Production of Citric Acid

Citric acid is a tricarboxylic acid, which contains three

carboxylic functional groups. It is a primary metabolic
product formed in the tricarboxylic acid (or Krebs) cycle
and is found in small quantities in virtually all plants and
animals, being isolated from lemon juice in 1784.

Figure: Chemical structure of citric acid

Citric acid is widely used in the food industry as an acidulant

and flavoring agent in beverages, confectionery and other
foods, and in leavening systems for baked goods. This
organic acid also has many non-food applications. They
include roles in maintaining metals in solution for
electroplating, as a cleaning and ‘pickling’ agent for metals,
and as a replacement for polyphosphates in the detergent
industry, along with several pharmaceutical uses.
Citric acid has become one of the world’s major
fermentation products, with an annual production of over
550 000 tonnes and a value approaching US$800 million.
The demand for citric acid is still increasing, particularly for
beverage applications.

Citric Acid Producing Microorganisms

Many microorganisms, including filamentous fungi, yeasts

and bacteria, can be used to produce this primary
metabolite. Filamentous fungi such as Aspergillus niger,
Aspergillus flavus, Aspergillus fumaricus, Absidia spp,
Acremonium spp, Mucor piriformis, Penicillium spp,
Trichoderma viride have been found to accumulate citric
acid.

Besides fungi, it is known that several yeasts also produce

citric acid especially species belonging to the genera
Candida, Hansenula, Pichia, Debaromyces, Torula,
Torulopsis, Saccharomyces and Zygosaccharomyces.
Although many microorganisms can be employed to
produce citric acid, A. niger is still the main industrial
producer.

Specific strains have been developed for various types of

fermentation processes, which are capable of generating
high yields of citric acid, often in excess of 70% of the
theoretical yield from the carbon source.

Industrial Production of Citric Acid

Raw Materials

Citric acid is mostly produced from sucrose-based medium

using submerged fermentation. Molasses is preferably used
as the source of sugar for microbial production of citric acid
due to its relatively low cost and high sugar content (40–55
%) in the form of sucrose, glucose and fructose.

Both beet and cane molasses are suitable for citric acid
production, however, beet molasses is preferred due to its
lower content of trace metals, supplying better production
yields than cane molasses. In the case of cane molasses,
generally it contains some metals (iron, calcium,
magnesium, manganese, zinc), which retard citric acid
synthesis and it requires some pretreatment for the
reduction of them.

A variety of agro-industrial residues and by-products such

as cassava bagasse, coffee husk, wheat bran, apple pomace,
pineapple waste, grape pomace, citrus waste etc. has also
been investigated with solid-state fermentation techniques
for their potential to be used as substrates for citric acid
production.

Fermentation Processes Used in Citric Acid Production

Citric acid production by fermentation is the most

economical and widely used way of obtaining this product.
Citric acid production by fermentation can be divided in
three phases, which include: (1) preparation and inoculation
of the raw material (2) fermentation and (3) recovery of
the product. The industrial citric acid fermentation can be
carried in three different ways: (1) surface fermentation (2)
submerged fermentation and (3) solid-state fermentation.

Surface fermentation
In the surface culture technique, sterile nutrient medium
containing sugar is placed into aluminium or stainless steel
trays which are arranged in shelves in sterile fermentation
chambers. The medium is formulated with relatively low
levels of iron, otherwise the citric acid yield is reduced. The
trays are inoculated by spraying with A. niger spores, either
a spore suspension or dry spores. The fungus then develops
on the surface of the medium.

Sterile air is blown over these cultures, which is important

for maintaining aerobic conditions, temperature control
and in lowering the CO2 level. Medium pH gradually falls to
below 2, at which point citric acid production begins. At
30°C, the fermentation takes about 8–12 days to complete.
After the fermentation is finished, the fermented liquor is
drained off and further processed for recovery of citric acid.
In some cases, the preformed mycelium is reused for one or
two rounds of fermentation.

Submerged fermentation
In this process, the nutrient media after inoculation are
subjected to vigorous, controlled aeration and agitation in
large fermenters. Unlike surface methods, vegetative
inoculum rather than spores are normally used. A 2-stage
submerged fermentation process involving a “growth stage”
and a “production stage” has also been developed. In this,
the growth medium is first inoculated with the spores and
after 3-4 days of growth, the mycelium is separated from
the solution and added to the fermentation medium. The
fermentation is the carried out for 3-4 days at 25-30 C. The
mother liquor after fermentation is drained off and citric
acid is extracted. More than 80% of the worldwide supply of
citric acid is produced using submerged batch fermentation.

Solid-state fermentation

Solid-state fermentation, also known by Koji fermentation,

is the simplest method for citric acid production and it has
been an alternative method for using agro-industrial
residues. The great advantage of solid-state fermentation
processes is the extremely cheap raw material used as main
substrate. The process uses a solid medium of steam-
sterilized wheat bran or sweet potato waste that has 70–
80% moisture content. This mash is inoculated with spores
of A. niger and then incubated in trays at 25 - 30 C for 6-7
days. After fermentation, the mash is extracted with water,
concentrated and then processed for citric acid
precipitation.

Citric Acid Recovery

Recovery of citric acid involves removal of fungal mycelium

from the culture medium via filtration. The resulting
clarified solution is heated and lime (CaO) is added to form
a precipitate of calcium citrate. This is separated by filtration
and treated with sulfuric acid to generate citric acid and a
precipitate of calcium sulfate. Following filtration, the
dilute citric acid solution is decolorized with activated
carbon and evaporated to produce crystals of citric acid.
These crystals are recovered by centrifugation, then dried
and packaged.

Biochemistry of Citric Acid Production

Several unusual nutrient conditions are required in
combination for overproduction of citric acid e.g., excess of
carbon source, hydrogen ions and dissolved oxygen and
suboptimal concentrations of certain trace metals and
phosphate), which synergistically influence the yield of
citric acid.

Glycolysis pathway is inhibited by accumulation of citric

acid but in case of A. niger, citric acid overproduction
occurs by an active glycolytic pathway. The protein
breakdown under manganese deficiency results in a high
intracellular NH4+ concentration. This increase is able to
counterbalance the inhibition exerted by citric acid on
phosphofructokinase. High concentrations of NH4+ and
glucose also repress the synthesis of α-ketoglutarate
dehydrogenase, inhibiting the citric acid catabolism via the
Krebs cycle, leading to its accumulation.
An important aspect concerns the need that the Krebs cycle
can be completed to support the continuous production of
citric acid. To address the lack of cycle intermediates
consequent to the metabolic dysfunction responsible for
the accumulation of citric acid, pyruvic acid produced from
glucose is not only decarboxylated to acetyl-CoA by the
pyruvate dehydrogenase complex but it is also partially
carboxylated to oxaloacetic acid by the action of pyruvate
carboxylase.
Pyruvate + CO2 + H2O + ATP → Oxaloacetate + ADP + Pi

This reaction is not the only anaplerotic reaction used to

replenish the Krebs cycle. Depending on the organism,
more oxaloacetic acid can be produced from
phosphoenolpyruvate and CO2 by phosphoenolpyruvate
carboxykinase.

Phosphoenol pyruvate + ADP +CO2 → Oxaloacetate + ATP

The glyoxylate cycle acts as another source of oxaloacetate

for citrate synthesis. Acetyl CoA condenses with glyoxylate
and the reaction is catalyzed by malate synthetase.

Glyoxylate + Acetyl CoA → Malate + Coenzyme A

The glyoxylate required for the synthetase reaction is

supplied by the isocitritase reaction as shown

Isocitrate → Succinate + Glyoxylate

Accumulation of Citric Acid

It has been proposed that the accumulation of citric acid

requires deactivation of the Krebs cycle enzymes responsible
for its degradation, aconitase and/or isocitrate
dehydrogenase. But there are evidences that during the
production of citric acid, the Krebs cycle is active in the
production of intermediates required for biomass
formation. Therefore citric acid accumulation may more
likely be the result of enhanced (deregulated) biosynthesis
rather than inhibited degradation.

An alternative hypothesis to explain the accumulation of

citric acid is associated to tricarboxylate transporter activity,
which competes with aconitase for citric acid. Under
conditions in which its affinity for citric acid is greater than
that of aconitase, this enzyme ejects citric acid out of the
mitochondria without inhibition of enzymes of the cycle.