D. Y.

PATIL UNIVERSITY, NAVI MUMBAI

SCHOOL OF BIOTECHNOLOGY AND BIOINFORMATICS
PRACTICAL MANUAL PREPARATION

Programme- M.Sc BT Semester- III

DOE DOS

Practical Title- Production and recovery of citric acid by fermentation at laboratory scale.

Experiment. No:6a

Aim- To produce citric acid using ASPERGILLUS NIGER grown in a suitable

medium

Theory: Citric acid, or 2-hydroxy-propane-1,2,3-tricarboxylic acid (C6H8O7.H2O) is

a naturally occurring weak organic acid found in all citrus fruits and it was
first isolated by karls scheels in 1874, from lemon juice.
It is widely used in the food, pharmaceutical, and beverage industries as an
acidifying and flavor-enhancing agent
The versatility and non-toxicity of citric acid are its main positive
characteristics. It is accepted globally as ‘generally recognized as safe’ (gras)
and has been endorsed by the fao/who committee on food additives
It is largely produced by microbial fermentation. The fungus aspergillus
niger is most commonly used for industrial production of citric acid. It is
considered to be superior to other microorganisms for the commercial
synthesis of citric acid because of the following reasons:
1. Can easily be cultivated.
2. Process uniform biochemical properties.
3. Produces only of small amount of oxalic acid under controlled conditions.
4. Yield large amount of citric acid.
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.

Requirements- I) MEDIA:
1) sterile citric acid propagation medium-100 ml
2) sterile citric acid production medium-100 ml

II) CULTURE: ASPERGILLUS NIGER

III) GLASSWARES:
1. 250ml erienmeyer flasks
2. Centriguge tubes/ test tubes
3. Sterile pipette-10ml

IV) INSTRUMENTS USED:

1. Shaker incubator at 33oc,80 rpm
2. Centrifuge
3, Incubator at 37oC

Result-
Conclusion /
Discussion

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 D. Y. PATIL UNIVERSITY, NAVI MUMBAI
SCHOOL OF BIOTECHNOLOGY AND BIOINFORMATICS
PRACTICAL MANUAL PREPARATION

Programme- M.Sc BT Semester- III

DOE DOS

Practical Title- Production and recovery of citric acid by fermentation at laboratory scale.

Experiment. No.6b

Aim- To estimate the concentration of citric acid produced by Aspergillus niger by

titrimetric method

Principle: Citric acid is one of the most important organic acid. Initially citric acid was
extracted from fruits but now it is largely produced by microbial fermentation.
Aspergillus niger and Aspergillus mentouis are widely used for commercial
production of citric acid. It is widely used in food, soft drinks, pharmaceuticals,
manufacturing of ink and dyes, leather industry, electroplating etc.

Many strains of Aspergillus niger cannot oxidize citric acid and hence accumulate
in the culture medium. The composition of the fermentation medium is critical for
obtaining high yield of citric acid and hence it is essential to limit the growth of
fungus so that high level of citric acid can accumulate in the medium. To determine
the efficiency of citric acid production by the given strain of Aspergillus niger, a
simple acid base titration-based experiment is performed. The filtrate obtained
from production medium is titrated against alkali of known strength (Normality),
using Phenolphthalein as an indicator. The end point is pale pink colour. The
volume of alkali used for titration is noted and used to calculate the “normality”
and the percentage of acid in the filtrate.
Requirements: 1) Burette
2) Pipette
3) Conical flask
4) 0.1N HCl solution
5) NaOH solution
6) Phenolphthalein
7) Filtrate sample

Procedure A] Standardization of Normality of NaOH:

1) Normality of the given NaOH solution was determined by standard acid-
base titration method against 0.1 N HCl.
2) The indicator used was phenolphthalein.
3) The end- point was determined at colour change from colourless to pale
pink.
4) About 2-3 drops of indicator was added to the 10 ml of given NaOH
solution and amount of 0.1N HCl required to obtain the pale pink colour
change was noted.
5) The above steps were repeated for two more times to obtain C. B. R.
B] Standardization of Known concentration of Citric acid:
1) Normality of standard 1% citric acid was determined in a similar method
against the ‘estimated NaOH’.
2) The indicator used was phenolphthalein.
3) The end- point was determined at colour change from colourless to pale
pink.
4) About 2-3 drops of indicator was added to the 10 ml of 1% standard Citric
acid solution and amount of 0.084N NaOH required to obtain the pale
pink colour change was noted (Normality of the NaOH solution used was
determined before).
5) The above steps were repeated for two more times to obtain C. B. R.

C] Estimation of Unknown concentration of Citric acid:

1) After filtration of the production medium, 10 ml of filtrate was titrated
against NaOH in burette.
2) The indicator used was phenolphthalein.
3) The end- point was determined at colour change from colourless to pale
pink.
4) About 2-3 drops of indicator was added to the 10 ml filtrate and amount
of NaOH required to obtain the pale pink colour change was noted.
5) The above step was repeated for two more times and Constant Burette
Reading was obtained for determination of the percentage of citric acid
produced by the given strain of Aspergillus niger in the production
medium used.
 6) Further calculation is required to determine the concentration of citric acid
obtained.

Result:

Conclusion/
Discussion:

Observations 1) Standardization of NaOH:

1) Solution in burette: 0.1 N HCl
2) Solution in conical flask: Given NaOH solution
3) Indicator: Phenolphthalein
4) End-point: Colourless to pink

Burette Readings Constant Burette Reading

1 2 3
Initial 0.0 ml 0.0 ml 0.0 ml
8.4 ml
Final 8.4 ml 8.2 ml 8.4 ml
Difference 8.4 ml 8.2 ml 8.4 ml

2) Standardization of Citric acid:

1) Solution in burette: 0.12 N NaOH
2) Solution in conical flask: 1% Citric acid solution
3) Indicator: Phenolphthalein
4) End-point: Colourless to pink

Burette Readings Constant Burette Reading

1 2 3
16.5 ml
Initial 0.0 ml 0.0 ml 0.0 ml
 Final 16.4 ml 16.6 ml 16.5 ml
Difference 16.4 ml 16.6 ml 16.5 ml

3) Estimation of unknown Citric acid samples:

1) Solution in burette: 0.12 N NaOH
2) Solution in conical flask: Filtrate solution obtained
3) Indicator: Phenolphthalein
4) End-point: Colourless to pink
Sample A:
Burette Readings Constant Burette Reading
1 2 3
Initial 0.0 ml 0.0 ml 0.0 ml
4.4 ml
Final 4.4 ml 4.3 ml 4.5 ml
Difference 4.4 ml 4.3 ml 4.5 ml

Sample B:
Burette Readings Constant Burette Reading
1 2 3
Initial 0.0 ml 0.0 ml 0.0 ml
4.2 ml
Final 4.5 ml 4.2 ml 4.2 ml
Difference 4.5 ml 4.2 ml 4.2 ml

Calculations:
1) Normality of NaOH= Normality of HCl * Volume of HCl
Volume of NaOH
=0.1 * 8.4
10
=0.084N

2) Known concentration of citric acid:

Normality of 1% Citric acid= Normality of NaOH * Volume of NaOH
Volume of Citric acid
=0.084 * 16.5
10
=0.14N

3) Unknown concentration of citric acid:

a) Normality of Citric acid= Normality of NaOH * Volume of NaOH
Volume of Citric acid
Sample A =0.084 * 4.4
10
=0.04N
Sample B =0.084 * 4.2
10
 =0.035N
b) Percentage of citric acid= Normality * Equivalent weight of citric acid *
100
Volume of Filtrate
[Note: Equivalent weight of Citric acid (monohydrous) = 70 mg/ mol.]
Sample A =0.04 * 70 * 100
10
=28%
Sample B =0.035 * 70 * 100 =24.5%
10

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