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Saturday, August 4, 2012

Citric Acid Production by Aspergillus niger, Estimation of Citric acid


Citric acid is an important organic acid and it was initially being extracted from citrus fruits. Nowadays it is largely produced by microbial fermentation. Citric acid is commercially used in foods, soft drinks, pharmaceuticals, leather tanning, electroplating etc. Aspergillus niger is the most commonly used species for the production of citric acid.
Most strains of  Aspergillus niger which are mutants cannot oxidize citric acid and hence accumulate in culture medium. The composition of the culture medium is critical for obtaining high yield of citric acid. Its is essential to limit the growth of the fungus, so that high yield of citric acid accumulates in the medium. this can be accomplished by keeping trace metal deficiency in the medium. Acid is added to achieve low pH of 3.5. Sucrose serves as a carbon source for the production of citric acid. Ammonium nitrate is used to prevent the fermentation of oxalic acid glutamic acid.Fermentation is aerobic and can be carried out by submerged culture method.

Citric acid is an important organic acid and it was initially being extracted from citrus fruits. Nowadays it is largely produced by microbial fermentation. Citric acid is commercially used in foods, soft drinks, pharmaceuticals, leather tanning, electroplating etc. Aspergillus niger is the most commonly used species for the production of citric acid.

Citric Acid Production Principle
Most strains of  Aspergillus niger which are mutants cannot oxidize citric acid and hence accumulate in culture medium. The composition of the culture medium is critical for obtaining high yield of citric acid. Its is essential to limit the growth of the fungus, so that high yield of citric acid accumulates in the medium. this can be accomplished by keeping trace metal deficiency in the medium. Acid is added to achieve low pH of 3.5. Sucrose serves as a carbon source for the production of citric acid. Ammonium nitrate is used to prevent the fermentation of oxalic acid & glutamic acid. Fermentation is aerobic and can be carried out by submerged culture method.

Citric Acid Production Medium
Sucrose - 150 gm
Ammonium nitrate - 2.5 gm
Potassium Dihydrogen Orthophosphate - 1.0 gm
Magnesium sulphate heptahydrate - 0.25 gm
Distilled water - 1L
pH - 3.5

Culturing Aspergilus niger
  1. prepare the citric acid medium & dispense about 50ml in 250ml conical flask.
  2. Autoclave and allow it cool.
  3. Inoculate the medium with spores of Aspergillus niger & incubate it on a shaker water bath at 25oC with gentle shaking for 3-5 days.
  4. After Incubation, filter the mycelium using double layered muslin cloth & measure the amount of citric acid in the filtrate by colorimetric and titrimetric methods.
Estimation Citric Acid by Titrimetric Method
The filtrate obtained is titrated against an alkali of known strength using phenolphthalein as indicator.The end point is the formation of pale pink colour. The volume of alkali used for neutralization is used to find the normality and the percentage of acid in the sample.

Estimation of Citric acid using phenolphthalein as indicator
  1. 100ml of the filtrate is pipetted into a conical flask and 2-3 drops of phenolphthalein indicator is added to it.
  2. This is titrated against 0.1N NaOH taken in the burette till a pale pink colour is formed.
  3. The titration is repeated till concordant values are obtained.
Calculation:
Example of Titration Results
No
Initial Burette Reading
Final Burette Reading
Vol of NaOH used
1
0
8.0
8.0
2
0
8.1
8.1
3
0
8.1
8.1

Normality of Citric acid  =  [N(NaOH) * V(NaOH)] / V(Citric acid)
                                      = [0.1 * 8.1]/ 10
                                      = 0.081

% of Citric acid = [Normality * Equivalent wt of citric acid * 100] / Volume of filtrate
                        = [0.081*96*100] / 10
                        = 77.76%

N - Normality, V - Volume.
Equivalent wt of Citric acid - 96.