J ournal of Pharmacy Research Vol.3.I ssue 6.

J une 2010
Madhusudan chaturvedi et al. / J ournal of Pharmacy Research 2010, 3(6),1215-1222
1215-1222
Research Article
ISSN: 0974-6943 Available online through
www.jpronline.info
*Corresponding author.
Madhusudan chaturvedi
E-mail:madhumanojiopmku@gmail.com
INTRODUCTION
Citric acid production from cane molasses using submerged fermentation by Aspergillus
niger ATCC9142
Chaturvedi Madhusudan, Singh Manoj,Chugh M Rishi
SBCBE, VIT University, Vellore, T.N.
Research Scholar,Department of Marine and Coastal Studies, School of Energy Sciences, Madurai Kamaraj University, Madurai.
Institute of Pathology, ICMR, Safdarjang Hospital campus, New Delhi - 110029
Received on: 17-03-2010; Revised on: 15-04-2010; Accepted on:13-05-2010
ABSTRACT
Citric acid, C
3
H
4
OH (COOH)
3,
was first isolated from lemon juice by a Swedish chemist, Carl Wilhelm Scheele, in 1784. Citric acid is manufactured by
fermentation of cane sugar or molasses in the presence of a fungus, Aspergillus niger ATCC 9142. Fermentation of sugar by the mold Aspergillus niger ATCC
9142 is the chief commercial source of the acid. Fermentation results in the breakdown of complex organic substances into simpler ones through the action of
catalysis. This project involves the production of citric acid from fungal strain of Aspergillus niger ATCC 9142, using various sources like cane molasses, beet
molasses, sweet potato and grape sugar by employing various methods such as submerged and surface fermentation. The recovery of citric acid from fermented
broth is generally performed through three procedures precipitation, extraction and adsorption (mainly using ion-exchange resins). The main aim of the project
is to achieve a cost reduction in citric acid production by using less expensive substrates.
Keywords: Aspergillus niger, citric acid, potato dextrose agar
History of citric acid:
Citric acid fermentation was first observed as a fungal product
by Wehmer in 1893 by a culture of Penicillium glaucum on sugar me-
dium. It was the work of Currie which opened up the way for successful
industrial production of citric acid. In 1916, he found that numerous
strains of Aspergillus niger produced significant amounts of citric acid.
The most important finding was that A. niger grew well at pH values
around 2.53.5 and high concentrations of sugars favour citric acid
production. The first citric acid fermentations were carried out in surface
cultures. In general, citric acid is commercially produced by submerged
microbial fermentation of molasses; the fermentation process using As-
pergillus niger is still the main source of citric acid worldwide. Although
methods were well developed to synthesise citric acid using chemical
means, better successes were achieved using microbial fermentations,
and over the period of time, this technique has become the method of
ultimate choice for its commercial production over chemical synthesis
(1). It was necessary to consider raw material much more carefully. Sev-
eral works were dedicated to the optimization of the conditions for the
utilization of cheap material like sugar cane molasses, beet molasses,
starch and hydrolysate starch (2).
Citric acid

At room temperature, citric acid is a white crystalline powder. It can exist
either in an anhydrous (water-free) form, or as a monohydrate that contains one
water molecule for every molecule of citric acid. Chemically, citric acid shares the
properties of other carboxylic acids Citric acid is a weak organic acid found in citrus
fruits and vegetables, but it is most concentrated in lemons and limes, where it can
comprise as much as 8% of the dry weight of the fruit. Citric acid i.e. 2-hydroxy 1,2,3
propane tricarboxylic acid (CH
2
COOH.COH.COOH.CH
2
COOH) is ubiquitous in
nature and exists as an intermediate in the citric acid cycle (Krebs cycle) when
carbohydrates are oxidized to carbon dioxide. Molasses is a desirable raw material for
citric acid fermentation because of its availability and relatively low price. The present
investigation deals with the study of citric acid fermentation by Aspergillus niger
ATCC 9142. Cane-molasses and few other substrates were employed as the basal
fermentation media under the surface and submerged fermentation conditions. The
study revealed the nutritional status of the organism and basic fermentation parameters.
Microbial Production of Citric Acid
Microorganisms
A large number of microorganisms including fungi and bacte-
ria such as Arthrobacter paraffinens, Bacillus licheniformis and Coryne-
bacterium ssp., Aspergillus niger, A. aculeatus, A. carbonarius, A.
awamori, A. foetidus, A. fonsecaeus, A. phoenicis and Penicillium
janthinellum; and yeasts such as Candida tropicalis, C. oleophila, C.
guilliermondii, C. citroformans, Hansenula anamola and Yarrowia
lipolytica have been employed for citric acid production (3-8). Among
the mentioned strains, the fungus A. niger has remained the organism of
choice for commercial production because it produces more citric acid
per time unit. The problem in the production of citric acid for yeasts is
the simultaneous formation of isocitric acid. The main advantages of
using A. niger are its ease of handling, its ability to ferment a variety of
cheap raw materials, and high yields.
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The improvement of citric acid producing strains has been
carried out by mutagenesis and selection. The most employed tech-
nique has been by inducing mutations in parental strains using mu-
tagens (6,7,9). Mutants of Aspergillus niger are used for commercial
production (10). To obtain hyper-producer strains, UV treatment can
frequently be combined with some chemical mutagens. The single- -
spore technique and the passage method are the principal methods of
selecting strains. The first one has the disadvantage that mineral acid
and organic acids (gluconic and oxalic acids) simulate the presence of
citric acid (5-7, 11).
Different methods of fermentation can lead to different yields
of citric acid production by the same strain. Thus, a strain which pro-
duces good yields in the solid fermentation or liquid surface is not
necessarily good producer in the submerged fermentation. In that way,
each method and raw material of industrial interest should be tested
with known producer strains (8).
In any technique used in citric acid production the inoculation
of microorganism is done by means of spores which are added into the
fermentation medium (8). Spores can be inoculated either mixing them
with the air, which is introduced in substrate, or in form of a spore
suspension. Spores are produced in glass bottles on solid substrates at
optimum temperature (6). The type of sporulation medium and time of
incubation affect spore viability and citric acid production by mycelia
grown from A. niger. It was mentioned that potato dextrose agar gives
high citric acid yields. Viability increases with time of incubation, but
higher production of citric acid was achieved in less than 7 days of
spore incubation (12).
Aspergillus niger

Aspergillus niger is a fungus and one of the most common species of
the genus Aspergillus.
Aspergillus niger is cultured for the industrial production of
many substances. Various strains of A. niger are used in the industrial
preparation of citric acid and gluconic acid and have been assessed as
acceptable for daily intake by the World Health Organisation.
Aspergillus is utilized industrially in a number of ways. Most
sodas and soft drinks contain citric acid as a main ingredient. It is too
expensive to isolate the citric acid from citrus fruits so it is produced in
large-scale fermentation vats utilizing Aspergillus niger.
REVIEW OF LITERATURE:-
Citric acid is a 6-carbon containing tricarboxylic acid which
was first isolated from lemon juice. It is a natural component of many
citrus fruits, and was crystallized from lemon juice by Scheele in 1784.
Approximately 70% of citric acid produced is used in the food and
beverage industry for various purposes, 12% in pharmaceuticals and
about 18% for other industrial uses (13).
The estimated world production of citric acid was reported as
350.000 tons/year in 1986 (14). It however was recently reported that the
world market requirement of citric acid is around 500.000 tons/year (15).
Although mainly A. niger has been used in the citric acid production
process, other strains of fungi apart from A. niger, various kinds of
yeast and some bacteria are known to accumulate citric acid in the
medium (16). Steel et al. (17) recommended that between 120x103 and
280x103 pellets per liter (obtained from spore inoculated shake flasks) is
a suitable inoculums level, although Kristiansen (18) indicated that the
final concentration of citric acid and dry weight is not related to inocu-
lum size, as long as it was kept below 106 spores/ml culture. Yigitoglu
(19) therefore employed a less complex method for inoculum.
There is a general agreement in the literature that the pelleted
form is desirable for acid production. An ideal pellet configuration, pel-
lets of 1.2 to 2.5 mm diameter after five days, was described early (20).
Gomez et al. (21) showed that the pelleted form is favorable due to pellet
cultures have low culture viscosity causing improving bulk mixing and
aeration conditions and lower oxygen consumption than in the cultures
composed mainly of filamentous (dispersed) forms.Strain improvement
by mutation in order to achieve higher yields and higher trace metal
tolerance is a continual aim of industrial producers. Its importance can
be illustrated by the 500 fold increase in penicillin production from Peni-
cillium chrysogenum due to mutation (22).
McKay et al. (23) increased the production of citric acid yields
from glucose by Yarrowia lipolytica IFO 1658 two fold and by Candida
guillermondii NRRL Y-448 from galactose, six fold via ultra-violet mu-
tagenesis and subsequent selection. James et al. (24) produced a mutant
via multiple X-ray and UV irradiation of spores, and mutant strains
showing a six fold increase in citric acid yield compared to the parent
strain.
The biochemical pathways related to citric acid accumulation
and the role of the tricarboxylic acid cycle (TCA) in fungi has been well
established (13). Citric acid accumulation can be divided into three pro-
cesses (14):
1. The breakdown of hexoses to pyruvate and acetyl-CoA by glycoly-
sis,
2. Formation of oxaloacetate,
3. Condensation of acetyl-CoA and oxaloacetate to citric acid.
As citric acid synthesis involves the condensation of an acetyl
unit with oxaloacetate, it is quite important to generate sufficient oxalo-
acetate in order for production to continue. Regeneration of oxaloac-
etate involves fourmechanisms (16):
1. The direct carboxylation of pyruvate catalyzed by malic enzyme pro-
vides malate which is readily oxidized into oxaloacetate through malic
dehydrogenase;
2. The carboxylation of pyruvate catalyzed by pyruvate carboxylase,
Mg+
Pyruvate + CO
2
+H
2
O+ATP Oxaloacetate +ADP+Pi
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3. The carboxylation of phospho-enol pyruvate (PEP) catalyzed by PEP
carboxykinase,
PEP + ADP+CO
2


Oxaloacetate +ATP
4. via the glyoxylate cycle involving the key enzymes isocitrate lyase
and malate synthase,
Isocitrate

Succinate + Glyoxalate


Co-enzyme A + malate

Glyoxalate + Acetyl-Co-A

Oxaloacetate
Pyruvate carboxylase is an important enzyme for citric acid
production. It is poorly regulated, only weakly inhibited by 2-Oxoglutarate
and not influenced by acetyl-CoA (25). Phosphofructokinase was the
regulatory enzyme of citric acid production in A. niger (26).
The enzyme was inhibited by high concentrations of citrate
and ATP but activated by ADP, AMP, inorganic phosphate and ammo-
nium ions. During citric acid production ammonium ions overcome the
inhibition of PFK by citrate and ATP. Aconitase and isocitrate dehydro-
genase are very important key enzymes in citric acid fermentation. The
activity of these enzymes decrease to very low levels during the pro-
duction stage which cause faulty operation of the cycle whilst the activ-
ity of citrate synthase increases (13)
MATERIALS AND METHODS:-
Production of citric acid using fermentation technology (and
not the chemical synthesis) by Aspergillus niger ATCC 9142. The work
basically includes two types of fermentation viz. surface and submerged
fermentation. The entire work has been performed on a small lab scale
level (and not on an industrial level).
METHODOLOGY
Raw material used: Cane molasses, Beet molasses, Brewery waste,
Sugar.
Culture selection and maintenance
The mother culture of Aspergillus niger ATCC 9142 is ob-
tained from the National Collection Of Industrial Microorganisms (Na-
tional Chemical Laboratory) Pune, Maharashtra. The sub cultured cul-
tures of Aspergillus niger are maintained on sterilized potato dextrose
agar medium (Diced potato 200 g/l, Dextrose 20 g/l and Agar 15 g/l), pH
4.5 and stored at 5C in the refrigerator (the sub culturing method has
been described below). All the culture media, unless other wise stated,
are sterilized in autoclave at 15-lbs/inch
2
pressure (121C) for 15 min.
Although mainly A. niger is used in the citric acid production process,
the reason for choosing A. niger over other potential citrate producing
organism are: cheap raw materials (molasses) used as substrate; easily
available; cost effective; high consistent yields etc.
Sub culturing of Aspergillus niger
First of all, 500 ml of potato dextrose agar medium is made. For
making the PDA, the following method is followed.
Boil finely diced potatoes in water until thoroughly cooked and filter it
through cheesecloth. After filtration add water to filtrate to make the
volume to 0.5 L. Heat the filtrate to dissolve the added agar and add the
glucose before sterilization.
Composition of Potato Dextrose Agar:-
Diced potatoes.............150.0 g
Glucose..................... 10.0 g
Agar........................ 7.5 g
Distilled water..............0.5 L
1.The prepared media is distributed into 6 test tubes (up to half of the
capacity of each test tube). Autoclave at 121
0
C for 15 minutes. Keep in
slanting position and allow them to solidify. Incubate two of the slants
(as blank) to check if any type of contamination is there or not. Incubate
the slants at 20-25
0
C for 4 days.
2.After cross checking of contamination is over, the remaining slants are
sub-cultured using the mother culture. A loop full quantity from the
mother culture is taken on an inoculation loop and streaked (rubbed in
zigzag fashion) onto the prepared slants in an aseptic environment.
Now, incubate these slants at 20-25
0
C for 4-5 days.
It is to be noted that subcultures (from the mother culture)
can not be used more than five times.
Treatment of raw material
Cane molasses is the main raw material used in the present
study. Cane molasses contains 20% water, 62% sugar contents, 10%
non-sugar contents, and 8% inorganic salts (ash contents), making a
blackish homogenous liquid with high viscosity. Ash contents include
ions such as Mg, Mn, Al, Fe and Zn in variable ratio. Sugar content is
diluted to about 25% sugar level.
a.The cane molasses is heated to boiling temperature by keeping in
autoclave for 1-2 hours to prevent fermentation that might be caused
due to contamination.
b.Then the solution is cooled and filtered through normal filter paper.
c.1N H
2
SO
4
is added to the molasses, the same boiled for half an hour,
cooled, neutralized with lime-water (CaO) and was left to stand over
night for clarification. H
2
SO
4
was added to breakdown complex sugars
in juice to simpler sugars so that microorganisms can utilize it.
d.Filter the solution again using normal filter paper to remove the impu-
rities of the first phase. The following elements are added in the flask
containing the molasses.
KH
2
PO
4
- 2.5 gm
NH
4
NO
3
- 1.05 gm
MgSO
4
.7H
2
0 - 0.5 gm
FeCl
3
- 0.01 gm
ZnSO
4
- 0.0025 gm
e.Then the pH of the solution is adjusted using H
2
SO
4
since no further
breakdown is required. The pH was finally adjusted to 5.
f.Distribute the sample into two flasks with each containing 500 ml of the
cane molasses.
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g.Again autoclave the cotton plugged flasks at 110
0
C for 10 minutes.
This sterilization step was carried out in horizontal autoclave.
Inoculums preparation
1.500 ml of molasses medium (Sugar 15%, pH 6.0) in 1.0 L cotton wool
plugged Erlenmeyer flask, is now sterilized.
2. 5 ml of 0.9 % normal saline is transferred to well sporulated Aspergil-
lus niger slants. The inoculation needle is rubbed to ensure proper
mixing of the culture with saline.
3.Small amount of mixed solution from the well sporulated slant is asep-
tically transferred to the flask containing raw material (molasses). Nor-
mal saline is used so as to prevent the death of the spores.
Fermentation
One of the flasks is incubated at 30
0
C in a rotary incubator
shaker at 300 rpm for 5 days. This is for submerged fermentation.

The other flask was incubated in cooling incubator at 28
0
C for
5 days. This is done for surface fermentation.
It is to be noted here that the final concentration of citric acid
and dry weight is not related to inoculum size, as long as it was kept
below 10
6
spores/ml culture.
Growth form
The pelleted form is desirable for acid production. An ideal
pellet configuration, pellets of 1.2 to 2.5 mm diameter after five days, was
described early. The pelleted form is favorable due to pellet cultures
have low culture viscosity causing improving bulk mixing and aeration
conditions and lower oxygen consumption than in the cultures com-
posed mainly of filamentous (dispersed) forms. Furthermore, problems
of wall growth and pipe blockage are reduced and separation of biom-
ass from culture liquid by filtration is considerably enhanced by the
pelleted growth form.
Steps followed after fermentation
1.Take out both the flasks from the shaker incubator and cooling incu-
bator.
2.Heat both of them up to 90 95
0
C to kill the mycelium.

3.Filter the same using muslin cloth and then again filter using filter
paper.
4.Add Ca(OH)
2
or CaCO
3
to the filtrate until the solution gets neutral-
ized.
5.Filter again and collect the precipitate this time. Wash the same with
distilled water and again filter through Whattman 42 filter paper. Keep
the flasks in shaker again for continued washing for 24 36 hours. Filter
again using filter paper.
6The precipitate is filtered and washed with water several times. It is
then treated with H
2
SO
4
. The solution is again filtered to remove CaSO
4
.
The mother liquor containing citric acid is decolourized by charcoal and
passed through ion exchange resin columns. The liquor is concentrated
in vacuum and finally run into low temperature crystallizers where citric
acid crystallizes as citric acid monohydrate, the details of which have
been described below.






Charcoal Treatment
The filtrate is collected after treating with Sulphuric acid. In a
beaker, add 20 gms of charcoal powder to it, mix properly and again filter
with filter paper. Charcoal absorbs the free acidic part and other impuri-
ties. The filtrate is then passed through the filter paper again and then
finally through the chromatography column.

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Preparation of chromatography column
A 50 ml burette, ion exchange resin 244 and glass wool are
taken. Now, a little amount of glass wool is put into the burette. Distilled
water is added to it. Distilled water is passed through the burette to
settle down the glass wool. Resin powder is mixed in a separate beaker
with distilled water & allowed to settle down. The top layer (superna-
tant) is discarded and solid part (precipitate) is put into the burette till 43
mark and again distilled water is added from top. Distilled water is added
till the column is properly settled and colorless drops start passing out
from the burette.
Ion exchange Resin treatment
Now, the filtrate after the charcoal treatment is passed through
this column. The clear solution obtained is now taken to Rotor evapora-
tor for vacuum distillation. We finally obtain the citric acid powder which
is then dried by using vacuum oven.

Identification & Assay:-
Identification of citric acid is initially done on the basis of solubility.
This can be done in the following two ways:-
1.Water solubility test: - Little amount of citric acid is taken in a test tube and
distilled water is added to it. If it is very soluble in it, then it passes the
solubility test. Otherwise, it is some other acid.
2.Ethanol solubility test: - Little amount of citric acid is taken in a test tube and
ethanol is added to it. If it is freely soluble in it, then it passes the solubility
test. Otherwise, it is some other acid.
Note: Citric acid is very soluble in water, freely soluble in ethanol and
sparingly soluble in ether.
3.Calcium chloride test: - To a little amount of citric acid precipitate, water is
added. Then pH is adjusted to 7 by using 1N NaOH and 0.1 % CaCl
2
is added.
If precipitate dissolves, then it confirms the presence of citric acid and if it
does not dissolve then citric acid is absent.
4.Potassium permanganate test: - To a little amount of citric acid precipitate,
add 25 ml of water. Then 0.5ml H
2
SO
4
and 3ml of KMnO
4
are added. Warm the
solution on a burner. If the color of KMnO
4
disappears, then it confirms the
presence of citric acid.
Purity and Yield Calculation
Citric acid (CA) is determined titrimetrically by using 0.1 N NaOH
and phenolphthalein as indicator and calculated in % according to the fol-
lowing formula:-

RESULTS:-
A successful process depends both on an appropriate strain
and optimization of fermentation parameters. In the present work, cul-
tural conditions such as sugar concentration, time profile of citric acid
synthesis, incubation temperature, initial pH, agitation intensity and air
supply were optimized by Aspergillus niger ATCC 9142 in a laboratory
scale.
In the present study, the strain of Aspergillus niger ATCC
9142 supported maximum production of citric acid (106.65 g/l) without
supplements which is substantial. The addition of nitrogen sources and
minerals like iron and phosphate may further increase the production of
citric acid, as required for an industrial process.
ALKEM LABORATORIES LIMITED, R&D CENTRE, TALOJ A, NAVI MUMBAI
(Analysis Report)
Citric Acid Monohydrate
Formula :C
6
H
8
O
7
.H
2
O
Mol. Wt :210.13 (192.13 + 18)
Description :A white, crystalline powder or colorless crystals.
Solubility :Very soluble in water, freely soluble in etha nol, spar
ingly soluble in ether
Functional use :Acidulant, dispersing agent, sequestrant
Flowsheet of the standard presipitation method of citric acid recovery
from fermentation broth
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Packing & storage : 25 kg in composite paper bag with inner PE
bag.Kept in cool and dry place.
Analysis : Clarity and color of solution - Test Pass
Identification - Test Pass
Solubility - Test Pass
Organic volatile impurities - Test pass
Calcium Chloride test - Test Pass
KMnO
4
test - Test Pass
Mesh : Granular : 12-30 mesh
Fine granular : 24-120 mesh
% Citric Acid (Yield): 39.44 %

DISCUSSION
Nitrogen is a limiting factor in the citric acid production. Nitro-
gen is usually supplied in the form of ammonium nitrate, which was
completely metabolized during fermentation periods. Citric acid started
to appear when the nitrogen concentration fell below a low limiting
value. It appears that the citric acid was produced by carbon-storing
cells. Low dry weight might have been caused by the drastic reduction
and denaturation of some enzymes active in the accumulation of carbon
in the used pH range. The sugar concentration decreased throughout
the fermentation period. This indicated that the cells were still viable.
Therefore, there seems to be a link between the storage of carbon and
the production of citric acid. Similarly, other factors that affect the fer-
mentation process have been detailed below.
Factors affecting the fermentation Process:-
Medium Constituents:-
Trace elements: Trace element nutrition is one of the most important
factors affecting the yields (grams citric acid per gram sugar) of citric
acid fermentation. In particular, the levels of manganese, iron, copper
and zinc are quite critical. If the levels of these trace elements are correct
other factors have less pronounced effects. Conversely, medium will
not allow high production unless the trace element content is controlled
carefully.
Manganese: Manganese (Mn
2+
ions) in the nutrient medium plays a key
role in the accumulation of large amount of citrate by A. niger. When the
Mn
2+
concentration is maintained below 0.02 mM (which does not af-
fect growth rate or biomass yield) large amounts of citric acid are pro-
duced. Manganese deficiency leads to significantly lower lipid content
in A. niger whereas there is elevated lipid level in manganese sufficient
cultures.
Iron: - Up to 1 mg iron per liter medium is essential for high yields of citric
acid by A. niger, but that amounts in excess of this interferes with citric
acid accumulation. Partial deficiency of iron is necessary for citric acid
production. The presence of excess iron favors the production of oxalic
acid.
Copper: Copper ions play an important role in reducing the deleterious
effect of iron on citric acid production. Copper ions can successfully
counteract addition of manganese to citric acid fermentation media and
are inhibitors of cellular manganese uptake. Copper is an essential re-
quirement for citric acid production and optimum concentration of Cu
2+
is 40 ppm for high yield.
Zinc: Low concentrations of zinc in the fermentation medium are gener-
ally favored in most citric acid production media. Zinc plays a role in the
regulation of growth and citrate accumulation. At high zinc levels (about
2 M) the cultures are maintained in growth phase, but when the me-
dium becomes zinc deficient (below 0.2 M) growth is terminated and
citric acid accumulation begins. Addition of zinc to citrate accumulating
cultures results in their reversion to growth phase.
Sugars: Due to their rapid assimilation by fungus the usual carbon
sources are glucose, fructose, or sucrose for high final yield of citric
acid. Some sugars such as galactose and arabinose inhibit citric acid
production. In most cases, sucrose or its cheaper commercial source
molasses is used.
Nitrogen source: Usually ammonium sulfate or ammonium nitrate is
used as a nitrogen source. Physiologically, acid ammonium compounds
are preferred since their consumption lowers the pH of the medium to
below 2 which is an additional prerequisite of citric acid fermentation.
The optimal concentration of ammonium sulfate is 5 kgm
-3
. However the
best initial ammonium sulfate level is 3 kgm
-3
by a series of fermentation
which were carried out at varying initial concentration of ammonium
sulfate between 0.5 and 4 kgm
-3
. When the concentration of intracellular
ammonium ion is between 2 and 3 mmol/g cell the production rate of
citric acid is the highest. However when the concentration of intracellu-
lar ammonium ion is decreased below 1 mmol/g cell, the citric acid pro-
duction gets stopped.
Phosphate: The effect of phosphate is not very pronounced but the
balance between manganese, zinc and phosphate is critical. In any cases
of trace metal contamination, phosphate limitation can have a beneficial
effect on citric acid yield. Requirement of phosphate for fungal growth
is 0.1 to 0.2%. However the presence of copper in the medium could
reduce the optimum phosphate concentration. Phosphate plays a key
role in secondary metabolite production. When 0.005% phosphate is
added to beet molasses, 5-ketogluconic and gluconic acid replaces ox-
alic acid as secondary products. In addition fermentation time gets sig-
nificantly reduced.
Magnesium: Magnesium is essential for growth and citric acid produc-
tion due to its role as a cofactor in a number of enzyme reactions in the
cell. The optimum concentration of magnesium sulphate to produce
maximum citric acid varies from 0.02 to 0.025%.
Environmental conditions:-
Aeration: Aeration has a critical effect on the submerged citric acid
production process. Aeration should be 0.6 vvm (liter air per liter me-
dium per minute). The citric acid concentration rises from 30.3 to 48.7
kgm
-3
by increasing air flow rate from 0.9 to 1.3 vvm. Citric acid produc-
tion is also related to oxygen pressure. The yield of citric acid increases
by increasing the flow rate of air and the oxygen pressure up to 1.7
atmospheres using pure oxygen for pressures of 1 atm and greater,
beyond which citric acid production decreases.
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Agitation: Agitation in stirred tank fermenters is critical. Increasing agi-
tator speed breaks up pellets, leading to dispersion of more than 95% of
pellets, resulting in higher yields of citric acid. Maximum yield is at
agitator speeds between 400 and 700 rpm. 500 rpm is the optimal agita-
tion speed for citric acid production. However the higher yield of citric
acid 28 kgm
-3
is generally obtained in culture agitated at lower impeller
speed (300 rpm).
pH: citric acid yield increases with increasing pH. The optimal initial pH
is 6.5. A pH of 2.5 is a clear optimum for final product concentration in a
10 dm
3
STR. The optimal final pH for batch fermentation is 1.7. It is
recommended that pH should be kept low (below 2.0). According to this
at higher pHs, A. niger accumulates gluconic acid, especially when the
pH is around 4.0.

Incubation temperature: The incubation temperature should be in the
range to 28 to 32C, while 30C is the optimum for citric acid production.
Duration of fermentation: The citric acid fermentation is completed in 8
days. Extension of the fermentation period does not increase the yields
of citric acid. Generally incubation period of about 6-9 days is preferred.
Other factors:-
Alcohols: Addition of lower alcohols, methanol, ethanol, n-propanol, to
crude carbohydrate raw materials increases the yield of citric acid. Opti-
mal concentration of methanol, which is said to be more effective than
ethanol, varies from 1 to 4% by volume. However addition of methanol
to highly purified, high yielding substrates is deleterious to acid yields.
The exact mechanism of the alcohol effect however is unexplained,
though it is postulated that addition of methanol increases the tolerance
of fungi to Fe
2+
, Zn
2+
and Mn
2+
.
Lipids: Addition of natural oils with a high content of unsaturated fatty
acids and oleic acid at 2% (v/v) to fermentation media led to increase in
the yield by 20% without affecting dry weight of mycelium. A concentra-
tion of fatty acid of 0.05 to 0.3% has to be maintained during the fermen-
tation.
Vitamins: Ascorbic acid and p-amino benzoic acid, at all concentra-
tions, inhibited growth of citric acid. The presence of thiamine (3x10
-5
M) and riboflavin (4x10
-5
M) stimulates the citric acid formation to the
extent of 59% and 50% respectively. Biotin (3x10
-5
M) produces the
greatest enhancement, stimulating growth and increasing the produc-
tion of citric acid by 66.4%.
Amino acids: The presence of glutamic acid (4x10
- 3
M) and aspartic acid
(3x10
-3
M) stimulates citric acid production by 79 and 76.7% respec-
tively. Presence of lysine (5x10-3 M) and serine (4x10
-3
M) also influence
the formation of citric acid by 62.3 and 50.4%. The effect of cysteine (in
all concentrations) is found to be detrimental.
Toxic chemicals: There is slight increase in citric acid formation in the
presence of phenol (20 ppm) and b-naphthol (20 ppm). But hydroquinone
(with 30 ppm) and o-cresol (with 15 ppm) exhibit maximum citric acid
stimulation i.e. 85 and 80 kgm
-3
respectively. Acid formation in the pres-
ence of resorcinol (with 50 ppm) is 78 kgm
-3
. The increase in citric acid
production may be due to either the direct effect of these phenols on the
growth process i.e., metabolism of A. niger, or due to the inhibition of
enzymes involved in further metabolism of citric acid.
Applications of Citric Acid:-
Citric acid is a universally used alimentary additive. It is accepted world-
wide as GRAS (generally recognized as safe), approved by the Joint
FAO/WHO Expert Committee on Food Additives (27,28,29). The food
and pharmaceutical industries utilize citric acid extensively because of
its general recognition of safety, pleasant acid taste, high water solubil-
ity and chelating and buffering properties. In addition to its carboxyl
and hydroxyl groups permit the formation of a variety of complex mol-
ecules and reactive products of commercial interest. Table 4 presents
the main applications of citric acid (27-29, 30).
C.R. SOCCOL et al.: Citric Acid Production, Food Technol. Biotechnol.
44 (2) 141149 (2006) 147
Applications Industry function
Beverages Wines andCiders Prevents browning in some white wines. Prevents
turbidity of wines and ciders. Used in pH adjustment.
Soft drinks and Provides tartness. Stimulates natural fruit flavour. As
syrups acidulant in carbonated and sucrose based beverages.
Food Jellies,jams and Used in pH adjustment. Acts as acidulant. Provides
preservatives the desired degree of tartness, tang and flavour.
Increases the effectiveness of antimicrobial
preservatives.
Dairy Products As emulsifier in ice creams and processed cheese.
Acidifying agent and antioxidant in many cheese
products.
Candies Acts as acidulant. Provides tartness. Minimizes
sucrose inversion. Produces dark colour in hard
candies. Prevents crystallization of sucrose
Frozen fruit Protects ascorbic acid byinactivating trace metals.
Lowers pH to inactivate oxidative enzymes.
Fats and Oils Synergist for other antioxidants, as sequestrant.
Stabilizing action.
Animal Feed Feed complementation
Agriculture Micronutrient evaluation in fertilizers. Enhances
Pavailability in plants
Pharmaceutics Pharmaceuticals As effervescent in powders and tablets in
combination with bicarbonates. Anticoagulant.
Provides rapid dissolution of active ingredients.
Acidulant in mildly astringent formulation.
Cosmetics and Buffering agent. pH adjustment. Antioxidant as a
toiletries metallicion chelator.
Other Industrial Acts as buffer agent. Sequestring metal ions.
applications Neutralizes bases. Used in nontoxic, noncorrosive
and biodegradable processes that meet current
ecological and safety standards.
Metal cleaning Removes metal oxides from the surface of ferrous
and nonferrous metals, for operational cleaning of
iron and copper oxides. In electroplating, copper
plating, metal cleaning, leather tanning, printing inks,
bottle washing compounds, floor cement, textiles,
photographic reagents, concrete, plaster, refractories
and moulds, adhesives, paper, polymers, tobacco,
waste treatment, chemical conditioner on teeth
surface, ion complexation in ceramic manufacture.
J ournal of Pharmacy Research Vol.3.I ssue 6.J une 2010
Madhusudan chaturvedi et al. / J ournal of Pharmacy Research 2010, 3(6),1215-1222
1215-1222
Source of support: Nil, Conflict of interest: None Declared
CONCLUSION:
Citric acid is the largest produced organic acid measured in
tonnage. Till now its annual production has reached upto 1.5 million
tones and continues to increase more each year because of its enor-
mous application in food, beverage, pharmaceutical and agricultural
industries.in traditional processes, submerged fermentation with the help
of fungus aspergillus niger, is mostly used for its mass production.
However, different new techniques of its production are continuously
being used for showing new methods for production of citric acid.Upon
the basis of work done so far, it can be predicted that citric acid produc-
tion by fermentation of molasses and other liquid citrus cannery wastes
is entirely feasible.
ACKNOWLEDGEMENT:
We would like to thanks Dr. D Dhanshekhran, Mr. M D Tiwari and
Dr. L K Yerneni, for their help.
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