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# CHAPTER III # 59 CHAPTER III Isolation and Identification of cold-active α-amylase producing microorganisms

Transcript of CHAPTER III - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/13985/8/08...# CHAPTER III # 62...

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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

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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

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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

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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

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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

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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

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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.

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> 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

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> 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

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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.

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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

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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

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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.

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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

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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)

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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.