Poppy Diana Sari, Bambang Dwi Argo*, J. Bambang Rahadi
W.*
* Department
of Agricultural Engineering,
Faculty of Agricultural Technology,
Brawijaya University
(INDONESIA)
ABSTRACT
This
study aimed to obtain the
optimal conditions of cellulase enzyme production
by fungus Trichoderma reesei and Aspergillus niger using rice
straw as a substrate
and being done by Solid State Fermentation
System. Optimization divided into two phases,
the
first phase using 2 variables,
the
first variable is 3 levels of pH treatment, pH 4,
pH 5 and pH 6,
the second variable is 10
days of observation time.
The second phase of treatment with 2 variables,
the first variable is pH level as 6 levels, namely pH x, X.2 pH, pH x.4, x.5
pH, pH X.6, and pH x.8, the second variable is the observation time
up to 10 days.
Observations
were made every 24 hours Each treatment performed 3 replicates and carried out with 2
types of fungus, that obtained 180 units and 360 units of treatment for the first
and second phase sequentially. The value of x is revere
the pH value of the
first phase which produced the
highest enzyme activity.
Research on the optimal conditions obtained enzyme production by Trichoderma reesei in the
first phase was at pH 5 by
incubation for 4.68 days,
generating optimal enzyme activity of 1.8 IU / ml,
whereas enzyme production by Aspergillus niger is on pH 4.82,
with incubation
for 12.1 days, produce optimal enzyme activity of 1.78 IU / ml.
The
second phase obtained optimal conditions of
enzyme production by Trichoderma reesei at pH 5.4 with 4.69 days of
incubation, produces an optimal enzyme activity of 1.8 IU / ml.
Whereas on second
phase of enzyme production by
Aspergillus niger is at pH 4.83 and incubation
for 11.4 days,
produces an
optimal enzyme activity of 1.80 IU / ml.
Keywords: Cellulase Enzymes,
rice
straw, Trichoderma reesei, Aspergillus niger,
pH
INTRODUCTION
Cellulose is a natural polymer bio-materials
and
is a potentially important source for
producing useful industrial materials such
as fuels and chemicals.
Cellulosic
materials can be directly converted
into biofuel using chemicals,
enzymes or
both [1, 2]. Chemical methods is
less good and not economical when
compared to enzymatic hydrolysis due to produce by-products at high temperatures and is a
concern for the environment [3].
Multi-enzyme
cellulase is formed
by several proteins.
Converting
cellulose to glucose in the enzymatic hydrolysis for ethanol production process [4].
Cellulase
enzyme itself has a very
important role in the hydrolysis
of cellulose to produce glucose,
which
is sold in the market and are
needed for various
purposes both for the
manufacture of chemicals which other higher economic
value such as ethanol,
acetone and organic
acids, as well as used as a carbon
source for the production of
microbial utilization of
enzymes and antibiotics [5, 6,
7].
The main obstacle in the development of industrial-scale use
of enzymes is the high cost of production. To that end, the use of rice straw
as a substrate fermentation media which contains cellulose for growth of
microorganisms has a bright prospect in the future, as it provides a lower cost
alternative when compared to the manufacture of enzymes using synthetic chemicals
as growth media mkroorganisme . Production of cellulase enzymes by using rice straw
substrates containing cellulose will also produce other products that are
useful to humans such as glucose, ethanol, single cell protein, and others [8].
Trichoderma reesei
produces enzyme activity higher and
faster than the Aspergillus niger.
Strains
of Trichoderma
reesei and Aspergillus niger produces the
highest enzyme activity were almost
the same. In a study conducted, the
highest enzyme activity of
enzyme cellulase by Trichoderma
reesei is 1.66 IU / ml on day
6, while the
highest enzyme activity of
enzyme cellulase by Aspergillus
niger is 1.69 IU / ml on day 8 [9].
The purpose of this study is to obtain an optimal condition
of cellulase enzyme production in terms of pH and incubation time. Observations
were made of the enzyme activity that is produced in solid state fermentation
(SSF) by using an economical material is rice straw as a substrate by Trichoderma reesei and Aspergillus niger.
MATERIALS AND METHODS
The
study was conducted from July 2012
to March 2013.
The
study was conducted in the Mechatronics
laboratory of Brawijaya University - Malang,
Central
Laboratory of Biological Sciences of
Brawijaya University - Malang and Biomolecular
and Genetics Laboratory of Biology Faculty
of the Islamic
State University of Maulana Malik Ibrahim – Malang,
Indonesia.
In this
study, there are several materials
used, such as Trichoderma reesei and Aspergillus
niger obtained from Microbiology laboratory PAU Food and Nutrition Gadjah
Mada University Indonesia, nutrient solution (aquades, yeast extract, Bacteriological peptone,
(NH4)2SO4, KH2PO4, FeSO4·7
H2O, CMC 1 %), tween 80, NaOH and HCl. This study is divided into
two phases, namely preparation of
materials (substrates) and
optimization of cellulase enzyme production. The
sections include:
Preparation of materials (substrates)
Rice
straw
used in this study is Ciherang. That is
because Ciherang an easy varieties found in
the region of Java. Rice straw
used in the study obtained from the Pakis -
Malang, because the majority of farmers in the area
grow Ciherang.
Rice
straw
that had been obtained,
then cleaned up
sticks rice straw obtained. Then dried in
the sun to dry. Once dried rice straw sticks
then cut ± 2
cm, and milled
using diskmill. Having
obtained the milled and then sieved with a 100
mesh sieve. Straw
powder was then used for the production of cellulase enzymes.
Cellulase
Enzyme Production
Erlenmeyer 250
ml, added 5
grams of powdered rice straw and 25
ml of nutrient solution [10] with pH conditions
in accordance with the treatment (Phase 1: pH 4, pH 5 and pH 6. Phase 2: pH x, X.2 pH, pH x.4, pH x.5, pH X.6, and pH x.8, the value of
x is the optimal
pH value of production
of cellulase enzymes at phase 1). pH was measured again when
the substrate was mixed with a nutrient solution so that pH changes
are known to occur.
Then the sludge of
rice straw covered with cotton,
aluminum foil and
rubber which is then sterilized using autoclave
for 15 minutes. Inoculum
fungi Trichoderma reesei and Aspergillus niger put as much as 2%
at a density of 2x108 cfu/ml to 2.5x108 cfu/ml into the mud
of rice straw and
covered with cotton, aluminum foil and rubber.
Incubation was performed at 30oC
with pH according
to treatment conditions, harvesting
is done by using
a 1% tween 80. Incubation was performed for 10 days, and
observations of enzyme activity is done every 24 hours
with CMCase DNS
method. Data were collected for enzyme activity and an
increase in the pH condition of the solution.
Research
Design
Research
carried out by
simple randomized block design. The research is divided into two
phases, the first phase is the
production of cellulase enzymes with
2 factors, namely pH treatment
with 3 levels treatments which are pH 4,
pH 5 and pH 6 and 10 days observations,
to obtain 30 observations
with three times
repetition and being done to 2 different kind of fungus in order to obtain 180 data.
Then the second phase is the production of cellulase enzymes with 2 factors, namely pH x, pH X.2, pH x.4, pH x.5, pH X.6, and pH x.8 where x is the pH value of the pH in the first phase where the highest cellulase enzyme activity obtained,
and observations made
during the 10 days, up to 60 observations
obtained with 3
repetitions and being done to 2 different kind of fungus
in order to obtain 360 data.
Research
Optimization
Research optimization calculations performed using Response
Surface Methods using Minitab program.
RESULT AND DISCUSSION
Materials
preparation
rice
straw
is dried and cut into
pieces along approximately 2
cm in order to facilitate
the work of grinding, then
ground and sieved with a 100 mesh sieve size. The entire
series of treatments in order to obtain rice straw powder 100 mesh.
Then tested levels of lignin, hemicellulose and cellulose. Thus obtained:
Table 1.
Content of rice straw
Component
|
Percentage (%)
|
Hemiselulosa
Selulosa
Lignin
|
18.495
30.38
7.935
|
Source
: [11]
Cellulase
Enzyme Production
Cellulase
enzyme produced in this study is the enzyme cellulase from fungi Trichoderma reesei and Aspergillus niger rice straw as the
substrate. The phase of the cellulase enzyme production begins with the
selection of microbes that being used which is Trichoderma reesei with the consideration that the type of microbes
capable of producing endo-β-1.4-glucanase
and exo-β-1.4-glucanase up to 80% and
Aspergillus niger to produce β-glucosidase high. Furthermore microbes
are cultured on PDA (Potato Dextrose Agar) slant in a zig-zag and incubated at
a temperature of ± 30°C for 7 days.
Subsequently the culture inoculated in inoculums solution for 3 days and then
suspended into the media in the form of rice straw fermentation and nutrient
solution, which where the sludge was sterilized first by using autoclave.
Enzyme extracting process is done by separating sludge and liquid fermentation
using a centrifuge with a speed of 4000 rpm for 30 min at 4°C to obtain the
enzyme liquid (supernatant). Cellulase enzyme production is then performed with
the enzyme activity measured using CMCase method and cellulase enzyme activity
obtained in accordance the data below:
Table 2. Cellulase
enzyme activity in phase 1.
Day
|
Trichoderma reesei
|
Aspergillus niger
|
pH 4
|
pH 5
|
pH 6
|
pH 4
|
pH 5
|
pH 6
|
1
|
0.502
|
0.833
|
0.886
|
0.437
|
0.487
|
0.491
|
2
|
1.023
|
1.263
|
1.122
|
0.593
|
0.666
|
0.658
|
3
|
1.255
|
1.575
|
1.392
|
0.711
|
0.776
|
0.78
|
4
|
1.655
|
1.688
|
1.621
|
0.86
|
0.958
|
0.879
|
5
|
1.795
|
1.833
|
1.711
|
0.939
|
1.008
|
0.985
|
6
|
1.636
|
2.000
|
1.848
|
1.016
|
1.08
|
1.031
|
7
|
1.553
|
1.777
|
1.735
|
1.365
|
1.613
|
1.232
|
8
|
1.442
|
1.556
|
1.616
|
1.852
|
2.042
|
1.495
|
9
|
1.297
|
1.415
|
1.506
|
1.86
|
1.989
|
1.468
|
10
|
0.765
|
1.323
|
1.373
|
1.552
|
1.852
|
1.278
|
From
the data
above it can be seen that the Trichoderma reesei
cellulase enzyme can produce with enzyme
activity of 2.00 IU/ml at pH 5 to 6 days of incubation time, while
Aspergillus niger
cellulase enzymes can generate with
an enzyme activity of 2.042 IU/ml at pH 5 with 8 days of incubation time. Table 2 shows that the pH conditions
which produced the
highest sellulase enzyme activity was pH 5, then the pH conditions used
in the second phase
of the study. Referring to the results of this study are not consistent with the statement Nadiem Anwar et al (2010), the enzyme activity of Aspergillus niger is 2,042 IU / ml higher than the enzyme activity of Trichoderma
reesei which is 2,000 IU / ml. On
the second phase, the pH condition was set at pH 5, pH 5.2, pH 5.4, pH 5.5, pH
5.6 and pH 5.8 with 10 days of incubation, the observation was done every 24
hours. From the second phase, obtained the result of cellulase enzyme activity
as below :
Table 3. Cellulase enzyme activity in phase 2.
Day
|
Trichoderma reesei
|
Aspergillus niger
|
pH 5
|
pH 5.2
|
pH 5.4
|
pH 5.5
|
pH 5.6
|
pH 5.8
|
pH 5
|
pH 5.2
|
pH 5.4
|
pH 5.5
|
pH 5.6
|
pH 5.8
|
1
|
0.738
|
0.730
|
0.784
|
0.825
|
0.822
|
0.822
|
0.456
|
0.498
|
0.483
|
0.483
|
0.468
|
0.472
|
2
|
1.156
|
1.198
|
1.286
|
1.324
|
1.327
|
1.346
|
0.658
|
0.681
|
0.650
|
0.669
|
0.650
|
0.654
|
3
|
1.533
|
1.552
|
1.556
|
1.575
|
1.578
|
1.575
|
0.738
|
0.780
|
0.715
|
0.719
|
0.742
|
0.734
|
4
|
1.704
|
1.708
|
1.719
|
1.727
|
1.746
|
1.750
|
0.844
|
0.939
|
0.829
|
0.844
|
0.856
|
0.882
|
5
|
1.803
|
1.803
|
1.837
|
1.860
|
1.848
|
1.871
|
0.932
|
1.000
|
0.989
|
0.978
|
1.000
|
0.989
|
6
|
1.917
|
1.944
|
1.959
|
1.993
|
1.982
|
1.985
|
1.023
|
1.084
|
1.061
|
1.050
|
1.061
|
1.054
|
7
|
1.761
|
1.772
|
1.784
|
1.788
|
1.780
|
1.780
|
1.605
|
1.616
|
1.597
|
1.613
|
1.609
|
1.616
|
8
|
1.529
|
1.540
|
1.540
|
1.567
|
1.567
|
1.578
|
2.058
|
2.103
|
2.084
|
2.061
|
2.061
|
2.065
|
9
|
1.445
|
1.430
|
1.426
|
1.438
|
1.438
|
1.430
|
2.012
|
2.042
|
2.016
|
1.997
|
1.985
|
1.982
|
10
|
1.320
|
1.350
|
1.331
|
1.350
|
1.350
|
1.335
|
1.871
|
1.955
|
1.906
|
1.890
|
1.852
|
1.845
|
In the second
phase can be seen that the highest cellulase enzyme activity of the fungi Trichoderma reesei was 1.993 IU/ml at pH 5.5 with a long incubation of 6
days, while the highest
cellulase enzyme activity of the fungi Aspergillus niger was 2.103 IU/ml at pH 5.2 with a long incubation of 8
days.
Fig.1. Contour plot of enzyme cellulose activity in 1st phase
a. Obtained from fungus Trichoderma
reesei based on pH and incubation time. b.
Obtained from fungus Aspergillus niger based on pH and incubation
time.
Figure 1.a shows that the cellulase enzyme production by Trichoderma reesei, conditions of pH and
incubation time which gained high cellulose enzyme activity was in the range of
pH 4.5 to pH 6.5 with incubation period of 3 to 10 days. While the figure 1.b
shows that the longer the incubation time, the higher the enzyme activity sellulase
obtained, but the pH of the enzyme activity sellulase produce high is in the
range of pH 3.5 to pH 6.
Based
on the analysis of
variance, the production of
cellulase enzymes significantly
different in stage 1 between pH conditions,
both
in the production of
cellulase enzymes with 1 type
of fungi or the other.
On
cellulase enzyme production of Trichoderma reesei phase 1,
significantly
different pH conditions both on day 1,
to 3,
to 6,
to 7,
to 8,
to 9 and to 10,
then carried LSD 5%
and 1%.
Whereas
in the enzyme production of Aspergillus niger sellulase phase 1,
significantly
different pH conditions both on day 1,
to 2,
to 3,
to 4,
7th,
8th,
9th and 10th,
then made LSD 5%
and 1 %.
Fig.2. Contour plot of enzyme cellulose activity in 2nd phase.
a. Obtained from fungus Trichoderma
reesei based on pH and incubation time. b.
Obtained from fungus Aspergillus niger based on pH and incubation
time.
Figure 2.a shows that the
cellulase enzyme production of Trichoderma reesei,
pH and
time of incubation conditions which obtained high
cellulase enzyme activity was in the
range of pH 4.5 to pH 6 with
an incubation period of 3
to 10 days,
in
accordance with the contour plot in phase 1.
While fig
2.b shows that the
longer the incubation time,
the
higher the enzyme activity sellulase obtained,
in
accordance with the stage 1,
but the pH
of the enzyme
activity sellulase produce high is in the
range of pH 5 to pH 5.6.
Based
on the analysis of
variance, there are no real differences that
occur in the study sellulase enzyme production using fungus
Trichoderma reesei or by using Aspergillus niger in
phase 2.
Optimization
Of Cellulase Enzyme Production
Research carried
out optimization calculations,
the results
of measurements to
obtain optimal treatment
conditions. Optimization of enzyme production sellulase performed to
obtain the pH and optimal treatment time to
obtain optimal values of
enzyme activity. In using Response Surface Methods with Minitab program,
the
condition is said optimal
if D-optimal
(desirability
Optimization)
is
equal to 1.0000.
Fig.3. Optimization of
Cellulase Enzyme Production 1st phase
a. Produce By Trichoderma reesei. b. Produce By Aspergillus niger
Figure 3.a shows that
the optimal conditions
for cellulase enzyme production of Trichoderma reesei is at pH 5 by
incubation for 4.6771 days,
with
a D-value
of 1.0000 produces optimal enzyme activity of 1.8 IU / ml.
By cellulase
enzyme production using fungus Trichoderma reesei on stage 1 by
treatment with pH 4,
5 and 6 obtained
by the equation:
Y
= 1.83333 – 0.08726X1 + 0.29063X2 + 0.47054X3
– 0.44060X22 – 0.70552X32 +
0.15217X2X3, from the equation, the value of X1 is a group of simple randomized block design calculations, X2 is pH
treatment and X3 is time. While fig 3.b shows that the
optimal conditions of cellulase enzyme production from Aspergillus niger is at pH 4.8180 and incubation
12.0711 days, the
D-optimal value
of 1.0000 produces
the enzyme activity of 1.7833 IU / ml. By cellulase
enzyme production using Aspergillus
niger on stage 1 by treatment with pH
4, 5 and 6 obtained by the
equation: Y = 1.008 – 0.02855X1 + 0.03115X2 +
0.66855X3 – 0.15723X22 – 0.08773X32
- 0.06275X2X3, from the equation, the value of X1 is a group of simple randomized block design calculations, X2 is pH
treatment and X3 is time.
Fig.4. Optimization of
Cellulase Enzyme Production 2nd phase
a. Produce By Trichoderma reesei. b. Produce By Aspergillus riger.
Figure 4.a shows that
the optimal conditions
for cellulase enzyme production of Trichoderma reesei is at pH 5.4 by
incubation for 4.6893 days,
with
a D-value
of 1.0000 produces optimal enzyme activity of 1.8 IU / ml.
By cellulase enzyme production using fungus Trichoderma reesei on stage 2
obtained an equation : Y = 1.83667 – 0.15712X1
+ 0.01495X2 + 0.54295X3 – 0.13923X22
– 0.75790X32 + 0.00292X2X3, from the equation, the value of X1 is a
group of simple randomized block design calculations, X2 is pH treatment and X3 is time.
While fig 3.b shows
that the optimal conditions
of cellulase enzyme production from
Aspergillus niger
is at pH 4.8343 with incubation 10.6782 days,
the D-optimal
value of 1.0000
produces the enzyme activity of 1.8 IU
/ ml. By cellulase
enzyme production using Aspergillus
niger on stage 2 obtained an equation: Y = 0.989000 –
0.001881X1 + 0.005488X2 + 0.785072X3 –
0.014062X22 – 0.042729X32 -
0.006583X2X3, from the equation, the value of X1 is a group of simple randomized block design calculations, X2 is pH
treatment and X3 is time.
CONCLUSION
From
the results of
the research which has been done, can be
drawn some of conclusions among others, where for the first conclusion is the optimal conditions for the
cellulase enzymes production from the fungus Trichoderma reesei for the 1st
phase was formed at pH 5, with incubation for 4.68
days and produce
optimal enzyme activity
of 1.80 IU/ml
with D-optimal
value of 1.0000.
Whereas in the 2nd phase, the
optimal conditions of cellulase enzyme production
from Trichoderma
reesei fungus was obtain at pH 5.4
and 4.69 days
incubation and produce optimal enzyme activity
of 1.80 IU/ml
with D-optimal
value of 1.0000. While the optimal conditions for the
production of cellulase enzymes from
Aspergillus niger
in the 1st phase is at pH 4.82, with incubation
for 12.1 days and produce optimal
enzyme activity of
1.78 IU/ml with
D-optimal value
of 1.0000. Whereas in the
2nd phase, optimal conditions
obtained in cellulase enzyme production from
Aspergillus niger
is at pH 4.83
and incubation for 11.4 days and produce optimal enzyme
activity of 1.80
IU/ml with D-optimal value of 1.0000.
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1.
