microbial production of citric acid, koji process, surface and submerged fermentation, recovery , purification and applications of citric acid

1. CITRIC ACID
PRODUCTION

2. INTRODUCTION
• Citric acid (2-hydroxy-1,2,3- propane tri carboxylic acid) is the most
important commercial product, which is found in almost all plant &
animal tissues.
• Citric acid is the most important organic acid produced in tonnage and is
extensively used in food and pharmaceutical industries.
• Citric acid is a weak organic acid found in citrus fruits(lemon). It is good
,natural preservative and is also used to add an acidic taste to food and
soft drinks. More than million tonnes are produced every year by
fermentation.

3. HISTORY
• Citric acid was first isolated in 1784 by the Swedish chemist carl
Wilhelm Scheele, who crystallize it from lemon juice.
• Industrial scale citric acid production began in 1890 based on the
Italian citrus fruit industry.
• In 1893, C. Wehmer discovered penicillin mold could produce citric
acid from sugar. However, microbial production of citric acid did not
become industrially important until world war I disrupted Italian
citrus exports.

4. APPLICATIONS OF CITRIC ACID:
• 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.

5. APPLICATIONS OF CITRIC ACID:
• 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.

6. MICROBIAL STRAINS FOR CITRIC
ACID PRODUCTION:
Many microorganisms can produce citric acid. The fungus
aspergillus niger is most commonly used for industrial production
of citric acid. The other organisms (although less important)
include A. Clavatus, A. Wentii, penicillium luteum, Candida
catenula, C. Guilliermondii and corynebacterium sp.
Only mutants of A. Niger and yeast genus Candida have almost
exclusively been utilized.

7. ADVANTAGES OF USING THIS MICRO-
ORGANISM ARE:
• Its ease of handling.
• Its ability to ferment a variety of cheap raw materials.
• It provides high yield

8. BIOSYNTHESIS OF CITRIC ACID:
• Citric acid is a primary metabolic product (of primary
metabolism) formed in the tricarboxylic acid (Krebs) cycle.
Glucose is the predominant carbon source for citric acid
production. The biosynthetic pathway for citric acid
production involves glycolysis wherein glucose is converted
to two molecules of pyruvate. Pyruvate in turn forms acetyl
CoA and oxaloacetate which condense to finally give citrate.

9. CARBOHYDRATE SOURCE
• A wide range of raw materials can be used for the supply of
carbohydrates. These include molasses (sugar cane or sugar
beet), starch (from potatoes), date syrup, cotton wastes, banana
extract, sweet potato pulp, and brewery waste and pineapple
waste water.
• A high yield of citric acid production occurs if the sugars that
are rapidly metabolised are used e.g. sucrose, glucose, maltose.
At present, cane molasses and beet molasses are commonly
used.

10. • The concentration of carbohydrate significantly influences
citric acid production. Ideally, the sugar concentration
should be 12-25%. At a concentration less than 5% sucrose,
citric acid formation is negligible.

11. • Trace metals:
• Certain trace elements (Fe, Cu, Zn, Mn, Mg, Co) are essential for the
growth of A. Niger. Some of the trace metals particularly Mn2+, Fe3+ and
Zn2+ increase the yield of citric acid. These ions promote glycolysis and
reduce respiration; both these processes promote citric acid production.
• pH:
• pH of the medium is kept around 5 when molasses is used and at pH 3
when sucrose used. Advantage with low pH is that the risk of
contamination is very minimal, since many organisms cannot grow at this
pH.

12. • Nitrogen source:
• Ammonium salts, nitrates and urea are the nitrogen
sources used in the media for citric acid production. All the
three compounds are equally good sources, as long as they
do not adversely affect the pH of the medium.

13. Fermentation
process
1 koji or solid state fermentation
process
2 Liquid surface culture process
3 Submerged culture process

14. KOJI PROCESS
• It also refers to as “Solid-state fermentation”. The koji process was first introduced
in Japan. It is related to the use of agro-industrial residues for citric acid
production. In Koji process, raw materials like apple pomace, sugar cane, beet
molasses etc. can be used. These raw materials are utilized by the Aspergillus
niger.
• The pH and moisture content of the raw material is adjusted to 4-5 and 70%
respectively. Then the raw material is cooled at 30-60 degrees Celsius and after
that, inoculate A. niger. After inoculation, the medium is transferred into large
trays of 3-5cm depth and incubated at 25-30 degrees Celsius for 3-7 days.
• At last, the citric acid is extracted from the fermentation tank. The starch content
of the raw material is degraded into citric acid by the amylase enzyme of the
Aspergillus niger. The koji process does not require the substrate pre-treatment
because the trace elements do not affect the production of citric acid.

15. SURFACE CULTURE PROCESS
• It also refers to as “liquid surface fermentation”. Surface culture fermentation was the first
method introduced for citric acid production in 1919. In liquid surface fermentation, the
culture medium (molasses and salts) (5-6 pH) is added to the aluminium shallow trays up to
5-20 cm deep. The process is carried out in the fermentation chamber which provides
uniform air circulation, maintains relative temperature and humidity.
• First, the spores of A.niger is blown onto the surface of the culture medium for about 5-6
days and then dry air is passed. Now, the pH of the culture medium is adjusted between 1.5-
2 pH. After 24 hours, the spores start to germinate and the growth of white mycelium is
observed on the surface of the culture medium.
• After the utilization of sugar content by the mould, the remaining liquid is separated from
the mycelial mat. In the surface culture process, a small amount of citric acid is produced as
the primary metabolites by the A. niger.

16. SUBMERGED CULTURE PROCESS
• It also refers as “Submerged culture fermentation”. About 80% of the citric acid
production is carried out through submerged fermentation method. Submerged
fermentation makes the use of black Aspergillus i.e. A. japonicus. It is performed
in a bioreactor made of stainless steel compiled with proper aeration, cooling
system, impellers etc.
• For carbon source, substrate like beet molasses, corn starch etc. are used as a
substrate. For the nitrogen source, ammonia is used. The substrate used in this
method requires pre-treatment like the addition of nutrients, sterilization etc.
• The A. japonicus is inoculated into the culture medium and maintained at
30degrees Celsius. Submerged fermentation is mostly carried out in a batch
bioreactor in which 1500kg of citric acid and 500kg of biomass can be produced
from the 2500kg glucose and 860kg of oxygen.

18. RECOVERY OF CITRIC ACID
• The product formed after fermentation is the fermented liquor looks hazy due to the
presence of antifoaming agents, mycelia etc. Therefore to separate these things, a slurry of
calcium hydroxide i.e. Ca (OH)2 to form a precipitate of calcium citrate.
• The precipitate of calcium citrate is filtered and washed. After filtration, a filtrate forms
which is treated with the sulphuric acid for the precipitation of calcium as “Calcium
sulphate” (CaSO4). Calcium sulphate is then treated with the activated carbon by which it
gets demineralized after passing it consecutively from the ion exchange bed.
• The solution obtained from this is subjected to the circulating crystallizers. The crystals
formed as a result of crystallization are then removed by centrifugation. After the
completion of these steps, the remaining solvent is dried, sieved and then packed. The
remaining mother liquor is again recovered by the same process.

20. USES
• The production of citric acid is necessary for its versatile uses in the different
fields that are given below:
• Food industries: Citric acid is used for the production of jams, jellies, candies,
frozen fruits etc. In certain foods, citric acid is used as an “Artificial flavouring
agent”.
• Beverage industries: Citric acid is used for the production of soft-drinks and
distilled beverages like wine.
• Hospitals: Citric acid is used as “Effervescent agent” at the time of blood
transfusion.
• Cosmetic industries: Citric acid is used in cosmetic products like astringent
lotions, hair gels etc.

21. FACTORS AFFECTING THE
PRODUCTION
• There are some factors which can directly or indirectly affect the
production process or the fermentation of citric acid.

22. CARBON SOURCE CONCENTRATION:
• For most of the industrial production, carbon sources like glucose and sucrose
are recommended as they provide a good source of carbon for the growth of the
biomass. Galactose acts as an alternative source of glucose and sucrose, but it
allows low microbial growth and thus not favours the cumulation of citric acid.
• Other carbon sources like cellulose, starch etc. allows limited growth and may
slow down the growth rate of the microorganisms, which will lead to minimal
production. The optimal sugar concentration ranges between 10-14% and the
sugar concentration below 2.5% will not produce citric acid.

23. NITROGEN SOURCE CONCENTRATION:
• Ammonium salts like urea, ammonium sulphate etc. results in a
decrease in pH, which is crucial for citric acid production. The
nitrogen concentration must be in between 0.1-0.4 N/L. High nitrogen
source will increase the microbial growth which will consume more
sugar and in turn decrease the yield of citric acid.

24. PHOSPHOROUS SOURCE CONCENTRATION:
• For the best production and growth of fungi, potassium dihydrogen
phosphate is considered best for the phosphorous source. The
concentration of phosphorous must be in between 0.5-5.0 g/l for the
maximum yield. If phosphorous present in excess, it leads to the
production of sugar acids which will decrease the carbon dioxide
fixation and thus, stimulate the fungal growth.

25. PRESENCE OF TRACE ELEMENTS:
• Divalent metals such as iron, zinc, manganese, copper etc. produce at
the time of fermentation. If KH2PO4 is added to the zinc, it favours
the production but the trace elements like manganese, iron, and high
concentration of zinc leads to a reduction in yield.

26. LOWER ALCOHOLS CONCENTRATION:
• Lower alcohols like ethanol, methanol etc. enhance the citric acid
fermentation. The concentration of lower alcohol must be in between
1-3%. Lower alcohols not only stimulate the citric acid production
but also promotes the sporulation of microorganisms.

27. MISCELLANEOUS COMPOUNDS:
• Calcium fluoride, sodium fluoride etc. are the miscellaneous
compounds which accelerate the citric acid production whereas
potassium ferrocyanide decreases the yield.

28. PH CONCENTRATION:
• The pH of the culture medium is directly related to the growth of
microorganisms and their metabolic activities. A pH changes to acidic
by the microbial activity, like for Aspergillus, the pH drops to 3.0. The
pH below 2.0 is optimal for the production of citric acid and a pH of 2.2
is best for the growth of mould.

29. AERATION SYSTEM:
• Aeration increases the dissolved oxygen concentration in the culture
medium and thus increases the yield of citric acid. The aeration
system also reduces the fermentation time. The concentration of
oxygen must be above 25% saturation.