Production and estimation of Citric acid

Aim

To demonstrate and estimate citric acid production by Aspergillus niger

 

Principle

Citric acid is the key intermediate of tricarboxylic acid cycle. Citric acid is used as acidulant in food and pharmaceutical industry for the production of carbonated beverages and used as plasticizer. Commercially, citric acid is produced by surface fermentation using Aspergillus niger. Beet molasses medium containing 10-20% sucrose is used in the commercial production. Doelger Prescott medium is used in this experiment to produce citric acid using Aspergillus niger. 

Estimation of citric acid is done by titration against standard NaOH using phenolphtahelin indicator. The amount of citric acid produced is expressed in gram per 100ml.

 Materials Required

1. Culture: Aspergillus niger

2. Media: Doelger Prescott broth

3. Routine microbiological facilities, Burette, pipette, conical flask, measuring cylinder

4. Reagents: 0.1 N NaOH, Phenolphthalein

 Procedure

1. 300 ml of Doelger Prescott media was prepared. pH was adjusted to 4.0 using 0.1 N HCL.

2. The medium was autoclaved and inoculated with a loopful of Aspergillus niger culture.

3. The flask was incubated at room temperature for 7 days.

4. After incubation for 7 days, the pH of the medium was noted.

5. The amount of citric acid in the culture broth was estimated by titration.

Estimation of Citric acid by titration

1. 1 ml of phenolphthalein indicator was added to 200 ml hot boiled water in a 250 ml flask.

2. 1 ml of culture filtrate was added to the above flask and titrated against 0.1  NaOH to the endpoint, in a well illuminated white background.

3. The initial and final readings were noted to calculate the volume of standard NaOH

 Result

The pH of the media was 4 before inoculation of Aspergillus niger culture and it decreased to 2 after 7 day incubation. This is due to the production of citric acid by the fungal cylture.

The amount of citric acid was estimated to be 0.30 g/100ml

  

Observation (left side)

1 N NaOH stock solution was prepared by dissolving 40 g NaOH in distilled water. From this stock solution, 0.1 N NaOH was prepared by mixing 50 ml stock solution with 450 ml distilled water.

Citric acid estimation

Conical flask: 200 ml hot boiled water + 1 ml of phenolphthalein + 1 ml of culture filtrate

Burette: 0.1 N NaOH

Endpoint: Appearance of distinct pale pink colour

	1	2	3
Initial burette reading	50	48	46
Final burette reading	49.5	47.6	45.5
Volume of 0.1 N NaOH rundown (ml)	0.5	0.4	0.5

 

Calculation (left hand side)

Citric acid (g/100ml) =

Volume of 0.1N NaOH × Normality × Equivalent molecular weight of citric acid ×100

Volume of sample taken

0.47 × 0.1 × 0.064 × 100/1

Citric acid (g/100ml) =         0.30 g/100 ml