Levan production by Bacillus licheniformis NS032 using sugar beet molasses Gojgic-Cvijovic Gojgic Cvijovic Gordana1*, Jakovljevic Dragica1, Kekez Bran

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8 Levan production by Bacillus licheniformis NS032 using sugar beet molasses Gojgic-Cvijovic Gojgic Cvijovic Gordana1*, Jakovljevic Dragica1, Kekez Branka2, Beskoski Vladimir2, Vrvic M. Miroslav2 1Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Njegoseva 12, Belgrade, Serbia, 2Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia * ggojgic@chem.bg.ac.rs Table 1 Box-Behnen experimental design and results Introduction Levan is microbial exopolysaccharide composed of fructose units mainly linked by -(2,6)- glycosidic bonds with some -(2,1)-linked branch chains. This polysaccharide has great potential as novel functional biopolymer in foods, feeds, pharmaceutical and chemical industries due to its many outstanding properties like high water solubility, strong adhesivity, low viscosity and good biocompatibility [1]. Molasses is a by-product of the final crystallization stage in the process of sugar production. Because molasses is one of the best economical carbon source, it is widely used substrate in microbial fermentations including production of polysaccharides [2-4]. Objectives The aim of this study was to investigate the possibility of use of molasses as substitute for sucrose in levan production process. Run a A: B: C: Crude polysaccharide Sucrose, Molasses, Yeast observed, extract Crude polysaccharide predicted, Levan Levan Observed Predicted, b Methods For levan production Bacillus licheniformis NS032 was used [5]. Fermentations were conducted in base medium, BM (: sucrose 200, yeast extract 1.5, K2HPO4 3, KH2PO4 3, NaCl 1, MgSO4 x 7H2O 0.2, FeSO4 x 7H2O 0.001; ph 7.0) at 37 C without agitation for 4 days. Molasses used in this study was obtained from Sugar Factory, Senta, Serbia and has the following composition: total solids 77 8% sucrose 49.4%, 77.8%, 49 4% betain 5.9%, 5 9% total nitrogen 1.64%, 1 64% ash 6.3%, chloride 1.25%, potassium 1.89%, sodium 1.1%, Fe 70 ppm, Zn 4.2 ppm, Mn 2.5 ppm, Ni1.9 ppm, Cu 0.71 ppm. Evaluation of the effect of molasses addition to sucrose cultivation medium was made using response surface methodology with BoxBehnen design and Design Expert software. Crude polysaccharide content was determined by ethanol precipitation and gravimetric method. Levan content in crude polysaccharide was determined using Megazyme Fructan Assay Kit after hydrolysis of crude polysaccharide by 0.1% oxalic acid. Results The production of levan by B. licheniformis NS032, depends on the sucrose concentration in the fermentation medium. Maximum productivity ( g levan/g sucrose) by this culture was achieved in BM with sucrose [5]. Complete replacement of sucrose in cultivation medium by sugar beet molasses shows an inhibitory effect on the production of levan and growth of B. licheniformis, even when pretreated molasses is used. However, in preliminary experiments, it was observed that the polysaccharide can be synthesized by partial substitution of sucrose with molasses. In the present work, optimization of medium composition with respect to sucrose/molasses ratio and sucrose total concentration was studied using RSM. Box Box-Behnen Behnen design, incorporating three variables variablessucrose, molasses and yeast extract was used in order to compare crude polysaccharide and levan yield during fermentation. The design matrix of the variables is given in Table 1 along with experimental and predicted values of the responses. There was a considerable variation in crude polysaccharide content depending on the fermentation conditions with a maximum yield of 72.6 (run 11 ) and minimum yield of 13.9 (run 13 ). At the same time, maximum levan yield is achived in run 6 (62.6 ) and minimum in run 13 (11.6 ). Fig. 1 Response surface plots showing the effect of significant variables on crude polysaccharide (a) and levan yield (b) According to the results obtained, two RSM models were suggested: linear for the crude polysaccharide response (F value 23.09, P< ) and quadratic for the levan response. In the linear model two factors were significant: sucrose and molasses (P< ). Increasing the sucrose concentration together with increasing concentration of molasses, leads to maximum production of crude polysaccharide (Figure 1a). In the quadratic model (F value 6.04, P ) two following terms were significant: sucrose (P ) ) and molasses (P ) ) as was one quadratic coefficient (molassses2, P ) (Figure 1b). In order to determine the optimum values of the test variables the numerical optimization function from Design Expert software was used. The optimum conditions for levan production were sucrose 120, molasses 114 and yeast extract 1.5. Conclusion On the basis of the quadratic response surface model in medium that contains 120 sucrose, 114 molasses and 1.5 yeast extract maximum concentration of levan (57.4 ) is predicted. References 1. Srikanth et al., Carbohydr Polym 120 (2015) Kucukasik et al., Appl Microbiol Biotechnol 89 (2011) Roukas, Process Biochemistry 33 (1998) de Oliveira et al., Biochem Eng J 37 (2007) Kekez et al., Appl Biochem Biotechnol 1(2015) Acknowledgments This work was supported by the Ministry of Education and Science of the Republic of Serbia through Project III

CHEMISTRY OF LIFE 05 FEBRUARY 2014

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