Ahmad M. Khalid

Phylogenetic analysis of different isolates of Sulfobacillus spp. isolated from uranium-rich environments and recovery of genes using integron-specific primers

The isolation and phylogenetic characterization of acidophilic moderate thermophilic bacteria from different locations of uranium mines and a uranium processing mill in Pakistan is reported. The dominant culturable bacteria found were related to Sulfobacillus thermosulfidooxidans in all the samples analyzed. Different strains displayed different levels of identity (95–97%) to16S rDNA of known strains of this species, indicating group heterogeneity. Genomic DNA from five isolates was subjected to amplification using integron-specific primers HS286 and HS287. Recovery of different integron-linked genes from one of the isolates indicated the usefulness of this approach for gene mining in place of traditional gene recovery methodologies.

An improved solid medium for isolation, enumeration and genetic investigations of autotrophic iron- and sulphur-oxidizing bacteria

An improved solid medium using Gelrite as a supporting gel for isolation and enumeration of acidophilic chemolithotrophic Thiobacillus strains was tested. Dark-brown circular colonies, which were robust and well-differentiated, developed on this medium within 72–96 h. The plating efficiency of T. ferrooxidans strains was 92.1±5.8%. Linear correlations with an optical density (OD) at 400 nm of 0.5, corresponding to 7·106 and 5·107 cells·ml−1 for strains ATCC 13661 and TMB, respectively, were obtained. Linear correlations between OD, total bacterial protein, dry biomass and cell number were also determined. These data can be used for modelling and scale-up design of microbial leaching operations. With this plating technique, one prerequisite for selection of different mutants of T. ferrooxidans has been fulfilled.

Technical communication: Determination of cuprous ions in bacterial leachates and for environmental monitoring

A sensitive and precise spectrophotometric method has been developed for the determination of copper(I) in bacterial leach liquors produced by the action of Thiobacillus ferrooxidans and T. thiooxidans on copper ores. In this method bicinchoninic acid (BCA) has been used as the chromogenic reagent which produces a stable purple complex with Cu(I) which was found to obey Beers Law and with λmax at 560 nm. The coloured complex has a molar extinction coefficient (ε) value of 6.6 × 103 l mol−1 cm−1; specific absorptivity (α) value of 0.104 ml−1 g cm−1 and the Sandell sensitivity (S) value was 0.0096 μg cm2. Optimal conditions for development of coloration/sensitivity were determined. Interferences due to cations and anions were investigated and various masking agents for alleviating their inhibition were studied. The method has been found very useful in determining ratios of Cu(I) to Cu(II) in bacterial leach liquors and should play a significant role in determining the reaction mechanisms of biological leaching and for environmental monitoring.

Phylogenetic Analysis of Bacterial Isolates from Man-Made High-pH, High-Salt Environments and Identification of Gene-Cassette–Associated Open Reading Frames

Environmental samples were collected from high-pH sites in Pakistan, including a uranium heap set up for carbonate leaching, the lime unit of a tannery, and the Khewra salt mine. Another sample was collected from a hot spring on the shore of the soda lake, Magadi, in Kenya. Microbial cultures were enriched from Pakistani samples. Phylogenetic analysis of isolates was carried out by sequencing 16S rRNA genes. Genomic DNA was amplified by polymerase chain reaction using integron gene-cassette–specific primers. Different gene-cassette–linked genes were recovered from the cultured strains related to Halomonas magadiensis, Virgibacillus halodenitrificans, and Yania flava and from the uncultured environmental DNA sample. The usefulness of this technique as a tool for gene mining is indicated.

A simple apparatus for measuring hardness of agar and Gelrite plates

A simple apparatus for measuring hardness of Gelrite and agar plates, that can be easily fabricated, is described. It gives reproducible results comparable with those obtained with expensive commercial equipment. It has been successfully used to improve enumeration ofThiobacillus ferrooxidans andT. thiooxidans on solid media plates.