Acidithiobacillia class members originating at sites within the Pacific Ring of Fire and other tectonically active locations and description of the novel genus 'Igneacidithiobacillus'.

Recent studies have expanded the genomic contours of the Acidithiobacillia, highlighting important lacunae in our comprehension of the phylogenetic space occupied by certain lineages of the class. One such lineage is 'Igneacidithiobacillus', a novel genus-level taxon, represented by 'Igneacidithiobacillus copahuensis' VAN18-1T as its type species, along with two other uncultivated metagenome-assembled genomes (MAGs) originating from geothermally active sites across the Pacific Ring of Fire. In this study, we investigate the genetic and genomic diversity, and the distribution patterns of several uncharacterized Acidithiobacillia class strains and sequence clones, which are ascribed to the same 16S rRNA gene sequence clade. By digging deeper into this data and contributing to novel MAGs emerging from environmental studies in tectonically active locations, the description of this novel genus has been consolidated. Using state-of-the-art genomic taxonomy methods, we added to already recognized taxa, an additional four novel Candidate (Ca.) species, including 'Ca. Igneacidithiobacillus chanchocoensis' (mCHCt20-1TS), 'Igneacidithiobacillus siniensis' (S30A2T), 'Ca. Igneacidithiobacillus taupoensis' (TVZ-G3 TS), and 'Ca. Igneacidithiobacillus waiarikiensis' (TVZ-G4 TS). Analysis of published data on the isolation, enrichment, cultivation, and preliminary microbiological characterization of several of these unassigned or misassigned strains, along with the type species of the genus, plus the recoverable environmental data from metagenomic studies, allowed us to identify habitat preferences of these taxa. Commonalities and lineage-specific adaptations of the seven species of the genus were derived from pangenome analysis and comparative genomic metabolic reconstruction. The findings emerging from this study lay the groundwork for further research on the ecology, evolution, and biotechnological potential of the novel genus 'Igneacidithiobacillus'.

Plasmonic and Photothermal Effects of CuS Nanoparticles Biosynthesized from Acid Mine Drainage with Potential Drug Delivery Applications.

In this work, the plasmonic and photothermal effects of CuS nanoparticles biosynthesized from acid mine drainage (AMD) were studied. CuS were formed by delivering the H2S generated by a sulfidogenic bioreactor to an off-line system containing the AMD. The precipitates collected after contact for an hour were washed and physico-chemically characterized, showing a nanoparticle with a mean diameter of 33 nm, crystalline nature and semiconductor behavior with a direct band gap of 2.2 eV. Moreover, the CuS nanoparticles exhibited localized surface plasmonic resonance in the near infrared range, with a high absorption band centered at 973 nm of wavelength, which allowed an increase in the temperature of the surrounding media under irradiation. Finally, the cytotoxicity of the CuS nanoparticles as well as their potential use as part of drug delivery platforms were investigated.

Optimization of the chemolithotrophic denitrification of ion exchange concentrate using hydrogen-based membrane biofilm reactors.

A H2-based membrane biofilm reactor (MBfR) was used to remove nitrate from a synthetic ion-exchange brine made up of 23.8 g L-1 NaCl. To aid the selection of the best nitrate management strategy, our research was based on the integrated analysis of ionic exchange and MBfR processes, including a detailed cost analysis. The nitrate removal flux was not affected if key nutrients were present in the feed solution including potassium and sodium bicarbonate. Operating pH was maintained between 7 and 8. By using a H2 pressure of 15 psi, a hydraulic retention time (HRT) of 4 h, and a surface loading rate of 13.6 ± 0.2 g N m-2 d-1, the average nitrate removal flux was 3.3 ± 0.6 g N m-2 d-1. At HRTs of up to 24 h, the system was able to maintain a removal flux of 1.6 ± 0.2 g N m-2 d-1. Microbial diversity analysis showed that the consortium was dominated by the genera Sulfurimonas and Marinobacter. The estimated cost for a 200 m3/h capacity, coupled ion exchange (IX) + MBfR treatment plant is 0.43 USD/m3. This is a sustainable and competitive alternative to an IX-only plant for the same flowrate. The proposed treatment option allows for brine recycling and reduces costs by 55% by avoiding brine disposal expenses.

Bio-recovery of CuS nanoparticles from the treatment of acid mine drainage with potential photocatalytic and antibacterial applications.

In the present work, CuS nanoparticles were biorecovered from a real acid mine drainage (AMD) and its photocatalytic and antibacterial activities were studied. CuS were formed by delivering biogenic H2S produced by a continuous sulfidogenic bioreactor to an off-line vessel containing the AMD. The main physico-chemical properties of CuS nanoparticles were analyzed by UV-vis spectroscopy, TEM, FE-SEM, XRD and XPS. Moreover, its photocatalytic activity on the photodegradation of organic dyes in water and its antibacterial activity against several bacterial strains were studied and compared with CuS nanoparticles synthetized from a CuSO4 aqueous solution based on the same synthesis method. CuS nanoparticles from the real AMD showed similar physico-chemical properties and photocatalytic and antibacterial activities in comparison to CuS nanoparticles formed with the copper solutions. These results open the way to recover valorous CuS nanoparticles from AMD with potential industrial applications using a metal bioremediation process based on sulfidogenic bioreactors.

Electrotrophic perchlorate reduction by a psychrotolerant acidophile isolated from an acid rock drainage in Antarctica.

A new extremophilic isolate (USS-CCA7) was obtained from an acidic environment (pH âˆ¼ 3.2) in Antarctica phylogenetically related to Acidithiobacillus ferrivorans; its electrotrophic capacities were evaluated in a three-electrode electrochemical cell. Cyclic voltammetry showed cathodic peaks of -428 mV, -536 mV, and -634 mV (vs. Ag/AgCl; pH = 1.7; 3 M KCl) for nitrate, oxygen, and perchlorate, respectively. The catalytic role of this microorganism was also observed by a decrease in the charge transfer resistance registered via electrochemical impedance spectroscopy. Five-day chronoamperometry of culture at pH = 1.7, USS-CCA7 showed a perchlorate removal rate of 19.106 ± 1.689 mgL-1 day-1 and a cathodic efficiency of 112 ± 5.2  %. Growth on electrodes was observed by epifluorescence and scanning electron microscopy. Interestingly, the results showed that toward higher pH, the cathodic peak of perchlorate is reduced in the voltammetric profiles. This study highlights the use of this psychrotolerant acidophile for the bioremediation of harsh perchlorate-pressured terrestrial under acidic conditions.

Sulfidogenic Bioreactor-Mediated Formation of ZnS Nanoparticles with Antimicrobial and Photocatalytic Activity.

The use of sulfidogenic bioreactors is a biotechnology trend to recover valuable metals such as copper and zinc as sulfide biominerals from mine-impacted waters. In the present work, ZnS nanoparticles were produced using "green" H2S gas generated by a sulfidogenic bioreactor. ZnS nanoparticles were physico-chemically characterized by UV-vis and fluorescence spectroscopy, TEM, XRD and XPS. The experimental results showed spherical-like shape nanoparticles with principal zinc-blende crystalline structure, a semiconductor character with an optical band gap around 3.73 eV, and fluorescence emission in the UV-visible range. In addition, the photocatalytic activity on the degradation of organic dyes in water, as well as bactericidal properties against several bacterial strains, were studied. ZnS nanoparticles were able to degrade methylene blue and rhodamine in water under UV radiation, and also showed high antibacterial activity against different bacterial strains including Escherichia coli and Staphylococcus aureus. The results open the way to obtain valorous ZnS nanoparticles from the use of dissimilatory reduction of sulfate using a sulfidogenic bioreactor.

Comparison of two acidophilic sulfidogenic consortia for the treatment of acidic mine water.

Sulfate-reducing bioreactors are a biotechnological alternative for the treatment of acid mine drainage (AMD). In this study, two separate bioreactors with pH and temperature-controlled (Bio I and II) were operated with two different acidophilic microbial consortia to determine their efficiencies in sulfate removal from a synthetic acidic mine water. The bioreactors were operated for 302 days in continuous flow mode under the same parameters: fed with a sulfate solution of ∼30 mM with a pH of 2.5, the temperature at 30°C, stirred gently at 40 rpm and using a continuous stream of nitrogen to help remove the H2S produced in the bioreactor. The glycerol consumption, acetate production, and sulfate removal were monitored throughout the course of the experiment. The community composition and potential metabolic functional groups were analyzed via 16S rRNA partial gene sequencing. Bio I consortium reduced the sulfate, achieving a range of sulfate concentration from 4.7 to 19 mM in the effluent liquor. The removal of sulfate in Bio II was between 5.6 and 18 mM. Both bioreactors' communities showed the presence of the genus De sulfosporosinus as the main sulfate-reducing bacteria (SRB). Despite differences in microbial composition, both bioreactors have similar potential metabolism, with a higher percentage of microorganisms that can use sulfate in respiration. Overall, both bioreactors showed similar performance in treating acidic mine water containing mostly sulfate using two different acidophilic sulfidogenic consortia obtained from different global locations.

Draft Genome Sequence of the Novel, Moderately Thermophilic, Iron- and Sulfur-Oxidizing Firmicute Strain Y002, Isolated from an Extremely Acidic Geothermal Environment.

We report the draft genome sequence of the Firmicute strain Y002, a facultatively anaerobic, acidophilic bacterium that catalyzes the dissimilatory oxidation of iron and sulfur and the reduction of ferric iron. Analysis of the genome (2.9 Mb; G+C content, 46 mol%) provided insights into its ability to grow in extremely acidic geothermal environments.

High-Rate Sulfate Removal Coupled to Elemental Sulfur Production in Mining Process Waters Based on Membrane-Biofilm Technology.

It is anticipated that copper mining output will significantly increase over the next 20 years because of the more intensive use of copper in electricity-related technologies such as for transport and clean power generation, leading to a significant increase in the impacts on water resources if stricter regulations and as a result cleaner mining and processing technologies are not implemented. A key concern of discarded copper production process water is sulfate. In this study we aim to transform sulfate into sulfur in real mining process water. For that, we operate a sequential 2-step membrane biofilm reactor (MBfR) system. We coupled a hydrogenotrophic MBfR (H2-MBfR) for sulfate reduction to an oxidizing MBfR (O2-MBfR) for oxidation of sulfide to elemental sulfur. A key process improvement of the H2-MBfR was online pH control, which led to stable high-rate sulfate removal not limited by biomass accumulation and with H2 supply that was on demand. The H2-MBfR easily adapted to increasing sulfate loads, but the O2-MBfR was difficult to adjust to the varying H2-MBfR outputs, requiring better coupling control. The H2-MBfR achieved high average volumetric sulfate reduction performances of 1.7-3.74 g S/m3-d at 92-97% efficiencies, comparable to current high-rate technologies, but without requiring gas recycling and recompression and by minimizing the H2 off-gassing risk. On the other hand, the O2-MBfR reached average volumetric sulfur production rates of 0.7-2.66 g S/m3-d at efficiencies of 48-78%. The O2-MBfR needs further optimization by automatizing the gas feed, evaluating the controlled removal of excess biomass and S0 particles accumulating in the biofilm, and achieving better coupling control between both reactors. Finally, an economic/sustainability evaluation shows that MBfR technology can benefit from the green production of H2 and O2 at operating costs which compare favorably with membrane filtration, without generating residual streams, and with the recovery of valuable elemental sulfur.

Characteristics of an Iron-Reducing, Moderately Acidophilic Actinobacterium Isolated from Pyritic Mine Waste, and Its Potential Role in Mitigating Mineral Dissolution in Mineral Tailings Deposits.

Reactive pyritic mine tailings can be populated by chemolithotrophic prokaryotes that enhance the solubilities of many metals, though iron-reducing heterotrophic microorganisms can inhibit the environmental risk posed by tailings by promoting processes that are the reverse of those carried out by pyrite-oxidising autotrophic bacteria. A strain (IT2) of Curtobacterium ammoniigenes, a bacterium not previously identified as being associated with acidic mine wastes, was isolated from pyritic mine tailings and partially characterized. Strain IT2 was able to reduce ferric iron under anaerobic conditions, but was not found to catalyse the oxidation of ferrous iron or elemental (zero-valent) sulfur, and was an obligate heterotrophic. It metabolized monosaccharides and required small amounts of yeast extract for growth. Isolate IT2 is a mesophilic bacterium, with a temperature growth optimum of 30 °C and is moderately acidophilic, growing optimally at pH 4.0 and between pH 2.7 and 5.0. The isolate tolerated elevated concentrations of many transition metals, and was able to grow in the cell-free spent medium of the acidophilic autotroph Acidithiobacillus ferrooxidans, supporting the hypothesis that it can proliferate in acidic mine tailings. Its potential role in mitigating the production of acidic, metal-rich drainage waters from mine wastes is discussed.

A membrane-biofilm system for sulfate conversion to elemental sulfur in mining-influenced waters.

A system of two membrane biofilm reactors (MBfRs) was tested for the conversion of sulfate (1.5 g/L) in mining-process water into elemental sulfur (S0) particles. Initially, a H2-based MBfR reduced sulfate to sulfide, and an O2-based MBfR then oxidized sulfide to S0. Later, the two MBfRs were coupled by a recirculation flow. Surface loading, reactor-coupling configuration, and substrate-gas pressure exerted important controls over performance of each MBfR and the coupled system. Continuously recirculating the liquid between the H2-based MBfR and the O2-based MBfR, compared to series operation, avoided the buildup of sulfide and gave overall greater sulfate removal (99% vs 62%) and production of S0 (61% vs 54%). The trade-off was that recirculation coupling demanded greater delivery of H2 and O2 (in air) due to the establishment of a sulfur cycle catalyzed by Sulfurospirillum spp., which had an average abundance of 46% in the H2-based MBfR fibers and 62% in the O2-based MBfR fibers at the end of the experiments. Sulfate-reducing bacteria (Desulfovibrio and Desulfomicrobium) and sulfur-oxidizing bacteria (Thiofaba, Thiomonas, Acidithiobacillus and Sulfuricurvum) averaged only 22% and 11% in the H2-based MBfR and O2-based MBfR fibers, respectively. Evidence suggests that the undesired Sulfurospirillum species, which reduce S0 to sulfide, can be suppressed by increasing sulfate-surface loading and H2 pressure.

Acidiferrimicrobium australe gen. nov., sp. nov., an acidophilic and obligately heterotrophic, member of the Actinobacteria that catalyses dissimilatory oxido-reduction of iron isolated from metal-rich acidic water in Chile.

A novel acidophilic member of the phylum Actinobacteria was isolated from an acidic, metal-contaminated stream draining from an abandoned underground coal mine (Trongol mine), situated close to Curanilahue, Biobío Region, Chile. The isolate (USS-CCA1T) was demonstrated to be a heterotroph that catalysed under aerobic conditions the oxidation of ferrous iron and the reduction of ferric iron under anaerobic conditions, but not the oxidation of sulfur nor hydrogen. USS-CCA1T is a Gram-positive, motile, short rod-shaped, mesophilic bacterium with a temperature growth optimum at 30 °C (range 20-39 °C). It was categorized as an extreme acidophile growing between 1.7 and 4.5 and optimally at pH 3.0. The G+C content of the chromosomal DNA of the isolate was 74.1 mol%, which is highly related to Aciditerrimonas ferrireducens IC-180T , (the most closely related genus; 94.4 % 16S rRNA gene identity), and higher than other acidophilic actinobacteria. The isolate (USS-CCA1T) was shown to form a distinct 16S rRNA clade from characterized acidophilic actinobacteria, well separated from the genera Acidimicrobium, Ferrimicrobium, Ferrithrix, 'Acidithrix' and Aciditerrimonas. Genomic indexes (ANIb, DDH, AAI, POCP) derived from the USS-CCA1T draft genome sequence (deposited at DDBJ/ENA/GenBank under the accession WJHE00000000) support assignment of the isolate to a new species and a new genus within the Acidimicrobiaceae family. Isolate USS-CCA1T is the designated type strain of the novel species Acidiferrimicrobium australe (=DSM 106828T,=RGM 2506T).

Influence of operating conditions on sulfate reduction from real mining process water by membrane biofilm reactors.

Two H2-based membrane biofilm reactor (H2-MBfR) systems, differing in membrane type, were tested for sulfate reduction from a real mining-process water having low alkalinity and high concentrations of dissolved sulfate and calcium. Maximum sulfate reductions were 99%, with an optimum pH range between 8 and 8.5, which minimized any toxic effect of unionized hydrogen sulfide (H2S) on sulfate-reducing bacteria (SRB) and calcite scaling on the fibers and in the biofilm. Although several strategies for control of pH and gas back-diffusion were applied, it was not possible to sustain a high degree of sulfate reduction over the long-term. The most likely cause was precipitation of calcite inside the biofilm and on the surface of fibers, which was shown by scanning electron microscopy coupled with energy dispersive spectroscopy (SEM-EDS) analysis. Another possible cause was a decline in pH, leading to inhibition by H2S. A H2/CO2 mixture in the gas supply was able to temporarily recover the effectiveness of the reactors and stabilize the pH. Biomolecular analysis showed that the biofilm was comprised of 15-20% SRB, but a great variety of autotrophic and heterotrophic genera, including sulfur-oxidizing bacteria, were present. Results also suggest that the MBfR system can be optimized by improving H2 mass transfer using fibers of higher gas permeability and by feeding a H2/CO2 mixture that is automatically adjusted for pH control.

Enrichment and isolation of acid-tolerant sulfate-reducing microorganisms in the anoxic, acidic hot spring sediments from Copahue volcano, Argentina.

The geothermal Copahue-Caviahue (GCC) system (Argentina) is an extreme acidic environment, dominated by the activity of Copahue volcano. Environments characterised by low pH values, such as volcanic areas, are of particular interest for the search of acidophilic microorganisms with application in biotechnological processes. In this work, sulfate-reducing microorganisms were investigated in geothermal acidic, anaerobic zones from GCC system. Sediment samples from Agua del Limón (AL1), Las Máquinas (LMa2), Las Maquinitas (LMi) and Baño 9 (B9-2, B9-3) were found to be acidic (pH values 2.1-3.0) to moderate acidic (5.1-5.2), containing small total organic carbon values, and ferric iron precipitates. The organic electron donor added to the enrichment was completely oxidised to CO2. Bacteria related to 'Desulfobacillus acidavidus' strain CL4 were found to be dominant (67-83% of the total number of clones) in the enrichment cultures, and their presence was confirmed by their isolation on overlay plates. Other bacteria were also detected with lower abundance (6-20% of the total number of clones), with representatives of the genera Acidithiobacillus, Sulfobacillus, Alicyclobacillus and Athalassotoga/Mesoaciditoga. These enrichment and isolates found at low pH confirm the presence of anaerobic activities in the acidic sediments from the geothermal Copahue-Caviahue system.

Evaluation of the bio-protection mechanism in diffusive exchange permeable reactive barriers for the treatment of acid mine drainage.

This research studied the bio-protection mechanism based on chemical gradients in diffusive exchange permeable reactive barriers, evaluating the thickness of the reactive layers in the treatment of concentrated acid mine drainage (AMD). Six bench-scale reactors were constructed with reactive layer thicknesses of 2.5, 5, and 7.5 cm in duplicate. The reactors were first fed a sulfated solution for 55 days, followed by concentrated AMD for 166 days. The change of feed to AMD mainly affected the reactors with thinner 2.5 cm layers in comparison to the reactors with 5 and 7.5 cm layers. Cu and Zn removal efficiency was practically 100% in all the reactors; however, in the thinner layer reactors, metal breakthrough occurred towards the end of the experiment concurrently with inhibitory metal concentrations in the reactive layers. On the contrary, the reactors with layer thicknesses of 5 and 7.5 cm evaluated did not present toxic concentrations of these metals at any of the monitoring points. The bio-protection criterion qD correctly predicted that the thin-layer reactor would be the most affected by the toxicity of AMD. The criterion also indicated that all the reactors should fail. Nevertheless, the fault in the thinner layer reactor registered in the effluent after >150 days; therefore, the possibility of failure in the 5 and 7.5 cm thickness reactors is not rejected, as it could have occurred if the experiment had continued.

Recent Developments for Remediating Acidic Mine Waters Using Sulfidogenic Bacteria.

Acidic mine drainage (AMD) is regarded as a pollutant and considered as potential source of valuable metals. With diminishing metal resources and ever-increasing demand on industry, recovering AMD metals is a sustainable initiative, despite facing major challenges. AMD refers to effluents draining from abandoned mines and mine wastes usually highly acidic that contain a variety of dissolved metals (Fe, Mn, Cu, Ni, and Zn) in much greater concentration than what is found in natural water bodies. There are numerous remediation treatments including chemical (lime treatment) or biological methods (aerobic wetlands and compost bioreactors) used for metal precipitation and removal from AMD. However, controlled biomineralization and selective recovering of metals using sulfidogenic bacteria are advantageous, reducing costs and environmental risks of sludge disposal. The increased understanding of the microbiology of acid-tolerant sulfidogenic bacteria will lead to the development of novel approaches to AMD treatment. We present and discuss several important recent approaches using low sulfidogenic bioreactors to both remediate and selectively recover metal sulfides from AMD. This work also highlights the efficiency and drawbacks of these types of treatments for metal recovery and points to future research for enhancing the use of novel acidophilic and acid-tolerant sulfidogenic microorganisms in AMD treatment.

A novel "in-feed" delivery platform applied for oral DNA vaccination against IPNV enables high protection in Atlantic salmon (Salmon salar).

BACKGROUND: DNA vaccination has emerged as a promising tool against infectious diseases of farmed fish. Oral delivery allows stress-free administration that is ideal for mass immunization and of paramount importance for infectious pancreatic necrosis (IPN) and other viral disease that affect young salmonids and cause economic losses in aquaculture worldwide. METHODS: We describe the development and in vivo assessment of an "in-feed" formulation strategy for oral immunization with liposomal DNA vaccines, by delivering a vaccine construct coding for an immunogenic region of the VP2 capsid protein. A challenge against IPNV was carried out to determine the vaccine efficacy, by comparing the mortality of pre-smolt Atlantic salmons immunized and non-immunized with the oral vaccine. The antibody response (ELISA) and hematological parameters after immunization were examined, as well as the vaccine effect on the growth and internal structures of fry salmons (histological analysis). The vaccine distribution in the experimental tank after oral administration was investigated by HPLC and PCR amplification. RESULTS: The oral vaccine induced detectable levels of VP2-specific antibodies and conferred significant protection following IPNV challenge, with relative percent survivals (RPS) of 58.2%, for single dose (1mgpDNA/kgfish⋅d), and 66% for double dose (2mgpDNA/kgfish⋅d). We further provide evidence in favour of the vaccine safety to fish and demonstrated absence of pDNA in the tank water, but presence of vaccine residues in faeces and unconsumed feed sediments (solid wastes). CONCLUSION: The delivery platform for liposomal DNA vaccination via feed was successfully proved against IPNV in Atlantic salmon, showing the oral vaccine to be immunogenic and safe for fish, and providing significant protection after oral administration. The "in-feed" technology for oral DNA vaccination holds potential to be applied against IPNV and other pathogens that currently threaten the aquaculture worldwide.

Draft Genome Sequence of a Novel Acidophilic Iron-Oxidizing Firmicutes Species, "Acidibacillus ferrooxidans" (SLC66T).

Here, we present the draft genome sequence of the type strain of "Acidibacillus ferrooxidans," a mesophilic, heterotrophic, and acidophilic bacterium that was isolated from mine spoilage subjected to accelerated weathering in humidity cell tests carried out by the former U.S. Bureau of Mines in Salt Lake City, UT.

Isolation and characterisation of mineral-oxidising "Acidibacillus" spp. from mine sites and geothermal environments in different global locations.

Eight strains of acidophilic bacteria, isolated from mine-impacted and geothermal sites from different parts of the world, were shown to form a distinct clade (proposed genus "Acidibacillus") within the phylum Firmicutes, well separated from the acidophilic genera Sulfobacillus and Alicyclobacillus. Two of the strains (both isolated from sites in Yellowstone National Park, USA) were moderate thermophiles that oxidised both ferrous iron and elemental sulphur, while the other six were mesophiles that also oxidised ferrous iron, but not sulphur. All eight isolates reduced ferric iron to varying degrees. The two groups shared <95% similarity of their 16S rRNA genes and were therefore considered to be distinct species: "Acidibacillus sulfuroxidans" (moderately thermophilic isolates) and "Acidibacillus ferrooxidans" (mesophilic isolates). Both species were obligate heterotrophs; none of the eight strains grew in the absence of organic carbon. "Acidibacillus" spp. were generally highly tolerant of elevated concentrations of cationic transition metals, though "A. sulfuroxidans" strains were more sensitive to some (e.g. nickel and zinc) than those of "A. ferrooxidans". Initial annotation of the genomes of two strains of "A. ferrooxidans" revealed the presence of genes (cbbL) involved in the RuBisCO pathway for CO2 assimilation and iron oxidation (rus), though with relatively low sequence identities.

Solid and liquid media for isolating and cultivating acidophilic and acid-tolerant sulfate-reducing bacteria.

Growth media have been developed to facilitate the enrichment and isolation of acidophilic and acid-tolerant sulfate-reducing bacteria (aSRB) from environmental and industrial samples, and to allow their cultivation in vitro The main features of the 'standard' solid and liquid devised media are as follows: (i) use of glycerol rather than an aliphatic acid as electron donor; (ii) inclusion of stoichiometric concentrations of zinc ions to both buffer pH and to convert potentially harmful hydrogen sulphide produced by the aSRB to insoluble zinc sulphide; (iii) inclusion of Acidocella aromatica (an heterotrophic acidophile that does not metabolize glycerol or yeast extract) in the gel underlayer of double layered (overlay) solid media, to remove acetic acid produced by aSRB that incompletely oxidize glycerol and also aliphatic acids (mostly pyruvic) released by acid hydrolysis of the gelling agent used (agarose). Colonies of aSRB are readily distinguished from those of other anaerobes due to their deposition and accumulation of metal sulphide precipitates. Data presented illustrate the effectiveness of the overlay solid media described for isolating aSRB from acidic anaerobic sediments and low pH sulfidogenic bioreactors.