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Optimization of SCP production ofAspergillus niger using different fruit peels

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 International Journal of Bioinformatics and Biological Science: v.1 n.1 p.1-8. March, 2013

Optimization of SCP production of

Aspergillus niger using different fruit peels
Akhilesh Bind* Manish Kumar and Divya Singh

Department of Biochemistry & Biochemical Engineering, Sam Higginbottom

Institute of Agriculture Technology & Sciences (Deemed-to-be-University)
Formerly Allahabad Agricultural Institute Allahabad-211007, India
*
Corresponding Author: Akhilesh Bind akhileshbind@gmail.com
Received: 24 December 2012; Accepted: 03 January 2013

ABSTRACT
An attempt was made to apply solid state fermentation for the production of single
cell protein using fruit peels of Banana, orange and papaya peels respectively and
their different combinations were used for optimization of single cell protein
production of Aspergillus nige at different pH and incubation period. Substrate of
fruit peel was used in different combination to maximize protein content in A.niger.
4th combination gave the best result of maximum production of 1.352 mg/ml of
protein on 8 days of incubation and at pH 7 which is directly related to maximum
fungal growth.
Keywords: Single cell protein (SCP), Aspergillus niger

BACKGROUND
SCP is a term coined to embrace microbial biomass products which were produced
by fermentation. SCP production by technology arose as a promising way to solve
the problem of the worldwide shortage of protein. They evolved as bioconversion
process which turned waste into products which added nutritional and market value.
Intensive research into fermentation science and technology for biomass production
as well as feeding has resulted in a profound body of knowledge, the benefits of
which now span far beyond the field of SCP production. These agricultural products
out marketed SCP on the grounds of lower price. The combination of sophisticated
products with food processing technology yielded a new generation of SCP products
which may be used as meat substitutes, texture providing agents and flavor enhancers.
Future application of heterologous protein expression may further develop the
potential of this food line resulting in precisely tailored products which meet specific
dietary requirements or stimulate high added value specialty products(Ugalde and
Castrillo,2001).
In year (Anupama,2001) studied the bioconversion of agricultural and industrial
waste to protein rich food and fodder stock. SCP production has the potential for
feeding the ever increasing world population at cheaper rates(Najafpour,2007)
 Bind et al.

Suggested the potential of SCP to overcome shortage of food in the world. The
present investigation was carried out to utilize various combinations of fruit wastes
in the production of SCP by using standard food fungi Aspergillus niger.

MATERIALS AND METHODS

Collection of culture
Pure culture of Aspergillus niger (MCCB-0201) was collected from research
laboratory, Department of Microbiology and Fermentation Technology, JSBB,
SHIATS, Allahabad. The culture was maintained on PDA media. The slants were
grown at 28±2°C for 7 days.

Pretreatment of substrate
Banana, orange and papaya peels were collected from the local market. It was
pretreated to expose cellulose fibers to cellulolytic attack by the organism. The
pretreatment process was carried out by adding 1% sodium hydroxide to 10g of
fruit waste and autoclaving at 121°C and at 15 psi for 30 min. Pretreated material
was allowed to cool, subsequently filtered and washed to neutral pH.. Then it was
dried at 60°C in an oven for 12 h. Later on it was used for solid state
fermentation(Anupama,2001).

Media and cultivation for solid state fermentation

The fermentation was carried out in 250ml flask containing 10g of solid substrate
in various combinations of fruit waste. There were four different combinations used
in different ratios (Table 1). The content of the flask were mixed thoroughly and
autoclaved at 121°C for 20 min. Each experiment was done in triplicate.
Table 1: Various combinations of substrate used during the experiment

Combinations Ratio of substrates Orange Banana Papaya

1st combination 1:1:1 3.33g 3.33g 3.33g

2nd combination 1:1:2 2.5g 2.5g 5.0g
3rd combination 2:1:1 5.0g 2.5g 2.5g
4th combination 1:2:1 2.5g 5.0g 2.5g

Inoculation: After cooling, the production media was inoculated with the 106 spores/
ml of inoculums and incubated under constant condition at 30°C for 4 days.
Buffer extraction: 50 ml of carbonate bicarbonate buffer (0.1 M at pH 10.6) was
added to the flask and then culture was homogenized for 30 minute using rotator
shaker at 250 rpm then the content was squeezed through the distilled water wet
muslin cloth. The filtrate was centrifuged at 5000 rpm for 10 minutes then supernatant
was filtered through a whatman no.1 filter paper(Anupama,2001)

2 International Journal of Bioinformatics and Biological Science: v.1 n.1 p.1-8. March, 2013
 Optimization of SCP production of Aspergillus niger using different fruit peels

Optimization: Various combinations of substrates were made of different ratios,

the fermentation media was incubated for 2days, 4 days, 6 days, 8 days and 10 days
and fermentation media was incubated also at different pH of 4, 6, 7 and 8. Since
Aspergillus niger grows best within the pH range of 5-7 to see the effect of incubation
time on the single cell protein.
Protein estimation: Total protein estimation was done according to Lowry
method(Lowry,1951).

RESULTS AND DISCUSSION

The concentrations stated in the present investigation was very much similar to the
values revealed by(Ojokoh and Uzeh,2005) for papaya peel(Yalemtesfa et al) for
orange and(Yabaya and Ado ,2008) for banana peel.
Table 2: SCP (mg/ml) content without fermentation

Concentrations Orange peel Banana peel Papaya peel

0.1 ml 0.363 0.349 0.358

0.2 ml 0.416 0.570 0.458

Effect of different combinations on single cell protein:

Various combinations of different ratios of substrates were taken to check its effect
on overall protein production. There were four different combinations used during
the experiment.
Table 3: SCP (mg/ml) content in combinations of fruit peels without fermentation

Concentrations 1st combination 2nd combination 3rd combination 4th combination

0.1 ml 0.124 0.118 0.146 0.159

0.2 ml 0.194 0.204 0.226 0.205

Out of all, 4th combination showed greater protein content thus, the results shown
by these combinations can be of greater interest in producing SCP and will show
potential results on its application as feed or food. The maximum SCP production
of various combinations (Table 3) for 0.1ml and 0.2ml volume of sample were
given as followed:
Table 4: SCP (mg/ml) content in combinations of fruit peels after fermentation

Concentration 1st combination 2nd combination 3rd combination 4th combination

0.1 ml 0.774 0.790 0.608 0.840

0.2 ml 1.340 1.130 1.126 1.352

(Table 4) It determined that maximum SCP production occurred in the 4th combination
i.e. 1.352 mg/ml in 0.2 concentrations of sample aliquots. All the combinations

International Journal of Bioinformatics and Biological Science: v.1 n.1 p.1-8. March, 2013 3
 Bind et al.

showed more SCP production than pure substrates during the investigation. This
finding can be applied on large scale up and low cost technology alternatives should
be explored intensively. Furthermore, the biomass produced from fruit waste can
be evaluated for its suitability to be used either as a biofertilizer or as animal feed.
It should be noted that the 4th combination where banana peel greater ratio in
comparison to orange and papaya peels (2:1) showed more favorable results than
rest of the combinations(Yousufi,2012) showed more SCP production when banana
peel was in combination with other substrates.

Effect of incubation period

Increase in protein production was initiated at early stages of incubation and reached
a peak of maximum production at 8th day. It was observed that SCP declined with
prolonged incubation which could be due to loss of nutrient and accumulation of
toxic compound in the medium. It was found that protein of pure substrates as
banana, papaya and orange showed maximum productions on 3 day i.e. 72 hours
(Yalemtesfa et al ,2010).
Table 5: Effect of incubation period on SCP (mg/ml) production using fruit peels

Incubation time Orange peel Banana peel Papaya peel

0.1 ml 0.2 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml

1st day 0.321 0.382 0.109 0.156 0.308 0.360
2th day 0.328 0.463 0.258 0.317 0.413 0.521
3rd day 0.620 0.984 0.613 0.766 0.630 0.812
4th day 0.384 0.528 0.358 0.378 0.457 0.584

The present investigation reveals similar results shown by some researcher’s as in

the case of orange peel (Yalemtesfa et al ,2010) and (Khan and Dahot,2010) showed
production of SCP on orange peel was 0.75 g/l and 0.44 mg/ml whereas in the
present investigation it was 0.62 mg/ml. Then in case of banana peel(Kandari and
Gupta ,2012) showed the biomass production in banana peel was 0.365 mg/ml and
current result was found to be 0.613 mg/ml of the sample. This investigation had
more protein content because of the type of strain used. In papaya peel protein
content was found to be 0.630 mg/ml, similarly,( Khan et al ,2010)revealed 0.595
mg/g of substrate. The change either increase or decrease in the concentration is
due to the change in substrates and conditions during the experiment.
It should be noted that the protein content on the 1-2 days is not much thus, the
fungi is in its lag phase and then there is a sudden growth of the organism leading to

4 International Journal of Bioinformatics and Biological Science: v.1 n.1 p.1-8. March, 2013
 Optimization of SCP production of Aspergillus niger using different fruit peels

growth in biomass on the 3rd day. And at the 4th day it shows the declining phase
where the biomass produced decreases due to low concentration of substrates
(Yalemtesfa et al ,2010) . It (Table 6) consists of SCP production on the mentioned
combinations (as given in Table 3) for different incubation period i.e. 2, 4, 6, 8 and
10 days. The following results are obtained below:
Table 6: Effect of incubation period on SCP (mg/ml) production using different
combinations of substrates

Incubation 1st combination 2nd combination 3rd combination 4th combination

time
0.1ml 0.2ml 0.1ml 0.2ml 0.1ml 0.2ml 0.1ml 0.2ml
2th day 0.128 0.197 0.125 0.207 0.143 0.248 0.161 0.198
4th day 0.162 0.274 0.142 0.180 0.118 0.190 0.158 0.286
6th day 0.429 0.889 0.655 0.981 0.541 0.936 0.434 0.732
8th day 0.714 1.342 0.764 1.126 0.595 1.130 0.841 1.352
10th day 0.639 1.122 0.547 0.910 0.496 0.940 0.618 1.126

It determined that the exponential fungal growth was observed from the 6th day
which showed maximum protein enrichment was found on 8th day and after that on
10th day the declining phase started due to low substrate concentration. The optimum
time for maximum protein production was determined to be on 8th day.

Effect of pH
On fermentation of the substrates and its combinations at different pH of 4, 6, 7 and
8, it was found that at 7 pH protein production was at its maximum(Yalemesfa et al
,2010) also reported the maximum production at pH 7. However, the best growth
for Aspergillus niger lies within the range of 5-7.
Table 7: Effect of pH on SCP (mg/ml) production using fruit peels

pH Orange peel Banana peel Papaya peel

0.1 ml 0.2 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml

4 0.344 0.690 0.580 0.720 0.390 0.773
6 0.533 0.814 0.605 0.686 0.544 0.815
7 0.590 0.906 0.610 0.733 0.740 0.970
8 0.465 0.730 0.330 0.450 0.520 0.740

It should be noted that, on optimizing the pure peels (substrate) at different pH

showed maximum production favored at pH 7 and then at pH 6 showing the second
best growth. It determined that the strain Aspergillus niger grows best within the
pH range of 5-7 (Yalemtesfa et al ,2010)

International Journal of Bioinformatics and Biological Science: v.1 n.1 p.1-8. March, 2013 5
 Bind et al.

Table 8: Effect of pH on SCP (mg/ml) production using different combinations of substrates

pH 1st combination 2nd combination 3rd combination 4th combination

0.1 ml 0.2 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml 0.1 ml 0.2 ml

4 0.380 0.632 0.443 0.889 0.392 0.785 0.436 0.741
6 0.393 0.636 0.456 0.896 0.504 0.890 0.537 0.943
7 0.713 0.974 0.718 1.120 0.595 0.982 0.841 1.126
8 0.455 0.890 0.644 0.981 0.541 0.936 0.645 1.001

During the experiment pH 4, 6, and 8 was taken into consideration with reference
to the neutral pH 7. Thus, maximum protein content was seen at pH 7 then at pH 8.
Thus, it clearly showed that Aspergillus niger strain best grows at basic pH and
mostly at neutral pH i.e. 7.
On fermentation of various combinations (Table 3) it again reveals that Aspergillus
niger grows best within the pH range of 5-7(Yalemtesfa et al ,2010) showed
maximum growth with the same strain at pH 7 and so in the present investigation
which also clearly depict the same showing maximum growth at pH 7 and then at
pH 8 and so on. Knowing that, supplementation of the substrate with nitrogen source
can easily be done for potential result in producing SCP.
Comparison of SCP (mg/ml) production between best incubation time and pH:
The 4th combination gives the maximum production of SCP due to its cellulose and
carbohydrate content which makes it acts as a good carbon source. Usually all the
combinations produced more SCP in comparison between incubation time and pH,
from the table below (Table 9) showed that combinations when used may prove to
be favorable for the fungal biomass production or SCP production as a whole.

Figure 1: SCP (mg/ml) production of various combinations of substrates

6 International Journal of Bioinformatics and Biological Science: v.1 n.1 p.1-8. March, 2013
 Optimization of SCP production of Aspergillus niger using different fruit peels

Figure 2: SCP (mg/ml) production of fruit peels

Table 9: Comparison of SCP (mg/ml) production between best incubation time and pH

Substrates used for Protein concentration Protein concentration

fermentation (mg/ml)at different (mg/ml) at different pH
incubation period

1st combination 1.342 (8th day) 0.974 (7)

2nd combination 1.126 (“) 1.120 (7)
3rd combination 1.130 (“) 0.982 (7)
4th combination 1.352 (“) 1.126 (7)
Orange peel 0.984 (3rd day) 0.906 (7)
Banana peel 0.766 (“) 0.733 (7)
Papaya peel 0.812 (“) 0.970 (7)

Aspergillus niger on 4th combination gave the best result of maximum production
of 1.352 mg/ml of protein on 8 days of incubation and at pH 7and maximum
fungal growth was observed which directly relates to protein content of A.niger .
Fruit peels which are considered as waste product that causes environment pollution.
So fruit peels can be used in different combinations to produce SCP in enhanced
nutritive value i.e. protein in different ratio as compared to individual fruit peels.
Combination show enhanced protein content in biomass due to available substrates
from different fruit peels. Fungal biomass utilizes different pathways to utilize
different substrate to the fullest which is established by different combinations.
The degree of mycelia biomass growth depends on the substrate utilized.
Combinations provide greater protein content as compared to pure substrates. Out
of the four combinations the best result was shown by the 4th combination which
contained orange, banana and papaya peels in 1:2:1 ratio and the amount of total
protein content in SCP of the 4th combination was 1.352mg/ml of the sample.

International Journal of Bioinformatics and Biological Science: v.1 n.1 p.1-8. March, 2013 7
 Bind et al.

Thus present investigation might be of great help in making use of the fruit waste
for sustainable management of fruit peels for economical and replenishment of
food (nutrition scarcity) for poor and populous country like India.

CONCLUSION
From the present finding we conclude that the biomass production using different
parameters (incubation time, and pH ) and fruit peels combinations showed better
results in comparison to fruit peels individually . Hence attempt for optimizing
different combinations of fruit peels for SCP production seems to be successful and
has a potential to be used for SCP for various applications and sustainable
development.

ACKNOWLEDGEMENT
We are thankful to Honorable Vice-chancellor of Sam Higginbottom Institute of
Agriculture, Technology & Sciences, Allahabad, for providing all necessary facilities
to conduct the experiment.

REFERENCES
Anupama, P. Ravindra 2001. Studies on production of single cell protein by Aspergillus niger
in solid state fermentation of rice bran. An International Journal: Brazilian Archives
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Kandari, V., Gupta, S. 2012. Bioconversion of vegetables and fruit peels waste in viable
products. Journal of Microbiology and Biotechnology Research. 2 (2012): 308-312.
Khan, M., Khan, S.S., Ahmed, Z., Tanvee, A. 2009. Production of fungal single cell protein
using Rhizopus oligosporus grown on various fruit waste. Biological Forum: An
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Khan, M.Y., Dahot, U. 2010. Effect of various agriculture waste and pure sugars on the
production of single cell protein by Penicillium expansum. World Applied Science
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Lowry, O.H. 1951. Modified Lowry protein assay. Journal of Biological Chemistry. 193:
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Najafpour, B.D. 2007. Single cell protein. Journal of Biochemical Engineering and
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8 International Journal of Bioinformatics and Biological Science: v.1 n.1 p.1-8. March, 2013

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