Isolation and phylogenetic characterization of acidophilic microorganisms indigenous to acidic drainage waters at an abandoned Norwegian copper mine.

The biodiversity of culturable acidophilic microbes in three acidic (pH 2.7-3.7), metal-rich waters at an abandoned subarctic copper mine in central Norway was assessed. Acidophilic bacteria were isolated by plating on selective solid media, and dominant isolates were identified from their physiological characteristics and 16S rRNA gene sequences. The dominant iron-oxidizing acidophile in all three waters was an Acidithiobacillus ferrooxidans-like eubacterium, which shared 98% 16S rDNA identity with the type strain. A strain of Leptospirillum ferrooxidans was obtained from one of the waters after enrichment in pyrite medium, but this iron oxidizer was below detectable levels in the acidic waters themselves. In two sites, there were up to six distinct heterotrophic acidophiles, present at 10(3) ml(-1). These included Acidiphilium-like isolates (one closely related to Acidiphilium rubrum, a second to Acidiphilium cryptum and a third apparently novel isolate), an Acidocella-like isolate (96% 16S rDNA identity to Acidocella facilis) and a bacterium that shared 94.5% 16S rDNA identity to Acidisphaera rubrifaciens. The other numerically significant heterotrophic isolate was not apparently related to any known acidophile, with the closest match (96% 16S rDNA sequence identity) to an acetogen, Frateuria aurantia. The results indicated that the biodiversity of acidophilic bacteria, especially heterotrophs, in acidic mine waters may be much greater than previously recognized.

Chromosomally encoded arsenical resistance of the moderately thermophilic acidophile Acidithiobacillus caldus.

Arsenical resistance is important to bioleaching microorganisms because these organisms release arsenic from minerals such as arsenopyrite during bioleaching. The acidophile Acidithiobacillus caldus KU was found to be resistant to the arsenical ions arsenate, arsenite, and antimony via an inducible, chromosomally encoded resistance mechanism. Because no apparent alteration of the toxic ions was observed, Acidithiobacillus (At.) caldus was tested to determine if it was resistant as a result of decreased accumulation of toxic ions. Reduced accumulation of arsenate and arsenite by induced At. caldus cells supported this hypothesis. It was also found that, with the addition of an energy source, induced At. caldus could transport arsenate and arsenite out of the cell against a concentration gradient. The lack of efflux in the absence of an added energy source and in the presence of inhibitors suggested that efflux was energy dependent. Induced At. caldus also expressed arsenate reductase activity, indicating that At. caldus has an arsenical resistance mechanism that is analogous to previously described systems from other Bacteria. Southern hybridization analysis showed that At. caldus and other gram-negative acidophiles carry an Escherichia coli arsB homologue on the chromosome.

A fast and simple turbidimetric method for the determination of sulfate in sulfate-reducing bacterial cultures.

A standard turbidimetric assay for the determination of sulfate in water was modified with the objective of achieving a quick and simple method for monitoring the decrease of sulfate in cultures of sulfate-reducing bacteria. The effects of sulfate concentration, mixing time and the ratio of sample to conditioning reagent were optimized using a central composite face-centered response surface model design. The results suggested that a mixing time of 30 s resulted in smaller absorbance variance, the variance in absorbance measurements tended to increase with concentration of sulfate and that the ratio between the amount of conditioning reagent and sample had no significant influence on the absorbance variance. The modified assay thus developed is simple and quick, and covers a comparatively large sulfate concentration range (0-5 mM) compared to the standard turbidimetric assay.

Multiple Serotypes of the Moderate Thermophile Thiobacillus caldus, a Limitation of Immunological Assays for Biomining Microorganisms.

Phylogenetic and phenotypic analysis indicates that a moderately thermophilic isolate, C-SH12, from Australia belongs to the species Thiobacillus caldus. Antiserum generated against whole cells of T. caldus KU recognized protein antigens common to cell lysates of the three T. caldus strains KU, BC13, and C-SH12 but did not recognize whole cells of isolate C-SH12. Differences in the lipopolysaccharide (LPS) of strain C-SH12 and those of the other two T. caldus strains were found, and the anti-KU antiserum did not recognize the LPS from strain C-SH12. These data indicate that this T. caldus isolate belongs to a serotype different from that of strains KU and BC13.

Reduced sulfur compound oxidation by Thiobacillus caldus.

The oxidation of reduced inorganic sulfur compounds was studied by using resting cells of the moderate thermophile Thiobacillus caldus strain KU. The oxygen consumption rate and total oxygen consumed were determined for the reduced sulfur compounds thiosulfate, tetrathionate, sulfur, sulfide, and sulfite in the absence and in the presence of inhibitors and uncouplers. The uncouplers 2,4-dinitrophenol and carbonyl cyanide m-chlorophenyl-hydrazone had no affect on the oxidation of thiosulfate, suggesting that thiosulfate is metabolized periplasmically. In contrast, the uncouplers completely inhibited the oxidation of tetrathionate, sulfide, sulfur, and sulfite, indicating that these compounds are metabolized in the cytoplasm of T. caldus KU. N-Ethylmaleimide inhibited the oxidation of tetrathionate and thiosulfate at the stage of elemental sulfur, while 2-heptyl-4-hydroxyquinoline-N-oxide stopped the oxidation of thiosulfate, tetrathionate, and elemental sulfur at the stage of sulfite. The following intermediates in the oxidation of the sulfur compounds were found by using uncouplers and inhibitors: thiosulfate was oxidized to tetrathionate, elemental sulfur was formed during the oxidation of tetrathionate and sulfide, and sulfite was found as an intermediate of tetrathionate and sulfur metabolism. On the basis of these data we propose a model for the metabolism of the reduced inorganic sulfur compounds by T. caldus KU.

Characterization of Thiobacillus caldus sp. nov., a moderately thermophilic acidophile.

Two isolates of a novel, moderately thermophilic Thiobacillus species have been studied. The isolates, KU and BC13, are Gram-negative, motile bacteria having a pH optimum for growth of 2-2.5 and an optimum growth temperature of 45 degrees C. Both isolates are capable of chemolithotrophic growth on reduced sulfur substrates. They can also use molecular hydrogen as an electron donor. These two isolates can grow mixotrophically with sulfur or tetrathionate and yeast extract or glucose. The G+C content is 63.1-63.9 mol% and the isolates exhibit no significant DNA homology to any other Thiobacillus species. Strains KU and BC13 both contain ubiquinone Q-8. 16S rRNA analysis indicates that these strains belong to a group of bacteria which includes other chemolithotrophic sulfur oxidizers such as T. ferrooxidans and T. thiooxidans. These characteristics distinguish KU and BC13 from any other species described previously and they thus represent the first acidophilic, thermophilic Thiobacillus species, named T. caldus sp. nov., to be described. The type strain, referred to as strain KU in this paper, has been deposited in the Deutsche Sammlung von Mikroorganismen, Braunschweig, FRG, with the accession number DSM 8584.

Oxidative dissolution of arsenopyrite by mesophilic and moderately thermophilic acidophiles.

The purpose of this work was to determine solution- and solid-phase changes associated with the oxidative leaching of arsenopyrite (FeAsS) by Thiobacillus ferrooxidans and a moderately thermoacidophilic mixed culture. Jarosite [KFe(3)(SO(4))(2)(OH)(6)], elemental sulfur (S), and amorphous ferric arsenate were detected by X-ray diffraction as solid-phase products. The oxidation was not a strongly acid-producing reaction and was accompanied by a relatively low redox level. The X-ray diffraction lines of jarosite increased considerably when ferrous sulfate was used as an additional substrate for T. ferroxidans. A moderately thermoacidophilic mixed culture oxidized arsenopyrite faster at 45 degrees C than did T. ferroxidans at 22 degrees C, and the oxidation was accompanied by a nearly stoichiometric release of Fe and As. The redox potential was initially low but subsequently increased during arsenopyrite oxidation by the thermoacidophiles. Jarosite, S, and amorphous ferric arsenate were also formed under these conditions.

An immunological assay for detection and enumeration of thermophilic biomining microorganisms.

A specific, fast, and sensitive nonradioactive immunobinding assay for the detection and enumeration of the moderate thermophile Thiobacillus caldus and the thermophilic archaeon Sulfolobus acidocaldarius was developed. It employs enhanced chemiluminescence or peroxidase-conjugated immunoglobulins in a dot or slot blotting system and is very convenient for monitoring thermophilic bioleaching microorganisms in effluents from industrial bioleaching processes.

Solid-phase products of bacterial oxidation of arsenical pyrite.

Bacterial leaching of an As-containing pyrite concentrate produced acidic (pH < 1) leachates. During the leaching, the bacteria solubilized both As and Fe, and these two elements were distributed in solution-phase and solid-phase products. Jarosite and scorodite were the exclusive crystalline products in precipitate samples from the bacterial leaching of the sulfide concentrate.

Chemical inactivation of microorganisms on rigid gas permeable contact lenses.

The efficiency of four contact lens disinfectant formulations was tested against three types of bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, and Serratia marcescens) and one fungus (Aspergillus niger). The bacteria were tested both as free suspensions and after attachment on surfaces of rigid gas permeable contact lenses (RGPCL). The disinfection data were used to calculate the death rates and decimal reduction times for the test organisms. P. aeruginosa was the most sensitive and A. niger was the most resistant organism to the disinfectants. Scanning electron micrographs showed that P. aeruginosa occurred mostly as single cells, with little extracellular material, on the lens surface. In contrast, S. marcescens produced copious amounts of capsular material layered on the lens surface, promoting cell aggregation. Transmission electron micrographs revealed that bacterial cells were physically separated from the lens surface by a space barricaded with capsular material.

Biological degradation of 2,4-dichlorophenoxyacetic acid: chloride mass balance in stirred tank reactors.

A mass balance was developed for the degradation of 2,4-dichlorophenoxyacetic acid by a mixed culture. Batch culture experiments showed the degradation to be an acid-producing step. Inorganic chloride concentration consistently correlated with the expected value and with base consumption to maintain a constant pH.