International Journal of Engineering Trends and Technology (IJETT) Volume 9 Number 1 - Mar 2014

Selective Synthesis of Citric Acid from Molasses

using Aspergillus Niger
Jayant.K.Pohnerkar#1 , S.A.Desai#2

1 Tatyasaheb Kore Institute of Engg. & Tech.,Warananagar, Dist.Kolhapur India

2 Prof. in chemical Engg.Deptt. Of chemical Engg.,T.K.I.E.T.Warananagar, Dist. Kolhapur, India

Abstract - Selective synthesis of Citric Acid from local cane
molasses using Aspergillus niger was studied. A local stain of
A.niger isolated from lemon tree leaves & cultured. A.niger stain
ATCC-595 were separately studied for the Bioconversion of local
molasses into citric acid. The molasses fermentation was carried
out at 30C in a flask. The results showed increased efficiency for
citric acid production when compared with wild type. It produced
210.12 gm/lit of citric acid as compared to wild stain 102.5 gm/lit.
The maximum citric acid production is on the 9th day of
fermentation by used stain and 11th day by the wild type. This
investigation is one of the ecofriendly technology can be used for
economic development & environmental protection in india. By
recycling & reusing waste material from cane Industries Citric acid
production can be easily done.
Keywords: Citric acid; Fermentation; Aspergillus niger ; Surface
culture
I. INTRODUCTION
Citric acid (CH2COOH.COH.COOH.CH2COOH) is a
carboxylic acid, soluble in water with a pleasant taste is the
important acid used in the food Industries. It exists in nature
when carbohydrates are oxidized to CO2 , Carbon dioxide.
Because of its high solubility ,palatability & low toxicity it
can be used in food, bio chemical & pharmaceutical
industries. It is needed for inhibition of oxidization,
conservation of pickles, neutralization etc.
These uses have placed greater stress on
increased Citric acid production& search for more efficient
fermentation process. The worldwide demand of citric acid is
above 80 Lac. tons/year & it is bound to increase day by day.
Various processes are used for Citric acid production. Surface
culture process is still being used form 1940s, but not
effective due to low output of Citric acid. New methods of
submerged fermentation improved the production of Citric
acid.
Different other methods were being used for Citric acid
production , Extraction of Citric acid from fruits & its
chemical synthesis, Citric acid from whey & other dairy
product wastes, Citric acid from beet molasses as substrates
etc. But most commercially used method for production of
Citric acid is by Aspergillus niger using cane molasses as an
example of fungal overflow metabolism.
Many microorganisms such as fungi &
bacteria can produce Citric acid but Aspergillus niger
remained the organism of choice for production of Citric acid
due to its genetic stability , high yields , capacity of using
cheaper raw material (like cane molasses) & absence of
undesirable reactions.
|According to various researches the
mutant stains might show several fold increase in citrate
production compared to wild type.
Cane molasses is still the substrate of choice of Citric
acid production by Aspergillus niger . In the middle eastern
region (Jordan) large amounts of whey are produced as a by
product of cheese & other dairy products manufacturing.
From this using Aspergillus niger Citric acid can be
produced. In the country like |Republic of China, Sugar is
produced from beet. The beet molasses are used for citric
acid production using Aspergillus niger in China.
In India sugar cane factories are large in number & local
cane molasses are easily available due to their waste disposal
problems. Using A.niger from local cane molasses Citric acid
production can be done using different stains. India spents lot
of money for the importation of citric acid. Alsot there is
disposal problems of cane molasses. Both problems can be
solved by citric acid production. Thus our work consists of
search for stain of A.niger & fermentation process which is
very efficient for the bioconversion of these deposits of local
molasses to citric acid to insure the self supply of this acid to
local industries, & the environment protection.

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 International Journal of Engineering Trends and Technology (IJETT) Volume 9 Number 1 - Mar 2014

II.METHODS & MATERIALS drops under a more/less vigorous agitation.

A. Isolation of Aspergillus niger

The used A.niger (wild type) was
isolated from leaves of Citrous lemon & an A.niger ATCC-
595 stockculture was reactivated &cultivated by streaking a
loopful of culture on petridishes containing Potato Dextrose
Agar (PDA) & incubated at 25-30 C for 10-15 days.
Different colonies that appeared were biochemically &
morphologically characterized & used as inoculums for the
fermentation process.

B. Fermentation Conditions
Fermentation was carried out in a Bioreactor with
0.71 working volume. |The surface culture was used & the
inocubating temperature was 30C . PH adjusted to 6 with
NaOH (1gm/lit). Incubation periods are 4days, 6days,
8,10,12,14 days.

C. Pretreatment of substrate
Cane molasses obtained from Sugar Factory was used
depending on the volume of used flasks, 200gm of molasses
were diluted in 180ml of Distilled water, PH was adjusted to
6 with NaOH. To inhibit proliferation of micro-organisms
,mixture was sterilized at 121C for 15min.

D. Inoculation of Substrate & sampling

The cellular suspensions of A.niger were prepared
& transferred to the flasks. The fermentation was carried at
30C .

E. Measurement of Citric acid content

Citric acid was determined titrimetrically by using 0.1N
NaOH & Phenolfthalein as indicator & calculated as %
according to following formula:

% Citric Acid = Normality * Vol. of NaOH* Equivalent

Weight of Citric acid/ Weight of sample (gm)*10

Using a graduated pipette ,1ml of supernatant was diluted in

49ml of distilled water to increase discoloration &5drops of
phenolphthalein were used to serve as color indicator. The
Citric acid was titrated by addition of NaOH(1gm/lit) in

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 International Journal of Engineering Trends and Technology (IJETT) Volume 9 Number 1 - Mar 2014
III.RESULTS & DISCUSSION
After 5days of incubation , the whole surface of culture
media was invaded by a mixture of whitish & blackish
colonies of mycelia.
Individualized & cultivated , the black mushroom formed the
granular colonies with balck spores whereas the mycelia of
white mushroom still remained on the surface of the culture
media. Different measurements were done during
fermentation within the intervals of time which were equal.
|The precipitates observed during the fermentation would
have been caused by the accumulation of debris from
digested substrate. Consumption of sugars contained in
molasses was very intense from 2nd day to 5th day of
fermentation. In the initial days the enzyme showed slow
sugar compared with the wild parent. Fermentation must be
stopped before the ageing of the mold to prevent those
reactions. The temperature of 30C played a great role in
optimizing the production of this acid.
ISSN: 2231-5381 http://www.ijettjournal.org
ACKNOWLEDGEMENT
The author gratefully acknowledges the N.C.I.M. National
Collection of Industrial Microorganisms a division of
National Chemical Laboratory (N.C.L.) Pune, India for
supplying |Aspergillus niger srain used in this study. Our
sincere thanks to the authorities of the Tatyasaheb Kore
Institute of Engg. & Technology, Warananagar, Dist.
Kolhapur, India for their unlimited hospitality and their
priceless moral as well as material support during this
study.Also we are greatly indebted to the Deptt. Of
Biotechnology , Tatyasaheb Kore Institute of Engg. &
Technology, Warananagar, Dist. Kolhapur, India for allowing
us to use their laboratories.
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