Agricultural Wastes 16 (1986) 103-111

Enzymatic Saccharification of Sodium Hypochlorite

Treated Bagasse and Cotton Stalks

N a d i a N a i m , A b d e l - M o h s e n S, lsmail, Lotfy A. Sallam & Abdel-

M o n e m H. E1-Refai

Laboratory of Microbial Chemistry,National ResearchCentre, Dokki, Cairo, Egypt

A BSTRA ('T

Attention was devoted to the delign(fication of the eru&, bagasse ( CB) and
cotton stalks (CS) by cheap commercial sodium hypochlorite solution.
Optimum delign(fieation o/'these suhstrates was achieved by treatment
with O"7 g Cl g - ~ suhstrate (pH 7.0)Jbr 20 h at room temperature, with a
solid.'liquid (S:L) ratio 1.20.
The delign(fied CB and CS were sacehar(lied with an enzyme prepara-
lion j)*om a potent local mutant of Trichoderma viride (253 M16). ,4
sati.~'/aetory saechar(fication was obtained with most of the treatments
used. The highest conversion yield (93%) was recorded with CB treated
with sodium hypoehlorite solution (S:L ratio 1.20),/ollowed hv N a O H
(o.05 M).

INTRODUCTION

Tile crystalline structure of native cellulose as well as the presence of lig-

nin in the cellulosic residues limits cellulose hydrolysis by either acids or
enzymes (Mandels et al., 1974; Linko, 1977; Neese et al., 1977; Tanaka et
al., 1978}. Therefore, pretreatment of these residues seemed to be an
indispensable step to achieve economical conversion of their cellulosic
constituents to fermentable syrup (Andren et al., 1976: Cowling & Kirk,
1q76).
Physical treatment, such as crushing by ball mill, or chemical treat-
ment with acid or alkali is effective in promoting enzymatic cellulose hy-
drolysis (Toyama & Ogawa, 1972: Mandels el al., 1974; Millett et ell.,
1975; Ghose & Kostick, 1969; Fan et al., 1981).
103
A~,ricuttural Wastes 0141-4607/86/$03-50 ( Elsevier Applied Science Publishers Ltd.
England, 1986. Printed in Great Britain
104 Nadia Naim et al.

Relatively few studies have been devoted to delignification of cellu-

losic residues with active chlorine, and most of these were carried out to
give optimal pulp or paper properties rather than to give optimal yields
of assimilable sugars after enzymatic hydrolysis (Rydholm, 1965).
In our laboratory emphasis has been placed on optimizing saccharifi-
cation of Egyptian sugar-cane bagasse (E1-Refai et al., 1982, 1983).
Ismail et al. (1985) achieved maximal enzymatic conversion of bagasse
cellulose (about 90% yield) using sodium chlorite-treated sample with
Trichoderma viride 253-M16. However, the utilisation of sodium chlor-
ite seems to be uneconomic, and in the present investigation efforts have
been focused to replace this expensive agent with the cheap and readily
available sodium hypochlorite solution.

METHODS

Microorganism

The microorganism employed in the present study is a mutant strain of

Trichoderma viride: T. viride 253-M16. The fungus was maintained on
potato-dextrose medium.

Cellulose substrates

The crude sugar-cane bagasse (CB) and the cotton stalks (CS) were air
dried and ground to 149-250/tin. Sodium hypochlorite (NaOC1)-treated
CB or CS were used as cellulosic substrates.

Chemical analysis

Methods of analysis were: for moisture, ash (Abdel-Fattah et al., 1977);

holocellulose (Mansour, 1963); lignin (Adams, 1965).

Sodium hypochlorite treatments

Pretreatments of CB or CS were carried out using commercial sodium

hypochlorite solution. Its available chlorine was determined by the iodo-
metric method (Vogel, 1951). The wet CB or CS was treated with sodium
hypochlorite/acetic acid mixture at room temperature. The chlorine con-
 Enzymatic sacchar(fication q/hagasse and cotton stalks 105

centration, the solid:liquid ratio as well as the treatment time course

were, however, varied in a series of experiments.

Saeeharification of sodium hypochlorite-treated cellulosic samples

E?IZyHIC sourc( ~
Crude enzyme preparations were obtained from culture of T. viride 253
MI6 grown in 250ml Erlenmeyer flasks containing 50ml portions of
medium containing (g litre-~): (NH,)2SO 4, 2.4: KH2PO4, 2-0; calcium
citrate, 2.0; glucose, 0-5; peptone, 0.1; CB, 20.0; initial pH value, 5-5. The
inoculated flasks were shaken for 7 days at 3 0 C on a rotary shaker
(150rev min-1). Cultures were harvested, centrifuged and the clear
supernatant liquid was then treated with 70% saturated ammonium sul,-
phate followed by separation and dialysis of the protein precipitate. A
known concentration of this protein was then used as source of the
enzyme.

Saccharification
Eighteen milligrams (on holocellulose basis) of each dry, delignified
sample (CB or CS) were soaked in 1 ml of distilled water overnight:
2.5ml of the enzyme preparation (0-48mg protein ml ~) to give an
enzyme:substrate ratio of 1:15, and 2"5 ml of 0.2 m acetate buffer, pH 5-0,
were added and 0-1 ml of 0"05 M NaN 3 was added to the mixture as pre-
servative. The saccharification was carried out in 25 ml conical flasks,
under shaken conditions at 45 C for 4 days. A similar reaction mixture,
using heat inactivated enzyme solution, was prepared as control.
The reaction mixture was filtered through G-3 sintered glass and
diluted to 10ml. The reducing sugars released m the saccharification
medium were determined as glucose plus xylose by the methods of
Sornogyi (1945) and Nelson (1944). The saccharification percentage was
calculated from the weight of reducing sugars formed and the weight of
holocellulose in the substrate.

RESULTS AN[) DISCUSSION

Chemical analysis of crude bagasse and cotton stalks

The results (Table 1) show that holocellulose was the major constituent
in both CB and CS samples. Lignin was detected in relatively higher con-
centration in CS.
106 Nadia Naim et al.

TABLE 1
Some Constituents of Crude Bagasse (CB) and Cotton Stalks (CS)
(% to dry weight)

Component Sugar-cane bagasse Cotton stalks

(%) (%)

Moisture 4.9 1.4

Ash 3.4 4.0
Holocellulose 69.4 72.0
Lignin 19"0 23.0

Delignification with sodium hypochlorite

The effect of treating the substrates with NaOC1 of different chlorine
concentrations is shown in Table 2. Complete delignification of CB and
CS was achieved upon treatment with NaOC1 solutions of 0"7 and 0.9 g
C1 (g CB)- ~ or (g CS)- ~. With lower chlorine levels the loss in the sub-
strate weights seemed to be due to the removal of the lignin. However,
with higher chlorine levels (0"7 and 0'9 g%), increasing amounts of sub-
strate constituents other than lignin appeared to be dissolved.

TABLE 2
The Percentage Loss of Dry Weight and Lignin Contents of CB and CS
Due to Treatment with NaOCI of Different Chlorine Concentrations

Chlorine Sample % Loss in % Loss in

concentration dr)' weight lignin
(g/reaction mixture)"

0-3 CB 9'6 59.5

CS I 1"3 53"0
0"5 CB 15.6 88.8
CS 13.2 78.5
0.7 CB 21.6 100.0
CS 25.7 100.0
0.9 CB 26-0 100-0
CS 26-3 100.0

" Reaction mixture: 1 g sample (CB or CS) + different concentrations of

available CI (0.3-0.9g/mixture) dissolved in 100ml water; pH was
adjusted to 7.0 using glacial acetic acid: incubation at room temperature
(25°C) for 10h. The mixture represents the quantity of available CI
used for delignification samples.
 Eno,matic sacchar(fication of bagasse and cotton stalks 107

TABLE 3
The Percentage Loss of Dry Weight and Lignin Con-
tents of CB and CS Due to Treatment with NaOC1 for
Different Times ~

Time Sample %, Loss in % Loss in

(h) ~#y weight lignin

5 CB I 15 588
CS 18.4 63.6
10 CB 24-4 86-1
CS 24-5 897
15 CB 30.1 966
CS 25"0 91 8
20 CB 31-8 100.0
CS 26.9 100-0

"Reaction mixture: 1 g sample (CB or CS) + 0.7 g CI

in 100ml water: pH was adjusted to 7.0 using glacial
acetic acid: incubation at room temperature.

The loss in sample weight may be due to the formation of soluble oxi-
dation products and the initial products of oxidation may be also further
oxidized or hydrolysed (Marsh, 1956; Epstein & Lewin, 1961).
The delignification process was affected by the time of exposure of the
substrates to the added hypochlorite (Table 3), and 15-20 h was suffi-
cient to delignify CB and CS almost completely. However, there was
greater loss of the CB than of the CS weight.
To determine the optimum ratio of the substrate to sodium hypo-
chlorite (solid:liquid (S:L) ratio). Certain amounts of the substrates were
treated with different volumes of NaOC1 solution having varied chlorine
concentrations. The data given in Table 4 showed that complete deligni-
fication of the substrates was attained with the different volumes of
NaOC1 solutions of 0.7 and 0.9 g CI g- 1 substrate. However, the treat-
ment with 0-7 g C1 in a total volume of 10 or 20 ml of the treatment mix-
ture represented optimal conditions, since it provided the utilization of
minimal solution volume and allowed complete removal of lignin with
no extra loss in the substrate weight. With the highest hypochlorite level.
a loss in weight of about 45% was recorded on conducting the treatment
process of CB in 10 ml total volume. The over-oxidation of cellulose with
higher chlorine levels has been previously recorded by Marsh (1956) and
Davidson & Nevell (1957).
108 Nadia Naim et ai.

TABLE 4
The Percentage Loss of Dry Weight and Lignin Contents of CB and CS Due to Treat-
ment with NaOCI of Different Chlorine Concentrations at Different Solid:Liquid (S:L)
Ratios

Chlorine S: L ratio Sample % Loss in % Loss in

concentration (w/v) dry weight lignin
(g/reaction mixture) a

l:10 CB 16"4 13"5

CS 17'7 17"2
1:20 CB 16.2 14'6
CS 18'6 19"3
0'5 1:30 CB 16'4 13"3
CS 18.5 19"5
1:50 CB 14.2 25'3
CS 19-3 16"1
1:100 CB 15.6 11"0
CS 13"2 21"3

1: 10 CB 20.8 100"0
CS 26.1 100"0
1:20 CB 22-0 100"0
CS 23.6 100"0
0'7 1:30 CB 22.2 100"0
CS 22.5 100"0
1:50 CB 22"8 100-0
CS 22'0 100-0
1:100 CB 21.6 100"0
CS 25"6 100"0

1:I 0 CB 45.2 100"0

CS 28.3 100"0
1:20 CB 43' 1 100"0
CS 28.4 100.0
0'9 1:30 CB 4.2.3 100-0
CS 26.4 100"0
1:50 CB 41 "4 100.0
CS 27.6 100'0
1:100 CB 31.8 100"0
CS 26.9 100"0

a Reaction mixture: l g sample (CB or CS+different C1 concentrations (0.5~)-9g/

mixture) dissolved in different volumes of water (10-100ml); pH was adjusted to 7.0
using glacial acetic acid; incubation at room temperature for 15 h.
 Enzymatic saccharification o f bagasse and cotton stalks 109

Saccharification of the treated substrates

The NaOCl-treated bagasse and cotton stalks were separately treated by

T. viride 2 5 3 - M 1 6 e n z y m e f r a c t i o n s a l t e d o u t at 7 0 % a m m o n i u m sul-
p h a t e s a t u r a t i o n ( T a b l e 5). P o o r s a c c h a r i f i c a t i o n yields w e r e a c h i e v e d
w i t h the c r u d e ( u n t r e a t e d ) s a m p l e s , s h o w i n g the i n a c c e s s i b i l i t y o f their
cellulosic c o n s t i t u e n t s t o e n z y m e a t t a c k . O n the o t h e r h a n d , the N a O C 1
t r e a t m e n t r e n d e r e d the t e s t e d s u b s t r a t e s m o r e a t t a c k a b l e b y t h e f u n g a l
cellulase s y s t e m . H o w e v e r , the c o n v e r s i o n v a r i e d c o n s i d e r a b l y w i t h t h e
t y p e o f s u b s t r a t e as well as w i t h the c o n c e n t r a t i o n o f the c h l o r i n e u s e d in
the t r e a t m e n t s . G e n e r a l l y , b e t t e r e n z y m i c h y d r o l y s i s w a s r e c o r d e d w i t h

TABLE 5
Saccharification of CB and CS and NaOCl-treated CB and CS with an Enzyme Fraction
from T. viride 253 M 16

Treatment conditions ~ Substrate Sacchari- Enzyme

fication b activity"
(%)

Soaking with l ml water overnight CB 1.5 0-23

CS 0.6 0.09
Soaking with 1 ml water overnight, CB 36-9 5.54
treatment with 0-3 g CS 15.9 2.39
Cl(g substrate) - ~e
Soaking with 1 ml water overnight, CB 57.2 8-58
treatment with 0.5 g Cl(g substrate)- 1 CS 22-0 3.30
Soaking with 1 ml water overnight, CB 66.7 10-01
treatment with 0.7 g Cl(g substrate)- 1 CS 28-7 4.31
Soaking with I ml water overnight, CB 59.0 8.85
treatment with 0.9 g Cl(g substrate)- 1 CS 25.9 3.88
Soaking with 1 ml water overnight, CB 93.5 14.03
treatment with 0.05 M NaOH (0.2 ml)
for 24 h, after treatment with 0.7 g
Cl(g substrate)- 1

"All treatments were at room temperature (25 C).

h Saccharification mixture: 18mg treated substrate (as holocellulose)+25ml enzyme
solution (0"48 mg protein ml 1) + 2-5 ml 0.2 g acetate buffer, pH 5, + 0.1 ml 0.05 M N a N ¢
saccharification period 4 days.
Specific enzyme activity=reducing sugars released in reaction mixture (mg enzyme
protein)-
Substrate treatments with NaOC1 were at S:L ratio 1:20 (w/v) for 15 h.
l l0 Nadia Naim et al.

the treated bagasse. The highest conversion (66.7%) was achieved with
the bagasse sample pretreated with NaOC1 solution of 0.7 g C1 level. On
the contrary, relatively limited enzymatic hydrolysis (29% saccharifica-
tion yield) was observed with CS treated under identical conditions.
Such variations of the enzymatic activity with the different substrates
might be correlated with a probable difference in the physicochemical
structures of their cellulosic constituents and enzymic substrate speci-
ficity (Rautela, 1967; Mansour & Seif E1-Dien, 1972).
A remarkable improvement in conversion (93.5 %) was found with the
CB sample pretreated with NaOC1 followed by NaOH. The stimulatory
effect of NaOH may be explained by its role as a swelling agent loosening
the crystalline region of cellulose and increasing the initial specific sur-
face areas, thus increasing the enzymatic activity (Schurz, 1978; Fan et
al., 1980, 1981, 1982).

ACKNOWLEDGEMENT

This work is a part of a program supervised by Professor A. H. E1-Refai,

NRC, Cairo, to utilise Egyptian sugar-cane bagasse for feeding purposes.

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