Bioresource Technology 102 (2011) 6095–6103
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Optimization, purification, characterization and antioxidant activity of an extracellular polysaccharide produced by Paenibacillus polymyxa SQR-21 Waseem Raza, Kousar Makeen, Yang Wang, Yangchun Xu, Shen Qirong ⇑ Jiangsu Provincial Key Lab for Organic Slid Waste Utilization, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
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Article history: Received 11 November 2010 Received in revised form 7 February 2011 Accepted 8 February 2011 Available online 16 February 2011 Keywords: Antioxidation activity Monosaccharide composition Paenibacillus polymyxa SQR-21 Polysaccharides
a b s t r a c t The optimization, purification and characterization of an extracellular polysaccharide (EPS) from a bacterium Paenibacillus polymyxa SQR-21 (SQR-21) were investigated. The results showed that SQR-21 produced one kind of EPS having molecular weight of 8.96 105 Da. The EPS was comprised of mannose, galactose and glucose in a ratio of 1.23:1.14:1. The ratio of monosaccharides and glucuronic acid was 7.5:1. The preferable culture conditions for EPS production were pH 6.5, temperature 30 °C for 96 h with yeast extract and galactose as best N and C sources, respectively. The maximum EPS production (3.44 g L1) was achieved with galactose 48.5 g L1, Fe3+ 242 lM and Ca2+ 441 lM. In addition, the EPS showed good superoxide scavenging, flocculating and metal chelating activities while moderate inhibition of lipid peroxidation and reducing activities were determined. These results showed the great potential of EPS produced by SQR-21 to be used in industry in place of synthetic compounds. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Many microorganisms secrete extracellular polysaccharides (EPS) and get attached to solid surfaces which help them to grow as colonies termed biofilms (Liu et al., 2010a,b). These EPS either remain attached to the cell surfaces or get released into the extracellular medium. Due to their many interesting physico-chemical and rheological properties, the microbial EPS has a wide range of industrial applications such as the production of textiles, detergents, adhesives, cosmetics, pharmaceuticals, food additives as well as applications in brewing, microbial enhanced oil recovery, wastewater treatment, dredging and various downstream processing processes, cosmetology, pharmacology and as food additives (Sutherland, 2002). The EPS also contribute to various physiological activities in human beings as anti-tumor, anti-viral and antiinflammatory agents and can act as inducers for interferon, platelet aggregation inhibition, and colony stimulating factor synthesis (Lin and Zhang, 2004). Microbial EPS are water-soluble polymers and may be ionic or non-ionic in nature. The repeating units of these EPS are very regular, branched or unbranched, and interconnected by glycosidic linkages. In the recent years, a major emphasis has been laid on the search for novel microbial EPS and a wide variety of microbial strains are reported to produce polysaccharides with varied compositions and interesting and useful properties (Sutherland, 2002). The different biopolymers which have been extensively studied and being currently marketed as commercial ⇑ Corresponding author. E-mail address:
[email protected] (S. Qirong). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.02.033
products include xanthan from Xanthomonas campestris, gellan from Sphingomonas paucimobilis, bacterial alginates secreted by Pseudomonas species and Azotobacter chrococcum, bacterial cellulose from Acetobacter xylinium, hyaluronic acid from Streptococcus equii and succinoglycan from Rhizobium (Sutherland, 2002). Paenibacillus polymyxa has also attracted great interest because of its great biotechnological potential in different industrial processes and sustainable agriculture. The genus Paenibacillus consists of over 89 species of facultative anaerobes, endospore-forming, neutrophilic, periflagellated heterotrophic and low G + C Grampositive bacilli (Raza et al., 2008). The P. polymyxa strains exist at different places such as forest trees, soils, roots, rhizosphere of various crop plants and marine sediments (Raza et al., 2008) and produce a wide variety of different polysaccharides with diverse physiological and biotechnological functions (Han and Clarke, 1990; Lee et al., 1997; Liu et al., 2009). The P. polymyxa strain P13, was reported as an EPS producer which exhibited significant biosorption capacity of Cu2+ produced in several industries (Acosta et al., 2005). Bioflocculation of high-ash Indian coals using P. polymyxa showed 60% decrease in ash, suggesting that selective flocculation of coal is possible (Vijayalakshmi and Raichur, 2002). The P. polymyxa strain JB115 was isolated from Korean soil as a glucan producer for the development of animal feed additives showing activities of biological response modifiers, natural immuno-modulators and a potential anti-tumor agent for livestock (Jung et al., 2007). Until now, there are only some reports on the culture conditions for the production of EPS from P. polymyxa (Han and Clarke, 1990; Lee et al., 1997). The production of EPS is not species specific and
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each strain of same species produces different kinds of EPS with different biotechnology properties. For that reason, maximum strains should be assessed to find out EPS with extraordinary physiological and biotechnological properties. Therefore, objectives of the present study were to optimize, purify and characterize the EPS produced by P. polymyxa strain SQR-21. The SQR-21 was isolated from the rhizosphere of a healthy cucumber plant in Fusarium wilt diseased field. This strain produces fusaricidin type antifungal compounds and hydrolytic enzymes and effectively controls the Fusarium wilt of cucumber and watermelon (Raza et al., 2009). The EPS isolated from SQR-21 was purified and its chemical composition was determined. For the optimization of EPS production first, single factor experiments were carried out, later, considering the effective factors, response surface methodology (RSM) was employed which is a well-known method applied in the optimization of medium compositions and other critical variables responsible for the production of bio-molecules (Raza et al., 2010a,b). The antioxidation potential of EPS was also investigated. 2. Methods 2.1. Bacterial strain The bacterial stain P. polymyxa SQR-21 (GenBank Accession No. FJ600406; China General Microbiology Culture Collection Center (CGMCC), Accession No. 1544) was provided by Jiangsu Provincial Key Lab of Organic Solid Waste Utilization, Nanjing, China. The SQR-21 bacterial culture was maintained on Luria Bertani (LB) agar plates and stored at 80 °C in tryptic soya broth (TSB) containing 20% glycerol for further use. 2.2. Culture conditions and EPS analysis For the EPS production by SQR-21, tryptone medium (tryptone, 10; NaCl, 5 and sucrose, 10 g L1; pH 7.5) was used. For the quantification of EPS, the cell free liquid cultures were mixed with two volumes of cold ethanol to avoid the quantification of residual sugar contents of the culture medium. The precipitates were collected by centrifugation and dissolved in distilled water. The EPS concentrations were determined by the phenol–sulfuric acid method using glucose as standard (Dubois et al., 1956). 2.3. One-factor-at-a-time experiments For the optimization of EPS production, factors affecting cell growth and EPS production were investigated using one-factorat-a-time method. The time course experiment was carried out in 1-L flask containing 250 ml of the culture medium up to seven days. To determine the optimum initial pH for EPS production, the pH (5–8) of the medium was adjusted by the addition of 1 M HCl and 1 M NaOH before sterilization. To find out the optimum temperature for EPS production, liquid cultures were incubated at 20, 22, 24, 26, 28, 30 32, 34 and 36 °C, respectively. To optimize the medium composition, different kinds of carbon and nitrogen sources were chosen to supplement the basal medium. In all above experiments, the sterilized medium was inoculated with 100 ll of over night culture of SQR-21 in tryptone broth. In previous reports, seven metal ions (Ca2+, Ni2+, Mn2+, Cu2+, Mg2+, Zn2+ and Fe3+) were evaluated for their effects on EPS production (Raza et al., 2010a,b). According to these results, four metal ions (Ca2+, Ni2+, Cu2+ and Fe3+) were chosen for fractional factorial design experiment. 2.4. Fractional factorial design (FFD) Six factors, having effect on EPS production, were identified by one factor-at-a-time experiments. Among these, the factors having
most significant effect on EPS production were screened by FFD. The parameters for six factors (galactose, yeast extract, Ni2+, Fe3+, Ca2+ and Cu2+) were chosen as main variables and designated as x1, x2, x3, x4, x5 and x6, respectively. The low, middle and high levels of each variable were designated as 1, 0, and +1. According to this design, a total 19 experiments including three replications were conducted. FFD was based on the following first-order polynomial model:
Y ¼ b0 þ
X
ð1Þ
bixi
where Y was the predicted response (EPS production), b0 was the model intercept, bi was the linear coefficient and xi was the level of independent variable. From the regression analysis, the variables significant at 95% of confidence level (P < 0.05) were considered to have significant effects on EPS production. 2.5. Path of steepest ascent experiment The variables screened by FFD (galactose, Fe3+, Ca2+) were further optimized by using steepest ascent design. The experiment was moved along the steepest ascent path in which the concentrations of galactose, Fe3+ and Ca2+ were increased according to their coefficients in the above mentioned first-order model until the response was not increased any more. This point would be near the optimal point and could be used as a center point to optimize the medium by CCD. 2.6. Central composite design (CCD) In the end, central composite design was employed to optimize the three most significant variables (galactose, Fe3+ and Ca2+). These three independent variables were studied at five different levels (1.68, 1, 0, +1, +1.68) and 20 experiments, containing six replications at the center point, were conducted for estimating the purely experimental uncertainty variance in triplicates. The behavior of the system was explained by the following seconddegree polynomial equation:
Y ¼ B0 þ
n X
Bi xi þ
i¼1
n X
Bij xi xj þ
i
