Abstract:
The present study is concerned with the isolation of thermoduric microflora from the hot
springs of Azad Kashmir. A total of seventy two water samples mixed with some mud
were collected from nine hot springs in Tatta Pani (Azad Kashmir) at four different time
intervals depending upon seasonal variation, and were analyzed in-situ and ex-situ in
terms of pH, temperature, soluble anions and cations, biological oxygen demand,
chemical oxygen demand and electric conductivity. The temperature and pH of Tatta
Pani hot springs (Azad Kashmir) ranged from 38-110 o C and 6.82-7.18, respectively.
Thirty seven pure cultures were isolated from the said hot springs. Based on
morphological, physiological and biochemical characterization, the isolates were divided
into seven groups. One representative isolate from each group was further subjected to
molecular characterization. All isolates showed thermoduricity, TP-1, TP-2, TP-3, TP-4
and TP-5 isolates tolerated 100 o C for 30 min, 105 o C for 20 min and 110 o C for 10 min
while isolates TP-33 and TP-37 tolerated 110 o C for 30 min and 115 o C for 10 min.
Isolate TP-1 was facultative anaerobic bacterium that formed pale yellow, round,
smooth, flat and slimy colonies while the cells were Gram positive rods, about 3.5-5.0
μm in length to 0.6-0.7 μm in width and were motile. It showed growth within the
temperature range of 35-80 o C with optimum growth observed at 65 o C. It grew within the
pH range of 5.5-8.5 with optimal growth observed at pH 7.0. It tolerated NaCl within the
range of 0-4.5% (w/v) with optimum growth observed at 1%. It showed growth on
maltose, fructose, lactose, starch, xylan and CMC used as sole carbon source. It was
oxidase and catalase positive and gave positive tests for o-nitro phenyl β-D-
galactopyranoside, gelatin hydrolysis and produced acid from maltose. Almost complete
16S rRNA gene sequence analysis showed that it had 97% similarity with Geobacillus
pallidus.
TP-2 isolate was aerobic, Gram positive, motile, rod shaped bacterium that ranged
in size from about 2.1-3.6 μm to 0.2-0.3 μm in width. It formed cream colored, round,
smooth, flat and slimy colonies. The temperature and pH range for growth was found to
be 45-75 o C and 5.5-8.5, respectively with optimum growth observed at 65 o C and pH 7.0.
It showed growth within the NaCl concentration of 0-3.5% (w/v) with optimal growthxii
observed at 0.5%. It was capable of growing on CMC, lactose, sucrose, starch, glucose,
maltose, xylan, fructose and filter paper used as sole carbon source. It was catalase and
oxidase positive and gave positive test for o-nitro phenyl β-D-galactopyranoside, gelatin
hydrolysis and nitrate reduction and produced acid from glucose, maltose and sucrose.
Based on 16S rRNA gene sequence analysis, isolate TP-2 gave low level of similarity
(89%) with Geobacillus debilis.
Isolates TP-3 and TP-4 were facultative anaerobic, Gram positive, catalase and
oxidase negative, motile, rod shaped bacteria that showed growth within the temperature
range and pH range of 45-75 o C and 5.5-9.0, respectively with optimal growth observed at
70 o C and pH 7.0. Isolates TP-3 and TP-4 showed optimal growth at 1.5% and 1.0% NaCl
concentration, respectively and produced acid from maltose, sucrose and mannose while
isolate TP-3 produced acid from glucose also. Isolate TP-3 utilized glucose, maltose,
fructose, lactose, sucrose, starch, CMC, wheat bran extract and filter paper for growth
while isolate TP-4 showed growth on maltose, fructose, lactose, sucrose, starch, wheat
bran extract, xylan and CMC. 16S rRNA gene sequences showed that isolates TP-3 and
TP-4 displayed 94% and 96% similarity, respectively with Geobacillus vulcani.
Isolate TP-5 was facultative anaerobic, motile, Gram positive, catalase positive,
oxidase negative, rod shaped bacterium, 2.7-3.8 μm in length to about 0.6-0.7 μm in
width. It formed whitish, round, smooth, convex and slimy colonies. It grew optimally at
70 o C and pH 7.0 and tolerated NaCl concentration of 0-4% (w/v) with optimum growth
observed at 0.5%. Isolate TP-5 utilized all the carbon sources (glucose, maltose, fructose,
lactose, sucrose, starch, xylan, wheat bran extract, CMC and filter paper) for growth. It
gave positive results for gelatin hydrolysis and nitrate reduction and produced acid from
glucose, maltose, sucrose and mannose. 16S rRNA gene sequence analysis displayed
94% similarity with Geobacillus stearothermophilus.
Based on phenotypic (morphological, physiological and biochemical) and
genotypic (16S rRNA gene sequence analysis) characterization, and taking phylogenetic
analysis into consideration, it was concluded that isolates TP-1, TP-3, TP-4 and TP-5
belonged to the genus Geobacillus while isolate TP-2 was quite distinct in its characters
from the known Geobacillus species as well as to other established genra, so it mayxiii
represent a novel strain. Further, intracellular protein profiling of bacterial isolates using
SDS-PAGE analysis with the type strain of Geobacillus pallidus displayed that
intracellular protein pattern of isolate TP-1 was most closely related to the intracellular
protein pattern of Geobacillus pallidus ATCC 51176 (type strain), isolates TP-3, TP-4
and TP-5 displayed intermediate level of differences in protein pattern in comparison to
that of type strain while the intracellular protein pattern of isolate TP-2 was most distant
as compared to type strain.
Isolates TP-33 and TP-37 were anaerobic archaea that formed off-white, round
colonies. The cells were Gram negative cocci having diameter of 0.7-1.5 and 0.7-1.7 μm
for isolates TP-33 and TP-37, respectively. Isolates TP-33 and TP-37 grew optimally at
80 o C and 75 o C, respectively and at pH 7.0. The optimal NaCl concentration for growth
was determined to be 0.5% and 0.3% for isolates TP-33 and TP-37, respectively. Isolates
TP-33 and TP-37 grew on complex proteinaceous substrates i.e, peptone, tryptone and
yeast extract while were unable to grow in the absence of cystine. Isolate TP-33 utilized
maltose and starch for growth while isolate TP-37 was unable to grow on any of the
carbon sources tested in the absence of proteinaceous substrates but grew on xylan and
glucose in the presence of 0.1% peptone. 16S rRNA gene sequence analysis showed that
isolates TP-33 and TP-37 displayed 97% and 95% similarity with Thermococcus
waiotapuensis and Thermococcus zilligii, respectively. Based on morphological,
physiological and molecular characterization as well as phylogenetic analysis it was
found that isolates TP-33 and TP-37 belonged to the genus Thermococcus.
Isolate TP-1 produced extracellular α-amylase, CMCase, xylanase, lipase and
protease, isolate TP-2 produced extracellular α-amylase, CMCase, FPase, xylanase,
lipase and protease, isolate TP-3 gave extracellular activities for α-amylase, CMCase,
FPase, lipase, protease and phytase enzymes, isolate TP-4 gave extracellular activities for
α-amylase, CMCase, lipase, protease and phytase and isolate TP-5 gave extracellular
activities for α-amylase, CMCase, FPase, xylanase, lipase and protease. Intracellular
CMCase activity was recorded for isolates TP-1, TP-2, TP-3, TP-4 and TP-5 while
intracellular FPase activity was observed for isolates TP-2, TP-3 and TP-5. Isolate TP-33
gave positive result for extracellular α-amylase while isolate TP-37 gave positive resultxiv
for extracellular xylanase and protease. Maximum α-amylase activity (0.993 U/ml/min)
was given by isolate TP-5, isolate TP-1 gave maximum production of extracellular
CMCase (0.091 U/ml/min), intracellular CMCase (0.025 U/g/min), extracellular xylanase
(0.587 U/ml/min), extracellular lipase (0.23 U/ml/min) and extracellular protease (0.314
U/ml/min). Maximum extracellular FPase activity (0.021 U/ml/min) was given by isolate
TP-5 while isolate TP-3 gave maximum production of intracellular FPase (0.009
U/g/min) and extracellular phytase (0.023 U/ml/min).