Result and Discussion

PRODUCTION AND UTILIZATION OF CELLULASE FROM

BIOTROPIA NO. 25, 2005 : 50 – 59

TRICHODERMA VIRIDE

R. HIDAYAT1, S. WULANDARI2, K.G. WIRYAWAN3 and SURYAHADI3

1Department of Animal Nutrition and Feed Science, Padjadjaran University,
Bandung, Indonesia.

2Faculty of Animal Science, Jember Agricultural Polytechnique, Jember, Indonesia.
3Department of Animal Nutrition and Feed Science, Bogor Agricultural University,

Bogor, Indonesia.

ABSTRACT

An appropriate preservation technology for forage such as silage needs to be developed in order to
overcome the shortage of tropical forage during the dry season. A good quality of silage is obtained by
decreasing the fibre contents (Neutral Detergent Fibre, Acid Detergent Fibre and lignin). The research
was conducted in two stages: 1). Production and activity test of crude enzymes from Trichoderma viride
and 2). Comparative test using crude enzymes from Trichoderma viride strain QM 9414 (CETV) and
commercial cellulase (Cellulase “Onozuka R-10”, heidelberg) on rice straw silage. The treatments were
arranged as follows : P0=Untreated rice straw silage, P1=Treated with commercial cellulase at 10 IU kg-1,
P2= Treated with CETV at 3.8 IU kg-1, P3=Treated with CETV at 7.6 IU kg-1 and P4= Treated with
CETV at 11.4 IU kg-1 of fresh rice straw. All treatments were enriched with 5% molasses. The result of
the research indicated that: 1). The activity of crude enzymes from Trichoderma viride strain QM 9414
(CETV) was 1.52 IUml-1. 2). The addition of both enzymes did not decrease dry matter of rice straw
silage; however, organic matter significantly decreased (p<0.05) by addition of commercial cellulase. 3).
The addition of both enzymes decreased fibre contents (NDF), furthermore increased (p<0.05) in sacco
digestibility of dry matter and organic matter of rice straw silage.

Keywords : Cellulase/Trichoderma viride/Rice straw/Silage/Fibre

INTRODUCTION

The main problem of improving ruminant production in Indonesia is the
shortage of high quality forage during dry season causing the animal production
become inefficient, because of an increase in feed cost. On the other hand, the
forage is abundant during the wet season.
Application of forage preservation technology in the farm may solve the
problem, as the forage becomes more available all the year. There are two forage
preservation techniques namely dry preservation (hay) and wet preservation (silage).
For some matters, ensiling has many advantages, as forage quality may be
maintained for a few months. However, the silage production process still has
weakness, as the dry matter decreases and the fibre content of the forage is not
influenced by the process.
Rice straw is one of the agricultural by-products which is a very potential
source of energy for ruminants. Availability of rice straw increases as the rice straw
silage production increases. Rice straw is usually abundant at the same time as the

period of high forage production occurring in the wet season. Farmers choose forage
as feed rather than rice straw. Farmers store rice straw to be used during dry season
when forage production is low. Rice straw as feed has been used generally in the
tropic and sub-tropical areas (Jackson 1977), especially in the hot season. In
Indonesia, rice straw has been used for feed at least 31-39%, but most of it was burnt
or used as fertilizer (36-62%) and the rest is used for industries (Komar 1984).

BIOTROPIA NO. 25, 2005

Rice straw has low nutritive values. Rice straw contains 80% organic material
which can be potentially digested; but in reality, it can be digested by ruminants
only 45-50% (Lubis 1963; Jackson 1977). According to Doyle et al. (1986), rice
straw nutrient content is generally low consisting of crude protein 2.2-9.5%,
hemicelluloses 21-29%, cellulose 35-49%, with coefficient digestibility value of
organic materials 31-59%, and lignin 4-8%. Dry storage condition (hay) makes the
quality of rice straw becomes worse, therefore, a better method is needed to store
the rice straw.
The present techniques in silage production are focused mainly on feed stuff
preservation rather than improvement of its quality. It needs an effort to improve the
process, and ensiling may be improved by both preservation and increase of the
quality of forage.
Addition of enzymes as an active substance may disperse cell wall (NDF and
ADF compounds) (Stokes and Chen 1994), increase digestibility and release the
soluble carbohydrate portion of the forage. Enzymatic pre-hydrolysis of forage
component may promote bacterial growth, enhance lactic acid production and
decrease pH of silage (Bolsen and Sapienza 1993).
The present experiment is focused on application of commercial cellulase
enzyme and crude enzyme produced by Trichoderma viride to improve nutritive
value of rice straw.

MATERIALS AND METHODS

Materials

The materials used in this experiment were: Rice straw (cultivar IR 64), a
commercial cellulase (Onozuka R-10 from Trichoderma viride with activity 1.0
Umg-1 produced by SERVA Electrophoresis GmbH D-69115 Heidelberg Carl-Benz-
str.7) and Trichoderma viride strain QM 9414.

Growth Media for Trichoderma viride

Growth media composition that were used as substrates for Trichoderma
viride to produce raw enzymes were : Media I, one litre media contains : 5 g
NH4NO3, 0.5g KCl, 0.01g FeSO4. 7H2O, 0.5g MgSO4.7H2O, 0.001g CuSO4.2H2O,
1g yeast extract and 6g pepton ,the pH of media was 5.45. Media II, one litre media
contains : 5g NH4NO3, 0.5g KCl, 0.01g FeSO4 .7H2O, 0.5g MgSO4.7H2O, 0.001g
CuSO4.2H2O, 1g yeast extract and 15g rice straw. (Montesqrit 1998).

Experimental Design

Production and utilization of cellulose from Trichoderma viride – R. Hidayat et al.

The research was conducted in two stages as follows:

Stage I: Production and activity test of crude enzymes from Trichoderma
viride.
One loop of Trichoderma viride was cultured in a reaction tube containing 3-5 ml
media I for four days and then it was transferred to an Erlenmeyer glass containing
250 ml media II and incubated for 14 days with shaking. The crude enzymes were
obtained by centrifugation at 4000 rpm, at 50 C for 30 minutes and the supernatant
was used as an additive in the production of rice straw silage.
Stage II: A comparative test using crude enzymes from Trichoderma viride
strain QM 9414 (CETV) and a commercial cellulase (cellulase “Onozuka R-10”,
Heidel-berg) on rice straw silage.
The experiment used completely randomized design with 5 replicates. A total
of 25 silos with 1 kg per silo of fresh material were prepared. The treatments were
based on enzyme activity and arranged as follows : P0=Untreated rice straw silage,
P1=Treated with commercial cellulase at 10 IU kg-1, P2= Treated with CETV at 3.8
IU kg-1, P3=Treated with CETV at 7.6 IU kg-1 and P4= Treated with CETV at 11.4
IU kg-1 of fresh rice straw. All treatments were enriched with 5% molasses to
provide ready available carbohydrate (RAC) as a stimulant fermentation. The pH
before ensiling was normal.

Silage Preparation

The rice straw silage was made as many as 1 kg for each sample. The ensiling
procedure is as follows: The rice straw was chopped into pieces of ± 5 cm length.
The fresh and chopped rice straw was mixed with either commercial enzymes or
crude enzymes from Trichoderma viride (spraying). The mixture was pressed and
compacted by hand, and placed into a silo (glass jar). Then the silo was closed
tightly so that the condition became free from O2, air and waterproof.

Chemical Analysis

Silage samples were collected for chemical analysis on the 30th day of
ensiling. The physical characteristics; colour, presence of fungi and smell of the
silage were studied. Hence, the following parameters were measured : dry matter
(DM), organic matter (OM), crude protein (CP) according to proximate analysis, pH
(pH-Beckman model Φ 40). NDF, ADF and lignin content were analysed according
to Van Soest and Wine (1967). Lactic acid was determined with an HPLC technique
(Shimazu, Japan 3081-09202-20ATD-E). DM and OM digestibility were determined
by nylon bag technique (in sacco) in a fistulated buffalo. The size of nylon bag is
140 x 90 mm. One gram sample of silage was placed into nylon bag and incubated
in the rumen for 24 hours. One week before and during the experiment, the buffalo
was fed on 40 kg elephant grass and 5 kg rice bran per day.

Statistical Analysis

BIOTROPIA NO. 25, 2005

The data were subjected to Analysis of Variance (ANOVA). When the F- test
was significant (P<0.05), the contrast orthogonal test for paired comparisons was
used.

RESULTS AND DISCUSSIONS

Production and Activity Test of Crude Enzymes from Trichoderma viride

On medium I, fungal growth was visible at the second day of cultivation. On
medium II, fungal growth was detected at the second day which was shown by the
mycelial growth in the media after transferred into Erlenmeyer glass. On the fourth
day, the growth of mycelium was maximum and it was harvested. From 25 glasses
of Erlenmeyer, 21 of them were harvested and others were thrown away because
they were contaminated by unexpected micro-organisms or had imperfect growth.
The cellulase activity of crude enzymes from Trichoderma viride (CETV) was
1.52 IUml-1. This result was higher than previous results (Montesqrit 1998) by the
same procedure, which showed that the CMC-ase activity from T. viride fermented
for 14 days was 0.673 IUml-1. This was caused by a different strain of T. viride used,
while the present experiment used T. viride strain QM 9414.

Comparative Test Using Crude Enzymes From Trichoderma viride (CETV) and
Commercial Cellulase on Rice Straw Silage.

The influence of treatments on the physical quality of rice straw silage

Generally, rice straw silage had the same physical characters, either in colour,
smell, texture or the presence of fungi. Rice straw silage had the same colour that
was yellow brownish and moulds were seen on the top of the silage. The colour of
silage was not different from the fresh rice straw.
The silage had good smell, fresh and crumb texture for all treatments. It
showed that the fermentation had taken place by producing lactic acid. The
utilization of dry rice straw as silage material also had positive impact on silage
quality i.e. the silage liquid was little and there was not much soluble nutrient
wasted into silage liquid. Other reports indicated that making silage in wet condition
produced much liquid (McDonald et al.1991), so dryer condition was needed to
make easy handling and produced higher and drier silage.

The influence of treatments on the chemical quality of rice straw

Production and utilization of cellulose from Trichoderma viride – R. Hidayat et al.

1. Nutrients content of rice straw silage

Table 1 presents the nutrients content of rice straw silage. The dry matter
content of rice straw silage treated with enzymes (P1, P2, P3 and P4) was not
significantly different from dry matter of untreated rice straw silage. This result was
different from rice straw directly treated by T. viride in which it caused degradation
of rice straw dry matter 8.4% (Soetjiharto 1997). However, dry matter of rice straw
silage treated by commercial enzymes was significantly (p<0.05) reduced compared
to that of silage treated by CETV.

Table 1. Nutrient content of rice straw silage

Treatments
Nutrients

P0 P1 P2 P3 P4

Dry matter (%) 24.3±0.4 23.9±0.6 24.6±0.1 24.6±0.2 24.9±0.6
Organic Matter (%) 79.1±0.2 79.2±0.2 79.2±0.3 79.6±0.1 79.5±0.4
Crude protein (%) 5.9±0.5 5.8±0.3 5.4±0.2 5.3±0.3 5.6±0.3

Organic matter content of rice straw silage treated with enzymes (P1, P2, P3
and P4) was 79.4 %, and higher (p<0.05) compared with control (P0=79.1 %). This
result was different from direct microbiological treatment as stated by Djunaidi
(1988) that organic matter content of rice straw silage put on the ground was
descending. Organic matter content of rice straw silage treated with commercial
enzymes (79.2 %) was not significantly different from organic matter of rice straw
silage treated with CETV (P2, P3, and P4 = 79.4%). It was different from Stokes
and Chen (1994) observation that the increase of commercial cellulase addition to
corn silage decreased the silage organic matter content.
Crude protein of rice straw silage treated with enzymes (P1, P2, P3 and P4)
was 5.5 % and significantly lower (p<0.05) than crude protein of untreated rice
straw silage (P0=5.9%). This was caused by CETV which still contained protease,
so that some crude protein of rice straw was degraded to become ammonia. A
different result was reported by Stokes and Chen (1994) in which crude protein
content of corn silage treated with commercial cellulase was increased. This was
caused by microorganism growth as source of protein.

2. Fibre content of rice straw silage

The effects of addition of commercial cellulose and CETV to fibre content of
rice straw silage are presented in Table 2. The Neutral Detergent Fibre (NDF)

content of rice straw silage treated with enzymes (P1, P2 ,P3 and P4) was 72.3 %,
and significantly lower (p<0.05) compared to untreated silage (P0=79.0%).
Reduction of NDF content of rice straw silage treated with CETV followed the
equation of Y= 77.75-1.37X. Corn silage treated with cellulase had lower NDF
content (46.7%) compared to untreated corn silage (53.1 %) (Stokes and Chen
1994).

BIOTROPIA NO. 25, 2005

There is no significant difference (p<0.05) between Acid Detergent Fibre
(ADF) content of rice straw silage treated with enzymes (P1, P2 ,P3 and P4=56.2 %)
and control (P0=59.5%), but the ADF content of rice straw silage treated with
enzyme tended to be lower (p<0.05) than ADF content of untreated rice straw.
Enzyme treatment decreased ADF content of rice straw silage about 5.6% from
control.

Table 2. Fibre content of rice straw silage

Treatments

P0 P1 P2 P3 P4 Significance

NDF (%) 79.0±3.4 76.4±1.2 74.2±2.7 71.2±3.1 67.4±2.9 P < 0,05
ADF (%) 59.5±4.4 59.2±2.9 57.6±7.6 53.6±2.7 54.2±2.1 NS

Lignin (%) 7.7±.0.8 8.4±.1.7 6.6±.1.7 6.2±.1.5 5.7±.0.5 NS

NDF= Neutral Detergent Fibre, ADF=Acid Detergent Fibre, P0=Untreated rice straw silage, P1=Treated
with commercial cellulase at 10 IU kg-1, P2= Treated with CETV at 3.8 IU kg-1, P3=Treated with CETV
at 7.6 IU kg-1 and P4= Treated with CETV at 11.4 IU kg-1 of fresh rice straw.

The Acid Detergent Fibre (ADF) content of rice straw silage treated with
CETV (P2, P3, and P4) was 55.1 %, and it was not significantly different from ADF
content of rice straw silage treated with commercial enzyme (P1=59.2%). This result
was different from that reported by Stokes and Chen (1994), that ADF content of
corn silage treated by commercial enzyme decreased about 11 – 13 %. It appears
that ADF content of rice straw silage treated with CETV was lower compared with
commercial enzyme treatment. This means that CETV had higher potential than
commercial cellulase in degrading ADF of rice straw silage. Rice straw is difficult to
be degraded because rice crop is harvested at mature stage with high cell wall
content and perfect lignification level.
The lignin content of rice straw silage treated with enzymes (P1,P2,P3 and P4=
6.6%) was not different from control (7.7%). However, the content of rice straw
silage lignin treated by commercial enzyme (P1=8.4%) was significantly higher
compared with that treated by CETV (6.2 %).
The decrease in lignin content by CETV treatment might be caused by: crude
enzymes CETV which possess many enzymatic activities including lignase, and
production of organic acid during ensilage.

Lignin can be depolymerised by microbial enzymes produced by Trichoderma
sp. (Paterson 1986). On the other hand, commercial enzyme is purely cellulase
enzyme that has no lignase activities, so increasing the level of commercial enzyme
did not affect the lignin content of rice straw silage.

Production and utilization of cellulose from Trichoderma viride – R. Hidayat et al.

3. ph and lactic acid production of rice straw silage

There were no differences between pH of rice straw silage treated with enzyme
(P1, P2, P3 and P4 = 3.72) than that of control (P0 = 3.72). This result is in line with
Stokes and Chen (1994) who reported that enzyme application has no effect to corn
silage pH. Nevertheless, rice straw silage pH treated with commercial enzyme (P1 =
3.68) had significantly lower (P<0.05) pH than rice straw silage treated by CETV
(3.73). The effect on the addition of commercial cellulase and CETV to pH and
lactic acid production of rice straw silage is presented in Table 3.

Table 3. pH and Lactic Acid Production of rice straw Silage

Treatments

P0 P1 P2 P3 P4 Significance

Lactic Acid (%) 0.23±0.05 0.35±0.04 0.27±0.01 0.26±0.03 0.25±0.04 P < 0.05

pH 3.72±0.03 3.68±0.02 3.71±0.01 3.75±0.01 3.74±0.02 NS

pH of rice straw silage treated with commercial enzyme had the lowest pH.
This was related to silage lactic acid production, where silage treated with
commercial enzyme had the highest concentration of lactic acid (0.35 %). Rice straw
silage pH treated with CETV tended to increase as the enzyme levels were
increased. This might be due to a carry-over of some buffers from the media of
Trichoderma viride during centrifugation for enzyme preparation. In addition,
protease activity of CETV could degrade protein into ammonia. The increase in pH
of rice straw silage treated with CETV follows the linear equation of Y=3.696+
0.0072X. In general, rice straw silage pH was very good, i.e. under 4. This means
that rice straw silage can be stored in silos for a long time. Good silage had pH
between 3.5 and 4.0 (AAK 1983).
Lactic acid production of rice straw silage treated with enzyme (P1, P2, P3 and
P4) was 0.29 % higher than the control (P0=0.25 %). Addition of enzyme could
improve lactic acid production of rice straw silage. Low moisture content probably
influenced the relative growth of homofermentative and heterofermentative lactic
acid bacteria during ensiling, which in turn lowered the pH of silage, minimizing

deterioration (McDonald et al. 1991). A different result was reported by Man and
Wiktorsson (2002) in which lactic acid production of cassava and gliricidia tops
silage was 0.95-0.99 %, respectively.

BIOTROPIA NO. 25, 2005

Lactic acid concentration of rice straw silage treated with commercial enzyme
(P1=0.35 %) was significantly higher than lactic acid concentration of rice straw
silage treated with CETV (P2,P3 and P4=0.26 %). The lactic acid concentration was
parallel to rice straw silage pH. The higher production of lactic acid, the lower
became the silage pH. pH of rice straw silage treated with commercial enzyme was
the lowest. While the pH of rice straw silage treated with CETV increased parallelly
to CETV dosages.

Digestibility of Dry Matter and Organic Matter of Rice Straw Silage

The digestibility of dry matter (DDM) of rice straw silage treated with enzyme
(P1, P2, P3 and P4=37.5%) was significantly higher than the digestibility of dry
matter of untreated rice straw silage (P0=28.0%). A similar result was obtained for
digestibility of organic matter (DOM) (Table 4.). The increased degradation might
have to do with the increased damage of feed surface caused by exogenous enzyme
treatment of feed before feeding (Nsereko et al. 2000). Hong et al. (2003) stated
that nutrient degradation rate and effective degradability of DM increased by
addition of enzyme mixture consisting of mainly cellulase and xylanase. The
enzyme applied to forages immediately before in vitro incubation also improved
digestion of DM and NDF, suggesting that fibrolitic enzymes applied for feeding
(direct fed) may enhance digestion of forage by cattle (Feng et al. 1996).
Beauchemin et al. (2001) reported that applying fibrolytic enzymes prior to feeding
enhances ruminal fibre digestion by altering the structure of the feed, thereby
making it more susceptible to degradation. Exogenous enzymes treatment, prior to
ingestion, increase bacterial colonization and thereby improves DM disappearance
of forage (Yang et al. 1999) because exogenous enzymes increased microbial
attachment of ruminal microbes to feed and increased activity of enzymes associated
with feed particles (Wang et al. 2001).
The digestibility of dry matter of rice straw silage treated by CETV (P2, P3 and
P4=37.1%) was not significantly different from the digestibility of dry matter of rice
straw silage treated with commercial enzyme (P1=38.8%).
The improvement in the digestibility of silage treated with enzymes was caused
by decreasing NDF, ADF and lignin content in the silage. Decreasing fibre content
(ADF and NDF) and lignin of rice straw silage made the silage easier to be degraded
by rumen microbes.
The increase of DDM of rice straw silage treated with CETV followed the
linear equation of Y=21.31+3.14X. Meanwhile, DOM followed the equation of
Y=17.43+3.46X. It showed that CETV dosage had not reached the maximum level
as the digestibility was still increasing.

Table 4. Digestibility of Dry Matter and Organic Matter of Rice Straw Silage

Production and utilization of cellulose from Trichoderma viride – R. Hidayat et al.

Treatments Digestibility of Dry Matter (%) Digestibility of Organic Matter (%)

P0 28.6±3.3 25.6±3.6
P1 38.8±2.5 36.8±2.6
P2 28.3±2.0 25.0±2.5
P3 38.9±1.3 37.0±1.7
P4 44.0±3.5 42.3±4.0

P0=Untreated rice straw silage, P1=Treated with commercial cellulase at 10 IU kg-1, P2= Treated with
CETV at 3.8 IU kg-1, P3=Treated with CETV at 7.6 IU kg-1 and P4= Treated with CETV at 11.4 IU kg-1
of fresh rice straw. Digestibility of dry matter (DDM) and organic matter (DOM) of rice straw silage
treated with enzyme (P1, P2, P3) was significantly higher than digestibility of dry and organic matter of
untreated rice straw silage (P0).

The present study clearly indicates that the most promising method to improve
the nutritive value of agricultural by-products in terms of increasing the digestibility
is to treat rice straw with cellulase enzyme addition during ensiling.

CONCLUSIONS

Fungi of Trichoderma viride strain QM9414 produced crude enzymes with
high cellulolytic activity i.e. 1.52 IU/ml. CETV was able to improve the biological
value of rice straw silage and it was better than commercial cellulase. Addition of
commercial cellulase (Onozuka R10 from Trichoderma viride ) and crude enzymes
from Trichoderma viride (CETV) have positive influence on physical quality of rice
straw silage and did not cause negative effect on dry matter and organic matter
content of rice straw silage.

ACKNOWLEDGEMENT

The researcher would like to thank SEAMEO SEARCA for financing this
research.

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R. HIDAYAT1, S. WULANDARI2, K.G. WIRYAWAN3 and SURYAHADI3
ABSTRACT
INTRODUCTION
The main problem of improving ruminant production in Indonesia is the shortage of high quality forage during dry season causing the animal production become inefficient, because of an increase in feed cost. On the other hand, the forage is abundant during the wet season.
Silage Preparation
Chemical Analysis

RESULTS AND DISCUSSIONS
Production and Activity Test of Crude Enzymes from Trichoderma viride
The influence of treatments on the physical quality of rice straw silage
1. Nutrients content of rice straw silage
2. Fibre content of rice straw silage
Hong, S.H., B.K. Lee, N.J. Choi, S.S. Lee, S.G. Yun and J.K. Ha. 2003. Effect of enzyme application method and levels and pre-treatment times on rumen fermentation, nutrient degradation and digestion in goats and steers. Asian-Aust.J.Anim.Sci. Vol 16, No. 3:389-393.
Jackson, M.G. 1977. The alkali treatment of straw, Anim. Feed Sci. and Tech. 2:105-130.
Komar, A. 1984. Technology of rice straw preservation as feed. Yayasan Dian Grahita. Indonesia, Bandung.
Lubis, D.A. 1963. Feed Science. Yayasan Pembangunan Jakarta.
Man, N.V. and H. Wiktorsson. 2002. Effect of molasses on nutritional quality of cassava and gliricidia tops silage. Asian-Aust. J. Anim. Sci. 5(9):1294-1299.
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