this is a concise matter & predetermined that reader should have initial knowledge about it.

1. BIOTECHNOLOGY OF CITRIC ACID
PRODUCTION
CONCLUDED BY-
Musharraf Ali
M.Tech(BCE)-IIT(BHU)

2. 1- INTRODUCTION-
• Citric acid is ( 2-
hydroxy-1,2,3
propane tricarboxylic
acid)-
• Molecular weight:
192g.
• It is naturally found in
fruits such as lemon,
orange, pineapple,
plum, and pear

3. • Citric acid was first commercially obtained from lemon juice, crystallized
in 1784 by Carl Wilhelm Scheele, a Swedish chemist.
• In 1893 Wehmer investigated citric acid production from sucrose with
strains of Mucor and Penicillium.
• In 1917 Currie described the first production of citric acid with Aspergillus
niger.
• In 1952, America Miles Company (Miles Laboratories, Inc., Elkhart, IN)
used the submerged fermentation process in a pilot program on a plant
scale to produce citric acid.
• After 1952, many countries used glucose or beet and cane molasses as
substrates to produce citric acid.

4. . Two methods are commonly used for the production
of citric acid by A. niger: surface fermentation and
submerged culture fermentation.
• The current annual world production of citric acid
among 35 countries is ~600,000 tons.
• USA is still a net importer of citric acid, while Western
Europe is a net exporter.
• Citric acid is used in the food and beverage industries
(70%), in pharmaceuticals (12%), and in other
industries and applications (18%).

5. 2- MICROORGANISMS USED FOR THE
PRODUCTION OF CITRIC ACID:-

6. 3- BIOSYNTHESIS OF CITRIC ACID

8. 4- FACTORS AFFECTING CITRIC ACID
PRODUCTION
1- Nutrient-
Aspergillus niger grows well in media containing carbohydrates(sucrose,
glucose, fructose, maltose, mannose, and starch), nitrogen (as ammonium
or nitrate ions), phosphate, low amounts of potassium, magnesium, sulfate,
and trace metals such as iron, manganese, zinc, and copper.
The nitial sugar concentration plays an important role. The highest citric acid
concentrations were observed in cultures grown at high initial sugar
concentrations (15−20% w/v).Further increase of sugar concentration (i.e.,
250 g/L)) resulted in a decrease of acid concentration by 15%. The
decreased concentration of acid encountered with the highest concentration
treatment was probably due to osmotic effects.
In addition to carbohydrates, nitrogen and the phosphate concentrations have a
strong influence on citric acid production. Generally, a nitrogen or phosphate
concentration less than 0.2% (w/v) in the medium appears to be adequate.

9. 2- Inhibitors and Stimulants
The most important stimulants used forimproving citric acid yield
by A. niger are methanol & ethanol.These chemicals have
been found to retard growth, delay sporulation, & increase (by
30–50%) citric acid production. Addition of ethanol resulted in a
twofold increase in CS activity, and a 75% decrease in ACH
activity.
Other stimulants such as fats and oils significantly increased the
production of citric acid.
Addition of some inhibitors such as calcium fluoride, sodium
fluoride, potassium fluoride, hydrogen peroxide,
naphthaquinone, methylene blue, sodium malonate, potassium
ferricyanide, iodoacetate, sodium azide, and sodium arsenate
to the different media increased (by 30–40%) citric acid
concentration.

10. 3- Inoculum:
To prepare inoculum, A. niger is grown in standard media for
molds. It is usually cultivated on potato dextrose agar (PDA)
slants or in petri dishes at 28–30°C for 3–5 days. The spores
obtained are suspended in sterile water containing 0.1% Tween
80.
When mycelia pellets are used, they are grown in submerged
fermentation for 2–3 days in medium that has the same
composition as the production medium. The production
medium is then inoculated at a concentration of 5–10% (v/v).
When spores are used as inoculum, the substrate is inoculated
with 0.5–1.0% (v/v) of the inoculum to give a final concentration
of ~106
spores/mL.
In solid-state fermentation, the medium is inoculated with 5%
(v/w) of the inoculum containing 108
spores/mL to give a
concentration of 0.5*107
spores/g wet substrate.

11. 4 Fermentation Time
The optimum time for the maximum production of citric
acid depends on the strain used, the chemical
composition of the medium, the fermentation system,
and generally, the conditions under which
fermentation takes place.
In the surface culture, fermentation time is usually
completed in 10–20 days, while in the submerged
culture incubation time is much shorter (5–10 days).
In solid-state fermentation the fermentation time
depends strongly on the amount of inoculum used,
the moisture content of the substrate, the initial pH,
the temperature, and the particle size of the medium.

12. 5- Temperature
Asperillus niger and other fungi used in the production of citric
acid from synthetic media or molasses in submerged
fermentation have an optimum temperature between 25 &
30'C.
Also, no significant differences were noted in biomass yield,
specific biomass production rate, and specific sugar uptake
rate among cultures grown at 25 and 30°C. Increasing the
fermentation temperature from 25 to 40°C significantly
affected specific citric acid production rate (0.157 to 0.111 g
citric acid/g biomass dry weight/d) and biomass dry weight
(20.5 to 30g/L).
Perlman and Sih reported that a two stage process, where A.
niger was incubated at 28–30°C for 2–3 days and then for a
week at 20°C, resulted in higher yields than when the
temperature was maintained at 28–30°C.

13. 6- pH
When A. niger is used for the production of citric acid,
the initial pH is dependent on the medium employed.
In synthetic media the initial pH of the medium is
usually adjusted to 2.5–3.5, while in the case of
molasses the initial pH must be neutral or slightly
acidic in order for germination and growth of the
microorganism to occur.
The pH of the medium is adjusted with HCl, H2SO4, or
NaOH. The pH of the substrate decreases during
fermentation (from 7.0 to 2.0) due to the production of
citric acid and other acids generated in the TCA cycle.
When yeasts are used for the production of citric acid,
the pH is often adjusted to 6.0–7.0 by addition of lime,
calcium carbonate, or sodium hydroxide.

14. 7- Aeration and Agitation
Aspergillus niger is an aerobic microorganism and therefore requires oxygen.
Aerating and agitating the fermentation broth normally satisfies the oxygen
demand of a fermentation
process.Agitation is important for adequate mixing, mass transfer, and heat
transfer. It not only assists mass transfer between the different phases
present in the culture, but also
maintains homogeneous chemical and physical conditions in the culture by
continuous mixing.
The concentration of dissolved oxygen increased with the increase of speed of
agitation. In the cultures agitated at 300, 400, 500, and 600 rpm the
concentration of dissolved oxygen from the second to the 12th day
remained at ~22%, 30%, 40%, and 50% of the initial saturation level,
respectively. It fell rapidly during the first 2 days of fermentation after which
it increased more slowly due to the rapid increase of biomass concentration
(6.0–9.0 g/L) observed at the same time.

15. 5- SUBSTRATES USED FOR THE PRODUCTION
OF CITRIC ACID-

17. 6-Simplified Flow chart for citric acid
production

18. 7- RECOVERY OF CITRIC ACID
• The fermentation broth obtained either from surface or submerged
fermentation is filtered to remove mycelia or cells and other suspended
impurities.
• Citric acid is precipitated from the filtrate as calcium citrate by the addition
of lime slurry at 95°C for 1 h or 50°C for 20min.
• The precipitate is washed to remove soluble impurities and treated with
sulfuric acid to precipitate calcium sulfate and regenerate the citric acid.
• The solution is then filtered to remove CaSO4. The liquid is decolorized
with charcoal and ion exchangers.
• The purified solution is concentrated by evaporation and run into low
temperature crystallizers.
• Finally, the crystals are removed by centrifugation. Citric acid is marketed
as an anhydrous crystalline chemical, as a monohydrate or as a crystalline
sodium salt.