Production of Xanthan Gum

Introduction –
Xanthan gum with molecular formula (C35H49O29)n was discovered by Allene Rosalind Jeanes
and her research team in the early 1960s at the Department of Agriculture.Hetero polysaccharide,
sugar residues Glucose, Mannose Glucouronic acid, Acetyle Group, Pyruvyl groups. Polyanionic
heteropolysaccaharide. Molecular weight (2.0 × 106–2.0 × 107 Da). Highly viscous, water soluble non-
toxic.

Produce BY
Xanthan gum is produced by Xanthomonas spp. such as Xanthomonas
campestris, Xanthomonas pelargonii, Xanthomonas phaseoli and Xanthomonas
malvacearum during aerobic fermentation. . Among xanthomonas species X. campestris is
commercially used for xanthan gum production and is widely studied. X. campestris is a plant
pathogen that can infect many plants, including some important crops like cabbage, alfalfa and
beans. Members of the Xanthomonas species are single straight rod-shaped, Gram-negative,
0.4–0.7 μm wide and 0.7–1.8 μm long.

Microbial Production of Xanthan Gum

The major operations involved in the microbial production of xanthan are as follows:

l Organism and inoculum preparation

2 Media preparation

3 Fermentation

4 Downstream processing

Organism and Inoculum Preparation

Xanthan gum is produced by the bacterium X. campestris, maintained on a sucrose–tryptone–yeast-
extract agar (STYA) slant. An inoculum of 5–10% is needed for optimum polysaccharide production. The
inocula are prepared by transferring cells from an STYA slant (at 28 C) to a tube containing 7 ml STYA
medium (pH 7.0), and incubating at 28 C and 160 rpm for 24 h. The inoculum then is transferred to 250
ml flasks and, 24 h later, to the laboratory fermenters.

Media Preparation ;
1 Carbon Source (2-5%), Glucose, corn starch or syrup, citrus waste
 2 Nitrogen Source (0.05-1%), Ammonium nitrate, peptone, Yeast extract

3 Enhancer (0.09 -0.18%) citric Acid.{chelating agent} It prevents the heat presipitation of xanthan

The addition of corn steep liquor at 1 g

l

1 increases the yield and viscosity of xanthan gum produced by cells grown in sucrose. Corn steep
liquor also shortens the cultivation time and promotes better sugar utilization

Process parameters :
PH of medium 6-7.5

During the production phase organic acids are synthesized due to which Ph of medium drops below 5 if
this happens there will production of xanthan. Thus careful monitoring and addition of antifoam agents
is very essential.

2 Potassium hydroxide solution generally is used for the control of pH in the fermentation: It serves as
both a titrant and a potassium source, and although it is more expensive than sodium hydroxide, it is
preferred because it yields xanthan gum containing the desirable Kþ forms of mannose and glucuronic
acid. Dissolved oxygen is another par

Temperature (30-33Celcius)

Plays a role on the molecular weight of xanthan. Temperature regulates the acetylation , higher
temperature higher acetylation, higher the molecular weight.

Oxygen profile (0.03 vvm) weigh Xanthan is an obligate aerobe, and it requires coutinous
aeration What happens it during the later phases when higher amount of xanthane is produced, the
media become viscous and thus aeration is problem at that point time. Care must be taken to
continously aerate the mixture.

Fermentation time (48 hours)

Bioreactor design :- Stirrred tank bioreactor ,Immobilized bioreactor(support matrix)

Viscosity The higher the pyruvic acid content in the xanthan gum, the greater the viscosity and
thermal stability.

Downstream Processing
Separate the Cell

Pasteurization
Ultrafiltration

Alcohol precipitation

(95% Ethanol) and washing. The addition of an electrolyte, usually potassium chloride (1%), lowers the
isopropanol requirement by about 30%. The addition of salts in sufficient concentration causes precipitation or
complex coacervation due to the binding of the cation salt to the ionized groups in the molecule, thus reducing the
amount of alcohol necessary for precipitation

Centrifugation or filtration

Drying (drum drying)or any suitable drying method

The dried precipitate finally is milled, to obtain a product of mesh size 40–200. The milled product
contains no viable X. campestris cells so that it can conform to the FDA criteria for food-grade products.
Great care during milling is required, to avoid excessive heating, which can lead to the darkening or
degradation of the polymer. Owing to the hygroscopic nature of the milled product, containers with a
low permeability to water are used for packaging. The absorption of moisture can cause clumping during
redissolution and, in some cases, hydrolytic degradation of the product

Isolation of Xanthomonas Compestris

A Pathogen that causes common bacterial blight in beans from infected leaveSterilize hands using
ethanol. To isolate xanthomonas compestris you will need to collect infect leaves from the Field.

Cut OFF the infected parts, showing the typical symptom of the disease.These pieces will need to be
sterilized to Kill any other organisms

Add the infected material to a petri dish using sterile Forceps. Immerse the material with 2% NaCIO and
leave for 2 min. After, rinse off the excess NaCIO using distilled water.Repeat this process the all NaCIO
removed.

→ The material is now ready for isolation. → Using the balance weigh the cut leaf material.

→ cut the leaf material into smaller pieces using sterilized blade. Place cut pieces into 30ml bottle.

Pippete 2ml/g of phosphate buffer Saline and add into leaf material. In this instance, 500ul of PBS is
added to 1/4g of infected leaf material.

Leave the infected material to soak in PBS for 16-24 hour. The next step will be to create serial dilutions
of the homogenate. Add 4.5ml of PBS to four 30ml bottles. Label the four tubes 1,2,3,4. Vortex the leaf
homogenate at 100rpm for 1-2minutes. Pipette 500ul of the leaf homogenate into the 1 tube and vortex
the solution at 100rpm for 1-2min.Pipette 500ul from tune 1 to tube 2.Now vortex the tube labeled 2 for
1-2min.
Repeat the process for the remaining tubes (i.e. add 500ul from tube 2 to 3,etc).Plate the homogenate
onto MXP media.( see the CIAT pathology protocol. foMXPmedia preparation).Pipette 100ul of each of
the diluents onto a separate MXP plate(only two dilute solutions are shown here)

Spread evenly onto each plate using a sterile spreader.Ensure that the spreader is sterilized between
each dilute solution.Using sealing tape, seal off the petri dishes for each of the concentration.Incubate
the plate for 5-6 days at 28c.Label the plate with date media used and pathogen ( xanthomonas
compestris pv phaseoli-XCP).After 5 to 6 days remove the cultures.

Open a plate sterilize the loop using a flame and allow it to cool. Select one yellow mucoid colony with a
translucent area surrounding it, and streak the surface of YDC media. ( refer the CIAT pathology protocol
for YDC media preparation). Seal the YDC petri dish securely using sealing tape. Label the YDC plate with
the date and pathogen culture. Incubate for 1-2 days at 28c.

Flood the plate with phosphate buffer at a PH of 7. Using sterile forceps, scrape the bacteria off the
media to mix with the buffer.Pour the bacteria homogenate into a flask. At this stage the cell
concentratiom is high, so you need to dilute it further.Add a little more buffer and shake the mixture
lightly. (Before inoculating the plants you should adjust the cell concentration of the bacterial mixture)

We can use the haemocytometer to dilute the inoculum to 5 10 ki power 7cfu per ml. you can use a
colorimeter or( Vis spectrophotometer) at a wavelength of 620mm, to adjust the concentration.

The inoculum is now ready