Satheeshkumar et al Journal of Drug Delivery & Therapeutics. 2019; 9(4-A):9-14
Available online on 30.08.2019 at http://jddtonline.info
Journal of Drug Delivery and Therapeutics
Open Access to Pharmaceutical and Medical Research
© 2011-18, publisher and licensee JDDT, This is an Open Access article which permits unrestricted
non-commercial use, provided the original work is properly cited
Open Access Research Article
Utilization of Fruit Waste f or the Production of Citric Acid by using
Aspergillus Niger
Satheeshkumar Subramaniyan1, Sivagurunathan Paramasivam2, Muthulakshmi Kannaiyan3, Uma
Chinnaiyan*
Department of Microbiology, Faculty of Science, Annamalai University, Annamalai nagar, Chidambaram-608002, India
ABSTRACT
The main organic acids in industrial used were citric, acetic, tartaric, malic, lactic and gluconic acid. The most utilized organic acid is citric acid
or tricarboxylic acid. Chemical synthesis of citric acid is more costly than fermentation. The citrus processing industry generates tons of waste
such as peel and segment membranes resulting from the extraction of citrus juice in industrial plants. About 6 isolates were obtained from the
fruit waste dumped soil and they were subjected to screening for citric acid production. A. niger over other potential citric acid-producing
organism towards efficient utilization of agroindustry residues and by-products for citric acid production. It was grown on PDA plates at a
temperature of 28°C for 5 to 7 days. Fruit wastes like orange peel, sweet lime peel, banana peel and pineapple peel as a potential substrate for
the production of citric acid using A. niger isolates. The nitrogen source of the fermentation medium will have a direct effect on the yield of citric
acid. Among the four fruit wastes, citric acid production was maximum from orange peel followed by sweet lime peel, pineapple peel and
banana peel respectively. Orange peel contains soluble sugars and pectin as the main components. Study the impact of different carbon sources
on citric acid., the basal media were supplemented with glucose, sucrose, fructose, maltose and the fermentation were carried up to 5 days at
30°C. Nitrogen sources on the fermentation were studied by incorporating ammonium phosphate, potassium hydrogen phosphate and peptone.
The effect of lower alcohol incorporation on the fermentation of citric acid was studied.
Article Info: Received 09 July 2019; Review Completed 21 Aug 2019; Accepted 25 Aug 2019; Available online 30 Aug 2019
Cite this article as:
S. Satheeshkumar, P. Sivagurunathan, K. Muthulakshmi, C. Uma, Utilization of Fruit Waste for the Production of Citric Acid
by using Aspergillus Niger, Journal of Drug Delivery and Therapeutics. 2019; 9(4-A):9-14
http://dx.doi.org/10.22270/jddt.v9i4-A.3487
*Address for Correspondence:
Uma Chinnaiyan, Department of Microbiology, Faculty of Science, Annamalai University, Annamalai nagar, Chidambaram-608002, India
INTRODUCTION production6. India positions sixth place in the production of
citrus fruits in the world and the citrus fruit wastes are also
The citric acid (2-hydroxy-propane 1, 2, 3-tricarboxylic acid) generated in million tons for every year7. The citrus
derives its name from the Latin word citrus, the fruit of the processing industry generates tons of waste such as peel and
citrus tree which resembles a lemon. Citric acid is a segment membranes resulting from the extraction of citrus
tricarboxylic acid with a molecular weight of 210.14 Da 1. juice in industrial plants. The management of these wastes,
With an estimated annual production of about 10,00000 which produce odor and soil pollution, represents a
tons, citric acid is one of the fermentation products with the noteworthy issue for the food industry8.
most elevated level of production world wide2. A
considerable amount of citric acid is required in several Orange peel contains soluble sugars and pectin as the main
industrial processes3.The food industries consume about components. According to to9, the orange peel is constituted
70% of the total citric acid production, while other by soluble sugars, starch, fiber, (cellulose, 9.21% wt;
industries (Pharmaceuticals, Beverages, etc) consume the hemicelluloses, 10.5% wt; lignin, 0.84% wt and pectins,
remaining 30%4. Chemical synthesis of citric acid is more 42.5% wt) ashes, 3.50 % wt; fats, 1.95 % wt; and proteins,
costly than fermentation. Various kinds of yeast, fungi and 6.50% wt10. Peels of sweet lime contain a higher
some bacteria are known to produce citric acid5.The reason concentration of phenolics, flavonoids, ascorbic acids,
for choosing A. niger over other potential citric acid- carotenoids and reducing sugars11. Pineapple peel is a by-
producing organism are cheap raw materials used as product resulting from the processing of pineapple into
substrate; high consistent yields. There has been an slices and represents about 10% (w / w) of the weight of the
increasing trend towards efficient utilization of agro- original fruit. Pineapple peel is rich in cellulose,
industrial residues and by-products for citric acid hemicellulose and other carbohydrates12.
ISSN: 2250-1177 [9] CODEN (USA): JDDTAO
, Satheeshkumar et al Journal of Drug Delivery & Therapeutics. 2019; 9(4-A):9-14
Banana is one of the most important major fruit crops grown Five grams of each substrate were taken in separate flasks
in India. In respect of area and production, it ranks second and sterilized at 121°C, 15 lbs pressure for 15 min.
only to mango. The banana culture in India is as old as Autoclaving of the substrate was done to provide proper
Indian civilization. The peel of banana was a rich source of cooking of the substrate and to kill the microorganisms.
water, sugar, protein, fat, fiber, tannin, carotene, pigments13. After sterilization, the media were inoculated with strains of
Due to the impact of dumping fruit wastes into the A. niger (10⁸ spores/ml) and incubated at 30°C for 5 days.
environment and about the wide applications of citric acid, Methanol 3% (w/v) was added to the medium before
we carried out studies to test the potential of fruit wastes inoculation. After fermentation, the media were diluted with
like orange peel, sweet lime peel, banana peel and pineapple the addition of 100 ml of distilled water and then filtered by
peel as a substrate to produce citric acid. using Whatman No.1 filter paper. The resulting filtrate was
used for subsequent analysis.
MATERIALS AND METHODS
Determination of citric acid
Isolation of fungi
Citric acid was determined titrimetrically (AOAC, 1995) by
Determination of fungal load from fruit waste dumped using 0.1 N NaOH and phenolphthalin as an indicator. The
soil percentage of citric acid was calculated according to the
Fungal isolates were obtained from the fruit waste dumped following formula:
soil. The soil samples were collected in sterile polythene Normality X Volume of NaOH X Equiv.Wt. of citric acid
bags and brought to the laboratory for further analysis. The % Citric acid = -----------------------------------------------------------------------
soil sample was serially diluted, spread plate method was Weight of sample (g) X 10
performed using the dilutions 10-3, 10-4 and 10-5 in RBA Optimization studies
medium and incubated at 30°C for 5 days. After incubation,
Parameter optima are important in any type of fermentation
the black color colonies were suspected as Aspergillus sp and
process, because the production may get an increase or
selected for the screening of citric acid production.
decrease in respect to media components, pH, temperature,
Screening of fungal isolates for citric acid production media components, etc. To study the effect of above-said
parameters, the following experiments were carried out.
The fungal isolates were subjected to primary screening for
the production of citric acid as per the method described i. Effect of medium components on citric acid
by12. Czapek-Dok (CD) agar medium was prepared with production
bromocresol green indicator and plated on to the sterile
Study the impact of different carbon sources on citric acid.,
Petri plates. After solidification, 0.5 ml of spore suspension
the basal media were supplemented with glucose, sucrose,
of fungal isolates were transferred to each of the Petri plates
fructose, and maltose (5 to 15% (w/v)) and the fermentation
and incubated at 30°C for 5 days. Formation of yellow zones
was carried up to 5 days at 30°C. The culture filtrate
around the colony is the indication of citric acid producers.
obtained after fermentation was analyzed for citric acid
The isolates that produced the widest yellow zone were
production.
selected for further studies.
ii. Effect of nitrogen sources
Identification of fungal isolates
The effect of different nitrogen sources on the fermentation
The citric acid-producing fungal isolates were subcultured
on to PDA plates, incubated at room temperature for five was studied by incorporating ammonium phosphate,
potassium hydrogen phosphate and peptone (0.5%). The
days. After incubation, the isolates were identified based on
culture filtrate was taken after fermentation was analyzed
their cultural characteristics and microscopic observation
for citric acid production.
using Lactophenol Cotton Blue (LPCB) staining method.
Preparation of substrate for citric acid production iii. Effect of lower alcohols
Collection and pretreatment of fruit waste samples The effect of lower alcohol incorporation on the
fermentation of citric acid was studied. The media were
The fruit waste samples viz., orange peel, sweet lime peel, added with methanol and ethanol at a different
banana peel and pineapple peel were collected from local concentration ranging from 1 to 4%. The resulting culture
fruit market and juice shop located around Chidambaram filtrate after fermentation was used to estimate citric acid
Taluk. The collected fruit waste samples were dried in a hot production.
air oven at 60°C for 2 hrs. Then the dried samples were cut
iv. Effect of trace elements
into 2 mm sized particles, were used as substrate for citric
acid production. To study the effect of trace elements, the media were
introduced with copper, iron and zinc at a concentration of
Production of citric acid
10 ppm. The culture filtrate was analyzed for citric acid
Inoculum preparation production.
The strains of A. niger were grown on PDA plates at a v. Effect of pH.
temperature of 28°C for 5 to 7 days. After incubation, the
To study the effect of pH on the fermentation of citric acid,
plates were washed with sterile 0.1% of Tween 80 solution
the media were adjusted with different pH ranging from 2 to
to collect the spores. The number of spores was counted
6. The culture filtrate obtained after fermentation was
using haemocytometer. The suspension containing 10⁸
analyzed for citric acid production.
spores/ml were citric acid is used as an ingredient for
production7. vi. Effect of temperature
Fermentation The influence of temperature on the production of citric acid
was determined. The fermentation media were incubated at
The basic medium for fermentation was prepared by adding
different temperature levels viz., 20°C, 25°C, 30°C and 35°C.
5 g (5%) pretreated substrate in 100 ml of distilled water.
ISSN: 2250-1177 [10] CODEN (USA): JDDTAO
Available online on 30.08.2019 at http://jddtonline.info
Journal of Drug Delivery and Therapeutics
Open Access to Pharmaceutical and Medical Research
© 2011-18, publisher and licensee JDDT, This is an Open Access article which permits unrestricted
non-commercial use, provided the original work is properly cited
Open Access Research Article
Utilization of Fruit Waste f or the Production of Citric Acid by using
Aspergillus Niger
Satheeshkumar Subramaniyan1, Sivagurunathan Paramasivam2, Muthulakshmi Kannaiyan3, Uma
Chinnaiyan*
Department of Microbiology, Faculty of Science, Annamalai University, Annamalai nagar, Chidambaram-608002, India
ABSTRACT
The main organic acids in industrial used were citric, acetic, tartaric, malic, lactic and gluconic acid. The most utilized organic acid is citric acid
or tricarboxylic acid. Chemical synthesis of citric acid is more costly than fermentation. The citrus processing industry generates tons of waste
such as peel and segment membranes resulting from the extraction of citrus juice in industrial plants. About 6 isolates were obtained from the
fruit waste dumped soil and they were subjected to screening for citric acid production. A. niger over other potential citric acid-producing
organism towards efficient utilization of agroindustry residues and by-products for citric acid production. It was grown on PDA plates at a
temperature of 28°C for 5 to 7 days. Fruit wastes like orange peel, sweet lime peel, banana peel and pineapple peel as a potential substrate for
the production of citric acid using A. niger isolates. The nitrogen source of the fermentation medium will have a direct effect on the yield of citric
acid. Among the four fruit wastes, citric acid production was maximum from orange peel followed by sweet lime peel, pineapple peel and
banana peel respectively. Orange peel contains soluble sugars and pectin as the main components. Study the impact of different carbon sources
on citric acid., the basal media were supplemented with glucose, sucrose, fructose, maltose and the fermentation were carried up to 5 days at
30°C. Nitrogen sources on the fermentation were studied by incorporating ammonium phosphate, potassium hydrogen phosphate and peptone.
The effect of lower alcohol incorporation on the fermentation of citric acid was studied.
Article Info: Received 09 July 2019; Review Completed 21 Aug 2019; Accepted 25 Aug 2019; Available online 30 Aug 2019
Cite this article as:
S. Satheeshkumar, P. Sivagurunathan, K. Muthulakshmi, C. Uma, Utilization of Fruit Waste for the Production of Citric Acid
by using Aspergillus Niger, Journal of Drug Delivery and Therapeutics. 2019; 9(4-A):9-14
http://dx.doi.org/10.22270/jddt.v9i4-A.3487
*Address for Correspondence:
Uma Chinnaiyan, Department of Microbiology, Faculty of Science, Annamalai University, Annamalai nagar, Chidambaram-608002, India
INTRODUCTION production6. India positions sixth place in the production of
citrus fruits in the world and the citrus fruit wastes are also
The citric acid (2-hydroxy-propane 1, 2, 3-tricarboxylic acid) generated in million tons for every year7. The citrus
derives its name from the Latin word citrus, the fruit of the processing industry generates tons of waste such as peel and
citrus tree which resembles a lemon. Citric acid is a segment membranes resulting from the extraction of citrus
tricarboxylic acid with a molecular weight of 210.14 Da 1. juice in industrial plants. The management of these wastes,
With an estimated annual production of about 10,00000 which produce odor and soil pollution, represents a
tons, citric acid is one of the fermentation products with the noteworthy issue for the food industry8.
most elevated level of production world wide2. A
considerable amount of citric acid is required in several Orange peel contains soluble sugars and pectin as the main
industrial processes3.The food industries consume about components. According to to9, the orange peel is constituted
70% of the total citric acid production, while other by soluble sugars, starch, fiber, (cellulose, 9.21% wt;
industries (Pharmaceuticals, Beverages, etc) consume the hemicelluloses, 10.5% wt; lignin, 0.84% wt and pectins,
remaining 30%4. Chemical synthesis of citric acid is more 42.5% wt) ashes, 3.50 % wt; fats, 1.95 % wt; and proteins,
costly than fermentation. Various kinds of yeast, fungi and 6.50% wt10. Peels of sweet lime contain a higher
some bacteria are known to produce citric acid5.The reason concentration of phenolics, flavonoids, ascorbic acids,
for choosing A. niger over other potential citric acid- carotenoids and reducing sugars11. Pineapple peel is a by-
producing organism are cheap raw materials used as product resulting from the processing of pineapple into
substrate; high consistent yields. There has been an slices and represents about 10% (w / w) of the weight of the
increasing trend towards efficient utilization of agro- original fruit. Pineapple peel is rich in cellulose,
industrial residues and by-products for citric acid hemicellulose and other carbohydrates12.
ISSN: 2250-1177 [9] CODEN (USA): JDDTAO
, Satheeshkumar et al Journal of Drug Delivery & Therapeutics. 2019; 9(4-A):9-14
Banana is one of the most important major fruit crops grown Five grams of each substrate were taken in separate flasks
in India. In respect of area and production, it ranks second and sterilized at 121°C, 15 lbs pressure for 15 min.
only to mango. The banana culture in India is as old as Autoclaving of the substrate was done to provide proper
Indian civilization. The peel of banana was a rich source of cooking of the substrate and to kill the microorganisms.
water, sugar, protein, fat, fiber, tannin, carotene, pigments13. After sterilization, the media were inoculated with strains of
Due to the impact of dumping fruit wastes into the A. niger (10⁸ spores/ml) and incubated at 30°C for 5 days.
environment and about the wide applications of citric acid, Methanol 3% (w/v) was added to the medium before
we carried out studies to test the potential of fruit wastes inoculation. After fermentation, the media were diluted with
like orange peel, sweet lime peel, banana peel and pineapple the addition of 100 ml of distilled water and then filtered by
peel as a substrate to produce citric acid. using Whatman No.1 filter paper. The resulting filtrate was
used for subsequent analysis.
MATERIALS AND METHODS
Determination of citric acid
Isolation of fungi
Citric acid was determined titrimetrically (AOAC, 1995) by
Determination of fungal load from fruit waste dumped using 0.1 N NaOH and phenolphthalin as an indicator. The
soil percentage of citric acid was calculated according to the
Fungal isolates were obtained from the fruit waste dumped following formula:
soil. The soil samples were collected in sterile polythene Normality X Volume of NaOH X Equiv.Wt. of citric acid
bags and brought to the laboratory for further analysis. The % Citric acid = -----------------------------------------------------------------------
soil sample was serially diluted, spread plate method was Weight of sample (g) X 10
performed using the dilutions 10-3, 10-4 and 10-5 in RBA Optimization studies
medium and incubated at 30°C for 5 days. After incubation,
Parameter optima are important in any type of fermentation
the black color colonies were suspected as Aspergillus sp and
process, because the production may get an increase or
selected for the screening of citric acid production.
decrease in respect to media components, pH, temperature,
Screening of fungal isolates for citric acid production media components, etc. To study the effect of above-said
parameters, the following experiments were carried out.
The fungal isolates were subjected to primary screening for
the production of citric acid as per the method described i. Effect of medium components on citric acid
by12. Czapek-Dok (CD) agar medium was prepared with production
bromocresol green indicator and plated on to the sterile
Study the impact of different carbon sources on citric acid.,
Petri plates. After solidification, 0.5 ml of spore suspension
the basal media were supplemented with glucose, sucrose,
of fungal isolates were transferred to each of the Petri plates
fructose, and maltose (5 to 15% (w/v)) and the fermentation
and incubated at 30°C for 5 days. Formation of yellow zones
was carried up to 5 days at 30°C. The culture filtrate
around the colony is the indication of citric acid producers.
obtained after fermentation was analyzed for citric acid
The isolates that produced the widest yellow zone were
production.
selected for further studies.
ii. Effect of nitrogen sources
Identification of fungal isolates
The effect of different nitrogen sources on the fermentation
The citric acid-producing fungal isolates were subcultured
on to PDA plates, incubated at room temperature for five was studied by incorporating ammonium phosphate,
potassium hydrogen phosphate and peptone (0.5%). The
days. After incubation, the isolates were identified based on
culture filtrate was taken after fermentation was analyzed
their cultural characteristics and microscopic observation
for citric acid production.
using Lactophenol Cotton Blue (LPCB) staining method.
Preparation of substrate for citric acid production iii. Effect of lower alcohols
Collection and pretreatment of fruit waste samples The effect of lower alcohol incorporation on the
fermentation of citric acid was studied. The media were
The fruit waste samples viz., orange peel, sweet lime peel, added with methanol and ethanol at a different
banana peel and pineapple peel were collected from local concentration ranging from 1 to 4%. The resulting culture
fruit market and juice shop located around Chidambaram filtrate after fermentation was used to estimate citric acid
Taluk. The collected fruit waste samples were dried in a hot production.
air oven at 60°C for 2 hrs. Then the dried samples were cut
iv. Effect of trace elements
into 2 mm sized particles, were used as substrate for citric
acid production. To study the effect of trace elements, the media were
introduced with copper, iron and zinc at a concentration of
Production of citric acid
10 ppm. The culture filtrate was analyzed for citric acid
Inoculum preparation production.
The strains of A. niger were grown on PDA plates at a v. Effect of pH.
temperature of 28°C for 5 to 7 days. After incubation, the
To study the effect of pH on the fermentation of citric acid,
plates were washed with sterile 0.1% of Tween 80 solution
the media were adjusted with different pH ranging from 2 to
to collect the spores. The number of spores was counted
6. The culture filtrate obtained after fermentation was
using haemocytometer. The suspension containing 10⁸
analyzed for citric acid production.
spores/ml were citric acid is used as an ingredient for
production7. vi. Effect of temperature
Fermentation The influence of temperature on the production of citric acid
was determined. The fermentation media were incubated at
The basic medium for fermentation was prepared by adding
different temperature levels viz., 20°C, 25°C, 30°C and 35°C.
5 g (5%) pretreated substrate in 100 ml of distilled water.
ISSN: 2250-1177 [10] CODEN (USA): JDDTAO
