Xanthan Gum Production

Industrial production of Xanthan gum started in 1970’s. Over the years it became one
of the most important industrially produced anionic hetero polysaccharide. Reason
for its prime importance is its wide use in food, cosmetics, paper making, medicine,
paint and petroleum industry. It is used as an emulsifier, thickener and friction and
water mobility reducer. Xanthan gum is convenient to use over other
polysaccharides due to some of its properties such as of pH resistance, salt
resistance, stability towards many enzymes, viscosity properties at low
concentrations and resistance to shear degradation. Xanthan gum is industrially
produced by fermentation. Recombinant DNA mediated technique is used for
enhancing production of xanthan gum.

1. INTRODUCTION

Xanthan gum is a microbial exopolysaccharide. It is mostly excreted by the bacteria of

genus Xanthomonas, for example Xanthomonas Campestris NRRL B-1003, NRRL B1454,
NRRL B-1043 and sphingomonas. [1] It comprises of repeating units of pentasaccharide,
which is made up of Glucose, Mannose and Glucuronic acid in the molar ratio 2:2:1.  Its
primary chain comprises of beta-d-glucose monomers and its secondary chain comprises of
one glucuronic acid between two molecules of mannose. Secondary chain links primary
chain at every second glucose moiety. Precursors required for production of xanthan gum
includes UDP-Glucose, UDP-Glucuronic acid and GDP-Mannose. Glycosyl moieties are
transferred to lipid carriers with the help of glycosyltransferase. Once glycosyl moieties are
transferred to the lipid carrier, acetyl and pyruvyl residues are added to it. Length of the
xanthan chain varies depending upon 1. Bacterial strain used 2. Composition of media used
3. Down-stream processing. Average Molecular weight of Xanthan Gum is 2 x
106 Daltons. It can be produced by fermentation of glucose, sucrose and lactose. Various
enzymes can be used for producing xanthan gum, which includes Transferase, Acetylase
and Ketalase. Vectors can also be used for enhancing production both qualitatively as well
as quantitatively. Xanthan cryogels are widely replacing Gelatin in the food industries and
thus helping vegetarians. Xanthan hydrogels can be used as pH sensors.

2. AEROBIC FERMENTATION OF XANTHAN GUM USING CHEMICALLY

DEFINED MEDIA:

2.1. ORGANISMS USED:

Xanthan gum can be produce by aerobic fermentation process. The organisms used for its
production includes Xanthomonas Campestris primarily, along with X. Campestris other
strains of xanthomonas such as X. Carotate, X. Paraveriocola, X. Translucens, X.
Vasoculoeum and X. Hederae can also be used.

The medium required for aerobic fermentation comprises of at least one carbon source from
glucose, maltose and starch, water soluble nitrogen source such as peptone or yeast
extract, magnesium salt, phosphate ions and other trace elements. Also a chelating agent
such as citric acid is added to maintain stability of the medium in presence of iron and other
trace elements. Optimum pH is 5.5-9.
2.2. FERMENTATION PROCEDURE:

A seed culture is prepared by inoculating Xanthomonas in the seed culture media

Seed culture is then added to the fermentation medium (5% of the total volume)

Fermentation medium is incubated at 28oC and it is agitated (agitation speed 500 to

800rpm)

Once the cell density of more than 1gm/litre is obtained, slowly the speed of agitation is
increased from 500rpm to 800rpm.

Medium is incubated further till the cell density of more than 2 gm. /liter is obtained. Once it
is obtained, fresh media is added to the fermenter and media from the previous
fermentation is removed.

Xanthan gum produced is precipitated by using ethyl or isopropyl alcohols.

It is further purified by cold press or filtration or centrifugation.

Presence of Xanthan gum is confirmed by Mass spectroscopic analysis or NMR

The residues of the remaining media are protein rich and can be used as animal feed.

The optimum conditions required for process mentioned above are as follows:

1. Optimum pH: 5.5-8

2. Optimum temperature:28oC
3. Agitation rate: 0.2-2:vol./vol.-min
4. Air rate: 0.5 vol./vol./min.
 5. Dissolved Oxygen: 90%

2.3 COMPOSITION OF THE FERMENTATION MEDIA:

Composition of seed culture media: Composition of the production media:

1. Glucose : 5g/l 1. Glucose : 50g/l
2. Defatted Soybean meal: 5.4g/l 2. Nitrogen content: 0.32g/l
3. NH4NO3: 2.1g/l 3. KH2PO4
4. Trace elements: 0.05g/l 4. MgSO4.7H2O
5. Water 5. Trace elements: 0.05g/l
6. Water

3. PRODUCTION OF XANTHAN GUM USING SOLID OR SEMI SOLID MEDIA:

There may be a question in everyone’s mind. What is the need of developing a solid or a
semi-solid medium for producing xanthan gum? The answer to this question is, if we use
liquid media, it becomes more viscous once the production of exopolysaccharide starts.
This leads to problem in gaseous exchange. More amount solvents (Ethanol, Isopropyl
alcohol, KCl) are needed for precipitation of the Xanthan gum. More amount of water is
required for preparing media. Energy required for aeration is more as compared to the
semi-solid medium. All these factors make submerged fermentation more costly and energy
utilized is more. In contrast to this a semi-solid and solid media requires less amount of
water, solvents required for precipitating xanthan gum are less and energy required for
aeration is less. All these factors make the procedure more economical and less energy
consuming.

3.1 USE OF POTATO OR POTATO WASTE MEDIA:

Potato or potato waste can be used to prepare a solid or a semi-solid medium. Other than
potato alternative sources such as Apple pomace, spent malt grains, citrus waste, olive-mill
waste and sugar beet pulp can be used for fermentation. [6]In this type of fermentation
substrate provides complex starch medium which is utilized by the organisms. Solid
medium is prepared by cutting potato or potato waste into small pieces of size ranging from
¼ inches to /3 inches. [6] If required this medium is hydrated to certain level. However in case
of a semi-solid medium, the total solid content of the medium should be 6.5% or higher.
3.1.1 Composition of solid and semi-solid medium:

Ingredients Solid medium Semi-solid medium

1. Potato waste 50g 50g

2. K2HPO4 0.25g 0.25g

3. Yeast extract 0g 0g

4. Water 0ml 25ml

5. Incubation at 5.0 rpm 100rpm

6. Xanthan after 4 days 0..565g Not available

Ref: [6]

3.1.2. Organisms used:

Organisms used for this type of fermentation includes Xanthomonas and related genetically
modified organism (described later in this article), which are capable of producing Xanthan
gum. Preferred strains for inoculation include Xanthomonas campestris NRRL B-1459,
NRRL B-1003, NRRL B-1043.These organisms are grown in seed culture for 3-4 days prior
to inoculation in production media.

3.1.3. Fermentation procedure:

Fermentation media is produced and sterilized prior to inoculation of bacteria. Bacteria are
inoculated in the medium and allowed to grow for about 72-144 hrs. This is because growth
of organisms does not begin to slow down till 72 hrs. and there is no significant decrease in
the growth rate before 144 hrs. Medium is either incubated on a rotary shaker or inside a
rolling bottle (5rpm). Incubating it inside a rolling bottle is more energy efficient. Once the
fermentation procedure is complete substrate is washed until all xanthan gum produced is
dissolved in it. This dissolved xanthan gum is then precipitated using either ethanol or
isopropanol.  Precipitate is then isolated either by centrifugation or filtration. Conformation of
production of xanthan gum is done by Mass spectroscopy or NMR.

3.2. USE OF DAIRY WASTE:

Dairy waste such as whey and dairy permeates can be used for producing xanthan gum.
Whey refers to the liquid recovered during manufacture of cheese. And permeate is filtrate
obtained when whey milk and other dairy products are filtered. Filtration helps in removal of
globular proteins such as Lacto globulin. Filters allow to pass lactose through it. Filtered
liquid contains about 5% of solids, from which 80% is lactose. For producing xanthan gum
using dairy waste, there is a need of organisms capable of utilizing lactose and producing
xanthan gum. Organisms are genetically engineered either by inducing mutation or by
introduction of exogenous genetic material into the genome of an organism in such a way
that, it will help in converting lactose to xanthan gum. Microorganisms useful for this type of
fermentation includes Xanthomonas campestris, X alblineans, X. frageriae, X. junglandis, X.
mannihotis, X gummoscidans and X. aranopodis. These organisms can be genetically
engineered for producing xanthan gum. X. campestris X59-1232(ATCC-55258) is known to
increase the production.

4. USE OF GENETICALLY ENGINEERED ORGANISMS FOR PRODUCTION OF

XANTHAN GUM.

Genetically engineered microorganisms can be used for enhanced production of xanthan

gum. This will help in enhancing quality as well as quantity of an exopolysaccharide
produced.

4.1. RECOMBINANT DNA USED FOR PRODUCING XANTHAN GUM:

Xanthomonas are obligate aerobes, which are used for production of xanthan gum. Aerobic
fermentation process requires more energy for agitation. The development of recombinant
plasmid helps in increasing production of xanthan gum and lowering the energy
requirement. DNA sequences required for production of xanthan gum have been identified
and inserted into a vector.[8] This vector is then allowed to express in a host bacterium such
as nitrifying bacterium, which do not necessarily needs aeration for its growth.

1. DNA sequences may be naturally obtained or can be created synthetically.

2. Preparation of DNA sequences as present naturally in the plasmid pRK 290-H336.
3. Insertion of DNA fragment into a cloning vector.
4. Insertion of vector into a host for cloning
5. Transfer of cloning vector into an expression host such as denitrifying bacteria.
6. Culturing of those bacteria
7. Precipitation of xanthan gum
8. Isolation of xanthan gum.

4.2. USE OF MULTICOPY RECOMBINANT DNA PLASMID FOR INCREASING

PRODUCTION OF XANTHAN GUM:

A multi copy recombinant plasmid is prepared from a cloning vector pRK293 and a 12.4kb
DNA fragment containing genes required for xanthan gum production. This recombinant
plasmid is first inserted into a host such as E-coli for cloning. Once cloning is complete,
plasmids are transferred into a xanthomonas via conjugal transfer, with the help of helper
plasmid pRK2013 (ATCC-37159). This plasmid is a high copy number plasmid, which
increase the production of xanthan gum.

5. ENZYMES USED IN PRODUCTION OF XANTHAN GUM:

Enzymes used in producing xanthan gum include Acetylase, Ketalase, Pyrophosphatase

and Transferase. The functions of these enzymes are mentioned in the figure below
It has been observed that non-acetylated and non-pyruvylated polymers are more viscous
than acetylated and pyruvylated polymers, even when used in smaller quantities. Certain
strains of Xanthomonas are genetically engineered to produce non-acetylated and non-
pyruvylated polymers. This helps in improving quality of final product.

6. USE OF PROTEASES AND LYSOZYMES IN RECOVERY OF XANTHAN GUM.

WHILE isolating xanthan gum from fermented broth it is first dissolved completely and then
precipitated using alcohols. The problem in this process is that, a clear solution is not
usually obtained while dissolving. Therefore for obtaining clear solution after dissolution,
Proteases and Lysozymes may be used. This helps in complete recovery of xanthan gum
without any loss. The broth is preheated prior to protease treatment. The broth is treated
with Lysozyme after treatment with protease. Once the clear solution is obtained Xanthan
gum may be precipitated using ethanol or isopropanol.

7. SPHINGOMONAS USED IN PRODUCTION OF XANTHAN GUM.

Xanthan gum can be produced by the strains of Sphingomonas. 12 genes required for
synthesizing xanthan gum. These genes include gum B,C,D,E,F,G,H,I,J,K,L,M. all these
genes are clustered on one DNA segment and then inserted in an expression vector. This
vector is then allowed to express in a strain of Sphingomonas. Xanthan gum produced by
Sphingomonas is indistinguishable from that of Xanthomonas.

8. CONCLUSION:

Xanthan gum is the one of the most important industrially produced microbial
exopolysaccharide. Different strategies have been used for enhancing production as well as
the down-stream processing of xanthan gum. Use of solid and semi-solid media is more
economical and energy efficient than the liquid media, as the energy required for aeration is
less and less alcohol is required for precipitation. [4] Potato waste and dairy permeates can
be used for producing xanthan gum. Xanthan gum obtained from dairy permeates is of
better quality than the xanthan obtained from potato waste. This is because absence of
globular proteins in the production media. Recombinant DNA technology can be used for
producing xanthan gum. We can insert a recombinant vector expressing genes for xanthan
gum production into a nitrifying bacterium. This helps in increasing production as well as
lowering the cost for production. Multicopy recombinant plasmids can be used for increasing
production of xanthan gum. These plasmids are first allowed to express in host such as E-
coli then they are transferred into xanthomonas by conjugation. Enzymes can be used for
enhancing the production. These enzymes include Acetylase, Ketalase, and
Acetyltransferase. Xanthan gum can be produced by using strains of Sphingomonas. 12
genes required for producing xanthan gum can be cloned into Sphingomonas. These genes
include gum B,C,D,E,F,G,H,I,J,K,L,M.