introduction: Citric acid (C6H8O7) is a weak organic tricarboxylic acid found in citrus fruits (lemons, oranges, grapes, tomatoes, beets etc.) Citric acid known as an intermediate of kreb's cycle, and hence present in all living organisms. Citric acid is produced by three method fermentation, chemical synthesis and extraction from citrus fruits. In 1782, Carl Wilhelm Scheele first obtained citric acid from lemon juice, but in 1923 Pfizer began operating a fermentation based process in the USA. Uses: Used as flavoring agent in food industries. Used in chemical industries (as an antifoam agent). In pharmaceuticals industries (as Tri-sodium citrate as blood preservative). In detergent industries (as strong cleaning agent). As chelating and sequestering agent. In production of carbonated beverages. Used as antioxidant in frozen fruits and vegetables. Various cosmetic product like lotion, shampoos, creams, and toothpaste. Biosynthesis: The metabolic pathway involved in citric acid biosynthesis the TCA cycle or the Krebs cycle. In TCA cycle critic acid is a intermediate product, glucose is predominant source of carbon for acid production. In glycolysis glucose is converted in 2 molecules of pyruvate. Pyruvate form Acetyl CoA and Oxaloacetate which finally convert in citrate. Citrate synthase is a regulatory enzyme for production of citric acid because the activity of this enzyme increases at the time of acid production, while activity of other enzymes that degrade the citrate are reduced. Pyruvate dehydrogenase is also a key enzyme that converts pyruvate to oxaloacetate in citrate production. type of fermentation: There are two types of fermentation: Surface fermentation - Characterized by growing microorganisms as a layer or film on a surface in contact nutrient medium, which may be solid or liquid. Submerged fermentation – In this process microorganisms are throughout the nutrient medium.

1. PRESENTED ON -
PRODUCTION OF CITRIC ACID

2. Introduction
 Citric acid (C6H8O7) is a weak organic tricarboxylic acid found in citrus fruits
(lemons, oranges, grapes, tomatoes, beets etc.)
 Citric acid known as an intermediate of kreb's cycle, and hence present in all
living organisms.
 Citric acid is produced by three method fermentation, chemical synthesis and
extraction from citrus fruits.
 In 1782, Carl Wilhelm Scheele first obtained citric acid from lemon juice, but
in 1923 Pfizer began operating a fermentation based process in the USA.

3. Citric acid
Chemical Formula-
 Chemical formula : C6H807
• IUPAC name : 2-Hydroxypropane-1,2,3-tricarboxylic acid

4. Uses of citric acid
 Used as flavoring agent in food industries.
 Used in chemical industries (as an antifoam agent).
 In pharmaceuticals industries (as Tri-sodium citrate as blood
preservative).
 In detergent industries (as strong cleaning agent).
 As chelating and sequestering agent.
 In production of carbonated beverages.
 Used as antioxidant in frozen fruits and vegetables.
 Various cosmetic product like lotion, shampoos, creams, and
toothpaste.

5. Biosynthesis
• The metabolic pathway involved in citric
acid biosynthesis the TCA cycle or the Krebs
cycle.
• In TCA cycle critic acid is a intermediate
product, glucose is predominant source of
carbon for acid production.
• In glycolysis glucose is converted in 2
molecules of pyruvate. Pyruvate form Acetyl
CoA and Oxaloacetate which finally convert
in citrate.

6.  Citrate synthase is a regulatory enzyme for production of citric acid because the activity of this
enzyme increases at the time of acid production, while activity of other enzymes that degrade
the citrate are reduced.
 Pyruvate dehydrogenase is also a key enzyme that converts pyruvate to oxaloacetate in citrate
production.

7. Microorganisms
 99% of world’s Citric acid is produced by microbial fermentation.
 Large number of micro-organisms including bacteria, fungi and yeasts have been
employed to produce citric acid.
 The main advantages of using these micro-organisms are:
a) Its easy of handling,
b) Its ability to ferment a variety of cheap raw materials,
c) High yields.
Fungi
Aspergillus niger
A. aculeatus
A. awamori
A. carbonorius
A wentii
A foetidus
Penicillium
janthinelum
Bacteria
Bacillus licheniformis
Arthrobacter paraffinens
Corynebacterium species
Yeast
Saccharomycopsis
lipotytica
Candida tropicalis
C. oleophila
C. guilliermondi
C. parapsilosis
C citroformans

8. Aspergillus niger
 Many fungi have been reported to possess the ability to produce citric acid.
 But only A. niger strains among these fungal strains are used for the following
reasons:
1. They are efficient (i.e. high-yielding) strains.
2. They possess fairly uniform biochemical properties.
3. They produce a negligible or small amount of oxalic acid, provided
fermentation conditions (e.g. pH and salts) have been adjusted to
favour the formation of citric acid.
4. They can easily be cultivated.

9. Type of production
There are two types of fermentation:
1. Surface fermentation - Characterized by
growing microorganisms as a layer or film on a
surface in contact nutrient medium, which may
be solid or liquid.
2. Submerged fermentation – In this process
microorganisms are throughout the nutrient
medium.

10. Liquid surface fermentation
 A. Niger floats on the surface of liquid medium.
 In aluminium tray sterile medium is filled.
 Mycelium develops on the surface and spores germinate.
 pH less than 2.
 The undesirable ions in sucrose syrup can be removed by addition of CaCO3 or calcium
phosphate etc.
 Duration 7-15 days at 26-28°C.
 Citric acid removed from the fluid by precipitation.

11. Solid surface fermentation
 The solid substrate such as wheat bran or pulp of potato used as culture
media.
 Spores of A. Niger is spread as a layer.
 pH 4-5.
 Incubate at 26-28°C and period 5-6 days.
 The growth of organism is accelerated by adding α – amylase enzyme,
although the fungus can hydrolyse starch with its own α – amylase.
 Hot water extraction and isolation of citric acid.

12. Submerged fermentation
 80% citric acid produced using submerged batch fed
(20% by surface).
 It is the process in which the fungal mycelium grows as
fungal pellets throughout the liquid medium in
fermenter.
 The process is done using either stirred bioreactor or
airlift bioreactor.
- Stirred tanks 40-200 meter cube
- Airlift fermenter 200-900 meter cube

13. Bioparameters of citrate production
(1). Preparation of inoculum –
 A. Niger are inoculated in media.
 At 25°C for 4-14 days – spores germinate.
(2). Production media –
Carbon source
 Sucrose and beet molasses are used as carbon source.
 Sucrose used for producing high yields of citric acid.
(sucrose concentration exceeding 15% should not be used, since the
excess amount of sugar less than 3% remained unconverted to citric
acid)
 Beet molasses required pretreatment, since it contains excessive
amount of trace metals (ferrocyanide or ferricyanide may be added to

14. Inorganic salts
 Trace metals nitrogen, potassium, phosphorus, sulphur and magnesium are needed in the
fermentation media.
 According to Currie (1917), the fermentation medium with the following chemical composition:
Components Gms./litre
Sucrose 125-150
NH4NO3 2.0-2.5
KH2PO4 0.75-1.0
MgSO4.7H2O 0.20-0.25
pH 3.4-3.5

15. pH
 According to Currie, the pH of the medium should be adjusted to 3.4 to 3.5 with HCl.
 Also, low pH value 1.6 – 2.20 have been found to most favorable by Doelger and Prescott.
 Low pH value in A. Niger for citric acid production are desirable for the following reasons:
(i). Sterilization of medium is more readily effected.
(ii). Formation of citric acid is favoured.
(iii). Formation of oxalic acid is suppressed.
(iv). The danger of contamination is minimized.
Temperature
 Temp. of incubation depends in fungal strain used and fermentation conditions.
 According to Doelger and Prescott, a temperature range considered is
26 to 28°C.

16. Αeration
 Rate of aeration either higher or lower, gives lower yields of citric acid.
 It is essential to determine the rate of air supply required for each new
apparatus installed.
Time
 Shallow pan method (surface culture process) the period required for in
the range of 7 to 10 days.
Ratio of surface area to volume
 The rate of bioconversion of sugar to citric acid depends on the ratio of
the surface area to the volume of the medium.
Surface Area ∝
1
𝑦𝑖𝑒𝑙𝑑 𝑜𝑓 𝑐𝑖𝑡𝑟𝑖𝑐 acid
Yield
• 60-80 gm Citric acid per 100gm incorporated sugar.

17. Purification and recovery
 The recovery operation for citric acid from the harvested fermentation broth is difficult
for the following important reasons:
(i) the presence of unconverted sugars,
(ii) the presence of other acid fermentation products (e.g. oxalic acid),
(iii) The presence of trace salts as impurities.
 Usually, recovery of citric acid is practiced as:
1. Recovery starts with filtration of culture broth and washing of mycelium.
2. Oxalic acid is unwanted bioproduct, it can be removed by precipitation by the addition
of milk of lime, forming the precipitation of calcium citrate.
3. The culture broth is then adjusted to pH 7.2 and temperature 70-
90◦
C for further purification.
4. Formation of the precipitate of calcium citrate.
5. Calcium citrate is dissolved in sulphuric acid, precipitate of calcium sulphate formed.
6. Further recovery is done by activated charcoal, cation-anion exchanger and crystallization.

18. Filtration(fermentation)
Treated with Ca(OH)2 (milk of lime)
precipitate of Ca citrate
Filter precipitate & wash
Precipitate + sulphuric acid
Supernatant of citrate
filter
Treat with activated charcoal
Evaporation & Crystallization
Recovery:-

19. Drying and packaging
• After the crystals are obtained, citric acid is dried in packaged.

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