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Food Sci. Biotechnol.

19(1): 201-206 (2010)

DOI 10.1007/s10068-010-0027-9

RESEARCH ARTICLE

Xanthan Gum Production of Xanthomonas spp. Isolated from


Different Plants
Tuncay Gumus, A. Sukru Demirci, Mustafa Mirik, Muhammet Arici, and Yesim Aysan

Received: 11 September 2009 / Revised: 18 November 2009 / Accepted: 19 November 2009 / Published Online: 28 February 2010
© KoSFoST and Springer 2010

Abstract Xanthan gum were produced from the following Introduction


Xanthomonas strains; standard strain Xanthomonas
campestris NRRL B-1459 and isolated strains Xanthomonas Xanthan gum is an important biopolymer that is produced
arbicola pv. juglandis, Xanthomonas axonopodis pv. efficiently by Gram-negative bacteria of the genus
vesicatoria, Xanthomonas axonopodis pv. begonia, Xanthomonas (1) and has been widely accepted commercially
Xanthomonas axonopodis pv. dieffenbachia. The viscosity (2) with an annual worldwide production of 30,000 tons,
features of the xanthan gums obtained were determined at corresponding to a market of $408 million (3-5). Xanthan
25-80ºC with different pH values and were compared to gum, a cream-colored, odorless, free-flowing powder,
commercial xanthan gum. Our results indicate that X. hydrates rapidly in cold and hot water to give a reliable
arbicola pv. juglandis showed the highest productivity viscosity even at low concentrations. It is highly resistant
(8.22±1.52 g/L gum). This was followed by X. axonopodis to enzymatic degradation, extremely stable over a wide pH
pv. begonia (7.74±1.30 g/L gum), and the control bacterial range, and forms highly pseudoplastic aqueous solutions
strain X. campestris NRRL B-1459 (7.46±0.28 g/L gum). (6). Xanthan gum, an extracellular high molecular weight
X. axonopodis pv. vesicatoria showed the lowest polysaccharide, has unique rheological properties which
productivity (6.40±0.55 g/L gum). No xanthan gum could have led to numerous applications such as stabilizing,
be obtained from X. axonopodis pv. dieffenbachia. viscosifying, emulsifying, thickening, and suspending
Xanthan gum produced by X. axonopodis pv. vesicatoria agent in many fields (7). There is considerable interest in
showed the highest viscosity value (428 mPa·sec at 1% increased industrial production of xanthan gum owing to
solution) in all Xanthomonas strains isolated from plants. its extensive applications (8,9) and wide use in a broad
range of industries, such as in food, toiletries, oil recovery,
Keywords: Xanthomonas spp., xanthan gum, viscosity cosmetics, water-based paints, etc (10). The major
feature applications of xanthan gum are in the food industry as a
suspending and thickening agent. It also has wide
applications in the chemical industry (6). In the industrial
production of xanthan gum, the growth medium should be
as economical as possible (11). Xanthan is produced by
Xanthomonas campestris, a plant-associated bacterium that
Tuncay Gumus ( ), A. Sukru Demirci, Muhammet Arici
Department of Food Engineering, Faculty of Agriculture, Namik Kemal is generally pathogenic for plants belonging to the family
University, 59030 Tekirdag, Turkey Brassicaceae. Xanthomonas causes a variety of diseases on
Tel: +90-282-293-1442; Fax: +90-282-293-1480 the leaves, stems, or fruits; an example is ‘black rot’ of
E-mail: tgumus@nku.edu.tr
crucifers such as cabbage, cauliflower, or broccoli (12).
Mustafa Mirik Bacterial leaf spot, caused by X. campestris pv. vitians
Department of Plant Protection, Faculty of Agriculture, Namik Kemal
University, 59030 Tekirdag, Turkey (Brown) Dye, is an important pathogen of lettuce (Lactuca
sativa L.) worldwide. Since 1995, serious outbreaks of the
Yesim Aysan disease occurred on lettuce cultivars grown in Ohio, USA
Department of Plant Protection, Faculty of Agriculture, Cukurova
University, Adana, Turkey (13).
202 T. Gumus et al.

It is important to screen new xanthan gum producers of (Model Biostat B; Sigma, Braun, Germany). As showed in
the Xanthomonas spp. with economical and functional the review of Rosalam and England (10), several researchers
properties, with studies to optimize the yields and indicated the optimal temperature of 28-30ºC, fermentation
productivity in the fermentative processes. time of 72 hr, stirring rate of 180 rpm, and initial pH of
In this research, we investigated xanthan gum production 7.2. Based on the results presented in the literatures,
yield from following the Xanthomonas strains: the standard fermentation operational conditions were determined as;
strain X. campestris NRRL B-1459, and the isolates temperature 30ºC, pH 7.0, stirrer speed 200 rpm, and air-
Xanthomonas arbicola pv. juglandis, Xanthomonas flow rate 1 mL/min for 72 hr. The productivity of the 5
axonopodis pv. vesicatoria, X. axonopodis pv. begonia, and species of Xanthomonas by fermentation is compared with
X. axonopodis pv. dieffenbachia. The viscosity of the gums the fixed fermentation condition.
were analyzed at 25-80ºC, at different pH values and
compared to commercial xanthan gum. Fermentation medium The medium consisted of (g/L)
glucose 40.0, citric acid 2.1, KH2PO4 2.866, MgCl2 0.507,
Na2SO4 0.089, H3BO3 0.006, ZnO 0.006, FeCl3 ·6H2O
Material and Methods 0.020, and CaCO3 0.020. Glucose was used as carbon
source for the fermentation studies (19).
Organisms Xanthomonas arbicola pv. juglandis (Ceviz
1), X. axonopodis pv. vesicatoria (XCVA3-1), X. axonopodis Xanthan concentration The final fermentation broth
pv. begonia (Xcb-9), and X. axonopodis pv. dieffenbachia was centrifuged at 10,000 × g for 30 min at 4oC to remove
(Xad-2) were isolated from walnut (Juglans regia L), the cells. The supernatant was collected and the gum was
pepper (Capsicum annuum L), begonia (Begonia X tuber precipitated by using isopropanol (1:2 v/v) then filtered
hybrid), and anthurium (Anthurium andraenum), respectively. through a filter paper. The gum was dried at 50-60oC
Identification of the strains was initially confirmed by overnight. The dry weight of the gum was determined
morphological, biochemical, and physiological tests including (20,21).
potassium hydroxide solubility for Gram reaction, catalase
reaction, oxidative/fermentative metabolism, and hypersensitive Preparation of gum solution Water solutions of xanthan
reaction on tobacco leaves. Identification of the strains was of 0.25, 0.50, and 1%(w/w) were prepared as described in
confirmed by fatty acid methyl ester (FAME) analysis. The Doğan et al. (22).
4 strains of Xanthomonas spp. were isolated in Turkey (14-
17). Viscosity measurements In this study the viscosity of
X. campestris NRRL-B 1459 was obtained from the the samples were measured using vibro-viscometer (SV-
United States Department of Agriculture, Research- Education 10; A & D Co., Ltd., Tokyo, Japan). This type viscometer
and Economics Agricultural Research Service, Microbial measures viscosity by controlling the amplitude of the
Genomics, and Bioprocessing Research Unit. sensor plates immersed in a sample and measuring the
The bacteria were grown on glucose yeast extract agar electric current to drive the sensor plates. To perform a
medium containing (g/L): glucose 20.0, yeast extract 10.0, measurement, firstly the thin sensor plates were immersed
calcium carbonate 20.0, agar 14.0 at 30oC for 48 hr, stored into a sample. When the spring plates were vibrated with
at 4oC and subcultured every 3 weeks. a uniform frequency, the amplitude varied in response to
the quantity of the frictional force produced by the viscidity
Inoculum preparation Actively growing cells from a between the sensor plates and the sample. The vibro-
newly prepared slant were inoculated into 100 mL yeast viscometer controls the driving electric current to vibrate
extract malt (YM) liquid medium (Difco, Benton the spring plates in order to make uniform amplitude. Since
Dickenson Co., Sparks, MD, USA) containing (g/L) malt the frictional force of viscidity is directly proportional to
extract 3.0, peptone 5.0, yeast extract 3.0, glucose 20.0 at the viscosity, the driving electric current (driving power)
pH 7.0 in a 250-mL Erlenmeyer flask. The culture was for vibrating the spring plates with a constant frequency to
incubated at 28-30oC for 36-48 hr in an incubator shaker. make uniform amplitude is also directly proportional to the
The liquid culture was used to inoculate the final viscosity of each sample. The vibro-viscometer measures
fermentation medium. For the gum production test, 10% the driving electric current to vibrate the sensor plates with
(v/v) of the inoculum was added to the production medium a uniform frequency and amplitude, and then the viscosity
(18). is given by the positive correlation between the driving
electric current and the viscosity (23).
Production of xanthan gum Production of xanthan gum
was carried out in a fermentor with 1.5-L work volume Statistical analysis The yield data of gum production of
Xanthan Gum Production by Xanthomonas spp. 203

Xanthomonas spp. was obtained from 5 replicates and highest gum production was 12 g/L. Lopez et al. (2) tested
viscosity data of the gums was obtained from 3 replicates. 4 strains of X. campestris to produce xanthan gum using
The data were analyzed by analysis of variance (ANOVA) olive mill wastewaters (OMW). The most valuable strain
using the SPSS statistical package program, and differences was X. campestris NRRL B-1459 S4LII because of its
among the viscosity means were compared using the ability to produce xanthan using 7% of OMW as the
Duncan’s multiple range test (24). nutrient source, producing 7 g/L of xanthan gum. Whey
was also used in earlier studies to produce xanthan gum
reaching a maximum xanthan gum production (1.2 g/100
Results and Discussion mL of whey) with X. campestris XLM 1521. Another
study by Molina et al. (34) described the use of whey as a
Production of xanthan gum The productivities, after carbon source and the authors reported that the maximum
fermentation, of the 5 different strains of Xanthomonas gum yield was 14 g/L. In these studies, xanthan gum
spp. isolated from different plants are given in Table 1. production from different agricultural waste products as
Of the 5 different bacterial species used, X. arbicola pv. substrates was compared with the polymer produced using
juglandis showed the highest productivity (8.22±1.52 g/L glucose and sucrose as substrate. According to Rosalam
gum), X. axonopodis pv. begonia produced an average of and England (10), many attempts have been reported for
7.74±1.30 g/L gum while the control bacterial strain X. optimizing variables of the xanthan gum fermentation and
campestris NRRL B-1459 produced an average of 7.46± glucose is still the best in term of the products yield,
0.28 g/L gum. X. axonopodis pv. vesicatoria produced an supply, and the product quality.
average of 6.40±0.55 g/L gum and showed the lowest
productivity. On the other hand, X. axonopodis pv. Viscosity of xanthan gum The effect of gum concentration,
dieffenbachia couldn’t produce any xanthan gum. The pH, and temperature on the viscosity features of xanthan
xanthan gum production of Xanthomonas spp. followed the gum produced using different Xanthomonas spp. and
sequence: X. arbicola pv. juglandis > X. axonopodis pv. commercial xanthan gum are given Table 2.
begonia >X. campestris NRRL B-1459> X. axonopodis pv. Viscosity measurements for the xanthan gums obtained
vesicatoria > X. axonopodis pv. dieffenbachia. Rottova et in this study and commercial gum were carried out in the
al. (25) tested 10 strains of Xanthomonas to produce temperature range of 25-80oC, and at different pH values,
xanthan gum in standard medium and the best ones in using 0.25, 0.50, and 1%(w/v) aqueous xanthan gum solutions
terms of productivity were X. campestris pv. mangi- (Table 2). Apparent viscosity (AV) values exhibited
feraeindicae 1230 (8.93 g/L), X. campestris pv. campestris considerable differences at low concentrations for both the
254 (9.49 g/L), and X. campestris pv. campestris 1078 commercial gum and the gums produced in the laboratory
(9.67 g/L). Other reports indicate xanthan gum production at the same pH and temperatures. Generally, the AV of
of 15.65 (26), 30 (27), 19 (28), 30 (29), 2.3-8.3 (30), and solutions decreased with increasing temperature at all pH
17.7 g/L (31). The productivity obtained in this study is values. The effect of pH and temperature on viscosity
comparable to those reported by Moreira et al. (30). features of water (the control liquid) are shown in Table 3
Some research related to xanthan gum production using and was determined to be statistically insignificant (p>0.05).
different substrates has been reported in literature. Moreno At a concentration of 0.25%(w/v) gum solutions, the
et al. (32) studied xanthan gum production by X. gum produced from X. axonopodis pv. vesicatoria reached
campestris NRRL B-1459 using melon as a substrate and maximum viscosity (66.0 mPa·sec) at 25oC and pH 5.0.
the maximum gum production obtained was 1.6 g/L. The lowest average viscosity value was determined as 4.50
Bilanovic et al. (33) investigated the use of citrus waste as mPa·sec with gum obtained from X. campestris at 80oC
a low cost substrate for xanthan gum production and and pH 3.5 (p <0.05).

Table 1. Productivity (g/L) of the 5 species of Xanthomonas spp. by fermentation processes


Trial X. juglandis X. begonia X. vesicatoria X. diffenbachia X. campestris NRRL B-1459
1 7.8 6.4 7.0 - 7.4
2 6.1 7.3 5.5 - 7.6
3 7.9 7.4 6.6 - 7.0
4 10.1 7.7 6.5 - 7.7
5 9.2 9.9 6.4 - 7.6
Average 8.22±1.52A1) 7.74±1.30B 6.40±0.55D 0 7.46±0.28C
1)Standard errors of the mean (n =5); A,BMeans within the same line with different superscript letters are significantly different (p <0.05).
204 T. Gumus et al.

Table 2. Effect of gum concentration, pH, and temperature on viscosity features of xanthan gum produced from different
Xanthomonas spp. and commercial xanthan gum (mPa·sec)
X. gum pH Temp. X. juglandis X. begonia X. vesicatoria X. campestris Commercial
conc. (%) (oC)
25 14.9±0.2bC1) 33.5±0.3cB 58.5±0.1bA 07.5±0.1bD 15.3±0.1fC
3.5 40 10.6±0.3eC 20.0±0.2gB 48.5±0.1dA 05.7±0.3dB 10.9±0.3hC
60 08.4±0.1fC 14.5±0.2jB 38.0±0.3fgA 05.7±0.0dB 08.0±0.1jc
80 05.7±0.2hD 10.9±0.1kB 25.3±0.1iA 04.5±0.2eE 07.2±0.1kC
25 15.0±0.3bD 38.0±0.1aB 66.0±0.3aA 07.9±0.2abE 27.2±0.1aC
40 11.6±0.3dD 31.3±0.0dB 55.4±0.1cA 07.3±0.0BcE 22.3±0.1cC
0.25 5 60 07.5±0.5gD 27.6±0.2eB 47.0±0.1eA 06.5±0.2cdE 20.1±0.0dC
80 05.0±0.1hE 18.5±0.1hB 37.4±0.0gA 05.8±0.1dD 12.8±0.1gC
25 17.0±0.1aD 36.0±0.1bB 65.5±0.1aA 08.4±0.1aE 26.1±0.2bC
40 13.2±0.1cD 23.1±0.0fC 48.0±0.3dA 07.6±0.0abE 26.5±0.1abB
7 60 10.0±0.1eC 16.8±0.1iB 38.3±0.1fA 07.3±0.1bcD 17.1±0.2eB
80 07.3±0.1gC 10.5±0.2kB 27.0±0.1hA 06.5±0.2cdD 10.1±0.1lB
25 44.0±0.3cD .109±0.5cB .174±0.4bA 43.5±0.3dD 91.0±0.3cA
3.5 40 37.6±0.3dD 68.5±0.3iC .131±0.6eA 34.5±0.1hE 77.8±0.1eB
60 33.4±0.1fD 54.3±0.2jC .102±0.2hA 28.7±0.2jE 69.7±0.2fB
80 22.1±0.1iD 39.8±0.2kC 67.2±0.1kA 22.5±0.4kD 53.7±0.1jB
25 45.6±0.1dB .117±0.6bB .170±0.6cA 44.7±0.4cE 84.1±0.4cC
40 36.9±0.2dE 88.6±0.3eB .122±0.8fA 41.1±0.3eD 80.4±0.4dC
0.5 5 60 31.2±0.5gE 75.1±0.1gB 93.7±0.3iA 37.3±0.1gD 66.1±0.5hC
80 19.4±0.3jE 53.6±0.5jB 63.8±0.1lA 28.7±0.1jD 50.8±0.3kC
25 50.8±0.4aE .137±0.6aB .180±0.8aA 51.7±0.3aD 91.3±0.2aC
40 43.4±0.3cE .100±0.5dB .145±0.5dA 45.7±0.1bD 85.3±0.2bC
7 60 35.3±0.5eE 85.8±0.3fB .121±0.6gA 38.2±0.3fD 67.5±0.1gC
80 25.8±0.1hE 70.8±0.5hB 79.8±0.3jA 31.7±0.1iD 57.7±0.2iC
25 .140±0.5bD .342±0.9cC .415±0.6bB .110±0.6bE .450±0.7bA
3.5 40 85.6±0.2fE .265±0.8fC .361±0.8eB 92.7±0.4dD .421±0.4dA
60 60.8±0.1hE .194±0.5iC .294±0.7hB 77.6±0.3fD .351±0.6gA
80 41.2±0.1kE .111±0.8lC .184±0.5kB 51.3±0.4kD .276±0.7kA
25 .135±0.7cE .385±0.5bC .428±0.4aB .148±0.8aD .445±0.6cA
40 89.0±0.4eE .283±0.6eC .372±0.7dB 96.8±0.3cD .406±0.4fA
1 5 60 58.0±0.2iE .235±0.9hC .310±0.9gB 68.3±0.7hD .344±0.7iA
80 39.3±0.2lE .157±0.8kC .211±0.6jB 57.0±0.4jD .284±0.2jA
25 .149±0.6aD .409±0.5aB .374±0.6cC 87.8±0.2eE .452±0.5aA
40 91.7±0.5dD .302±0.5dC .320±0.6fB 71.7±0.3gE .415±0.6eA
7 60 69.5±0.4gD .261±0.8gB .255±0.5iC 64.1±0.7iE .350±0.3hA
80 43.0±0.5jD .192±0.7jB .133±0.4lC 38.6±0.5lE .276±0.6kA
1)Means within the same lineA,B and column a,b with different superscript letters are significantly different (p <0.05) (n =3); Data are mean±SE.

The apparent viscosity values of xanthan gum solutions and pH 5.0 (p <0.05). For the 1% gum solutions, the
at 0.25% concentration exhibited viscosity values higher viscosity was in the range of 38.6 (X. campestris NRRL B-
than commercial gum, while the viscosity value of the gum 1459, at 80oC and pH 7.0) to 428 mPa·sec (X. axonopodis
produced from X. campestris NRRL B-1459 was lower pv. vesicatoria, at 25oC and pH 5.0). Additionally, the
than commercial gum. Similarly, the maximum viscosity of average viscosity value was determined as 450 mPa·sec for
aqueous solutions containing 0.5% of xanthan gum was the commercial gum at 25oC and pH 3.5 (p <0.05).
180 mPa·sec with gum produced from X. axonopodis pv. The viscosity of xanthan solution depends on both the
vesicatoria at 25oC and pH 7.0. At this concentration, the temperature at which the viscosity is measured and the
lowest average viscosity was determined as 19.4 mPa·sec temperature at which the xanthan is dissolved. The
with gum obtained from X. arbicola pv. juglandis at 80oC viscosity decreases with increasing temperature. This
Xanthan Gum Production by Xanthomonas spp. 205

Table 3. Effect of pH and temperature on the viscosity features isolated from different plants and the X campestris NRRL
of water B-1459 control strain were determined. A comparison of
pH Temperature ( C)
o
Viscosity (mPa·sec) the viscosity values obtained was made with a commercially
25 00.85±0.0 1) obtained gum. This research has shown that using different
40 0.95±0.0 strains Xanthomonas isolated from plants it is possible to
3.5 produce xanthan gum economically in a laboratory setting.
60 0.73±0.0
80 0.57±0.0 While xanthan gum produced from some strains
25 1.34±0.0 reported in the current study exhibited higher viscosity than
40 0.73±0.0 commercial gum, the gum obtained using some other
5 strains showed lower viscosity. These gums produced can
60 0.73±0.0
80 0.48±0.0 find applications in the food industry as emulsifiers,
25 1.03±0.0 stabilizers, and suspension agents. However, further studies
40 1.05±0.0 are needed to investigate the rate of production, rheological
7 and chemical properties of xanthan gum.
60 0.79±0.0
80 0.65±0.0
Data are mean±SE (n =3).
1)

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