This document discusses citric acid production. It is mainly produced through submerged fermentation using Aspergillus niger. Key factors that affect production include the carbon source, nitrogen source, pH, aeration, and trace elements. Citric acid has various applications in food, pharmaceuticals, chemicals, and other industries due to its pleasant taste and ability to complex with metals.

1. CITRIC ACID PRODUCTION
Dr. Esther Shoba R
Assistant Professor
Kristu Jayanti College
Bangalore

2. INTRODUCTION
• Citric acid is the most important organic acid produced in tonnage and
is extensively used in food and pharmaceutical industries.
• It is produced mainly by submerged fermentation using Aspergillus
niger or Candida sp. from different sources of carbohydrates, such as
molasses and starch based media.
• However, other fermentation techniques, e.g. solid state fermentation
and surface fermentation, and alternative sources of carbon such as
agro-industrial residues have been intensively studied showing great
perspective to its production.

3. • Citric acid (C6H8O7, 2 - hydroxy - 1,2,3 - propane tricarboxylic acid), a
natural constituent and common metabolite of plants and animals, is
the most versatile and widely used organic acid in the field of food
(60%) and pharmaceuticals (10%).
• It has got several other applications in various other fields.
• There is constant increase (3.5-4%) each year in its consumption,
showing the need of finding new alternatives for its manufacture.

4. HISTORICAL DEVELOPMENTS
• Citric acid was first isolated by Karls Scheels in 1874, in England, from
the lemon juice imported from Italy.
• In 1923, Wehmer observed the presence of citric acid as a by-product of
calcium oxalate produced by a culture of Penicillium glaucum.
• Other investigations showed the isolation of two varieties of fungi
belonging to genus Citromyces (namely Penicillium). However, industrial
trials did not succeed due to contamination problems and long duration
of fermentation (Rohr et al., 1983).
• The industrial process was first open by Currie, in 1917, who found that
Aspergillus niger had the capacity to accumulate significant amounts of
citric acid in sugar based medium.

5. MICROORGANISMS USED
• A large number of micro-organisms including bacteria, fungi and yeasts
have been employed to produce citric acid.
• Most of them, however, are not able to produce commercially acceptable
yields.
• This fact could be explained by the fact that citric acid is a metabolite of
energy metabolism and its accumulation rises in appreciable amounts only
under conditions of drastic imbalances
• Among these, only A. niger and certain yeasts such
as Saccharomycopsis sp. are employed for commercial production.
• However, the fungus A. niger has remained the organism of choice for
commercial production. The main advantages of using this micro-organism
are: (a) its ease of handling, (b) its ability to ferment a variety of cheap raw
materials, and (c) high yields.

7. STRAIN IMPROVEMENT
• The most employed technique to improve citric acid producing strains
has been by inducing mutations in parental strains using mutagens.
• Among physical mutagens, g-radiation have often used.
• To obtain hyper-producer strains, frequently UV treatment could be
combined with some chemical mutagens, e.g. aziridine, N-nitroso-N-
methylurea or ethyl methane-sulfonate (Musilkova et al., 1983).
• By using a suitable selection technique on model medium with non-
specific carbon sources, a strain yielding high amounts of citric acid
from unusual substrates can be obtained from the mutants produced.

8. • Diploids displayed higher citric acid yields compared to their parent
haploids, but tended to be less stable (Bonatelli and Azevedo, 1983).
• Protoplast fusion appeared to be a promising tool to extend the range
of genetic manipulation of A. niger with respect to citric acid
production.
• Kirimura et al. (1988a) studied protoplast fusion of production strains.
They were able to obtain fusants with acid production capacities
exceeding those of the parent strains in solid state fermentation, but
not in submerged fermentation.
• However, there is no single effective technique to achieve hyper-
producing mutants and much remains to be done in this area.

9. Raw materials
• Although citric acid is mostly produced from starch or sucrose based
media using liquid fermentation, a variety of raw materials such as
molasses, several starchy materials and hydrocarbons have also been
employed.
• Rohr et al. (1983) classified raw materials used for citric acid production
in to two groups: (i) with a low ash content from which the cations
could be removed by standard procedures (e.g. cane or beet sugar,
dextrose syrups and crystallized dextrose);
• (ii) raw materials with a high ash content and high amounts of other
non sugar substances (e.g. cane and beet molasses, crude unfiltered
starch hydro-lysates).

10. Factors affecting production
• Carbon source: Citric acid accumulation is strongly affected by the
nature of the carbon source.
• The presence of easily metabolized carbohydrates has been found
essential for good production of citric acid.
• Hossain et al. (1984) showed that sucrose was the most favourable
carbon source followed by glucose, fructose and galactose.
• initial sugar concentration was critical for citric acid production and
other organic acids produced by A. niger. Xu et al. (1989) reported
that A.niger strains needed an initial sugar concentration of 10-14%
as optimal; no citric acid was produced at sugar concentration of less
than 2.5%.

11. Nitrogen Source
• Citric acid production is directly influenced by the nitrogen source.
Physiologically, ammonium salts are preferred, e.g. urea, ammonium
sulfate, ammonium chlorure, peptone, malt extract, etc.
• Nitrogen consumption leads to pH decrease, which is very important
point in citric acid fermentation (Rohr et al., 1983, Kubicek and Rohr,
1986).
• However, it is necessary to maintain pH values in the first day of
fermentation prior to a certain quantity biomass production.
• Urea has a tampon effect, which assures pH control (Raimbault, 1980).
The concentration of nitrogen source required for citric acid
fermentation is 0.1 to 0.4 N /liter. A high nitrogen concentration
increases fungal growth and the consumption of sugars, but decreases
the amount of citric acid produced

12. Phosphorus source
• Presence of phosphate in the medium has a great effect on the yield of
citric acid.
• Potassium dihydrogen phosphate has been reported to be the most
suitable phosphorous source.
• Shu and Johnson (1948) reported that phosphorous at concentration of
0.5 to 5.0 g/L was required by the fungus in a chemically defined
medium for maximum production of citric acid.
• Phosphate is known to be essential for the growth and metabolism of
A. niger (Shankaranand and Lonsane, 1994).

13. Trace Elements
• Trace element nutrition is probably the main factor influencing the
yield of citric acid.
• A number of divalent metals such as zinc, manganese, iron, copper
and magnesium have been found to affect citric acid production by A.
niger.

14. pH
• The pH of a culture may change in response to microbial metabolic
activities.
• The most obvious reason is the secretion of organic acids such as citric,
acetic or lactic acids, which will cause the pH to decrease.
• Changes in pH kinetics depend highly also on the micro-organism.
• With Aspergillus sp., Penicillium sp. and Rhizopus sp., pH can drop very
quickly until less than 3.0.
• Generally, a pH below 2.0 is required for optimum production of citric
acid. A low initial pH has the advantage of checking contamination and
inhibiting oxalic acid formation. A pH of 2.2 was reported to be
optimum for the growth of the mould as well as for the production of
citric acid

15. Aeration
• The yield of citric acid production substantially increases when the
dissolved O2 tension is higher.
• This can be achieved by strong aeration or by sparging with pure O2.
• It has been observed that sudden interruptions in O2 supply (as
occurs during power breakdowns) cause drastic reduction in citric
acid production without harming the growth of the organism.

17. Production techniques
• The industrial citric acid production can be carried in three different
ways:
• Surface fermentation
• Submerged fermentation
• Solid-state fermentation

18. Surface Fermentation
• liquid as nutrient medium is the oldest method for citric acid production.
• It is still in use due to a simple technology, low energy costs and higher
reproducibility.
• Further, the interference of trace metals and dissolved O2 tension are
minimal.
• The labour costs are however, higher since the manpower requirements are
more for cleaning the systems.
• About 20% of the citric acid in the world is produced by surface processes.
• The nutrient supply for surface fermentation normally comes from beet
molasses.
• The fermentation is usually carried out in aluminium trays filled with sterile
nutrient medium. The inoculum in the form of spores is sprayed over the
medium. A sterile air is passed for supplying O2 as well as cooling. The
temperature is maintained around 30°C during fermentation.

19. • As the spores germinate (that occurs within 24 hours of inoculation), a
layer of mycelium is formed over the medium.
• The pH of the nutrient medium falls to less than 2, as the mycelium
grows in size and forms a thick layer on the surface of the nutrient
solution.
• The fermentation is stopped after 7-15 days.
• The mycelium and nutrient solution are separated. The mycelium is
mechanically pressed and thoroughly washed to obtain maximum
amount of citric acid.
• The nutrient solution is subjected to processing for the recovery of citric
acid. The final yield of citric acid is in the range of 0.7-0.9 of per gram of
sugar

20. Submerged process
• Around 80% of the world’s supply of citric acid is produced by
submerged processes. This is the most preferred method due to its
high efficiency and easy automation. The disadvantages of submerged
fermentation are — adverse influence of trace metals and other
impurities, variations in O2 tension, and advanced control technology
that requires highly trained personnel.
• Two types of bioreactors are in use— stirred tanks and aerated towers.
The vessels of the bioreactors are made up of high-quality stainless
steel. The sparging of air occurs from the base of the fermenter.

21. • The success and yield of citric acid production mainly depend on the
structure of mycelium.
• The mycelium with forked and bulbous hyphae and branches which
aggregate into pellets is ideal for citric acid formation.
• On the other hand, no citric acid production occurs if the mycelium is
loose and filamentous with limited branches.
• An adequate supply of O2 (20-25% of saturation value) is required for
good production of citric acid.
• The ideal aeration rate is in the range of 0.2-1 vvm (volume/ volume/
minute).

22. • The submerged fermenters have the problem of foam formation
which may occupy about 1 /3rd of the bioreactor.
• Antifoam agents (e.g. lard oil) and mechanical antifoam devices are
used to prevent foaming.
• Nutrient concentration is very important in the industrial production
of citric acid.

23. Solid State fermentation
• 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%.

25. Recovery
• 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.

26. • 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

28. APPLICATIONS
• 1. Citric acid, due to its pleasant taste and palatability, is used as a flavoring
agent in foods and beverages e.g., jams, jellies, candies, desserts, frozen
fruits, soft drinks, wine. Besides brightening the colour, citric acid acts as an
antioxidant and preserves the flavors of foods.
• 2. It is used in the chemical industry as an antifoam agent, and for the
treatment of textiles. In metal industry, pure metals are complexed with
citrate and produced as metal citrates.
• 3. In pharmaceutical industry, as trisodium citrate, it is used as a blood
preservative. Citric acid is also used for preservation of ointments and
cosmetic preparations. As iron citrate, it serve as a good source of iron.

29. • 4. Citric acid can be utilized as an agent for stabilization of fats, oils or
ascorbic acid. It forms a complex with metal ions (iron, copper) and
prevents metal catalysed reactions. Citric acid is also used as a
stabilizer of emulsions in the preparation of cheese.
• 5. In detergent/cleaning industry, citric acid has slowly replaced
polyphosphates.