Journal of Microbiology and Biotechnology Research€¦ · work was done according to the methods described in Bergeys Manual of determinative bacteriology 9 th edition. EPS Production

Journal of Microbiology and Biotechnology Research

Scholars Research Library

J. Microbiol. Biotech. Res., 2013, 3 (3):24-31 (http://scholarsresearchlibrary.com/archive.html)

ISSN : 2231 –3168

CODEN (USA) : JMBRB4

24 Available online at www.scholarsresearchlibrary.com

Isolation, screening and optimization of exopolysaccharide producing bacterium from saline soil

*Sunil T. Pawar, Amarsinh A. Bhosale, Trishala B. Gawade and Tejswini R. Nale

Department of Microbiology, Tuljaram Chaturchand College, Baramati, Pune, MS(India)

_____________________________________________________________________________________________ ABSTRACT Exopolysaccharides (EPS) are high-molecular-weight polymers that consists sugar residues which are widely vary in structure and function. Many bacteria possess an ability to synthesize and excrete exopolysaccharides. Attempt has been made to isolate and identify the EPS producing bacteria from saline soil of Baramati region of Maharashtra. Five different organisms were isolated from saline soil to explore the EPS production. Bacteria were identified by their morphological, biochemical characteristics as well as 16S rRNA gene sequencing. Out of five isolates, isolate no-4 was found to be potential isolates for EPS production. EPS production of isolate no-4 was optimized in nutrient broth containing 2 percent sucrose for different environmental and nutritional conditions viz. incubation periods, pH, temperature, carbon source and different sodium chloride (NaCl) conc. Sucrose was found to be the suitable carbon source to produce EPS. Three days of incubation period was found to be an optimum for production of maximum yield of EPS in nutrient broth containing 4% sucrose. The optimum pH, temperature and NaCl for EPS production were found to be 7.5, 300C and 80 mM, respectively. The EPS was characterized a Fourier Transform Infrared Spectrometer. The FTIR spectrum of EPS showed a broad intense band at 4000 cm-1 and sharp band at 2980.89 cm-1, which strongly suggests the presence of broad stretching of O-H (carboxylic acid and H-bonded) group. The peak at 1038.92 cm-1 corresponds to stretching of C-O (Alcohol, Ether and Phenol) group. Key words: Exopolysaccharide, Salinity, FTIR. _____________________________________________________________________________________________

INTRODUCTION

Exopolysaccharides (EPS) are high-molecular-weight polymers that compose sugar residues. Many bacteria, yeasts, fungi and algal cells possess an ability to synthesize and excrete exopolysaccharide [1, 24, 6, and 11]. Also referred an extracellular polysaccharides or EPS material, these complex carbohydrates are widely varied in structure and function. The synthesis of exocellular investments of a polysaccharide nature by bacterial cells is generally considered as directly related to environmental constraints on the producing microorganisms. Exopolysaccharides (EPS) produced by Lactic acid bacteria possess the possibility of replacing stabilizer and thickeners, currently produced commercially by non-food grade bacteria [4, 19]. Microbial exopolysaccharides have found a wide range of applications in the food, the pharmaceutical and other industries, due to their unique structure and physical properties. Some of these applications include their use as emulsifiers, stabilizers, binders, gelling agents, coagulants, lubricants, film formers, thickening and suspending agents [21]. Exopolysaccharides are generally composed of monosaccharide and some non carbohydrate substituent, like acetate, succinate. These EPS possess regular structures and thus have unique rheological properties and these molecules are highly pure. EPS could be acidic or basic in nature [23].

Sunil T. Pawar et al J. Microbiol. Biotech. Res., 2013, 3 (3):24-31 ______________________________________________________________________________


Various Anabaena and Nostoc species from freshwater habitat showed their ability to excrete EPS. The EPS of Anabaena inaequalis, A. lutea and Nostoc commune were consists of rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, and uronic acids [8]. Soil salinity is a part of the natural ecosystem in arid and semi-arid regions and an increasing problem in an agricultural soils the all over world. Salinity of the habitat could be an important ecological niche, generating an environmental stress on micro flora survived in it [18, 20]. Capsular exopolysaccharides can protect pathogenic bacteria and contribute to their pathogenicity. Attachment of nitrogen-fixing bacteria to plant roots and soil particles are important for colonization of rhizosphere and roots and for infection of the plant, can be mediated by exopolysaccharides [22]. Cyanobacterial and bacterial Exopolysaccharides (EPS) have been reported to play a significant role in providing protection to the cell as a boundary layer [3], contributing to soil aggregation due to its gluing properties [12] and binding heavy metals due to the presence of several active functional groups onto it [7, 14]. Therefore, it is important to isolate and characterize EPS producing microorganisms from saline soil. It might be helpful for reclamation of such habitat.

MATERIALS AND METHODS Collection of soil samples To find the saline area in Baramati and nearby sub-urban areas, we visited the Agriculture Development Trust (Krishi Vigyan Kendra), located at Sharadanagar, Tal-Baramati, Dist-Pune, Maharashtra, India. All necessary information about the salinity, saline areas in Baramati and nearby sub-urban areas was collected. Using Survey report of KVK, soil samples were collected from diverse agro-ecological regions of Baramati. Soil samples were collected as per standard protocol. All soil samples were collected in sterile plastic bags and carried to the laboratory for further investigation. Physicochemical properties of soil Soil pH was measured in a 1:2.5 soil-water suspension using a glass electrode (Systronics, Digital pH meter, India). Soil electrical conductivity (EC) was determined by measuring the electrical conductance of soil-saturation extract with a conductivity meter (Equip-Tonics, India). EC and pH were determined in the Department of Microbiology, T. C. College, Baramati. Organic carbon and organic nitrogen were analyzed at Krishi Vigyan Kendra, Sharadanagar, Baramati. Screening and isolation of Exopolysaccharide producing bacteria Serially diluted saline soil sample were used for microbial isolation. 0.1ml soil suspensions were spread on nutrient agar plates. Plates were incubated at 48 hrs at room temperature. Isolates were maintained on nutrient agar plates. Mucoid colonies were screened and re-streaked on another nutrient agar plate to obtain pure culture. Isolated cultures were identified on the basis of morphological, biochemical and microscopic observations. The identification work was done according to the methods described in Bergeys Manual of determinative bacteriology 9th edition. EPS Production Isolated organisms were used for production of exoplysaccharides. Bacterial isolates were maintained on Nutrient agar slant and stored in refrigerator. Production was carried out in 250 ml flasks containing 50 ml of medium [23]. The medium consisting of the following components (g/l): peptone 10 gm, meat extracts 3 gm, sodium chloride 5 gm and sucrose 2%. Media were sterilized at 1210C for 20 min. The pH was adjusted to 6.5. The flasks were incubated on a rotary shaker at room temperature for 72 hrs. Isolation and Extraction of EPS Cells were harvested by centrifugation for 20 min at 10,000 rpm. After centrifugation, two volumes of ice cold Isopropanol were added into it and stored overnight at 40C. Precipitated material was collected by centrifugation (20 min at 10,000 rpm) and the pellets were dried at 1000C. After drying weigh the pellet to know which organism were showed high production of exoplysaccharides and the best strain which showed higher production was used for the optimization of exopolysaccharide production at different environmental conditions.



Optimization of exopolysaccharide production To study the effect of different parameters, 1% inoculum containing 5×106 cells/ml were inoculated in 100 ml of production medium. EPS production was optimized under different environmental and nutritional conditions viz. incubation period (1 to 5 days), pH (6.5, 7, 7.5, 8), Temperatures (20, 30, 37, 450C), carbon sources (glucose, lactose, fructose, sucrose, mannitol), salt concentrations (80, 100, 120, 140, 160, 180 mM NaCl) and different sucrose concentrations (1, 2, 3, 4 and 5 %) also as per [19, 11]. Estimation of Carbohydrate and Protein content of crude EPS The total carbohydrate content was estimated by phenol sulphuric acid method [5]. The amount of protein present in the EPS was estimated by the Lowry et al. [10]. Qualitative tests for carbohydrate Monosaccharide, oligosaccharide and polysaccharide were detected by Molish test, Fehling’s test, Benedicts test and Barfoed test respectively [16]. FTIR spectroscopy analysis of EPS The EPS was characterized by using a Fourier Transform Infrared Spectrometer (PerkinElmer Spectrum Version 10.03.06). The dried EPS was ground with KBr powder and passed into pellet for FTIR spectroscopy between frequency range 650 to 4000 cm-1 with resolution 4 cm-1 and 2 scans.

RESULTS AND DISCUSSION

Many bacteria are present in the soil. A wide range of bacteria are known to produce exopolysaccharides. As per survey report of KVK, 12 different sites were selected on the basis of their physicochemical nature from different regions of Baramati, Dist- Pune (MS). All sites are located about 3 to 15 km from Baramati. Selection of saline soils was made on the basis of pH and conductivity measurement. Samples were collected accordingly. Out of 12 sites, 4 sites were found to be saline and others were non saline. It was found that soil pH was slightly alkaline in saline soil than non saline soil. Physicochemical analysis was done in triplicate and the figures in Table 1 shows average values.

Table1. Physicochemical properties of soil

Sample No. pH Electrical conductivity (dS/m) 1 7.9 4.2 2 7 0.91 3 8.4 6.1 4 7.5 0.37 5 6.8 0.11 6 7.1 0.18 7 8.1 3.9 8 7.2 0.56 9 6.9 0.69 10 7.8 5.2 11 7.3 0.49 12 7.6 0.83

Table 2. Morphological Characters

Characters Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5 Size 2 mm 2 mm 2 mm 3 mm 2 mm Shape Circular Circular Circular Circular Circular Color Colorless Greenish blue Yellow Pale yellow Yellow Margin Convex Plain Plain Convex Convex Elevation Irregular Regular Entire Entire Entire Opacity Opaque Transparent Opaque Opaque Opaque Consistency Mucoid moist Moist Mucoid Mucoid

Bacteria were isolated by using standard plate method. Serially diluted samples were spread on sterile nutrient agar plates. These plates were incubated for 48 hrs at room temperature. The isolates were initially differentiated on the



basis of their cultural and cellular morphological, biochemical characters, which are given in Table 2, 3, and 4, respectively.

Table3. Microscopic Characters

Characters Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5 Motility Motile Non motile Motile Motile Motile Gram Staining Negative rods Negative rods Positive rods Negative rods Negative rods Spore staining Non spore forming Non spore forming Non Spore forming Non spore forming Non spore forming Capsule staining capsulated Non capsulated Capsulated capsulated Non capsulated

Table4. Biochemical Characters

Carbohydrate utilization and fermentation

Sugars Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5 Glucose U, F U, F U, F U, F U, F Sucrose U, F U, F U, F U, F U, F Mannitol U, F U, F U, F U, F U, F Fructose U, F U, F U, F U, F U, F Lactose U, F U, F U, F U, F U, F

(U) - Sugar utilization, (F) - Sugar fermentation Hydrolysis tests

Tests Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5 Starch - - + + + Gelatin - + + - +

(-) - Negative test, (+) - Positive test

Other tests

Tests Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5 Oxidase + + + + + Catalase + + + + + Nitrate reduction + + + + +

(-) - Negative test, (+) - Positive test Identification of isolates was done on the basis of their morphological, microscopic and biochemical characters with reference to the key of Bergeys manual of determinative bacteriology (9th edition). The isolates may be 1. Azotobacter spp. 2. Pseudomonas Spp. 3. Agrobacterium spp. 4. Alpha Proteobacterium group and 5. Xanthomonas spp. These isolates were further used for the screening of high amount of exopolysaccharide production.

Fig.1. Screening of the isolates for EPS production



The purpose of this experiment was to obtain the efficient strain isolated from saline soils which produce high amount of EPS. Five different isolates from saline/alkaline soil of Baramati region were screened for EPS producing activity. [11] Studied the different fungal strains for the production of exopolysaccharides. They screened different lactic acid bacterial strains for their exopolysaccharide production [2]. Out of five isolates, isolate no-4 (Fig. 1) produced higher amount of exopolysaccharide as compared to other isolates. Identification of isolate no-4 was confirmed by their molecular characterization. Therefore isolate no-4 was used for the further work. The cultures subjected to genomic DNA isolation and PCR amplification by Colony PCR; sequencing reaction by using Big Dye Terminator v3.1 sequencing kit. Sequences were assembled, edited and identified by using the sequence analysis tool- Blast and Clustal W. The phylogenetic tree generated using the neighbouring joining method. The matrix used was Blossum 62. According to the phylogenetic tree, interested organism may belong to Alpha Proteobacterium group.

Fig.2. (a) - Sequence of a isolate no-4 strain for EPS production, (b) - Phylogenetic tree of a isolate no-4 strain for EPS production.

The exopolysaccharide production was optimized under different environmental conditions (Table 5). EPS production was the maximum in Nutrient Broth, contains 2% sucrose. The EPS production from Ganoderma alppanatum were also reported [9]. EPS production was determined during different periods of incubation (1, 2, 3, 4 and 5 Days). The EPS production was highest in 3 days incubation period (5.2 g/l). For A. alternata, [11] found that ninth day incubation period was optimum for EPS production. The pH and temperature of the culture medium is a vital factor that governs cell growth and EPS production [14, 19]. We found that, 7.5 pH and 300C temperature were optimum for EPS production. The production rate of EPS was decreased rapidly with increasing the pH and temperature. [14, 19] reported that pH 3 and 300C temperature would be suitable for EPS production.

Table5. Optimization of exopolysaccharide

Parameter Values EPS g/l Parameter Values EPS g/l

Incubation Period (Days)

1 2.2

Sucrose concentration (%)

1 3.4 2 4.1 2 4.1 3 5.4 3 4.6 4 3.8 4 5.3 5 3.4 5 4.5

Temperature (0C)

20 2.9

pH

6.5 3.1 30 5.1 7 3.7 37 3.9 7.5 4.9 45 3.5 8 3.5

Carbon source

Glucose 3.6

Salt concentration (NaCl mM)

80 4.9 Lactose 2.8 100 4.3 Sucrose 5.1 120 3.8 Fructose 3.1 140 3.1 Mannitol 3.3 160 2.4



Fig.3. Optimization of exopolysaccharide

Carbohydrates are the major component of the cytoskeleton and an important nutritional requirement for the growth and cell development [17]. The effects of different carbon sources on the production of EPS were studied. In this study, it was found that sucrose was the suitable carbon source (Fig. 4). However, [15] it was reported that glucose was suitable carbon source for EPS production. As mentioned earlier, the sucrose was the suitable carbon source for the EPS production. 4% sucrose concentration in medium showed higher production. The EPS production was found to higher at 80mM salt concentration.

Fig. 4. Optimization of Exopolysaccharide production at different environmental conditions



We have partially characterized the isolated polymer. A chemical analysis of the polymer reveals the presence of proteins and carbohydrates. The protein content in EPS was in the range of 9.4%. Whereas, carbohydrates accounted for more than 68%. A spot test for carbohydrates indicates the presence of reducing sugar (i.e. Glucose and Fructose).

Fig.5. FTIR spectroscopy analysis of EPS

Functional group was detected by using FTIR spectrum from crude EPS. The IR spectrum of polymer has shown a broad intense band at 4000 cm-1-650 cm-1 with resolution 4 cm-1. (Fig. 5) The spectrum shown in Fig. 5, sharp band at 2989.89 cm-1 strongly suggests a presence of broad stretching (O-H, carboxylic acid and H- bonded) groups. The peak at 1038.92 cm-1 corresponds to stretching of (C-O, alcohol, ether and phenol) groups. Polysaccharides possessing carboxyl group is reported previously [13, 6]. The IR spectrum of polymer proved the presence of carboxyl group which may serve as a binding site for divalent cations. This carboxyl group may also work as a functional moiety to generate an innovative new or modified polymer, by using different novel approaches like synthetic polymers.

CONCLUSION

Five different isolates from saline/alkaline soil of Baramati region were screened for EPS producing activity. Out of five isolates, isolate no-4 produced higher amount of exopolysaccharide. The exopolysaccharide production was optimized under different Nutritional and environmental conditions. EPS production was found to be maximum in Nutrient broth in presence of 4% sucrose as a carbon source and 80mM salt concentration, at room temperature within three days of incubation. A chemical analysis of the polymer reveals the presence of proteins and

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carbohydrates. EPS was characterized by partially FTIR spectrum, which shows the presence of carboxylic acid group and H- bonded group. Acknowledgement Authors are thankful to UGC, New Delhi, for providing financial support to major research project [F.No.39-199/2010 (SR)] and also grateful to Hon’ble principal Dr. Chandrashekar Murumkar for providing necessary facilities and constant help during the research period.

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Journal of Microbiology and Biotechnology Research€¦ · work was done according to the methods described in Bergeys Manual of determinative bacteriology 9 th edition. EPS Production