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.