R.Gopinadh et al.

/ Journal of Pharmacy Research 2012,5(1),374-376

Research Article Available online through
ISSN: 0974-6943 www.jpronline.info
Statistical experimental design for citric acid production using Madhuca indica
*R. Gopinadh a , Prof C. Ayyannab, Narayana Saibaba K. Va , R.Jagadhic
a
Department of biotechnology, Gitam institute of Technology (GIT), GITAM University,Visakhapatnam -530045, Andhra Pradesh, India
b
Director, Al-Ameer College of Engg, Visakhapatnam, Andhra Pradesh, India
c
Department of Pharmaceutical Sciences, Visakhapatnam, Andhra Pradesh, India
Received on:20-09-2011; Revised on: 15-10-2011; Accepted on:10-12-2011

ABSTRACT
Statistical Experimental Design was employed for the improvement of citric Acid production from Madhucaindica through submerged fermentation process
using Aspergillus niger MTCC 282. The results obtained from the two-level full factorial design with six factor combinations show effects free from
confounding, that is, all effects are distinguishable from other effects by doing fewer runs or with all possible combinations . The results obtained from two-
level full factorial design showed that Mg 0.05mg/ml, Amm.Sul 0.2 g/l,EDTA 0.2g/l, KH2 PO4 2 g/l were found to be major factors for the production of citric
acid. The Statistical analysis showed that maximum production of citric acid (70.0187g/l) was obtained at pH 4.

Keywords: biofuels, green house gas emissions, Biomass, second generation, and biogas

1. INTRODUCTION
Citric acid is a biodegradable - environmentally accepted - versatile organic
acid produced by fermentation. Citric acid derives its name from the Latin
citrus, the citron tree, the fruit of which resembles a lemon. It is a weak
carboxylic organic acid soluble in water with a pleasant taste. Being an im-
portant intermediate in the Krebs cycle it plays a vital role in the metabolism
of virtually all living things. It can also be used as an environmentally benign
cleaning agent.
Citric acid exists in substantial amounts in a variety of fruits and vegetables,
mostly in citrus fruits . The concentrations of citric acid in citrus fruits range
depend upon the type of fruit. For example, fruits such as oranges, grape-
fruits have 0.005 mol/L and lemons, limes have a higher concentration i.e.
0.30 mol/L. Other factors which determine the amount of acid present in the
fruits are cultivar and also the circumstances in which the fruit was grown.
Citric acid (2-hydroxy-1,2,3-propanetricarboxylic acid) is extensively pro-
duced by Aspergillus niger and widely used in food, beverage, cosmetic,
pharmaceutical, chemical, textile and electroplating industry [1,2]. Currently,
it is commercially produced by submerged (liquid state) fermentation (SmF)
of starch or sucrose based media (sucrose or glucose syrups) using Aspergil-
lus niger [2, 3]. However, the global demand of citric acid is growing faster
than its production implying that more economical process is required. Sub-
merged state bioconversion (SSB) involves the growth of microorganisms on
moist substrates in the absence or near-absence of free flowing water and the
submerged substrate acts as a source of carbon, nitrogen, minerals and carrier
necessary for microbial growth [4,5]. As the microorganisms in a submerged
fermentation are growing under the conditions similar to their natural habitat,
they can produce certain enzymes, metabolites, proteins and spores [6]. The
*Corresponding author.
R.Gopinadh
Assistant Professor
Department of Biotechnology,
Gitam institute of technology
Gitam University
Visakhapatnam-530045,
Andhra Pradesh, India
Tel +919866264077
Email: gopinaadh@yahoo.com
major chemical parameters which influence the growth of Aspergillus niger
and its production of citric acid on a submerged substrate are nutrient sources,
moisture content, particle sizes, incubation temperature, pH and inoculum
density [2,6,7]. The accumulation of citric acid is strongly affected by fer-
mentation conditions and higher production of citric acid was recorded by the
optimization of fermentation process conditions besides optimization of
media, [2]. Global demand for citric acid is high while the cost of existing raw
materials is increasing thus requiring an alternative economical process with
new substrates is necessary.
Submerged fermentation (liquid state bioconversion) has been selected by the
world’s largest citric acid manufacturers due to the lower capital and labora-
tory or cost. Other than the utilization of glucose as major substrate, various
cheaper substrates such as insulin [8] , date fruit syrup [9], carob pod [10],
sugar cane molasses [11] have been used for citric acid production by As-
pergillus niger. Some other microbes (bacteria and yeast) have been used in
the industrial process for citric acid from sugars and n-alkanes [12,13]. Citric
acid can also be obtained synthetically from acetone or glycerol, but chemical
synthesis is no cheaper than fungal fermentation [14].
Factors affecting the production of citric acid by fermentation include the
nutritional composition of the Mahua flower, available carbon source, envi-
ronmental conditions, pH, and dissolved oxygen. The influence of types and
concentrations of sugars, ammonium nitrate, and aeration on citric acid pro-
duction by Aspergillus niger has also been studied [9, 13, 15, 16]. Most of
the studies have been done using single factor optimization which has limita-
tions to maximize the products due to neglect of the interaction effects of the
factors. Statistical optimization is a better solution to optimize the operating
factors of the processes considering the linear, quadratic, and interaction
effects in the treatment. Several researches have been done using statistical
optimization for different bioprocess engineering applications [17, 18, 19].
The objective of the present study was, therefore, to evaluate citric acid
production by Aspergillus niger using submerged fermentation process hold-
ing the sugar-rich Substrate periodically supplemented with nutrient solu-
tion. The sugar-rich byproduct was simulated by using Madhuca indica wet-
ted with a nutrient solution. The chemical fermentation parameters Fe, Mg
, Amm.Sul , Ethanol ,EDTA , KH2 PO4 was stastically designed using full

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factorial method and interactions between different variables were evaluated Table 1: Levels of Variables tested in Factorial experimental Design
by choosing a design, and meaningful results were obtained. S.No Variable Parameter Coded Level
-1 0 1
2. MATERIALS AND METHODS 1 A Fe 0.5 2.25 4
2 B Mg 0.05 0.15 0.25
2.1 Microorganism and culture conditions 3 C Amm Sulphate 0.2 0.4 0.6
4 D Ethanol 0.5 1.5 0.25
Aspergillus niger MTCC 281 was obtained from the microbial type culture 5 E EDTA 0.2 0.4 0.6
collection, Chandigarh, India was used in the study. The culture was main 6 F KH 2 PO 4 2 2.5 3
tained on PDA (Potato Dextrose Agar) Slants at 32o C and is sub cultured Table 2. The experimental design using the Factorial experimental
every month. Inoculam was prepared by adding 10ml of sterilized Tween-80 Design for screening of medium components
solution (0.1%).Spores were scrapped superficially and used as inoculums
S.N0 A B C D E F Yield CA g/l
for fermentation. Experiments for optimizing medium constituents were
conducted at Ph 4, Temperature 30o c and Substrate concentration of 15% (w/ 1 -1 -1 -1 1 1 1 65.9435
2 1 -1 -1 -1 -1 1 70.0187
v). 3 1 -1 1 -1 1 -1 58.1991
4 -1 -1 1 1 -1 -1 63.2518
2.2 Production Medium 5 0 0 0 0 0 0 63.2092
Madhuca indica (MI) was used as Carbon source for fermentation. Juice of 6 0 0 0 0 0 0 63.7831
MI was obtained by grinding and weighed amount of MI was dissolved in 7 -1 1 1 -1 -1 1 53.124
8 1 1 -1 1 -1 -1 62.0388
water and sterilized at 121o C for 15 minutes. Ph of the medium was adjusted 9 0 0 0 0 0 0 63.124
by adding acid or Alkali. Spore suspension (1ml) was prepared from a 7 day 10 -1 1 -1 -1 1 -1 60.0287
11 1 1 1 1 1 1 51.4323
old culture and inoculated to 100 ml of medium in a 250 ml Erlenmeyer flask. 12 1 -1 -1 -1 -1 1 69.5275
Medium was incubated in orbital shaking incubator at 150 revolutions per 13 -1 -1 1 1 -1 -1 61.8684
14 1 1 1 1 1 1 51.8157
minute. Samples were collected for every 24 hours. 15 0 0 0 0 0 0 63.6879
16 1 -1 1 -1 1 -1 58.9962
17 -1 1 1 -1 -1 1 53.7305
2.3 Analytical Methods 18 1 1 -1 1 -1 -1 62.8258
Citric Acid was estimated as described by [20] .Reducing sugars were deter- 19 -1 -1 -1 1 1 1 64.7405
mined using [21] 20 -1 1 -1 -1 1 -1 59.9536

The experimental design for screening of medium components is shown in

2.4 Stastical Experimental Design Tables 1 and 2. Each variable was set at two levels, that is, high level and low
2.4.1 Two-level full factorial designs level. The high level of each variable was set far enough from the low level to
Stastical design of Experiments help to improve the processes. Different identify which ingredients of the media have significant
Factors are screened to determine interactions between different variables Influence on the Citric acid production.
which are important for explaining process variation. Process characteriza-
tion is used to reduce the number of input variables by identifying the key 3. RESULTS AND DISCUSSION
input variables or process conditions that affect product quality. Factorial The first optimization step was using a 20-run Two-level full factorial designs
designs allow for the simultaneous study of the effects that several factors to identify the significant factors for Citric acid production by Aspergillus
niger MTCC 282. According to the resulting effects of these Six variables on
may have on a process [22]. Factorial designs helps to understand how
citric acid concentration and the associated significant levels presented in
factors interact and drive the process to produce optimal process perfor-
Table 3, it can be seen that concentration of mg, ammonium sulfate, EDTA,
mance [21]. KH2 PO4 , all have positive effects on Citric acid . Fe, Ethanol showed
negative effects on Citric acid production. With the help of relative ranking,
Preliminary investigations indicated that the substrate concentrations, am- mg, ammonium sulfate, EDTA, KH2 PO4 within the tested limits were selected
monium Sulphate, , Fe, Mg , Ethanol ,EDTA , KH2 PO4 are highly significant for further optimization, which had the most significant effects on Citric acid
variables for citric acid production. Thus these variables were chosen and production.
designated as A, B, C, D, E, F respectively. The purpose of the first step in
the experiment was to identify the significance of the ingredients of the media Table 3. Factorial Fit: Yield CA versus A, B, C, D, E, F
for the production of citric acid [23]. Two-level full factorial designs was
employed in screening of the major constituents of the media that has signifi- Estimated Effects and Coefficients for Yield (coded units)
cant effect on the response of citric acid. A 26 factorial experiment was
Term Effect Coef SE Coef T P
designed with 4 axial points with (a = 0.05) and Two replicates and four
centre points leading to a total of 20 experiments. This design was employed Constant 60.468 0.1256 481.53 0.000
to study of interactions between the factors. Interactions are the driving A 0.277 2.983 0.1256 1.10 0.292
B -7.200 -3.600 0.1256 10.62 0.000
force in many processes. Without the use of factorial experiments, important C -7.832 -3.916 0.1256 -31.19 0.000
interactions may remain undetected. The combinations of factor levels repre- D -3.159 -1.580 0.1256 -12.58 0.000
sent the conditions at which responses will be measured. E -0.854 -0.427 0.1256 -3.40 0.005
F 1.037 0.519 0.1256 4.13 0.001
Coding of the variables was done according to the Following Equation (1)... Ct Pt 0.138 0.2808 -28.67 0.000

S = 0.502303 R-Sq = 99.43% R-Sq(adj) = 99.10%

i= 1, 2, 3…..k,------------------(1)
The positive effects of mg, ammonium sulfate, EDTA, KH2 PO4 were, prob-
ably, caused by the requirement of a large quantity of substrate to synthesize
real value of an independent variables at centre point and cells. It is possible that the high concentration of ammonium Sulphate could

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Fig 1: Pareto Chart of the Standardized Effects
contribute to the improvement of the glucose consumption rate, resulting in
both good cell growth and satisfactory Citric acid production.
The 2-level factorial design proved to be a powerful tool to rapidly determine
the effects of medium constituents on Citric acid production using Aspergil-
lus niger MTCC 282. . Further work need to be done to find out optimal
concentrations of medium components that significantly affect citric acid.
By choosing a design, meaningful results are obtained. The results obtained
from two-level full factorial design showed that Mg 0.05mg/ml, Amm.Sul
0.2g/l,EDTA 0.2g/l, KH2 PO4 2 g/l were found to be major factors for the
production of citric acid.
4. CONCLUSION
The Statistical analysis showed that maximum production of citric acid
(70.0187g/l) was obtained at ph 4 by Aspergillus niger MTCC 282 in sub-
merged fermentation using a Madhuca Indica (MI) as a low cost substrate.
This study is useful in understanding a mathematical model which helps in
selection of factors important for improving the yield in the reaction. Inter-
action can magnify or diminish main effects in the submerged fermentation
process and evaluating the interactions is extremely important in designing of
experiment.
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