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1 Available online at Life Science Archives (LSA) ISSN: Volume 3; Issue - 5; Year 2017; Page: DOI: /lsa Research Article OPTIMIZATION OF FERMENTATION CONDITIONS FOR THE PRODUCTION OF XANTHAN BY Xanthomonas campestris MTCC 2286 R. Krishnaveni 1 * and S. Balakumar 2, Department of Microbiology, A.V.C College (Autonomous), Mayiladuthurai, Tamil Nadu, India. Srinivasa Ramanujan Centre, SASTRA University, Kumbakonam, Tamil Nadu, India. Abstract Xanthan is an exopolysaccharide produced by the plant pathogenic bacterium Xanthomonas campestris. Owing to its unique physical and chemical properties, Xanthan gum has attracted particular attention in various industries as emulsifiers, stabilizers, binders, gelling agents, thickeners etc. In the present study, Xanthan gum was produced from Xanthomonas campestris MTCC 2286 and optimized using various parameters namely, the Carbon sources (Glucose, Sucrose, Fructose, Maltose and Starch soluble), the Nitrogen sources (Yeast extract, Ammonium chloride, Ammonium sulphate and Sodium nitrate) at various concentrations, ph, temperature, the size of the inoculums, the period of fermentation and the solvent used for recovery. With the optimized media components, the yield of Xanthan is found to be 22.5 gram per ml. Further, the so produced Xanthan was subjected to NMR and IR studies. Article History Received : Revised : Accepted: Introduction Exopolysaccharides produced by a variety of microorganisms are chemically well defined and have attracted worldwide attention due to their novel and unique physical properties. These exopolysaccharides find multifarious industrial applications in foods, pharmaceuticals and other industries as emulsifiers, stabilizers, binders, gelling agents, lubricants and thickening agents. * Corresponding author: R. Krishnaveni E.mail: rkvenimb@gmail.com Key words: Exopolysaccharides, Xanthan, Xanthomonas campestris and Optimization. These biopolymers are rapidly emerging as industrially important and are gradually becoming economically competitive with natural gums produced from marine algae and other plants. Their physical and chemical characteristics show little variability and they are not vulnerable to variations in climatic cultivation, production and pollution conditions. Besides, gums of microbial origin are susceptible to natural biodegradation, promoting little damage to the environment and diminishing pollution (Silvia and Crispin, 2006). Among those biopolymers, which is commercially produced on a large scale and which is subjected to extensive studies is the xanthan gum. This polymer represents the fastest growing segment of the polysaccharide industry. The

2 Krishnaveni/Life Science Archives (LSA), Volume 3, Issue 5, 2017, Page 1145 to largest growth is expected to be in the food applications (8.3 % per year), where demand for natural gums is decreasing. Worldwide consumption of Xanthan is approximately 23 million kg/year, estimated to grow continuously at an annual rate of 5 % - 10 %. Xanthan gum is a water soluble heteropolyscchaaride composed of glucose, mannose and glucuronic acid units that is commercially produced using glucose or sucrose (Sayyed Vahid et al., 2015). Xanthan gum is a GRAS (Generally accepted as safe) product and approved by FDA (Tahera Ghashghei et al., 2016). Xanthan gum is produced by a pure culture fermentation of a carbohydrate by a plant pathogenic bacterium, Xanthomonas campestris. The Xanthomonas campestris is a Gram negative bacteria, motile with a polar flagellum, chemoorganotrophic in nutrition, catalase positive and oxidase negative. The colonies are usually yellow in colour and this was due to the presence of Xanthomonadins or Bromynated aryl polyenes. Xanthan production was generally carried out as aerobic submerged fermentation process that runs for hrs at C with a ph of 7. The Xanthan gum finds various applications in food industry as suspending, thickening, stabilizing and gelling agent in juices, drinks, chocolates, agricultural industry improving the flow ability in fungicides, herbicides and insecticides formulations, petroleum industry in enhanced oil recovery and also used in ceramics, paper manufacturing, textiles, cosmetics, tooth pastes, paints and inks. Optimization of fermentation conditions for xanthan production is particularly important in view of wide industrial applicability of xanthan gum. Xanthan fermentation is probably the most complex fermentation process in terms of rheological property and associated mixing bottle neck in production xanthan gum. In order to improve the productivity and shorter fermentation time, extensive research work has been carried out in xanthan fermentation conditions. Most of the literature reported works with reference to Xanthomonas campestris NRRL B1459 and its derivatives. In the present study, a strain of Xanthomonas campestris MTCC 2286 from IMTECH (Institute of Microbial Technology), Chandigarh was obtained and screened for its ability to produce Xanthan gum. The purpose of this study was to optimize culture conditions to produce xanthan by Xanthomonas campestris MTCC 2286 with respect to several operating variables such as carbon source and concentration, nitrogen sources, ph, temperature, culture volume, period of fermentation and solvents used for recovery. A high recovery of xanthan was attempted in batch fermentation by employing optimized conditions. NMR and IR spectroscopic studies were carried on the xanthan gum produced in the optimized media. 2. Materials and Methods Bacterial Strain and maintenance Xanthomonas campestris MTCC 2286 strain was obtained from IMTECH (Institute of Microbial Technology), Chandigarh, India. The dried pellet in a sterile glass ampoule was dehydrated in the skim milk before being streaked on the YDC (Yeast Dextrose Calcium carbonate) agar slant. The composition of YDC agar is as follows: 10 g/l Yeast extract, 20 g/l Glucose, 20 g/l Calcium carbonate and 20 g/l agar. The agar slants were incubated at 28 C for 24 hrs. Then, they were stored at 4 C. The strain was maintained in an active and stable condition by transferring them every fortnight a month into a new agar slant. Gram staining is done periodically to check the purity of the culture. Inoculum media The YDC broth (Yeast Dextrose Calcium carbonate) - 10 g/l Yeast extract, 20 g/l Glucose and 20 g/l Calcium carbonate. Production media Yeast extract - 3 g/l, KH 2 PO 4-2 g/l, K 2 HPO 4-2 g/l, MgSO 4-1 g/l and Glucose - 20 g/l. Inoculum preparation Actively growing cells from a newly prepared slant was inoculated into the liquid medium into 250 ml Erlenmeyer flask. The culture was incubated at C for hrs in an

3 Krishnaveni/Life Science Archives (LSA), Volume 3, Issue 5, 2017, Page 1145 to Orbital shaker. The liquid culture was used to inoculate the final fermentation medium. Fermentation Fermentation experiments were carried out in 250ml Erlenmeyer flask at 28 C and 200 rpm at a ph of 7.0. About 5-10% v/v of inoculum was added to the fermentation medium and incubated for 72 hours. The shake flask experiments were carried out in triplicates. Analytical methods Determination of bacterial growth The bacterial growth was estimated when they reach an optical density (600 nm) of >0.8. Determination of Xanthan gum The final fermentation broth was centrifuged at 10,000-15,000 rpm for 30 minutes to remove the cells. The supernatant was collected and the gum was precipitated out of the supernatant fluid with approximately three volumes of isopropyl alcohol. Then, the gum was dried at 55 C until reaching constant weight. The production of Xanthan was evaluated by the weight of the dry product per liter of fermented broth and the average was expressed in g/l. Determination of Viscosity The ability of the bacteria to produce exopolysaccharide was determined by increase in the viscosity of the fermentation medium and hence the viscosity of the fermentation medium before and after fermentation was determined using Oswald s viscometer. NMR and IR analysis Xanthan was subjected to NMR studies. The spectroscopy work was done at SASTRA (Shanmuga Arts Science, Technology and Research Academy) University, Thanjavur, Tamil Nadu, India. About 10 mg of the sample dissolved in 5 ml of D 2 O solvent was taken in a Wilmad tube of 5 mm in diameter. The spectra were carried out at 30 C in D 2 O on a Bruker spectrometer. The spectra were recorded at a transmission frequency of 300 MHz with 16 runs. The magnetic strength and the running time were found to be 7.05 Tesla and 1.59 minutes respectively. The software used in this spectrometer was BSMS (Bruker Smart Magnetic System). IR spectroscopy on Xanthan was also performed at SASTRA University, Thanjavur, India. The IR spectroscopy of xanthan gum was run in KBr disc using Perkin - Elmer FT IR spectrophotometer in the frequency range of 4000 to 450 cm -1 and the spectra were recorded. 3. Results and Discussion Based on the results obtained, it was clear that the optimum production of Xanthan gum by Xanthomonas campestris MTCC 2286 was in a sucrose medium with a concentration of 3 % that runs for 96 hrs at 28 C with a ph of 6.5. The Xanthan gum in the fermented broth was recovered using Isopropyl alcohol. A maximum yield of about 22.5 g/l was obtained from the optimized media. Carbon source is the most important component of the media used from the production of Xanthan, because it directly affects the production yields, composition, structure and properties of Xanthan gum. In the present study different carbon sources namely glucose, sucrose, fructose, maltose and soluble starch were studied at a concentration ranging from 1 % to 3 %. But, best carbon source was found to be glucose and sucrose. To prove the above statement, it was seen that sucrose was found to be the best source of carbon for Xanthan production. Suresh and Prasad (2005) used sucrose as the sole source of carbon source at a concentration of 4.5 % and were able to achieve a maximum yield in their studies. By using Sucrose based medium, they were able to obtain a yield of about 54 g/l but the yield was found to be maximum at a 3 % concentration for Xanthan production in our work Similar to the present work, Krishna Leela and Gita Sharma (2000) did an extensive study on various parameters on Xanthan gum production. They took a wide range of parameters such as carbon source, nitrogen source, ph, temperature, age of inoculum, percentage of inoculum and period of fermentation. Their study showed that a concentration of 2 % of all sugars namely the glucose, sucrose, maltose, fructose and starch soluble tested were found to yield a Xanthan gum

4 Krishnaveni/Life Science Archives (LSA), Volume 3, Issue 5, 2017, Page 1145 to of about g/l, g/l, g/l, g/l and g/l respectively. Contradictory to their study, the Xanthan gum yield for glucose, sucrose, fructose, maltose and starch soluble was found to be 16.9 g/l, 17.8 g/l, 10.0 g/l, 14.3 g/l and 11.3 g/l respectively at a concentration of 3 %. Following carbon sources, nitrogen is the most important medium component for exopolysaccharide production. Abundant secretion of exopolysaccharide is usually noticeable when bacteria are supplied with abundant carbon source and minimal nitrogen source. Though nitrogen is important for various metabolic activities, a minimal concentration would be enough to give a good yield of Xanthan. In the present study, nitrogen sources namely yeast extract, salts of ammonium and nitrate were studied at a concentration of 0.2 to 0.4 %, with the yeast extract giving a maximum yield of about 16.3 g/l of Xanthan gum at 0.4 % concentrations. The other nitrogen sources were also able to give a precisely a good yield of Xanthan of about 16.1 g/l with sodium nitrate, followed by ammonium chloride and ammonium sulphate with an yield of about 15.3 g/l and 14.8 g/l. In this study, nitrate salt gave a good yield similar to yeast extract and this was supported by Fabien Lettisse et al. (2001). Bajaj et al. (2006) supported the present work stating that yeast extract was the best suited nitrogen source for the production of pollulan from Sphingomonas paucimobilis when compared to other nitrogen sources. A high yield of Xanthan of about 16.3 g/l was obtained when yeast extract was used at a concentration of 0.4 %. This statement was completely against the work of Yang Ming Lo et al. (1997) according to whom the Xanthan gum was maximum with an yield of about 32 g/l, when yeast extract was in the medium at a concentration of 0.3 %. Sanchez et al. (1997) used ammonium phosphate that gave a Xanthan of about 15.3 g/l at a concentration of 0.7 % whereas ammonium chloride and sulphate gave a polymer production of 15.3 and 14.8 g/l respectively. The ph influenced the physiology of the microorganism significantly by affecting the nutrient solubility and uptake, enzyme activity, cell membrane, morphology, by product formation and oxidative - reductive reactions. In the present work, the Xanthan yield was found to be good at ph 6.5. This coincides with the work of Ishwar Bajaj et al. (2007) who found out that the ph varies from 6.5 to 7 during the production of Gellan. Most of the work on Xanthan production supported a better yield at a ph ranging between 6.5 and 7. Garcia - Ochoa et al. (2000) stated that ph control was not necessary for Xanthan production. But, our work was supported by Silvia ad Crispin (2006) who demonstrated that the polysaccharide production increased from ph 5 to 7. Temperature is also an important parameter in Xanthan production. In the present work, the polymer production increased from 26 C to 30 C and decreased slightly at 32 C. The yield increased from 14.6 to 17.9 g/l and reduced to g/l respectively. Our work is supported by Krishna Leela and Gita Sharma (2000). Accordingly a temperature of about C was found to give a Xanthan production around 15 g/l. The work was also supported by Garcia - Ochoa et al. (2000) that for optimum gum production, a temperature range of about C was proved to be optimum. Two types of media were used for inoculum development namely the YDC media and LB media. When YDC media was used as inoculum media, the polymer production was found to be 17.7 g/l and the inoculum developed on LB gave equally a yield of about g/l. This was opposed by the work of Ashraf et al. (2008) as there was a very low production yield of 8.5 g/l on LB media. But our work on inoculum development on YDC was supported by Rosalam et al. (2008) giving a similar yield of about 20 g/l with respect to our work. The percentage of inoculum also affects the production of the Xanthan polymer. Normally a percentage of about 5 to 10 % v/v of inoculum is required. In our study a 10 % v/v of inoculum was

5 Krishnaveni/Life Science Archives (LSA), Volume 3, Issue 5, 2017, Page 1145 to used and gave a yield of about 20 g/l. This was supported by a number of authors. The work of Ashraf et al. (2008), Rajeswari et al. (1995) and Maria Rodrigues et al. (2000) clearly revealed that an inoculum of 10 % gave a good yield. Contradictory to our study, some work done by Garcia - Ochoa et al. (2000), Suresh and Prasad (2005), El-Tayeb and Khodair (2006) stated that a 5 % v/v of inoculum gave a higher yield of Xanthan gum. The work by Krishna Leela and Gita Sharma (2000) supported our results and they reported a yield around 15 g/l when a 10 % v/v of inoculum was used. The period of incubation also have an effect of Xanthan production. Most of the literature states that a period of 72 to 96 hrs was found to give a maximum yield. In the present work, the yield was found to be 21 g/l at the end of 96 hrs of fermentation period. This was supported by Krishna Leela and Gita Sharma (2000) who reported a value of a maximum yield at the end of 96 hrs of fermentation. In contrary to our work, Xanthan production by Maria et al. (2000) and Suresh and Prasad (2005) reported a maximum yield at 72 hrs. Even there are reports of maximum yield at 54 hours by Borges et al. (2008). Recovery of Xanthan from the fermented broth was achieved by using water miscible solvents namely acetone, isopropyl alcohol and ethanol. Most of the literature supported the use of Isopropyl alcohol as the best solvent for Xanthan recovery. In our work, use of Isopropyl alcohol resulted in the recovery of Xanthan gum of about 21.3 g/l. This statement was supported by the work done by Garcia - Ochoa et al. (2000) on Xanthan production with a maximum yield. Figure - 1: Gram staining of Xanthomonas campestris Figure 2: Capsule staining of Xanthomonas campestris Figure 3: Growth of Xanthomonas campestris on YDC agar with yellow pigmentation

6 Krishnaveni/Life Science Archives (LSA), Volume 3, Issue 5, 2017, Page 1145 to Figure - 4: Effect of Carbon sources on Xanthan gum production Figure 5: Effect of various Nitrogen sources on Xanthan gum production

7 Krishnaveni/Life Science Archives (LSA), Volume 3, Issue 5, 2017, Page 1145 to Figure 6: Effect of ph on Xanthan production Figure 7: Effect of Temperature on Xanthan production

8 Krishnaveni/Life Science Archives (LSA), Volume 3, Issue 5, 2017, Page 1145 to Figure 8: Effect of different Inoculum media on Xanthan production Figure 9: Effect of Volume of inoculum media on Xanthan production

9 Krishnaveni/Life Science Archives (LSA), Volume 3, Issue 5, 2017, Page 1145 to Figure 10: Effect of Period of inoculum on Xanthan production Figure 11: NMR Spectrum of Xanthan

10 Krishnaveni/Life Science Archives (LSA), Volume 3, Issue 5, 2017, Page 1145 to Figure 12: IR Spectrum of Xanthan Table - 1: Effect of solvents on Xanthan recovery Solvents Xanthan yield (g/l) Original Replica Average Acetone Ethyl acetate Isopropyl Table 2: Determination of viscosity in the production medium S. Viscosity of the medium in poise No Before fermentation After fermentation Table 3: Optimized production of Xanthan at ph 6.5, temperature 30 C using Sucrose at a concentration of 3 % S. Xanthan yield (g/l) No Original Replica Average

11 Krishnaveni/Life Science Archives (LSA), Volume 3, Issue 5, 2017, Page 1145 to Conclusion Optimized production of media components resulted in a good yield of about 22.5 g/l of Xanthan at 6.5 ph, 30 ºC for a period of 96 hours. The NMR and IR studies showed some variations in the structure of xanthan suggesting it to be a derivative of xanthan. Presence of impurities conveys that proper purification should be performed and focused. Future work was directed towards characterization of Xanthan gum. 5. References 1) Abdul Hafez, A. M., Hemmat M. Abdelhady, Sharaf, M. S and El-Tayeb, T. S. (2007). Bioconversion of various Industrial by products and agricultural wastes into pollulan. Journal of Applied Sciences Research, 3(11): ) Anil Lachke. (2000). Xanthan A Versatile Gum. Resonance, ) Ashraf, S., Soudi, M. R and Sadaghizadeh, M. (2008). Isolation of a novel mutated strain of Xanthomonas campestris for Xanthan gum production using whey as the sole substrate. Pakistan Journal of Biological Sciences, 11(3): ) Ashtaputre, A. A and Shah, A. K. (1995). Studies on the exopolysaccharide from Sphingomonas paucimobilis. Journal of Current Microbiology, 31: ) Bajaj, L. B., Soudagar, S., Singhel, R. S. and Pandey, A. (2006). Statistical approach to optimization of fermentative production of gellan gum from Sphingomonas paucimobilis. Journal of Bioscience and Bioengineering, 102: ) Becker, A., Katzen, F., Puhler, A and Ielpi, L. (1998). Xanthan gum biosynthesis and application: A biochemical and genetic perspective. Applied Microbiology and Biotechnology, 50: ) Borges, C. D., Moreira, A., Vendruscolo, C.T and Ayub, M. A. Z. (2008). Influence of agitation and aeration in Xanthan production by Xanthomonas campestris pv pruni strain 101. Revista Argentina de Microbiologia, 40: ) Fabien Letisse, Paule Chivallereau, Jean Luc Simon and Nic D.Lindly. (2001). Kinetic analysis of growth and Xanthan gum production with Xanthomonas campestris on sucrose using sequentially consumed nitrogen sources. Applied Microbial Biotechnology, 55: ) Francisco Cacik, Rodolfo G. Dondo and Dardo Marques. (2001). Optimal control of a batch bioreactor for the production of Xanthan gum. Computers and chemical Engineering, 25: ) Garcia Ochoa, F., Santos, V. E., Casas, J. A and Gomez, E. (2000). Xanthan gum: production, recovery and properties. Biotechnology Advances, 18: ) Ishwar B. Bajaj, Shrikant A. Survase, Parag S. Saudagar and Rekha S. Singhal. (2007). Gellan gum: Fermentative production, Downstream processing and applications. Food Technology and Biotechnology, 45(4): ) Krishna Leela, J and Gita Sharma. (2000). Studies on Xanthan production from Xanthomonas campestris. Bioprocess Engineering, 23: ) Maria Nitschke and Vanessa Rodrigues. (2000). Effect of virulence and serial transfers of Xanthomonas campestris on Xanthan gum production. Brazilian Journal of Microbiology, 31: ) Rajeswari, K. V., Prakash, G and Ghosh, P. (1995). Improved process for Xanthan gum production using modified media and intermittent feeding strategy. Letters in Applied Microbiology, 21: ) Rosalam, S and England, R. (2000). Review of Xanthan gum production from unmodified starches by Xanthomonas campestris sp. Enzyme and Microbial Technology, 39: ) Sanchez, A., Ramirez, M. E., Tornes, L. G and Galindo, E. (1997). Characterization of Xanthan from selected a Xanthomonas strain cultivated under constant dissolved O 2. World Journal of Microbiology and Biotechnology, 13(4): ) Sayyed Vahid Niknezhad. (2015). Optimization of Xanthan gum production

12 Krishnaveni/Life Science Archives (LSA), Volume 3, Issue 5, 2017, Page 1145 to using cheese whey and response surface methodology. Bioscan, 24(2): ) Taherah Ghashghaei. (2016). Optimization of Xanthan gum production from grape juice concentrate using Plackette Burman design and response surface methodology, Life Science Archives, 3(1): ) Yang Ming Lo, Shang Tian Yang and David B. Men. (1997). Effects of yeast extract and glucose on Xanthan production and growth in cultures of Xanthomonas campestris. Applied Microbial Technology, 47: Quick Response Code Access this Article in Online Website DOI Number DOI: /lsa How to Cite this Article: R. Krishnaveni and S. Balakumar Optimization of Fermentation conditions for the production of Xanthan by Xanthomonas campestris MTCC Life Science Archives, 3 (5): DOI: /lsa