Download vii Abstract Heat-loving microbes or thermophiles are

Abstract
Heat-loving microbes or thermophiles are among the best studied of the
extremophiles. In the present study, thermophilic bacteria were isolated from
Tattapani hot spring (Himachal Pradesh) by enrichment of the water and sediment
samples. Nineteen thermophilic bacterial isolates (PW1-13 from water and PS3, PS4,
PS5, PS7, PS9 and PS10 from the sediment) were isolated. The microbial isolates
showed optimum growth at 80ºC and at pH 6-7. Both Na+ and K+ ions were detected
at 0.11 M and 1.7 mM respectively in the hot spring water. Consequently, 50% of the
bacterial isolates showed growth in the presence of 0.5M KCl, whereas PW12 isolate
could grow in the presence of 0.75 M KCl or 0.5 – 0.75 M NaCl. All the bacterial
strains were Gram’s positive that were comparatively small, medium, and long rods.
All the strains were non-motile, except PS3. 14 bacterial isolates were oxidase
positive, whereas 16 bacterial isolates were nitrate reducer. All the bacterial isolates
were analyzed by randomly amplified polymorphic DNA (RAPD) using three random
primers and produced PCR fragments ranging from 250 – 6000 bp in size. Bacterial
isolates of water and soil sediment are phylogenitically different and evolved in close
association. Bacterial isolates PW11, PW12, PW13 and PS4 used starch, raffinose,
and trehalose as a carbon source and peptone as a nitrogen source for their optimum
growth. Thermophilic bacterial isolates PW11, PW12, PW13 and PS4 showed growth
in the presence of different heavy metal ions (Ni2+, Ca2+, Mn2+, Mg2+, Zn2+, Co2+,
Cu2+, Hg2+, Fe2+, Cd2+), and solvents (n- butanol, toluene, ethanol and cyclohexane).
Further, PW11, PW12, PW13 and PS4 were analyzed by PCR amplification of gene
encoding 16S rDNA. 16S rDNA sequence analysis by BLAST search identifies PW13
as Geobacillus thermoleovorans, PW11 as Geobacillus kaustrophilus, PS4 and PW12
as Geobacillus toebii with GenBank accession number KF751757, KF751758,
KF751759, and KJ509869 respectively. G. kaustrophilus strain PW11,
G.
thermoleovorans strain PW13, and G. toebii strain PS4 produce varied levels of
extracellular industrially important thermophilic amylase, protease, lipase, cellulase
and glutaminase; while G. toebii strain PW12 produces lipase and cellulase only.
These thermophilic enzymes are functional at broad range of temperature (50-100°C),
pH (5-9), with maximum activity at 80-90°C and pH7-8. These thermoenzymes
retained activity in the presence of heavy metal ions (Ni2+, Ca2+, Mn2+, Mg2+, Zn2+,
Co2+, Cu2+,
Hg2+, Fe2+, Cd2+), solvents (n- butanol, toluene, ethanol and
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cyclohexane,) and chatropic agents (SDS, Triton X 100 and EDTA). Moreover, these
enzymes are thermostable even at 100°C. The combined hydrolytic activity of some
thermophilic bacterial isolate can be used for bioconversion of organic materials to
useful products. Owing to their ability to remain functional in extreme conditions,
such as high temperatures, offering important biotechnological potential.
Genomic library of G. thermoleovorans strain PW13 was screened for gene
encoding glutaminase. The clone PC4 showed thermophilic glutaminase activity.
Maximum extracellular glutaminase activity of 63U/mg was observed in the cell free
spent medium at 65°C. An ORF of 184 amino acids showed homology with
Glutaminase of Geobacillus sp. A8.
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