Biomineralization Abilities of Cupriavidus Strain and Bacillus subtilis Strains In Vitro Isolated from Speleothems,Rani Cave,Chhattisgarh, India

In vitro culture experiments using three bacterial strains CSJC1, CSJC2, and CSJC3 isolated from speleothems, Rani cave, Chhattisgarh, India, were studied to examine their biomineralization potential. These speleothems showed high microbial cell enumerations on nutrient agar and iron agar (9 × 10⁴ CFU/g) followed by thiosulfate agar (7 × 10⁴ CFU/g), and 60 diverse strains were isolated. The BLASTn sequence search of 16S rRNA sequences with the NCBI database to establish the identity of CSJC1, CSJC2, and CSJC3 strains yielded similarity scores of ≥99% with the respective organisms, and the strains were identified as CSJC1 – Bacillus sp., CSJC2 – Cupriavidus sp., CSJC3 – Bacillus sp. The phylogenetic analysis of CSJC2 strain suggests that it formed a separate major cluster with Cupriavidus sp. and Cupriavidus necator. The phylogenetic analysis of CSJC1 and CSJC3 strains revealed that it formed a major cluster with several strains of Bacillus sp. and Bacillus subtilis. The biominerals induced by Cupriavidus sp. CSJC2 strain imaged with an ultra high-resolution field emission scanning electron microscope (FE-SEM) were seen as calcified coccoid shells that transformed into calcified dumbbells. FE-SEM imaging of biominerals induced by B. subtilis CSJC1 and CSJC3 tested both on B4 media and sheep blood agar individually showed that the precipitates formed calcified dumbbells that were almost similar but not identical phenotypically, indicating that strain-specific morphologies and crystal formation is easier when Ca is present in the media. This is the first comprehensive report on the possible evidences about the role of Cupriavidus sp. in calcite precipitation isolated from speleothems in the Indian caves. These results allow us to postulate that the identified strains may have a role in the biogenic influences in mineral formations at Rani cave.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

Biomineralization of 2,3,4,6-Tetrachlorophenol by Bacillus sp. and Staphylococcus sp. Isolated from Secondary Sludge of Pulp and Paper Mill

Three 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP)-mineralizing bacteria were isolated from the secondary sludge of a pulp and paper industry. The isolates used 2,3,4,6-TeCP as a source of carbon and energy and were capable of degrading this compound, as indicated by stoichiometric release of chloride and biomass formation. Based on 16S rRNA gene sequence analysis, the bacteria were identified as Bacillus megaterium (CL3), Staphylococcus suciri (CL10), and Bacillus thuringensis (CL11). High-performance liquid chromatography (HPLC) analysis revealed that these isolates were able to degrade 2,3,4,6-TeCP at higher concentrations (600 mg/L or 2.5 mM). A consortia of the isolates completely removed 2,3,4,6-TeCP from the sludge obtained from a pulp and paper mill within 2 weeks when supplemented at a rate of 100 mg/L or 0.43 mM. A bacterial consortium also significantly reduced absorbable organic halogen (AOX) and extractable organic halogen (EOX) by 63% and 68%, respectively, from the sludge. These isolates have a high potential to remove 2,3,4,6-TeCP and may be used for remediation of pulp paper mill waste containing 2,3,4,6-TeCP.     

Laboratory studies on biomachining of copper using Staphylococcus sp.

The possibility of using bacteria to drill metallic surfaces has been demonstrated using Staphylococcus sp., a facultative anaerobic bacterium, isolated from corroded copper piping. The experiment involved exposure of copper coupons (25 mm × 15 mm × 3 mm) to a culture of Staphylococcus sp. for a maximum period of 7 days. Coupons exposed to sterile bacterial growth medium were used as controls. Exposed coupons were removed intermittently and observed microscopically for the extent of drilling. The total pit area and volume on these coupons were determined using image analysis. The results showed that both the biomachined area and volume increased with the duration of coupon exposure. In the drilling experiment, a copper thin film 2 μm thick was perforated by this bacterium within a period of 7 days. In conclusion, the results suggested that bacteria can be used as a tool for machining metallic surfaces.     

Isolation and characterization of bacterial strains with the ability to utilize high concentrations of levulinic acid,a platform chemical from inedible biomass

Nineteen levulinic acid (LA)-utilizing bacteria were isolated from environmental samples. Following examination of the use of 80 g/L LA by some isolated strains, Brevibacterium epidermidis LA39–2 consumed 62.6 g/L LA following 8 days incubation. The strain also utilized both 90 and 100 g/L LA, with consumption ratio of 84.3 and 53.3%, respectively, after 10 days incubation.     

Template synthesis,spectroscopic, antibacterial,and antifungal studies of trivalent transition metal ion macrocyclic complexes

A novel series of complexes of the type [M(C₃₆H₂₂N₆)X]X₂, where M = Cr(III), Mn(III), Fe(III); X = Cl^?, NO₃^?, CH₃COO^?; and (C₃₆H₂₂N₆) corresponds to the tetradentate macrocyclic ligand, have been synthesized by condensation of 1,8-diaminonaphthalene and isatin in the presence of trivalent metal salts in methanolic medium. The complexes have been characterized by elemental analysis, conductance and magnetic measurements, and UV/Vis, IR, and mass spectroscopy. On the basis of these studies, a five coordinate square pyramidal geometry for all of these complexes is proposed. All synthesized macrocyclic complexes have been tested for in vitro antimicrobial activities against some pathogenic bacterial strains, viz. Staphylococcus aureus, Bacillus subtilis (Gram-positive), Escherichia coli, Pseudomonas aeruginosa (Gram-negative), and two fungal strains, viz. Aspergillus niger, Aspergillus flavus. The MICs shown by the complexes against these microbial strains have been compared with MICs shown by standard antibiotic ciprofloxacin and the antifungal drug amphotericin-B.     

Chromium-resistant bacteria and cyanobacteria: impact on Cr(VI) reduction potential and plant growth

Two chromium-resistant bacterial strains, Bacillus cereus S-6 and Ochrobactrum intermedium CrT-1, and two cyanobacterial strains, Oscillatoria sp. and Synechocystis sp., were used in this study. At initial chromate concentrations of 300 and 600 μg K₂CrO₄ mL⁻¹, and an inoculum size of 9.6×10⁷ cells mL⁻¹, B. cereus S-6 completely reduced Cr(VI), while O. intermedium CrT-1 reduced Cr(VI) by 98% and 70%, respectively after 96 h. At 100 μg K₂CrO₄ mL⁻¹, Synechocystis sp. MK(S) and Oscillatoria sp. BJ2 reduced 62.1% and 39.9% of Cr(VI), respectively, at 30°C and pH 8. Application of hexavalent chromate salts adversely affected wheat seedling growth and anatomical characters. However, bacterial inoculation alleviated the toxic effects, as reflected by significant improvements in growth as well as anatomical parameters. Cyanobacterial strains also led to some enhancement of various growth parameters in wheat seedlings.

     

MEDIUM OPTIMIZATION OF ANTIFUNGAL ACTIVITY PRODUCTION BY Bacillus amyloliquefaciens USING STATISTICAL EXPERIMENTAL DESIGN

In order to overproduce biofungicides agents by Bacillus amyloliquefaciens BLB371, a suitable culture medium was optimized using response surface methodology. Plackett–Burman design and central composite design were employed for experimental design and analysis of the results. Peptone, sucrose, and yeast extract were found to significantly influence antifungal activity production and their optimal concentrations were, respectively, 20 g/L, 25 g/L, and 4.5 g/L. The corresponding biofungicide production was 250 AU/mL, corresponding to 56% improvement in antifungal components production over a previously used medium (160 AU/mL). Moreover, our results indicated that a deficiency of the minerals CuSO₄, FeCl₃ · 6H₂O, Na₂MoO₄, KI, ZnSO₄ · 7H₂O, H₃BO₃, and C₆H₈O₇ in the optimized culture medium was not crucial for biofungicides production by Bacillus amyloliquefaciens BLB371, which is interesting from a practical point of view, particularly for low-cost production and use of the biofungicide for the control of agricultural fungal pests.     

Biomineralization Potential of Bacillus subtilis,Rummeliibacillus Stabekisii and Staphylococcus Epidermidis Strains In Vitro Isolated from Speleothems,Khasi Hill Caves,Meghalaya, India

Microorganisms were isolated and identified from speleothems at Khasi hill caves, Meghalaya. The aim was to understand their biomineralization potential. Analyses of the speleothems from Krem Soitan, Krem Mawpun, and Krem Lawbah using scanning electron microscope (SEM) showed evidences for microbe–mineral interactions. SEM showed microbial reticulate and beaded filaments, cells, fiber calcites, and clusters of coccoid-like structures. A total of 113 bacterial strains were isolated and identified by a combination of conventional and molecular based tools. 105 strains that were sequenced belonged to the genus: Bacillus, Rummeliibacillus, Staphylococcus, and Brevibacterium. The BLASTn sequence search of 16S rRNA sequences with the National Centre for Biotechnology Information database to establish the identity of the strains yielded similarity scores of ≥99% with the respective organisms. The strains were identified as Bacillus simplex, Bacillus gaemokensis, Bacillus subtilis, Bacillus thuringiensis, Bacillus albus, Bacillus cereus, Bacillus anthracis, Bacillus weihenstephanensis, Rummeliibacillus stabekisii, Bacillus wiedmannii, Staphylococcus epidermidis, Rummeliibacillus pycnus, Kurthia zopfii, and Brevibacterium frigoritolerans. These strains were tested for biomineralization on B-4 medium. Five strains (B. subtilis, R. stabekisii, Staphylococcus epiderdimis, B. cereus, and B. wiedmannii) had the capability to precipitate biominerals in vitro. B. subtilis, R. stabekisii, and S. epidermidis precipitated 0.24, 0.36, and 0.35 g/L of biominerals at 22°C at the end of the four week experiment period. These strains increased the pH of the medium from 7 to 8.95. The precipitated biominerals were imaged using an ultra-high resolution field emission SEM. X-ray diffraction of the biomineral precipitated by R. stabekisii showed that it was composed of vaterite and jungite. Whereas S. epidermidis showed that it was composed of calcite, vaterite, and jungite. B. subtilis produced small, circular calcite crystals. This is the first comprehensive report on the possible evidences about the role of R. stabekisii and S. epidermidis in calcite precipitation isolated from speleothems in the Indian caves. These results allow us to postulate that the identified strains have biomineralization potential. Further evidences of the coexistence of exopolysaccharides, whisker fiber calcites, microbial filaments, and coccoid-like forms point to biogenic inputs in the cave mineral formations.     

Characterization of a methane‐utilizing strain and its application for monitoring methane

Aims: To explore new resources of methane‐utilizing micro‐organism and develop a microbial biosensing system for monitoring methane released from natural and semi‐natural ecosystems. Methods and Results: A methane (CH₄)‐utilizing bacterial strain was isolated from paddy soil using CH₄ as the sole carbon source and identified as Klebsiella sp. ME17 by phenotyping and 16S rDNA sequence analysis. The efficiency of CH₄ utilization of strain ME17 was 83·2% by gas chromatography analysis. A microbial biosensing system for CH₄ detection was developed by combining immobilized cells of strain ME17 with a dissolved oxygen sensor. It was found that response time of the system to CH₄ was <90s. The dissolved O₂ consumption increased with increasing CH₄ from 0% to 16·0% (v/v) demonstrating a positive linear relationship with a low detection limit of 0·2% (v/v). The relative standard deviation is 3·48%. Conclusions: Klebsiella sp. ME17 isolate is capable of utilizing CH₄. The microbial biosensing system of strain ME17 has been successfully applied to measure standard CH₄ sample with satisfactory results. Significance and Impact of the Study: This study suggests that certain strains of Klebsiella genus are capable of utilizing CH₄. Our proposed method appears very attractive for CH₄ measurement in coal mine.     

Optimization of extracellular alkaline protease production from species of <Emphasis Type="Italic">Bacillus</Emphasis>

Thirty-five strains capable of secreting extracellular alkaline proteases were isolated from the soil and waste water near the milk processing plant, slaughterhouse. Strain APP1 with the highest-yield alkaline proteases was identified as Bacillus sp. The cultural conditions were optimized for maximum enzyme production. When the initial pH of the medium was 9.0, the culture maintained maximum proteolytic activity for 2,560 U ml⁻¹ at 50°C for 48 h under the optimized conditions containing (g⁻¹): soyabean meal, 15; wheat flour, 30; K₂HPO₄, 4; Na₂HPO₄, 1; MgSO₄·7H₂O, 0.1; Na₂CO₃, 6. The alkaline protease showed extreme stability toward SDS and oxidizing agents, which retained its activity above 73 and 110% on treatment for 72 h with 5% SDS and 5% H₂O₂, respectively.     

Induction of Calcium Carbonate by Bacillus cereus

The purpose of this research was to study how the bacteria Bacillus cereus (DCB1) utilizes calcium ions in a culture medium with carbon dioxide (CO₂) to yield calcium carbonate (CaCO₃). The bacteria strain DCB1 was a dominant strain isolated from dolomitic surfaces in areas of Karst topographies. The experimental method was as follows: a modified beef extract-peptone medium (beef extract 3.0 g, peptone 10 g, NaCl 5.0 g, CaCl₂ 2.0 g, glass powder 2.0 g, distilled water 1 L, and a pH between 6.5 and 7.5) was inoculated with B. cereus to attempt to induce the synthesis of CaCO₃. The sample was then processed by centrifugation every 24 h during the 7-day cultivation period. The pH, carbonic anhydrase (CA) activity, and the concentrations of both HCO^- ₃ and Ca²⁺ in the supernatant fluid were measured. Subsequently, precipitation in the culture medium was analyzed to confirm, or otherwise, the presence and if present, the formation, of CaCO₃. Methods used included X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Energy Dispersive Spectroscopy (EDS). Meanwhile, the carbon source in the carbonate was classified by its isotope composition. Results showed that B. cereus can improve its pH value in this culture medium; concentrations of HCO^- ₃ and Ca²⁺ showed a significant decline over the duration of the cultivation period. CA activity reached its maximum during the second day; XRD, SEM, TEM, and isotope analysis all revealed the presence of CaCO₃ as a precipitate. Additionally, these results did not occur in an aseptic control group: no detectable level of CaCO₃ was produced therein. In conclusion: B. cereus can metabolize active materials, such as secretase, by its own growth and metabolism, and can either utilize atmospheric CO₂, or respire, to induce CaCO₃ production. Experimental evidence is offered for a concomitant CO₂ reduction and CaCO₃ induction by microorganisms.     

Production of Laccase and Optimization of Its Production by Bacillus sp. Using Distillery Spent Wash as Inducer

A bacillus sp. isolated from the sediments of a distillery mill was used for laccase production under optimized culture conditions. The distillery effluent was used as an inducer for overproduction of laccase by employing the Taguchi approach. Screening of different medium components and their effect on laccase production was studied using an M-16 orthogonal array. The formation of laccase was considerably increased by addition of 1 mM copper sulfate (51.95 U/ml), which was further enhanced by the use of different inducers. The usefulness of the Taguchi method for optimization of culture conditions was investigated with five selected factors at four levels, and it was observed that the optimized medium resulted in a 9-fold increase in extracellular laccase production compared with the control. The optimized medium composition for laccase production was dextrose (1%), tryptone (0.1%), CuSO₄ (1 mM), and an inducer (distillery effluent 10% [v/v]) at pH 7, which altogether resulted in 107.32 U/ml extracellular laccase activity. Hence, the Taguchi approach proved to be a reliable tool in optimizing culture conditions and achieving the best possible combination for enhanced laccase production.     

Plant regeneration of the arsenic hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L<Emphasis Type="Italic">.</Emphasis> from spores and identification of its tolerance and accumulation of arsenic and copper

An in vitro plant regeneration system was established from the spores of Pteris vittata and identification of its tolerance, and accumulation of gametophytes and callous, to arsenic (As) and copper (Cu) was investigated. The highest frequency (100%) of callus formation was achieved from gametophyte explants treated with 0.5 mg l^?1 6-benzylaminopurine (6-BA) + 0.5 mg l^?1 gibberellin acid (GA). Furthermore, sporophytes were differentiated from the callus tissue derived from gametophyte explants on MS medium supplemented with 0.5 mg l^?1 6-BA, 0.5–1.0 mg l^?1 GA and additional 300 mg l^?1 lactalbumin hydrolysate (LH) for 4 weeks. The optimum combination of ½ MS + 1.0 mg l^?1 GA + 0.5 mg l^?1 6-BA + 300 mg l^?1 LH promoted sporophyte formation on 75 ± 10% of the callus. Every callus derived from gametophyte explants could achieve 3–4 sporophytes. The in vitro growth of gametophyte and callus was accelerated in the medium containing Na₃AsO₄ lower than 0.5 mM, but this growth was inhibited with 2 mM Na₃AsO₄. And with the increase of Na₃AsO₄ in the culture medium from 0 to 2 mM, the As accumulation in gametophytes and callus increased and achieved a level of 763.3 and 315.4 mg kg^?1, respectively. Gametophytes and calluses transplanted to culture medium, supplemented with different concentrations of CuSO₄, are similar to those in Na₃AsO₄, and the Cu accumulation in gametophytes could achieve 7,940 mg kg^?1 when gametophytes were subcultured in medium containing 3 mM CuSO₄. These results suggested that the high efficiency propagation system could be a useful and rapid means to identify other heavy metal tolerance and accumulation. Further, the regeneration ability of callus made it possible for genetic transformation of this fern.     

Partial Purification and Characterization of Chromate Reductase of a Novel Ochrobactrum sp. Strain Cr-B4

Hexavalent chromium contamination is a serious problem due to its high toxicity and carcinogenic effects on the biological systems. The enzymatic reduction of toxic Cr(VI) to the less toxic Cr(III) is an efficient technology for detoxification of Cr(VI)-contaminated industrial effluents. In this regard, a chromate reductase enzyme from a novel Ochrobactrum sp. strain Cr-B4, having the ability to detoxify Cr(VI) contaminated sites, has been partially purified and characterized. The molecular mass of this chromate reductase was found to be 31.53 kD, with a specific activity 14.26 U/mg without any addition of electron donors. The temperature and pH optima for chromate reductase activity were 40°C and 8.0, respectively. The activation energy (E_a) for the chromate reductase was found to be 34.7 kJ/mol up to 40°C and the activation energy for its deactivation (E_d) was found to be 79.6 kJ/mol over a temperature range of 50–80°C. The frequency factor for activation of chromate reductase was found to be 566.79 s^?1, and for deactivation of chromate reductase it was found to be 265.66 × 10³ s^?1. The reductase activity of this enzyme was affected by the presence of various heavy metals and complexing agents, some of which (ethylenediamine tetraacetic acid [EDTA], mercaptoethanol, NaN₃, Pb²⁺, Ni²⁺, Zn²⁺, and Cd²⁺) inhibited the enzyme activity, while metals like Cu²⁺ and Fe³⁺ significantly enhanced the reductase activity. The enzyme followed Michaelis–Menten kinetics with K_m of 104.29 µM and a V_max of 4.64 µM/min/mg.     

Gluconic Acid Fermentation by Pullularia pullulans

During the study on the sugar metabolism of molds, several strains of Pullularia pullulans were found to produce large amounts of gluconic acid from glucose. Thirty seven strains of P. pullulans were then tested for their acid-producing abilities. Seven strains did not produce any amount of gluconic acid. However, all of the other strains were shown to be capable of producing this acid. The superior strains produced yiclds of gluconic acid as high as about 90%, based on glucose available, in shaking cultures at 30°C after 2 days. The yields were increased up to approximately 100% during later stages. In addition to high yields, gluconic acid was produced exclusively by these strains. Glutamic acid and inorganic ammonium salts, such as (NH₄)₂SO₄, NH₄Cl and (NH₄)₂HPO₄, were favorable nitrogen sources for acid production. In the case of (NH₄)₂SO₄, the optimum concentration was 0.05%. The addition of CaCO₃ was essential for gluconic acid production by P. pullulans and a 3% concentration of CaC0₃ appeared to be desirable for the maximum conversion to gluconic acid in a medium containing 10% glucose.     

Absolute Configuration of Hydroxycitric Acid Produced by Microorganisms

Optical resolution for (2S,3R) and (2R,3S)-hydroxycitric acid (HCA) enantiomers was developed using chiral column chromatography. HCA from Bacillus megaterium G45C and Streptomyces sp. U121, newly isolated in our previous study, was analyzed to determine the absolute configuration. These results indicate that both strains generate optically pure (2S,3R)-hibiscus type HCA enantiomer.     

Bioactive Pimarane Diterpenes from the Arctic Fungus Eutypella sp. D‐1

Two new pimarane diterpenes, libertellenone M ( 1 ) and libertellenone N ( 2 ), together with five known compounds were isolated from the culture extract of Eutypella sp. D‐1 derived from high‐latitude soil of the Arctic. The structures of these compounds were determined by spectroscopic data as well as experimental and calculated electronic circular dichroism (ECD) analysis. Antimicrobial and cytotoxic activities of the isolated compounds were evaluated. Compound 3 exhibited weak antibacterial activity against Escherichia coli, Bacillus subtilis, and Vibrio vulnificus, each with MIC values of 16 μg/mL. Compounds 2 and 3 showed moderate cytotoxic activity against K562 and MCF‐7 cell lines with IC₅₀ values of 7.67 and 9.57 μm , respectively.     

Phytoremediation affects microbial development on a limestone quarry

Phytoremediation was used to regenerate a limestone quarry area. Plant growth mixed medium added over the quarry surface, consisting of a mixture of pyrolusite byproducts, natural soil, sand, and rice husk. Three different plant species: pine, cypress, and broom were planted at 9 randomized plots in order to assess the effects of vegetation on the microbial development, which was measured for the following 3 years. Substrate samples were analyzed for organic carbon content (C_org), microbial biomass (C_mic), basal CO₂ respiration activity (BR), alkaline phosphatase (ALP), and acid phosphatase activities at each plant specie and year. Furthermore, the ratio C_mic/C_org, the metabolic quotient (qCO₂), and the C mineralization quotient (qM) were determined. The highest survival rates occurred for broom (93.52%), followed by cypress and pine (82.41%) at the final year, while the content of C_mic, BR, and ALP was increased significantly under plants (pine, cypress, and broom) compared with control. C_mic content and BR was plant dependent. Cypress sites had the highest values of C_mic (214.9 μgCg^?1) and BR (112.8 μgCO₂-Cg^?1d^?1) at the 3^rd year. The plant root environment clearly enhances and regulates the microbial community, in correspondence to the species used. Below ground enhanced activity could fulfill the scope of phytoremediation strategies.     

Isolation and Characterization of Iron and Sulfur-Oxidizing Bacteria from Maiduk Copper Mine at Shahrbabk Province in Iran

Microbial oxidation of iron and sulfur are important steps in biogeochemical cycles in mining environments. The aim of this study was the enrichment and identification of two important groups of bacteria that are involved in bioleaching of copper ores. Some soil samples were collected from the Maiduk copper mine. Iron-oxidizing bacteria were enriched in 9K medium containing ferrous sulfate, and sulfur oxidizers were enriched in 9K medium containing powdered sulfur instead of ferrous sulfate as energy source. After three subcultures, autotrophic bacteria were isolated on 9K agarose medium with appropriate energy sources. The autotrophic bacteria from the enrichments were identified by amplification of 16S rRNA gene and sequencing. Bioleaching experiments were performed in 100 ml of 9K medium containing 5 g of low-grade copper ore instead of ferrous sulfate. Twelve different iron and sulfur-oxidizing bacteria were isolated from the collected soil samples of Maiduk copper mine. Molecular identification indicated that two prevalent strains in the ore enrichments could be assigned to the Acidithiobacillus ferooxidans strain HGM and the Thiobacillus thioparus strain HGE. These two strains reached their logarithmic phase of growth after 8 days of incubation in their respective media at 30°C. Of these two cultures, strain HGM leached more copper ore (300 ppm) from the Maiduk copper ore than did strain HGE (200 ppm). Application of these two strains to the Maiduk copper ore in situ and to ore heaps should improve the leaching process.