Production of Citric acid

Citric acid is formed as an intermediate in the Kreb’s cycle, but it is accumulated in greater
quantities in the fungus Aspergillus niger, may be due to metabolic abnormality. This aspect of
the fungus is being exploited for the fermentative production of citric acid. It is also found as a
natural product in many fruits especially citrus fruits. Prior to the development of fermentation
technology, citric acid was extracted from the juice of these fruits. Mexico contributes 1% of
world production of citric acid.

Citric acid can also be produced from glycero, but it is very expensive. Though Aspergillus
wentii, A. clavatus, Penicillium divaricatum, P. citrinum, P. luteum, Mucor pyriformis,
Citromyces pleffencinus, Candida guilleirmondii, Saccharomyces lipolytica, Trichoderma viride,
Arthrobacter paraffinicius and Corynebacterium spp. can produce citric acid, A. niger is
employed extensively.

Chemical Structure of Citric Acid:

Structurally, citric acid is a hydroxypropane-1, 2, 3 tricarboxylic acid.

Fermentation Process of Citric Acid:

Various fermentation processes used for the manufacture of citric acid.

Aspergillus niger is employed in most of the processes for the following reasons:

1. Can easily be cultivated.

2. Process uniform biochemical properties.
3. Produces only of small amount of oxalic acid under controlled conditions.
4. Yield large amount of citric acid.

Surface culture process employing solid medium and submerged culture process employing
stirred reactor are briefly described here:
1. Surface Culture Process: Though it is an old process, it is still employed. It is a kind of
stationary fermentation process. This process consists of four phases:

(i) Inoculum production,

(ii) Preparation of medium,

(iii) Fermentation process and

(iv) Harvest and recovery.

(i) Inoculum Production: Spore suspension is used as inoculum for the production of citric
acid. Suitable and high yielding strain of A. niger is selected from a stock culture. The stock
culture is inoculated on to the surface of a sporulating medium present in glass bottles. The
bottles are incubated for 10-14 days at 25°C. The composition of trace elements like salts of
manganese, zinc or iron in sporulating medium should be suitably maintained; otherwise they
will affect the yield of citric acid in the actual fermentation. Suspension of spores is obtained by
suspending the grown up spores in a suitable diluents such as water containing a wetting agent,
sodium lauryl sulphate. Besides the total number, the viability of spore crop is critical.

(ii) Preparation of Medium: The medium, used in the production of citric acid, should have
carbohydrate source and inorganic salts. A variety of materials can be used as carbon source.
But, generally sucrose and beet molasses are used as carbon source. Sucrose is the best source of
carbon among different organic substances tested. A medium with less than 15% sucrose is
reported to give high yield of citric acid. Reduced yield of citric acid is observed when a part of
sucrose was substituted by fructose or glucose. Commercially, beet molasses is also extensively
used as carbon source in the production of citric acid employing A.niger. Apart from sugars, beet
molasses also contains excessive amounts of inorganic salts. To eliminate these excessive
inorganic salts, it is treated with ferrocyanide or ferricyanide before it is employed in the medium
preparation. Alternatively, the inorganic salts are removed by passing the beet molasses through
a cation exchange resin. The elements like nitrogen, potassium, phosphorus and magnesium are
also needed in the medium, apart from carbon source. They are added in the form of ammonium
nitrate, potassium dihydrogen phosphate or potassium monohydrogen phosphate and magnesium
sulphate into the medium in minimum quantity as given in table.

The presence of these elements at higher concentration lowers the yield of citric acid and
increases the yield of oxalic acid.
(iii) Fermentation Process: The production medium is placed in shallow pans in such a way
that a thin layer of medium with a depth of 1 to 2.5 cm is formed. The spores of inoculum are
added to the medium to keep them floating on its surface. This is achieved by suitable
modulating devices. Incubation is done in the incubation chambers at 30-40°C. Figure 4.3 shows
the layout of the typical fermentation.

The temperature is kept constant at 30°C during the fermentation. Air current ventilation is also
important for gas exchange because the rate of citric acid production falls if CO in the
atmosphere increases to 10%. Within 24 hrs after inoculation the germinating spore form a thin
layer of mycelium on the surface of the nutrient solution.

As a result of the uptake of ammonium ions, the pH in the culture liquid falls to 1.5 to 2.0. After
30 hrs of fermentation, if the iron concentration is more oxalic acid and yellowish pigment is
formed which in turn hinders the recovery process. The fully developed mycelium floats as thick
convoluted white layer on the liquid medium. The fermentation is stopped after 8-14 days.

The rate of bioconversion of sugar to citric acid depends on the ratio of surface area to the
volume of the medium. There will be higher yield of citric acid if the ratio is lower. In this
shallow pan method, the ratio of surface area to the volume of the medium is lower due to which
large surface area of the mycelial mat is exposed to shallow layer of the medium.

Under these conditions more and more of sugar is converted into citric acid. That is why this
process is considered to be superior to the submerged culture process. Yield per hour from this
process amounts to 1.2 – 1.5 kg citric acid monohydrate per square meter of fermentation
surface.

(iv) Harvest and Recovery: The mycelium is separated from the fermentation broth. Any
intracellular citric acid present in the mycelium is obtained by pressing the mycelium. The
filtered broth is treated with calcium hydroxide. It is filtered and washed. It is then treated with
equal volume of sulphuric acid to liberate citric acid. Calcium sulphate is formed as a precipitate
in this process. The precipitate is separated by filtration. An impure solution of citric acid is
obtained which is decolorized by treating with activated carbon and also demineralized. Finally
pure citric acid crystals are produced by evaporation. It is also recovered alternatively by counter
current extraction method.
2. Submerged Culture Process: In this method A. niger is made to grow uniformly dispersed
throughout the liquid production medium. Fermentation is generally carried out in large
fermenters having 4klt a capacity of thousands of gallons and are provided with mechanical
agitator and sparges. Eighty percent of the world’s supply of citric acid is produced by this
process. Cost of production decreases by 25% by this method. It involves low labour cost, longer
incubation period, more energy consumption and sophisticated techniques.

Three factors are important for production in submerged culture process. They are quality of the
metal used for the construction of fermenter, mycelium structure and oxygen supply.

(i) Inoculum Production: Mycelial mats called pellets are used as inoculum for
fermentation in this process. Suitable and high yielding strains of A. niger are selected
from a stock culture. The spores are induced to germinate in a seed fermenter. A
nutrient solution containing 15% sugar from molasses is used in this seed fermenter.
To induce the formation of mycelial pellets, cyanide ions are added to the medium.

Pellet formation largely depends upon the concentration of cyanide ions in the medium. Lower
yield of citric acid occurs if the cyanide ions are in less concentration. This is because lower
concentration of cyanide ions induces formation of normal mycelium instead of pellets. The
spores germinate at 32°C and form pellets of 0.2 — 0.5 mm diameter within 24 hrs. During this
period the pH falls to 4.3. These pellets are then used as inoculum for production fermenters.

(ii) Preparation of Medium: The medium, employed for surface culture process is also
employed in this process.
(iii) Fermentation Process: Mostly fermenters used for citric acid production are
constructed in the range of 10 — 220 klt. They must be made of stainless steel to
prevent leaching of heavy metals. Normal steel, if it is used in the construction of
fermenters, at low pH level of 1-2 may inhibit the formation of citric acid. Small
fermenters with a capacity upto 1000 lt should have plastic lining even though they
are made of stainless steel because of large surface/volume ratio. However, such
plastic lining is not necessary for large stainless steel fermenters.

The mycelia pellets developed in the seed tank are transferred aseptically to the fermenters and
incubated at a constant temperature, 30°C. The structure of the mycelium that forms in the
fermenter is vital to a successful production process. Little citric acid is produced if the
mycelium is loose and filamentous with limited branches and no chlamydospores.

Optimal citric acid is formed if the mycelium is in the form of pellets. The ratio of iron to copper
in the medium determines the nature of mycelium. In some cases, production fermenters are
inoculated directly with spores. Although A. niger requires relatively little oxygen, it is sensitive
to oxygen deficiency. There must be minimum oxygen concentration of 20 to 25% of the
saturation value throughout the fermentation process. Short interruptions in the oxygen supply
cease the production irreversibly. The aeration rate should be 0.2-1.0 volume per min. during the
acid production phase. Due to low viscosity, stirring is not necessary. Thus, although some plants
use stirred fermenters, airlift reactors can also be used. Foaming is a problem in the submerged
culture process. However, it can be controlled by adding antifoam agents such as lard oil at
frequent intervals.

Uses of Citric Acid in Industries:

Industrially, citric acid is used in the following ways:

1. It is used in the production of carbonated beverages.

2. As a chelating and sequestering agent in the tanning and textile industry.
3. Citrate esters are used as plasticizer.
4. It is abundantly used in food industry as an acidulent in the preparation of food items like
jams, preserved fruits and fruit juices etc.
5. It is used in frozen foods to prevent its change in colour and flavour.
6. Metal painting industry
7. In pharmaceutical industry
8. In the manufacture of astringent, hair rinsers and hair setting fluids.
9. In beverage industry as a preservative to prevent oxidation of alcohol, emulsifier of dairy
products like cheese and ice creams
10. It is used as preservative and to prevent change in colour, flavor and in the oxidation of
alcohol.