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Bioscience Discovery, 8(4): 643-649, October - 2017

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Research Article

Isolation, Screening and Characterization of Marine Bacteria for

Exopolysaccharide Production

Rahul Chaudhari, Murtaza Hajoori*, Manish Suthar and Sagar Desai

Bhagwan Mahavir College of Science & Technology,

Bharthana-Vesu, Surat.

*Email: murtazahajoori@yahoo.com.

Article Info

Abstract

Received: 10-06-2017,

Revised: 05-08-2017,

Accepted: 19-08-2017

Exopolysaccharides are polymers of carbohydrates secreted by some bacteria

and fungi outside their cell walls. Marine microorganisms have significant

osmotic tolerance and leading to produce exopolysaccharide even at higher salt

concentration. The exopolysaccharide producing bacteria were isolated from

marine environment. The marine soil samples and marine water samples were

collected from sea coast of Dandi, Navsari and Tithal, Valsad. Total 13

morphological distinct colonies were obtained and 05 isolate were selected

having mucoid colony characterization. All the 05 isolate were screened using

string test and congo red agar test. Isolate VS2B was screened and evaluated to

be more potent for exopolysaccharide production of 5.60 mg/ml in Zobell

marine broth on 4th day of incubation. The molecular identification of VS2B

isolate was carried out by 16S rRNA sequencing and identified as Bacillus

subtilis. Thus, this study demonstrates VS2B isolate may prove to be potential

strain for commercial production of exopolysaccharide.

Keywords:

Exopolysaccharide, Zobell

marine broth, String test,

Congo red agar test, Bacillus

subtilis.

INTRODUCTION

The marine environment covers almost 70%

of surface of earth surface and they contain vast

biological diversity which accounts more than 90%

of whole biosphere and offer a great source of novel

compounds (Aneiros, 2004). Bacteria from marine

environment grow in extreme environmental

condition such as high pressure, low temperature,

high salinity and depletion of micronutrients.

Despite of such extreme conditions, such

microorganism grows and produces bioactive

compound (De Carvalho and Fernandis, 2010).

Halophilic organisms can be broadly classified on

the basis of their optimum salinity for growth into

mild halophiles (1-6%, w/v NaCl), moderate

halophiles (7-15%) and extreme halophiles (15-

30%) (Madigan and Martinko, 2006).

Exopolysaccharide are polymers of mainly

carbohydrates excreted outside their cell by some of

the microorganisms as a part of their routine

metabolism. It may exhibits as capsule or as slime

layer (Sutherland, 1972). Exopolysaccharide may be

formed of either homopolysaccharide or

heteropolysaccharide along with the number of

different organic and inorganic substituent’s

(Byrom, 1987). Marine microorganisms have

significant osmotic tolerance leading to their

capability of polysaccharide production at higher salt

concentration, which is very much desired for

development of an economically feasible process for

polysaccharide production (Poli et al., 2010).

The exopolysaccharide secreted by the

bacteria impart protective role in the natural

environment against desiccation, phagocytosis,

predation, antibiotics, toxic metal ions and osmotic

stress (Looijesteijn, 2001). Microbial

exopolysaccharides seem to be a promising

alternative as they can effectively act as strong

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Rahul Chaudhari et al.,

reducing as well as stabilizing agent for metal

nanoparticle production (Huang and Yang, 2004).

Silver nanoparticle has been synthesized by several

microbial polysaccharides such as gellan gum and

pullulan, which exhibited a strong antimicrobial

activity (Dhar, 2012). Few reports were available on

production of silver nanoparticles (AgNPs) using

polysaccharides such as heparin, hyaluronic acid,

cellulose, starch, alginic acid etc. (Kemp et al., 2009;

Raveendran et al., 2003).

MATERIALS AND METHODS

Sample Collection:

About 10g of marine soil samples and 5 litres of

marine water samples were collected from sea coast

of Dandi, Navsari and Tithal, Valsad in sterile

containers. All samples were stored at 4ºC until

further use.

Enrichment & Isolation of marine bacteria:

1g of soil sample was inoculated in a flask containing

Zobell marine broth medium supplemented with 5%

NaCl. 1ml of marine water sample was inoculated in

a flask containing Zobell marine broth medium

supplemented with 5% NaCl. The inoculated flasks

were incubated on Rotary Shaker at 100 rpm for 3

days at 37ºC temperature. The enriched culture was

serially diluted to prepare aliquots ranging from 10-1

to 10-6 using normal saline as diluent. 100 µl from

each aliquot were spreaded onto Zobell marine agar

plate supplement with 5% NaCl. The plates were

kept at 37 ºC for 24 hours. Morphologically distinct

colonies were picked and subcultured to obtain pure

culture of isolates. Each isolates were streaked onto

ZMB slant and kept at 4ºC until further utilization.

Screening for exopolysaccharide production by

Isolates:

Exopolysaccharide producing bacteria were

screened on the basis of colonial characteristic,

string test and congo red agar test.

a. Colonial characteristic and string test:

Mucoid colonies have a glistening and slimy

appearance on agar plates were the primary criteria

for selection of bacteria (Fusconi and Godinho,

2002). The colonies when extended with wire loop

form a long filament (Vescovo et al., 1989).

Exopolysaccharide production was confirmed

visually or through the string test (Fang et al., 2004)

by formation of a string (>5 mm) upon lifting of the

loop indicates positive result.

b. Congo red agar plate method:

The isolates were streaked onto Zobell marine agar

medium supplement with 0.08% Congo red. The

positive result determined by black, smooth, humid

with mucoid type colony (Freeman et al., 1989).

Recovery and evaluation of exopolysaccharide

production by isolates:

The screened isolates were further evaluated

for exopolysaccharide production. The inoculum

was prepared by transferring bacterial colony into

250 ml conical flask containing 50 ml of Zobell

marine broth supplement with 5% NaCl. Inoculated

flasks were incubated on shaker at 100 rpm for 96

hours at 37ºC temperature. The Zobell marine broth

was made cell free by centrifugation at 10,000 rpm

for 20 minutes. Cold absolute ethanol was added to

supernatant in ratio of 1∶3 (v/v) and kept at 4°C for

24 hours (Kim and Yim, 2007) for precipitation of

exopolysaccharide. The precipitated were recovered

by centrifugation and purified by washing with Milli

Q water. Finally, the exopolysaccharide was again

precipitated by 1:3 volume of cold absolute alcohol

and exopolysaccharide pellet were dried at 60 0C.

The precipitate was dissolve in 2 ml milli Q water for

quantification of exopolysaccharide (Rodrigues and

Bhosle, 1991) determined in terms of total

carbohydrate analysis by Phenol sulphuric acid

method (Dubois et al., 1956).

Morphological, Colonial and Biochemical

characterization of bacterial Isolate:

The isolate was identified according to Gram

staining, colonial characteristics and biochemical

characterization (Rakesh and Kiran Patel, 2004).

Molecular Identification of exopolysaccharide

producing Isolate:

a. Extraction of bacterial genomic DNA

Bacteria was grown in Zobell marine broth for 24 hrs

at 37 0C. Activated culture was collected in 2 ml

microfuge tube and centrifuge at 10,000 rpm for 10

minute at 4 0C. Further DNA extraction,

precipitation and purification were carried out as per

manufacture protocol of HiPer Bacterial Genomic

DNA Extraction Kit (Himedia Laboratory Pvt. Ltd.

Mumbai). DNA from isolate was electrophoreses in

0.8 % Agarose gel and visualized on a Gel

Documentation System (Genie, Bangalore).

b. 16S rRNA sequencing for identification of

Isolate:

The selected isolate was send to Gujarat State

Biotechnology Mission (GSBTM), Gandhinagar,

Gujarat, India, for 16S rRNA sequencing. Similarity

search of 16S rRNA sequence of isolate VS2B was

carried out by nucleotide BLAST 2.2.31 (blastn)

software available at NCBI (Johnson et al., 2008).

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Bioscience Discovery, 8(4): 643-649, October - 2017

The identification of isolate was made by evaluating

similarity index, total score, query coverage and E-

value (Relman, 1993). The 16S rRNA sequence

obtained was submitted to National Centre for

Biotechnology Information through Sequence

Submission Wizard available at NCBI

(https://submit.ncbi.nlm.nih.gov/subs/genbank).

Phylogenetic tree using neighbor joining algorithm

was constructed to determine taxonomic position of

the isolate (Sievers et al., 2011).

RESULTS AND DISCUSSIONS:

Enrichment and Isolation of Bacteria:

Total 13 different bacterial colonies were isolated

from marine soil and water samples on the basis of

unique colonial characteristics on Zobell marine agar

plate. Out of 13 different isolates, 05 isolates were

selected on the basis of mucoid colony

characteristics. All the 05 selected isolates were

screened for exopolysaccharide production by string

test and congo red agar plate, the isolate VS2B shows

both the test positive. The results of screening

represented as Table 1.

Table 1: Results of screening for exopolysaccharide production

Bacterial Isolates Colony Characteristics String Test

(mm)

Congo Red Agar Plate

DW1S1 Mucoid <5 mm -

DS1C1 Mucoid <5 mm -

VW1B Mucoid <5 mm -

VS1C1 Mucoid <5 mm -

VS2B Mucoid >5 mm +

All the 05 isolate shows mucoid colonial

characteristics. The mucoid colonies characteristics

serve as the selection criteria for exopolysachharide

producing bacteria (Fusconi and Godinho, 2002).

Among these 05 isolates, VS2B show ropy type

characteristics by lifting a colony by wire loop, it

forms a string of length more than >5 mm. The

results are in accordance with the method adoptd by

past researchers for selection of exopolysaccharide

producing bacteria (Vescovo et al, 1989). Isolate

VS2B shows mucoid, humid type, smooth, black

colour growth on congo red gar plate indicative of

positive test for exopolysaccharide production. The

results are in accordance with past author (Freeman

et al., 1989). The string test is shown as figure 1(a)

and congo red agar plate as figure 1(b).

Evaluation of exopolysaccharide production by

isolates: All the 05 isolates viz., DW1S1, DS1C1,

VW1B, VS1C1 and VS2B were utilized to evaluate

exoploysaccharide production in Zobell marine

broth. The production of exopolysaccharide was

found to be elevated on 3rd day i.e., 4.65 mg/ml and

increase upto 5.60 mg/ml on 4th day of incubation by

VS2B. The exopolysaccharide production by

VS1C1, DS1C1, DW1B was 0.23 mg/ml, 0.20

mg/ml and 0.10 mg/ml respectively on 4th days of

incubation. However, VW1B shows

exopolysaccharide production of 0.13 at 3rd day of

incubation. The quantification was carried out as

described by various authors (Kim and Yim, 2007;

Rodrigues and Bhosle, 1991; Dubois et al., 1956).

Morphological, Colonial and Biochemical

characterization of Bacterial isolate VS2B.

Morphological, colonial and biochemical

characterization helps in partial identification of

microorganism. Morphological and colonial

characteristics of VS2B isolate were shown as Table

2. Biochemical characteristics of VS2B isolate were

shown as Table 3. The colony of VS2B isolate on

nutrient agar shown as Figure 2.

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Rahul Chaudhari et al.,

Figure 1 Screening of VS2B isolate (a) String Test, (b) Modified congo red Agar Plate Test

Table 2 Characterization of VS2B Isolate

Isolate VS2B

Morphological characteristics

Gram reaction. Gram Positive

Size, Shape and Arrangement Long rods shape bacteria occurring in chain of two.

Colonial characteristics

Shape Round

Colony Size Intermediate

Margin Entire

Pigmentation No Pigmentation

Elevation Flat

Surface Mucoid

Consistency Moist

Opacity Opaque

a b

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Bioscience Discovery, 8(4): 643-649, October - 2017

Table 3. Biochemical characterization of VS2B isolate

Name of Test Result Name of Test Result Name of Test Result

Indole production test - Glucose utilization + (A, G) Gelatinase +

Methyl red test - Fructose utilization + (A, G) Lipase -

Voges-Proskauer test - Maltose utilization + (A, G) Amylase +

Citrate utilization test - Sucrose utilization + (A, G) Protease +

H2S production test - Xylose utilization + (A, G) Cellulase +

Nitrate reduction test - Mannitol utilization - Phenylalanine

deaminase

-

Urea hydrolysis test - Lactose utilization + (A, G) Catalase test +

Decarboxylase test + Dehydrogenase test +

Keys:(-) =Negative result; (+)=Positive result, A=Acid production, G=Gas production

Figure 2. Isolate VS2B colony on Nutrient agar plate

Molecular Identification of VS2B isolate by 16S

rRNA sequencing

DNA isolation were performed and validated by

agarose gel electrophoresis shown as Figure 3(a).

The 16S rRNA sequencing was conducted using

forward primer (8F: 5’-

AGAGTTTGATCCTGGCTCAG-3’) and reverse

primer (1492R: 5’-GGTTACCTTGTTACGACTT-

3’). The 16S rRNA sequence of VS2B shows 100%

identical with Bacillus subtilis strain I3_19 16S

rRNA gene, partial sequence having accession no.

KT873853.1. The total and maximum score obtained

of alignment was 1378 with 100% query coverage

and 0.0 E-value. Thus, isolate VS2B was identified

as Bacillus subtilis. The earlier researcher also

demonstrated that Bacillus subtilis had ability to

produce exopolysaccharides of sorptive nature that

play role in sorption of charged molecule. It is been

demonstrated that Bacillus subtilis produces poly--

glutamate as polysachharide. (Marvasi et al., 2010).

Phylogenetic tree of the strain of Bacillus subtilis

with closest similarity using neighbor joining

method was analyzed by clustal omega (Sievers et

al., 2011) shown as Figure 3(b). The 16S rRNA

sequence was published at NCBI with accession

number KY941130.1 shown as Figure 4.

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Rahul Chaudhari et al.,

Figure 3 (a) Agarose gel electrophoresis of Genomic DNA of isolates V2SB, (b) Bacillus subtilis

phylogenetic view using NJ method

Figure 4 16S rRNA sequence of Bacillus subtilis submitted to NCBI.

ACKNOWLEDGEMENT

The authors would like to thanks the Bhagwan

Mahavir College of Science and Technology, Surat

for providing laboratory facility to conduct research

work.

a b

VS2B Control VS2B

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Bioscience Discovery, 8(4): 643-649, October - 2017

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How to cite this article

Rahul Chaudhari, Murtaza Hajoori, Manish Suthar and Sagar Desai, 2017. Isolation, Screening and Characterization of Marine Bacteria for Exopolysaccharide Production. Bioscience Discovery, 8(4): 643-649.