influence of nitrogen sources and agitation in xanthan gum - BioIT ...

INFLUENCE OF NITROGEN SOURCES AND AGITATION IN XANTHAN GUMon a variety of nutrients, particularly the nitrogenand carbon sources, with glucose and sucrose asthe most frequently used carbon sources [6,7].Most commercial production method for xanthangum uses glucose or invert sugars, and mostindustries prefer batch processes than continuousprocesses [8]. Other substrates have also beentested, such as sucrose, hydrolyzed rice, barley,corn flour, acid whey, sugar cane molasses,coconut juice, sugar cane, etc., but glucose is stillthe best in terms of product yield, supply, andproduct quality [5, 9-11]. It has been found thatthe production and the properties of xanthan gumare influenced by bacterial strain [12-13], culturemedium [8, 14-15], temperature [16-17], pH [18],time of fermentation [19], and agitation rate [20-21].The purpose of this study was to optimize thexanthan gum production by Xanthomonascampestris with respect to nitrogen sources andeffect of agitation.[II] MATERIALS AND METHODS2.1. Microorganism and inoculum preparationXanthomonas campestris NCIM 2961 wasobtained from NCIM, Pune, and used throughoutthis study. The strain was maintained on nutrientagar slant containing (g/L) glucose 10; Maltextract 3; Yeast extract 3; and Peptone 5; pH=7grown at 30 °C for 24 hours and stored at 4 °C.Actively growing cells from a newly preparedslant were inoculated into the liquid medium in250 ml Erlenmeyer flask. The culture wasincubated at 30-35 °C for 24 hours in anincubator shaker. The liquid culture was used toinoculate the final fermentation medium.2.2. Fermentation experimentsAll the fermentation experiments were conductedin a 2L bioreactor (Biostat-B, Sartorius,Germany). The production medium wascomposed of glucose (25 g/L), yeast extract (3.0g/L), KH 2 PO 4 (2.0 g/L), MgSO 4 (1.0 g/L) andantifoam agent (0.1 mL/L). The fermentationmedium without the carbon source was sterilizedin the fermentation vessel. The carbon source wassterilized separately and then asepticallyintroduced into the vessel. During fermentationruns, the dissolved oxygen concentration wasmaintained at 10-30 % of saturation value byincreasing the stirrer speed as needed, whilekeeping constant 1 vvm (air volume/mediumvolume/minute) airflow rate. Temperature wasmaintained constant at 28 °C for 24 h. The pH ofthe medium was held at pH=7.0 by adding 1 MHCl/1M NaOH. During the process, theconcentrations of the cells, glucose, and xanthangum were measured in the culture medium in fivesamples of 50 mL each. All fermentation runswere conducted in triplicate.2.3. Determination of biomass and xanthangum concentrationThe fermentation broth produced by the batchprocesses were diluted using distilled water tolower the viscosity, and then 20 mL aliquots weretransferred into micro centrifuge tubes. Themicro-centrifuge tubes containing aliquots werecentrifuged at 10,000 rpm for 30 minutes at 4 °C.After centrifugation, two fractions were formed,supernatant containing xanthan gum, and biomassdeposited as a pellet. The biomass pellet wasresuspended with water for washing and thenrecentrifuged to reprecipitate the biomass. Thebiomass deposited at the bottom of tubes wasdried in the oven at 60 °C for two hours andweighed to get the dry mass to show the relativeperformance of the cotton fibre in retaining thecells. Supernatants were mixed with 2/3 (v/v)isopropanol, re-centrifuged at 10,000 rpm for 45minutes at 4 °C to completely precipitate xanthangum before removing the solvent and water fromthe top portion. The precipitated xanthan gumcollected from all samples was dried overnight inthe oven at 50 to60 °C in the pre weighed micro-centrifuge tube.Aarthy Palaniraj et al. 306