SEROLOGICAL AND MOLECULAR STUDIES OF BACTERIAL POPULATION DEVELOPED FOR BIODESULFURIZATION OF COAL HEAPS

Abstract

Polyclonal anti sera against whole cells of locally isolated acidophilic bacterial isolates i.e (HC-AF2), (KC-AT2), and Sulfobacillus thermosuljidooxidans strains (MT13, THl ) used in coal heap biodesul furi zation process were successfully raised in rabbits. Among these bacteria, KC-A T2 was observed to be highly immunogenic while THl was least immunogeni c. The specificity of each of the developed polyclonal antisera was determined again st many other acidophilic isolates (TF9, TFlO, MT9, MTIO, MT16, MT17) by the serological tests including agglutination reaction, fluorescent antibody labeling reaction and enzyme linked immunosorbent assay (ELISA). The po lyclonal anti sera against mesophilic isolates HC-AF2 and KC-AT2 were found to be more specific as they ex hibited no cross reactivity with other mesophilic and moderate thermophi lic acidophilic isolates. While polyclonal antisera developed for THI and MT13 strains showed cross reactivity against other isolates (MT9, MTIO, MT16, MT17) of the same species. Populat ion of these bacteria in coal heap set for biodesulfurizat ion was determined by quantitative ELISA, which indicated that moderate thermophiles were dominated bacteria in leach liquor while in heap both mesophiles and moderate thermophiles were detected. PCR amplification of 16S rDNA of genomic DNA from I-ICAF2 and KC-AT2 ind icated that they belong to At. ferrooxidans (98%) and At. thiooxidans (95%), respectively. A phylogenetic tree of these bacteria was constructed by aligning 16S rDNA sequences of other acidophilic bacteria obtained from NCBI database using various softwares available at our laboratory. Integron linked genes were amplified in these bacteria using integron specific primers HS286 and HS287. Two types of DNA bands of approximately 396 bp and 220 bp were obtained in case of both, the HC-AF2 and moderate thermophiles. Similarly in case of mixed culture the bands of same size were obtained. However, in case of pure KC-AT2 no amplification product, was observed after agarose gel electrophoresis with the same set of primers.