ISSN : 0974 - 7435 Volume 6 Issue 3
Trade Science Inc.
BioTechnology
An Indian Journal
Full Paper
BTAIJ, 6(3), 2012 [93-96]
Citric acid production by Aspergillus niger through solid state
fermentation using fruit wastes
K.Hariveeran Goud, A.Srilakshmi, A.Praveen Kumar, G.Narasimha*
Applied Microbiology Laboratory, Department of Virology, Sri Venkateswara University,
Tirupati-517502, Andhra Pradesh, (INDIA)
Email: gnsimha123@rediffmail.com; dr.g.narasimha@gmail.com
Received: 10th April, 2012 ; Accepted: 10th May, 2012
ABSTRACT
In this study, a fungal strain was isolated from soil contaminated with fruit wastes and screened for citric acid
production and it was identified as Aspergillus niger. The solid wastesubstrates like fruit peels (grapes, mo-
sambi peel) and bagasse with varying particle sizes of M1- (0. 250mm), M2 (0.150mm) and M3 (0.63mm) and
4% methanol, 70% moisture level was maintained throughout the solid state fermentation process for the citric
acid production from the fungal culture. The Aspergillus niger produces the higher yields of citric acid where
grape peel with particle size 0.63mm as a substrate in the medium. Higher fungal biomass and protein contents
estimated in grapes (M3), mosambi (M1) and bagasse (M3)- wastes substrates respectively in the solid state fer-
mentation. © 2012 Trade Science Inc. - INDIA
INTRODUCTION logical procedures, mainly based on the use of
microfungi, which are currently the most widely
Citric acid (2-hydroxy2, -propanetricarboxylic used[3]. One of the most important fungi used in
acid) is one of the most versatile and important the industrial microbiology Aspergillus niger has
carboxylic acid intermediate of metabolism in been employed for many years in the production
most plants and animals. Due to its innocuous na- of citric acid. Citric acid is produced from bulk
ture, citric acid is extensively used in food prepa- hydrated materials and as a by-product of sugar
rations, pharmaceuticals and cosmetics. About production by Aspergillus niger[4]. In recent years,
70% citric acid is used in food industry and re- considerable interest has been shown in solid state
maining 30% in other industries[1]. Now a day, production of citric acid by Aspergillus niger us-
citric acid is also increasingly utilized as a mono- ing agro residues like bagasse, corncob, carob pod
mer for the manufacture of biodegradable poly- and waste of food processing industries like apple
mers which are widely used in various medical and grape pomace and fruit peel due to its several
applications[2]. Citric acid (CA) is found in a vari- advantages like solid waste management, biomass
ety of acidic fruit juices, particularly in the citric energy conservation, production of high value
ones, although its extraction from natural sources, products and little risk of bacterial contamination
[5]
primarily lemon, was gradually replaced by bio- . Variety of substrates, such as sucrose, starch
94 Citric acid production by Aspergillus niger through solid state BTAIJ, 6(3) 2012
Full Paper
from various sources, cane and beet molasses, ap- 0.150mm, (M3)0.63mm. Bagasse of desired parti-
ple pomace, orange / pine apple wastes, banana cle size was taken in 250ml Erlenmeyer flasks
waste have been utilized for the economical pro- and 70% moisture was maintained in the follow-
duction of citric acid[6,7]. The present objective of ing composition (gm/l). Sucrose 15, NH4NO3
the study is to screen and identify the potent fun- 0.25, MgSO4 0.025, H2PO4 0.1, CuSO4 0.004 and
gal strain for citric acid production using cheaply the medium pH was adjusted with pH 4.0. To set
available substrates through solid state fermenta- the desired moisture levels media were sterilized
tion (SSF). at 1210C for 15 min.
For grape and mosambi substrates, the peels
MATERIAL AND METHODS were dried in an oven at 700 C for 2 days
grounded and screened to collect the particles of
The soil sample contaminated with fruit waste the size M1, M2, M3. Five gram grounded fruit
was collected from local Fruit Market of Tirupati waste was taken in 250ml conical flask and mois-
Town, Chittoor District, Andhra Pradesh, India. tened with distilled water to desired moisture
Isolation, identification and screening of level 70%. Since mosambi peel waste contained
citric acid producing fungi less sugar it was moistened with sucrose solution
The fungal culture was enumerated by soil to increase the sugar level to 31.8 g/100g of dry
serial dilution technique and the isolates were solid (Previously optimized). The flasks contain-
maintained on Czapek-Dox agar slants. Accord- ing medium were sterilized at 1210C for 15 min.
ing to Kareem et al, the fungal cultures were After sterilization, the flasks containing medium
screened[8] for citric acid production. The potent were allowed to cool to room temperature and
citric acid producing fungal culture was identified inoculated with 1ml of spore suspension (2x107
based on its macroscopic and microscopic charac- spores/ml) of A.niger followed by proper mixing.
teristics as followed by Narasimha et al[9] by re- After inoculation flasks were incubated at 30oC in
ferring to the standard book entitled a humidity controlled incubator. Methanol (4%v/
“Compendium of soil fungi”[10]. w) was added to the medium before inoculation
and after sterilization of the medium.
Inoculum
Spore suspension of A. niger with concentra- Extraction and analytical methods
tion of 2 x 107 spores/ml was prepared by adding Fermented material of the flask was dried in
25ml of sterile distilled water with Tween-80 an oven at 50o C and extracted by the addition of
(0.1%) on Czapek-Dox agar slant and was shaken 100ml of distilled water. The mixture was agi-
vigorously for a minute on vortex mixer. It was tated on a rotary shaker for 2 hour and then fil-
suitably diluted to obtain a spore concentration of tered through Whattman filter paper No.1. The
2 x 107 spores/ml. supernatant was used for the estimation of total
residual sugar and citric acid. Total reducing
Collection of substrate samples
sugar was determined[11] and citric acid was esti-
Two different fruits wastes mosambi peel and mated by the acetic anhydride and pyridine
Grape residues was collected from local fruit mar- method[12], Protein estimation[13] and pH and Bio-
ket of Tirupati. Baggase was collected from Sri mass was also estimated [14].
Venkateswara Sugar Factory, Gajulamandyam,
Chittoor, Andhra Pradesh, India. RESULT AND DISCUSSION
Solid State Fermentation
The fugal isolated from soil contaminated
The solid state fermentation was carried out with fruit waste was screened according to the
with Bagasse, Grape and mosambi peels were method of Kareem et al[8]. In this method the for-
dried in an oven (700C) and cut in to small pieces. mation of yellow zone surrounding the fungal col-
Grounded and screened to collect three fractions ony in the plate is the indication for the citric acid
of different particle sizes (M1)0.250mm, (M2) ability of the culture and the fungal culture was
BioTechnology
An Indian Journal
BTAIJ, 6(3) 2012 G.Narasimha et al. 95
Full Paper
identified as A.niger based on its macroscopic and strates (bagasse, mosambi, grape) with parti-
microscopic characteristics listed in the TABLE 1. cle sizes M 1 (0.250mm), M 2 (0.150mm) M 3
Similarly the fungal culture A.niger was isolated (0.63mm) and the results were represented in
from soil contaminated with effluents of cotton gin- TABLES 3-5. In the present study, maximum
ning mill[9] and dairy industries[15] were identified. citric acid production was observed in grape
The maximum biomass and protein content as substrate in the medium (34.4g/Kg) with
was observed in grape substrate with particle size particle size of M 3 (0.63mm). The increase in
of M1 (3.2g, 4.56µg/ml), M2 (3.4g, 4.24 µg/ml) citric acid production and biomass values
respectively (TABLE 2). The pH value main- was accompanied with steady decrease in
sugar along the incubation time [8] . The paral-
TABLE 1: Macroscopic and microscopic characteristic lel relationship between citric acid produc-
of Aspergillus niger
TABLE 3: Citric acid production by A.niger on differ-
Colony
ent particle sizes of bagasse substrate z
Color Texture Diameter
Macroscopic (cm)
Bagasse substrate Citric acid
Black Filamentous 10.2 sugar
different particle production
Conidiophores Conidiophores Conidia consumption (%)
length (µm) diameter (µm) shape
size (g/kg)
Microscopic
3.31 3.21 Globose M1(0.250mm) 89.2 23.6
TABLE 2 : pH, Biomass and protein content of Asper- M2(0.150mm) 92 24.9
gillus niger on different substrates with particle size of
bagasse, grape and mosambi M3(0.63mm) 88.3 22.1
Biomass Protein *Values represented in the table are mean of duplicates
Substrates Particle size pH
(gm) (µg/ml)
M1(0.250mm) 3.2 2.82 0.78 TABLE 4: Citric acid production by A.niger on differ-
ent particle sizes of mosambi substrate
Bagasse M2(0.150mm) 3.1 2.53 0.60
M3(0.63mm) 3.0 2.20 0.64 Mosambi
Sugar Citric acid
substrate
M1(0.250mm) 2.40 3.20 4.56 consumption production
different
Grape M2(0.150mm) 2.08 3.40 4.24 (%) (g/kg)
particle size
M3(0.63mm) 1.5 3.0 3.20 M1(0.250mm) 94.1 28.6
M1(0.250mm) 2.14 3.12 4.16 M2(0.150mm) 92 26.7
M3(0.63mm) 89.5 23.3
Mosambi M2(0.150mm) 2.70 3.40 3.12
M3(0.63mm) 2.80 3.10 2.80 *Values represented in the table are mean of duplicates
TABLE 5 : Citric acid production by A.niger on differ-
tained at the beginning of fermentation was im- ent particle sizes of grape substrate
portant for a specific biomass formation. Nor-
mally, citric acid production occurred after 24 h Grape
of fermentation, this study shows that as incuba- Sugar Citric acid
substrate
tion time increased more citric acid is produced consumption production
different
and pH values decreased. Thus, the drop in pH (%) (g/kg)
particle size
observed during the process was due to the forma- M1(0.250mm) 94.3 29.8
tion and accumulation of citric acid[8]. M2(0.150mm) 96.6 31.9
The fungal culture A.niger was used for M3(0.63mm) 99.2 34.4
the citric acid production on different sub-
*Values represented in the table are mean of duplicates
BioTechnology
An Indian Journal
96 Citric acid production by Aspergillus niger through solid state BTAIJ, 6(3) 2012
Full Paper
tion and the consumption of sugar was also [3] Ana Maria Torrado, Sandra Cortes, Jose Manuel
observed (TABLES 3-5). These results were Salgado, Belen Max, Noelia Rodriguez, Belinda
agreed with the report of El-Holi and Al- P.Bibbins, Attilio Converti, Jose Manuel
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[4] M.Y.Lu, I.S.Maddox, J.D.Brooks; Bio Resource
microbe, resulting in lower citric acid pro-
Technology, 54, 235-239 (1995).
duction. In contrast to Kumar et al [17] re- [5] Ashish Kumar, V.K.Jain; African Journal of Bio-
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