OPTIMIZATION OF FERMENTATION CONDITIONS FOR · PDF filePRODUCTION OF XANTHAN BY Xanthomonas...

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Life Science Archives (LSA)

ISSN: 2454-1354

Volume – 3; Issue - 5; Year – 2017; Page: 1145 – 1156

DOI: 10.22192/lsa.2017.3.5.1

©2017 Published by JPS Scientific Publications Ltd. All Rights Reserved

Research Article

OPTIMIZATION OF FERMENTATION CONDITIONS FOR THE

PRODUCTION OF XANTHAN BY Xanthomonas campestris MTCC 2286

R. Krishnaveni1* and S. Balakumar

2,

Department of Microbiology, A.V.C College (Autonomous), Mayiladuthurai, Tamil Nadu, India.

Srinivasa Ramanujan Centre, SASTRA University, Kumbakonam, Tamil Nadu, India.

Abstract

Xanthan is an exopolysaccharide produced by the plant pathogenic bacterium Xanthomonas

campestris. Owing to its unique physical and chemical properties, Xanthan gum has attracted particular

attention in various industries as emulsifiers, stabilizers, binders, gelling agents, thickeners etc. In the present

study, Xanthan gum was produced from Xanthomonas campestris MTCC 2286 and optimized using various

parameters namely, the Carbon sources (Glucose, Sucrose, Fructose, Maltose and Starch soluble), the

Nitrogen sources (Yeast extract, Ammonium chloride, Ammonium sulphate and Sodium nitrate) at various

concentrations, pH, temperature, the size of the inoculums, the period of fermentation and the solvent used

for recovery. With the optimized media components, the yield of Xanthan is found to be 22.5 gram per ml.

Further, the so produced Xanthan was subjected to NMR and IR studies.

Article History Received : 15.06.2017

Revised : 12.07.2017

Accepted: 10.08.2017

Key words: Exopolysaccharides, Xanthan,

Xanthomonas campestris and Optimization.

1. Introduction Exopolysaccharides produced by a variety

of microorganisms are chemically well defined

and have attracted worldwide attention due to their

novel and unique physical properties. These

exopolysaccharides find multifarious industrial

applications in foods, pharmaceuticals and other

industries as emulsifiers, stabilizers, binders,

gelling agents, lubricants and thickening agents.

* Corresponding author: R. Krishnaveni

E.mail: [email protected]

These biopolymers are rapidly emerging as

industrially important and are gradually becoming

economically competitive with natural gums

produced from marine algae and other plants.

Their physical and chemical characteristics show

little variability and they are not vulnerable to

variations in climatic cultivation, production and

pollution conditions. Besides, gums of microbial

origin are susceptible to natural biodegradation,

promoting little damage to the environment and

diminishing pollution (Silvia and Crispin, 2006).

Among those biopolymers, which is

commercially produced on a large scale and which

is subjected to extensive studies is the xanthan

gum. This polymer represents the fastest growing

segment of the polysaccharide industry. The

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Krishnaveni/Life Science Archives (LSA), Volume – 3, Issue – 5, 2017, Page – 1145 to 1156 1146


largest growth is expected to be in the food

applications (8.3 % per year), where demand for

natural gums is decreasing. Worldwide

consumption of Xanthan is approximately 23

million kg/year, estimated to grow continuously at

an annual rate of 5 % - 10 %. Xanthan gum is a

water soluble heteropolyscchaaride composed of

glucose, mannose and glucuronic acid units that is

commercially produced using glucose or sucrose

(Sayyed Vahid et al., 2015).

Xanthan gum is a GRAS (Generally

accepted as safe) product and approved by FDA

(Tahera Ghashghei et al., 2016). Xanthan gum is

produced by a pure culture fermentation of a

carbohydrate by a plant pathogenic bacterium,

Xanthomonas campestris. The Xanthomonas

campestris is a Gram negative bacteria, motile

with a polar flagellum, chemoorganotrophic in

nutrition, catalase positive and oxidase negative.

The colonies are usually yellow in colour and this

was due to the presence of Xanthomonadins or

Bromynated aryl polyenes. Xanthan production

was generally carried out as aerobic submerged

fermentation process that runs for 78 - 96 hrs at 28

– 30 °C with a pH of 7. The Xanthan gum finds

various applications in food industry as

suspending, thickening, stabilizing and gelling

agent in juices, drinks, chocolates, agricultural

industry improving the flow ability in fungicides,

herbicides and insecticides formulations,

petroleum industry in enhanced oil recovery and

also used in ceramics, paper manufacturing,

textiles, cosmetics, tooth pastes, paints and inks.

Optimization of fermentation conditions

for xanthan production is particularly important in

view of wide industrial applicability of xanthan

gum. Xanthan fermentation is probably the most

complex fermentation process in terms of

rheological property and associated mixing bottle

neck in production xanthan gum. In order to

improve the productivity and shorter fermentation

time, extensive research work has been carried out

in xanthan fermentation conditions.

Most of the literature reported works with

reference to Xanthomonas campestris NRRL

B1459 and its derivatives. In the present study, a

strain of Xanthomonas campestris MTCC 2286

from IMTECH (Institute of Microbial

Technology), Chandigarh was obtained and

screened for its ability to produce Xanthan gum.

The purpose of this study was to optimize culture

conditions to produce xanthan by Xanthomonas

campestris MTCC 2286 with respect to several

operating variables such as carbon source and

concentration, nitrogen sources, pH, temperature,

culture volume, period of fermentation and

solvents used for recovery. A high recovery of

xanthan was attempted in batch fermentation by

employing optimized conditions. NMR and IR

spectroscopic studies were carried on the xanthan

gum produced in the optimized media.

2. Materials and Methods

Bacterial Strain and maintenance

Xanthomonas campestris MTCC 2286

strain was obtained from IMTECH (Institute of

Microbial Technology), Chandigarh, India. The

dried pellet in a sterile glass ampoule was

dehydrated in the skim milk before being streaked

on the YDC (Yeast Dextrose Calcium carbonate)

agar slant. The composition of YDC agar is as

follows: 10 g/L Yeast extract, 20 g/L Glucose, 20

g/L Calcium carbonate and 20 g/L agar. The agar

slants were incubated at 28 C for 24 hrs. Then,

they were stored at 4 C. The strain was

maintained in an active and stable condition by

transferring them every fortnight a month into a

new agar slant. Gram staining is done periodically

to check the purity of the culture.

Inoculum media

The YDC broth (Yeast Dextrose Calcium

carbonate) - 10 g/L Yeast extract, 20 g/L Glucose

and 20 g/L Calcium carbonate.

Production media Yeast extract - 3 g/L, KH2PO4 - 2 g/L,

K2HPO4 - 2 g/L, MgSO4 - 1 g/L and Glucose - 20

g/L.

Inoculum preparation

Actively growing cells from a newly

prepared slant was inoculated into the liquid

medium into 250 ml Erlenmeyer flask. The culture

was incubated at 28 – 30 C for 36 - 48 hrs in an



Orbital shaker. The liquid culture was used to

inoculate the final fermentation medium.

Fermentation Fermentation experiments were carried

out in 250ml Erlenmeyer flask at 28C and 200

rpm at a pH of 7.0. About 5 -10% v/v of inoculum

was added to the fermentation medium and

incubated for 72 hours. The shake flask

experiments were carried out in triplicates.

Analytical methods

Determination of bacterial growth

The bacterial growth was estimated when

they reach an optical density (600 nm) of >0.8.

Determination of Xanthan gum

The final fermentation broth was

centrifuged at 10,000 - 15,000 rpm for 30 minutes

to remove the cells. The supernatant was collected

and the gum was precipitated out of the

supernatant fluid with approximately three

volumes of isopropyl alcohol. Then, the gum was

dried at 55 C until reaching constant weight. The

production of Xanthan was evaluated by the

weight of the dry product per liter of fermented

broth and the average was expressed in g/L.

Determination of Viscosity

The ability of the bacteria to produce

exopolysaccharide was determined by increase in

the viscosity of the fermentation medium and

hence the viscosity of the fermentation medium

before and after fermentation was determined

using Oswald’s viscometer.

NMR and IR analysis

Xanthan was subjected to NMR studies.

The spectroscopy work was done at SASTRA

(Shanmuga Arts Science, Technology and

Research Academy) University, Thanjavur, Tamil

Nadu, India. About 10 mg of the sample dissolved

in 5 ml of D2O solvent was taken in a Wilmad tube of 5 mm in diameter. The spectra were

carried out at 30 C in D2O on a Bruker

spectrometer. The spectra were recorded at a

transmission frequency of 300 MHz with 16 runs.

The magnetic strength and the running time were

found to be 7.05 Tesla and 1.59 minutes

respectively. The software used in this

spectrometer was BSMS (Bruker Smart Magnetic

System). IR spectroscopy on Xanthan was also

performed at SASTRA University, Thanjavur,

India. The IR spectroscopy of xanthan gum was

run in KBr disc using Perkin - Elmer FT IR

spectrophotometer in the frequency range of 4000

to 450 cm-1

and the spectra were recorded.

3. Results and Discussion

Based on the results obtained, it was clear

that the optimum production of Xanthan gum by

Xanthomonas campestris MTCC 2286 was in a

sucrose medium with a concentration of 3 % that

runs for 96 hrs at 28 C with a pH of 6.5. The

Xanthan gum in the fermented broth was

recovered using Isopropyl alcohol. A maximum

yield of about 22.5 g/L was obtained from the

optimized media.

Carbon source is the most important

component of the media used from the production

of Xanthan, because it directly affects the

production yields, composition, structure and

properties of Xanthan gum. In the present study

different carbon sources namely glucose, sucrose,

fructose, maltose and soluble starch were studied

at a concentration ranging from 1 % to 3 %. But,

best carbon source was found to be glucose and

sucrose. To prove the above statement, it was seen

that sucrose was found to be the best source of

carbon for Xanthan production. Suresh and

Prasad (2005) used sucrose as the sole source of

carbon source at a concentration of 4.5 % and

were able to achieve a maximum yield in their

studies. By using Sucrose based medium, they

were able to obtain a yield of about 54 g/L but the

yield was found to be maximum at a 3 %

concentration for Xanthan production in our work

Similar to the present work, Krishna Leela and

Gita Sharma (2000) did an extensive study on

various parameters on Xanthan gum production.

They took a wide range of parameters such as

carbon source, nitrogen source, pH, temperature,

age of inoculum, percentage of inoculum and

period of fermentation. Their study showed that a

concentration of 2 % of all sugars namely the

glucose, sucrose, maltose, fructose and starch

soluble tested were found to yield a Xanthan gum



of about 14.74 g/L, 13.23 g/L, 12.321 g/L, 5.232

g/L and 12.10 g/L respectively. Contradictory to

their study, the Xanthan gum yield for glucose,

sucrose, fructose, maltose and starch soluble was

found to be 16.9 g/L, 17.8 g/L, 10.0 g/L, 14.3 g/L

and 11.3 g/L respectively at a concentration of 3

%.

Following carbon sources, nitrogen is the

most important medium component for

exopolysaccharide production. Abundant secretion

of exopolysaccharide is usually noticeable when

bacteria are supplied with abundant carbon source

and minimal nitrogen source. Though nitrogen is

important for various metabolic activities, a

minimal concentration would be enough to give a

good yield of Xanthan. In the present study,

nitrogen sources namely yeast extract, salts of

ammonium and nitrate were studied at a

concentration of 0.2 to 0.4 %, with the yeast

extract giving a maximum yield of about 16.3 g/L

of Xanthan gum at 0.4 % concentrations.

The other nitrogen sources were also able

to give a precisely a good yield of Xanthan of

about 16.1 g/L with sodium nitrate, followed by

ammonium chloride and ammonium sulphate with

an yield of about 15.3 g/L and 14.8 g/L. In this

study, nitrate salt gave a good yield similar to

yeast extract and this was supported by Fabien

Lettisse et al. (2001). Bajaj et al. (2006) supported

the present work stating that yeast extract was the

best suited nitrogen source for the production of

pollulan from Sphingomonas paucimobilis when

compared to other nitrogen sources. A high yield

of Xanthan of about 16.3 g/L was obtained when

yeast extract was used at a concentration of 0.4 %.

This statement was completely against the work of

Yang Ming Lo et al. (1997) according to whom

the Xanthan gum was maximum with an yield of

about 32 g/L, when yeast extract was in the

medium at a concentration of 0.3 %. Sanchez et al.

(1997) used ammonium phosphate that gave a

Xanthan of about 15.3 g/L at a concentration of

0.7 % whereas ammonium chloride and sulphate

gave a polymer production of 15.3 and 14.8 g/L

respectively.

The pH influenced the physiology of the

microorganism significantly by affecting the

nutrient solubility and uptake, enzyme activity,

cell membrane, morphology, by product formation

and oxidative - reductive reactions. In the present

work, the Xanthan yield was found to be good at

pH 6.5. This coincides with the work of Ishwar

Bajaj et al. (2007) who found out that the pH

varies from 6.5 to 7 during the production of

Gellan. Most of the work on Xanthan production

supported a better yield at a pH ranging between

6.5 and 7. Garcia - Ochoa et al. (2000) stated that

pH control was not necessary for Xanthan

production. But, our work was supported by Silvia

ad Crispin (2006) who demonstrated that the

polysaccharide production increased from pH 5 to

7.

Temperature is also an important

parameter in Xanthan production. In the present

work, the polymer production increased from 26

C to 30 C and decreased slightly at 32 C. The yield increased from 14.6 to 17.9 g/L and reduced

to 15.05 g/L respectively. Our work is supported

by Krishna Leela and Gita Sharma (2000).

Accordingly a temperature of about 28 - 30 C

was found to give a Xanthan production around 15

g/L. The work was also supported by Garcia -

Ochoa et al. (2000) that for optimum gum

production, a temperature range of about 28 - 30

C was proved to be optimum.

Two types of media were used for

inoculum development namely the YDC media

and LB media. When YDC media was used as

inoculum media, the polymer production was

found to be 17.7 g/L and the inoculum developed

on LB gave equally a yield of about 16.85 g/L.

This was opposed by the work of Ashraf et al.

(2008) as there was a very low production yield of

8.5 g/L on LB media. But our work on inoculum

development on YDC was supported by Rosalam

et al. (2008) giving a similar yield of about 20 g/L

with respect to our work.

The percentage of inoculum also affects

the production of the Xanthan polymer. Normally

a percentage of about 5 to 10 % v/v of inoculum is

required. In our study a 10 % v/v of inoculum was



used and gave a yield of about 20 g/L. This was

supported by a number of authors. The work of

Ashraf et al. (2008), Rajeswari et al. (1995) and

Maria Rodrigues et al. (2000) clearly revealed that

an inoculum of 10 % gave a good yield.

Contradictory to our study, some work done by

Garcia - Ochoa et al. (2000), Suresh and Prasad

(2005), El-Tayeb and Khodair (2006) stated that a

5 % v/v of inoculum gave a higher yield of

Xanthan gum. The work by Krishna Leela and

Gita Sharma (2000) supported our results and they

reported a yield around 15 g/L when a 10 % v/v of

inoculum was used.

The period of incubation also have an

effect of Xanthan production. Most of the

literature states that a period of 72 to 96 hrs was

found to give a maximum yield. In the present

work, the yield was found to be 21 g/L at the end

of 96 hrs of fermentation period. This was

supported by Krishna Leela and Gita Sharma

(2000) who reported a value of a maximum yield

at the end of 96 hrs of fermentation. In contrary to

our work, Xanthan production by Maria et al.

(2000) and Suresh and Prasad (2005) reported a

maximum yield at 72 hrs. Even there are reports of

maximum yield at 54 hours by Borges et al.

(2008).

Recovery of Xanthan from the fermented

broth was achieved by using water miscible

solvents namely acetone, isopropyl alcohol and

ethanol. Most of the literature supported the use of

Isopropyl alcohol as the best solvent for Xanthan

recovery. In our work, use of Isopropyl alcohol

resulted in the recovery of Xanthan gum of about

21.3 g/L. This statement was supported by the

work done by Garcia - Ochoa et al. (2000) on

Xanthan production with a maximum yield.

Figure - 1: Gram staining of Xanthomonas

campestris

Figure – 2: Capsule staining of Xanthomonas

campestris

Figure – 3: Growth of Xanthomonas campestris

on YDC agar with yellow pigmentation



Figure - 4: Effect of Carbon sources on Xanthan gum production

Figure – 5: Effect of various Nitrogen sources on Xanthan gum production



Figure – 6: Effect of pH on Xanthan production

Figure – 7: Effect of Temperature on Xanthan production



Figure – 8: Effect of different Inoculum media on Xanthan production

Figure – 9: Effect of Volume of inoculum media on Xanthan production



Figure – 10: Effect of Period of inoculum on Xanthan production

Figure – 11: NMR Spectrum of Xanthan



Figure – 12: IR Spectrum of Xanthan

Table - 1: Effect of solvents on Xanthan recovery

Solvents Xanthan yield (g/L)

Original Replica Average

Acetone 19.2 20.7 19.9

Ethyl acetate 11.8 12.8 12.3

Isopropyl 20.9 21.3 21.1

Table – 2: Determination of viscosity in the production medium

Table – 3: Optimized production of Xanthan at pH 6.5, temperature 30 C using Sucrose at a

concentration of 3 %

S.

No

Viscosity of the medium in poise

Before

fermentation

After

fermentation

1 0.0019 0.003

S.

No

Xanthan yield (g/L)

Original Replica Average

1 21.5 23.6 22.5



4. Conclusion

Optimized production of media

components resulted in a good yield of about 22.5

g/L of Xanthan at 6.5 pH, 30 ºC for a period of 96

hours. The NMR and IR studies showed some

variations in the structure of xanthan suggesting it

to be a derivative of xanthan. Presence of

impurities conveys that proper purification should

be performed and focused. Future work was

directed towards characterization of Xanthan gum.

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DOI Number DOI: 10.22192/lsa.2017.3.5.1

How to Cite this Article:

R. Krishnaveni and S. Balakumar. 2017. Optimization of Fermentation conditions for the

production of Xanthan by Xanthomonas campestris MTCC 2286. Life Science Archives, 3 (5):

1145 – 1156.

DOI: 10.22192/lsa.2017.3.5.1

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