Optimization of Xanthan Gum Production by Xanthomonas Campestris Grown in Molasses
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Process Biochemistry 39 (2003) 249Á/256 www.elsevier.com/locate/procbio Optimization of xanthan gum production by Xanthomonas campestris grown in molasses Stavros Kalogiannis a, Gesthimani Iakovidou a, Maria Liakopoulou-Kyriakides b, Dimitrios A. Kyriakidis c, George N. Skaracis a,* a Department of Plant Breeding and Biotechnology, Hellenic Sugar Industry, Thessaloniki 57400, Greece b Department of Chemical Engineering, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece c Faculty of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece Received 27 September 2002; received in revised form 19 December 2002; accepted 12 February 2003 Abstract Xanthan gum production by Xanthomonas campestris ATCC 1395 using sugar beet molasses as carbon source was studied. The pre-treatment of sugar beet molasses and the supplementation of the medium were investigated in order to improve xanthan gum production. Addition of K2HPO4 to the medium had a significant positive effect on both xanthan gum and biomass production. The medium was subsequently optimized with regard to molasses, K2HPO4 concentration and initial pH. Maximum xanthan gum production (53 g/l) was observed after 24 h at 175 g/l molasses, 4 g/l K2HPO4 and at neutral initial pH. Results indicate that K2HPO4 serves as a buffering agent as well as a nutrient for the growth of X. campestris. Sugar beet molasses appears to be a suitable industrial substrate for xanthan gum fermentations. # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Xanthan gum; Biomass; Molasses; X. campestris; Fermentation 1. Introduction production, recovery and properties of xanthan gum has been reviewed recently [3]. Xanthan gum, the exopolysaccharide from Xantho- Molasses is a co-product of sugar production, both monas campestris pv campestris, is one of the major from sugar beet as well as from sugar cane, and is commercial biopolymers produced with an annual defined as the runoff syrup from the final stage of world wide production of 30 000 tons, corresponding crystallization, from which further crystallization of to a market of $408 million [1,2]. Because of its unique sugar is uneconomical [4]. Despite their similarities, structure, xanthan displays special pseudoplastic prop- beet and cane molasses exhibit significant differences erties, high viscosity and solubility, enhanced stability with regards to nitrogenous compounds, fermentable over a wide range of pH values and temperatures, as sugars, ash and vitamin content [5]. Sugar beet molasses, well as compatibility with many salts, food ingredients therefore, is a solution of sugar, organic and inorganic and other polysaccharides used as thickening agents. matter in water with a dry substance of 74Á/77% (w/w). These characteristics contribute to the employment of Total sugars (mainly sucrose) constitute approximately xanthan in a wide range of applications especially in the 47Á/48% (w/w) of molasses, ash 9Á/14% (w/w) and total food industry as a thickening and stabilizing agent, in nitrogen containing compounds (mainly betaine and cosmetics, in the paper milling, textiles and the pharma- glutamic acid) 8Á/12% (w/w). Variations in composition ceutical sector and also in enhanced oil recovery. The do occur between years and sugar plants [5]. Sugar beet molasses is widely used as a substrate in fermentations since it constitutes a valuable source of growth sub- stances such as pantothenic acid, inositol, trace elements * Corresponding author. Tel.: /30-310-79-8610; fax: /30-310-79- 8726. and, to a lesser extent, biotin [5]. However, the E-mail address: [email protected] (G.N. Skaracis). occurrence of undesired volatile nitrogenous pyrazines, 0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00067-0 250 S. Kalogiannis et al. / Process Biochemistry 39 (2003) 249Á/256 pyrroles as well as furans and phenols have been 2.3. Fermentations described [5]. Other contaminants such as heavy metals, biocides and organic acids were also detected but most Experiments were carried out in 500 ml Erlenmeyer of them at concentration levels lower than that needed flasks with 100 ml of medium containing sugar beet to affect process inhibition [5].Nevertheless, in processes molasses under the same conditions as the pre-cultures with molasses as a sole substrate or when used in high for 3 days. Cultures were grown in duplicates and sterile amounts as a co-substrate, specific inhibitors are partly additives were added under aseptic conditions after removed upon pre-treatment [5]. autoclaving. Aliquots of approximately 12 ml were To our knowledge, sugar beet molasses has only been withdrawn aseptically from the cultures every 24 h. used in one earlier study to produce xanthan [6],in which a number of industrial substrates were screened for xanthan gum production. In the present study the 2.4. Molasses and resins optimization of xanthan gum production from sugar beet molasses in terms of medium composition and Sugar beet molasses were supplied by the Platy Sugar molasses pre-treatment was performed. Sugar beet Plant of Hellenic Sugar Industry S.A. The industrial molasses proved to be an excellent substrate since under grade resins used for the pretreatment of molasses were optimized conditions both xanthan gum production and kindly offered by Rohm & Haas S.A. biomass formation were enhanced. 2.5. Xanthan gum estimation 2. Materials and methods Xanthan gum was assayed according as previously 2.1. Microorganism and growth conditions reported [7] with the difference that the potassium chloride solution was supplemented with EDTA to X. campestris ATCC 1395, a wild-type strain, was achieve a final EDTA concentration of 4 mM. Account used throughout this study. The strain was adapted to was taken for the precipitates derived from non-inocu- high molasses concentrations as described below and lated media and values were corrected accordingly. All maintained in submerged cultures on LB100S broth (in assays were performed in duplicates and means were g/l: yeast extract 5, tryptone 10, NaCl 10, sucrose 100) based on the values derived from the duplicate cultures. stored at /73 8C. For the first two adaptation passages X. campestris was cultured overnight in an orbital shaking incubator (250 rpm) at 30 8C in LB medium containing 25 and 100 g sucrose, respectively, whereas 2.6. Molecular weight and pyruvate content of xanthan for the next three passages it was cultured under the same conditions in 200 g/l sugar beet molasses. At each Molecular weight was determined according to Papa- passage biomass and xanthan gum were measured and gianni et al. (2001) [8]. Pyruvate content was determined the best results were obtained at the above fifth passage by HPLC following hydrolysis with HCl at 80 8Cand (20.0 and 40.5 g/l, respectively). extraction with ethyl acetate as previously reported [8]. 2.2. Inoculum 2.7. Experimental design and statistical analysis Slants consisting of LB25S (sucrose 25 g/l) were inoculated from the submerged stored culture by The PlackettÁ/Burman experimental design [9,10] was streaking. The slants were incubated at 289/1 8C for used to evaluate the relative importance of various 36Á/40 h and they were used to inoculate 20 ml of LB25S nutrients for xanthan gum production based on the first order model: in 100 ml Erlenmeyer flasks. The initial absorbance at X 600 nm (A600) of the medium was adjusted to approxi- Y b b x mately 0.1 and flasks were incubated in a rotary shaking 0 i i incubator (289/1 8C, 200 rpm) for 7 h until their A600 with no interaction among the factors used. Five reached approximately 1.4. Aliquots of 10 ml were used variables were screened in eight experiments with two to initiate 100 ml cultures in 500 ml Erlenmeyer flasks dummy variables, each variable being a medium con- containing a final concentration of sugar beet molasses stituent. of 105 g/l and cultivated under identical conditions for The regression coefficients and t-values were calcu- 17 h. Xanthan gum production cultures were subse- lated by compatible analysis of the data obtained from quently inoculated with a 5% (v/v) inoculum from the the duplicate cultures on xanthan gum and biomass last culture. production using the MINITAB calculation software. S. Kalogiannis et al. / Process Biochemistry 39 (2003) 249Á/256 251 3. Results gum production obtained from each culture are listed in Table 1, where it is shown that none of these pretreat- 3.1. Growth adaptation of X. campestris in molasses ments had a positive effect either on xanthan gum or on biomass production. In order to achieve fast growth and thereupon higher efficiency, the organism was adapted to high molasses 3.4. Selection of additives environments by consecutive subculturings. The result- ing pure culture possessed enhanced characteristics in An initial set of five media components were identi- terms of growth and xanthan gum production. fied as potentially affecting xanthan gum and biomass production. The arrangement of factor levels, according to the PlackettÁ/Burman experimental plan, is provided 3.2. Modification of the xanthan gum assay in Table 2. The data on xanthan gum and biomass production at 24 h of fermentation showed a wide During biomass precipitation when molasses were variation from 11.9 to 47.5 g/l for xanthan gum and used as a substrate it was observed that part of xanthan from 12.6 to 31.7 for biomass production. From the co-precipitated with biomass, even when the samples responses (xanthan gum and biomass production) of the were diluted to a dilution factor of up to 10 [7]. Hence, eight experiments reported in Table 2, the effect of each the method was modified as described in Section 2 in of the five variables was calculated and it was clearly order to avoid the observed co-precipitation of xanthan indicated that the addition of K2HPO4 had a positive gum with biomass during centrifugation.