Jordan Journal of Agricultural Sciences, Volume 12, No.

4 2016

Production and Optimization of Cellulolytic Enzymes from Trichoderma Isolates

under Solid State Fermentation
Weldesemayat Gorems*

ABSTRACT

Agricultural wastes were used as sole carbon sources for the production of cellulase by Trichoderma isolates in
solid state fermentation (SSF). The study aimed to identify and optimize the potential agricultural wastes for
cellulase production by Trichoderma isolates under SSF. Carboxymethyl cellulose (CMC) and Congo Red were
used to screen four isolates that had stronger ability to produce cellulase for further study. Cellulase production was
assayed by measuring the amount of glucose liberated in μmol/ml/min by using the dinitrosalisalic acid (DNS)
assay method at 540 nm. Maximum cellulase was recorded between 5-11 days incubation. The experiment found
that wheat bran, Rice bran and wheat straw were comparatively better for high cellulase production whereas cotton
seed, coffee pulp and barely bran relatively showed the least cellulase production in SSF. Trichoderma isolates,
AUT1 produce the highest carboxymethyl cellulase on wheat straw (5.68 U/g), AUT5 on rice bran (8.15 U/g),
AUT2 and AUT4 on wheat bran, their enzymatic activities were 4.92 U/g and 7.01 U/g, respectively. However, the
isolate AUT5 gave the highest carboxymethyl cellulase (8.15 U/g) on rice bran whereas isolate AUT2 produce the
least this enzyme on cotton seed (1.02 U/g). The maximum amount of cellulase was observed between 55% to 65%
moisture contents. It is evident from the present study agricultural wastes were better carbon source for the
production of cellulase by Trichoderma isolates under solid state fermentation.

Keywords: Agricultural wastes, cellulase, production and optimization, solid state fermentation,

Trichoderma isolates.

INTRODUCTION al., 2006). The main components of agricultural wastes

are cellulose, hemicelluloses, pectin and lignin (Tahoun
The agricultural wastes and industrial residues and Ibrahim, 1999). Cellulose is the most abundant
adversely affect human and animal health and also biopolymer in nature and constitutes a large pool of
pollute the environment (Belewu and Babalola, 2009). carbon source for the microorganisms responsible for the
The increasing expansion of agro-industrial activity has decomposition of organic matter in soil (Shankar et al.,
led to the accumulation of a large quantity of 2011 and Shin et al., 2000). Lignocelluloses residues are
lignocellulosic residues all over the world (Albores et very crucial for the production of cellulolytic,
hemicellulolytic and ligninolytic by solid state
* Addis Ababa University, Pobox-128 Shashemen, Ethiopia. fermentation (Sanchez, 2009).

[email protected]
Received on 3/6/2015 and Accepted for Publication on Cellulase is a family of O-glycoside enzymes that
20/3/2016. hyrolyse β,1-4 glycosidic bonds of native cellulose and
other related cello-oligosaccharide derivatives (Shafque

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Production and Optimization… Weldesemayat Gorems

and Bajwa, 2009). They are extracellular, inducible, and Furthermore, attempts to use these enzymes in the
hydrolyzes the conversion of cellulose into disaccharides degradation of cellulosic wastes have not been
and simpler sugars. The complete degradation of successful for several reasons such as low enzymatic
cellulose to glucose requires the action of at least three yields, low specific activities, heterogeneity of wastes
types of enzymes (Gow and Gadd, 1996): endo-β-1,4 - and end product inhibition of the enzymes. Therefore, it
glucanase, exo-β-1,4-glucanase (cellobiohydrolase) and is a prerequisite to design a set of optimal process
β-glucosidase (Zahri et al., 2005; Aneja, 2005; operating conditions to achieve high enzyme production
Miettinen-Oinonen, 2007). Cellulolytic enzymes have (Bhat, 2000).
been applicable in many industries such as food Enzymes could be produced by submerged state
industries, animal feed industries, brewing and wine fermentation (SmF) and solid-state fermentation (SSF).
making, agriculture biomass refining, pulp and paper SSF is a fermentation process performed on a non-
industries, textile and laundry industries and ethanol soluble material that acts both as physical support and
production (Nakari & Pentilla, 1996). However, the cost source of nutrients in absences of free flowing liquid.
of cellulase production and optimization profoundly Generally, SSF holds tremendous potential for the
influences the economics of the entire production production of enzymes (Pandey, 1992).
process (Bhat, 2000). Currently, these enzymes account The exploitation of agricultural by-products such as
for approximately 20% of world enzyme market wheat straw and bran, rice bran, barley bran, peanut
(Murphy and Horgan, 2005; Bhat and Hazlewood, shell, and sawdust, by fermentation is a very interesting
2003). Cellulases chiefly produced by microorganisms biotechnological approach for the production of
such as fungi, bacteria and actinomycetes. Trichoderma cellulases due to their high cellulose content. This paper
species is one of the best-known cellulolytic organisms describes screening Trichoderma isolates for their
(Chinedu and Okochi, 2003). Commercially speaking, cellulolytic activity and then testing the isolates for their
the main production organisms are strains of ability to produce cellulases in a solid substrate
Trichoderma reesei (Murphy and Horgan, 2005). fermentation process using agricultural wastes. The
Trichoderma are filamentous fungi belonging to a group objective of the present study is designed to optimize the
of largely asexually reproducing soil fungi that includes production and optimization of cellulase enzyme from
a wide spectrum of microorganisms that range from very Trichoderma isolates under solid state fermentation and
effective soil colonizers with high biodegradation to find out the potential agricultural wastes for cellulase
potential to facultative plant symbionts that colonize the production in the aforementioned condition.
rhizosphere (Chet and Baker, 1981; Kubicek, 2004).
Although the use of cellulases in various industries MATERIALS AND METHODS
has been increasing very rapidly, the cellulases used Source of Trichoderma isolates
hitherto have mainly been crude mixtures causing Seven isolates of Trichoderma were obtained from
unacceptable losses of fabric strength and weight. Mycology Laboratory, Department of Microbial, Cellular
Furthermore, the un-optimized cellulase composition of and Molecular Biology, College of Natural Sciences,
commercial preparations and non-optimal dosage of the Addis Ababa University. Further studies have been done
enzymes has led to low reproducibility of the processes. in Mycology Laboratory, Addis Ababa University. All

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Jordan Journal of Agricultural Sciences, Volume 12, No.4 2016

Trichoderma isolates used in this study were previously the culture was extracted by adding 100 ml distilled
isolated from soil collected from Jimma zone. All the water, filtering (Whatman No.1 filter paper) and
isolates were designated as AUT1 to AUT7 which stands centrifuged at 10,000 rpm for 15 min. The filtrates were
for Addis Ababa University Trichoderma isolates. used to assay enzyme activity (Ghose, 1987). The same
Preparation of inoculants procedure was followed for coffee pulp, rice bran, cotton
Potatoes dextrose agar (PDA) (Oxiod)) was prepared seed, wheat bran and barley bran.
and poured into the Petri dishes. The preserved Extraction of enzyme
Trichoderma isolates were transferred into PDA (pH The enzyme was extracted by adding 100 ml of
o
5.6) and incubated at 30 C. Cultures were grown distilled water to the fermented substrate in each flask.
aerobically for 7 days and then after 7 days of cultivation The flasks were rotated on a rotary shaker at 121 rpm for
on PDA, the isolates of Trichoderma were transferred 1 hr at room temperature (25oC) (Ul-Huque, 1992). The
into CM-cellulose containing media for screening, fermented broth was filtered by using Whatman No.1
optimization and evaluation of the potential filter paper and centrifuged at 10,000 rpm for 15 min to
Trichoderma isolates for the production of cellulase remove fungal biomass. The filtrates were used to assay
enzyme. cellulase enzymes activity (Ghose, 1987).
Screening and evaluation of Trichoderma isolates Effect of additives for cellulase enzyme
for the production of cellulase production
To screen the potential cellulase producing WS, RB and WB were supplemented with different
Trichoderma isolates, enrichment procedure was done in glucose, fructose, maltose, lactose and cellulose as
minimal CMC medium comprising (NaNO3; 2 g, carbon source at a concentration of 5% (w/w) and yeast
K2HPO4; 1 g, MgSO4 7H2HO; 0.5 g, KCl; 0.5 g, CMC; 5 extract, sodium nitrate, ammonium sulphate, peptone as
g and peptone; 2 g with 15 g agar pH 5.5) (Aneja, 2005). nitrogen sources at a concentration of 1% (w/w) and the
After incubation for 3 to 5 days at 30°C, the plates were effect of these additives on the level of cellulase
flooded with 0.1% Congo Red for 15 min. Again the production were evaluated. The enzyme was extracted
plates were distained with 1M NaCl for 30 min. The on the optimum time of growth and its activity was
Trichoderma isolates that showed a clearing zone around measured following the standard assay procedure
the colony were isolated as cellulase producing potential. (Ghose, 1987).
Solid state fermentation Optimization of moisture content of the solid
Wheat straw (WS), wheat bran (WB), barley bran media
(BB), rice bran (RB), coffee pulp (CP) and cotton seed The effect of moisture content on enzyme production
(CS) were used for the production of cellulase from was studied by varying the percentage of water in the
Trichoderma isolates under SSF. To ten gram of wheat medium from 45% to 80% (with an interval of 10%). All
straw in 250ml Erlenmeyer flask capacity, 10ml stock the liquid added into the flask and original moisture
mineral salt solution (K2HPO4 0.05 g, MgSO4.7H2O 0.02 content of the WB (6.2%), RB (6.5%) and WS (6.1%)
g, NH4NO3 0.1 g, CaCl2.2H2O 0.01 g and 1ml of 1% was taken into consideration in calculating the
o
FeCl3 ) was added and sterilized for 15 min, at 121 C percentage of water in the medium. After 12 days of
o
(Ul-Huque, 1992). After 12 days of incubation at 30 C, incubation the enzyme was extracted and assayed

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Production and Optimization… Weldesemayat Gorems

following the standard assay procedure (Ghose, 1987). (version 16). Statistically significant differences between
Effect of time course for cellulase enzyme means were tested by analysis of variance and post hoc
production test by using ANOVA software. The differences
The optimum time course for cellulase production by between means were considered statistically significant
Trichoderma isolates in SSF was determined by inoculating when the test yielded a value P < 0.05. The results of
10 g of WB, WS and RB with AUT1, AUT2, AUT4 and the experiment were presented as mean ± SE (standard
AUT5 fungal isolates and incubated at room temperature mean error) (Raghunathan, 2013).
o
(25 C) over a period of 14 days. An Erlenmeyer flask (250
ml) containing minimal medium using cellulose as sole RESULTS
carbon source, pH 5.5, were inoculated with two plugs (0.5 Screening and evaluation of cellulase production
mm diameter) of Trichoderma isolates. Samples were from Trichoderma isolates
withdrawn from inoculated flasks at 2 days intervals. The All Trichoderma isolates were subjected to CMC
samples were extracted by adding 100 ml distilled water agar for isolation of potential cellulase producing
and then followed by filtration and centrifugation at 10,000 isolates. Growth of each test isolate of Trichoderma was
rpm for 15 min to remove fungal biomass were assayed to observed after 3 days of incubation at 30oC. All isolates
determine reducing sugars using DNS method (Ghose, of Trichoderma were positive for CMCase. However,
1987). isolates were differing in their ability to produce
Cellulase activity assay cellulose degrading enzymes (Table 1). The isolate
Carboxymethyl cellulase (CMCase) was assayed by (AUT5) was showed the highest hallow zone on the
using a modified method described by Mandel et al. CM-cellulose agar media (75 mm) whereas AUT7
(1976). The activity was determined by mixing 0.1 ml showed the least clear zone diameter (9 mm). It is
of enzyme solution with 0.9 ml of 0.5% CMC in 50 mM evident from Table 1 that AUT1 (32 mm), AUT2 (30
of sodium acetate buffer in a 14 ml of test tube, at pH 5, mm), AUT4 (54 mm) and AUT5 (75 mm) were the most
o
vortexed for 1 min, incubated for 30 min at 50 C. The efficient isolates selected for further studies according to
reaction was stopped by adding 2 ml of dinitrosalicyclic their high clear zone diameter on CMC agar. Moreover,
acid (DNS) reagent in the above mixture. The mixture this experiment was confirmed again by DNS method
o
was boiled for 15 min (95-100 C) in a boiling water bath (Table 1). From qualitatively assay of AUT5 produced
and cooled in cold water. The formation of reducing 0.33U/ml cellulase enzyme. It produced small amount of
sugars was measured by DNS reagents (Ghose, 1987) enzyme activity (0.33 U/ml) when compared to other
spectrophotometerically (JENWAY, 6405UV/Vis. isolates. Similarly, isolates AUT1 and AUT2 were
Spectrophotometer, UK) at 540 nm. One unit of enzyme showed the highest enzymatic activity, 0.4 U/ml and
activity in each case was defined as the amount of 0.41 U/ml, respectively. The isolates AUT3, AUT6 and
enzyme which released 1μm of glucose per minute AUT7 were produced very small amount of cellulolytic
(Ghose, 1987). activity, 0.12, 0.11 and 0.14 U/ml respectively.
Statistical Analysis
All experiments and enzyme assays were performed
in duplicates, statistically evaluated by excel and SPSS

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Jordan Journal of Agricultural Sciences, Volume 12, No.4 2016

Table 1. Screening of potential cellulase producing cellulase activity. The combination of fungal isolates and
ability of Trichoderma isolates on cellulose media substrates were AUT2 (WB), AUT4 (WB), AUT5 (RB)
Clear zone (mm) Enzyme activity and AUT1 (WS) (Table 3 and 4, Fig. 1).
Isolates
Mean (U/ml) As indicated in Table 1. (a and b) the production of
AUT1 32 0.4 cellulase from Trichoderma isolates were significantly
AUT2 30 0.41 affected by substrates. AUT1, AUT2, AUT4 and AUT5
AUT3 15 0.12 showed a significant difference for cellulase production
AUT4 54 0.37 on WS, RB, CS and BB whereas the isolates showed no
AUT5 75 0.33 significant difference on WB and CP. AUT1 produced
AUT6 10 0.11 large amount of cellulase on WS, this is significantly
AUT7 9 0.14 different from the cellulase obtained from CS and CP.
However, there was no significant difference among
Solid state fermentation for cellulase production
WB, RB, and BB. Isolate AUT2 was showed maximum
Different agricultural wastes were employed for the
amount of cellulase on WB followed by BB and RB. No
production of cellulase from Trichoderma isolates. The
significant differences observed among WB, BB, RB,
experiment found that WS, WB and RB were showed
CP and WS; WS and CS but they were significant
maximum cellulase production after 12 days of incubation
differed with CS, except WS. Isolate AUT4 produced
at 30oC whereas CS, BB and CP were showed the least
large amount of cellulase on WB followed by WS and
amount of cellulase production by Trichoderma isolates
RB. No significant differences were observed among the
(Table 2). The maximum cellulase production was recorded
substrates except BB, and BB was not significantly
by AUT1 (5.68±0.06 U/g) on WS, AUT2 (4.92±0.16 U/g)
different from CP. Isolate AUT5 produced maximum
and AUT4 (7.01±0.055 U/g) on WB and AUT5
amount of cellulase on RB followed by CS and WB.
(8.15±0.065 U/g) on RB. The minimum cellulase
There were no significant differences among RB, CS
production was observed by AUT2 (1.05±0.08 U/g) on CS
and WB; BB and WS; CP, CS and WB but significant
and by AUT5 (1.26±0.02 U/g) on WS. Therefore,
difference were observe among RB, BB, CP and WS.
optimization and other experiments were done only on four
fungal isolates and four solid wastes that showed maximum

Table 2. Evaluation of different solid substrates for the production of cellulase by Trichoderma isolates
a. Comparison among isolates on the same substrates/comparison across the raw
Isolates/ Enzyme Activity U/g
substrates AUT1 AUT2 AUT4 AUT5
a a a
WB 4.98± 0.17 4.92±0.16 7.01±0.055 5.99±0.055a
WS 5.68±0.06a 2.95±0.065b 6.62±0.105a 1.26±0.02b
RB 5.32±0.17a 4.08±0.065b 5.95±0.1a 8.15±0.065b
CS 2.43±0.065b 1.05±0.08b 5.81±0.015s 6.26±0.105a

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Production and Optimization… Weldesemayat Gorems

Isolates/ Enzyme Activity U/g

substrates AUT1 AUT2 AUT4 AUT5
b b b
CP 2.13±0.04 3.72±0.06 4. 68±0.04 3.81±0.055b
BB 3.49±0.055ac 4.42±0.045a 2.80±0.03ac 1.66±0.075c

b. Comparison among substrates by inoculating the same fungi/comparison across the column
Enzyme Activity U/g
Isolates
AUT1 AUT2 AUT4 AUT5
ac a a
WB 4.98± 0.17 4.92±0.16 7.01±0.055 5.99±0.055ad
WS 5.68±0.06a 2.95±0.065ac 6.62±0.105a 1.26±0.02bc
RB 5.32±0.17a 4.08±0.065a 5.95±0.1a 8.15±0.065a
CS 2.43±0.065b 1.05±0.08bc 5.81±0.015s 6.26±0.105ad
CP 2.13±0.04 b 3.72±0.06a 4. 68±0.04ac 3.81±0.055dc
BB 3.49±0.055bc 4.42±0.045a 2.80±0.03bc 1.66±0.075bc
NB: The same letter indicates no significant difference and different letter indicates there is significant difference. ±SE

The effect of additives on cellulase production Trichoderma isolates were also significantly affected by
under SSF different nitrogen sources (Table 3). The combination of
The effect of different additives was evaluated for the wheat straw and sodium nitrate showed the highest
production of cellulase by Trichoderma isolates (Table 3). cellulase production by isolate AUT1 (5.54±0.05 U/g)
Comparatively cellulose (5.95+0.06 U/g) and lactose and significantly different from yeast extract and
(5.59+0.07 U/g) showed the highest cellulase production by peptone but not Ammonium sulphate. On the other hand,
AUT4, whereas, glucose showed the least amount of the combination of rice bran and peptone showed the
cellulase production as compared to the control. Similarly, highest cellulase production by isolate AUT5
cellulase was not produced in the presence of fructose and (8.955±0.135 U/g) and this is significantly different
maltose by isolates AUT1, AUT4 and AUT5. However, from yeast extract and sodium nitrate but not
cellulase production by AUT2 was not significantly Ammonium sulphate; and peptone and wheat bran
affected by the presence of carbon sources except glucose. showed highest cellulase production by AUT2 and
Therefore, cellulose and lactose increased the activity of AUT4, their enzyme activity were 6.46±0.11,
cellulase whereas glucose decreased the activity of cellulase 6.795±1.465 U/g, respectively. No significant
as compared to the control. differences were observed among the nitrogen sources
Similarly, the production of cellulase by for isolates AUT2 and AUT4.

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Jordan Journal of Agricultural Sciences, Volume 12, No.4 2016

Table 3. The effect of carbon and nitrogen sources on the production of cellulase under solid state fermentation
(SSF) by Trichoderma isolates.
Enzyme activity U/g of Trichoderma isolates
Sources of substrates
AUT1 + WS AUT2 + WB AUT4 + WB AUT5 + RB
ac ab ab
Carbon Control 3.68±0.06 4.92±0.16 4.01±0.05 3.15±0.06ab
a a a
Glucose 1.1±0.03 3.34±0.01 2.61±0.03 1.93±0.04a
fructose None 6.42 ± 0.22b None None
ab
Maltose None 5.82 ±0.05 None None
bc ab b
Lactose 4 ±0.05 5.33 ±0.08 5.59 ±0.07 5.57±0.09b
Cellulose 3.94 ±0.05bc 4.99 ±0.06ab 5.95 ±0.06b 4.66 ±0.07b
Nitrogen Yeast extract 2.87±0.07ac 5.00 ± 0.09a 5.92±0.03a 3.72±0.03a
Sodium nitrate 5.54±0.05b 5.19±0.08a 4.90±0.08a 3.81±0.15a
Ammonium sulphate 4.36±0.03bc 5.64±0.05a 6.11±0.03a 5.73±0.09ab
ac a a
Peptone 2.645±0.05 6.46±0.11 6.79±1.46 8.95±0.13b
± SE

45% or > 70% were not suitable for high cellulase

The effect of moisture levels
production. On the other hand isolate AUT1 did not show
The cellulase produced by Trichoderma isolates were any enzyme activities at the moisture contents 45% and
affected by the moisture content of the substrate as 80%, isolates AUT4 and AUT5 did not show any
indicated in Fig. 1. The maximum amount of cellulase was
enzymatic activities at the moisture level 80%. Isolates
recorded between 55% to 65% moisture contents. At 65% AUT2, AUT4 and AUT5 showed no significant differences
moisture content, isolates AUT5 and AUT1 showed between 55% and 65% moisture contents but significant
maximum enzyme activity being 7.34 U/g and 5.37 U/g ,
difference was showed by isolate AUT1 between 55% and
respectively whereas isolates AUT2 and AUT4 showed 65% moisture content.
maximum enzyme activity at 55% moisture content being
5.92 U/g and 4.65 U/g , respectively. Moisture contents <

Figure 1. The effect of moisture level on the production cellulase from Trichoderma isolates.

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Production and Optimization… Weldesemayat Gorems

The effect of time course on the production of after 5 days incubation on wheat bran at 30oC; isolate
cellulase under SSF AUT2 showed maximum enzyme production
o
The time course of maximum cellulase production (8.305+0.06 U/g) at 9 days incubation at 30 C; isolates
under SSF was varied depend up on the substrates and AUT1 and AUT5 showed maximum cellulase enzyme
isolates employed (Table 4). The isolate AUT4 showed production being 5.47+0.06 U/g and 7.675+0.03 U/g
maximum enzyme production peak (9.96+0.03 U/g) after 11 days incubation on WS and RB, respectively.

Table 4. Time course of cellulase enzyme production under solid state fermentation (SSF)
Enzyme activity U/g
Isolates/days
3 5 7 9 11 13

AUT1+WS 2.745±0.05a 4.19±0.12ab 4.31±0.13ab 3.92±0.03ab 5.47±0.06b 3.73±0.07ab

AUT2 +WB 2.52±0.06a 3.73±0.1a 6.68±0.04b 8.31±0.07b 7.36±0.06b 6.99±0.1b
AUT4 + WB 7.47±0.43a 9.96±0.03a 9.12±0.18a 8.15±0.87a 7.17±0.12a 7.33±0.08a

AUT5+RB None None 1.49±0.03b 5.74±0.06a 7.68±0.04a 7.19±0.08a

± SE

were able to produce CMCase while cultivated on the

It is evident from table 4 cellulase production agricultural wastes. Wheat straw, wheat bran and rice bran
significantly affected by time course of enzyme were comparatively better for cellulase production by
production. Cellulase produced by isolate AUT1 at day 3 AUT1, AUT2 and AUT4, and AUT5 isolates, respectively.
was significantly different with at day 11. Similarly, However, the activity of cellulase was varied in agricultural
cellulase produced by isolate AUT2 at days 3 and 5 were wastes. The cellulase activity of AUT1 was (5.68±0.06
significantly different with days 7, 9, 11and 13. U/g) on WS, AUT5 was (8.15±0.065 U/g) on RB, AUT2
Cellulase produced by AUT5 at day 7 was significantly and AUT4 were (4.92±0.16 U/g and 7.01±0.055 U/g) on
different from days 9, 11 and 13. However, in the case WB. There is a significant difference between AUT1 and
of isolate AUT4 no significant differences were AUT5; AUT2 and AUT5; and AUT2 and AUT4. However,
observed among the days. there is no significant difference between AUT1 and
AUT2; and AUT1 and AUT4. This may be due to the
DISCUSSION adsorption of enzymes and the formation of enzyme-
Agricultural wastes (wheat bran, wheat straw, rice substrate complexes are considered to be critical steps in the
bran, barley bran, cotton seed and coffee pulp) were enzymatic hydrolysis of cellulose. Cellulose fibers contain
tested and fermentation parameters were optimized for both amorphous and crystalline regions. Crystalline regions
production of cellulase by Trichoderma isolates in SSF are considered to be more difficult to be degraded than the
medium of wheat bran, wheat straw, rice bran and amorphous regions (Abo-State et al., 2010). Moreover,
results have been discussed as under: Balaraju et al., (2010) reported that WB and RB were better
The study revealed that all isolates used in this study for cellulase production by Oudemansiella radicata under

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Jordan Journal of Agricultural Sciences, Volume 12, No.4 2016

SSF. It might be due to the fact that WB contains adequate incubation. Masbah et al. (1983) observed that T.
amount of nutrients like proteins 1.32%, carbohydrates koningii the cellulase activity reached to maximum after
69%, fats 1.9%, fibers 2.6%, ash 1.8% Ca 0.05%, Mg 16 days of incubation under SSF. Khare and Upadhyay
0.17%, P 0.35%, K 0.45%, S 0.12%, various amino acids (2011) have reported that the maximum production of
and porosity for oxygen supply. However, this work cellulases by T. viride was observed after 6 days of
appeared to contradict with the previous results reported by incubation. Whereas, Sun et al. (2010) observed that the
Ravindran et al., (2010) reported that cotton seed under enzyme activity from apple pomace by Trichoderma spp
SSF condition showed maximum enzyme production at was maximum at 120 h in SSF. This is probably due to
high alkaline pH by Chaetomium spp. the cease of the growth, the release of simpler sugar and
For studying the effect of nitrogen source, proteases into the medium during the later growth phase
supplementation of the fermentation medium with different (Ishaque and Kluepfel, 1980).
nitrogen sources was carried out. A significant increase in
the enzyme productivity by the tested isolates was recorded CONCLUSIONS
in the presence of 1% peptone and sodium nitrate as Trichoderma isolates produced high level of
compared to the control. A combination of peptone with cellulase in solid state fermentation and agricultural
wheat bran and rice bran were comparatively better for wastes were better media for the production of cellulase
cellulase production by AUT2 and AUT4, and AUT5, by the isolates. The activities were comparable with
respectively. This result well agreement with the study some of the fungal strains reported so far. Wheat straw,
conducted by Mrudula and Murugammal (2011), good wheat bran and rice bran were comparatively better
cellulase production can be obtained with peptone as the substrate for cellulase production by isolates. The
organic nitrogen source in SSF. In the case of isolate production of cellulase was affected by in the presence
AUT1, a combination of wheat straw and sodium nitrate of different additives, carbon and nitrogen sources.
was better for cellulase production. Cellulose and lactose were the preferred carbon sources
The results indicated that when moisture level whereas peptone was the preferred nitrogen source for
increased beyond a certain limit the enzyme activity cellulase production under solid state fermentation.
started decreasing. This decline may be attributed to
poor aeration in SSF and partial adsorption of enzyme to ACKNOWLEDGEMENTS
the substrate. Xia et al., (1999) studied the cellulase The authors greatly acknowledge the Departments of
production by solid state fermentation on lignocellulosic Microbial, Cellular and Molecular Biology, College of
waste and reported that water content of solid substrate Natural Sciences of the Addis Ababa University for the
is one of the key factors in cellulase production kind assistance in providing the laboratory facilities and
experiments. The present study nearly similar to the the required consumables and equipment during the
study conducted by Xia et al., (1999) SSF at a water whole period of this research works. National
contents ranging between 55-65% was found to be the Agricultural Research Fund (NARF), Ethiopian
most suitable for cellulase production. Agricultural Research Institute is also acknowledged for
The experiment found that maximum cellulase providing funds and purchasing the laboratory culture
production obtained ranging between 5-11 days of media, and solvents during the study.

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Production and Optimization… Weldesemayat Gorems

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Biotechnology 1: 21-28. cultural factors on production of cellulase and β-1, 3-
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‫…‪Production and Optimization‬‬ ‫‪Weldesemayat Gorems‬‬

‫اﻧﺗﺎج اﻧزﻳم اﻟﺗﺣﻠﻝ اﻟﺳﻠﻳﻠوزي ﻣن ﻓطرﻳﺎت ﺗراﻳﻛودﻳرم ﻳﺎﺳﺗﺧدام طرﻳﻘﺔ ‪ SSF‬ﻋﻠﻰ اﻟﻣﺧﻠﻔﺎت اﻟزراﻋﻳﺔ‬

‫وﻟدﻳﺳﻳﻣﺎﻳﺎت ﺟورﻳﻣز*‬

‫ملخـص‬

‫استخدمت المخلفات الزراعية كمصدر كربوني وحيد إلنتاج انزيمات النحلل السيليولوزي بوساطة فطر ترايكوديرم‬
‫المعزول بطريقة تخمرات الحالة اﻟﺻﻠﺑﻪ‪ .‬تھدف الدراسة الى التحديد االمثل الستخدام المخلفات الزراعية إلنتاج انزيم‬
‫التحلل السيليولوزي بطريقة تخمرات الحالة الصلبة‪ .‬تم استخدام الكاربوكسي ميثل سليسلوز والكونكو رد للكشف عن‬
‫اربع عزالت والتي اثبتت القدرة على انتاج السليولوز ‪ .‬تم تقدير انتاج انزيم السليسلوز عن طريق قياس كمية الجلوكوز‬
‫المحرر )مايكرومول‪/‬مل‪/‬دقيقة( حيث استخدم طريقة داينايتروسلسالك اسد على طول موجي ‪ 540‬نانومتر‪ ،‬تم الحصول‬
‫على اعلى تركيز لالنزيم بعد ‪ 11-5‬يوم من التخمر‪ .‬اعطت عزالت ِ‪ AUT1‬تفوقا ملوحظا النتاج الكاربكوميثل سليلوز‬
‫)‪( 5.6U/g‬في قش القمح ونخالة الرز ‪ (8.15 U/g) AUT5‬و ‪ AUT2‬و‪ AUT4‬في نخالة القمح حيث كانت الفاعلية‬
‫االنزيمية ‪ 4.92 U/g‬و ‪ 7.01 U/g‬للعزالت على التوالي‪ .‬ومع ذلك اعطت عزالت ‪ AUT5‬اعلى الكاربكوميثل سليلوز‬
‫)‪ (8.15 U/g‬في عزالت نخالة الرز بينما اعطت عزالت ‪ AUT2‬اقل تركيز لالنزيم في بذور القطن )‪(1.02 U/g‬‬
‫لوحظ ان اعلى تركيز لالنزيم كانت على رطوبة ‪ %55‬الى ‪ . %65‬اثبتت الدراسة ان المخلفات الزراعية كانت افضل‬
‫مصدر كربوني لعزالت الفطر ترايكوديرم باستخدام طريقة ‪ SSF‬على المخلفات الزراعية‪.‬‬
‫الكلمات الدالة‪ :‬المخلفات الزراعية‪ ،‬السيليولوزي‪ ،‬انتاج االنزيمات‪.‬‬

‫‪‬‬
‫* ﺟﺎﻣﻌﺔ ادﻳس اﺑﺎﺑﺎ‪ ،‬اﺛﻳوﺑﻳﺎ‪[email protected] .‬‬
‫ﺗﺎرﻳﺦ اﺳﺗﻼم اﻟﺑﺣث ‪ 2015/6/3‬وﺗﺎرﻳﺦ ﻗﺑوﻟﻪ ‪.2016/3/20‬‬

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