Optimization and Characterization of Exopolysaccharide from Marine Soil Bacteria

 

P. Maheswari¹, K. Arjun Kumar¹, S. Sankaralingam², N. Sivakumar³

¹Department of Microbiology, Ayya Nadar Janaki Ammal College, Sivakasi, Tamil Nadu, India.

²Department of Botany, Saraswathi Narayanan College, Madurai, Tamil Nadu, India.

³School of Biotechnology, Department of Molecular Microbiology, Madurai Kamaraj University,

Madurai, Tamil Nadu, India.

 

ABSTRACT:

 

 

INTRODUCTION:

The environment had a fair interaction with the biotic community that was subjected to bring more beneficial effects for the human welfare. Thus, it had been a great concept of interaction of the microorganisms with the environment and humans, gives the fine effect to environment, human health by producing various compounds and metabolites (Maeda et al., 2000). Some of the bacterial species produces the metabolites intracellular and extracellular to protect from the unfavourable environment (Nadia et al., 2016). These are said to be the bacterial polysaccharides which have various applications in many fields. The extracellular secretion of the polysaccharides by the bacterial cells are said to be the exopolysaccharides (Zhemning et al., 2005).

 

Exopolysaccharides are the long chained compounds composed of sugar residues and secreted by microorganisms into the surrounding environment (Liu et al., 2011). Diverse structural, functional and valuable polysaccharides are synthesized by bacteria of all taxa and secreted into the external environment (Dogsa et al., 2005). They may either be homopolymeric or heteropolymeric in composition and of diverse high molecular weights. Bacterial EPS as a complex mixture of macro molecular poly electrolytes including polysaccharides, proteins and nucleic acids, each comprising variable molecular mass and structural properties (Uchechukwuet al., 2012).

 

The notable functions of exopolysaccharides as they act as nutrition source like carbon and nitrogen to the biofilm forming community (Madhuriket al., 2014). Protective barriers as act as the anti-microbial agents against various microorganisms (Burgulaet al., 2007). Immunomodulation are also noted. Degradation of oils and acts as surfactants.Tumour reduction and noted in the studies of anti-tumour activities in human carcinoma (Dattaet al., 2016).

 

MATERIALS AND METHODS:

Sample description and collection (Ahmed et al., 2012):

The marine sediment soil region was considered for the isolation of the polysaccharide producing bacteria. The slimy, mucoid colony was isolated and was biochemically characterized. The bacterium was cultured in mass level. The polysaccharide was recovered with methanol overnight and was precipitated.

 

Optimization of cultural conditions for EPS production (Maheswari, 2009):

Some properties of the EPS obtained by cultivating the selected strains under the optimized conditions were investigated. The factors like pH, temperature, carbon, nitrogen, incubation time, amino acids, inoculum concentration, NaCl concentration and metal ions concentration which were expected to affect the production of EPS by the selected strain was optimized by selecting one parameter at a time.

 

Carbohydrate estimation of exopolysaccharides (EPS):

The total carbohydrate content will be estimated by Phenol sulphuric acid method proposed by (Sheetal Sonawdekar et al., 2016).

 

Gel permeation chromatography in Sephadex G-100 column:

Gel permeation chromatography in Sephadex G-100 column was carried out for the preliminary purification of the EPS.

 

Genomic DNA extraction from isolate:

The genomic DNA content of the isolated bacterium was analysed using the agarose gel electrophoresis.

 

Characterization of EPS:

The bacterial exopolysaccharide was characterized by using Fourier transform infrared Spectrophotometer, Gas chromatographic-Mass spectrophotometric and X ray diffraction (XRD). X ray diffraction is one of the most important non-destructive tools to analyse all kinds of matter - ranging from fluids, to powders and crystals.

 

RESULTS:

The isolation and biochemical characterization of the isolate

Table 1: biochemical characterization

Table 2: Carbohydrate fermentation test

 

Estimation of total carbohydrate and protein:

Glucose was taken as the standard to compare with the sample. The total carbohydrate present in the sample was found to be 0.46+0.008165mg/ml. The total percentage of protein present in the sample was found to be 0.086 +0.0047mg/ml.

 

Optimization for the isolate:

The optimization shows that pH 7 and temperature 50 C was suitable for the growth of bacterium. The bacterium utilized mannose as carbon source, yeast extract as nitrogen source, leucine as amino acid source, sodium sulphate as metal ion. 2 percentage of sodium chloride and 6 percentage of inoculum were required for the effective growth of bacterium. It took 120 hrs for the enormous production of exopolysaccharide in media.         

 

Mass production:

Table. 3. Mass production and yield of polysaccharide

 

Characterization of the polysaccharide:

Agarose Gel Electrophoresis:

The DNA bands were observed and the molecular weight of the DNA was observed which was compared to the standard DNA ladder. (Fig. 1).

 

Fig. 1.Agarose Gel electrophoresis of the isolate

 

Fourier Transform Infra-Red analysis of polysaccharide:

The purified polysaccharide was analysed for knowing the functional groups in it. FT- IR spectrum of the polysaccharide was observed (Fig. 2) and (Table 4).

 

GC-MS analysis of polysaccharide:

The GCMS analysis of the polysaccharide was done after the purification process. There are several peaks have been obtained resembling the sugar compounds.

 

Fig.2. FT IR spectrum of polysaccharide

 

Fig. 3.GC MS analysis of the polysaccharide

 

Table 4. FTIR analysis of the polysaccharide.

 

The compounds said to be present in the polysaccharide were Piperazine, [1, 3] Benzimidazo, 2-Phenazinecarboxylic acid, 2, 5-Cyclohexadien-1-one, 2-Anilino-4-chloroquinoline, 2-[4-(2-Methyl-piperidine-1-sulfate, 2-Methyl-3, 5-dinitrobenzyl alcohol, 1, 2-Dihydropyrido, Chalcone, 2-Propen-1-one, 6-Nitro-2-p-tolyl-2H-indazole, 9H-Fluorene-4-carboxylic acid, 4-Phenyl-3, 4-dihydroisoquinoline, 1, 4-Bis(trimethylsilyl)benzene, 1, 4-Bis(trimethylsilyl) benzene, Cyclotrisiloxane (Fig. 3).

 

Fig. 4. XRD analysis of the polysaccharide.

 

X -Ray diffraction of the Polysaccharide:

The geometrical measurements of the polysaccharide was analysed by XRD. The values of the peaks were noted. (Fig. 4). Calculations have been performed for the polysaccharide. The average value of the dimensions was 151.8+0.85 for the x ray diffracted polysaccharide. The shape of the polysaccharide resembles amorphous crystal in nature. Thus, the purified polysaccharide resembles like crystalline in nature showed K-Alpha wavelength of 1.54443 and K-Beta wavelength of about 1.39225.

 

DISCUSSION:

Polysaccharides are the polymeric compounds having more applications in various fields. In the present study the polysaccharide producing bacteria was isolated from the marine sediment region. In the present work the bacterial polysaccharide was estimated for carbohydrate and protein content. 0.46+0.008165mg/ml of carbohydrate and 0.086+0.0047mg/ml of protein was present in the exopolysaccharide. In the same way Philippis et al., (2013) reported that the carbohydrate content of the exopolysaccharide producing Bacillus sp. was found to be 0.53+0.2mg/ml and the protein content was found to be 0.25+0.43mg/ml for the same species.

 

Relatively Bueno et al., (2011) reported that the polysaccharide producer was to be optimized for the higher production of yield. Similarly Kawai et al., (1992) observed that the pH of 7 is effective for the growth of isolated polysaccharide producing bacteria. The temperature range of 30^oC is suitable for the bacterial growth by utilizing the sucrose as the carbon source. In the present study it was found that the pH value of 7 was suitable for the growth of bacteria. The growth, turbidity increases at temperature of 40^oC.

In the same way Souw, (2009) reported that the peptone was used by the bacteria for its higher growth rate. The bacterium requires magnesium chloride and the aspartate as its nitrogen and amino acid source respectively. The bacteria require 8% of inoculum concentration for the growth in the optimized broth medium.

 

Williams., (2007) reported that it requires sucrose as it carbon source, sodium nitrate as its nitrogen source, sodium sulphate as its metal ion source for the growth and production of the polysaccharide in the broth medium. In the present study mannose was most utilized by the bacteria for its growth revealing that mannose was the major carbon source. The peptone acts as the fine source of nitrogen and leucine provides the production of secondary metabolites. Sodium sulphate was the major source of metal ions provider for the bacteria.

 

In the present study the DNA were isolated from the polysaccharide producing bacteria and bands were clearly viewed. In the similar way Helling (2009) reported that B. subtilis DNA in agarose gels results in the appearance of a number of bands indicating the total quantity of the DNA of the bacteria.

 

Relatively Corpe et al., (1970) reported that the polysaccharide of marine isolate have FTIR exhibit a broad 0—H stretching band at 3309 cm^-1 and sharp C—H stretching band at 2895 cm ^-1. The high frequency side at 1652 cm ^-1 and 1469 cm^-1bands could be assigned to have the presence of carboxylic groups. In addition, a carbohydrate bands were present between 1000 to        1200 cm^-1. The bands at 1080 cm^-1 and 1128 cm^-1 indicate the presence of C-0 stretching of the carbohydrates. In the present study the bacterial polysaccharide was observed to have the peak         569.93 cm^-1, 622.96 cm^-1, 671.18 cm^-1 represents C–Br stretch (alkyl halides), 782.08 cm^-1 representing C–H (aromatics), 1054.99 cm ^-1 representing C–N stretch (aliphatic amines), 1175.53 cm^-1 shows C–H (–CH2X) of alkyl halides, 1364.54 cm^-1 represents C–H rock alkanes etc.

 

In the present study it was reported that the exopolysaccharide which was given for the GC-MS analysis have observed to contain several compounds. Piperazine, [1, 3] Benzimidazo, 2-Phenazinecarboxylic acid, 2, 5-Cyclohexadien-1-one, 2-Anilino-4-chloroquinoline, 2-[4-(2-Methyl-piperidine-1-sulfate, etc., were the chief compounds present. Thus piperazine was guessed to hold the antimicrobial activity. Similarly Helling et al., (2009) reported that gas chromatography coupled with mass spectrometry was one of the instrumentation to analyse the elements in the mixture of compounds. The polysaccharide of Bacillus velezens found to contain -Methyl-3, 5-dinitrobenzyl alcohol, 1, 2-Dihydropyrido, Chalcone, 2-Propen-1-one,, 4-Bis(trimethylsilyl) benzene, Cyclotrisiloxane.

 

In the present study it was reported that the polysaccharide analysed for the XRD have Original K-Alpha1 wavelength of 1.5406 and K-Alpha1 wavelength = "1.54060". The Original K-Alpha2 wavelength of "1.54443"and K-Alpha2 wavelength of "1.54443" and Original K-Beta wavelength = "1.39225", K-Beta wavelength is "1.39225". Acar 2001 similarly reported that, XRD pattern of EPS produced by using B.licheniformis B22 bacterium have an extremely vast height that shows especially of amorphous in nature.

 

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Accepted on 10.12.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(6):2540-2544.