Journal of Agricultural Chemistry and Environment, 2014, 3, 20-25
Published Online April 2014 in SciRes. http://www.scirp.org/journal/jacen
http://dx.doi.org/10.4236/jacen.2014.32B004
How to cite this paper: Al-Shoaily, K., et al. (2014) Multivariate Study and Analysis of the Production of Citric Acid from
Dates by Surface Method. Journal of Agricultural Chemistry and Environment, 3, 20-25.
http://dx.doi.org/10.4236/jacen.2014.32B004
Multivariate Study and Analysis of the
Production of Citric Acid from Dates by
Surface Method
Khalid Al-Shoaily*, Mansoura Al-Amri, Fouzia Al-Rawahi, Muhammad Al-Sidrani,
Amal Al-Ghafri
Food Processing Research Laboratory, Ministry of Agriculture & Fisheries, Muscat, Oman
Received January 2014
Abstract
The production of citric acid from dates of the date palm Phoenix dactilifera was studied using a
full factorial design with two design variables; Aspergillus niger strain, and starter juice treatment.
Analyses of data have shown that mould type and the interaction between Aspergillus niger strain
+ juice treatment had significant effect (P ≥ 0.05) on the final total acidity. It was also found that all
design variables and the interaction between them had significant effect (P ≥ 0.05) on the final pH
and Total Soluble Solids. The highest total acidity expressed as citric acid was achieved with a
commercial strain of Aspergillus niger, and filtered and centrifuged date juice.
Keywords
Dates; Citric; Aspergillus nige r
1. Introduction
Dates continue to be the most abundantly produced fruit in Oman; the annual total yield according to ministry of
agriculture and fisheries (MAF) Oman 2010 estimation was 276,405 tons. Although potentially there is very
valuable raw material for many industries, the almost exclusive use of dates is still direct consumption. The high
content of monosaccharides (glucose, and fructose) (Al-Farsi, Alasalvar et al. 2007) [1] makes the dates suitable
for fermentation to produce various organic acids such as acetic acid and citric acid.
Citric acid is an organic acid that naturally occurs in fruits such as lemons oranges, and may be synthesised
from glycerol (Kristiansen Bjorn, Mattey et al. 1999) [2]. It is also produced via fermentation by microorgan-
isms action, many microorganisms accumulate citric acid, among them is Aspergillus niger, A. awamori, Absi-
dia sp, Acremonium sp. ...etc. Nowadays Aspergillus niger is almost exclusively used to produce citric acid at
industrial scale (Kristian sen Bjorn, Mattey et al. 1999) [2]. Citric acid has a wide range of use in food and beve-
rage, in preparation of numerous industrial products, pharmaceuticals, and as a cleaning agent (Lotfy Walid A,
Ghanem Khaled M. et al. 2007) [3]. Factorial experimental des ign and multivariate an alysis is a useful tool used
*
Corresponding author.

K. Al-Shoaily et al.
21
to estimate the impact a parameter (a design variable) has on a response ( non design variable ), this design is
used for screening purpose to study main effects and interactions between combinations between design va-
riables (Esbensen, Guyot et al. 2004) [4].
In the work reported here, we attempt to study the effects of two design variables (juice treatment, and mould
type) on three non design variables (pH, total acidity, and total soluble solids TSS) in an experiment to produce
citric acid by surface method from date juice.
2. Materials and Method
2.1. Microorganism
Two mould types were used in this study: a commercial strain of Aspergillus niger supplied by Culti-loops (the
code is kept), and the other is a local strain of Aspergillus niger isolated from local dates surface.
2.2. Inoculum
Loops from pre prepared pure slants of Aspergillus nig er (local or commercial) were streaked (Figure 1) on po-
tato dextrose agar (Supplied by Himedia-India), and used to inoculate 18°brix date juice, then placed in a shaker
incubator at 30˚C for 48 hours to produce pellets (Abid M. Al-, Al-Amri M. et al. 2010) [5].
2.3. Preparation of Date Juice
Dates sugars were extracted with water as explained in (Abid M. Al-, Al-Amri M. et al. 2010) [5], the final TSS
was ad jus ted at 18˚brix. The juice was subsequently treated in one of two filtration ways:
1) Filtration with whatman No.1 filter paper or
2) Filtration with whatman No.1 filter paper followed by centrifugation at 4000 RPM.
2.4. Inoculation with Aspergillus niger
Two types of Aspergillus niger, a commercial strain, and a local strain isolated from the surface of dates were
used to inoculate the date juice.
2.5. Treatment with Tricalcium Phosphate (TP)
To eliminate in hibition caused b y metals the date j uice was treated with Tr icalcium Phosphate Treatment to che-
late minerals as explained in (Roukas T. and P.) [6] and (A b i d M . Al -, Al-Amri M. et al. 2010) [5].
2.6. Analytical Metho ds
Total acidity was by titration with NaOH, pH was measured with pH meter (WTW, supplied by, Germany), TSS
by bench t op refractometer BS supplied by Stanley & Bellingham, UK
2.7. Experimental Design
A full factorial design was implemented with 2 design variables and 3 non design variables as shown in Figure
2 & Table 1, the treatments were performed in triplicates:
2.8. Data analysis
All analysis of data were performed with MINITAB14.
3. Results and Discussions
Total acidity, pH, and total soluble solids were monitored daily in order to determine the pattern at which the
changes occur.
Acidity of fermenting medium was used as an indication of acid production. The total acidity increased slowly
up to day eight then exponentially up to day 14 and leveled (Figure 3).

K. Al-Shoaily et al.
22
Figure 1. Aspergillus niger growth on PDA.
Table 1. Design variables (factors) and non design variables used.
Design variables Non design variables
1) Filtration treatment:
a) Filtration through whatman filter(T1).
b) Centrifugation at 4000 RPM (T2).
2) Inoculum type:
a) Local strain Aspergillus niger (S1).
b) Commercial strain Aspergillus niger (S2).
1) pH
2) Total soluble solids TSS
3) Total acidity.
3.1. Optimization of Fermentation Conditions
A full factorial design was implemented as sh own in Figure 2 & Table 1, this design is used for screening de-
sign variables ( juice treatment and Aspergillus niger strain) in order to find out about main effects they have on
non design variables or responses (pH, total acidity, and total soluble solids TSS) of the final product, in addi-
tion to interaction effects of the design variables (Esben se n , Guyot et al. 2004) [4].
3.2. Effect of Mould Type on Final Total Acidity
The final analysis results are shown in Tab le 2. The mould type significantly affected th e total acidity (P ≥ 0.05),
total acidity increased from 0.37, to 3.75, and from 1.18 to 3.58% by changing the mould type from local (S1), to
commercial (S2) strains respectively.
3.3. Effect of Mould Type on Final pH
The final pH was significantly affected by mould type (P ≥ 0.005), the final pH (Figure 4) was reduced from
3.74, to 2.57, and from 2.9 to 2.58 by changing the mould type from local (S1), to commercial (S2) strains re-
spectively.
3.4. Effect of Mould Type on Final TSS
The final total solub le solids TSS of the final product w as used as an indication of sugar as similation by Asper-
gillus niger (Figure 5). The mould type significantly (P ≥ 0.005), affect ed the TSS. The read ings of TS S of the
final product Table 2 show decrease in TSS values from 14 to 13.8, and from 15.5 to 13.03 with change of
mould type from local (S1), to commercial (S2) strains respective ly.
3.5. Effect of Juice Treatment on Final Total Acidity
As can be seen in Table3 the juice treatment had insignificant effect alon e on total acidity (P ≥ 0.05), but the in-
teraction with the mould type was significant (P ≥ 0.05) (see Table 3).

K. Al-Shoaily et al.
23
Figure 2. A process flow diagram showing design variables and
non design variables.
Figure 3. Changes in acidity of fermented medium. T1S1 juice treatment 1 and
mould type 1; T1S2 juice treatment 1 and mould type 2; T2S1 juice treatment
2and mould type 1; T2S2 juice treatmen t 2 and mould type 2.
Table 2. Final results of acidity, pH, and TSS.
% Acidity pH TSS °brix
T1S1
0.37
3.74
14.00
T1S2
3.75
2.57
13.80
T2S1
1.18
2.90
15.50
T2S2
3.58
2.58
13.03
Prepare date juice
18°brix
Adjust pH to 7
Add TP 2%
Add ammonium
nitrate 2%
Filter whatmanCentrifuge at 4000
rpm for 20 min
Full factorial design to study the Production of citric acid from dates
by surface method
Design variables Non Design variables
Monitor pH, TSS, and citric acid
Inoculate with
Aspergillus niger
(local)
Inoculate with
Aspergillus niger
(commercial)

K. Al-Shoaily et al.
24
Figure 4. Changes in pH of fermented medium.
Figure 5. Changes in TSS of fermented medium.
3.6. Effect of Juice Treatment on Final pH
The final pH was significantly affected by juice treatment (P ≥ 0.005) (see Table 3), the final pH was reduced
from 3.74, to 2.57, and from 2.9 to 2.58 Table 2 by changing the juice treatment from local (T1), to centrifuga-
tion (T2) respectively.
3.7. Effect of Juice Treatment on Final TSS
The readings of TSS of the final product (Table 2) show decrease in TSS values from 14 to 13.8, and from 15.5
to 13.03 with change of juice filtration treatment fro m (T1), to (T2) respectively. Th e decrease was significant (P
≥ 0.005), which shows the need for thorough filt ra tion techni que.
3.8. Analysis of Variance (One Way ANOVA)
Analysis of variance (one way ANOVA) Table 4 shows that both parameters (main effects) used were signifi-
cant, further more the interaction between mould type and juice treatment was significant as well.
4. Conclusion
The results revealed the significance of mould type and filtration technique in the production of citric acid from
date juice. Despite the result the mould was more significant (P ≤ 0.005) than juice treatment (P ≥ 0.05); the
combined effect of interaction between the two was significant (P ≤ 0.05). The maximum total acidity achieved

K. Al-Shoaily et al.
25
Table 3. P values for non design variables versus design variables.
Non design variables (responses) Design variable (factors) Total acidity pH TSS
Aspergillus niger strain 0.000 (**) 0.000 (**) 0.020 (*)
Juice treatment 0.810 (NS) 0.000 (**) 0.005 (*)
2 way interaction: Aspergillus niger strain + Juice treatment 0.041 (*) 0.000 (**) 0.020 (*)
(**) P ≤0.001, (*) P ≤0.05, (NS) not significant.
Table 4. Analysis of var ian ce ( one way ANOVA).
Non design variables (responses) Design variable (factors) Total acidity pH TSS
Main effects
0.000 (**) 0.000 (**) 0.004 (*)
2 way interaction 0.041 (**) 0.000 (**) 0.020 (*)
(**) P ≤ 0.001, (*) P ≤ 0.05, (NS) not significant.
was 3.75% by using the commercial strain of Aspergillus niger and centrifugation of the juice pr ior to fermenta-
tion. Using of factorial design showed the importance of interaction effect that the parameters had on the res-
ponses. Dates can be a promising material for production of citric acid.
References
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[2] Kristiansen, B., e t al. (1999) Citric Acid Biotechnology. Taylor & Frances, London.
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