SINET: Ethiop. J. Sci.

, 37(2):131–142, 2014
© College of Natural Sciences, Addis Ababa University, 2014 ISSN: 0379–2897

PRODUCTION AND OPTIMIZATION OF CELLULASE FROM TRICHODRMA

ISOLATES UNDER LIQUID STATE FERMENTATION (LSF)

Weldesemayat Gorems and Tesfaye Alemu *

Department of Microbial, Cellular and Molecular Biology, College of Natural Sciences, Addis Ababa
University, Addis Ababa, Ethiopia. Email: [email protected]; [email protected]

ABSTRACT: Pure cellulose was used as sole carbon source for the production of cellulase by
Trichoderma isolates under liquid state fermentation (LSF). Carboxymethyl cellulose (CMC) and
Congo Red were used and considered only four Trichoderma isolates with stronger ability to
produce and optimize cellulase. Cellulase production was assayed by measuring the amount of
glucose liberated in μmol/ml/min by using the dinitrosalicylic acid reagent (DNS) assay method at
540nm absorbance. To maximize cellulase production, the critical parameters such as carbon source,
nitrogen source, cellulose concentration, cultivation temperature and pH on enzyme production
were optimized using LSF. The highest cellulase activity was observed after 12 days of incubation on
media containing, yeast extract (1%), cellulose concentration (1%) and pH (5.5) from seven
Trichoderma isolates under LSF. Cellulase synthesis was repressed in the presence of glucose and
fructose while it was induced in the presence of maltose and lactose under LSF. It is evident from the
present study that the cellulase production extracted to maximum level from Trichoderma isolates
was active at temperature ranges of 40–60°C and pH values 4.5–6.5. Yeast extract was the preferred
nitrogen source to produce cellulase under LSF; and shaking of the culture improved cellulase
production by about 2–3 factors higher than a static culture. This indicated that oxygen supply is
the critical factor for the growth and enzyme production by Trichoderma isolates. Therefore, these
cellulase producing Trichoderma isolates can be used in food industries, animal feed industries,
brewing and wine making, agriculture biomass refining, pulp and paper industries, textile and
laundry industries and ethanol production.

Key words/phrases: Cellulase, CMC, Submerged state fermentation, Trichoderma isolates

INTRODUCTION plant symbionts (Chet and Baker, 1981 and

Kubicek, 2004). Cellulolytic enzymes have been
Cellulases are a group of hydrolytic enzymes applicable in many industries such as food
which are capable of depolymerizing cellulose to industries, animal feed industries, brewing and
smaller molecules (glucose, fructose, etc). The wine making, agriculture biomass refining, pulp
complete degradation of cellulose to simpler and paper industries, textile and laundry
sugars requires the action of at least three types industries and ethanol production. However, the
of enzymes (Gow and Gadd, 1996): endo-β-1,4- cost of cellulase production and optimization
glucanase, exo-β-1,4-glucanase (cellobiohy- profoundly influences the economics of the entire
drolase) and β-glucosidase (Aneja, 2005; Zahri et production process (Bhat, 2000). T. reesei have
al., 2005; Miettinen-Oinonen, 2007). Cellulases developed strains for commercial scale produc-
chiefly produced by microorganisms such as tion of cellulase (Murphy and Horgan, 2005).
fungi, bacteria and actinomycetes. Trichoderma Currently, these enzymes account for
species is one of the best-known cellulolytic approximately 20% of world enzyme market
organisms (Chinedu and Okochi, 2003). Tricho- (Bhat, 2003). Enzymes could be produced by
derma spp are filamentous fungi belonging to a liquid state fermentation (LSF) and solid-state
group of largely asexually reproducing soil fermentation (SSF); the study used LSF. LSF
fungi; includes a wide spectrum of microor- involves the production of enzymes by microor-
ganisms that range from high biodegradation ganisms in a liquid nutrient media. Although the
potential for instance (T. reesei) to facultative use of cellulases in various industries has been

*
Author to whom all correspondence should be addressed.
132 Weldesemayat Gorems and Tesfaye Alemu

increasing very rapidly, the cellulases have KCl; 0.5g, CMC; 5g and peptone; 2g with 15g agar
mainly been crude mixtures causing unaccept- pH 5.5 per litter) (Aneja, 2005). After incubation
able losses of fabric strength and weight. for 3 to 5 days at 30°C on the above mentioned
Furthermore, the un-optimized cellulase compo- medium, the plates were flooded with 0.1%
sition of commercial preparations and non- Congo Red for 15 minutes. Then after the plates
optimal dosage of the enzymes have led to low were distained with 1M NaCl for 30 minutes. The
reproducibility of the processes. Furthermore, isolates that showed clearing zones around the
attempts to use these enzymes in the degradation colony were considered as cellulase producing
of cellulosic wastes have not been successful for Trichoderma isolates.
several reasons such as low enzymatic yields,
low specific activities and end product inhibition Liquid state fermentation
of the enzymes. The aim of this study is to Cellulase production was carried out using
produce and optimize cellulase from Trichoderma cellulose as a carbon source under LSF. The
isolates under LSF. Therefore, this study has composition of the medium was in g/L:
initiated to optimize cultivation conditions for (Na2NO3; 2g, K2HPO4; 1g, MgSO4 7H2HO; 0.5g,
maximum cellulase production, to evaluate the KCl; 0.5g, Cellulose; 5g and peptone; 2g) in 250ml
effect of different carbon and nitrogen sources Erlenmeyer flask (Aneja, 2005). In all conditions,
and to determine the optimum working the culture media were autoclaved for 15min, at
conditions to achieve high enzyme production. 121°C. The autoclaved media was inoculated
with two plugs (5mm diameter) of Trichoderma
isolates from 7 days old culture and incubated
MATERIALS AND METHODS under continues shaking at 121rpm (Orbital
shaker, Gerhardt, Bonn) for 12 days. Then culture
Test fungal isolates broths were filtered off (Whatman No.1 filter
Seven Trichoderma isolates were obtained from paper) and centrifuged (Hermle, Germany), at
mycology laboratory, Department of Microbial, 10,000 rpm for 15 minutes to remove cell debris.
Cellular and Molecular Biology, College Natural The supernatants were used to assay cellulase by
Sciences, Addis Ababa University. The isolates of using 1% DNS (Dinitrosalicyclic Acid reagents)
Trichoderma used in this study were previously method (Ghose, 1987). The optical density (OD)
isolated, identified and characterized from soil was measured using UV-Spectrophotometer
collected from Jimma Zone (western Ethiopia). (JENWAY, 6405 UV/Vis. Spectrophotometer, UK) at
They were designated as AUT1-7 where AUT 540 nm.
stands for Addis Ababa University Trichoderma
isolate followed by numbers. Optimization of cellulase production
Effect of temperature
Preparation of inoculum
Trichoderma isolates were grown at different
Potato Dextrose Agar (PDA) (Oxoid) was temperature in cellulose broth pH 5.5 (15°C,
prepared and poured into the Petri dishes. The 20°C, 25°C, 30°C, 35°C and 40°C) for 12 days at
preserved Trichoderma isolates were transferred 121rpm on a shaking water bath (Precision
on to PDA at pH 5.6 and incubated at 30°C. Scientific Company, Chicago, USA); to determine
Cultures were aerobically grown for 7 days. After the optimal temperature.
7 days of incubation, isolates were transferred on
to CM-cellulose containing media for screening Effect of pH
the potential cellulase producing Trichoderma The effect of pH on cellulase production was
isolates. conducted by adjusting the cellulose broth at pH
3.5, 4.5, 5.5, 6.5, 7.5 and 8.5 using 1N NaOH and
Screening of cellulase producing Trichoderma 1N HCl before fungal inoculation.
isolates
To screen the potential cellulase producing Cellulose concentration
Trichoderma isolates, enrichment procedure was To study the effect of cellulose concentration
followed in minimal medium comprising on the production of cellulase by Trichoderma
(Na2NO3; 2g, K2HPO4; 1g, MgSO4 7H2HO; 0.5g, isolates, different concentration of cellulose were
SINET: Ethiop. J. Sci., 37(2), 2014 133

prepared (0.5–2%) with an interval of 0.5. Then photometer, UK), absorbance at 540 nm. One unit
the inoculated flasks were incubated at room of cellulase activity was defined as the amount of
temperature for 12 days at pH 5.5 on a shaking enzyme producing 1μm of glucose per minute
condition (Ul-Huque, 1992). under the specified assay conditions.

Effect of carbon sources Crude enzyme characterization

The effect of carbon sources on the production of For the determination of optimum temperature
cellulase by Trichoderma isolates were evaluated and temperature stability, activity was
by adding 0.2% of different carbon source determined by carrying out the above standard
(glucose, fructose, maltose and lactose) on a assay at several temperature values. To
growing media containing 0.3% cellulose, 0.2% determine optimum temperature for cellulase
NaNO3, 1% K2HPO4, 0.05% MgSO4 7H2O, 0.05% activity, the reaction mixture was incubated for
KCl, and 0.2% peptone. 30min in the temperature range of 20°C–80°C
(with an interval of 10°C). For temperature
Effect of nitrogen sources stability, each enzyme was incubated at different
The effect of nitrogen sources on the temperatures from 20°C–80°C (with an interval
production of cellulase by Trichoderma isolates of 10°C) for 30min before the addition of
were determined by replacing the existing substrate and the residual activity was measured
nitrogen with 1% of different nitrogen sources following the standard assay conditions. A
(peptone, ammonium sulphate, sodium nitrate simultaneously prepared enzyme-buffer mix was
and yeast extract). The control was prepared in stored at 4°C for 30min to be used as a control.
the absence of any nitrogen sources. Optimum pH for cellulase activity was
determined by assaying the cellulase at different
Time course of enzyme production pH values (3 to 10). The buffers used were citrate
phosphate buffer (pH 3.0 to 7.0), Tris buffer (7.0
To evaluate the effect of cultivation time on
to 9.0) and Glycine-NaOH buffer 50mM (pH 9–
cellulase production, Trichoderma isolates were
10) (with an interval of 1 in all pH ranges). For
grown at room temperature in shaking condition
the determination of pH stability, 100μL of
containing cellulose as the main carbon source
enzyme was mixed with 450μL buffers of varying
and the media was adjusted to pH 5.5. Samples
pH and incubated at room temperature for
were withdrawn from the culture broth at 2-day
30min. Residual activity was measured following
intervals over a period of 14 days after the
the standard assay conditions.
culture inoculated.

The effect of metal ions

Cellulase activity assay
To determine the effect of metal ions; salt
Carboxymethyl cellulase (CMCase) was assayed
solutions were used for source of the metal ions
using a modified method described by Mandels
NaCl, KCl, ZnSO4, MgSO4 and CaCl2. The
et al., in 1976 (cited in Dashtban et al., 2010). The
concentrations of the ions in the reaction mixture
activity was determined by mixing 0.1ml of
were 5mM. The enzymes were pre-incubated
enzyme solution with 0.9ml of 0.5% CMC in
50mM of sodium acetate buffer in a 14ml of test with different metal ions (Na+, K+, Zn+2 and Ca2+)
for 30min at room temperature, and then the
tube, pH 5, vortexed for 1min, incubated for 30
minutes at 50°C. The reaction was stopped by enzyme activity was measured under the
adding 2ml of dinitrosalicyclic acid (DNS). To standard assay conditions.
promote full colour development, the mixture
Statistical analysis
was boiled for 15 minutes (95–100°C) in a boiling
water bath and cooled in cold water. The All experiments and enzyme assays were
formation of reducing sugars was measured performed in duplicates. Data were statistically
using DNS reagents (Ghose, 1987); spectro- evaluated by Excel and SPSS (version 16)
photometeric (JENWAY, 6405 UV/Vis. Spectro programs and results have been presented as
mean ±SEM (standard error mean).
134 Weldesemayat Gorems and Tesfaye Alemu

RESULTS cellulose degrading enzymes (Figs 1 and 2). The

Trichoderma isolate (AUT5) was showed the highest
Screening of cellulase producing trichoderma hallow zone on the CMC agar media (75 mm)
isolates whereas AUT7 showed the least clear zone
All Trichoderma isolates were positive for CMCase diameter (9 mm) (Fig. 1). As a result only four
and able to grow in CMC agar media (Fig. 1) but the isolates AUT1, AUT2, AUT4 and AUT5 were
isolates were differing in their ability to produce considered for further studies.

0.45

0.4
Enzyme activity (U/ml)

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
AUT1 AUT2 AUT3 AUT4 AUT5 AUT6 AUT7

Trichoderma isolates

Figure 1. Screening and evaluation of potential cellulases producing Trichoderma isolates on CMC agar.

80

70

60
Clear zone in mm

50

40

30

20

10

0
AUT1 AUT2 AUT3 AUT4 AUT5 AUT6 AUT7

Trichoderma isolates
Figure 2. Screening and evaluation of potential cellulases producing Trichoderma isolates by DNS assay.
SINET: Ethiop. J. Sci., 37(2), 2014 135

Effect of temperature on cellulase production began to decrease when the temperature rising
The maximum cellulase activities were above 30°C.
recorded at 30°C for the selected isolates (Fig. 3).
The isolate AUT2 gave the highest cellulase Effect of pH on cellulase production
activity (0.544±0.011U/ml) on cellulose broth at The optimal pH was 5.5 for all isolates (Fig. 4).
30°C and pH 5.5. Similarly, isolates AUT1, AUT4 The cellulase activity of AUT1, AUT2, AUT4 and
and AUT5 produced 0.5025±0.0075 U/ml, AUT5, at PH 5.5 were 0.3905±0.0015,
0.4995±0.0045 U/ml and 0.4285±0.0115 U/ml 0.3935±0.0045, 0.5805±0.0085 and 0.314±0.0U/ml,
cellulase activity at 30°C and pH values 5.5, respectively. Trichoderma isolates AUT4 and AUT5
respectively. The enzyme activity increased as were showed the highest and the least cellulase
the temperature increase up to 30°C then after it activity at pH 5.5, respectively.

Figure 3. The effect of temperature on the production of cellulase by Trichoderma isolates.

Figure 4. The effect of different pH on the production of cellulase by Trichoderma isolates.

136 Weldesemayat Gorems and Tesfaye Alemu

Effect of cellulose concentration Time course of enzyme production

It is clearly indicated in Figure 5 that when the The cellulase activity increased upto12 days of
concentration of cellulose increased the enzyme incubation and decreased, thereafter (Fig. 6). The
activity also increased until the concentration maximum activity of cellulase was recorded at 12
reached at 1%. Maximum cellulase production days by all Trichoderma isolates but after that a
was obtained at 1% cellulose concentration by all steep decrease was observed by increasing the
Trichoderma isolates. Further, increase in cellulose fermentation time. The enzyme activities of the
concentration beyond the level of 1% the isolates AUT1, AUT2, AUT4 and AUT5 were
production of cellulase begun to decrease. The 0.3975±0.0055, 0.4033±0.0053, 0.3705±0.0045 and
enzyme activities of the isolates AUT1, AUT2, AUT4 0.3195±0.0045U/ml, respectively.
and AUT5 at 1% were 0.625±0.005, 0.609±0.004,
0.785±0.005 and 0.215±0.005U/ml, respectively.

Figure 5. Effect of cellulose concentrations on cellulase production by Trichoderma isolates.

Figure 6. Time course of cellulase production for Trichoderma isolates.

SINET: Ethiop. J. Sci., 37(2), 2014 137

Effect of Carbon sources Effect of nitrogen sources

Carbon source in the medium affects The nitrogenous sources have influenced the
considerably in the synthesis of cellulolytic production of cellulase by Trichoderma isolates
enzymes by Trichoderma isolate in liquid culture under LSF. It is evident from Figure 8 that all
(Fig. 7). Lactose and maltose were found to be the Trichoderma isolates on yeast extract medium was
good carbon source to induce the production of showed the highest cellulase production whereas
cellulase by four isolates but glucose and fructose on sodium nitrate medium showed the least
were reduce the production of cellulase. Lactose cellulase production. In the control, the cellulase
was a good inducer of cellulase production activity was detected.
followed by maltose by all isolates (AUT1, AUT2,
AUT4 and AUT5).

Figure 7. The effect of carbon sources on the production of cellulase by Trichoderma isolates.

Figure 8. The effect of nitrogen sources on the production of cellulase by Trichoderma isolates.
138 Weldesemayat Gorems and Tesfaye Alemu

Crude enzyme characterizations Temperature stability of cellulase

Optimum temperature for activity of cellulase The cellulase was stable at temperature under
The optimal temperature for the crude 40°C for all Trichoderma isolates. However,
cellulase was found to be 40°C for isolate AUT1, cellulase from AUT1, AUT2, AUT4 and AUT5
50°C for isolate AUT2 and 60°C for isolates AUT4 maintained 91%, 61%, 52% and 54% activity after
and AUT5 (Fig. 9). 30min of incubation at 40°C, respectively. All
isolates at 60°C and above; they retained less
than 50% of the enzyme activity as compared to
their original activity (Fig. 10).

Figure 9. Temperature profile of Trichoderma isolates cellulase, assayed at different temperatures.

Figure 10. The effect of temperature on the stability of cellulase secreted by Trichoderma isolates.
SINET: Ethiop. J. Sci., 37(2), 2014 139

Optimum pH for Activity of Cellulase pH lower than 3 and higher than 11, the enzyme
The maximum enzymatic activity was obtained was lost its activity completely.
at pH range 4.7– 5.4 with optimum pH at 5 for all
Trichoderma isolates (Fig. 11). Effect of metal ions on cellulase activity
The metal ions considerably affect the
pH stability of cellulase cellulolytic activity of Trichoderma isolates. Of all
Trichoderma isolates cellulase was stable in a the metal ions tested Zn++ and Mg++ brought
broad pH range with maximum stability in the about a decrease in cellulase activity whereas
pH range of 4.5–6.5 (Fig. 12). However, the Ca++, K+ and Na+ increase the cellulase activity
enzyme decreased its activity at pH values above (Table 1). Thus, Ca++, K+ and Na+ were the
7.5. More than 80% of the residual relative recommended cofactors to enhance the cellulase
activity retained in the pH ranges of 4.5–6.5. At activity of the Trichoderma isolates.

Figure 11. pH profile of Trichoderma isolates cellulase at 50°C.

Figure 12. The effect of pH on the stability of Trichoderma isolates cellulase extract.

Table 1. The effect of metal ions on the activity of cellulase by Trichoderma isolates.

Cellulolytic Activity (%of control)

Isolates
Control Ca++ K+ Mg++ Na+ Zn++
AUT1 112 104.8 87 114 99.4
AUT2 100% 99 108.4 78.6 112.3 78.6
AUT4 101 105 82.8 119.6 78.7
AUT5 125 104 79.9 121 73.2
140 Weldesemayat Gorems and Tesfaye Alemu

DISCUSSION have reported that glucose and fructose represses

the production of cellulase activity and lactose,
Trichoderma isolates are one of the most avicel (products of cellulose) and CMC induced
important microbial communities responsible for the production of cellulase by Trichoderma spp. In
cellulose degradation found abundantly in the contrast to the present study, Coban and Biyik
plant cell walls. The effect of environmental (2011) have reported that glucose gave the
factors such as temperature, pH and metal ions highest yield, followed by fructose, sucrose and
are found to be important parameters that ethanol. Glucose repression of the cellulase
influence enzyme activities. According to system overrides its induction, and de-repression
Tholudur et al. (1999), cellulase production in is believed to occur by an induction mechanism
cultures is growth associated and is influenced mediated by trans-glycosylation of glucose. Yeast
by various factors and their interactions can extract yielded the highest cellulase production
affect cellulase productivity. Li et al. (2009) also whereas sodium nitrite yielded the least cellulase
stated that optimal temperature for cellulase production. Ahamed and Vermette (2008) have
production depends on the strain variation of the revealed that yeast extract yielded the highest
microorganisms as well as the types of nutrient CMCase activity by Trichoderma reesei RUT-C30. In
composition. In the present study, the contrast to this study, the maximum production
Trichoderma isolates were able to grow over a of cellulase by T. viride was observed in the
broad range of temperature (15–40°C) and pH medium having NaNO3 as the nitrogen source
(3.5–8.5). However, maximum cellulase (Khare and Upadhyay, 2011). The organic
production was obtained at 30°C and 5.5 pH. compounds stimulated higher cellulase yields
This might be due to better growth of the isolates compared with inorganic compounds. It is
at these temperature and pH. This result is believed that simple and organic nitrogen
considerably similar to reported by Shafique and sources like peptone have a stimulatory effect on
Bajwa (2009) and Li et al. (2009) who have both the growth rate and cellulase synthesis and
indicated that the optimum temperature for thereby shortening the lag phase of the culture
maximum cellulase production for T. reesei was (Saxena et al., 2007). However, when the
30 ± 2°C and the optimum pH for T. viride was 5, organism was grown in liquid media containing
respectively. However, the result contradicts inorganic nitrogen sources cellulase productivity
previous results reported by Gautam et al. (2010) was reduced to some extent. This could be
who showed that the optimum temperature for attributed to the assumption that ammonium
cellulase production under LSF is between 40– salts may have an inhibitory effect on cellulase
50°C for T. viride. Li et al. (2009) has observed production (Saxena et al., 2007).
that the optimum temperature for cellulase Cellulose with medium viscosity has shown
enzyme production from T. viride was 50°C. better stimulating effect than cellulose with
Similarly, Voragena et al. (1980) have reported higher viscosity for cellulase production. High
that the optimum pH for maximum cellulase viscosity leads to retard cell division, resulted in
production from T. viride was ranges between 4.0 low production of metabolites and cellulase
and 5.5. Both high acidic and high basic pH show secretion (Ul-Haque, 1992). The study found that
negative effects, but a medium with low acidic addition of cellulose at 1% was optimal for
pH, 5.5 was ideal for enzyme production. This cellulase production. It was reported that the
might be due to the fact that fungal cultures optimal cellulose concentration for high cellulase
require slightly acidic pH for their growth and production for Aspergillus niger was 1% (Gautam
enzyme biosynthesis (Haltrich et al., 1996). et al., 2010), and T. reesei was at 0.5–1.5% (Rashid
According to Szakacs et al. (2006), cellulases are et al., 2009). Furthermore increasing the
often inhibited by the presence of high concentration of cellulose beyond the level of 1%
concentrations of their end products. In the resulted in lower cellulase production from
present study, lactose and maltose induced the Trichoderma isolates. This is probably due to the
production of cellulase whereas glucose and higher viscosity of the medium, which decreases
fructose repress the production of cellulase by the oxygen supply to the cells. Oxygen is
Trichoderma isolates under LSF. This study, agrees necessary for synthesis of cell membrane
with Szakacs et al. (2006) and Baig (2005) who components (sterols, non-saturated fatty acids).
SINET: Ethiop. J. Sci., 37(2), 2014 141

Incubation time is one of the most important ACKNOWLEDGMENTS

factors affecting the growth of Trichoderma
The authors greatly acknowledge the Department of
isolates as well as the production of cellulase.
Microbial, Cellular and Molecular Biology, College of
This study found that the highest yields of Natural Sciences, Addis Ababa University, for the kind
cellulase from the isolates were recorded on the assistance in providing the laboratory facilities and the
12thday in SMF using pure cellulose. Khare and required consumables and equipment during the
Upadhyay (2011) have reported that the whole period of this research work. The National
maximum production of cellulases by T. viride Agricultural Research Fund (NARF), Ethiopian
Agricultural Research Institute, is also acknowledged
was observed after 6 days of incubation. Sun et al.
for providing funds and purchasing the laboratory
(2010) have observed that the enzyme activity culture media, and solvents for this study.
from apple pomace by Trichoderma spp. was
maximum at 120 hr under LSF. This is probably
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