Citric Acid

 Citric Acid was first produced commercially by John and Edmund Storage Company in
UK in the year 1826. Scheele (1789) reported the isolation and crystallization of the four
constituents of lemon juice. Grimoux and Adams (1880) synthesized citric acid from
glycerol. Wehmner (1893) observed the occurrence of citric acid as a microbial product
by using Penicillium and Citromyces. It was Millard (1922) who recorded accumulation
of citric acid in culture of Aspergillus niger under condition of nutrition deficiency.
Meanwhile, Currie (1917) reported better yield while using A. niger. In 1923, Pfizer
began operating fermentation based process in USA.

Fermentation of Citric Acid:

 Aspergillus niger has been the choice for the production of this primary metabolite citric
acid for several decades. A large number of other microorganisms (fungi and yeast) such
as Aspergillus davatus, A. wentii, Penicillium luteum, P. citrinum, Mucor pyriforms,
Candida lipolytica, C. oleophila, C. guillermondis, Hensenulaspp. Torulopsis spp.,
Pichiaspp., Debaromyces daussenii etc. have also been used for citric acid production in
industries.
 The advantages of using yeast, rather than A. niger are the possibility of using very high
initial sugar concentration together with a much faster fermentations. This combination
gives a high productivity run to which must be added the reported insensitivity of the
fermentation to variations in the heavy metal content of the crude carbohydrates.
 From 1965 onwards, yeasts are used for citric acid production using carbohydrate and n-
alkanes. In all the processes, a variety of carbohydrates such as beet molasses, cane
molasses, sucrose, commercial glucose, starch hydrolysate etc. used in fermentation
medium.
 The starchy raw material is diluted to obtain 20-25% sugar concentration and mixed with
a nitrogen source (ammonium salts or urea) and other salts. The pH of the medium is kept
around 5 when molasses is used and at pH 3 when sucrose used.
The fermentation is carried out by any of the processes:
 Kofi process or solid state fermentation:
It is a Japanese process in which special strains of Aspergillus niger are used with the
solid substrate such as sweet potato starch.
Liquid surface culture process:
In this case, A. niger floats on the surface of a solution.
Submerged fermentation process:
It is the process in which the fungal mycelium grows throughout a solution in a deep
tank.
(а) Koji process:
 Mold is used in the preparation called Koji to which wheat bran was substituted in the
sweet potato material. The pH of the bran is adjusted between 4 and 5, and additional
moisture is picked up during steaming so as to get the water content of the mash around
70-80%.
 After cooling the bran to 30-60°C, the mass is inoculated with a koji which was made by
a special strain of A. niger which is probably not as possible to the presence of ions of
iron as the culture strains used in other process.
 Since bran contains starch which on saccharification by the amylase enzyme of A. niger
induces citric acid production. The bran after inoculation, is spread in trays to a depth of
3-5 cm and kept for incubation at 25-30°C. After 5-8 days, the koji is harvested and citric
acid is extracted with water.
(b) Liquid surface culture process:
 In this case aluminium or stainless steel shallow pans (5-20 cms deep) or trays are used.
The sterilized medium usually contains molasses and salts. The fermentation is carried
out by blowing the spores of A. niger over the surface of the solution for 5-6 days, after
which dry air is used.
 Spore germination occurs within 24 hours and a white mycelium grows over the surface
of the solution, eight or ten days after inoculation, the initial sugar concentration (20-
25%) reduced to the range of 1-3%.
 The liquid can be drained off and any portion of mycelial mat left becomes submerged
and inactivated. The small quantity of citric acid is produced during the growth phase.
This is called primary metabolite. The mycelium can also be reused.
 During the preparation of fermentable sugar from molasses, sucrose is the main
carbohydrate along with some glucose as well as protein, peptide, amino acids, and
 inorganic ions. This is to be subjected to heat; so it contains saccharic acids and related
compounds in traces.
 The initial sugar concentration is about 20-25%. The removal of metallic ions or
reduction in quantity of undesirable ions in sucrose syrup by adsorption with a
combination of CaCO3, colloidal silica, tricalcium phosphate and starch are other
important steps, The iron is also precipitated by addition of calcium ferrocyanide.
 Initially, the pH remains in the range of 5-6, but on spore germination, pH approaches the
range of 1.5-2 as ammonium ions are removed from the solution. It is important to
mention that at initial pH of 3-5 some oxalic acid is also produced.
 The presence of iron also favours oxalic acid production, and of yellow or yellow green
pigments in the mycelium sometimes secreted into the culture solution and is difficult to
remove during product recovery and purification (Fig. 20.12).

Submerged culture process:

 This process is quite economical. In this case, the organism (Aspergillus japonicus)
which is a black Aspergillus is slowly bubbled in a steam of air through a culture solution
of 15 cm depth. Since the organism shows subsurface growth and produces citric acid in
the culture solution, the yields are inferior in comparison to liquid surface culture
fermentation.
 The earlier workers used shaking culture and extracted Mollard’s phosphate deficiency
concept to submerged fermentation. But they could not realize the role of metallic ions
 which are commonly occurring as impurities in phosphate salts. Aeration is required for
the continuous fermentation.
 The addition of copper ion is must to ensure that the new growth is of the right
biochemical type. The antifoam agents are necessary. Such agents must be free of iron,
cobalt or nickel. Continuous culture techniques are not considered suitable for use in
citric acid product.

Recovery of Citric Acid:

 The culture filtrate used to be hazy due to the presence of residual antifoam agents,
mycelia and oxalate. The Ca(OH2) slurry is added to precipitate calcium citrate. After
filtrations the filterate is transferred and treated with H2SO4 to precipitate Ca as CaSO4.
 This is subjected to the treatment with activated carbon. It is demineralized by successive
passages through ion exchange beds and the purified solution is evaporated in a
circulating granulator or in a circulating crystallizers.
 The crystals are removed by centrifugation. The remaining mother liquor is returned to
the recovery stream. The solvent extraction can also be performed by adding 100 parts
tri-n-butyl phosphate and 5-30 parts n-butyl acetate or methyl isobutyl ketone which are
to be mixed with the filterate. The solvent is then extracted with water at 70-90°C. Citric
acid is further concentrated, decolorized and crystallized (Fig. 20.13).