Discovery of novel enormous extracellular polysaccharide (MCC EPS) from waxy corn rhizobacteria; Mitsuaria chitosanitabida strain CKP4/1 phere.
Extracellular polysaccharides (EPSs) from microorganisms are carbohydrate biopolymers which are synthesized and secreted out of cell close to their cell surface or loosely associated to their cell wall [1, 2]. EPS producing bacteria are widely spread in cropping areas especially in the rhizosphere of high sugar rich plants such as sugar cane and waxy corn which they are a bioresource providing a precious carbon source. Plants provide carbohydrate from the process of photosynthesis. Some of the carbohydrates are released together with other organic substances (amino acid, alcohol and vitamin) through their roots. Rhizobacteria consumes the releasing sugar and uses it as a substrate for synthesizing polysaccharide (biopolymer) . Several factors; carbon, nitrogen source, pH and temperature affect growth, EPS production, physical and chemical properties . Several bacteria can synthesize different types of EPS, which are dissimilar in biological functions and properties. Some are suitable for food processing, cosmetic, medical and environmental applications. They are presently important in producing an economical product . Several well-known EPS are synthesized from bacteria such as succinoglycan from Rhizobium sp., gellan gum from Sphingomonas paucimobilis, xanthan gum fromXanthomonas camperstiris, alginate from Pseudomonas sp., hyaluronic acid from Streptococcus equii and Natto cellulose from Acetobacter xylinium . The functionality of EPS depends on their composition and structure. Xanthan is a heteropolysaccharide which is composed of glucose, mannose, glucoronic acid, acetate and pyruvate. Xanthan has an anionic charge and has high viscosity and stability. They are used in many fields of industry, foods, petroleum, cosmetics, agriculture and pharmaceuticals. Another polysaccharide from bacteria as cellulose, it is a homopolysaccharide with only glucose in the molecule. Cellulose has a neutral charge, high crystallinity, insolubility in various solvents, moldability and high tensile strength properties. Because of these properties, they are used in foods as indigestible fiber and in biomedical for wound healing and tissue engineered blood vessels . Microbial EPS are suitable for various industries because their chemical and physical properties are constant and reproducible. EPS are used in the food industry as viscosifying agents, gelling agents, stabilizers, emulsifiers and water binding agents , and as a probiotic additive . Also as an antivirus, antitumor and immune stimulator  in the pharmaceutical industry and in the environmental industry as a bioremediator, as a waste water treatment for example, as a heavy metal remover due to their ions functions . As well in the cosmetics industry because their water retention ability and film-forming capacity . Since microbial EPS has many benefits, we are interested in screening microorganisms that efficiently produce biologically functional extracellular polymeric substances (EPS). Particularly bacterial EPS that are extensively studied and developed for commercial applications, because they are easily and quickly cultivated and also provide a high production rate of EPS . Therefore in this study, our main objective was to screen and isolate EPS producing bacteria from the rhizosphere of waxy corn grown Nakhon Pathom, Thailand. Characterization of extracted EPS was also used to develop new bio-materials, bio-functional, from the local bio-resource in Thailand.
MATERIALS AND METHODS
Screening of EPS producing bacteria:
Rhizophere microorganism were collected and isolated by sampling rhizosphere soil from waxy corn fields located in Nakhon Pathom, Thailand. Dilution plate count technique was performed for the isolation and screening
of EPS producing bacteria on modified Winogradsky's nitrogen free mineral agar medium, (MWA) containing 0.5% mineral mixed solution medium (5% K[H.sub.2] P[O.sub.4], 0.1% MgS[O.sub.4].7[H.sub.2]O, 2.5% NaCl, 0.1% FeS[O.sub.4].7[H.sub.2]O, 0.1% [Na.sub.2]Mo[O.sub.4].2[H.sub.2]O and 0.1% MnS[O.sub.4].4[H.sub.2]O), 1% Sucrose, 0.005% yeast extract, 0.01% CaC[O.sub.3]  and 2% agar, pH 6.2. EPS-forming colonies were selected by visual observation including its shape, colorless, transparent, sticky, mucilaginous, elastic and dome-like. A single colony was isolated and collected by plating on a MWA-plate and microbial strains were kept in glycerol at -80[degrees]C as a stock culture.
Identification and classification of EPS producing bacteria:
All selected EPS-producing bacteria were identified based on 16S rRNA gene sequencing. DNA extraction was done by using Isoplant II kit (Wako Pure Chemical Industrial Ltd, Japan) 16S rRNA gene was amplified by using polymerase chain reaction with Taq polymerase (Toyobo, Tokyo, Japan) and 16S rRNA universal primer (27F; 5'-AGAGTTTGATCCTGGCTCAG-3', forward primer) and 1525R (5'-AAAGGAGGTGATCCAGCC3', reverse primer). The 1500 bp of PCR products were sequenced by using ABI PRISM 310 Genetic Analyzer. The nucleotide sequences of 16S rRNA of the unidentified bacteria were compared to those of 16S rRNA in the Genebank database using the BLAST method to determine their approximate phylogenetic affiliation and their sequence similarities at the National Center for Biotechnology Information (NCBI), USA . The sequences of the related taxa were acquired from the same website. Nucleotide sequences were initially aligned using the CLUSTAL X program  and then manually adjusted. Distance matrices were calculated and a phylogenetic tree for the data setting was created according to the Mega4 (version 4.1) software package obtained from the web site .
Optimize of culture condition for growth and EPS production:
Effect of carbon source:
The effect of carbon sources on the growth of the selected EPS producing bacteria was investigated by culturing in the MW medium containing various 1% of sugar; sucrose, lactose, maltose, glucose and fructose. Cultivation condition was performed at 30[degrees]C, with shaking at 120 rpm. At the early stationary phase of growth (around 100 h), viscosity medium was harvested by centrifugation and EPS was extracted by alcohol precipitation (method defined in the extraction and purification section). The dry weight of EPS, in each condition, was measured (g/L). The best carbon source was chosen on the basis of the dry weight of EPS.
Effect of nitrogen source:
The effect of nitrogen sources on the growth and EPS production was examined. EPS producing bacteria were cultured in the MW medium containing the chosen carbon source and 0.05% of the various nitrogen compounds; yeast extract, tryptone, peptone, urea and NaN[O.sub.3]. Cultivation conditions were performed as above. The dry weight of EPS was recorded for each condition.
Effect of pH:
MW medium containing the best carbon and nitrogen sources was prepared at different pH (4, 5, 6, 7, 8, 9 and 10). The growth of EPS producing bacteria was determined every 2 h, until it reach a stationary phase, by measuring the optical density at 660 nm. The dry weight EPS at each pH condition was measured and compared.
Extraction and purification of EPS:
EPS extraction was carried out after inoculation the selected strain into MW medium. The culture was cultivated at 30[degrees]C, shaken at 120 rpm, on a rotary shaker, for 100 h. The viscous culture broth was pasteurized for 15 min at 70[degrees]C and then centrifuged at 8,000 g for 20 min. Precipitated cell was removed and supernatant was harvested. Precipitated EPS was collected by adding chilled 2-propanol to the supernatant in a 2:1 ratio. The derivable polysaccharide (MCC EPS) was collected by centrifugation and its aqueous solution was deprotenated by using Sevag reagent (1-butanol/chloroform, v/v = 1: 4) . Dialyzed againsts distilled water at 4[degrees]C several times. MCC EPS was freeze dried by using lyophilizer and the weight of the dried MCC EPS was recorded.
Determination of molecular weight (MW):
The molecular weight of MCC EPS was determined by HPLC using OH pak803 HQ column (8 mm x 300 mm, Shodex Europe office, Munich, Germany). EPS (1 mg) dissolved in milli Q water (1 mL) was injected into the column, eluted with milli Q water at a flow rate of 0.3 mL/min and temperature set at 50[degrees]C. The eluent was monitored with a refractive index detector (RID). Different molecular weights of pullulan standards type P-82 (Shodex) (P-800, P-400, P-200, P-100, P-50, P-20, P-10 and P-5) were used as references for determination of the molecular weight of the MCC EPS.
Analysis of monosaccharide compositions: By thin layer chromatography (TLC):
MCC EPS (3.5 mg) was hydrolyzed by using 2 M trifluoroacetic acid (TFA) at 120[degrees]C for 72 h., the hydrolyzate was co-concentrated with methanol 3-5 times. The hydrolyzed MCC EPS was dissolved in deionized water and then desalted with Amberlite MB4 (Organo, Tokyo, Japan). TLC was used for analyzing the result of the hydrolysis. MCC EPS hydrolysate was spotted on the TLC plate and compared with standard sugars. Eighty-five percent acetronitile following by 2-propanol: 1-butanol: [H.sub.2]O (12: 3: 4) was used for separation of the monosaccharide component. The spots of each digested product were visualized by naphthoresorcinol-ethanol-sulfuric acid spray reagent and then incubated at 100[degrees]C until the color of spot on TLC occurred.
By HPAEC-PAD analysis:
MCC EPS hydrolysated (as described above), was analyzed by a high performance anion exchange chromatography unit, using a pulsed amperometric detector (HPAEC-PAD), a CarboPac[TM] PA1 anion exchange column (4 x 250 mm; Dionex Co., Sunnyvale, CA, USA) and eluted by a 16 mM NaOH at a flow rate of 0.8 mL/min. Retention time of the sample was compared with the internal monosaccharide standard.
Physical properties of MCC EPS on zeta potential:
The MCC EPS surface charge was characterized by determining the zeta potential value using the electroacoustic technique with a zeta probe analyzer (Zeta Meter 3.0; Malvern Instruments Ltd; Worcestershire; UK) at room temperature .
Morphology of the MCC EPS on SEM:
The MCC EPS was characterized by using a scanning electron microscope (SEM, Camscan, MX2000, England) with Cressington Sputter Coater 108, Scientific and Technological Equipment Centre, Faculty of Science, Silpakorn University, Nakorn Pathom, Thailand.
RESULTS AND DISCUSSION
Isolation and identification of EPS producing bacteria:
Waxy corn soil samples were chosen for the screening of EPS producing bacteria on the hypothesis of soil ecology, soil microorganisms can use nutrients from the sugar generated by plants. The soil samples were serial diluted for dilution plate count. Screening was performed by spreading the samples on MWA plates, and then incubating at 30[degrees]C for 3-4 days. Sixty two isolates were collected as rhizobacteria, but only 23 isolates were EPS producing bacteria by morphological characterization on agar plates. Among the 23 isolates, one isolate, CKP4/1 phere showed the largest highly viscous colony on the MWA medium. Therefore, it was selected for this study. Isolate CKP4/1 phere was characterized to be an aerobic bacteria, gram negative, coccobacilli (Fig.la). 16S rRNA gene sequencing was blasted and aligned to identify EPS producing bacteria. Additionally, the phylogenetic tree, based on the 16S rRNA gene sequences was analyzed, results shown in Figure 1b. Results of BLASTX analysis showed a 99% identity match to the partial sequence of 16S rRNA gene of Mitsuaria chitosanitabida strain R8-376 16S rRNA gene (accession no. JQ659937. 1). Subsequently, 16S rRNA gene sequence of isolation CKP4/1 phere was deposited in the Genbank database as accession no. KR922044 of Mitsuaria chitosanitabida strain CKP4/1 phere. In accordance to Mitsuaria chitosanitabida, it had been reported to be an EPS producing bacteria .
Optimization of carbon source, nitrogen source and pH:
The capability of M. chitosanitabida strain CKP4/1 phere for MCC EPS production was studied. The result showed that MWB medium with adding sucrose gave the highest MCC EPS dried weight at 1.97 g/L, following by glucose at 1.87 g/L, maltose at 1.62 g/L, fructose at 0.78 g/L and lactose at 0.07 g/L as shown in Figure 2a. There are several researches reported that sucrose is an efficient carbon source for microorganisms. Particularly, Bacillus polymyxa produced EPS 18 g/L which is the highest experimental result using sucrose . Enterobacter sp. synthesized the highest EPS on sucrose with 2.6 g/L , Paenibacillus polymyxa EJS-3 produced EPS 22.82 g/L using sucrose as the main factor . And Rhodotorula acheniorum MC yeast strain also used sucrose for EPS production with 6.2 g/L . Also, it has been reported other suitable carbon sources such as mannitol used by Rhizobium isolate from Vigna mungo (L.) Hepper  Streptomyces violaceus MM72 prefers fructose as a carbon source than other sugars, for producing EPS . Paenibacillus polymyxa SQR-21 can produce the highest EPS 3.44 g/L using galactose as a carbon source  but Pseudomonas caryophylli CFR 1705 uses lactose to produce the highest EPS . Moreover, Azotobacter sp. SSB81 produced maximum EPS 2.52 g/L on glucose as a supplement , similar to Halomonas ventosae and Halomonas anticariensis which as halophilic bacteria produce the highest EPS from glucose .
Nitrogen sources also had an effect on MCC EPS production. In this study, urea was preferentially utilized by the organism for maximum production of MCC EPS (2.26 g/L) compared to other nitrogen sources, peptone (1.90 g/L), NaN[O.sub.3] (1.46 g/L), trptone (0.96 g/L) and yeast extract (0.53 g/L) (Fig. 2b). Alternately to other microorganisms such as using yeast extract by Paenibacillus polymyxa EJS-3 , Paenibacillus polymyxa SQR-21  and Streptomyces violaceus MM72 , using casamino acid as nitrogen source for EPS production from Azotobcter sp. SSB81 , peptone is the best nitrogen source for Enterobacter sp. , and the best nitrogen source for Bacillus polymyxa was potassium nitrate .
The optimized pH culture medium for MCC EPS production was investigated. MW medium at neutral pH, between pH 6 to pH 8 gave the highest dried weight MCC EPS at approximately pH 7 (Fig. 2c). While an acid pH range (pH 4-5) and an alkaline range (pH 8-10), EPS production was slightly decreased. This result was similar to Bacillus polymyxa that has a pH 7-8 as the optimal pH for producing EPS  and Paenibacillus polymyxa EJS-3 where a pH 8 is the optimum , Streptomyces violaceus MM72 produced the highest EPS at pH 7  similar to Pseudomonas caryophylli CFR 1705  but was not in agreement with Paenibacillus polymyxa SQR-21 with pH 6.5 as the maximum pH to produce EPS . Most bacteria preferred a neutral pH rather than acid or alkaline conditions for EPS production.
Figure 2d shows the growth profiles measured by optical density at 660 nm and the MCC EPS production during cultivation for 140 h. The result indicated that the bacteria synthesized and secreted MCC EPS as a secondary metabolite. It produced the highest MCC EPS at the stationary phase on the growth curve.
Determination of molecular weight (MW):
The molecular weight of MCC EPS was determined by HPLC detection using OH pak 803HQ column chromatography. According to the calibration curve with pullulan standards of the logarithms graph the molecular weight of MCC EPS was calculated to be 11.57 x 103 kDa. In a previous study, it reported the bacterial EPS consisted of high molecular weight, 10 to 30 kDa, not only in homopolysaccharide form but also heteropolysaccharide form . For example Paenibacillus polymyxa SQR-21 (8.96 x [10.sup.2] kDa) , Leuconostoc garlicum PR (20 x [10.sup.3] kDa) , Rhizobium isolate of Vigna mungo (L.) Hepper (750 kDa) , Halomonas ventosae (20 kDa), Halomonas anticariensis (46 kDa) , Rhizobium sp. N613 (35 kDa) , Streptomyces violaceus MM72 (8.96 x [10.sup.2] kDa) , Pseudomonas caryophylli CFR 1705 (1.1 x [10.sup.3] kDa) , Rhizobium radiobacter S10 (3.03 x [10.sup.3] kDa)  and Mitsuaria chitosanitabida strain KMBL5781 (54.7 kDa) . Indicating that, the MCC EPS form Mitsuaria chitosanitabida strain CKP4/1 phere was an enormous polysaccharide.
Analysis of monosaccharide compositions on TLC and HPAEC-PAD HPLC:
Acid hydrolysis was performed using 2M TFA for cleavage MCC EPS into monomer. Each of hours during hydrolysis, the hydrolysate was collected and spotted on TLC. Two developing solvent systems were used as mobile phase: twice of 85% acetonitrile and once of 2-propanol: 1-butanol: [H.sub.2]O (12:3:4) for developing. The result of monosaccharide composition was considered by comparison to moving distance (Rf value) on TLC of standard sugars (glucose, galactose, sucrose, fructose, mannose, rhamnose, arabinose and xylose). After 60 h completely hydrolyzation MCC EPS was broken down to be glucose; 0.60, galactose; 0.56, mannose; 0.65, rhamnose; 0.80, arabinose; 0.67 and unidentified moieties (shown in Fig 3a).
The result of the TLC analysis was confirmed by injection of 60 h hydrolysated EPS into HPAEC-PAD using a CarboPac[TM] PA1 anion exchange column (4 * 250 mm). The column was eluted by 16 mM NaOH with a flow rate of 0.8 ml/min. Compared with standard sugar injection, the chromatogram of hydrolyzed MCC EPS showed the retention times of 6.53, 10.23, 11.37, 14.73, 15.90 and 17.23 min; which were the retention times of fucose, rhamnose, arabinose, galactose, glucose and mannose, respectively. However, a number of peaks were not identifiable (Fig. 3b). Thereby, MCC EPS is a heteropolysaccharide. Differing to research by Charchoghlyan H and Park HD in 2013, they recorded that EPS from Mitsuaria chitosanitabida KMBL5781 consisted of glucose, mannose, galactose and fructose  but rare sugars such as rhamnose and fucose were not found. Fundamentally, most bacterial polysaccharides are composed of glucose, galactose and mannose in the molecule. There are only a few polysaccharides that contain rare sugars, namely rhamnose and fucose; for examples fucogel, clavan, rhamsan and welan . In this study, we discovered a novel enormous MCC EPS.
Morphological characterized of MCC EPS:
Morphological of dried MCC EPS was observed by scanning electron microscope. The result showed that MCC EPS appear as a fibrous sheet molecule (Fig. 4a).
Physical properties of MCC EPS on zeta potential (Zp):
The charge surface of the MCC EPS was analyzed (Fig. 4b). MCC EPS with neutral charge was found with a pH 2.0 with giving the highest zeta potential value (-1.97 mV) and then decreased from -1.97 to -47.3 mV, contrary with increasing pH from 2.0 to 6.0, respectively. In the alkaline pH range 7.0 to 11.0, Zp value was -38.5, -47.1, -44.3, -32.5 and -35.1 respectively, concluding that the MCC EPS had a negative charge in the molecule. The results related to galactomannan from Caesalpinia pulcherrima and Gleditsia triacanthos having negative Zp values which was not dependent on pH, different from sodium alginate and carrageenan that with higher pH values also have higher Zp values . Alginates are a natural anionic polysaccharide with sodium salt of alginic acid. This polysaccharide can be used as vicosifier, emulsifier, thickener and stabilizer in the food industry, used in medicines, paper coating and paint [36, 35]. Caragenan also are anionic polysaccharide because of sulphate group which they are normally used as thickener, stabilizer and gelling agent in food and pharmaceutical industries [37, 35], used as preclusion of unconscious agglomeration of the nanopaticles, and used as the integration of magnetic iron oxide nanoparticles [38, 35]. It is possible that MCC EPS can be used as absorbent or binding molecule to cations. Consequently, it is suitable for heavy metal remediation or waste water treatment application.
Mitsuaria chitosanitabida strain CKP4/1 phere from rhizosphere of waxy corn field synthesized the high viscous extracellular polysaccharide on MWA medium. The superior culture conditions for MCC EPS production in containing 1% sucrose as carbon source and 0.005% urea as nitrogen source were pH 6.0-7.0 at 30 [degrees]C for 100 h. The MCC EPS is heteropolysaccharide that it consisted of glucose, galactose, mannose, rhamnose, fucose, arabinose and two unidentified sugars. The molecular weight of the MCC EPS was determined be 11.57 x103 kDa and the surface of fibrous molecule contain an anionic charge.
Received 3 October 2015
Accepted 10 October 2015
Published Online 13 November 2015
This research was financially supported by graduated school of Kasetsart university. Furthermore, deeply thanks to Dr. Atsuo Kimura gave excellent suggestions and the great comments to this project, Dr. Weeranuch Lang contributed the knowledge and techniques in HPAEC-PAD analysis and Dr. Jintanart Wongchawalit suggested the idea for this research and greatly assisted the research, although commented and improved the manuscript. The authors would like to thank our colleagues from Hokkaido University who provided the several instruments involving in characterization of MCC EPS such as lyophilizer, and HPAEC-PAD. We thank for Miss Patcharapa Klahan and Miss Ananya Kittiwiwatkul for the grateful talked and discussed the results during doing research.
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(1) Nadanong Seeoob, (2) Weeranuch Lang, (2) Yasuyuki Hashidoko, (2) Atsuo Kimura, (1) Jintanart Wongchawalit
(1) Department of Microbiology, Faculty of Liberal Arts and Science, Kasetsart University Kamphaeng-Saen Campus, Nakhon Pathom, 73140, Thailand.
(2) Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Corresponding Author: Jintanart Wongchawalit, Department of Microbiology, Faculty of Liberal Arts and Science, Kasetsart University Kamphaeng-saen Campus, Nakhon Pathom, 73140, Thailand.
Tel: 0-3428-1105-7 ext. 7655, Fax: 0-3428-1057; E-mail: firstname.lastname@example.org
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|Author:||Seeoob, Nadanong; Lang, Weeranuch; Hashidoko, Yasuyuki; Kimura, Atsuo; Wongchawalit, Jintanart|
|Publication:||Advances in Environmental Biology|
|Date:||Oct 1, 2015|
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