Isolation and Characterization of Exopolysaccharide Producing Bacteria from Pak Bay (Mandapam)
Bacterial exopolysaccharides possess a wide variety of properties that may not be found in more
traditional polymers of plant or algal or animal origin. In the present study, the exopolysaccharide
producing bacteria was isolated and characterized from coastal (Mandapam) area of Pak bay,
Tamilnadu, India. Soil samples were collected and the exopolysaccharide producing strains were
screened and characterized by 16s rDNA sequencing method. The effect of different carbon sources
on exopolysaccharide production was examined. Then the exopolysaccharide was synthesized and
characterized by calorimetric, IR and HPLC method. Calorimetric analysis of exopolysaccharide
reveals the composition of exopolysaccharide and in the IR analysis the band at 1385 to 1380 cm-1
indicates the presence of C-H stretching. An absorbance at 1730 cm-1 indicating the presence of
carboxyl group. In addition, a small absorption at 1550 cm-1 indicating the presence of either
amino sugars or proteins were present in exopolysaccharide. Exiguobacterium sp. was isolated and
it produces considerable amount of exopolysaccharide when the medium was supplied with sucrose.
Received: December 04, 2012;
Accepted: March 11, 2013;
Published: April 16, 2014
Bacterial exopolysaccharides are ubiquitous in marine resources and are distributed
in the form of free living or associated forms, such as biofilm, microbial mats
etc. Microbial exopolymeric substances are produced by both prokaryotes and
eukaryotes. The sea has a plentiful source of biological and chemical diversity.
The ocean appropriately contains nearly 3 million described species, but nearly
about 99% of bacteria cannot be cultured (Lee et al.,
2007) and few mariner microbes are currently cultivated.
The fluctuation in the pressure, nutrients, salinity and pH in the marine leads
to the production of exopolysaccharide by the bacteria (Decho,
1990). Exopolysaccharide have a wide range of applications in ecological,
physiological and industrial fields. Exopolysaccharide protects the bacterial
cell from harsh environment such as desiccation (Passow,
2000), involve in the biofilm formation (Rodriguez-Valera
et al., 1981) and bioremediation activity (Allison,
1998). Therefore, the present study was conducted to isolate and characterize
the exopolysaccharide produced by marine bacteria.
MATERIALS AND METHODS
Isolation of bacteria: Soil samples were collected from (Mandapam area)
Pak bay. The soil samples were serially diluted and a known aliquot was plated
on to Zobell agar media incubated at 30°C for 7 days. Some strains exhibited
mucoid surface on the growth media. It indicates the production of exopolysaccharide
by bacteria (Ng and Hu, 1989). The strains were routinely
subcultured and maintained in Zobell agar slants as stock culture for further
Characterization of bacterial isolate: The 16S rDNA was amplified by
polymerase chain reaction (PCR) using the primers F-5' AGA GTT TGA TCC TGG CTC
AG 3' and R-5' GGT TAC CTT GTT ACG ACT T 3'. The cycle sequencing reaction was
performed using BigDye terminator V3.1 cycle sequencing Kit containing AmpliTac
DNA polymerase (from Applied Biosystems, P/N: 4337457). The sequencing reaction
- mix was prepared by adding 1 μL of BigDye v3.1, 2 μL of 5x sequencing
buffer and 1 μL of 50% DMSO. To 4 μL of Sequencing reaction-mix was
added 4 Pico moles of primer (2 μL) and sufficient amount of plasmid. The
constituted reaction was denatured at 95°C for 5 min. Cycling began with
denaturing at 95°C for 30 sec, annealing at 52°C for 30 sec and extension
for 4 min at 60°C and cycle repeated for a total 30 cycles in a MWG thermocycler.
The reaction was then purified on sephadex plate (Edge Biosystems) by centrifugation
to remove unbound labelled and unlabelled nucleotides and salts. The purified
reaction was loaded on to the 96 capillary tubes on ABI 3700 DNA analyzer and
electrophoresis was carried out for 4 h. DNA sequence was obtained using DNA
sequencer (ABI 310). The PCR product was sequenced using the same products were
primers and other internal primers to confirm the sequence. Blast program (www.ncbi.nlm.nih.gov/blast)
was used to assess the DNA similarities (Al-Nahas et
PHYLIP version (3.57) was used to assess the sequence data. Phylogenetic tree
was constructed by the neighbor joining method.
Isolation and purification of exopolysaccharide: For the isolation exopolysaccharide
producing strain was grown for 7 days at 32°C in a Zobell marine broth (Raguenes
et al., 2003). The culture was centrifuged at 10000 rpm for 15 minutes
and the supernatant was precipitated with 3 volumes of ice cold ethanol and
stored at 4°C overnight, before being centrifuged. The exopolysaccharide
was resuspended in distilled water and centrifuged. To remove excess salt from
exopolysaccharide the pellet was dissolved in distilled water and dialyzed (mol
wt cutoff 8000 dalton) against distilled water for 2 days. It was concentrated
and stored at room temperature until analysis.
Characterization of exopolysaccharide: The Total Protein content in
the sample was determined (Lowry et al., 1951)
with bovine serum albumin as a standard. The neutral carbohydrate content was
determined by the orcinol sulphuric acid (Titus et
al., 1995) and meta-hydroxydiphenyl method (Filisetti-Cozzi
and Carpita, 1991) was used to detect the uronic acid level in exopolysaccharide.
Effect of carbon source on growth and production of exopolysaccharide:
In order to determine the effect of different carbon sources (sucrose, glucose,
galactose and lactose) on the growth and exopolysaccharide production, the exopolysaccharide
producing strain was cultivated in Zobell marine broth for 24 h. From the 24
h culture 5 mL of culture aliquots was inoculated in 100 mL of basal medium
(peptone 5 g, yeast extract 3 g, malt extract 3 g, distilled water 500 mL and
sea water 500 mL pH 7.0) supplemented with different carbon sources (sucrose,
glucose. galactose, lactose) in different flasks. Then the growth was determined
by measuring the O.D value at 520 nm at regular time intervals and the exopolysaccharide
was extracted as mentioned above and the dry weight was measured (Lijour
et al., 1994).
FT-IR spectroscopy: Pellets for infra red analysis were obtained by
grinding a mixture of 2 g of exopolysaccharide with 200 g of potassium bromide.
FT-IR spectra was recorded with a resolution of 4 cm-1 in the 4000-400
cm-1 region (Omoike and Chorover, 2004).
HPLC Analysis of Basal exopolysaccharide: The exopolysaccharide (0.1
g) was hydrolyzed by treating with 1.25 mL of 72% sulphuric acid and was incubated
for 60 min at 30°C. Then 13.5 mL of distilled water was added and placed
it in a water bath for 4 h. After 4 h the mixture was cooled and 3.1 mL of 32%
sodium hydroxide was added. Then the hydrolyzed sample was dissolved in methanol.
The acid hydrolyzed exopolysaccharide sample was analyzed with a High Performance
Liquid Chromatography (HPLC) system (SHIMADZU LC 10 AT VP) equipped with Aqueous
GPC start up Kit column and eluted with distilled water at a flow rate of 1.0
mL min-1 at 20°C. (Vijayabaskar et al.,
Exopolysaccharide producing bacterial strain (S9) was isolated from marine
soil sample. After 7 days of incubation at 32°C on zobell agar medium a
circular convex colony with a mucoid tenure and orange colored colony was observed.
The most promising strain S9 was characterized as G+ve, rod shaped
motile bacteria. The 16S rDNA sequence analysis showed high percentage of similarity
to the genus Exiguobacterium sp. (Genbank Accession Number JF830805).
Figure 1 shows the phylogenetic relationship of Exiguobacterium
sp. based on 16S rDNA sequences. Phylogenetic analyses of the strain S9 showed
it was belonged to the Phylum Firmicutes and Bacillales Family XII and it was
closely related to the genus Exiguobacterium.
||Phylogenetic analysis of Exiguobacterium sp., s9. Phylogenetic
relationship among Exiguobacterium sp., S9 and selected marine bacteria.
The percentage number at the nodes indicates the level of bootstrap support
for the branch point in topology
The chemical composition of bacterial exopolysaccharide is presented in Fig.
2. The protein content is very low level (4%) and the amount of neutral
sugar is about 78% and contains uronic acids. The bacterial exopolysaccharide
contains protein and small amount of uronic acids which cannot be removed by
When compared with other sugars the highest yield of exopolysaccharide and
tremendous growth was observed, when sucrose was supplied as a whole source
of carbon (Fig. 3, 4).The exopolysaccharide
production was significantly influenced by the type of sugars as a carbon source.
The amount of exopolysaccharide production was not only determined by the Carbon:
Nitrogen ratio and concentration of sugars. It also influenced by the type of
sugars used as carbon source.
FT-IR spectra of the Exiguobacterium sp. (Fig. 5)
displayed a broad O-H stretching band above 3000 cm-1 and intense
absorptions between 1650 and 1050 cm-1 characteristic of polysaccharides.
The band at 835 to 805 cm-1 indicates the presence of substitution.
The band at 1385 to 1380 cm-1 indicates the presence of C-H vibration.
An absorbance at 1730 cm-1 indicated the presence of carboxyl groups
(Lijour et al., 1994). In addition, a small
absorption at 1550 cm-1 indicating the presence of either amino sugars
or proteins were present in exopolysaccharide.
The exopolysaccharide after being hydrolyzed and dissolved with methanol was
analyzed for its sugar composition by HPLC.
||Chemical composition of bacterial exopolysaccharide. Chemical
composition of bacterial exopolysaccharide, contains 78% sugars 18% of proteins
and 4% of uranic acids
||Effect of different sugars on the growth of Exiguobacterium
sp., S9. The sugar sucrose influences the better growth and yield of exopolysaccharide
||Dry weight of exopolysaccharide on using different carbon
source. The isolate S9 produced maximum amount of exopolysaccharide when
the media is supplied with sucrose on compared with other sugars
||FT-IR spectrum of exopolysaccharide. In the IR spectrum of
exopolysaccharide the band at above 3000 cm-1 and intense absorption
between 1650 and 1050 cm-1 are characteristic of polysaccharides
HPLC results bacterial exopolysaccharide.
The HPLC spectrum for exopolysaccharide shows the retention time obtained
was found to be 1.927 as myo-inositol, 2.073 as glucose, 2.410 as galactose
and 2.817 as fructose
By comparing the retention time found on with the standard retention time of
carbohydrates, the distinct peaks obtained were found to be 1.927 as myo-inositol,
2.073 as glucose, 2.410 as galactose and 2.817 as fructose (Fig.
According to Cambon-Bonavita et al. (2002) and
Junkins and Doyle (1992) the exopolysaccharide producing
strains are generally developed mucoid colonies. During the screening process
the presence of pigmentation in all strains was noticed. It provides resistant
to the bacteria (Rimington, 1931; Brown
and Lester, 1982).Bacterial strains isolated from marine soil produce extra
cellular polymer with an enhanced mucoid morphology. Results from 16s rDNA sequencing
indicates the strain was closely related and belongs to the genus Exiguobacterium
The concentration and the type of carbon source determined the exopolysaccharide
production by the bacteria. The production may be influenced by the metabolic
process of the bacteria to utilize different carbon sources.
Exopolysaccharide produced by Exiguobacterium sp. was analyzed calorimetrically
and FT-IR spectroscopy. Based on the IR spectrum of absorbance have been assigned
to different functional groups such as ether, sulphate, carboxylic etc. It confirms
the polysaccharides and low amount of ester sulphate group. Similar results
were observed previous studies of exopolysaccharide from Alteromonas
and Pseudoalteromonas by Passow (2000), Brown
and Lester (1982) and Pal et al. (1999).
The exopolysaccharide produced at different carbon sources are primarily composed
of carbohydrates. Some other organic compounds such as uronic acids, sulfates,
proteins may be found in bacterial exopolysaccharide (Quesada
et al., 2004; Sutherland, 2001). Production
of exopolysaccharide by a bacterial cell plays an important role in the aggregation
(Bejar et al., 1996; Chan
et al., 1984). When released into the water, a combination of biological,
chemical and physical forces causes this colloidal material to form aggregates
(Alldredge and Jackson.,1995; Passow,
2000; Kiorboe, 2001) and protect the cells from
environment stress such as osmotic pressure, pH variation etc. The stickiness
is an important in terms of the affinity of these exopolysaccharide for binding
to other cations such as dissolved metals (Biddanda, 1986).
Increased knowledge of the role of bacterial exopolysaccharide will also provide
insight into possible commercial uses for these novel polymers.
The FTIR spectrum of exopolysaccharide revealed characteristic functional groups,
such as stretching C-H at 3313.48, 2975.96 and 2935.46 cm-1 and a
weak COOH stretching peak at 3195.48 cm-1 .Further, stretching peak
was noticed at 1284.50 cm-1 which corresponds to amide. A broad stretching
of C-O-C, C-O at 1000-1200 cm-1 corresponds to the presence of carbohydrates
specifically, the peaks at 1114.78 cm-1 range ascertain the presence
of uronic acid,o-acetyl ester linkage bonds (Fig. 6). A comparison
of functional groups presents that exopolysaccharide having a higher number
of variable functional groups was more complex than the other exopolysaccharide
reported previously (Rougeaux et al., 1996).
The exopolysaccharide are widely distributed in marine an environment. It helps
the bacterial communities to survive in extreme environment and act as an anchor
between the bacterial cell and its immediate environment. Several exopolysaccharide
produced by bacteria in extreme marine habitats have a potential role.
The study is a first step towards understanding the sole of these exopolysaccharide
in the area of Pak bay. Complete study on the chemistry and the structure of
bacterial exopolysaccharide will provide the positive approach to employ this
novel biopolymer in several industries like pharmaceutical, food-processing
fields and environment protection.
Considering that the most of the marine bacteria and their metabolites were
unexplored, it is reasonable to state that the isolation and identification
of new microorganisms will provide wide opportunities in forth coming years.
We thank to management committee of Hajee Karutha Rowther Howdia College and
Department of Animal science, Bharathidasan University and Yaazh xenomics, Chennai
for providing the facilities to carry out this work.
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