Some of the methods used for commercial production of Citric acid

are as follows:
Citric acid (2-hydroxypropane-l, 2, 3, tricarboxylic acid) is a primary
metabolic product and is formed in the tricarboxylic acid cycle.
Chemically, citric acid was synthesized from the glycerol. It was Wehmer
(1893) who reported the wide occurrence of citric acid in the microbial
metabolites. Molliard (1922) confirmed the accumulation of citric acid in
the cultures of Aspergillus niger under the conditions of nutrient
deficiency.
(i) Koji Fermentation Process: There are three methods for
commercial production of citric acid:
This method is used in Japan which accounts for about one fifth of citric
acid produced per annum. Strains of A. niger are used in this method.
(ii) Liquid Surface Culture Fermentation Process:
In this method, liquid cultures are inoculated with the spores of A. niger
which germinate within 24 hours. Mycelia cover and float on the surface
of the solution.
(iii) Submerged Culture Fermentation Process:
In this case the mycelia of A. japonicum grow in the solution of about 15
cm depth in tanks. Citric acid produced in this way is inferior to that
produced by liquid surface culture fermentation.

For commercial production, strains of A. niger are selected from the

hybrids or mutants developed by certain procedure. The strains should
be such that could produce not less than 80 g citric acid per 100 g
glucose. For large scale production, continuous culture is not Suitable.
Therefore, a multitank system is required for continuous fermentation in
any process in which cell growth and metabolic products occur at
different times.
Culture medium contains carbohydrates, KH2PO4 (0.01-0.3%),
MgSO4.7H2O (0.25%) and a few trace elements. Solutions of
carbohydrates are concentrated cane juice, glucose or sucrose. If
substrate is molasses, ferrocyanide (20-150 mg/litre) can be added to
culture medium, before sterilization, to precipitate excess iron. Instead of
iron, clarified molasses can be passed through an ion exchange resin.
Under laboratory condition, highest yields are obtained by using sucrose
passed through an ion exchange resin.
At this stage, it is necessary to add copper (0.1-0.50 ppm) to the culture
medium only when the fermenter is made up of steel or some impervious
coating is applied (to ordinary fermenters) pH of the culture medium is
maintained to about 3.5 by adding ammonium nitrate (0.25) which avoids
the formation of oxalic and gluconic acids.
The culture medium is sterilized by passing through the pipes of steam
jacketed heat exchanger and cooled down to about 30 C in another
exchanger.

Inoculation of A. niger strain to the culture medium is followed by

aeration by bubbling air to allow maximum growth of the fungus.
Fermentation process is completed in about 5-14 days at 27-33 C. The
fermented broth acts as the source of citric acid. Lime (Ca (OH)2) is
added4o allow precipitation of citric acid in the form of calcium citrate.
Again the precipitate is treated with sulphuric acid to precipitate insoluble
calcium sulphate. It is then filtered.
The filtrate containing citric acid is purified by passing through column of
carbon granules pretreated with heat or washed with hydrochloric acid.
Passing through ion exchange bed solutions is demineralised, which is
then concentrated under vacuum to form crystals. Crystals are
recovered by centrifugation and mother liquor is returned to evaporator
to get remaining crystals.
Citric acid is made available in market in the form of anhydrous
crystalline chemical as powder.

Biochemistry of Fermentation:
Citric acid is produced during trophophase as a result of interruption of
tricarboxylic acid cycle. In A. niger about 78% sugar passes through
EMP pathway and results in production of acetyl CoA which condenses
with oxaloacetic acid to yield citric acid.
Further citric acid cycle is interrupted by inhibition of aconitase and
isocitric dehydrogenase either by copper or hydrogen peroxide.

However, iron is an essential cofactor of these enzymes. The Cu and Fe

in a ratio 0.3:2 (mg/litre) interrupt the activity of these enzymes at pH 2.0.
Aspergillus niger :
The cultures of Aspergillus niger were maintained on sterilized potato dextrose agar medium
(Diced potato 200 g/l, Dextrose 20 g/l and Agar 15 g/l), pH 4.5 and stored at 5C in the
refrigerator. All the culture media, unless other wise stated, were sterilized in autoclave at 15lbs/inch2pressure (121C) for 15 min.