Bioleaching of tennantite concentrate: influence of microbial community and solution redox potential.

Despite its growing importance as a Cu resource, studies on tennantite bioleaching are highly limited. One of the key challenges in processing such Cu-As sulfides is their refractoriness and the solubilisation of toxic As. The ultimate goal is to achieve selective bioleaching of Cu with simultaneous immobilisation of As in the leach residues. This study investigated the effectiveness of activated carbon (AC)-assisted bioleaching of tennantite concentrate using a mixed culture containing various "strong" and "weak" Fe-oxidising bacteria/archaea plus a S-oxidising bacterium, with particular emphasis on controlling the solution redox potential (Eh). In the initial flask bioleaching tests, a steady increase in Eh (up to 840 mV) was observed, reflecting the activity of "strong" Fe-oxidisers. In this situation, AC dosing effectively suppressed the Eh value and the highest Cu dissolution (70%) was obtained in the AC-0.01% system, while simultaneously immobilising As. In order to maximise Cu dissolution and As immobilisation, it was found preferable to target the Eh range of 650-700 mV during bioleaching. The next bioreactor tests used the mixed culture of the same origin, but had been subcultured a few generations further on tennantite concentrate. The Eh level remained unexpectedly low (~630 mV) for most of the leaching period, regardless of the AC dosage. It was later found that the bioreactor systems were almost exclusively dominated by Sb. thermosulfidooxidans, a "weak" Fe oxidiser with high Cu/As tolerance. In this case, there was no need to artificially suppress the Eh level by AC dosing and Cu leached readily to a final Cu dissolution of ~60% while As dissolution was suppressed to ~15%. Thus, depending on the microbial community that develops at the processing site, Eh control can be achieved either naturally by the activity of "weak" Fe-oxidisers as the predominant survivors under high Cu/As stress, or artificially by the addition of an Eh regulator such as a carbon catalyst.

Utilization of amino acid for selective leaching of critical metals from spent hydrodesulfurization catalyst.

While spent catalysts can cause serious environmental pollution, they can be considered an essential secondary metal source due to their high critical metal grades. The formation of the amino acid-metal complex is often seen in nature, and its potential application in hydrometallurgy can be foreseen. Alanine (Ala) was first screened as the most effective type of amino acid to be used for the selective leaching of spent hydrodesulfurization catalyst (consisting of MoS2 and Co3S4 supported on Al2O3, at 10% Mo and 2.4% Co grades). The sequential 3-step leaching (Step-1: Alkaline Ala leaching at 45°C, Step-2: Hot water leaching at 70°C, Step-3: Second alkaline Ala leaching at 45°C) was conducted where the role of Ala was found to be at least three-fold; 1) maintaining alkalinity by amino acid's buffering capacity to assist Mo leaching, 2) selectively precipitating Co by forming Co-Ala complex with a distinctive pink color, which can readily re-dissolve in hot water to be separated from spent catalyst particles. 3) Effectively suppressing unwanted dissolution of Al throughout the reaction without needing pH control. Consequently, highly metal-selective, two separate Co-rich (<1% Mo and 79% Co dissolved, Al not detected) and Mo-rich (96% Mo, 19% Co, and 2.1% Al dissolved) leachates were obtained. This study highlighted the potential utility of amino acids as non-toxic, alternative metal lixiviant as well as a metal precipitant for selective leaching of critical metals from spent hydrodesulfurization catalyst.

Bioremediation of highly toxic arsenic via carbon-fiber-assisted indirect As(III) oxidation by moderately-thermophilic, acidophilic Fe-oxidizing bacteria.

OBJECTIVE: To enable removal of highly toxic As(III) from acidic waters by inducing indirect microbial As(III) oxidation by Fe-oxidizing bacteria via carbon-assisted redox-coupling between As(III) oxidation and Fe3+ reduction. RESULTS: Carbon-fiber (CF) was shown to function as an electron-mediator to catalyze chemical (abiotic) redox-coupling between As(III) oxidation and Fe3+ reduction. Accordingly, by taking advantage of Fe3+ regeneration by Fe-oxidizing bacteria, it was possible to promote oxidative removal of As(III) as ferric arsenate at moderate temperature. This reaction can be of use under the situation where a high-temperature treatment is not immediately available. Arsenic once concentrated as ferric arsenate on carbon-fibers can be collected to undergo phase-transformation to crystalline scorodite as the next re-solubilization/re-crystallization step at a higher temperature (70 °C). CONCLUSIONS: While extremely acidophilic Fe-oxidizing bacteria are widely found in nature, the As-oxidizing counterparts, especially those grown on moderately-thermophilic and mesophilic temperatures, are hardly known. In this regard, the finding of this study could make a possible introduction of the semi-passive, low-temperature As-treatment using readily available Fe-oxidizing bacteria.

Production of highly catalytic, archaeal Pd(0) bionanoparticles using Sulfolobus tokodaii.

The thermo-acidophilic archaeon, Sulfolobus tokodaii, was utilized for the production of Pd(0) bionanoparticles from acidic Pd(II) solution. Use of active cells was essential to form well-dispersed Pd(0) nanoparticles located on the cell surface. The particle size could be manipulated by modifying the concentration of formate (as electron donor; e-donor) and by addition of enzymatic inhibitor (Cu2+) in the range of 14-63 nm mean size. Since robust Pd(II) reduction progressed in pre-grown S. tokodaii cells even in the presence of up to 500 mM Cl-, it was possible to conversely utilize the effect of Cl- to produce even finer and denser particles in the range of 8.7-15 nm mean size. This effect likely resulted from the increasing stability of anionic Pd(II)-chloride complex at elevated Cl- concentrations, eventually allowing involvement of greater number of initial Pd(0) crystal nucleation sites (enzymatic sites). The catalytic activity [evaluated based on Cr(VI) reduction reaction] of Pd(0) bionanoparticles of varying particle size formed under different conditions were compared. The finest Pd(0) bionanoparticles obtained at 50 mM Cl- (mean 8.7 nm; median 5.6 nm) exhibited the greatest specific Cr(VI) reduction rate, with four times higher catalytic activity compared to commercial Pd/C. The potential applicability of S. tokodaii cells in the recovery of highly catalytic Pd(0) nanoparticles from actual acidic chloride leachate was, thus, suggested.

Thiourea bioleaching for gold recycling from e-waste.

Recycling and reuse of electronic wastes (e-wastes) are becoming an increasingly critical strategy for securing metal resources as well as for minimizing environmental impacts. Thiourea leaching of gold (Au) from e-wastes can be considered an alternative to highly toxic cyanidation, provided that its reagents consumption can be largely reduced. While awareness of the use of biohydrometallurgical techniques in metal mining industries is increasing, the knowledge on microbiological precious metal solubilization is still limited. This led us to investigate and clarify for the first time the potential utility of microbiologically-mediated thiourea leaching (TU-bioleaching) of Au, with a special focus on reducing the reagents consumption while facilitating Au dissolution. Initial screening tests found that different Fe-oxidizing bacteria/archaea possessed varying degrees of thiourea tolerance (5-100 mM). When thiourea and PCB (Printed Circuit Boards) co-exist, Acidiplasma sp. Fv-Ap displayed the most robust Fe-oxidation. The Eh level during the reaction was first optimized by fluctuating the initial ratio of thiourea to Fe3+ (TU:Fe3+ = 2:1-40:1, by using 1 mM Fe3+ vs. 2-40 mM thiourea). The ratio precisely determined the Eh level during the TU-bioleaching and dictated the fate of thiourea decomposition and the resultant Au dissolution from PCB. Microbial contribution to Fe3+ regeneration was seen to support steady and continuous Au dissolution, enabling 98% Au dissolution while using low reagent concentrations of 1 mM Fe3+ and 10 mM thiourea under the microbial Eh control at around 490-545 mV. This novel TU-bioleaching process offers a new alternative approach for Au recycling from e-wastes and minimization of environmental hazards.

Palladium bionanoparticles production from acidic Pd(II) solutions and spent catalyst leachate using acidophilic Fe(III)-reducing bacteria.

The acidophilic, Fe(III)-reducing heterotrophic bacteria Acidocella aromatica PFBCT and Acidiphilium cryptum SJH were utilized to produce palladium (Pd) bionanoparticles via a simple 1-step microbiological reaction. Monosaccharide (or intracellular NADH)-dependent reactions lead to visualization of intra/extra-cellular enzymatic Pd(0) nucleation. Formic acid-dependent reactions proceeded via the first slow Pd(0) nucleation phase and the following autocatalytic Pd(II) reduction phase regardless of the presence or viability of the cells. However, use of active cells (with full enzymatic and membrane protein activities) at low formic acid concentration (5 mM) was critical to allow sufficient time for Pd(II) biosorption and the following enzymatic Pd(0) nucleation, which consequently enabled production of fine, dense and well-dispersed Pd(0) bionanoparticles. Differences of the resultant Pd(0) nanoparticles in size, density and localization between the two bacteria under each condition tested suggested different activity and location of enzymes and membrane "Pd(II) trafficking" proteins responsible for Pd(0) nucleation. Despite the inhibitory effect of leaching lixiviant and dissolved metal ions, Pd(0) bionanoparticles were effectively formed by active Ac. aromatica cells from both acidic synthetic Pd(II) solutions and from the actual spent catalyst leachates at equivalent 18-19 nm median size with comparable catalytic activity.

Microbial community profiling of the Chinoike Jigoku ("Blood Pond Hell") hot spring in Beppu, Japan: isolation and characterization of Fe(III)-reducing Sulfolobus sp. strain GA1.

Chinoike Jigoku ("Blood Pond Hell") is located in the hot spring town of Beppu on the southern island of Kyushu in Japan, and is the site of a red-colored acidic geothermal pond. This study aimed to investigate the microbial population composition in this extremely acidic environment and to isolate/characterize acidophilic microorganism with metal-reducing ability. Initially, PCR (using bacteria- and archaea-specific primers) of environmental DNA samples detected the presence of bacteria, but not archaea. This was followed by random sequencing analysis, confirming the presence of wide bacterial diversity at the site (123 clones derived from 18 bacterial and 1 archaeal genera), including those closely related to known autotrophic and heterotrophic acidophiles (Acidithiobacillus sp., Sulfobacillus sp., Alicyclobacillus sp.). Nevertheless, successive culture enrichment with Fe(III) under micro-aerobic conditions led to isolation of an unknown archaeal organism, Sulfolobus sp. GA1 (with 99.7% 16S rRNA gene sequence identity with Sulfolobus shibatae). Unlike many other known Sulfolobus spp., strain GA1 was shown to lack sulfur oxidation ability. Strain GA1 possessed only minor Fe(II) oxidation ability, but readily reduced Fe(III) during heterotrophic growth under micro-aerobic conditions. Strain GA1 was capable of reducing highly toxic Cr(VI) to less toxic/soluble Cr(III), demonstrating its potential utility in bioremediation of toxic metal species.

Microbial recovery of vanadium by the acidophilic bacterium, Acidocella aromatica.

OBJECTIVE: To investigate the bioreduction of toxic pentavalent vanadium [vanadate; V(V)] in the acidophilic, Fe(III)-reducing obligately heterotrophic bacterium, Acidocella aromatica PFBC. RESULTS: Although the initial lag-phase of growth became extended with increasing initial V(V) concentrations, the final cell density during aerobic growth of A. aromatica PFBC was unaffected by up to 2 mM V(V). While strain PFBC is an aerobe, growth-decoupled PFBC cell suspensions directly reduced V(V) using fructose, both micro-aerobically and anaerobically, under highly acidic (pH 2) and moderately acidic (pH 4.5) conditions. Bio reduced V(IV) was subsequently precipitated even under micro-aerobic conditions, mostly by encrusting the cell surface. An anaerobic condition at pH 4.5 was optimal for forming and maintaining stable V(IV)-precipitates. Recovery of approx. 70 % of V(V) from the solution phase was made possible with V(V) at 1 mM. CONCLUSIONS: The first case of direct V(V) reducing ability and its subsequent V recovery from the solution phase was shown in acidophilic prokaryotes. Possible utilities of V(V) bioreduction in acidic conditions, are discussed.

Bioreduction and immobilization of hexavalent chromium by the extremely acidophilic Fe(III)-reducing bacterium Acidocella aromatica strain PFBC.

The extremely acidophilic, Fe(III)-reducing heterotrophic bacterium Acidocella aromatica strain PFBC was tested for its potential utility in bioreduction of highly toxic heavy metal, hexavalent chromium, Cr(VI). During its aerobic growth on fructose at pH 2.5, 20 µM Cr(VI) was readily reduced to Cr(III), achieving the final Cr(VI) concentration of 0.4 µM (0.02 mg/L), meeting the WHO drinking water guideline of 0.05 mg/L. Despite of the highly inhibitory effect of Cr(VI) on cell growth at higher concentrations, especially at low pH, Cr(VI) reduction activity was readily observed in growth-decoupled cell suspensions under micro-aerobic and anaerobic conditions. Strain PFBC was not capable of anaerobic growth via dissimilatory reduction of Cr(VI), such as reported for Fe(III). In the presence of both Cr(VI) and Fe(III) under micro-aerobic condition, microbial Fe(III) reduction occurred only upon complete disappearance of Cr(VI) by its reduction to Cr(III). Following Cr(VI) reduction, the resultant Cr(III), supposedly present in the form of cationic Cr (III) (OH2) 6 (3+) , was partially immobilized on the negatively charged cell surface through biosorption. When Cr(III) was externally provided, rather than microbially produced, it was poorly immobilized on the cell surface. Cr(VI) reducing ability was reported for the first time in Acidocella sp. in this study, and its potential role in biogeochemical cycling of Cr, as well as its possible utility in Cr(VI) bioremediation, in highly acidic environments/solutions, were discussed.

Speciation of arsenic in a thermoacidophilic iron-oxidizing archaeon, Acidianus brierleyi, and its culture medium by inductively coupled plasma-optical emission spectroscopy combined with flow injection pretreatment using an anion-exchange mini-column.

The thermoacidophilic iron-oxidizing archaeon Acidianus brierleyi is a microorganism that could be useful in the removal of inorganic As from wastewater, because it simultaneously oxidizes As(III) and Fe(II) to As(V) and Fe(III) in an acidic culture medium, resulting in the immobilization of As(V) as FeAsO4. To investigate the oxidation mechanism, speciation of the As species in both the cells and its culture media is an important issue. Here we describe the successive determination of As(III), As(V), and total As in A. brierleyi and its culture medium via a facile method based on inductively coupled plasma-optical emission spectroscopy (ICP-OES) with a flow injection pretreatment system using a mini-column packed with an anion-exchange resin. The flow-injection pretreatment system consisted of a syringe pump, a selection valve, and a switching valve, which were controlled by a personal computer. Sample solutions with the pH adjusted to 5 were flowed into the mini-column to retain the anionic As(V), whereas As(III) was introduced into ICP-OES with no adsorption on the mini-column due to its electrically neutral form. An acidic solution (1 M HNO3) was then flowed into the mini-column to elute As(V) followed by ICP-OES measurement. The same sample was also subjected to ICP-OES without being passed through the mini-column in order to determine the total amounts of As(III) and As(V). The method was verified by comparing the results of the total As with the sum of As(III) and As(V). The calibration curves showed good linearity with limits of detection of 158, 86, and 211 ppb for As(III), As(V), and total As, respectively. The method was successfully applicable to the determination of the As species contained in the pellets of A. brierleyi and their culture media. The results suggested that the oxidation of As(III) was influenced by the presence of Fe(II) in the culture medium, i.e., Fe(II) enhanced the oxidation of As(III) in A. brierleyi. In addition, we found that no soluble As species was contained in the cell pellets and more than 60% of the As(III) in the culture medium was oxidized by A. brierleyi after a 6-day incubation.

pCGR2 copy number depends on the par locus that forms a ParC-ParB-DNA partition complex in Corynebacterium glutamicum.

AIMS: To characterize the par system of Corynebacterium glutamicum pCGR2 and to manipulate the par components to effectively manipulate plasmid copy number. METHODS AND RESULTS: ParB binds sequence specifically to centromere-binding sites around the parAB operon and serves as an autorepressor. A small ORF (orf4, later named parC) downstream of parAB encodes a protein with 23.7% sequence identity with ParB. ParB is also implicated in the repression of parC transcription. Nonetheless, this ParC protein does not bind to centromere-binding sites and is not essential for plasmid stability. Introduction of a frameshift mutation within ParC implicated the protein in regulation of both parAB and parC. Electrophoretic Mobility Shift Assay confirmed a previously unreported ParC-ParB-parS partition complex. ParC also interacts directly with ParB without the mediation of the centromere sites. Deletion of the par components resulted in different plasmid copy numbers. CONCLUSIONS: A previously unreported ParC-ParB-parS partition complex is formed in pCGR2, where interaction of ParC with ParB-parS may affect the level of repression by ParB. Modifying the par components and antisense RNA enables manipulation of plasmid copy number to varying degrees. SIGNIFICANCE AND IMPACT OF STUDY: Genetically manipulating the par components, in combination with deactivation of antisense RNA, is a novel approach to artificially elevate plasmid copy number. This approach can be applied for development of new genetic engineering tools.

Efficient markerless gene replacement in Corynebacterium glutamicum using a new temperature-sensitive plasmid.

Random chemical mutation of a Corynebacterium glutamicum-Escherichia coli shuttle vector derived from plasmid pCGR2 was done using hydroxylamine. It brought about amino acid substitutions G109D and E180K within the replicase superfamily domain of the plasmid's RepA protein and rendered the plasmid highly unstable, especially at higher incubation temperatures. Colony formation of C. glutamicum was consequently completely inhibited at 37°C but not at 25°C. G109 is a semi-conserved residue mutation which resulted in major temperature sensitivity. E180 on the other hand is not conserved even among RepA proteins of closely related C. glutamicum pCG1 family plasmids and its independent mutation caused relatively moderate plasmid instability. Nonetheless, simultaneous mutation of both residues was required to achieve temperature-sensitive colony formation. This new pCGR2-derived temperature-sensitive plasmid enabled highly efficient chromosomal integration in a variety of C. glutamicum wild-type strains, proving its usefulness in gene disruption studies. Based on this, an efficient markerless gene replacement system was demonstrated using a selection system incorporating the temperature-sensitive replicon and Bacillus subtilis sacB selection marker, a system hitherto not used in this bacterium. Single-crossover integrants were accurately selected by temperature-dependent manner and 93% of the colonies obtained by the subsequent sucrose selection were successful double-crossover recombinants.

Antisense-RNA-mediated plasmid copy number control in pCG1-family plasmids, pCGR2 and pCG1, in Corynebacterium glutamicum.

pCGR2 and pCG1 belong to different subfamilies of the pCG1 family of Corynebacterium glutamicum plasmids. Nonetheless, they harbour homologous putative antisense RNA genes, crrI and cgrI, respectively. The genes in turn share identical positions complementary to the leader region of their respective repA (encoding plasmid replication initiator) genes. Determination of their precise transcriptional start- and end-points revealed the presence of short antisense RNA molecules (72 bp, CrrI; and 73 bp, CgrI). These short RNAs and their target mRNAs were predicted to form highly structured molecules comprising stem-loops with known U-turn motifs. Abolishing synthesis of CrrI and CgrI by promoter mutagenesis resulted in about sevenfold increase in plasmid copy number on top of an 11-fold (CrrI) and 32-fold (CgrI) increase in repA mRNA, suggesting that CrrI and CgrI negatively control plasmid replication. This control is accentuated by parB, a gene that encodes a small centromere-binding plasmid-partitioning protein, and is located upstream of repA. Simultaneous deactivation of CrrI and parB led to a drastic 87-fold increase in copy number of a pCGR2-derived shuttle vector. Moreover, the fact that changes in the structure of the terminal loops of CrrI and CgrI affected plasmid copy number buttressed the important role of the loop structure in formation of the initial interaction complexes between antisense RNAs and their target mRNAs. Similar antisense RNA control systems are likely to exist not only in the two C. glutamicum pCG1 subfamilies but also in related plasmids across Corynebacterium species.

Ferrimicrobium acidiphilum gen. nov., sp. nov. and Ferrithrix thermotolerans gen. nov., sp. nov.: heterotrophic, iron-oxidizing, extremely acidophilic actinobacteria.

Two novel extremely acidophilic, iron-oxidizing actinobacteria were isolated, one from a mine site in North Wales, UK (isolate T23(T)), and the other from a geothermal site in Yellowstone National Park, Wyoming, USA (Y005(T)). These new actinobacteria belong to the subclass Acidimicrobidae, and in contrast to the only other classified member of the subclass (Acidimicrobium ferrooxidans), both isolates were obligate heterotrophs. The mine site isolate was mesophilic and grew as small rods, while the Yellowstone isolate was a moderate thermophile and grew as long filaments, forming macroscopic flocs in liquid media. Both isolates accelerated the oxidative dissolution of pyrite in yeast extract-amended cultures, but neither was able to oxidize reduced forms of sulfur. Ferrous iron oxidation enhanced growth yields of the novel mesophilic actinobacterium T23(T), though this was not confirmed for the Yellowstone isolate. Both isolates catalysed the dissimilatory reduction of ferric iron, using glycerol as electron donor, in oxygen-free medium. Based on comparative analyses of base compositions of their chromosomal DNA and of their 16S rRNA gene sequences, the isolates are both distinct from each other and from Acidimicrobium ferrooxidans, and are representatives of two novel genera. The names Ferrimicrobium acidiphilum gen. nov., sp. nov. and Ferrithrix thermotolerans gen. nov., sp. nov. are proposed for the mesophilic and moderately thermophilic isolates, respectively, with the respective type strains T23(T) (=DSM 19497(T)=ATCC BAA-1647(T)) and Y005(T) (=DSM 19514(T)=ATCC BAA-1645(T)).

Scanning the Corynebacterium glutamicum R genome for high-efficiency secretion signal sequences.

Systematic screening of secretion proteins using an approach based on the completely sequenced genome of Corynebacterium glutamicum R revealed 405 candidate signal peptides, 108 of which were able to heterologously secrete an active-form alpha-amylase derived from Geobacillus stearothermophilus. These comprised 90 general secretory (Sec)-type, 10 twin-arginine translocator (Tat)-type and eight Sec-type with presumptive lipobox peptides. Only Sec- and Tat-type signals directed high-efficiency secretion. In two assays, 11 of these signals resulted in 50- to 150-fold increased amounts of secreted alpha-amylase compared with the well-known corynebacterial secretory protein PS2. While the presence of an AXA motif at the cleavage sites was readily apparent, it was the presence of a glutamine residue adjacent to the cleavage site that may affect secretion efficiency.

Identification of new secreted proteins and secretion of heterologous amylase by C. glutamicum.

In this study, secreted Corynebacterium glutamicum proteins were investigated by two-dimensional gel electrophoresis. Around 100 spots observed in the pH range 4.5-5.5 had molecular masses that varied from 10 to 50 kDa. Upon N-terminal amino acid sequence analysis by Edman degradation, two of them were hits to two hypothetical proteins encoded by cgR_1176 and cgR_2070 on C. glutamicum R genome, respectively. Active-form alpha-amylase derived from Geobacillus stearothermophilus was successfully secreted by using the predicted cgR_1176 and cgR_2070 signal sequences, indicating that these hypothetical proteins were secreted proteins. Analysis using a disruption mutant of the twin-arginine translocation (Tat) export pathway machinery of C. glutamicum suggested that one is Tat pathway dependent secretion while the other is independent of the pathway. Our results demonstrate that C. glutamicum can secrete exoproteins by using its own signal sequences, indicating its potential as a host for protein productions.

"Bioshrouding": a novel approach for securing reactive mineral tailings.

A novel technique ("bioshrouding") for safeguarding highly reactive sulfidic mineral tailings deposits is proposed. In this, freshly milled wastes are colonised with ferric iron-reducing heterotrophic acidophilic bacteria that form biofilms on reactive mineral surfaces, thereby preventing or minimising colonisation by iron sulfide-oxidising chemolithotrophs such as Acidithiobacillus ferrooxidans and Leptospirillum spp. Data from initial experiments showed that dissolution of pyrite could be reduced by between 57 and 75% by "bioshrouding" the mineral with three different species of heterotrophic acidophiles (Acidiphilium, Acidocella and Acidobacterium spp.), under conditions that were conducive to microbial oxidative dissolution of the iron sulfide.

Differentiation and identification of iron-oxidizing acidophilic bacteria using cultivation techniques and amplified ribosomal DNA restriction enzyme analysis.

Acidophilic iron-oxidizing microorganisms are important both environmentally and in biotechnological applications. Although, as a group, they are readily detected by their ability to generate ferric iron (resulting in a distinctive color change in liquid media), these microbes highly diverse phylogenetically. Various other characteristics, such as optimum growth temperature, response to organic carbon sources, and cellular morphologies, facilitate, in some cases, identification of isolates to a genus or species level, although this approach has limitations and may give erroneous results. In this study, a combined approach of using physiological traits together with amplified ribosomal DNA restriction enzyme analysis (ARDREA) has been successful in identifying all known acidophilic iron-oxidizing bacteria to the species level. Computer-generated maps were used to identify restriction enzymes that allow the differentiation of the acidophiles, and these were confirmed experimentally using authentic bacterial strains. To test further the validity of this approach, six acidophilic moderately thermophilic iron-oxidizing bacteria isolated from Montserrat (West Indies) were analysed using the ARDREA protocol. Three of the isolates were identified as Sulfobacillus acidophilus-like, and one as Sulfobacillus thermosulfidooxidans-like bacteria. The fifth isolate gave DNA digest patterns that were distinct from all known strains of iron-oxidizing acidophiles. Subsequent sequencing of the 16S rRNA genes of these isolates confirmed the identity of the four Sulfobacillus isolates, and also that the fifth isolate was a novel species. Schematic diagrams showing how ARDREA may be used to rapidly identify all known acidophilic iron-oxidizing bacteria are presented.

Biooxidation of pyrite by defined mixed cultures of moderately thermophilic acidophiles in pH-controlled bioreactors: significance of microbial interactions.

The oxidative dissolution of pyrite (FeS2) by pure and mixed cultures of moderately thermophilic acidophiles was studied in shake flask cultures and in pH-controlled bioreactors, incubated at 45 degrees C. Various combinations of seven eubacteria (a Leptospirillum sp. (MT6), Acidimicrobium ferrooxidans, Acidithiobacillus caldus, an Alicyclobacillus sp. (Y004), and three Sulfobacillus spp.) and one archaeon (Ferroplasma sp. MT17) were examined. Pyrite dissolution was determined by measuring changes in soluble iron and generation of acidity, and microbial populations were monitored using a combined culture-dependent (plate counts) and culture-independent (fluorescent in situ hybridization) approach. In pure cultures, the most efficient pyrite-oxidizing acidophile was Leptospirillum MT6, which was unique among the prokaryotes used in being obligately autotrophic. Mixed cultures of Leptospirillum MT6 and the sulfur-oxidizer At. caldus generated more acidity than pure cultures of the iron-oxidizer, though this did not necessarily enhance pyrite dissolution. In contrast, a mixed culture of Leptospirillum MT6 and the obligate heterotroph Alicyclobacillus Y004 oxidized pyrite more rapidly and more completely than a pure culture of Leptospirillum MT6, in synchronized bioreactors. Although the autotroph, At. caldus, and the "heterotrophically inclined" iron-oxidizer, Am. ferrooxidans, were both ineffective at leaching pyrite in pure culture, a mixed culture of the two bacteria was able to accelerate dissolution of the mineral. Concentrations of dissolved organic carbon accumulated to >100 mg/L in some mixed cultures, and the most effective bioleaching systems were found to be consortia containing both autotrophic and heterotrophic moderate thermophiles. A mixed culture comprising the autotrophs Leptospirillum MT6 and At. caldus, and the heterotroph Ferroplasma MT17, was the most efficient of all of those examined. Mutualistic interactions between physiologically distinct moderately thermophilic acidophiles, involving transfer of organic and inorganic carbon and transformations of iron and sulfur, were considered to have critical roles in optimizing pyrite dissolution.

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.