Effect of carbon and nitrogen sources on the ...

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Scholars Research Library Archives of Applied Science Research, 2012, 4 (6):2507-2512 (http://scholarsresearchlibrary.com/archive.html)

ISSN 0975-508X CODEN (USA) AASRC9

Effect of carbon and nitrogen sources on the production of xanthan gum from Xanthomonas campestris isolated from soil. Kumara Swamy M*, Khan Behlol A, Rohit K C1, Purushotham B2 *Padmashree Institute of Management and Sciences, Kommagatta, Kengeri, Bangalore- 560060, India 1 Sapthagiri College of Engineering, #14/5, Hesaraghatta Main Road, Bangalore – 560057, India 2 East West College of Science, Vishwaneedam Post, Bangalore- 560091, India _____________________________________________________________________________________________ ABSTRACT This study evaluated the influence of various carbon and nitrogen sources on the production of xanthan gum by Xanthomonas campestris isolated from soil sample. Yeast extract as nitrogen source gave highest yield of xanthan gum (4.8g/l) compared to any other nitrogen sources (beef extract, ammonium sulphate, peptone and tryptone) used in the production medium. The biomass yield (24.5g/l) was also highest with the use of yeast extract. However found no significant variations with other nitrogen sources. Among the carbon sources, sucrose produced maximum xanthan gum (3.6g/l). Though galactose yielded higher biomass (26.6g/l), the xanthan gum was found to be lesser in the medium (2.6g/l). The results indicate that, the production of xanthan gum was very much influenced by the carbon and nitrogen sources used in the production media. Keywords: Xanthan gum, nitrogen, carbon, Xanthomonas, biopolymer. _____________________________________________________________________________________________ INTRODUCTION Xanthan or xanthan gum is one of the most important microbial polysaccharide [1]. It is produced commercially by using microbial fermentation with the bacterium Xanthomonas campestris. Xanthan structure is based on cellulose back bone having alternate glucosyl residues substituted by a triasaccharide chain of D-mannose, D-glucouronic acid and a terminal D-mannose. It is a natural polysaccharide and an important industrial biopolymer. It is an attractive alternative for the replacement of traditional gums obtained from plants and marine algae by chemical extraction process. Xanthan exhibits desirable properties like high viscosity at low concentrations, pseudoplasticity and insensitivity to a wide range of temperature, pH, and electrolyte concentrations. Because of its special rheological properties, xanthan is used in food, cosmetics, pharmaceuticals, paper, paint, textiles, and adhesives and otherwise in the oil and gas industry [1,2]. The toxicological and safety properties of xanthan gum for food and pharmaceutical applications have been extensively researched. Xanthan is non-toxic and does not inhibit growth. It is nonsensitizing and does not cause skin or eye irritation. On this basis, xanthan has been approved by the United States Food and Drug Administration (FDA) for use in food additive without any specific quantity limitations [2,3]. Worldwide consumption of xanthan is approximately 23 million kg/year estimated to grow continuously at an annual rate of 5%-10% [4]. Most of the work regarding to production of xanthan gum has been reported with strains from cultures collection mainly X. campestris NRRL-B 1459 or ATCC 13951 and their derivatives. Production of xanthan is effected by the

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Kumara Swamy M et al Arch. Appl. Sci. Res., 2012, 4 (6):2507-2512 ______________________________________________________________________________ use of various production media components [5, 6, 7, 8]. Most commercial production method for xanthan gum uses glucose or invert sugars, and most industries prefer batch processes than continuous processes [9]. Also production and the properties of xanthan gum are influenced by bacterial strain [4, 10]. The influence of temperature, pH, time of fermentation and rate of agitation also influences the production of xanthan gum [11, 12, 13, 14]. Hence the present investigation was undertaken to study the effect of various carbon and nitrogen sources on the production of xanthan gum by X. campestris. MATERIALS AND METHODS Microorganism The pure culture of Xanthomonas campestris isolated from soil was obtained from the Department of Microbiology, University of Mysore and subcultured regularly in our lab. The media used for the subculturing was Yeast dextrose calcium carbonate agar (YDCC). The composition of the media is as follows; Yeast extract: 10g, Calcium carbonate: 20g, Dextrose: 20g, Agar: 20g, Distilled water: 1000ml. The YDCC agar slants were made and they were kept for incubation for 24hrs at370C to check the contamination. The slants without contamination were selected and then inoculated with the pure culture of X. campestris and then incubated at 370C for 3 days. After 3 days, the agar slants were observed for orange colour growth of X. campestris. This culture was then used as the inoculum for xanthan gum production. Inoculum preparation and xanthan production The inoculum was prepared by transferring cells from 72 h YDCC agar slants incubated at 37°C to 250 ml Erlenmeyer flasks containing 50 ml of YDCC broth (pH 7.0) and incubated at 370C for 48 hours. One ml of the cultures was then transferred to 49ml of production medium g/l (Glucose 20.0, Yeast extract 3.0, MgSO4 0.2, K2HPO4 5.0, pH 7.2) in 100ml Erlenmeyer flask. The cultures were then incubated at 370C for 96 hours. Samples were withdrawn every 24 hour and analyzed for concentrations of xanthan. Effect of nitrogen and carbon sources on xanthan production To study the effect of different carbon sources on xanthan production, the production medium was added with different carbon source (2%) such as glucose, galactose, sucrose, maltose and lactose for xanthan production. To study the effect of different nitrogen sources on xanthan production, nitrogen source (0.3%) such as beef extract, ammonium sulphate, peptone, tryptone and yeast extract were used in production medium. Biomass estimation Growth in the medium was estimated by measuring the dry weight of washed cell mass. 5ml broth was separated in a centrifuge at 10,000 rpm for 15 minutes. After centrifugation, two fractions were formed, supernatant containing xanthan gum, and biomass deposited as a pellet. The biomass pellet was resuspended with deionized water for washing and then recentrifuged to reprecipitate the biomass. The biomass deposited at the bottom of tubes was dried in the oven at 60 °C for two hours and weighed to get the dry mass. Xanthan estimation The polymer was recovered from the fermentation medium by centrifugation of 5ml broth at 10,000 rpm for 15 minutes. The pellet was decanted and the supernatant was precipitated with 2 to 3 volumes of isopropyl alcohol with shaking to precipitate out the polysaccharide. The precipitate was separated by centrifugation at 6000 rpm for 15 minutes. The residue was transferred to pre-weighed micro-centrifuge tube and dried for 18 hours in hot air oven at 600C. The micro-centrifuge tube was cooled to 300C for 1 hour and the dry weight gave the xanthan concentration of the fermented broth. The concentration of xanthan gum was determined as the dry weight of xanthan gum per litre culture medium. RESULTS AND DISCUSSION Xanthan gum is produced commercially produced by using microbial fermentation with the bacterium Xanthomonas campestris NRRL-B 1459 or ATCC 13951. Microbial production of xanthan is effected by the use of various production media components [5, 6, 7, 8], different bacterial strain [4, 10] and other biochemical parameters such as temperature, pH and rate of agitation [11, 12, 13, 14]. Culture conditions such as pH, temperature, source of carbon, nitrogen, protein and metal ions are known to influence the synthesis and secretion of extracellular enzymes by


Kumara Swamy M et al Arch. Appl. Sci. Res., 2012, 4 (6):2507-2512 ______________________________________________________________________________ microorganisms [15]. Hence the present investigation was undertaken to study the effect of various carbon and nitrogen sources on the production of xanthan gum by X. campestris isolated from soil sample. X. campestris was cultivated in the production medium containing various nitrogen sources such as beef extract, ammonium sulphate, peptone, tryptone and yeast extract. The results are shown in the Figure 1. From the result it was found that, yeast extract was the best substrate for xanthan production by X. campestris when compared to other nitrogen sources. The use of yeast extract produced the highest xanthan production (4.8 g/l). Similarly Aarthy Palaniraj et al., have reported highest xanthan gum production when yeast extract was used as a nitrogen siource in the media [7]. The next best nitrogen source was found to be beef extract which resulted 3.2g/l of xanthan gum. The biomass yield was also highest with the use of yeast extract. But found no significant differences with other nitrogen sources (Figure 1 & 2).

Xanthan gum (g/l)

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Nitrogen source Figure 1: Production of Xanthan gum by Xanthomonas campestris with respect to different nitrogen sources.

X. campestris was cultivated in the production medium containing various carbon sources such as glucose, galactose, sucrose, maltose and lactose. When the cells were grown in the sucrose containing medium, the xanthan production (3.6 g/l) was the highest among those tested (Figure 3 & 4). This present result is in agreement with Souw and Demain (1979) and Kawahara and Obata (1998) who stated that, maximum xanthan production was obtained when sucrose was used as a carbon source using X. campestris NRRL-B 1459 and X. campestris pv. translucens, respectively [16, 17]. The use of glucose in the media produced 3.2g/l of xanthan gum. The least xanthan gum was found in the media containing lactose (2.2g/l). This may be due to the low activity of galactosidase produced by X. campestris MU1 to break lactose into glucose and galactose [18, 19]. Though galactose yielded less xanthan gum the biomass production (26.6g/l) was found to be the highest in the medium.


Kumara Swamy M et al Arch. Appl. Sci. Res., 2012, 4 (6):2507-2512 ______________________________________________________________________________

Biomass (g/l)

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Nitrogen source Figure 2: Biomass accumulation of Xanthomonas campestris with respect to different nitrogen sources.

Xanthan gum production (g/l)

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Carbon sources Figure 3: Production of Xanthan gum by Xanthomonas campestris with respect to different carbon sources.


Kumara Swamy M et al Arch. Appl. Sci. Res., 2012, 4 (6):2507-2512 ______________________________________________________________________________

Biomass (g/l)

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Carbon sources Figure 4: Biomass accumulation of Xanthomonas campestris with respect to different carbon sources.

CONCLUSION The optimization and production of the xanthan gum with different nitrogen sources such as the beef extract, ammonium sulphate, peptone, tryptone and yeast extract were compared and yeast extract showed highest xanthan gum production of 4.8g/l. Different carbon sources such as glucose, galactose, sucrose, maltose and lactose were optimized for xanthan gum production and found that sucrose as the best carbon source. Acknowledgements Authors gratefully acknowledge the Department of Microbiology, University of Mysore, India for providing the culture Xanthomonas campestris. REFERENCES [1] IW Sutherland, Extracellular polysaccharides. In: Biotechnology, vol 6, 2nd ed., (Rehm HJ and G Reed, Eds.) VCH, Weinheim, 1996, pp. 613-657. [2] JF Kennedy, IJ Bradshaw, Production, properties and application of xanthan. In:Bushell ME (ed) Progress in industrial microbiology, Elsevier, Amsterdam, 1984, 19: 319-371. [3] S Rosalam, R England, Enzyme Microbial Technology. 2006, 39(2), 197-207. [4] AS Moreira, JLS Vendruscolo, C Gil-Turnes, CT Vendruscolo, Food Hydrocol. 2001, 15, 469-474. [5] F Garcia-Ochoa, E Gomez, Biochem. Eng.1998, 1, 1-10. [6] F Garcia-Ochoa, VE Santos, JA Casas, E Gomez, Biotechnol. Adv. 2001, 18, 549-579. [7] Aarthy Palaniraj, Vijayakumar Jayaraman, Sekar Babu Hariram, International Journal of Advanced Biotechnology And Research. 2011, 2(3), 305-309. [8] AS Amanullah, S Satti, AW Nienow, Biotechnol. Prog. 1998, 14, 265-269. [9] F Letisse, P Chevallereau, JL Simon, ND Lindley, Appl. Microbiol. Biotechnol. 2001, 55, 417-422. [10] H Rodriguez, L Aguilar, J. Ind. Microbiol. Biochem. 1997, 18(4), 232-234. [11] CH Shu, ST Yang, Biotechnol. Bioeng. 1990, 35, 454-468. [12] ME Esgalhado, JC Roseiro, CMT Amaral, Process Biochem. 1995, 30(7), 667-671.


Kumara Swamy M et al Arch. Appl. Sci. Res., 2012, 4 (6):2507-2512 ______________________________________________________________________________ [13] F Cacik, RG Dondo, D Marques, Comput. Chem. Eng. 2001, 25, 409-418. [14] HU Peters, H Herbst, PGM Hesselink, H Lunsdorf, A Schumpe, WD Deckwer, Biotechnol. Bioeng. 1989, 34, 1393-1397. [15] It Jamilah, Anja Meryandini, Iman Rusmana, Antonius Suwanto, Nisa Rachmania Mubarik, Microbiol. Indones. 2009, 3(2), 67-71. [16] P Souw, AL Demain, Appl. Environ. Microbiol. 1979, 37, 1186-1192. [17] H Kawahara, H Obata, Appl. Microbiol. Biotechnol. 1998, 49, 353-358. [18] JF Fu, TH Tseng, Appl. Environ. Microbiol. 1990, 56, 919-923.
