Alkaline Fe(III) reduction by a novel alkali-tolerant Serratia sp. isolated from surface sediments close to Sellafield nuclear facility, UK

Extensive denitrification resulted in a dramatic increase in pH (from 6.8 to 9.5) in nitrate-impacted, acetate-amended sediment microcosms containing sediment representative of the Sellafield nuclear facility, UK. Denitrification was followed by Fe(III) reduction, indicating the presence of alkali-tolerant, metal-reducing bacteria. A close relative (99% 16S rRNA gene sequence homology) to Serratia liquefaciens dominated progressive enrichment cultures containing Fe(III)-citrate as the sole electron acceptor at pH 9 and was isolated aerobically using solid media. The optimum growth conditions for this facultatively anaerobic Serratia species were investigated, and it was capable of metabolizing a wide range of electron acceptors including oxygen, nitrate, FeGel, Fe-NTA and Fe-citrate and electron donors including acetate, lactate, formate, ethanol, glucose, glycerol and yeast extract at an optimum pH of c. 6.5 at 20 °C. The alkali tolerance of this strain extends the pH range of highly adaptable Fe(III)-reducing Serratia species from mildly acidic pH values associated with acid mine drainage conditions to alkali conditions representative of subsurface sediments stimulated for extensive denitrification and metal reduction.     

Reduction of Soluble Iron and Reductive Dissolution of Ferric Iron-Containing Minerals by Moderately Thermophilic Iron-Oxidizing Bacteria

Five moderately thermophilic iron-oxidizing bacteria, including representative strains of the three classified species (Sulfobacillus thermosulfidooxidans, Sulfobacillus acidophilus, and Acidimicrobium ferrooxidans), were shown to be capable of reducing ferric iron to ferrous iron when they were grown under oxygen limitation conditions. Iron reduction was most readily observed when the isolates were grown as mixotrophs or heterotrophs with glycerol as an electron donor; in addition, some strains were able to couple the oxidation of tetrathionate to the reduction of ferric iron. Cycling of iron between the ferrous and ferric states was observed during batch culture growth in unshaken flasks incubated under aerobic conditions, although the patterns of oxidoreduction of iron varied in different species of iron-oxidizing moderate thermophiles and in strains of a single species (S. acidophilus). All three bacterial species were able to grow anaerobically with ferric iron as a sole electron acceptor; the growth yields correlated with the amount of ferric iron reduced when the isolates were grown in the absence of oxygen. One of the moderate thermophiles (identified as a strain of S. acidophilus) was able to bring about the reductive dissolution of three ferric iron-containing minerals (ferric hydroxide, jarosite, and goethite) when it was grown under restricted aeration conditions with glycerol as a carbon and energy source. The significance of iron reduction by moderately thermophilic iron oxidizers in both environmental and applied contexts is discussed.Moderately thermophilic acidophilic bacteria that catalyze the dissimilatory oxidation of ferrous iron are distinct both phylogenetically and in aspects of their physiology. They differ from the known acidophilic mesophilic iron oxidizers (gram-negative, nonsporulating chemolithotrophic bacteria) and the extremely thermophilic iron oxidizers (certain archaea) in several fundamental ways, including cellular morphology (they are gram-positive rods that often form endospores) and growth temperature optima, which are typically 45 to 55°C (15). In addition, the moderately thermophilic iron-oxidizing acidophiles characteristically have a highly versatile metabolism (18) and may grow as autotrophs (e.g., in media containing ferrous iron or reduced sulfur), heterotrophs (e.g., on yeast extract), mixotrophs (e.g., in media containing both ferrous iron and glucose, in which both CO₂ and glucose are used as carbon sources), or chemolithoheterotrophs (e.g., in ferrous iron-yeast extract medium, in which iron acts as the energy source and yeast extract is the carbon source). Isolates have been obtained from a range of thermal acidic environments, such as geothermal areas, self-heating mine waste spoils, and commercial mineral-processing operations (2a, 5, 14). There are currently two recognized genera of these bacteria. All but one Sulfobacillus species are iron- and sulfur-oxidizing, gram-positive, sporulating rods. Two such species have been described, Sulfobacillus thermosulfidooxidans and Sulfobacillus acidophilus, which may be distinguished by their different chromosomal DNA base compositions and by their abilities to grow autotrophically on reduced sulfur (16). The genus Acidimicrobium currently contains a single species, Acidimicrobium ferrooxidans. This organism differs from Sulfobacillus spp. by its greater capacity to fix CO₂, by its lower tolerance of ferric iron, by its apparent lack of spore formation (although it is also gram positive), and by its chromosomal DNA base composition (4). Analysis of 16S rRNA sequences has also differentiated this moderate thermophile from Sulfobacillus spp. (9).The small amount of energy associated with the oxidation of ferrous iron (−30 kJ mol⁻¹ at pH 2) can serve as the exclusive source of energy for moderately thermophilic iron-oxidizing acidophiles when they are growing autotrophically with oxygen as the terminal electron acceptor. Under limited aeration conditions, ferric iron, which is often abundant and present in a soluble form in extremely acidic environments, is a thermodynamically attractive alternative electron sink (electrode potential [E′], +780 mV). Ferric iron reduction by mesophilic chemolithotrophic and heterotrophic acidophiles has been observed previously (5, 7, 17). Some moderately thermophilic, acidophilic, heterotrophic bacteria (Alicyclobacillus-like isolates) (5a) and the extremely thermophilic archaeon Sulfolobus acidocaldarius (3) can also reduce iron. While many neutrophilic microorganisms are also able to reduce ferric iron, the ability to conserve energy to support growth by coupling organic matter oxidation exclusively to ferric iron reduction appears to be more restricted among neutrophilic bacteria (11).In this paper, we describe the dissimilatory reduction of ferric iron by representative isolates of different species of iron-oxidizing moderate thermophiles with both an organic electron donor (glycerol) and an inorganic electron donor (tetrathionate), and we also describe the reductive dissolution of ferric iron-containing minerals by a Sulfobacillus isolate.     

Identification and characterization of a novel acidotolerant Fe(III)-reducing bacterium from a 3,000-year-old acidic rock drainage site

Acidic, ochre-precipitating springs at Mam Tor, East Midlands, UK, are analogous to sites impacted by acid mine drainage over prolonged periods of time, and were studied for the presence of Fe(III)-reducing bacteria. From enrichment cultures inoculated with Mam Tor sediment, a facultative anaerobe capable of reducing Fe(III) at pH values as low as three was isolated. 16S rRNA gene analysis showed that this bacterium is a close relative of Serratia species and not previously shown to respire using Fe(III) as an electron acceptor. Direct cell counts of the isolate grown with Fe(III)-NTA coupled with protein assays suggest that this bacterium is able to conserve energy for growth through Fe(III) reduction.     

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.     

Novel thermo-acidophilic bacteria isolated from geothermal sites in Yellowstone National Park: physiological and phylogenetic characteristics

Moderately thermophilic acidophilic bacteria were isolated from geothermal (30-83 degrees C) acidic (pH 2.7-3.7) sites in Yellowstone National Park. The temperature maxima and pH minima of the isolates ranged from 50 to 65 degrees C, and pH 1.0-1.9. Eight of the bacteria were able to catalyze the dissimilatory oxidation of ferrous iron, and eleven could reduce ferric iron to ferrous iron in anaerobic cultures. Several of the isolates could also oxidize tetrathionate. Six of the iron-oxidizing isolates, and one obligate heterotroph, were low G+C gram-positive bacteria ( Firmicutes). The former included three Sulfobacillus-like isolates (two closely related to a previously isolated Yellowstone strain, and the third to a mesophilic bacterium isolated from Montserrat), while the other three appeared to belong to a different genus. The other two iron-oxidizers were an Actinobacterium (related to Acidimicrobium ferrooxidans) and a Methylobacterium-like isolate (a genus within the alpha -Proteobacteria that has not previously been found to contain either iron-oxidizers or acidophiles). The other three (heterotrophic) isolates were also alpha-Proteobacteria and appeared be a novel thermophilic Acidisphaera sp. An ARDREA protocol was developed to discriminate between the iron-oxidizing isolates. Digestion of amplified rRNA genes with two restriction enzymes ( SnaBI and BsaAI) separated these bacteria into five distinct groups; this result was confirmed by analysis of sequenced rRNA genes.     

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.     

Isolation and characterization of Acidicaldus organivorus, gen. nov., sp. nov.: a novel sulfur-oxidizing, ferric iron-reducing thermo-acidophilic heterotrophic Proteobacterium

Thermo-acidophilic prokaryotes isolated from geothermal sites in Yellowstone National Park were identified as novel α-Proteobacteria, distantly related (~93% 16S rRNA gene identity) to the mesophilic acidophile Acidisphaera rubrifaciens. One of these isolates (Y008) was shown to be more thermophilic than all previously characterized acidophilic proteobacteria, with a temperature optimum for growth between 50 and 55°C and a temperature maximum of 65°C. Growth was observed in media maintained at pH between 1.75 and 3.0 and was fastest at pH between 2.5 and 3.0. The G + C content of Y008 was 71.8±0.9 mol%. The acidophile was able to grow heterotrophically on a range of organic substrates, including various monosaccharides, alcohols and amino acids and phenol, though growth on single organic compounds required the provision of one or more growth factors. The isolate oxidized sulfur to sulfuric acid in media containing yeast extract, but was not capable of autotrophic growth with sulfur as energy source. Growth occurred under aerobic conditions and also in the absence of oxygen via anaerobic respiration using ferric iron as terminal electron acceptor. Based on these genotypic and phenotypic traits, it is proposed that Y008 represents the type species of Acidicaldus organivorus, gen. nov., sp. nov.     

Acidocella aromatica sp. nov.: an acidophilic heterotrophic alphaproteobacterium with unusual phenotypic traits

Three obligately heterotrophic bacterial isolates were identified as strains of a proposed novel species of extremely acidophilic, mesophilic Alphaproteobacteria, Acidocella aromatica. They utilized a restricted range of organic substrates, which included fructose (but none of the other monosaccharides tested), acetate and several aromatic compounds (benzoate, benzyl alcohol and phenol). No growth was obtained on complex organic substrates, such as yeast extract and tryptone. Tolerance of the proposed type strain of the species (PFBC) to acetic acid was much greater than that typically reported for acidophiles. The bacteria grew aerobically, and catalyzed the dissimilatory reductive dissolution of the ferric iron mineral schwertmannite under both micro-aerobic and anaerobic conditions. Strain PFBC did not grow anaerobically via ferric iron respiration, though it has been reported to grow in co-culture with acid-tolerant sulfidogenic bacteria under strictly anoxic conditions. Tolerance of strains of Acidocella aromatica to nickel were about two orders of magnitude greater than those of other Acidocella spp., though similar levels of tolerance to other metals tested was observed. The use of this novel acidophile in solid media designed to promote the isolation and growth of other (aerobic and anaerobic) acidophilic heterotrophs is discussed.     

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.     

Utilization of Iron Gallate and Other Organic Iron Complexes by Bacteria from Water Supplies

The degradation of four soluble organic iron compounds by bacteria isolated from surface waters and the precipitation of iron from these complexes by the isolates was studied. All eight isolates brought about the precipitation of iron when grown on ferric ammonium citrate agar. Three isolates were able to degrade ferric malonate, and three others degraded ferric malate with iron precipitation. Only three isolates, two strains of Pseudomonas and one of Moraxella, were able to degrade gallic acid when this was supplied as the sole carbon source. One strain of Pseudomonas was found to be active in degrading ferric gallate. Electron microscopy of cells of this bacterium after growth in ferric gallate as the sole carbon source yielded results indicating uniform deposition of the iron on or in the bacterial cells. Seven of the isolates could degrade the iron gallate complex if supplied with additional carbon in the form of yeast extract.     

Evidence that the potential for dissimilatory ferric iron reduction is widespread among acidophilic heterotrophic bacteria.

Nineteen characterized strains and isolates of acidophilic heterotrophic bacteria were screened for their abilities to catalyse the reductive dissolution of the ferric iron mineral schwertmannite, under oxygen-limiting conditions. Acidocella facilis, Acidobacterium capsulatum, and all of the Acidiphilium, Acidocella and Acidobacterium-like isolates that grew in liquid cultures were able to reduce iron. In contrast, neither Acidisphaera rubrifaciens nor three Acidisphaera-like isolates tested were found to have the capacity for dissimilatory iron reduction. One of two iron-oxidizing Frateuria-like isolates also reduced iron under oxygen-limiting conditions. Microbial dissolution of schwertmannite was paralleled with increased concentrations of soluble ferrous iron and sulfate in microbial cultures, together with increased pH values and decreased redox potentials. While dissimilatory ferric iron reduction has been described previously for Acidiphilium spp., this is this first report of this capacity in Acidocella and the moderate acidophile Acidobacterium. The finding has significant implications for understanding of the biogeochemistry of acidic environments.     

Growth and iron oxidation by acidophilic moderate thermophiles

Abstract Most of the moderately thermophilic, acidophilic iron-oxidizing bacteria which have been isolated required a source of reduced sulphur for growth on iron. One isolate (strain ALV) utilized sulphate as the sole source of sulphur. All of the isolates were capable of chemolitho-heterotrophin growth on iron in the presence of yeast extract. Autotrophic growth has been confirmed in all strains except one previously described, but now re-isolated, moderate thermophile (TH3).     

Effect of cultivation conditions on the growth and activities of sulfur metabolism enzymes and carboxylases of Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41

The moderately thermophilic acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41 is capable of utilizing sulfides of gold-arsenic concentrate and elemental sulfur as a source of energy. The growth in the presence of S0 under auto- or mixotrophic conditions was less stable compared with the media containing iron monoxide. The enzymes involved in oxidation of sulfur inorganic compounds--thiosulfate-oxidizing enzyme, tetrathionate hydrolase, rhodonase, adenylyl sulfate reductase, sulfite oxidase, and sulfur oxygenase--were discovered in the cells of Sulfobacillus grown in the mineral medium containing 0.02% yeast extract and either sulfur or iron monoxide and thiosulfate. Cell-free extracts of the cultures grown in the medium with sulfur under auto- or mixotrophic conditions displayed activity of the key enzyme of the Calvin cycle--ribulose bisphosphate carboxylase--and several other enzymes involved in heterotrophic fixation of carbonic acid. Activities of carboxylases depended on the composition of cultivation media.     

Reduction of ferric iron by acidophilic heterotrophic bacteria: evidence for constitutive and inducible enzyme systems in Acidiphilium spp

AIMS: To compare the abilities of two obligately acidophilic heterotrophic bacteria, Acidiphilium acidophilum and Acidiphilium SJH, to reduce ferric iron to ferrous when grown under different culture conditions. METHODS AND RESULTS: Bacteria were grown in batch culture, under different aeration status, and in the presence of either ferrous or ferric iron. The specific rates of ferric iron reduction by fermenter-grown Acidiphilium SJH were unaffected by dissolved oxygen (DO) concentrations, while iron reduction by A. acidophilum was highly dependent on DO concentrations in the growth media. The ionic form of iron present (ferrous or ferric) had a minimal effect on the abilities of harvested cells to reduce ferric iron. Whole cell protein profiles of Acidiphilium SJH were very similar, regardless of the DO status of the growth medium, while additional proteins were present in A. acidophilum grown microaerobically compared with aerobically-grown cells. CONCLUSIONS: The dissimilatory reduction of ferric iron is constitutive in Acidiphilium SJH while it is inducible in A. acidophilum. SIGNIFICANCE AND IMPACT OF THE STUDY: Ferric iron reduction by Acidiphilium spp. may occur in oxygen-containing as well as anoxic acidic environments. This will detract from the effectiveness of bioremediation systems where removal of iron from polluted waters is mediated via oxidation and precipitation of the metal.     

Enumeration of acetylene-reducing bacteria in strip-mined reclamation sites in a temperate grassland

Summary From 36 to 71% of bacteria, depending on the sampling site, that were isolated from the soil or rhizosphere of undisturbed prairie soil or reclamation sites of strip-mined grassland areas in western North Dakota were capable of reducing acetylene. These bacteria generally could be divided into two populations; one capable of acetylene reduction under aerobic conditions and another capable of acetylene reduction under anaerobic conditions. The reclamation site to which no topsoil had been applied, pH 8.5, had a bacterial population which generally was capable of higher levels of acetylene reduction than individual bacteria isolated from other sites.     

Isolation and physiological characterisation of Thiobacillus aquaesulis sp. nov., a novel facultatively autotrophic moderate thermophile

A moderately thermophilic, facultatively chemolithoautotrophic thiobacillus isolated from a thermal sulphur spring is described. It differs from all other species currently known to be in culture. It grows lithoautotrophically on thiosulphate, trithionate or tetrathionate, which are oxidized to sulphate. Batch cultures on thiosulphate do not produce tetrathionate, but do precipitate elemental sulphur during growth. In autotrophic chemostat cultures the organism produces yields on thiosulphate, trithionate and tetrathionate that are among the highest observed for a Thiobacillus. Autotrophic cultures contain ribulose bisphosphate carboxylase. Heterotrophic growth has been observed only on complex media such as yeast extract and nutrient broth. It is capable of autotrophic growth and denitrification under anaerobic conditions with thiosulphate and nitrate. It grows between 30 to 55° C, and pH 7 to 9, with best growth at about 43°C and pH 7.6. It contains ubiquinone Q-8, and its DNA contains 65.7 mol% G+C. The organism is formally described and named as Thiobacillus aquaesulis.Now the Department of Biological Sciences     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium.

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Grazing of acidophilic bacteria by a flagellated protozoan

A biflagellated protozoan was isolated from an acidic drainage stream located inside a disused pyrite mine. The stream contained copious amounts of acid streamer bacterial growths, and the flagellate was observed in situ apparently grazing the streamer bacteria. The protozoan was obligately acidophilic, growing between pH 1.8 and 4.5, but not at pH 1.6 or 5.0, with optimum growth between pH 3 and 4. It was highly sensitive to copper, molybdenum, silver, and uranium, but tolerated ferrous and ferric iron up to 50 and 25 mM, respectively. In the laboratory, the protozoan was found to graze a range of acidophilic bacteria, including the chemolithotrophs Thiobacillus ferrooxidans, Leptospirillum ferrooxidans, and the heterotroph Acidiphilium cryptum. Thiobacillus thiooxidans and Thiobacillus acidophilus were not grazed. Filamentous growth of certain acidophiles afforded some protection against being grazed by the flagellate. In mixed cultures of T. ferrooxidans and L. ferrooxidans, the protozoan isolate displayed preferential grazing of the former. The possibility of using acidophilic protozoa as a means of controlling bacteria responsible for the production of acid mine drainage is discussed.Offprint requests to: Dr. D. B. Johnson.     

Sulfidogenesis in low pH (3.8-4.2) media by a mixed population of acidophilic bacteria

A defined mixed bacterial culture was established which catalyzed dissimilatory sulfate reduction, using glycerol as electron donor, at pH 3.8-4.2. The bacterial consortium comprised a endospore-forming sulfate reducing bacterium (isolate M1) that had been isolated from acidic sediment in a geothermal area of Montserrat (West Indies) and which had 94% sequence identity (of its 16S rRNA gene) to the Gram-positive neutrophile Desulfosporosinus orientis, and a Gram-negative (non sulfate-reducing) acidophile (isolate PFBC) that shared 99% gene identity with Acidocella aromatica. Whilst M1 was an obligate anaerobe, isolate PFBC, as other Acidocella spp., only grew in pure culture in aerobic media. Analysis of microbial communities, using a combination of total bacterial counts and fluorescent in situ hybridization, confirmed that concurrent growth of both bacteria occurred during sulfidogenesis under strictly anoxic conditions in a pH-controlled fermenter. In pure culture, M1 oxidized glycerol incompletely, producing stoichiometric amounts of acetic acid. In mixed culture with PFBC, however, acetic acid was present only in small concentrations and its occurrence was transient. Since M1 did not oxidize acetic acid, it was inferred that this metabolite was catabolized by Acidocella PFBC which, unlike glycerol, was shown to support the growth of this acidophile under aerobic conditions. In fermenter cultures maintained at pH 3.8-4.2, sulfidogenesis resulted in the removal of soluble zinc (as solid phase ZnS) whilst ferrous iron remained in solution. Potential syntrophic interactions, involving hydrogen transfer between M1 and PFBC, are discussed, as is the potential of sulfidogenesis in acidic liquors for the selective recovery of heavy metals from wastewaters.