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CHAPTER III
Isolation and Identification
of cold-active α-amylase producing microorganisms
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3.1. INTRODUCTION
Life under low-temperature conditions was identified as early as 1840 by Hooker,
who observed that algae were associated with sea ice (Hoyoux et al., 2004). In 1887, Forster
was the first who reported that microorganisms isolated from fish could grow well at 0°C
(Forster, 1887). The term “psychrophilic” was first used in 1902 by Schmidt-Nielsen to
describe such cold-adapted organisms (Schmidt, 1902). Psychrophile is defined as an
organism, prokaryotic or eukaryotic, living permanently at temperatures close to the
freezing point of water in thermal equilibrium with the medium. But the classical definition
of psychrophiles was given by Morita (1975) which is frequently used in the literature. This
definition proposes that psychrophilic microorganisms have optimum growth temperatures
of <15°C. But a psychrotrophic term is used for those cold-adapted organisms that have an
optimum growth temperature of ~15-20°C but are able to grow up to 30°C. Margesin et al.
(2002) reported that psychrophiles are numerous and are widely distributed, including large
range of species of gram-positive and gram-negative bacteria, yeast, algae, marine
invertebrates, insects and polar fish. Various adaptation mechanisms against low
temperature were developed by psychrophiles including a huge range of structural and
physiological adjustments in order to cope with the deleterious effect of low temperatures.
Indeed, they display metabolic fluxes at low temperatures that are more or less comparable
to those exhibited by closely related mesophiles living at moderate temperatures (Morita,
1975; Feller et al., 1994b; Russel, 2000). Capability of psychrophilic organisms to live
successfully in such low temperature environments is because of production of “cold-
active” enzymes which are able to cope with the reduction of chemical reaction rates
induced by low temperatures. However, most cellular adaptations to low temperatures and
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the underlying molecular mechanisms are not fully understood and are still being
investigated. Moreover, a study of proteins and enzymes from cold-adapted organisms is not
only useful in the understanding of some general processes related to the protein structure
and function but also in protein folding investigations. In addition, cold-active and heat-
labile psychrophilic enzymes possess an interesting biotechnological potential.
Cold-adapted microorganisms prosper at temperatures close to the freezing point of
water and have successfully colonized permanently cold habitats such as polar and alpine
regions or deep-sea waters (Bowman et al., 1997; Margesin and Schinner, 1997; Morita et
al., 1997). These cold habitats constitute more than three-quarters of the earth’s surface and
are exposed to temperatures that are more or less permanently below 5°C. Microbial growth
and metabolic activities have been recorded beneath 3–6 meters of ice in permanently frozen
lakes, in sub-glacial ice and sediments, in surface snow at the South Pole where the highest
summer temperatures remain well below zero (at least -10°C). Moreover, biological
activities have been recorded in the brine veins of sea ice at temperatures as low as -20°C
(Deming, 2002). More often, psychrophilic microorganisms are not only adapted to low
temperatures but also to other environmental constraints. In deep sea water, for example,
they have to be adapted to extremely high pressure and therefore must be, at the same time
are both psychrophiles and piezophiles (Yayanos, 1995).
Low temperature strongly inhibit the rate of chemical reactions and moreover in
addition to various cellular adaptations, the main adaptation strategy of psychrophiles is the
modification of enzyme kinetics, allowing the emergence of metabolic rates compatible to
life at low temperatures. The temperature dependence of chemical reactions is described by
the Arrhenius equation k=Ae-Ea/RT, in which A is the pre-exponential factor (related to steric
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factors and molecular collision frequency), Ea is the activation energy, R is the gas constant
(8.314 JK–1 mol–1) and T is the absolute temperature in Kelvin. Thus, any decrease in
temperature will induce an exponential decrease in the reaction rate and for most biological
systems; a decrease of 10°C in temperature decreases the rate of chemical reactions by a
factor of 2 to 3 termed as temperature quoficient (Q10), which expresses the ratio of reaction
rates measured at an interval of temperature of 10°C. Nevertheless, psychrophiles succeeded
to maintain an appropriate rate for enzyme-catalyzed reactions that are involved in essential
cellular processes by synthesizing cold-active and heat-labile enzymes with an activity that
is up to 10 times higher at low temperatures than that of their mesophilic homologues. The
synthesis of such cold-active or psychrophilic enzymes is the main physiological adaptation
at the enzyme level (Hoyoux et al., 2004).
More than 100 species of psychrophiles have been identified and reported which
comprises of both Gram-negative and Gram-positive bacteria from various habitats ranging
from soil, sandstone, fresh water and marine lakes, sea ice and oceans. Various species
within the genera Alcaligenes, Alteromonas, Aquaspirillum, Arthrobacter, Bacillus,
Bacteroides, Brevibacterium, Gelidibacter, Methanococcoides, Methanogenium,
Methanosarcina, Microbacterium, Micrococcus, Moritella, Octandecabacter, Phormidium,
Photobacterium, Polaribacter, Polaromonas, Psychroserpens, Shewanella and Vibrio have
been reported to be psychrophilic (Morita and Moyer, 2001). The psychrophilic and
barophilic bacteria belong to α-Proteobacteria which includes Shewanella, Photobacterium,
Colwellia, Moritella and Alteromonas haloplanktis. For the first time, Leifsonia aurea,
Sporosarcina macmurdoensis and Kocuria 62olaris (Reddy et al., 2003a; 2003b) have been
reported from Antarctica. A psychrophilic and slightly halophilic methanogen,
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Methanococcoides burtonii was isolated from perennially cold, anoxic hypolimnion of Ace
Lake, Antarctica. Archaea such as Methanogenium, Methanococcoides and Halorubrum
species, yeast like Candida and Cryptococcus species, fungi such as Penicillium and
Cladosporium and microalgae (Chloromonas) can be found in these environments and
display striking cold-adaptive characteristics (Morita, 1975; Russell, 1990; Gounot, 1991;
Allen et al., 2001; Deming, 2002; Margesin, 2002).
A wide range of cold-adapted bacteria producing cold-active enzymes have been
described. However, most of the cold-adapted enzymes that so far been characterized
originated from the Antarctic terrestrial and the Antarctic sea water (Gerday et al., 1997 and
Russell, 1998). Specific activity of cold enzymes from wild strains and some of their
recombinant forms have been determined in organisms of Antarctic and arctic regions
including alcohol dehydrogenase (Feller et al., 1994a), α-amylase (Vazquez et al., 1995),
Aspartate transcarbamylase (Feller et al., 1992), Ca+2/Zn+2 protease (Villeret et al., 1997)
Citrate synthetase (Gerike et al., 1997) α-lactamase, Malate dehydrogenase, Subtilisin
(Narinx, 1997), Triose phosphate isomerase (Alvarez et al., 1998) and Xylanase (Reddy et
al., 2003b).
Alpha-amylases are ubiquitous enzymes produced by plants, animals and microbes,
where they play a dominant role in carbohydrate metabolism. Amylases from plant and
microbial sources have been employed for centuries as food additives. Barley amylases have
been used in the brewing industry. Fungal amylases have been widely used for the
preparation of oriental foods (Burhan et al., 2003). Among bacteria, Bacillus sp. is widely
used for α-amylase production to meet industrial needs. B. subtilis, B. stearothermophilus,
B. licheniformis and B. amyloliquefaciens are known to be good producers of α-amylase and
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these have been widely used for commercial production of the enzyme for various
applications. Similarly, filamentous fungi have been widely used for the production of
amylases for centuries. As these moulds are known to be prolific producers of extracellular
proteins, they are widely exploited for the production of different enzymes including α-
amylase. Fungi belonging to the genus Aspergillus have been most commonly employed for
the production of α-amylase.
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3.2. MATERIALS AND METHODS
3.2.1. Collection of soil samples and Microbiological analysis
Soil samples were collected from twenty five different ecological niches of Gangotri
glacier, Western Himalaya, India for the isolation of cold-adapted amylase producing
microorganisms. The glacier is situated in between 3056′N and 7904′-7915′E and around
30 km in length covering an area of 143 km2 flowing northwest and having temperature 2-
5ºC in summer and subzero in winter. Soil samples were collected during early winter in the
month of October, 2009. The samples were collected from 10-14 cm depth with a sterile
spatula in sterile poly-bags. For rhizosphere soil, plants were uprooted carefully and the
excess of soil was removed by shaking the plant thoroughly and adhering soil was collected.
The samples were brought to the laboratory under cold condition. The collected samples
were stored at –20°C.
3.2.2. Total viable counts (viable plate count method)
For enumeration of the bacterial load in the soil samples, one gram of respective
samples were homogenized in 9 ml of cold sterilized double distilled water and the
suspension was serially diluted up to 10-6. The diluted suspension (0.1 ml) of various
dilutions were inoculated in triplicate for each dilution on nutrient agar media (Appendix 1)
and incubated for 48 hours at 15+2ºC. For colony counting plates, appropriate dilutions
which contain colonies in the range of 30 to 300 was selected. The numbers of colonies
appearing on dilution plates were counted and the bacterial load was recorded as CFUs per
gram of soil by applying the formula:
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Mean plate count X Dilution factor Number of cells/g soil =
Weight of soil
3.2.3. Total amylolytic counts
In order to detect cold-adapted amylolytic microbes, one gram of soil sample was
suspended in 9 ml of cold, sterile distilled water and after appropriate dilution suspensions
were plated on starch agar media (Appendix 1) in triplicate. For cold-adapted amylolytic
counts, the plates were incubated for 72-96 hours at 15+2ºC, respectively. The individual
colonies showing clear zone of hydrolysis on flooding I2/KI solution, was counted as
amylolytic. The total amylolytic count per gram of soil was calculated by using following
formula:
Mean amylolytic count X Dilution factor Amylolytic count/g soil =
Weight of soil
Morphologically distinct colonies showing starch degradation were isolated to obtained pure
cultures and stored at 4ºC for subsequent experiments.
3.2.4. Screening of cold-active α-amylase producing bacteria
Total 120 bacterial isolates were screened for production of cold-adapted extra-
cellular amylase on starch agar media at low temperature (15±2°C). On the basis of larger
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clear zone formation around the colonies, fourteen isolates were selected for production of
cold-active amylase.
3.2.5. Selection of strains
Six potential isolates of cold-adapted amylase producing bacteria were tested for the
enzyme production by using amylase producing broth media (Appendix 1). The above
medium (100 ml) was inoculated at 1% (v/v) with a 48 hour old culture (OD, 0.6) and
incubated at 15±2ºC in a refrigerator shaker (120 rpm) for 48 hours. The growth cultures
were then centrifuged at 10,000×g (If needed RCF was converted into RPM – Appendix 2)
for 10 min at 4ºC and the supernatant used for amylase assay as described below. On the
basis of maximum amylase production, isolates GA2 and GA6 were taken for detailed
investigations.
3.2.6. Assay of amylolytic activity
Amylolytic activity with starch (Himedia) as a substrate was assayed by the method
of Swain et al. (2006). This amylase assay was based on reduction in blue color intensity
resulting from enzyme hydrolysis of starch. The reaction mixture consisted of 0.2 ml
enzyme (cell free supernatant), 0.25 ml of 0.1% soluble starch solution and 0.5 ml of
phosphate buffer (0.1M, pH 6.0) incubated at 50°C for 10 min. The reaction was stopped by
adding 0.25 ml of 0.1N HCl and color was developed by adding 0.25 ml Gram’s iodine
solution. The optical density of blue color solution was determined by using
spectrophotometer at 690 nm. The activity was expressed in units. One unit of enzyme
activity is defined as the quantity of enzyme that causes 0.01% reduction of blue color
intensity of starch-iodine solution at 50°C in one min per ml.
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3.2.7. Identification of potential isolates
The potential cold-adapted amylolytic bacterial isolates were identified by studying
morphological and biochemical characteristics according to the Bergey’s Manual of
Determinative Bacteriology (Holt et al., 1989). The identity was further confirmed by 16S
rRNA analysis.
3.2.7.1. Isolation of genomic DNA for 16S rRNA and PCR amplification
Total genomic DNA was extracted from the cells by using phenol-chloroform
method (Ruzzante et al., 1996). Isolated DNA was checked for its quality and concentration
by Agarose gel electrophoresis on UV transilluminator. 16S rRNA region was amplified
with universal forward and reverse bacterial primers. The PCR amplification was performed
using a PTC-150 Mini cycler (MJ Research), with a primary heating step for 2 min at 95°C,
followed by 30 cycles of denaturation for 20 sec at 95°C, annealing for 60 sec at 55°C, and
extension for 2 min at 72°C, then followed by a final extension step for 7 min at 72°C. Each
25 µl reaction mixture contained 2 µl of genomic DNA, 14.25 µl of MilliQ water, 2.5 µl of
10X buffer (100 mM Tris-HCl, pH 8.3; 500 mM KCl), 1.5 µl of MgCl2 (25 mM), 2.5 µl of
dNTP’s mixture (dATP, dCTP, dGTP, dTTP at 10 mM concn.), 1.0 µl of each primer (20
pmoles/ml), and 0.25 µl of Taq DNA polymerase. The PCR-amplified product was analyzed
on 1% Agarose gel containing ethidium bromide (0.5 mg/ml) and 1 kb DNA molecular
weight marker and documented using a gel documentation system. The PCR amplicon for
the partial 16S rRNA gene was further processed for sequencing. Sequencing was carried
out using the same set of primers in both the directions to check the validity of the sequence.
Sequencing was done from the DNA Sequencing Services, Ocimum Biosolutions,
Bangalore and before sequencing, samples were passed pre-sequencing quality check by gel
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electrophoresis. After sequencing, final DNA sequences were analyzed by bioinformatics
tool.
3.2.8. Characterization of identified isolates
The identified isolates were further characterized with respect to their antibiotic
sensitivity test. Isolates were also checked for their plasmid mediated characteristics,
whether amylolytic characters were plasmid borne traits or chromosomal DNA trait, by
curing of plasmid.
3.2.8.1. Antibiotic susceptibility
The antibiotic susceptibility of the bacteria was studied on Muller-Hinton agar
medium (Appendix 1) by disc diffusion methods (Bauer et al., 1966). Muller-Hinton agar
was prepared according to the media composition and poured into sterilized petriplates.
After solidification, it was inoculated with test organisms by swabbing and disc of
antibiotics (which are economically important and commonly used) were placed. The
inoculated plates with discs were incubated at 20±2ºC and examined for inhibition of
growth. Zone of inhibition was recorded after 24-72 hours when lawn of bacteria was
visible against clear zone of growth inhibition around the discs. Interpretation of results was
done on the basis of manufacturer’s instructions (HiMedia Pvt. Ltd., India). The antibiotics
(mcg) used were Penicillin (10), Ampicillin (10), Chloramphenicol (30), Streptomycin (10),
Tetracyclin (30) and Doxycycline (30).
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3.2.8.2. Plasmid curing
For plasmid curing, 10 ml of peptone water supplemented with 20-100 g/ml
ethidium bromide was inoculated with 0.1 ml of overnight broth culture and incubated at
optimum growth temperature for 24 hour (Trevors, 1986). Appropriate dilutions of the
culture were plated on nutrient agar to obtain single colony isolates. After overnight
incubation at 20±2ºC, resulting colonies were tested for loss of amylase production on
starch agar plates. The colonies, which are not forming clear zone around it, were regarded
as the cured ones. Colonies suspected of plasmid loss can then be examined for plasmids
using agarose gel electrophoresis. The percentage of curing was determined by the formula:
No. of organism cured % Curing = X 100
No. of organism tested
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3.3. RESULTS AND DISCUSSION
3.3.1. Microbiological analysis of soil and isolation of bacteria
Microbiological studies revealed the presence of bacteria in soil samples of Gangotri
glacier. Total viable bacterial counts and total amylolytic counts from soil samples of
different ecological niches of Gangotri glacier are summarized in Table 3.1. As shown in
Table 3.1, total viable bacterial count and total amylolytic count varied from 3.9542 to
6.9542 and 2.9452 to 6.1020 log CFUg-1 of soil, respectively at 15±2°C. Out of total
hundred and twenty bacterial colonies, fifty two positive bacterial colonies producing
extracellular amylase on starch agar media at 15±2ºC were isolated from 25 soil samples of
Gangotri glacier.
The results suggested that the soil of Gangotri glacier could be an affluent source of
technologically significant microbial pool. In microbial ecosystem of cold regions,
psychrophilic and psychrotrophic organisms play a major role in the biodegradation of
organic matter. These organisms are efficient not only in permanently cold areas but also in
habitats, which experience seasonal variation in temperature during late fall and spring.
Suman et al. (2010) reprted cold-active amylase producing β-proteobacteria from soil of
Roopkund glacier, Himalayan Mountain ranges, India. These glaciers are supposed to be
highly diverse in bacterial population surviving at low temperature. Various proteolytic
bacteria such as Pseudomonas aeroginosa, Bacillus subtilis and Bacillus liceniformis were
also isolated from the soil sample of Gangotri glacier. These isolates were capable of
producing extracellular protease at alkaline pH and at temperatures ranging from 10-37ºC
(Baghel et al. 2005). The high density of amylolytic bacteria in glacier may be due to
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increasing human activities in the form of pilgrims and scientific expedition that causes
environmental pollution and ecological disturbances. The study of these bacterial isolates
and their enzymes will be useful to develop desired microbial consortium for organic waste
treatment at cold regions and also they may find applications in other industries.
Table 3.1. Microbial analysis of soil samples collected from Gangotri glacier
Sample No. Sampling Site
log CFU g-1 soil
TVC at TAC at 15±2ºC 15±2ºC
Gaumukh (Right side)
1 50 feet down stream from Gaumukh 4.9912 4.4624
2 50 feet down stream sediments 5.3979 5.0891
3 50 feet down stream near water 5.2787 5.0342
4 50 feet down stream near shrubs 4.5314 3.5630
5 50 feet down stream near rocks 5.9030 5.5792
6 75 feet down stream 4.3617 3.9060
7 75 feet down stream sediment 3.9542 2.9452
8 100 feet down stream near shop 5.9294 4.4310
9 100 feet down stream near rocks 5.000 4.7262
10 100 feet down stream shop drainage 5.1139 3.8724
Bhojwasa
11 Potato vegetations 4.3424 3.6554
12 Ashram drainage 6.9542 5.5000
13 Beneath bolder 5.8450 4.2672
14 Kitchen waste 5.9030 5.5090
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15 Potato rhizosphere 6.9030 6.1020
16 Shrub rhizosphere 5.0000 4.2420
17 Plants rhizosphere 4.3010 4.2552
Jaspur band
18 Cabbage rhizosphere 5.7481 3.8672
19 Apple rhizosphere 5.9590 4.7681
20 Road side 4.3222 3.5099
21 Road side near hotel 4.6812 4.3120
22 Cabbage rhizosphere near hotel 5.0000 4.8989
23 Restaurant drainage 5.0413 4.7324
24 Road side superficial soil 4.9822 3.9450
25 10 Km down from Jaspur band 5.9777 5.2461
TVC: Total viable counts; TAC: Total amylolytic counts
3.3.2. Screening and selection of cold-active amylase producing bacteria
Total fifty two positive bacterial isolates, producing extracellular amylase were
isolated from soil sample of Gangotri glacier at 15±2°C, on starch agar media. Out of 52
positive isolates (Table 3.2), 14 showed good zone of hydrolysis when flooded with Grams
iodine solution (Table 3.3). On the basis of diameter of hydrolysis zone on starch agar
media at 15±2°C, six potential isolates, designated as GA2, GA4, GA6, GA10, GA11 and
GA12 were selected as potent amylase producing strains. The amylase production of six
potential isolates was further quantified at neutral pH by using amylase assay method. The
results are given in Table 3.4. On the basis of maximum amylase production at neutral pH,
two isolates GA2 and GA6 (Figure 3.1) were taken for further optimization and detailed
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investigations. These isolates producing amylase were selected because they can be valuable
in food processing industry, in detergent industry, in desizing and for bioremediation in cold
regions.
Table 3.2. Screening of microorganisms for amylolytic activity at low
temperature (15ºC)
Culture No. Diameter of colony growth (a)
(mm)
Diameter of hydrolysis zone (b)
(mm)
Ratio
(b/a)
1 15 18 1.20
2 15 30 2.00
3 8.5 10 1.17
4 10.5 12.5 1.19
5 11.4 13 1.14
6 5.5 6.5 1.18
7 10.2 11.8 1.15
8 10 14 1.40
9 20 30 1.52
10 11 15 1.36
11 10 20 2.00
12 12 15 1.25
13 10.4 12.4 1.19
14 8 9.5 1.18
15 10.1 11.4 1.12
16 8.6 9.4 1.09
17 11 13 1.18
18 12.4 14.6 1.17
19 9 10.5 1.16
20 13 14.5 1.11
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21 11.5 12 1.04
22 6.5 7.2 1.10
23 7.5 7.9 1.05
24 6 7 1.16
25 10 10.8 1.08
26 8.5 9 1.05
27 12 13.5 1.12
28 12 15 1.25
29 10 13 1.30
30 10 10.5 1.05
31 6.8 7.4 1.08
32 7.1 7.5 1.05
33 9.2 10 1.08
34 10 10.6 1.06
35 15 24 1.60
36 10 18 1.80
37 10 20 2.0
38 11 12 1.09
39 8.5 9.5 1.11
40 5 5.8 1.16
41 5.8 6.5 1.12
42 8 9 1.12
43 12 13 1.08
44 5.8 6.5 1.12
45 7 8.2 1.17
46 7.2 8 1.11
47 7.4 8 1.08
48 11 14 1.27
49 10 15 1.50
# CHAPTER III # 76
50 5 5.8 1.16
51 10 10.6 1.06
52 9.5 10.5 1.10
* Values are mean of 3 replications after 3-4 days incubation at low temp. (15ºC)
Table 3.3. Diameter of hydrolysis zone on starch agar media of selected strains at
15±2ºC.
S. No. Culture No.
and Source
Renaming Diameter of hydrolysis zone (mm)
1 1 (Gaumukh) GA1 1.2
2 2 (Gaumukh) GA2 2.0
3 8 (Bhojwasa) GA3 1.4
4 9 (Bhojwasa) GA4 1.52
5 10 (Gaumukh) GA5 1.36
6 11 (Jaspur band) GA6 2.0
7 12 (Jaspur band) GA7 1.25
8 28 (Jaspur band) GA8 1.25
9 29 (Gaumukh) GA9 1.3
10 35 (Gaumukh) GA10 1.6
11 36 (Gaumukh) GA11 1.8
12 37 (Jaspur band) GA12 2.0
13 48 (Jaspur band) GA13 1.27
14 49 (Bhojwasa) GA14 1.5
# CHAPTER III # 77
Table 3.4. Production of amylase at 15ºC after 48 hour incubation
S. No. Culture No. and Source Amylase activity (Units), pH 7.0
1 GA2 (Gaumukh) 3572
2 GA4 (Bhojwasa) 2858
3 GA6 (Jaspur band) 5000
4 GA10 (Gaumukh) 2500
5 GA11 (Gaumukh) 2110
6 GA12 (Jaspur band) 1254
(a) (b)
Figure 3.1. Screening of microorganisms for amylolytic activity at low temperature
(15ºC) (a) screening of positive colony: GA2 (b) screening of positive
colony: GA6
# CHAPTER III # 78
3.3.3. Identification of potential isolates
3.3.3.1. Morphological and biochemical identification
The isolates were identified by studying morphological and biochemical
characteristics as per Bergey’s Manual of Determinative Bacteriology (Holt et al., 1989).
Detailed morphological, physiological and biochemical tests of the isolates are given in
Table 3.5.
Table 3.5. Morphological, physiological and biochemical characteristics of the isolates
Morphological Tests GA2 GA6
Colony morphology
Configuration
Shape
Irregular
Circular
Irregular
Circular
Margin Entire Undulate
Elevation Convex Flat
Surface Smooth, moist & glistening Dull & dry
Pigment Translucent yellow Creamish white
Opacity
Structure
Size (nm)
Opaque
Amorphous
2-4
Opaque
Amorphous
3-5
Gram’s reaction Positive Positive
Cell shape Rod Rod (mostly as diplobacillus)
Spore(s) Positive Positive
# CHAPTER III # 79
Motility Non motile Non motile
Fluorescence (UV) Negative Negative
Physiological tests GA2 GA6
Growth at temp.
4ºC + +
10ºC + +
20ºC ++ ++
28ºC +/- +/-
30ºC +/- +/-
37ºC +/- +/-
45ºC - -
Growth at pH
pH 4.0 - -
pH 5.0 +/- + /-
pH 6.0 +/- +/-
pH 7.0 +/- +/-
pH 9.0 ++ +/-
pH 10.0 +/- ++
pH 12.0 +/- +/-
Growth on NaCl (%)
2.0 ++ ++
4.0 + +
6.0 + +
8.0 +/- -
10.0 - -
Growth under anaerobic condition
Strictly aerobic Facultative aerobic
+ + = highest growth, + = good growth, +/- = average growth, - = no growth
# CHAPTER III # 80
Acid production from carbohydrates:
Tests GA2 GA6
Arabinose
Glucose
Glycogen
Ribose
Positive
Positive
Negative
Positive
Negative
Positive
Positive
Negative
Dextrose Positive Positive
Maltose Positive Positive
Biochemical Tests GA2 GA6
Indole test Positive Negative
Methyl red test Positive Negative
Citrate utilization Negative Negative
Starch hydrolysis Positive Positive
Urea hydrolysis Positive Negative
H2S production Negative Negative
Catalase test Positive Positive
# CHAPTER III # 81
3.3.3.2. Analysis of DNA Sequences
Homology of the partial 16S rRNA gene sequence of the strains with reference to
16S rRNA sequences was analyzed using the BLAST algorithm in GenBank
(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic and molecular evolutionary analysis
was conducted using MEGA version 4.1 (Tamura et al., 2007). Only the highest-scored
BLAST result was considered for phylotype identification. BLAST showed that the strain
GA2 which is of 1450 bp linear DNA have maximum homology (99%) with
Microbacterium foliorum with nucleotide base count; Adenine: 348, Cytosine: 345,
Guanine: 472 and Thymine: 285. While strain GA6 which is of 1472 bp linear DNA have
maximum homology (98%) with Bacillus cereus GU812900 with nucleotide base count
Adenine: 378, Cytosine: 330, Guanine: 458 and Thymine: 306.
Both the DNA sequences were aligned using ClustalW. The forward and reverse
sequences of GA2 and GA6 which we got after sequencing were aligned with the maximum
homology sequence of Microbacterium foliorum and Bacillus cereus, respectively. The
phylogenetic tree was constructed using software MEGA 4.1, this software basically used to
estimate the parameters of genetic variation. A phylogenetic tree was constructed based on
bacterial 16S rRNA sequences, showed a close relationship between the strain GA2 with the
genus Microbacterium and between the strain GA6 with the genus Bacillus. For the tree
construction four different out groups were used which were shown in the Fig.3.2 and
Fig.3.3 with their accession numbers.
# CHAPTER III # 82
> Strain GA2 16S ribosomal RNA gene partial sequence (1450 bp)
GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGTGAACACGGAG
CTTGCTCTGTGGGATCAGTGGCGAACGGGTGAGTAACACGTGAGCAACCTACCC
CTGACTCTGGGATAAGCGCTGGAAACGGCGTCTAATACTGGATACGAGTGGCG
ACCGCATGGTCAGCTACTGGAAAGATTTATTGGTTGGGGATGGGCTCGCGGCCT
ATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGTCGACGGGTAGCCGGCCT
GAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGA
GGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCAACGCCG
CGTGAGGGATGACGGCCTTCGGGTTGTAAATCTCTTTTAGCAGGGAAGAAGCGA
AAGTGACGGTACCTGCAGAAAAAGCGCCGGCTAACTACGTGCCAGCAGCCGCG
GTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTA
GGCGGTTTGTCGCGTCTGCTGTGAAATCCGGAGGCTCAACCTCCGGCCTGCAGT
GGGTACGGGCAGACTAGAGTGCGGTAGGGGAGATTGGAATTCCTGGTGTAGCG
GTGGAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGATCTCTGGG
CCGTAACTGACGCTGAGGAGCGAAAGGGTGGGGAGCAAACAGGCTTAGATACC
CTGGTAGTCCACCCCGTAAACGTTGGGAACTAGTTGTGGGGTCCATTCCACGGA
TTCCGTGACGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAG
GCTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGCGGA
TTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATATACGAGAACGG
GCCAGAAATGGTCAACTCTTTGGACACTCGTAAACAGGTGGTGCATGGTTGTCG
TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGT
TCTATGTTGCCAGCACGTAATGGTGGGAACTCATGGGATACTGCCGGGGTCAAC
TCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTT
CACGCATGCTACAATGGCCGGTACAAAGGGCTGCAATACCGCGAGGTGGAGCG
AATCCCAAAAAGCCGGTCCCAGTTCGGATTGAGGTCTGCAACTCGACCTCATGA
AGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCG
GGTCTTGTACACACCGCCCGTCAAGTCATGAAAGTCGGTAACACCTGAAGCCGG
TGGCCTAACCCTTGTGGAGGGAGCCGTCCGTTAGAGTGGGATTCGGGATTAGGC
CG
# CHAPTER III # 83
> Strain GA6 16S ribosomal RNA gene partial sequence (1472 bp)
GGGGAAGCGGGGGGGCGGTGCTAATTACATGCAAGTCGAGCGAATGGATTAAG
AGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTG
CCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTT
GAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGAC
CCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGT
AGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACT
CCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGA
GCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGG
AAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAA
GCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTT
ATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTG
AAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGC
AGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGA
GGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCG
AAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACG
ATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATT
AAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGAC
GGGGGCCCGCACAAGCGGTGGAGCATGTGGGTTAATTCGAAGCAACGCGAAGA
ACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCG
GGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTT
GGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTT
GGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGT
CAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTA
CAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTT
CGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGG
ATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACA
CCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCC
GCTAAGGTGGACAAGATGATTGGTA
# CHAPTER III # 84
Figure 3.2. Phylogenetic tree showing the homology of strain GA2 with
Microbacterium foliorum.
Figure 3.3. Phylogenetic tree showing the homology of strain GA6 with Bacillus cereus.
# CHAPTER III # 85
GA2 TCGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGAC 300 gi|189483941|gb|EU714333.1| TCGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGAC 299
**************************************************
GA2 ACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGG 350 gi|189483941|gb|EU714333.1| ACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGG 349
**************************************************
GA2 CGCAAGCCTGATGCAGCAACGCCGCGTGAGGGATGACGGCCTTCGGGTTG 400 gi|189483941|gb|EU714333.1| CGCAAGCCTGATGCAGCAACGCCGCGTGAGGGATGACGGCCTTCGGGTTG 399
**************************************************
GA2 TAAATCTCTTTTAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAAA 450 gi|189483941|gb|EU714333.1| TAAACCTCTTTTAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAAA 449 ************************************************* GA2 AGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAG 500 gi|189483941|gb|EU714333.1| AGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAG 499
**************************************************
GA2 CGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGT 550 gi|189483941|gb|EU714333.1| CGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGT 549
**************************************************
GA2 CTGCTGTGAAATCCGGAGGCTCAACCTCCGGCCTGCAGTGGGTACGGGCA 600 gi|189483941|gb|EU714333.1| CTGCTGTGAAATCCGGAGGCTCAACCTCCGGCCTGCAGTGGGTACGGGCA 599
Figure 3.4. GA2 sequence with variation at position 405 where T replaces C when
comparing with Microbacterium foliorum strain 150
# CHAPTER III # 86
GA6 AGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAAC 800 gi|148613823|gb|EF584539.1| AGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAAC 798
**************************************************
GA6 GATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAAC 850 gi|148613823|gb|EF584539.1| GATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAAC 848
**************************************************
GA6 GCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAG 900 gi|148613823|gb|EF584539.1| GCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAG 898
**************************************************
GA6 GAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGGTTAATTCGAAG 950 gi|148613823|gb|EF584539.1| GAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAG 948
*************************************************
GA6 CAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGA 1000 gi|148613823|gb|EF584539.1| CAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGA 998
**************************************************
GA6 TAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTC 1050 gi|148613823|gb|EF584539.1|TAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTC 1048
**************************************************
GA6 AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTT 1100 gi|148613823|gb|EF584539.1|AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTT 1098
**************************************************
Figure 3.5. GA6 sequence with variation at position 939 where G replaces T when
comparing with Bacillus sp. JDM-2-1
# CHAPTER III # 87
16S rRNA gene sequence of strain GA2 showed 1% variation at nucleotide
position 405 where cytosine is replaced by thymine, when compared with highly closed
organism i.e. Microbactrium foliorum strain 150 (Figure 3.4). While 16S rRNA gene
sequence of strain GA6 showed 2% variation by single change at nucleotide position 939
where thymine is replaced by guanine, when compared with highly closed organism i.e.
Bacillus sp. strain JDM-2-1 (Figure 3.5).
After alignment the final sequences of GA2 and GA6 were submitted to Genbank at
National Centre for Biotechnology Information (NCBI) through Bankit. The Genbank
accession numbers of the partial sequence 16S ribosomal RNA gene for GA2 and GA6 are
HQ832574 and HQ832575 respectively (Table 3.6).
Table 3.6. Identification of bacterial strains
S. No. Strain designation Identity Accession number
1 GA2 Microbacterium foliorum GA2 HQ832574
2 GA6 Bacillus cereus GA6 HQ832575
3.3.4. Characterization of identified isolates
3.3.4.1. Antibiotic susceptibility
The results for antibiotic susceptibility are given in Table 3.7. When cultures were
subjected to the various antibiotics, it was found that Microbacterium foliorum GA2 is only
resistant to Penicillin (10 mcg) while it was sensitive to Tetracycline, Streptomycin,
Chloramphenicol, Ampicillin and Doxycycline.
# CHAPTER III # 88
Table 3.7. Antibiotic susceptibility pattern in amylolytic strains
Antibiotics Disc potency (mcg)
Resistant (R)/Sensitive (S)
M.foliorum GA2 B.cereus GA6
Penicillin 10 R R
Tetracycline 30 S S
Streptomycin 10 S S
Chloramphenicol 30 S S
Ampicillin 10 S R
Doxycycline 30 S S
But Bacillus cereus GA6 was resistant to both Penicillin (10 mcg) and Ampicillin (10 mcg).
However, with Tetracycline, Streptomycin, Chloramphenicol, and Doxycycline, it shows
strong sensitivity. Thus, it may be possible that genes resistant to above mentioned
antibiotics were present in respective strains.
3.3.4.2 . Plasmid curing
Curing of plasmid was done using curing agent ethidium bromide (10-100 g/ml),
which gets intercalated between the bases of DNA and inhibits replication of plasmid
without inhibiting chromosomal DNA replication. Such inhibition can lead to loss of
plasmid.
Out of 60 colonies tested on starch agar media after curing, Microbacterium
foliorum GA2 showed 100% curing, indicated that amylase production by M. foliorum GA2
# CHAPTER III # 89
is predominantly plasmid mediated characteristics while 70% colonies of Bacillus cereus
GA6 showed clear hydrolysis zone, indicated that the amylase production by B. cereus GA6
was not an absolute plasmid mediated characteristic (Table 3.8).
Table 3.8. Curing of amylolytic trait
Strain M.foliorum GA2 B.cereus GA6
Number of colony tested 60 60
Colony cured * 0 42
% Curing 100 70
*amylase negative (clear zone was not observed around the colony on starch agar media)
# CHAPTER III # 90
3.3.5. Conclusion:
The results concluded that M. foliorum GA2 (HQ832574) and B. cereus GA6
(HQ832575) were novel psychrotrophic bacterial strain producing excellent amount of cold-
active α-amylase. One more attractive feature of Microbacterium foliorum strain GA2 was
its 100% plasmid curing behavior in presence of strong curing agent, which reflects that
genes responsible for primary metabolites production such as amylase are plasmid borne.
Sequence determination of such regions of DNA could be useful in elucidating the type of
molecular interaction between ‘curing agents’ and DNA. Although low biodiversity of
psychrophilic/psychrotrophic microbes are explored so far, cold-active α-amylases from M.
foliorum GA2 and B. cereus GA6 of Gangotri glacier region may serve as promising
enzymes to replace the conventional synthetic processes in biotechnological industries.