Optimization of Xanthan Gum Production by Xanthomonas Campestris Grown in Molasses

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. The optimal effect on biomass and xanthan gum production. EDTA concentration for xanthan gum solubility with- Furthermore, cultures with molasses concentration 210 out affecting the biomass was determined by comparing g/l showed higher biomass and xanthan gum production the centrifugation and ethanol precipitates at increasing than those at a concentration of 140 g/l. All other EDTA concentrations. At EDTA concentrations up to 4 factors had a slightly negative effect on the three mM the centrifugation precipitate decreased sharply measured values. while the ethanol precipitate showed an equal increase The t-test for any individual effect allows an evalua- (Fig. 1). At higher concentrations up to 10 mM, both tion of a probability, P, which showed that the centrifugation and ethanol precipitates remained un- confidence level of the calculation of the effects of changed. Hence, at an EDTA concentration of 4 mM, K HPO and molasses was higher than 95%. xanthan gum recovery was considered to be maximal 2 4 and this concentration was used for subsequent biomass 3.5. Determination of optimal concentration of molasses and xanthan gum separation. Xanthan gum and biomass production of cultures 3.3. Pretreatment of molasses with increasing concentrations of molasses ranging from 105 to 245 g/l are shown in Fig. 2a and b, respectively. Several reagents and treatments were employed for Maximal xanthan gum production of 55 g/l was the pre-treatment of molasses aiming at the removal of obtained at a concentration of molasses of 210 g/l after growth inhibiting factors such as heavy metals [11], 48 h of incubation. The effect of molasses concentration volatile organic acids and alcohols with six or seven on biomass production was not as profound as that on carbon atoms [12,13]. The pretreaments used, their xanthan production (Fig. 2a). Maximum biomass was respective modes of action and the highest xanthan obtained by the cultures with molasses concentration higher than 175 g/l while biomass was lowest after 72 h of incubation in all cultures, indicating lysis of the cells. Molecular weight (MW) determination of xanthan gum produced under the above experimental conditions, 6 showed that it ranged from 0.8 to 1.4/10 Da. Xanthan gum maximum MW was obtained upon growth on a molasses concentration of 105 g/l. A decrease in the MW of xanthan was observed with increasing molasses concentration (Fig. 3).

3.6. Determination of optimal concentration of K2HPO4

X. campestris was grown in cultures containing the Fig. 1. Effect of EDTA concentration on the separation of xanthan optimal molasses concentration (175 g/l) and increasing gum from the biomass. concentrations of K2HPO4 ranging from 0 to 10 g/l. Fig. 252 S. Kalogiannis et al. / Process Biochemistry 39 (2003) 249Á/256

Table 1 Molasses pretreatments

Treatment Action Applications Xanthan production (g/l) at 24 and 48 h

No treatment 26.5/46.9 Aeration, aeration of hot mo- Removal of low carbon acids and Fattohi, [13] 29.2/30.3 lasses alkanols

K4Fe(CN)6 Reduction Citric acid fermentation [5] 14.9/14.3 Acidification (H2SO4) and pre- Removal of cations Baker’s yeast production 38.8/36.5 cipitation at pH 4.0 IRC 748, ion exchange resin Removal of cations, mainly heavy Metallurgy waste water treatment 15.1/15.9 metals XAD 761, ion exchange ab- Color removal, HMF, proteins, tan- Organis acids fermentations, pharmaceutical 20.5/23.3 sorption resin nins and iron complexes applications, starch hydrolysis XAD 1180, absorption resin Selective removal of organic com- Recovery and purification of antibiotics, ster- 18.8/16.8 pounds oids, enzymes etc Active carbon Absorption Heavy metals [11] 32.7/21.9

Acidification (H2SO4) and ac- Removal of cations and absorption 35.9/36.6 tive carbon

4 shows that maximum xanthan gum production was ing TrisÁ/buffer exhibited higher biomass and xanthan obtained at 4 g/l K2HPO4 concentration after 24 h while gum production than the ones without any additives. it was lower after 72 h in all cultures containing more The presence of K2HPO4 and KH2PO4 concentra- than 4 g/l K2HPO4. Maximum biomass production was tions did not have a significant effect on the average obtained at the same concentration of K2HPO4 also MW of the produced xanthan which remained in the 6 after 24 h. The experimentally determined optimal order of 1.1Á/1.3/10 Da. Concerning the initial pH concentration is comparable with the optimal phosphate values, it was found that xanthan gum with maximum concentration of 6 g/l observed in a previous study [14]. MW was obtained at a value of approximately 6.0 (data In order to investigate whether K2HPO4 improved obtained but not shown). Although pretreatments of productivity as phosphate ions were acting as a buffer- molasses did not improve xanthan gum production the 6 ing agent or because the essential phosphates for the MW was higher in all cases (/1.4/10 Da). Pyruvate growth of the microorganism were provided and also content was estimated in the order of 1.2Á/2.3%. whether potassium ions played an important role, cultures containing equimolar concentrations of 3.7. Determination of optimal initial pH K2HPO4 (4 g/l, approximately 23 mM), Na2HPO4 and

TrisÁ/buffer, pH 7.0 were compared in respect to The effect of the initial pH was studied in X. biomass and xanthan gum production (Fig. 5). Cultures campestris cultures with initial pH ranging between 5.1 with added Na2HPO4 exhibited identical production as and 7.7 (Fig. 6). The addition of phosphate salts to the the ones with K2HPO4 and higher than the ones sterilized molasses solutions affected greatly their pH containing TrisÁ/buffer. However, the cultures contain- values as buffer solutions of a significant concentration

Table 2

PlackettÁ/Burman matrix of five factors at two levels in eight fermentation flasks and the produced xanthan gum, biomass and the yield of xanthan (means of two experiments)

Number Factor (/, high level//, low level) Xanthan (g/l) Biomass (g/l)

K2HPO4 Yeast extract Dummy 1 Tap water Molasses Dummy 2 Triton 80

1 / / / / / / / 26.0 27.6 22.9 22.6 2 / / / / / / / 38.6 52.3 35.0 25.3 3 / / / / / / / 27.9 35.1 23.6 20.4 4 / / / / / / / 4.0 4.6 12.8 12.8 5 / / / / / / / 48.3 45.8 33.2 30.9 6 / / / / / / / 19.2 21.1 13.2 11.9 7 / / / / / / / 23.8 26.7 20.9 15.6 8 / / / / / / / 12.0 11.8 17.2 17.4

The respective high and low levels for the additives were; 0 and 10 g/l K2HPO4, 0 and 10 g/l yeast extract, 140 and 210 ml/l molasses, 0 and 0.1 g/l Triton 80, absence and presence of tap water. S. Kalogiannis et al. / Process Biochemistry 39 (2003) 249Á/256 253

Fig. 4. Kinetics of (a) xanthan gum and (b) biomass production at

Fig. 2. Effect of molasses concentration on (a) xanthan gum and (b) different concentrations K2HPO4. biomass production.

were created (23 mM). This particular property of the phosphate ions was exploited in order to achieve equivalent phosphate molarity but different initial pH values [15]. In all cultures with initial pH above 5.9 biomass production was higher than 30 g/l after 24 h (Fig. 6b) while the fluctuation of pH showed a similar pattern in all cultures. After 20 h the pH of the cultures dropped to approximately 6.0 whereas after 48 and 72 h it rose to approximately 7.0 (Fig. 6c). Maximum xanthan gum production was obtained after 24 h from the cultures with initial pH 6.6 (Fig. 6a).

Fig. 5. Obtained (a) xanthan gum and (b) biomass from cultures

Fig. 3. Dependence of xanthan gum average molecular weight on where Na2HPO4,K2HPO4 and TrisÁ/buffer pH 7.0 were used as molasses concentration of the medium. additives. 254 S. Kalogiannis et al. / Process Biochemistry 39 (2003) 249Á/256

Fig. 7. Obtained (a) xanthan gum and (b) biomass from cultures

inoculated and grown on from molasses (Á/"Á/), inoculated from a Fig. 6. Effect of initial pH on (a) xanthan gum, (b) biomass and (c) pH chemically defined medium and grown on molasses (Á/mÁ/) and fluctuation. inoculated and grown on a chemically defined medium ( Á/2Á/). 3.8. Comparison of media precipitation of xanthan gum and biomass due to the presence of polyvalent cations, especially calcium that is Fig. 7 provides a comparison between the LB and abundant in sugar beet molasses [5]. Polyvalent cations molasses used as fermentation and inoculum media. On such as calcium, aluminum, and quaternary ammonium molasses, growth and xanthan gum production were salts are especially effective in the precipitation of higher than on the synthetic substrate LB containing xanthan gum due to ion binding of the cations to the identical sucrose concentrations. Furthermore, the cul- ionized groups on the polyanionic polysaccharide [3]. tures inoculated from the molasses substrates showed Compared with other polysaccharides used as food higher xanthan gum production than the ones inocu- additives, xanthan gum has been shown to be among lated from LB. the ones that bind calcium the strongest [21]. Hence, supplementing the KCl solution, used in biomass precipitation with EDTA, resulted in improving 4. Discussion xanthan gum solubility (Fig. 1). Although beet molasses normally contain most sub- The adaptation of X. campestris in high molasses stances necessary for the nutrition of microorganisms, environments while maintaining its ability to produce depending on the fermentation, they can be supplemen- xanthan is reported. Success was realized in achieving ted with certain components such as nitrogen, phospho- growth at molasses concentrations higher than 140 g/l, rus or magnesium [5]. Owing to its low cost, the use of thus overcoming the difficulties encountered by re- tap water was investigated as it also can serveasan searchers earlier [16] who reported the inhibition of additional source of magnesium for the medium since it growth at such molasses concentrations. In a similar contained approximately 34 mg/l magnesium. Triton 80 fashion with these findings, the occurrence of sponta- has been reported to improve xanthan yield and neous high producing variants of X. campestris has been polymer quality by X. campestris grown on a chemically earlier demonstrated by a number of researchers [17Á/ defined medium [22]. However, in the present experi- 20]. Furthermore, the importance of strain adaptation ments, productivity was not improved (Table 3) prob- to molasses has also been stressed earlier [6]. ably because molasses already contains substances that The method for the determination of xanthan gum act as surfactants [5]. This is also indicated by the fact was modified in order to avoid the observed co- that foaming is frequently observed in molasses fermen- S. Kalogiannis et al. / Process Biochemistry 39 (2003) 249Á/256 255

Table 3

Experimental variables and their effects as a result of the PlackettÁ/Burman screening

Factor Xanthan Biomass

Effect Relative significance of t-test (confidence level) Effect Relative significance of t-test (confidence level)

K2HPO4 22.30 11.91 (99%) 11.51 6.13 (99%) Yeast extract /2.40 /1.28 /3.21 /1.71 Tap water /3.95 /2.11 /1.89 /1.00 Molasses 15.85 8.46 (99%) 4.54 2.41 (95%)

Triton 80 /2.20 /1.17 0.012 0.01 tations when the addition of an anti-foam agent is not duction and yield (61%) are among the highest yet sufficient. Yeast extract, which serves as an additional reported for xanthan gum fermentations on an indus- nitrogen source, had a negative effect on both xanthan trial or a laboratory medium [3]. In an earlier study a and biomass production (Table 3) probably because the xanthan gum production of 22.8 g/kg of medium from organic nitrogen content in molasses is sufficient to sugar beet molasses was reported [6]. Although, in support growth of X. campestris while further increase laboratory bioreactors, polysaccharide production by the addition of yeast extract had a detrimental effect. rarely exceeds 25 g/l [3], industrial processes can reach This is also supported by earlier findings that indicated a final xanthan gum content of approximately 50 g/l the inhibitory effect of high nitrogen concentrations [25,26]. However, Abd El Salam et al. (1993) reported a both on growth and xanthan gum production [23].In production of xanthan gum as high as 70.5 g/l from a conclusion, among the additives that were investigated, culture grown on sugar cane molasses using a relatively only phosphates proved to have a positive effect on high initial sugar concentration of 25% [24]. The xanthan gum production (Table 3). The optimum levels increase in production and growth observed using of molasses and phosphates were subsequently more molasses as a substrate are probably attributed partly accurately defined in experiments in the present study. to the high availability of aminoacids and especially Although, Abd El Salam et al. (1993) [24] reported the glutamate in molasses. Indeed, in an earlier study it was fortification with ammonium chloride of a medium shown that the best nitrogen source for X. campestris using sugar cane molasses as the carbon source, a was glutamate at a concentration of 15 mM (2.2 g/l) [27] comparison between their findings and those presented while in the present study the fermentation medium in this study, should be treated with care. Beet molasses contained approximately 1.3 g/l free and bound gluta- contains significantly more nitrogenous compounds and mate, with a total aminoacid content of 4.0 g/l. More- are expected to satisfy the nitrogen demand of the over, sugar beet molasses contains significant amounts organism to a greater extent but on the other hand they of organic acids [4], which could enhance xanthan gum also contain less biotin [5]. production as it has been observed earlier in the The investigation of the role of phosphates in the presence of metabolizable organic acids [28] and citric culture medium revealed that xanthan production was acid [27,28]. Hence, the combined effect of aminoacids lower in the presence of Tris than in the presence of and organic acids content of molasses along with the strain adaptation could be the reasons for the high K2HPO4 and Na2HPO4 but higher than in media with no additives. This indicated that the addition of production observed in the present study. phosphates enhanced xanthan gum production both Future work will focus mainly on the chemical and because the medium was deficient in phosphates and rheological characterization of the product as well as on their supplementation was required and because they purification and clarification methods. The scale up to serve as a buffering agent, reducing the pH fluctuations laboratory and pilot plant bioreactors is a prerequisite for the detailed study of the effects of time and of the culture. Hence, a K2HPO4 concentration of 4 g/l is the critical concentration at which the nutrient engineering parameters on xanthan production and requirements of the organism were met while pH is quality. maintained relatively constant. It is generally accepted that a near neutral pH is optimal for polysaccharide synthesis and the growth of the organism. The impor- tance of pH in fermentations of X. campestris has been References reviewed earlier [3]. [1] Sutherland IW. Novel and established applications of microbial In the present paper the production of approximately polysaccharides. Trends Biotechnol 1998;16:41Á/6. 53 g/l xanthan gum in 24 h is reported using as substrate [2] Demain AL. Small bugs, big business: the economic power of the sugar beet molasses, a food industry byproduct. Pro- microbe. Biotechnol Adv 2000;18:499Á/514. 256 S. Kalogiannis et al. / Process Biochemistry 39 (2003) 249Á/256

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