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In this article we will discuss about the principle, requirements and procedure for fermentation of citric acid.
Principle:
When A. niger is grown in a medium having higher concentration of sugar a and low (acidic) pH, citric acid is produced.
Requirements:
1. Starter culture of Aspergillus niger maintained on Czapek’s Dox Agar.
2. Test medium (pH 1.6-2.5):
3. Fermentation medium (pH 1.6-2.5):
4. One litre Erlenmeyer flasks.
5. Pipettes.
6. Inoculation needle.
7. Sterile distilled water.
8. Glass marking pencil.
9. Muslin cloth.
Procedure:
1. Grow Aspergillus niger on Czapek’s Dox agar for 120-144 hours at room temperature (28°-30°C).
2. Test the culture for its ability to produce acid by streaking it on test medium with bromocresol green which turns its colour to yellow due to the presence of acid produced by the culture.
3. Make a suspension of the spores and inoculate it at the rate of one ml for 100 ml medium in one litre capacity Erlenmeyer flasks. Inoculum should be added gently, so that the spores float on the surface of the medium.
4. Incubate in dark at room temperature (28°-30°C).
5. Withdraw the broth aseptically after every 24 hours and test titrable acidity and when it is maximum, harvest.
6. Filter the fermented broth through muslin cloth and measure the volume.
7. Add little warm distilled water to mycelial mat to obtain traces of citric acid sticking to it.
8. Use the filtrate for further analyses.
9. Chromatographically confirm the organic acid. Ascending chromatography with a solvent system of n-butanol formic acid: water (10:2:15) (use upper organic layer) is used. Concentrate broth in a porcelain dish on a water bath and spot along with standards. After running for 5—6 hours, dry the chromatogram and spray with 0.04% bromocresol green in ethanol.
Dry and calculate Rf values:
Rf = Distance travelled by solute / Distance travelled by solvent
This confirms that the fermented broth contains citric acid. Estimation of residual sugar is done by appropriate method.