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Small scale production and characterization of xanthan gum synthesized by

local isolates of Xanthomonas campestris

Article  in  Indian journal of experimental biology · March 2016

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Indian Journal of Experimental Biology
Vol. 54, February 2016, pp. 151-155
Note
Small scale production and characterization
of xanthan gum synthesized by local isolates
of Xanthomonas campestris
Rajesh Barua1, Md. Jahangir Alam1*, Mohammad Salim2
& Tamzida Shamim Ashrafee1
1
Dept. of Genetic Engineering and Biotechnology;
2
Dept. of Chemistry, Shahjalal University of Science and
Technology, Sylhet-3114, Bangladesh.
Received 22 June 2014; revised 12 November 2014
Xanthan gum is a commercially important microbial
exopolysaccharide (EPS) produced by Xanthomonas campestris.
X. campestris is a plant pathogen causing various plant diseases
such as black rot of crucifers, bacterial leaf blight and
citrus canker disease resulting in crop damage. In this study, we
isolated efficient local bacterial isolates which are capable
to produce xanthan gum utilizing different sources of carbon
(maltose, sucrose and glucose). Bacterial isolates from different
plant leaves and fruits were identified as Xanthomonas campestris
based on their morphological and biochemical characteristics.
Among the 23 isolates, 70% were capable of producing gum. Taro
plant, considered as new bacterial host, also have the capability to
produce xanthan gum. Production conditions of xanthan gum and
their relative viscosity by these bacterial isolates were optimized
using basal medium containing commercial carbon and nitrogen
sources and various temperature and rotation. Highest level of
xanthan gum (18.286 g/l) with relative viscosity (7.2) was
produced (Host, Citrus macroptera) at 28C, pH 7.0, 150 rpm
using sucrose as a carbon source at orbital shaker. Whereas, in lab
fermenter, same conditions gave best result (19.587 g/l gum) with
7.8 relative viscosity. Chilled alcohol (96%) was used to recover
the xanthan gum. FTIR studies also carried out for further
confirmation of compatibility by detecting the chemical groups.
Keywords: Citrus macroptera, Exopolysaccharides, FTIR, Gum,
Resin
Microbial exo-polysaccharides (EPS) or biopolymers
play important role in agro-chemistry, crude oil
recovery, and chemical, cosmetic, food, medical and
pharmaceutical industries1. The annual production of
xanthan gum as of 2012 according to Fufeng Group, one
of the largest manufacturers, is approx. 59000 metric tons2.
Xanthan gum is a water-soluble heteropolysaccharide
produced by fermentation using gram-negative
Xanthomonas spp., mainly X. Campestris3. X. campestris
——————
*Correspondence:
Phone: +88 0171 7318542
E-mail: mja.gebsust@yahoo.com
is a plant pathogen that damages the crop by diseases
such as bacterial leaf blight, black rot of crucifers, citrus
canker disease, etc.4.
The primary structure of xanthan gum consists of
repeated pentasaccharide units formed by two glucose
units, two mannose units and one glucuronic acid unit
in the molar ratio 2.8:2.0:2.0. Its main chain consists
of ß-D glucose units linked at the 1 and 4 positions.
Approximately, one-half of the terminal D-mannose
contains a pyruvic acid residue linked via keto group
to the 4 and 6 positions. Structural and superior
rheological properties of this polymer made it suitable
gelling and stabilizing agent in different industries5.
Xanthan is the most commercially accepted FDA
approved microbial polysaccharide has been
estimated to have a global demand of USD 987.7
million by 2020.3,6. In this context, it is important to
identify a local strain of Xanthomonas campestris and
optimize the fermentation conditions for xanthan gum
production. FTIR analysis can play a significant role
for detection chemical groups in xanthan gum7. In the
present study, we attempted to isolate the best
Xanthomonas campestris strain from local leaves and
fruits including satkora (Citrus macroptera) and
evaluate its capacity to produce xanthan gum.
Materials and Methods
Microorganisms and Media
Xanthomonas campestris was isolated from
different leaves and fruits viz., banana, bamboo,
batabi lemon, cabbage, carrot, gladiolus, jute, kagoji
lemon, kidney bean, malta, mango, orange, poaceae,
satkara, tomato, taro, water hyacinth, etc. The isolated
Xanthomonas strains were marked (e.g., XCa-
Xanthomonas isolated from cabbage, etc.) and grown
on Sucrose Peptone Agar (SPA) medium as described
by Opara and Odibo8. It was transferred every 14 days
to maintain good viability and stability of xanthan
production. Cultures of the bacterial strains were
maintained by glycerol stock and stored at −20C
(Wise CryoR, Korea). Production media was prepared
with (per 1000 mL) NH4H2PO4 1.5 g, K2HPO4 2.5 g,
MgSO4.7H2O 0.1 g and sucrose 50 g.
Inoculum and culture conditions
The inoculums required for xanthan gum
production were prepared by inoculating 10 mL of
152 INDIAN J EXP BIOL, FEBRUARY 2016
sterile Sucrose Peptone broth at tubes with the loopful
culture of different isolates separately. The tubes were
incubated at room temperature for 24 h (Lab
incubator of Digisystem Laboratory Instrument Inc.).
Xanthan gum production
Xanthan concentration was determined by a simple
modified method described by Leela and Sharma9.
For gum production, 10 mL of inoculum was added to
90 mL of production medium in a 500 mL
Erlenmeyer flask. The flasks were kept in orbital
rotary shaker for fermentation. After 96 h, samples
were collected, centrifuged for cell removal at 8000 g,
40 min at 4C. Suspended cell mass was removed
whilst supernatant was used for xanthan gum
isolation. Three volumes of chilled 96% alcohol were
added to the supernatant solution. After sometime,
xanthan gum was precipitated and settled down
(Fig. 1). This precipitated gum was left in an oven at
50-60C until constant weight was observed. Finally,
the constant weight of gum powder was taken. Then,
the best gum producing isolate was subjected to lab
fermenter (Electrolab, UK; serial no.-1204; batch no.
ref 166).
Cell density and relative viscosity measurement
The broth was centrifuged as described before. The
supernatant was discarded and the biomass was
washed once with 0.89% saline solution and
centrifuged again. The biomass was dried at 56°C to
constant weight, and expressed as grams of cells
(dry wt.) per liter of fermented broth (g/L)10. Relative
viscosity measurement was performed using
0.1% (w/v) gum solution with 1% KCl as described
by Wadhai & Dixit3.
Chemical group detection
FTIR spectra was analyzed by the dried powder of
xanthan with Fourier Transform Infrared to define the
functional group of synthesized xanthan gum. The dry
sample powder was mixed with potassium bromide
(KBr) and pressed into pellets under reduced pressure.
Fig. 1 — Gum precipitation after adding 96% alcohol
The FTIR spectra (SHIMADZU IR spectrometer,
Model No-dxp 400, Japan) results were obtained by
scanning between 4000 and 500 cm-1 7,11
Virulence test
For virulence test, 16 test tubes of SP broth were
inoculated with 16 different gum producing isolates
separately and kept for incubation at 30C for 24 h.
Two months old Allamanda plants (medicinal plant)
were placed in a wet condition for 24 h. These plants
were treated with broth inoculums and placed in a
room temperature to allow development of disease.
After 12 days, the percentages of lesion area of leaves
were measured.
Results and Discussion
Isolation and characterization of Xanthomonas campestris
According to Bergey’s Manual of Determinative
Bacteriology12; Opara & Odibo8; and Jabeen et al.13,
different biochemical tests were done. Identified
isolates showed positive results for different
biochemical tests (Gram staining; 4% NaCl test; Egg
Yolk reaction test; Starch hydrolysis; Glucose, Sucrose,
Maltose fermentation test; Citrate utilization test;
Gelatin Liquefaction; KOH test; Pigmentation and
colony colour test; and xanthan gum production test)
that strongly identified these bacteria as X. campestris.
Xanthan gum production
Among the 23 isolates of Xanthomonas campestris,
16 isolates (XMNg, XBD1, XKLD, XStl, XKI1,
XKI2, XMLTD, XKB, XKch, XCa, XJD1, XCrtD,
XGrD, XTo, XBTB and XGLA) showed their ability
to produce xanthan gum (Fig. 2 A-C). Borges et al.10
used Xanthomonas campestris pv. pruni 101 and
Wadhai & Dixit3 used X. campestris NRRL B- 1459
for reference strain comparing with other local citrus
canker diseased bacteria. Xanthomonas campestris
GK6 was isolated for gum, viscosity and biomass
analysis from infected cabbage leaves9. In this study,
XStl isolate appeared to be the best gum producer.
Another isolate, XCrtD also showed better response
(Fig. 2 A-C). In addition, new source Taro plant was
observed as host plant for xanthan gum producing
bacteria.
Effect of carbon sources
Xanthan gum production was observed in maltose
(Fig. 2A), glucose (Fig. 2B) and sucrose (Fig. 2C)
containing media separately (temperature 28C, pH
7.0 and rotation 150 rpm). Sucrose showed the best
result (18.286 g/L gum production, 7.2 relative
 BARUA et al.: PRODUCTION OF XANTHAN GUM BY XANTHOMONAS CAMPESTRIS 153

Fig. 2 — Production of xanthan gum on (A) maltose; (B) glucose; and (C) sucrose containing media (temp. 28C, pH 7.0 and rotation 150 rpm)

Table 1— Effects of pH, Temperature and Rotation (isolate-XStl;

Carbon source-Sucrose)
Gum conc. Relative Cell dry wt.
(g/L) viscosity conc. (g/L)
pH 7.0 18.286 7.2 2
(28C, 150 rpm)
7.5 16.321 6.2 40
(28C, 150 rpm)
Fig. 3 — Comparison of (A) flask and (B) lab fermenter
Temp. 28C 16.321 6.2 40 production of xanthan gum
(pH -7.0, 150 rpm)
30C 12.321 2.58 60 performed at 28C by XStl, but 60 g/L biomass was
(pH-7.0, 150 rpm) observed at 30C. Gilani et al.14 got good results at
Rotation 110 rpm 10.321 1.99 8 32C. Between two different rotations (150 rpm and
(30C, pH- 7.0)
110 rpm) used, maximum level of gum production
150 rpm 12.321 2.58 60
was observed after 96 h of cultivation in 150 rpm.
(30C, pH-7.0)
Nasab et al.15 used 240 rpm. Borges et al.10 produced
viscosity, 2 g/L biomass wt. for isolate XStl). 8.15 g/L gum using 200 rpm, whereas Wadhai & Dixit3
Glucose, sucrose, maltose, fructose, xylose, arabinose, obtained 6.47 g/L at 200 rpm. Some workers used
galactose, lactose, inositol, sorbitol, starch soluble highest rotation of 800 rpm to produce 30 g/L gum1.
and potato starch were used by Leela and Sharma9.
Borges et al.10 used only sucrose while Wadhai & Lab fermenter production
To observe the effects of lab fermenter, the best
Dixit3 used only glucose, and Gilani et al.13 used only
isolate from shaking flask production was taken to
molasses.
produce gum in lab fermenter at same conditions.
Effect of pH, Temperature and Rotation Between two scales of production, lab fermenter
In order to observe the effects of different pH, showed best result (19.587 g/L) which is slight more
media were adjusted to pH 7.0 and 7.5 where, 7.0 than that of flask production (Fig. 3). Nasab et al.15;
showed the best results for gum production (Table 1). Wadhai & Dixit3; and also Leela & Sharma9 achieved
Leela and Sharma9 found good performance at pH 7.2. highest rate of production after 96 h cultivation, as
On the other hand, best gum production was observed in the present study.
154 INDIAN J EXP BIOL, FEBRUARY 2016

FTIR spectra 1634, carboxyl group: 1557 and acetal group: 1055
To observe the Fourier transform infrared that recognized them suitable for industrial xanthan
spectroscopy, mainly Lii et al.16, protocol was used. gum (Fig. 4B).
The FTIR spectrum was used for detection of
different hydroxyl carboxyl, carbonyl, and acetal Virulence test
groups containing on xanthan gum (Table 2). Ramirez et al.17 suggested that virulence has some
According to Gilani et al.14 commercial xanthan gum relation with gum viscosity of fermentation broth. In
contains these chemical groups peak— hydroxyl this study, 50% gum producing isolates showed
group: 3386, carbonyl group: 1627, carboxyl group: infection to the medicinal plant Allamanda
1529 and acetal group: 1160. In the present study, all cathartica (Fig. 5). These are: XStl 6%, XCrtD
the isolates showed comparatively the same range of 2.5%, XMLTD 2%, XKch 2.5%, XBTB 1%, XGrD
peak for functional groups. The best isolate XStl 1%, XMNg 5% and XTo 3.5%. The virulence was
from Satkara contained hydroxyl group: 3383, calculated by percentage of lesion area. Wadhai &
carbonyl group: 1651, carboxyl group: 1558 and Dixit3 found 50-70% infection in lemon plant. Our
acetal group: 1055 (Fig. 4A); and gum produced by study showed less infection area, may be due to the
the isolate from Taro (Colocasia esculenta) plant fact that medicinal plant Allamanda has high
showed hydroxyl group: 3332, carbonyl group: antibacterial activity.
Table 2— FTIR spectral data of different functional groups for
sample product
Isolates Hydroxyl Carbonyl Carboxyl Acetal
XBD1 3379 1701 1560 1124
XCa 3340 1631 1558 1127
XBTB 3373 1660 1558 1053
XCrtD 3387 1660 1558 1134
XKB 3375 1849 1560 1060
XKch 3332 1634 1557 1055
XStl 3383 1651 1558 1055
XGrD 3387 1849 1560 1060
XJD1 3334 1848 1555 1055
XMLTD 3388 1770 1558 1052
XKLD 3350 1789 1559 1052
XTo 3360 1670 1560 1052
XKI1 3387 1632 1554 1057
XKI2 3307 1820 1530 1060
XGLA 3320 1829 1532 1060
XMNg 3327 1729 1529 1060 Fig. 5 — Hypersensitivity on medicinal plant Allamanda

Fig. 4 — FTIR Spectra of Xanthan gum; isolating source-Satkara (A) and Taro plant (B)
 BARUA et al.: PRODUCTION OF XANTHAN GUM BY XANTHOMONAS CAMPESTRIS
Conclusion
Among all, Xanthomonas campestris isolated from
satkora (Citrus macroptera) showed the best results.
Results from shaking flasks, lab fermenter and FTIR
studies suggested that the Xanthomonas campestris
isolated from satkora has potential for large scale
production.
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