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ELSEV I ER Resources, Conservation and Recycling 12 (1994) 195-200 a n d r e _____~,mmlltna
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Bioconversion of sugarcane bagasse into a

protein-rich product by white rot fungus
Samir A. E1-Sayed a'*, Mohamed T. Zaki b, Amal W. Abou E1-Khaira

"Agricultural Microbiology Research Department, Soils & Water Research Institute,

Agricultural Research Center, Giza, Egypt
hAnalytical chemistry Department, Faculty of Science, Ain-Shams University, Cairo, Egypt
Received 17 December 1992; revision received 3 January 1994; accepted 25 January 1994

Abstract

The fungus Pleurotus ostreatus NRRL-2366 degraded 56.7% and 45.9% of untreated and chemi-
cally pretreated (delignified) sugarcane bagasse, respectively, during 14-day incubation in a sub-
merged fermentation process. The biodegradation percentages of cellulose, hemicellulose and lignin
were 33.0%, 72.5% and 14.5%, respectively. An increment of 22.6% of crude protein content in the
residual fermented material was observed. Chemical composition of the end-product and its amino
acids profile were reported.

Keywords: Sugarcane; White rot fungus; Pleurotus ostreatus

1. Introduction

More than 2 million metric tonnes of lignocellulosic sugarcane residues are produced
every year in Egypt [ 1 ]. Much of these residues are disposed by burning, but such practice
has been subject to criticism because of the resulting air pollution and respiratory problems.
Sugarcane bagasse may be used as animal feed; however, its poor digestibility, low
protein content and high lignin concentration [2] limit its value as a fodder. Various
chemical and biochemical methods may be used to enhance the digestibility and improve
the nutritional qualities of sugarcane bagasse, however, such approaches can be expensive.
Hence, several workers [3-9] have attempted upgrading the nutritional quality of plant
residues. Here we describe a new submerged fermentation process based on employing the
fungus Pleurotus ostreatus, to modify the sugarcane bagasse to a protein-rich animal feed.

* Corresponding author.

0921-3449/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved

SSD10921-3449(94)00004-0
196 S.A. EI-Sayed et al. /Resources, Conservation and Recycling 12 (1994) 195-200

2. Materials and methods

The white rot fungus Pleurotus ostreatus NRRL-2366, was maintained on potato dextrose
agar (PDA) slants supplemented with 0.3% yeast extract.
The inoculum was prepared by using 100 mL of sterile nutrient glucose broth in 250 mL
Erlenmeyer flasks and incubated at 28°C on GFL-rotary shaker ( 150 rpm) for six days.
The fungal pellets were harvested under aseptic conditions and resuspended in 30 mL of
sterile distilled water. This suspension was used as inoculum.
Direct bioconversion of sugarcane bagasse was carried out in 250-mL conical flasks in
which 0.5 g ground (50 mesh) pretreated (chemically delignified using sodium chlorite,
according to Sidhu and Sandhu [ 10] ) or untreated substrate was placed along with 50 mL
of medium, containing (per litre distilled water): 2.0 g KHzPO4, 2.1 g (NH4)2SO4, 0.3 g
MgSOn.7H20, 0.5 g CaCIz, 0.5 g yeast extract, 0.005 g FeSO4.7H20, 0.00156 g
MnSOn.H20, 0.0014 g ZnSO4.7H20, 0.00266 g CaCI2, 6H20, adjusted to pH 5.0 [11].
The flasks were sterilized at 121°C for 20 min, cooled, and inoculated each with 3 mL of
the fungal pellets suspension. The flasks were incubated at 28°C on a GFL-rotary shaker
(150 rpm) for 14 days. The fungal growth and the residual material were harvested by
filtration through preweighed filter paper. The fermented residue and the fungal biomass
were washed repeatedly with distilled water and dried at 70°C to a constant weight.
The nitrogen content of the end-product and fungal mycelium was determined by the
Kjeldahl method and crude protein concentration was calculated (N × 6.25).
Analysis of the original substrate, as well as the fermented product for dry matter, total
ash, minerals and fat contents were carried out following the methods recommended by the
Association of Official Agriculture Chemists [ 12]. Lignin, and cellulose and hemicellulose
contents were estimated by the methods of Tanaka et al. [ 13] and Chahal et al. [ 14],
respectively. Calcium was determined according to Richards [ 15]. Amino acids of the
crude fungal protein were detected using Varian amino acid analyzer according to the
method of Bosi and Battaglini [ 16].

3. Results and discussion

Table 1 shows the bioconversion of sodium chlorite-pretreated and untreated sugarcane

bagasse into a protein-rich product by the white rot fungus Pleurotus ostreatus NRRL-
3266. The results revealed that the fungal agent successfully degraded both the chemically
pretreated and untreated bagasse. However, a maximum of 56.7% of the untreated waste
material was degraded compared to 45.9% of the sodium chlorite-pretreated substrate. These
results indicates that Pleurotus ostreatus is potentially useful for direct cultivation on
sugarcane bagasse without any chemical pretreatment as it produces cellulase enzymes
capable of breaking down crystalline cellulose [ 17,18] and it also readily decomposes
lignin [ 19]. For the untreated and pretreated bagasse, the crude protein contents of the
residual matter were 22.6% and 18.0% with a conversion efficiency of 26.4% and 23.6%,
respectively. These results are consistent with those obtained by Hatakka and Pirhonen
[20], who reported that high protein concentration (29.2%) was obtained with the white
rot fungus Fomesfomentarius when cultivated on granary wastes in a submerged culture.
 S.A. EI-Sayed et al. / Resources, Conservation and Recycling 12 (1994) 195-200 197

Table 1 Bioconversion of sodium chlorite pretreated and untreated sugarcane bagasse to protein-rich product by
Pleurotus ostreatus in a shake liquid culture

Incubation Total Protein Total Protein Net Net UndegradedDegraded Bioconversion

period biomass in protein from gained mycelium substrateb substratec efficiency~
(days) recovery residue obtained starter protein obtaineda (g) (g) (%)
(dry weight) (%) from culture (g) (g)
(g) residue and
(g) plant
residue
(g)

Untreated substrate
0 100.0 4.5 4.5 4.5 - - 100.0
7 91.0 5.5 5.0 4.5 0.5 1.5 89.5 10.5 5.0
14 86.0 22.6 19.4 4.5 14.9 42.7 43.3 56.7 26.4

Chemically treated substrate

0 100.0 4.5 4.5 4.5 - 100.0 - -
7 92.0 5.4 5.0 4.5 0.5 1.4 90.0 9.4 5.1
14 85.0 18.0 15.3 4.5 10.8 30.9 54.1 45.9 23.6
Calculation of net mycelia obtained based on mold content of 35% protein.
h Undegraded substrate= total residual substrate-net mycelia obtained.
c Degraded substrate = 100 - undegraded substrate.
a Bioconversionefficiency net gained protein × 100.
degraded substrate

Data showed that the crude protein content of the residual stuff and bagasse utilization
had a positive correlation with incubation period. Thus, the amounts of protein obtained on
the 7th day of incubation were reported to be 5.0 g with 10.5% and 9.4% degradation extent
of the untreated and chemically pretreated bagasse, respectively. On the other hand, the
protein increased to 19.4 g and 15.3 g when the substrate degradation rate reached 56.7%
and 45.9%, on the 14th day, respectively. A decrease of 21.3% in the yield of the total
protein gained within the residue of the originally pretreated substrate was detected after 14
days of incubation. Such a decrease was probably due to removal of water-soluble carbo-
hydrates, which were present in high proportion in the untreated sugarcane bagasse [21 ].
Moreover, the chlorite treatment produces methylated sugars, which probably imparts some
resistance to microbial degradation [ 22].
Chemical composition of the protein-rich fermentation product ( P R F P ) , presented in
Table 2, showed that the crude protein content of PRFP (22.6%) was much higher than
that of the original raw bagasse (2.7%). The cellulose and hemicellulose contents of the
resulting PRFP were less than those of the original substrate, which means that the weight
loss was mainly due to cellulose and hemicellulose consumption. Therefore, the biodegra-
dation percentages of cellulose and hemicellulose were 33.0% and 72.5%, respectively.
Similar results were obtained by Surinder and Suman [ 23 ], who found that 34.5% of paddy
straw were degraded within 24 days by P l e u r o t u s ostreatus.
A decrease of 14.5% in lignin content of the resulting material was recorded. Data listed
in Table 2 also reveal that the end-product contained more ash (5.7%) than the original
substrate (3.2%), which was derived from the mineral constituents in the medium and
198 S.A. EI-Sayed et al. / Resources, Conservation and Recycling 12 (1994) 195-200

Table 2 Chemical composition of the fermentation product of the chemically untreated sugarcane bagasse

Constituent content PRFP Initial

composition composition of
sugarcane
bagasse

Input sugarcane bagasse (g) 100.0 100.0

Output PRFP (g) 86.0
Crude protein (%) 22.6 2.7
Cellulose (%) 23.6 35.2
Hemicellulose ( % ) 6.5 23.6
Lignin (%) 18.2 21.3
Fat (%) 3.2 3.7
Ash (%) 5.7 3.2
Minerals (mg/100 g)
K÷ 2680.0 367.0
Na ÷ 85.0 46.0
Mg + + 245.0 50.0
Mn ÷ + 1.0 1.3
Cu ÷ ÷ 8.2 7.3
Fe + + 35.0 19.3
Ca ÷ ÷ 600.0 200.0
Zn + + 1.2 2.9
P 22.10 52.0
Cd - 0.4
Pb - 0.9

PRFP, protein-rich fermentation product.

might h a v e been incorporated into the microbial cells. Elemental analysis o f the ash denotes
the presence o f high levels o f potassium, calcium, magnesium, s o d i u m and phosphorus.
Results in T a b l e 3 s h o w the content o f a m i n o acids m a k i n g up the protein o f P l e u r o t u s
ostreatus m y c e l i a l pellets. H i g h levels o f valine + alanine, threonine + serine, aspartic acid
and tryptophane were observed. Other a m i n o acids were found in the f o l l o w i n g decreasing

Table 3 Amino acids content of Pleurotus ostreatus mycelial pellets protein

Amino acids g/100 g protein

Glycine 4.7
Valine + Alanine 17.4
lsoleucine + Leucine 7.1
Threonine + Serine 17.0
Aspartic acid 14.9
Methionine + Cystine 4.8
Histidine 3.1
Tyrosine 2.7
Glutamic acid 1.6
Phenylalanine, 3.9
Lysine 2.6
Arginine 7.6
Tryptophane 12.7
 S.A. EI-Sayed et aL / Resources, Conservation and Recycling 12 (1994) 195-200 199

order: arginine > isoleucine + leucine > methionine + cystine > glycine > phenylalanine >
histidine > tyrosine > lysine > glutamic acid.
B a s e d on the f o r e g o i n g results, s u b m e r g e d cultivation o f the white rot fungus P l e u r o t u s
o s t r e a t u s on sugarcane bagasse seems feasible. The protein-rich fermentation product has
a suitable a m i n o acids c o m p o s i t i o n for use as an animal feed. Further studies are needed to
optimize fermentation conditions. E x t e n s i v e animal feeding trials are also suggested to
ascertain the nutritional value and saftey o f the fermentation product.

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