Waste Management 30 (2010) 22232227

Contents lists available at ScienceDirect

Waste Management
journal homepage: www.elsevier.com/locate/wasman

Value addition of vegetable wastes by solid-state fermentation using

Aspergillus niger for use in aquafeed industry
N. Rajesh a, Imelda-Joseph c,*, R. Paul Raj b
a

Central Institute of Fisheries Education (Deemed University), Versova, Mumbai 400 061, India
Coastal Aquaculture Authority, Government of India, Ministry of Agriculture, Shastri Bhawan Annexe, 26, Haddows Road, Chennai 600 006, Tamil Nadu, India
c
Central Marine Fisheries Research Institute, Ernakulam North P.O., Cochin 682018, India
b

a r t i c l e

i n f o

Article history:
Received 2 April 2009
Accepted 17 December 2009
Available online 25 January 2010

a b s t r a c t
Vegetable waste typically has high moisture content and high levels of protein, vitamins and minerals. Its
value as an agricultural feed can be enhanced through solid-state fermentation (SSF). Two experiments
were conducted to evaluate the nutritional status of the products derived by SSF of a mixture of dried
vegetable waste powder and oil cake mixture (soybean our, wheat our, groundnut oil cake and sesame
oil cake at 4:3:2:1 ratio) using fungi Aspergillus niger S14, a mangrove isolate, and A. niger NCIM 616. Fermentation was carried out for 9 days at 35% moisture level and neutral pH. Signicant (p < 0.05) increase
in crude protein and amino acids were obtained in both the trials. The crude fat and crude bre content
showed signicant reduction at the end of fermentation. Nitrogen free extract (NFE) showed a gradual
decrease during the fermentation process. The results of the study suggest that the fermented product
obtained on days 6 and 9 in case of A. niger S14 and A. niger NCIM 616 respectively contained the highest
levels of crude protein.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
India is the second largest producer of vegetables in the world
and the current production level is over 71 million MT (TIFAC,
2004). According to India Agricultural Research Data Book 2004,
the losses in fruits and vegetables are to the tune of 30%. Taking
estimated production of fruits and vegetables in India at 150 million tones, the total waste generated comes to 50 million tones
per annum. This residual matter can be converted into value added
products either as raw materials for secondary processes, as operating supplies or as ingredients of new products by using an appropriate technology (Laufenberg et al., 2003). In many tropical
regions of Asia and Africa, wastes from crops are used in aquaculture as feed ingredients, as supplementary feeds or as pond fertilizers (Ravishankar and Keshavanath, 1986; Wohlfarth and Hulata,
1987; Subosa, 1992; Tacon, 1993, 1994). Most of the agro-industrial by-products and food industry wastes are poor in nutrients
such as proteins and vitamins and are rich in bres with low
digestibility and are not suitable for non-ruminant animals. In such
situations, a potential solution is available by the utilization of
microorganisms, mainly fungi, to convert agro-industrial wastes
to obtain products with higher nutritive value, especially in regard
to protein and vitamin contents, and with increased digestibility
* Corresponding author. Tel.: +91 484 2394867; fax: +91 484 2394909.
E-mail address: imeldajoseph@rediffmail.com (Imelda-Joseph).
0956-053X/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.wasman.2009.12.017

(Kuhad et al., 1997). Utilization of nonconventional resources for

animal feed has been made possible by the process of solid-state
fermentation (SSF) which involves fermentation in limited moisture levels (Pandey and Radhakrishnan, 1992; Pandey et al.,
2000, 2001). By SSF, the substrates get upgraded in protein (3.5
times increase in fungal protein content), fats, soluble sugars, vitamins and amino acids and thus can be used in animal feeds (Singh
et al., 1990; Puniya and Singh, 1995). Vegetable and fruit processing wastes contain mainly starch, cellulose, soluble sugars and organic acids which are utilized by some microorganisms, producing
biomass with high protein content (Stabnikova et al., 2005). The
hypothesis of the present study was that vegetable waste containing many complex polymers and/or anti-nutritional factors get
modied by the process of SSF using Aspergillus niger. The present
evaluation of the nutritional status of fermented vegetable waste
using two strains of A. niger will be useful in the future application
of the technology for value addition of agricultural by-products
and wastes for its use in animal and aquafeed industry.
2. Materials and methods
2.1. Microorganism
A. niger strain S14 isolated from the soil sample of Mangalavanam a mangrove swamp at Cochin (10030 N latitude and 76140
E longitude) and A. niger NCIM 616 obtained from National

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N. Rajesh et al. / Waste Management 30 (2010) 22232227

Collection of Industrial Microorganisms (NCIM), Pune, India were

used for the experimental studies. The cultures were tested not
to produce mycotoxins, which are harmful for cultured organisms.
The cultures were maintained in potato dextrose agar (PDA)
(Himedia, Mumbai) at 30 1 C with monthly sub culturing.
2.2. Substrate
Vegetable waste procured from Cochin vegetable market (Kerala, India) was used as a component of the substrate for solid-state
fermentation (SSF). The vegetable discards (cabbage, cauliower,
banana peel, banana, potato, carrot, beet root, ladies nger, peas,
beans, capsicum etc.) were chopped into about 5 cm size and sun
dried for 3 days and ground into ne powder less than 400 mm.
For SSF, the substrate was a mixture of vegetable waste powder
and oil cake mixture in the ratio of 4:1 respectively (mixed oil cake
was a combination of soybean, wheat our, groundnut oil cake and
sesame oil cake in the ratio of 4:3:2:1 respectively). The proximate
composition of the dried vegetable waste powder and mixed oil
cake were analyzed according to AOAC (1990).
2.3. Solid-state fermentation
Solid-state fermentation was carried out in 0.5 l conical asks
containing 50 g substrate with the moisture content adjusted to
35% (autoclaved at 121 C at 15 psi pressure for 1 h) for a period
of 10 days. An inoculum size of 2  107 spores 50 g1 substrate
was used for each ask and incubated at 30 2 C. Sampling was
done at every 24 h interval starting from days 0 to 9 for both the
experiments.
2.4. Product analysis
The products obtained after fermentation were dried to a constant moisture level in a hot air oven at 80 1 C and proximate
composition analyses were carried out (AOAC, 1990). Estimation
of crude protein was done using Kjeltec 2300 Analyzer Unit and
crude fat using Soxtec System 2043 Extraction Unit of Foss Tecator, Sweden. Fat free samples were used for the estimation of crude
bre using Fibertec 2010 System (Foss Tecator, Sweden). About
3 g of the sample was weighed in silica crucibles and was then ignited at 600 5 C (Mufe furnace, Barnstead, USA) for 3 h for
crude ash determination. The ash was digested with concentrated
hydrochloric acid and ltered to get residue that was again ignited
at 600 C for 3 h, cooled and weighed to determine acid insoluble
ash content.
2.5. Amino acid analysis
Amino acid analysis was carried out using acid hydrolysed (6 N
HCl) samples by reverse-phase high-performance liquid chromatography (HPLC) after pre-column derivatization by phenyl isothiocyanate (PITC) by a modied method adapted from Fierabracci et
al. (1991). HPLC was performed using Waters 1525 Binary HPLC
pump and Waters 2487 Dual Absorbance Detector. Data were processed and analyzed using Waters Breeze software. Operating conditions were: column temperature 38 C, column, pico-tag (waters,
pico-tag system); absorbance, 254 lm; pump pressure, 1500
1700 psi.
Tryptophan content in the samples was determined by spectrophotometry after alkaline hydrolysis (5% NaOH). Absorbance value
was measured at 500 nm (Genesis UV 10 Spectrophotometer) and
the concentration was calculated from standard values and expressed as g 100 g1 protein.

2.6. Statistical analysis

The proximate composition of the samples was statistically
analyzed using one-way ANOVA. Post hoc tests like Duncan multiple range test (DMRT) and least signicant differences (LSD) were
conducted to nd out whether variations existed between the
treatments. The glucosamine content was analyzed using oneway ANOVA. All statistical analyses were conducted using SPSS
for windows (Statistical Package for Social Science, Windows version, Chicago, IL, USA) and Microsoft Excel.
3. Results and discussion
Nutritional enrichment of feedstuffs and low-grade agricultural
wastes using fungal fermentation is a valuable biotechnological
approach for sustainable development with regional and national
impact (Singh et al., 1996). The present study has demonstrated
that the solid-state fungal fermentation enhanced the nutrient
composition of vegetable waste and oil cake mix. Fungi have the
ability to produce a variety of enzymes and their ability to degrade
cellulose has been reported (Iyayi and Losel, 2001). These enzymes
help to degrade the non-starch polysaccharides in the substrate to
soluble sugar (Hamlyn, 1998). The proximate composition of the
ingredients used for fermentation is given in Table 1 and that of
the fermented ingredient mix after SSF using A. niger S14 and A. niger NCIM 616 at different time intervals are given in Tables 2 and 3.
During fermentation, the crude protein content showed signicant
(p < 0.05) variations between days as well as between strains.
Using A. niger S14 a maximum increment of about 38% crude protein was recorded on day 8 compared to the initial. It has been reported that A. niger used in SSF produced at least 19 types of
enzymes, which are proteins, and of which amylase is produced
by as many as 28 strains (Pandey, 1995). A. niger was also found
to be the best cellobiase producer when cultivated in a moist
wheat bran and ground corn solid medium supplemented with
inorganic minerals (Tsao et al., 2000). In the present study, both
the strains improved the crude protein content with increasing
duration of post-inoculation, which is in agreement with previous
studies (Raimbault et al., 1985; Yang, 1988; Yang et al., 1993). The
double fold increase in protein content by A. niger 616 may be
attributed to the fact that it is an amylase producing industrial
strain, which has converted the carbohydrate fraction of the ingredient mix to fungal protein at a faster pace than the mangrove isolate. Since no external nitrogen was supplied in this study, there
could only be a conversion of some of the vegetable protein or
other nitrogenous compounds into fungal protein. Using Candida
sp. threefold increase in crude protein of apple pomace powder
on SSF for 4 days was reported by Joshi and Sandhu (1996). SSF
of biomass has been attempted as a means of elevating the total
protein content (Reade and Gregory, 1975; Rodriguez et al.,
1985). Singh et al. (1990) reported increase in crude protein and
decrease in energy content of the fermented potato process waste.
Ofuya and Nwanjiuba (1990) have demonstrated an increase of
185% in protein (5.616%) in cassava meal with the optimum duration of four days for break down of non-starch polysaccharides in

Table 1
Proximate composition of fermentation substrate (in %).
Parameters

Dried vegetable powder

Oil cake mix

Moisture
Crude protein
Crude fat
Crude bre
Crude ash
Nitrogen free extract

4.04 0.21
18.26 1.17
2.40 0.01
19.97 1.60
9.18 0.37
50.19 0.93

6.79 0.12
36.18 2.88
2.75 0.04
2.53 0.23
8.90 0.10
49.64 3.08

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N. Rajesh et al. / Waste Management 30 (2010) 22232227

Table 2
Proximate composition (in %) of unfermented mix and fermented products using A. niger S14.
Days
0
1
2
3
4
5
6
7
8
9

Moisture

Crude protein
a

Crude fat

5.17 0.36
5.31 0.28a
5.60 1.53a
5.67 0.53a
5.85 0.20b
7.03 2.63c
9.13 1.53d
7.04 0.93c
7.81 1.19e
7.26 0.86c

Crude bre
a

20.62 0.52
20.98 0.19a
21.57 0.33b
23.23 0.89c
24.01 0.73d
25.32 0.69e
27.51 0.56f
28.15 1.26g
28.38 1.43g
28.29 0.93g

Crude ash
a

1.89 0.03
1.98 0.05a
2.08 0.04a
2.35 0.37b
1.94 0.61a
1.64 0.02c
1.48 0.05c
1.49 0.17c
1.47 0.05c
1.21 0.04d

9.82 0.29
11.84 2.24b
12.52 0.34b
11.82 1.40b
10.07 0.85a
9.76 1.40a
10.56 1.24c
8.26 0.29d
8.25 0.15d
8.89 1.16d

NFE
a

53.28 0.45a
51.25 2.49b
48.79 0.45c
47.02 2.67d
47.26 2.40d
47.03 1.06d
40.70 1.53e
40.21 1.17f
39.96 2.65g
39.64 1.12g

11.49 0.07
11.51 0.12a
12.14 0.06a
12.76 0.32b
13.78 0.39c
14.55 0.49d
15.67 0.17e
15.62 0.49e
16.32 0.77e
16.63 0.77e

NFE nitrogen free extract; all values are average of triplicates SE; means within the same columns with different superscript letters are signicantly different (p < 0.05).

Table 3
Proximate composition of unfermented mix and fermented products using A. niger NCIM 616 (expressed in DM %).
Days

Moisture

Crude protein

Crude fat

Crude bre

Crude ash

NFE

0
1
2
3
4
5
6
7
8
9

1.61 2.25a
1.17 1.60a
2.26 2.33b
1.05 0.99a
1.91 0.21a
1.85 0.03a
1.31 0.89a
3.07 1.70c
2.12 0.61d
2.19 0.16e

21.81 0.42a
22.28 0.34a
22.64 0.13a
20.21 1.99a
23.29 0.21b
22.16 1.34a
24.26 1.95c
28.38 1.72d
26.04 1.10e
28.15 1.76f

1.89 0.14a
1.95 0.06a
1.95 0.06a
2.01 0.08a
2.07 0.02a
1.96 0.20a
2.52 0.30b
2.57 0.50c
2.21 0.17a
2.18 0.33a

12.57 0.11a
13.79 0.21a
13.56 0.26a
13.64 0.27a
14.20 0.05b
13.64 1.59a
15.89 0.37c
13.88 1.06a
14.70 1.03d
14.26 1.43e

9.83 0.09a
9.86 0.04a
9.92 0.17a
9.88 0.14a
9.90 0.24a
10.42 0.57a
11.47 0.43b
12.44 0.92c
12.34 0.07d
12.73 0.85e

51.29 2.57a
50.94 1.26a
49.67 2.22b
49.22 1.77b
48.49 0.48b
46.15 3.18c
44.54 2.12d
43.65 2.05e
42.57 2.75f
40.10 0.91g

NFE nitrogen free extract; all values are average of triplicates SE; means within the same columns with different superscript letters are signicantly different (p < 0.05).

course of fermentation for 9 days. Loss of lipid in palm kernel meal

during SSF due to its conversion into fungal protein biomass was
reported by Wing Keong et al. (2002). For fermented sweet potato
an increase in lipid concentration of 64% with A. niger was reported
by Abu et al. (2000). Fungi such as Mortierella isabellina, Mucor
cincinelloides and Penicillium spinulosm are capable of producing
65% of their dry weight as lipid (Weete and Ghandi, 1992).
Crude bre content showed a gradual increase with duration of
fermentation with A. niger S14. The initial bre content was
13.57 0.11% which had reached the maximum value of
15.89 0.37% on day 6. In the second trial of fermentation using
A. niger NCIM 616, the initial bre content was 9.82 0.29%, which
got reduced as fermentation proceeded. The increase observed

brewers dried grain, wheat offal and maize offal using A. niger, A.
avus and Penicillium sp.
In the present study, the crude fat content in the fermented
product gradually increased with the incubation period for A. niger
S14. The initial fat content in the substrate (1.89 0.14) had increased to the maximum of 2.57 0.50% on day 7, which was of
about 36% hike. After day 7, the values showed a decreasing trend
(Table 3). The initial fat content was 1.89 0.03% using A. niger
NCIM 616, which showed an increasing trend during the rst
4 days and reached the maximum on day 4 (2.35 0.36%), that
was 24% higher than the initial. Further reduction in crude fat during the course of fermentation is explained as assimilation of lipids
from the substrate possibly for biomass production during the

Table 4
Amino acid prole of the unfermented mix and fermented products using Aspergillus niger S14 (expressed in g 100 g1 protein).
Amino acid

Day 0

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Aspartic acid
Glutamic acid
Serine
Glycine
Histidine
Arginine
Threonine
Alanine
Proline
Tyrosine
Valine
Methionine
Cystine
Isoleucine
Leucine
Phenyl
alanine
Lysine
Tryptophan

9.99 0.12
22.09 0.15
5.84 0.10
4.08 0.12
2.93 0.15
6.91 0.14
4.75 0.17
4.30 0.14
4.54 0.11
3.82 0.15
4.72 0.14
2.34 0.12
0.93 0.12
3.95 0.14
6.56 0.15
6.56 0.16

11.32 0.10
22.11 0.11
6.27 0.12
4.38 0.13
2.85 0.11
5.93 0.13
4.43 0.15
4.40 0.17
4.48 0.12
3.72 0.13
4.55 0.14
2.24 0.23
0.86 0.18
3.47 0.15
7.12 0.14
6.01 0.13

11.49 0.22
21.86 0.21
6.40 0.12
4.46 0.18
2.67 0.17
6.28 0.14
4.41 0.10
4.68 0.18
4.54 0.21
3.64 0.13
4.50 0.15
2.20 0.16
0.75 0.14
3.42 0.17
7.08 0.18
5.94 0.21

11.40 0.11
21.55 0.11
6.41 0.14
4.32 0.17
2.51 0.10
6.90 0.21
4.65 0.14
4.85 0.17
4.48 0.16
3.51 0.12
4.47 0.11
2.19 0.17
0.71 0.13
3.42 0.14
7.18 0.15
5.86 0.14

11.02 0.18
21.45 0.17
6.49 0.15
4.72 0.17
2.48 0.15
6.72 0.15
4.31 0.11
4.96 0.14
4.81 0.12
3.57 0.13
4.65 0.14
1.96 0.16
0.65 0.19
3.53 0.12
7.27 0.10
5.80 0.11

11.93 0.11
21.01 0.18
6.72 0.17
4.79 0.10
2.20 0.11
6.72 0.24
4.13 0.12
5.02 0.17
4.67 0.14
3.49 0.17
4.77 0.14
1.76 0.14
0.64 0.10
3.65 0.11
7.22 0.21
5.76 0.24

11.54 0.14
20.75 0.21
6.90 0.22
4.62 0.21
2.50 0.10
6.77 0.10
5.20 0.17
5.51 0.18
4.19 0.15
3.28 0.12
4.78 0.15
1.80 0.19
0.49 0.09
3.81 0.12
7.36 0.11
5.01 0.24

11.59 0.12
19.64 0.13
6.74 0.14
5.10 0.21
2.45 0.10
6.82 0.17
5.22 0.13
5.34 0.17
4.25 0.19
3.21 0.14
5.85 0.19
1.58 0.16
0.41 0.18
4.52 0.18
7.30 0.11
4.85 0.10

11.60 0.18
19.80 0.12
6.13 0.15
4.79 0.11
2.40 0.17
6.69 0.21
5.47 0.16
5.64 0.18
5.01 0.21
3.20 0.18
5.42 0.17
1.56 0.15
0.29 0.17
4.75 0.10
7.31 0.12
4.74 0.15

11.24 0.13
19.76 0.14
7.51 0.10
2.70 0.19
2.94 0.17
7.68 0.18
5.65 0.21
6.00 0.24
4.37 0.10
3.18 0.12
5.19 0.11
1.04 0.14
0.26 0.16
4.31 0.15
7.71 0.10
4.75 0.12

3.41 0.11
2.30 0.10

3.38 0.17
2.49 0.14

3.34 0.23
2.33 0.24

3.34 0.14
2.25 0.17

3.31 0.19
2.30 0.17

3.30 0.19
2.22 0.21

3.29 0.13
2.21 0.15

3.28 0.14
1.85 0.17

3.28 0.18
1.92 0.19

3.42 0.11
2.31 0.21

All values are average of triplicates SE.

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N. Rajesh et al. / Waste Management 30 (2010) 22232227

Table 5
Amino acid proles of the unfermented mix and fermented products using Aspergillus niger NCIM 616 (expressed in %).
Amino acid

Day 0

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Aspartic acid
Glutamic acid
Serine
Glycine
Histidine
Arginine
Threonine
Alanine
Proline
Tyrosine
Valine
Methionine
Cystine
Isoleucine
Leucine
Phenyl alanine
Lysine
Tryptophan

11.69 0.10
20.60 0.13
6.13 0.12
5.33 0.13
1.98 0.12
7.02 0.21
4.76 0.14
5.55 0.19
5.36 0.05
3.01 0.14
6.03 0.14
1.98 0.15
0.74 0.17
4.57 0.15
7.71 0.19
4.02 0.16
2.56 0.09
0.94 0.25

11.79 0.16
19.14 0.05
5.70 0.17
4.53 0.19
2.51 0.18
6.23 0.13
4.59 0.15
4.86 0.10
4.42 0.12
3.86 0.14
6.50 0.13
3.05 0.14
0.89 0.17
4.99 0.19
8.05 0.16
4.15 0.14
2.82 0.17
1.92 0.14

11.53 0.12
19.5 5 0.21
5.94 0.15
4.97 0.19
2.07 0.17
5.71 0.10
4.63 0.25
5.05 0.18
4.28 0.10
3.37 0.12
6.80 0.14
2.85 0.11
0.51 0.17
4.78 0.21
8.32 0.15
4.56 0.15
3.46 0.17
1.62 0.19

12.76 0.20
18.87 0.21
6.24 0.17
4.96 0.10
2.29 0.14
5.59 0.16
4.93 0.13
5.18 0.21
4.14 0.24
3.10 0.17
6.55 0.21
2.38 0.14
0.71 0.14
4.72 0.18
7.92 0.14
4.82 0.16
3.38 0.17
1.44 0.15

11.640.21
16.36 0.14
6.01 0.17
5.00 0.15
2.81 0.14
7.38 0.17
5.01 0.21
5.35 0.17
4.84 0.24
3.50 0.10
6.25 0.11
2.44 0.21
0.85 0.18
5.13 0.17
7.41 0.19
5.12 0.17
3.50 0.10
1.39 0.24

12.20 0.21
17.83 0.14
6.18 0.17
5.24 0.18
2.24 0.19
6.56 0.14
4.84 0.13
5.43 0.14
4.71 0.14
2.98 0.16
6.08 0.17
2.50 0.17
0.35 0.15
4.80 0.15
7.56 0.16
5.22 0.17
3.85 0.15
1.42 0.18

10.96 0.12
15.01 0.19
5.90 0.17
5.05 0.18
2.82 0.12
6.66 0.14
4.94 0.18
5.30 0.15
4.93 0.14
3.90 0.16
6.87 0.17
2.34 0.15
0.66 0.14
5.27 0.16
7.85 0.15
5.40 0.18
4.55 0.24
1.58 0.22

11.65 0.14
15.47 0.21
6.52 0.14
4.64 0.18
2.69 0.17
6.53 0.10
5.04 0.11
5.05 0.21
3.53 0.21
3.69 0.15
7.03 0.16
2.62 0.18
0.95 0.17
5.28 0.15
8.01 0.17
5.03 0.14
4.96 0.16
1.32 0.17

12.32 0.18
16.35 0.12
6.16 0.11
4.22 0.14
2.06 0.13
7.42 0.15
4.71 0.17
4.93 0.15
3.17 0.17
3.56 0.11
6.45 0.17
2.32 0.15
0.65 0.14
5.25 0.13
7.99 0.15
5.12 0.13
5.50 0.17
1.83 0.25

13.57 0.15
17.41 0.13
6.35 0.16
4.28 0.17
1.59 0.15
6.88 0.11
4.60 0.12
5.01 0.17
2.98 0.18
3.54 0.17
6.52 0.12
2.26 0.14
0.15 0.17
4.96 0.16
8.11 0.17
4.64 0.19
5.30 0.21
1.84 0.27

All values are average of triplicates SE.

during initial phase of fermentation was due to the utilization of

available nutrients by the fungi and later reduction may be attributed to the break down of non-starch polysaccharide by the fungi
to fungal protein. The initial ash content was 9.83 0.09% on using
A. niger S14. This continued to increase and reached a maximum on
the day 9 (12.73 0.85%) with an increase of 29% (Table 2) from
initial. By A. niger NCIM 616, initial ash content was
11.49 0.07% and throughout the fermentation period it also
showed an increasing trend. The increase in ash content in fermented product during the course of fermentation can be explained by the loss in organic matter obtained through the
process of SSF. The percentage decrease in NFE from the initial value was 28% using A. niger S14, during the course of fermentation
and 22% using A. niger NCIM 616. Loss in NFE was observed in
the fermented products in both the trials. Loss of NFE in palm kernel meal during SSF due to its conversion into fungal protein biomass was reported by Wing Keong et al. (2002). In the present
investigation since A. niger is more rapidly amylolytic, a great
amount of carbohydrate might have been broken down during
the course of fermentation.
The changes in the amino acids composition in the fermented
products using A. niger S14 are given in Table 4. Essential amino
acids, which showed an increase in concentration, were valine
(23%), threonine (22%), isoleucine (20%), leucine (17%) and arginine
(11%). Those essential amino acids, which showed a reduction in
concentration, were methionine (55%), histidine (26%), phenylalanine (38%), lysine (4%) and tryptophan (27%). The non-essential
amino acids, which showed increase on fermentation, include alanine (39%), serine (28%), glycine (25%), aspartic acid (19%) and proline (10%).
The changes in levels of amino acids in fermented product using
A. niger NCIM 616 are given in Table 5. The fermented product contained 18 amino acids, including the 10 essential ones. Except
methionine, the level of essential amino acids increased as fermentation proceeded, methionine showed 51% reduction from the initial value. Other amino acids which showed increase during SSF
includes tryptophan (178%), lysine (139%), histidine (42%), phenylalanine (41%), valine (16%), isoleucine (14%), threonine (13%), leucine (8.3%) and arginine (5.6%). Most of the non-essential amino
acids got reduced except aspartic acid (18.4%), serine (20%) and
cysteine (28%) which showed increase on fermentation.
The fermentation process caused signicant increase in the concentration of essential amino acids, except methionine and phenylalanine in the case of A. niger S14 and arginine and methionine for

A. niger 616, when the results were expressed per 100 g feed (Tables 4 and 5). Methionine has been generally reported to be the
most limiting essential amino acid of the unicellular proteins (Aguire et al., 1976; Raimbault and Alazard, 1980). The level of amino
acids in cellobiases, the enzymes produced by certain strains of
A. niger showed high contents of aspartic acid, glutamic acid, threonine, serine, and glycine (Abdel-Naby et al., 1999). Among nonessential amino acids aspartic acid, serine and alanine got increased in the fermented product using A. niger S14 and aspartic
acid, serine, glycine and tyrosine got increased while using A. niger
616. The reduction in other non-essential amino acids may be due
to the utilization of these amino acids for the production of enzymes and other organic compounds by the fungal strain (Imelda-Joseph et al., 2008).
In both the trials, the dry matter loss was about 4% in the present study. Vijayakumar (2003) reported a 20% dry matter loss after
96 h of fermentation of mixed oil cakes with A. niger 616. In a similar study with wheat bran, Bhatnagar (2004) has also reported signicant (p < 0.05) reduction in dry matter throughout the
fermentation period for wheat bran using A. niger S14, suggesting
utilization of nutrients present in the substrate by fungi for its
growth and metabolic activities.

4. Conclusion
From the present study, it is evident that harvesting of the fermented product on day 6 and 10 in case of A. niger S14 and A. niger
616 respectively contained the highest levels of crude protein. At
these peak levels, the fermented product also contained a high level of bre and modest levels of NFE and ash. All of these changes
enhance the value of the vegetable waste as an animal feed, including aquafeed. The agricultural benet of these changes to the composition of the vegetable waste needs to be tested in animal feed.

Acknowledgements
The authors acknowledge the facilities provided by the Director,
Central Marine Fisheries Research Institute (CMFRI), Kochi, Kerala,
India for carrying out the work. The nancial support offered for
the rst author by Central Institute of Fisheries Education (CIFE),
Mumbai, India is gratefully acknowledged. We thank Mrs. G. Shylaja, Technical Ofcer, CMFRI for amino acid analysis.

N. Rajesh et al. / Waste Management 30 (2010) 22232227

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