OPTIMIZATION OF CELLULASE ENZYME PRODUCTION FROM Trichoderma reesei AND Aspergillus niger WITH RICE STRAW AS SUBSTRATE

Poppy Diana Sari, Bambang Dwi Argo*, J. Bambang Rahadi W.*

* Department of Agricultural Engineering, Faculty of Agricultural Technology, Brawijaya University

(INDONESIA)

p.diana.sari@gmail.com, dwiargo@ub.ac.id, b.rahadi@gmail.com

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 30^oC 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 1^st 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 2^nd 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 1^st 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 2^nd 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 1^st 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 2^nd 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 1^st 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 2^nd 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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