Acidophilic, Heterotrophic Bacteria of Acidic Mine Waters

Obligately acidophilic, heterotrophic bacteria were isolated both from enrichment cultures developed with acidic mine water and from natural mine drainage. The bacteria were grouped by the ability to utilize a number of organic acids as sole carbon sources. None of the strains were capable of chemolithotrophic growth on inorganic reduced iron and sulfur compounds. All bacteria were rod shaped, gram negative, nonencapsulated, motile, capable of growth at pH 2.6 but not at pH 6.0, catalase and oxidase positive, strictly aerobic, and capable of growth on citric acid. The bacteria were cultivatable on solid nutrient media only if agarose was employed as the hardening agent. Bacterial densities in natural mine waters ranged from approximately 20 to 250 cells per ml, depending upon source and culture medium. Ferric hydrates and stream vegetation contained from 1,500 to over 7 × 10⁶ cells per g.     

Assessment of the Microbial Community in a Constructed Wetland that Receives Acid Coal Mine Drainage

Constructed wetlands are used to treat acid drainage from surface or underground coal mines. However, little is known about the microbial communities in the receiving wetland cells. The purpose of this work was to characterize the microbial population present in a wetland that was receiving acid coal mine drainage (AMD). Samples were collected from the oxic sediment zone of a constructed wetland cell in southeastern Ohio that was treating acid drainage from an underground coal mine seep. Samples comprised Fe(III) precipitates and were pretreated with ammonium oxalate to remove interfering iron, and the DNA was extracted and purified by agarose gel electrophoresis prior to amplification of portions of the 16S rRNA gene. Amplified products were separated by denaturing gradient gel electrophoresis and DNA from seven distinct bands was excised from the gel and sequenced. The sequences were matched to sequences in the GenBank bacterial 16S rDNA database. The DNA in two of the bands yielded matches with Acidithiobacillus ferrooxidans and the DNA in each of the remaining five bands was consistent with one of the following microorganisms: Acidithiobacillus thiooxidans, strain TRA3-20 (a eubacterium), strain BEN-4 (an arsenite-oxidizing bacterium), an Alcaligenes sp., and a Bordetella sp. Low bacterial diversity in these samples reflects the highly inorganic nature of the oxic sediment layer where high abundance of iron- and sulfur-oxidizing bacteria would be expected. The results we obtained by molecular methods supported our findings, obtained using culture methods, that the dominant microbial species in an acid receiving, oxic wetland are A. thiooxidans and A. ferrooxidans.     

Acid mine drainage treatment with rotating biological contactors

A pilot scale rotating biological contactor (RBC) was set up near a coal mine at Hollywood, Penn. to evaluate ferrous iron, Fe(II), oxidation. Acid drainage from this mine entered the treatment unit which consisted of four sets of plastic disks affixed to a rotating central shaft. As the disks rotated half immersed in the flowing mine water, iron-oxidizing bacteria, presumed to be Thiobacillus ferrooxidans, colonized the disk surfaces with an average population of 70,000 cells/cm² and mediated the transformation of Fe(II) to the less soluble ferric state, Fe(III). Kinetics of microbial Fe(II) oxidation were established during an eleven month period of continuous pilot operation and were found to follow a concentration dependent first order relationship. Operating at an optimum disk rotation rate and hydraulic loadings of 2.7 and 5.4 gal/day-ft² (0.11 and 0.22 m³/day-m²) resulted in the oxidation of an average 240 mg/liter influent Fe(II) to produce effluent Fe(II) of 2 and mg/liter, respectively. The RBC appears potentially useful as a first step in the total treatment of acid mine drainage.     

Macroinvertebrate Responses to Algal and Bacterial Manipulations in Streams

Our study was designed to assess the relative importance of algae and bacteria as sources of energy for stream macroinvertebrates. In one experiment, we manipulated algae by artificially shading six sections in each of two streams, one stream with an open canopy (clear-cut drainage basin) and the other with a closed canopy (forested drainage basin); both streams were in Hubbard Brook Experimental Forest, New Hampshire, USA. Chlorophyll a concentrations were reduced from 0.2 to 0.05 μg/cm² in artificially shaded sections of both streams. However, macroinvertebrates showed no response to these algal manipulations in either the clear-cut or forested stream. Nutrient concentrations (N and P) were low and limiting to primary production in both the clear-cut and forested streams. Additionally, both streams had relatively low macroinvertebrate densities suggesting bottom-up controls were important in macroinvertebrate abundance. However, the forested stream did have higher macroinvertebrate densities presumably because of higher inputs of coarse particulate organic matter from the riparian vegetation. In a second experiment, in Augusta Creek, Michigan, we manipulated both algae and bacteria. To reduce algae, we artificially shaded experimental stream channels so that chlorophyll a was reduced from natural levels of 3.0–5.6 to 0.4–0.7 μg/cm². Half of the shaded channels had dissolved organic carbon (DOC – sucrose) dripped into them to raise DOC levels by 2–3 mg/l and thus stimulate bacterial abundance. Open channels, with higher algal abundance, had higher densities of Ephemerella, but only in November when nymphs were larger. Channels with increased DOC had higher bacterial abundances, higher densities of Chironomidae and lower densities of Heptageniidae. Several other macroinvertebrate taxa that were at relatively low abundance in our samples showed no significant response to these manipulations. Our results suggest that early instar Ephemerella may not rely as heavily on algae as later instars. Also, certain taxa were able to use the heterotrophic microbial community, especially chironomids which increased in numbers when bacterial density increased; thus, the bacterial carbon source may be more important to some stream macroinvertebrates than previous studies have suggested.     

Chemolithotrophic Sulfur-Oxidizing Bacteria from the Galapagos Rift Hydrothermal Vents

Three distinct physiological types of sulfur-oxidizing bacteria were enriched and isolated from samples collected at several deep-sea hydrothermal vents (2,550 m) of the Galapagos Rift ocean floor spreading center. Twelve strains of the obligately chemolithotrophic genus Thiomicrospira were obtained from venting water and from microbial mats covering surfaces in the immediate vicinity of the vents. From these and other sources two types of obligately heterotrophic sulfur oxidizers were repeatedly isolated that presumably oxidized thiosulfate either to sulfate (acid producing; 9 strains) or to polythionates (base producing; 74 strains). The former were thiobacilli-like, exhibiting a thiosulfate-stimulated increase in growth and CO₂ incorporation, whereas the latter were similar to previously encountered pseudomonad-like heterotrophs. The presence of chemolithotrophic sulfur-oxidizing bacteria in the sulfide-containing hydrothermal water supports the hypothesis that chemosynthesis provides a substantial primary food source for the rich populations of invertebrates found in the immediate vicinity of the vents.     

Performance and microbial community dynamics of a sulfate-reducing bioreactor treating coal generated acid mine drainage

The effectiveness of a passive flow sulfate-reducing bioreactor processing acid mine drainage (AMD) generated from an abandoned coal mine in Southern Illinois was evaluated using geochemical and microbial community analysis 10 months post bioreactor construction. The results indicated that the treatment system was successful in both raising the pH of the AMD from 3.09 to 6.56 and in lowering the total iron level by 95.9%. While sulfate levels did decrease by 67.4%, the level post treatment (1153 mg/l) remained above recommended drinking water levels. Stimulation of biological sulfate reduction was indicated by a +2.60‰ increase in δ³⁴S content of the remaining sulfate in the water post-treatment. Bacterial community analysis targeting 16S rRNA and dsrAB genes indicated that the pre-treated samples were dominated by bacteria related to iron-oxidizing Betaproteobacteria, while the post-treated water directly from the reactor outflow was dominated by sequences related to sulfur-oxidizing Epsilonproteobacteria and complex carbon degrading Bacteroidetes and Firmicutes phylums. Analysis of the post-treated water, prior to environmental release, revealed that the community shifted back to predominantly iron-oxidizing Betaproteobacteria. DsrA analysis implied limited diversity in the sulfate-reducing population present in both the bioreactor outflow and oxidation pond samples. These results support the use of passive flow bioreactors to lower the acidity, metal, and sulfate levels present in the AMD at the Tab-Simco mine, but suggest modifications of the system are necessary to both stimulate sulfate-reducing bacteria and inhibit sulfur-oxidizing bacteria.     

Microbial Communities in Subpermafrost Saline Fracture Water at the Lupin Au Mine, Nunavut, Canada

We report the first investigation of a deep subpermafrost microbial ecosystem, a terrestrial analog for the Martian subsurface. Our multidisciplinary team analyzed fracture water collected at 890 and 1,130 m depths beneath a 540-m-thick permafrost layer at the Lupin Au mine (Nunavut, Canada). ¹⁴C, ³H, and noble gas isotope analyses suggest that the Na–Ca–Cl, suboxic, fracture water represents a mixture of geologically ancient brine, ~25-kyr-old, meteoric water and a minor modern talik-water component. Microbial planktonic concentrations were ~10³ cells mL^?1. Analysis of the 16S rRNA gene from extracted DNA and enrichment cultures revealed 42 unique operational taxonomic units in 11 genera with Desulfosporosinus, Halothiobacillus, and Pseudomonas representing the most prominent phylotypes and failed to detect Archaea. The abundance of terminally branched and midchain-branched saturated fatty acids (5 to 15 mol%) was consistent with the abundance of Gram-positive bacteria in the clone libraries. Geochemical data, the ubiquinone (UQ) abundance (3 to 11 mol%), and the presence of both aerobic and anaerobic bacteria indicated that the environment was suboxic, not anoxic. Stable sulfur isotope analyses of the fracture water detected the presence of microbial sulfate reduction, and analyses of the vein-filling pyrite indicated that it was in isotopic equilibrium with the dissolved sulfide. Free energy calculations revealed that sulfate reduction and sulfide oxidation via denitrification and not methanogenesis were the most thermodynamically viable consistent with the principal metabolisms inferred from the 16S rRNA community composition and with CH₄ isotopic compositions. The sulfate-reducing bacteria most likely colonized the subsurface during the Pleistocene or earlier, whereas aerobic bacteria may have entered the fracture water networks either during deglaciation prior to permafrost formation 9,000 years ago or from the nearby talik through the hydrologic gradient created during mine dewatering. Although the absence of methanogens from this subsurface ecosystem is somewhat surprising, it may be attributable to an energy bottleneck that restricts their migration from surface permafrost deposits where they are frequently reported. These results have implications for the biological origin of CH₄ on Mars.     

Macroscopic Streamer Growths in Acidic, Metal-Rich Mine Waters in North Wales Consist of Novel and Remarkably Simple Bacterial Communities

The microbial composition of acid streamers (macroscopic biofilms) in acidic, metal-rich waters in two locations (an abandoned copper mine and a chalybeate spa) in north Wales was studied using cultivation-based and biomolecular techniques. Known chemolithotrophic and heterotrophic acidophiles were readily isolated from disrupted streamers, but they accounted for only <1 to 7% of the total microorganisms present. Fluorescent in situ hybridization (FISH) revealed that 80 to 90% of the microbes in both types of streamers were β-Proteobacteria. Terminal restriction fragment length polymorphism analysis of the streamers suggested that a single bacterial species was dominant in the copper mine streamers, while two distinct bacteria (one of which was identical to the bacterium found in the copper mine streamers) accounted for about 90% of the streamers in the spa water. 16S rRNA gene clone libraries showed that the β-proteobacterium found in both locations was closely related to a clone detected previously in acid mine drainage in California and that its closest characterized relatives were neutrophilic ammonium oxidizers. Using a modified isolation technique, this bacterium was isolated from the copper mine streamers and shown to be a novel acidophilic autotrophic iron oxidizer. The β-proteobacterium found only in the spa streamers was closely related to the neutrophilic iron oxidizer Gallionella ferruginea. FISH analysis using oligonucleotide probes that targeted the two β-proteobacteria confirmed that the biodiversity of the streamers in both locations was very limited. The microbial compositions of the acid streamers found at the two north Wales sites are very different from the microbial compositions of the previously described acid streamers found at Iron Mountain, California, and the Rio Tinto, Spain.     

Prokaryotic communities of the north-eastern Mongolian soda lakes

We have studied the activity and composition of several geochemically significant physiological groups of bacteria in more than twenty alkaline salt lakes of the north-east Mongolia steppe with water salinity from 3 to 390 g l^?1 and pH values ranging from 9.0 to 10.6. Active and diverse microbial communities have been found in most of the lakes. The methanotrophic bacteria were represented by the Type I members. Among the culturable forms of sulfur-oxidizing bacteria obligately chemolithoautotrophic and haloalkaliphilic representatives of the genera Thioalkalimicrobium and Thioalkalivibrio were detected in the sediments at high numbers (up to 10⁶ cells ml^?1). The largest population of anaerobic phototrophic bacteria was represented by purple sulfur bacteria of the Ectothiorhodospiraceae family. Salinity was the key factor in determining the activity and the composition of the microbial communities. The most diverse and active prokaryotic populations, including aerobic and anaerobic phototrophic, methanogenic, methanotrophic, sulfur-oxidizing, sulfate-reducing and nitrifying bacteria, were found in lakes with salinity less than 60 g l^?1. In hypersaline lakes with a salinity >100 g l^?1, the sulfur cycle remained active due to the activity of extremely halotolerant and alkaliphilic sulfur bacteria, while other important functional groups responsible for nitrification and methane oxidation processes were not detected. Overall, the prokaryotic communities of the Mongolian alkaline salt lakes represent an interesting new example of a diverse community of haloalkaliphilic bacteria well adopted to a broad salinity range.     

Radionuclides in plants growing on sludge and water from uranium mine water treatment

Chemical treatment of uranium mine acid drainage generates sludge containing high radioactivity levels with, for example, ²³⁸U and ²²⁶Ra concentrations at about 18 and 9 kBq kg⁻¹ dry weight, respectively. Spontaneous vegetation, such as grass (Polygonum sp.), reeds (Phragmites australis), and bullrush (Typha latifolia) growing in sludge dewatering ponds concentrated uranium and uranium daughter radionuclides. However, bullrush growing in natural wetlands by the stream receiving treated mine water discharges contained even higher radionuclide concentrations, e.g., 21 Bq kg⁻¹ dry weight of ²³⁸U, about four times higher than bullrush growing on sludge, probably because the uranium in water is more bioavailable to Typha than uranium in the chemical sludge. It is suggested that wetlands with plant species could be used as a secondary treatment to further reduce radioactivity in chemically treated uranium mine water and to improve water quality in streams receiving treated water discharges. Furthermore, it is concluded that vegetation both from sludge drying ponds and from streams receiving treated water discharges, due to the high radionuclide concentrations in the vegetation, is not suitable as cattle feeding.     

Characterization of the core microbiota of the drainage and surrounding soil of a Brazilian copper mine

The core microbiota of a neutral mine drainage and the surrounding high heavy metal content soil at a Brazilian copper mine were characterized by 16S rDNA pyrosequencing. The core microbiota of the drainage was dominated by the generalist genus Meiothermus. The soil samples contained a more heterogeneous bacterial community, with the presence of both generalist and specialist bacteria. Both environments supported mainly heterotrophic bacteria, including organisms resistant to heavy metals, although many of the bacterial groups identified remain poorly characterized. The results contribute to the understanding of bacterial communities in soils impacted by neutral mine drainage, for which information is scarce, and demonstrate that heavy metals can play an important role in shaping the microbial communities in mine environments.     

Heterotrophic Nitrification in an Acid Forest Soil and by an Acid-Tolerant Fungus

Nitrate was formed from ammonium at pH 3.2 to 6.1 in suspensions of a naturally acid forest soil; the maximum rates of formation occurred at ca. pH 4 to 5. Nitrate was also formed from soil nitrogen in suspensions incubated at 50°C. Autotrophic nitrifying bacteria could not be isolated from this soil. Enrichment cultures produced nitrate in a medium with β-alanine if much soil was added to the medium, and nitrite but not nitrate was formed in the presence of small amounts of soil. Nitrification by these enrichments was abolished by eucaryotic but not procaryotic inhibitors. A strain of Absidia cylindrospora isolated from this soil was found to produce nitrate and nitrite in a medium with β-alanine at pH values ranging from 4.0 to 4.8. Nitrate production by A. cylindrospora required the presence of sterile soil. Free and bound hydroxylamine, hydroxamic acids, and primary aliphatic nitro compounds did not accumulate during the conversion of β-alanine to nitrite by the fungus. The organism also formed nitrite from ammonium in a medium containing acetate. We suggest that nitrification in this soil is a heterotrophic process catalyzed by acid-tolerant fungi and not by autotrophs or heterotrophs in nonacid microsites.     

Monitoring Dental-Unit-Water-Line Output Water by Current In-office Test Kits

The importance of monitoring contamination levels in the output water of dental-unit-water-lines (DUWLs) is essential as they are prone to developing biofilms that may contaminate water that is used to treat patients, with opportunistic pathogens such as species of Legionella, Pseudomonas and others. Dentists and practice staff are also at risk of being infected by means of cross-infection due to aerosols generated from DUWL water. The unit of measurement for the microbial contamination of water by aerobic mesophilic heterotrophic bacteria is the colony-forming unit per millilitre (cfu/ml) of water. The UK has its own guidelines set by the Department of Health for water discharged from DUWL to be between 100 and 200 cfu/ml of water. The benchmark or accepted standard laboratory test is by microbiological culture on R2A agar plates. However, this is costly and not convenient for routine testing in dental practices. A number of commercial indicator tests are used in dental surgeries, but they were not developed for the dental market and serve only to indicate gross levels of contamination when used outside of the manufacturer’s recommended incubation period. The aim of this article is to briefly review the universal problem of DUWL contamination with microbial biofilms and to update dental professionals on the availability of currently available commercial in-office monitoring systems for aerobic mesophilic heterotrophic bacteria and to discuss their limitations for testing water samples in assuring compliance with recommended guidelines.     

Organic matter breakdown as a measure of stream health in New Zealand streams affected by acid mine drainage

Functional indicators of stream health have the potential to provide insights into stream condition that cannot be gained by traditional structural indices. We examined breakdown of leaves, wood, and cotton cloth strips at 18 sites along a gradient of effects of drainage from coal mines in New Zealand to determine the usefulness of these methods as functional indicators of stream health. The pH varied from 2.7 to neutral across the streams, and the more acidic streams typically had higher concentrations of aluminum, iron, zinc, and other metal ions. Precipitates of metal (mainly iron) hydroxides were present in most streams affected by mine drainage, especially in those with a pH of 4–5. Breakdown rates of all organic matter types were highest in several reference streams with neutral pH and lowest in sites with high rates of metal hydroxide deposition. Breakdown was relatively fast in the most acidic streams (pH < 3), in some cases as fast as at reference sites; these sites also had elevated nutrient concentrations. Shredding invertebrates were absent in litterbags from acidic streams and common at only 2 reference sites; their presence contributed to fast breakdown of leaves in the field and in lab microcosms. Microbial respiration was closely related to breakdown rates of leaves and wood; it was high at neutral and highly acidic streams, but lower at sites with pH 4–5, where metal hydroxides were precipitating onto solid surfaces. In these metal hydroxide-stressed streams, leaf and wood breakdown was slower, and associated biota, including microbes, were more affected than by water chemistry stressors (pH, dissolved metals) associated with mine drainage. Litter breakdown and microbial respiration provide insight into the functioning of streams, yielding different responses than traditional structural measures based on macroinvertebrates, which did not accurately distinguish impacts from acid mine drainage.     

Pristine but metal-rich Río Sucio (Dirty River) is dominated by Gallionella and other iron-sulfur oxidizing microbes

Whether the extreme conditions of acidity and heavy metal pollution of streams and rivers originating in pyritic formations are caused primarily by mining activities or by natural activities of metal-oxidizing microbes living within the geological formations is a subject of considerable controversy. Most microbiological studies of such waters have so far focused on acid mine drainage sites, which are heavily human-impacted environments, so it has been problematic to eliminate the human factor in the question of the origin of the key metal compounds. We have studied the physico-chemistry and microbiology of the Río Sucio in the Braulio Carrillo National Park of Costa Rica, 22 km from its volcanic rock origin. Neither the remote origin, nor the length of the river to the sampling site, have experienced human activity and are thus pristine. The river water had a characteristic brownish-yellow color due to high iron-dominated minerals, was slightly acidic, and rich in chemolithoautotrophic iron- and sulfur-oxidizing bacteria, dominated by Gallionella spp. Río Sucio is thus a natural acid-rock drainage system whose metal-containing components are derived primarily from microbial activities.

     

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.     

Development and Application of Small-Subunit rRNA Probes for Assessment of Selected Thiobacillus Species and Members of the Genus Acidiphilium

Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using ³²P radiolabels, probe specificity was characterized by hybridization dissociation temperature (T_d) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined T_ds. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris.     

Significance of microbial communities and interactions in safeguarding reactive mine tailings by ecological engineering

Pyritic mine tailings (mineral waste generated by metal mining) pose significant risk to the environment as point sources of acidic, metal-rich effluents (acid mine drainage [AMD]). While the accelerated oxidative dissolution of pyrite and other sulfide minerals in tailings by acidophilic chemolithotrophic prokaryotes has been widely reported, other acidophiles (heterotrophic bacteria that catalyze the dissimilatory reduction of iron and sulfur) can reverse the reactions involved in AMD genesis, and these have been implicated in the "natural attenuation" of mine waters. We have investigated whether by manipulating microbial communities in tailings (inoculating with iron- and sulfur-reducing acidophilic bacteria and phototrophic acidophilic microalgae) it is possible to mitigate the impact of the acid-generating and metal-mobilizing chemolithotrophic prokaryotes that are indigenous to tailing deposits. Sixty tailings mesocosms were set up, using five different microbial inoculation variants, and analyzed at regular intervals for changes in physicochemical and microbiological parameters for up to 1 year. Differences between treatment protocols were most apparent between tailings that had been inoculated with acidophilic algae in addition to aerobic and anaerobic heterotrophic bacteria and those that had been inoculated with only pyrite-oxidizing chemolithotrophs; these differences included higher pH values, lower redox potentials, and smaller concentrations of soluble copper and zinc. The results suggest that empirical ecological engineering of tailing lagoons to promote the growth and activities of iron- and sulfate-reducing bacteria could minimize their risk of AMD production and that the heterotrophic populations could be sustained by facilitating the growth of microalgae to provide continuous inputs of organic carbon.     

Biogeochemical cycling of iron and sulphur in leaching environments

Abstract: Bacterial dissimilatory reduction of iron and sulphur in extremely acidic environments is described. Evidence for reduction at two disused mine sites is presented, within stratified 'acid streamers' growths and in sediments from an acid mine drainage stream. A high proportion (approx. 40%) of mesophilic heterotrophic acidophiles were found to be capable of reducing ferric iron (soluble and insoluble forms) under microaerophilic and anoxic conditions. Mixed cultures of Thiobacillus ferrooxidans and Acidiphilium -like isolate SJH displayed cycling of iron in shake flask and fermenter cultures. Oxido-reduction of iron in mixed cultures was determined by oxygen concentration and availability of organic substrates. Some moderately thermophilic iron-oxidis- ing bacteria were also shown to be capable of reducing ferric iron under conditions of limiting oxygen when grown in glycerol/yeast extract or elemental sulphur media. Cycling of iron was observed in pure cultures of these acidophiles. Sulphate-reducing bacteria isolated from acid streamers could be grown in acidified glycerol/yeast extract media (as low as pH 2.9), but not in media used conventionally for their laboratory culture. An endospore-forming, non-motile rod resembling Desulfotomaculum has been isolated. This bacterium has a wide pH spectrum, and appears to be acid-tolerant rather than acidophilic.