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

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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


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.

[1] Kabera Justin, Ugirinshuti Viateur ,Mukantirenganya
Prudentienne (2010) , Use of nitrous acid mutant of Aspergillus niger
for citric acid production from local cane molasses, African Jornal of
Microbiology Research Vol.4(13) pp.1446-1452 , 4 July 2010

[2] Murad A.El-Holi and Khalaf S.Al-Delaimy (2003),Citric acid

production from Whey with Sugars and additives by Aspergillus
niger, African Jornal of Microbiology Research Vol.2(10) pp.356-
359 , October 2003

[3] Wang Jianlong,Wen Xianghua, Zhou Ding (2000), Production

of citric acid from molasses integrated with in-situ product
separation by ion-exchange resin adsorption, ELESEVIER
Bioresource Technology 75(2000) ,231-234

[4] Baryraktar E. Mehmetoglu U(2000) , Production of citric acid

using immobilized conidia of |Aspergillus niger, Appl. Biochem.
Biotechnol. 87:117-125
III.RESULTS & DISCUSSION [5] Gupta S (1994) , Continuous production of citric acid form
sugar cane molasses using a combination of submerged immobilized
After 5days of incubation , the whole surface of culture
and surface stabilized cultures of Aspergillus niger, KCU520,
media was invaded by a mixture of whitish & blackish
Biotechnol. Lett. 16:599-604.
colonies of mycelia.
Individualized & cultivated , the black mushroom formed the [6] Hossain M. Brooks JD.Moddax IS(1985) , Galactose inhibition
granular colonies with balck spores whereas the mycelia of of citric production from glucose by Aspergillus niger, Appl.
white mushroom still remained on the surface of the culture Microbiol. Biotechnol. 22:98-102.
media. Different measurements were done during
fermentation within the intervals of time which were equal. [7] Lu MY (1995) , Citric acid production by Aspergillus niger in
|The precipitates observed during the fermentation would solid substrate fermentation , Bioresour. Technol.54:235-573

have been caused by the accumulation of debris from

[8] Hendrickson R.Kersterson JW (1971) , Citrous molasses , FL
digested substrate. Consumption of sugars contained in Agr.Exp. Sta.Tech. Bulletin, p.677
molasses was very intense from 2nd day to 5th day of
fermentation. In the initial days the enzyme showed slow [9] Ikram-ul-Haq SA, Qadeer MA,Iqbal J(2001) , Citric acid
sugar compared with the wild parent. Fermentation must be fermentation by mutant strain of Aspergillus niger GCMC-7 using
stopped before the ageing of the mold to prevent those molasses based medium, Microbiol. Biotechnol.17(1):35-37
reactions. The temperature of 30C played a great role in
[10] Mattey M. Allan A (1990), Glycogen accumulation in
optimizing the production of this acid.
|Aspegillus niger, Transient Biochemical Solicits,18(5):1020-1022

[11] Rohr M. Kubicek CP,Kuminek J(1983), Citric acid In Rehm

HJ,Reed, G, (eds), Biotechnology, pp.3341-3352

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