (1997) at an acid mine drainage (AMD) site at Iron Mountain, Calif. Populations were quantified by fluorescence in situ hybridizations with group-specific probes. Probes were used for the domains Eucarya, Bacteria, and Archaea and the two species most widely studied and implicated for their role in AMD production, Thiobacillus ferrooxidans and Leptospirillum ferrooxidans. Results show that microbial populations, in relative proportions and absolute numbers, vary spatially and seasonally and correlate with geochemical and physical conditions (pH, temperature, conductivity, and rainfall). Bacterial populations were in the highest proportion (>95%) in January. Conversely, archaeal populations were in the highest proportion in July and September (~50%) and were virtually absent in the winter. Bacterial and archaeal populations correlated with conductivity and rainfall. High concentrations of dissolved solids, as reflected by high conductivity values (up to 125 mS/cm), occurred in the summer and correlated with high archaeal populations and proportionally lower bacterial populations. Eukaryotes were not detected in January, when total microbial cell numbers were lowest (<10⁵ cells/ml), but eukaryotes increased at low-pH sites (~0.5) during the remainder of the year. This correlated with decreasing water temperatures (50 to 30°C; January to November) and increasing numbers of prokaryotes (10⁸ to 10⁹ cells/ml). T. ferrooxidans was in highest abundance (>30%) at moderate pHs and temperatures (~2.5 and 20°C) in sites that were peripheral to primary acid-generating sites and lowest (0 to 5%) at low-pH sites (pH ~0.5) that were in contact with the ore body. L. ferrooxidans was more widely distributed with respect to geochemical conditions (pH = 0 to 3; 20 to 50°C) but was more abundant at higher temperatures and lower pHs (~40°C; pH ~0.5) than T. ferrooxidans.     

Genomic insights into the iron uptake mechanisms of the biomining microorganism Acidithiobacillus ferrooxidans

Commercial bioleaching of copper and the biooxidation of gold is a cost-effective and environmentally friendly process for metal recovery. A partial genome sequence of the acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans is available from two public sources. This information has been used to build preliminary models that describe how this microorganism confronts unusually high iron loads in the extremely acidic conditions (pH 2) found in natural environments and in bioleaching operations. A. ferrooxidans contains candidate genes for iron uptake, sensing, storage, and regulation of iron homeostasis. Predicted proteins exhibit significant amino acid similarity with known proteins from neutrophilic organisms, including conservation of functional motifs, permitting their identification by bioinformatics tools and allowing the recognition of common themes in iron transport across distantly related species. However, significant differences in amino acid sequence were detected in pertinent domains that suggest ways in which the periplasmic and outer membrane proteins of A. ferrooxidans maintain structural integrity and relevant protein-protein contacts at low pH. Unexpectedly, the microorganism also contains candidate genes, organized in operon-like structures that potentially encode at least 11 siderophore systems for the uptake of Fe(III), although it does not exhibit genes that could encode the biosynthesis of the siderophores themselves. The presence of multiple Fe(III) uptake systems suggests that A. ferrooxidans can inhabit aerobic environments where iron is scarce and where siderophore producers are present. It may also help to explain why it cannot tolerate high Fe(III) concentrations in bioleaching operations where it is out-competed by Leptospirillum species.     

Filamentous Hydrous Ferric Oxide Biosignatures in a Pipeline Carrying Acid Mine Drainage at Iron Mountain Mine,California

A pipeline carrying acidic mine effluent at Iron Mountain, CA, developed Fe(III)-rich precipitate caused by oxidation of Fe(II)_aq. The native microbial community in the pipe included filamentous microbes. The pipe scale consisted of microbial filaments, and schwertmannite (ferric oxyhydroxysulfate, FOHS) mineral spheres and filaments. FOHS filaments contained central lumina with diameters similar to those of microbial filaments. FOHS filament geometry, the geochemical environment, and the presence of filamentous microbes suggest that FOHS filaments are mineralized microbial filaments. This formation of textural biosignatures provides the basis for a conceptual model for the development and preservation of biosignatures in other environments.     

Sulfite oxidation by iron-grown cells of Thiobacillus ferrooxidans at pH 3 possibly involves free radicals, iron, and cytochrome oxidase

Thiobacillus ferrooxidans cells grown on ferrous iron oxidized sulfite to sulfate at pH 3, possibly by a free radical mechanism involving iron and cytochrome oxidase. A purely chemical system with low concentrations of Fe3+ simulated the T. ferrooxidans system. Metal chelators, ethylenediamine tetraacetic acid (EDTA), 4,5-dihydroxy-1-3-benzene disulfonic acid (Tiron), o-phenanthroline, and 2,2'-dipyridyl, inhibited both sulfite oxidation systems, but the T. ferrooxidans system was inhibited only after the initial brief oxygen consumption. EDTA and Tiron, strong chelators of Fe3+, inhibited the oxidation at lower concentrations than o-phenanthroline and 2,2'-dipyridyl, strong chelators of Fe2+. Inhibition of Fe3+-catalyzed sulfite oxidation by EDTA and Tiron was instant, but the inhibition by o-phenanthroline and dipyridyl was briefly delayed, presumably for the reduction of Fe3+ to Fe2+. Mannitol, a free radical scavenger, inhibited both systems to the same extent. Cyanide and azide inhibited only the T. ferrooxidans system, suggesting a role of cytochrome oxidase. It is proposed that sulfite is oxidized by a free radical mechanism initiated by Fe3+ on the cell surface of T. ferrooxidans. Cytochrome oxidase is possibly involved in the regeneration of Fe3+ from Fe2+ by the normal Fe2+-oxidizing system of T. ferrooxidans.     

Sedimentary diatom assemblages and their utility in computing diatom-inferred pH in Sudbury Ontario lakes

Surface sediment diatoms from 30 Sudbury lakes were analyzed to establish a relationship between diatoms and limnological characteristics of these lakes. Factor analysis of various chemical and physical variables suggested that most of the variance was associated with the pH, pH-metal, and conductivity factors. Factor analysis of common diatom taxa indicated that the distribution of most of these diatoms is closely related to lake water pH and/or pH related factors. Regression analysis of various pH indicator assemblages with measured lake water pH also demonstrated significant relationships. Among the transfer functions employed to compute diatom-inferred pH, multiple regressions using pH indicator assemblages proved to be the best. Using this method an accuracy of about 0.3 pH unit can be achieved, even for lakes which have received large trace metal inputs concurrent with increase in acidity. The presence of planktonic diatoms in acidic lakes suggests that the widely accepted hypothesis that planktonic diatoms are greatly reduced in waters at pH below 5.6 is not always true.     

Distribution and ecology of rotifer communities in high-altitude alpine sites — a multivariate approach

The distribution and ecology of benthic, periphytic and planktonic rotifers were investigated in a wide range of alpine waterbodies in Austria. A total of 162 substrate classified samples was taken at 60 sampling sites situated between 1824 m and 2753 m a.s.l. in the Central Alps (predominantly gneiss) and between 1290 m and 1643 m a.s.l. in the Northeastern Calcareous Alps. Multivariate analysis allowed the recognition of distinct sampling site groups. Different groups of taxa were identified on the basis of their total frequencies of occurrence and frequency of co-occurrence. Several species were shown to have distributions restricted to particular groups of habitats. Habitat complexity, as indicated by taxon diversity and density of submersed vegetation, pH, conductivity, and temperature seem to be the principal components affecting community composition and distribution of single species in alpine waterbodies. Highest diversities were measured in limestone solution lakes and acid bog ponds on primary bedrock.     

Bacterial Community in Copper Sulfide Ores Inoculated and Leached with Solution from a Commercial-Scale Copper Leaching Plant

Most copper bioleaching plants operate with a high concentration of sulfate salts caused by the continuous addition of sulfuric acid and the recycling of the leaching solution. Since the bacteria involved in bioleaching have been generally isolated at low sulfate concentrations, the bacterial population in ores leached with the high-sulfate solution (1.25 M) employed in a copper production plant was investigated. The complexity of the original population was assessed by the length pattern of the spacer regions between the 16S and 23S rRNA genes, observed after PCR amplification of the DNA extracted from the leached ore. Six main spacers were distinguished by electrophoretic migration, but they could be further resolved into eight spacers by nucleotide sequence homology. The degree of homology was inferred from the electrophoretic migration of the heteroduplexes formed after hybridization. One of the spacers was indistinguishable from that found in Thiobacillus thiooxidans, four could be related to Thiobacillus ferrooxidans, and three could be related to Leptospirillum ferrooxidans. Only five of the spacers in the original sample could be recovered after culturing in media containing different inorganic energy source. Altogether, the results indicate that the bacteria in the leached ore formed a community composed of at least three species: a fairly homogeneous population of T. thiooxidans strains and two heterogeneous populations of T. ferrooxidans and L. ferrooxidans strains.     

Detection of Thiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining.

An indirect fluorescent antibody (FA) staining technique was developed for the rapid detection of Thiobacillus ferrooxidans. The specificity of the FA stain for T. ferrooxidans was demonstrated with both laboratory and environmental samples. Coal refuse examined by scanning electron microscopy exhibited a rough, porous surface, which was characteristically covered by water-soluble crystals. Significant numbers of T. ferrooxidans were detected in the refuse pores. A positive correlation between numbers of T. ferrooxidans and acid production in coal refuse in the laboratory was demonstrated with the FA technique.     

Inhibition of growth, iron, and sulfur oxidation in Thiobacillus ferrooxidans by simple organic compounds.

Iron and sulfur oxidation by Thiobacillus ferrooxidans as well as growth on ferrous iron were inhibited by a variety of low molecular weight organic compounds. The influences of chemical structure of the organic inhibitors, pH, temperature, physical treatment of cells, and added inhibitory or stimulatory inorganic ions and iron oxidation suggest that a major factor contributing to the inhibitory effects on iron oxidation is the relative electronegativity of the organic molecule. The data also suggest that inhibitory organic compounds may (i) directly affect the iron-oxidizing enzyme system, (ii) react abiologically with ferrous iron outside the cell, (iii) interfere with the roles of phosphate and sulfate in iron oxidation, and (iv) nonselectively disrupt the cell envelope or membrane.     

Seasonal and spatial variations in microbial community structure and diversity in the acid stream draining across an ongoing surface mining site

This study examined the microbial community in an acidic stream draining across the Yun-Fu pyrite mine (Guangdong, China), where extremely acidic mine water is a persistent feature due to the intensive surface mining activities. Analysis of terminal restriction fragment length polymorphism (TRFLP) of 16S rRNA gene sequences showed that microbial populations varied spatially and seasonally and correlated with geochemical and physical conditions. After the stream moves from underground to the surface, the microbial community in the acidic water rapidly evolves into a distinct community close to that in the downstream storage pond. Comparisons of TRFLP peaks with sequenced clone libraries indicated that bacteria related to the recently isolated iron-oxidizer Ferrovum myxofaciens dominated the acidophilic community throughout the year except for the samples collected in spring from the storage pond, where Ferroplasma acidiphilum -like archaea represented the most abundant group. Acidithiobacillus ferrooxidans -affiliated organisms increased along the acid stream and remained common over the year, whereas Leptospirillum ferrooxidans -like bacteria were negligible or even not detected in the analyzed samples. The data indicate that changes in environmental conditions are accompanied by significant shifts in community structure of the prokaryotic assemblages at this opencast mining site.     

Detection of Thiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining.

An indirect fluorescent antibody (FA) staining technique was developed for the rapid detection of Thiobacillus ferrooxidans. The specificity of the FA stain for T. ferrooxidans was demonstrated with both laboratory and environmental samples. Coal refuse examined by scanning electron microscopy exhibited a rough, porous surface, which was characteristically covered by water-soluble crystals. Significant numbers of T. ferrooxidans were detected in the refuse pores. A positive correlation between numbers of T. ferrooxidans and acid production in coal refuse in the laboratory was demonstrated with the FA technique.     

Isolation and characterization of an acidophilic, heterotrophic bacterium capable of oxidizing ferrous iron.

A heterotrophic bacterium, isolated from an acidic stream in a disused pyrite mine which contained copious growths of "acid streamers," displayed characteristics which differentiated it from previously described mesophilic acidophiles. The isolate was obligately acidophilic, with a pH range of 2.0 to 4.4 and an optimum pH of 3.0. The bacterium was unable to fix carbon dioxide but oxidized ferrous iron, although at a slower rate than either Thiobacillus ferrooxidans or Leptospirillum ferrooxidans. Elemental sulfur and manganese(II) were not oxidized. In liquid media, the isolate produced macroscopic streamerlike growths. Microscopic examination revealed that the bacterium formed long (greater than 100 microns) filaments which tended to disintegrate during later growth stages, producing single, motile cells and small filaments. The isolate did not appear to utilize the energy from ferrous iron oxidation. Both iron (ferrous or ferric) and an organic substrate were necessary to promote growth. The isolate displayed a lower tolerance to heavy metals than other iron-oxidizing acidophiles, and growth was inhibited by exposure to light. There was evidence of extracellular sheath production by the isolate. In this and some other respects, the isolate resembles members of the Sphaerotilus-Leptothrix group of filamentous bacteria. The guanine-plus-cytosine content of the isolate was 62 mol%, which is less than that recorded for Sphaerotilus-Leptothrix spp. and greater than those of L. ferrooxidans and most T. ferrooxidans isolates.     

Sulfide oxidation by spheroplasts of Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is an acidophilic organism important to metal leaching of low-grade ores. The aforementioned importance is related to the ability of the bacterium to oxidize reduced iron and sulfur, principally found in nature as pyrite (FeS2). The present study dealt with sulfide oxidation at low pH values and the involvement of the cell envelope in the process of the inorganic oxidations. Sulfide oxidation was noted in spheroplasts of T. ferrooxidans prepared by enzymatic and chemical treatments and partially purified by differential centrifugation. No enzyme activities were noted in membrane fractions containing enrichments of lipopolysaccharide symbolic of outer membrane material or in membrane vesicles containing (or associated with) higher levels of proteins. Results to date indicate that in an acid milieu the envelope structure containing both the outer membrane and the intact inner cytoplasmic membrane is required for sulfide oxidation.