Effects of different parameters on cellulase production by Trichoderma harzianum TF2 using solid‐state fermentation (SSF)

Solid‐state fermentation is one of the easiest and cheapest methods for producing microbial bioactive com‐ pounds. Trichoderma harzianum has long been recognised as one of the potential fungi for this purpose. Trichoderma sp. were isolated from banana rhizosphere using the soil dilution method and later screened for their ability to produce cellulases using filter paper activity (FPase) and the carboxylmethyl cellulase (CMCase) test. Trichoderma sp. were also subjected to one factor change at a time to determine the effects of different parameters on cellulase production. It was observed that T. harzianum TF2 showed the ability to produce higher cellulase activity when wheat bran was used as the substrate. The results showed that 38.5 U/g of cellulase was produced with the use of wheat bran coupled with an incubation temperature of 28 °C and moisture content of 60%. T. harzianum TF2 showed good potential for use as a culture for cellulase production in this study due to its higher cellulase production under solid‐state fermentation, with the possibility of its application to industry.


Introduction
Cellulase has been well known for its economically im portant ability. Due to this, cellulase had been attract ing a lot of attention from researchers all over the world (Singhania et al. 2010; Darabzadeh et al. 2019; Han et al. 2020. Currently, cellulase derived from microorganisms are gaining their market shares rapidly, however fungal cellulase are more intensively studied because of their high enzyme productivity and also applicability in the indus tries (Darabzadeh et al. 2019). Production of cellulase from fungal can be done either using submerged fermen tation or solid state fermentation (SSF). However, due to the simplicity and economical way for enzymes produc tion, SSF was well accepted by the industry (Hölker and Lenz 2005).
The used of solid state fermentation was well em ployed and accepted in develop countries because it uses their excess agricultural wastes, which could be a source of pollution and turn it into a good source of substrate for enzyme production (Irfan et al. 2014). The use of these wastes will reduce the cost of production by 40%60% (Wen et al. 2005). The most used substrates were wheat bran, rice husk, rice bran, sawdust and others (Irfan et al. 2014; Tambichik et al. 2018. A few well know fungal that were widely used in cel lulase production are Trichoderma sp. and Aspergillus sp. (Kittanan et al. 2018; Triwahyuni et al. 2018; Wang et al. 2020. Sari et al. (2013) demonstrated the used of Tri choderma reesei and Aspergillus niger in producing cel lulases using rice straw from Indonesia. However, we be lieve that apart from T. reesei, T. harzanium can also be used to produce cellulases with higher or similar cellulase activity. Trichoderma harzianum can be found in abun dance in all soil and has been studied by many researchers for its broad potential as plant biological control, indus trial enzymes producers and composting (Pandey et al. 2014; Kumar et al. 2015; Triwahyuni et al. 2018. Haq et al. (2006) showed that T. harzianum can utilize several agricultural byproducts to produce cellulase by estimating the CMCase and FPase activities. Pandey and Srivastava (2015) and Zhang and Yang (2015), also showed that T. harzianum can be used to produce enzyme cellulase via SSF utilizing agriculture waste such as wheat bran by de termining both CMCase and FPase activities.
The aim of this study was to study the optimum sub strates, temperature and moisture content for used in SSF for the production of industrial important enzyme cellulase from Trichoderma sp. isolated from organic soil.

Isolation of Trichoderma sp. from soil
Trichoderma sp. was isolated from the banana rhizo sphere of an organic banana plot situated at MARDI Or ganic Farm at MARDI headquarters Serdang, Selangor, Malaysia. The soil samples were collected and put into a zip lock bag for transportation back to the laboratory. Upon reaching, 10 g of the soil sample was weighed and put into a 250 mL Erlenmeyer flask containing 100 mL of sterile distilled water and agitated for 1 h at 250 rpm. After 1 h, 150 µL of the soil suspension was pipetted to a fresh new Potato Dextrose Agar (PDA) plate. The plate was incubated at room temperature of 28±2 ºC for 7 days. Emerging Trichoderma sp. was picked up using an inocu lation loop and transferred to a new PDA plate.

Screening for cellulase activity produce by Trichoderma sp.
Trichoderma sp. was grown in Potato Dextrose Broth (PDB) for 12 days. Every day a flask would be used for the cellulase activity test until day 12 of the incubation. The culture filtrate was used for filter paper activity (FPase) and carboxylmethyl cellulase (CMCase) test according to the method recommended by Ghose (1987). For FPase ac tivity, Whatman number 1 filter paper was cut into a 1×5 cm strip after that 1.5 mL of the culture filtrate and 0.5 mL buffer (0.05 M citrate buffer, pH 4.5) were added to it and incubated for 1 h at 50 ºC. The hydrolysis process was terminated by the addition of 3 mL of dinitrosalicylic acid (DNS) solution, followed by 5 min of boiling. After 5 min, the solution was left to cool (Miller 1959). When it was cooled, 20 mL of distilled water was added and the absorbance was read at 540 nm using a nanodrop reader. While for CMCase activity, the method was the same, ex cept the filter paper used in the FPase was replaced with 5 mL of CMCase solution (1%). Tests were repeated for every day until day 12. Glucose was used as standard and enzyme activity was expressed as the amount of enzyme required to liberate 1 μmol of product at 50°C. One unit (U) of enzyme activity is defined as the amount of enzyme required to liberate 1 μmol of product at 50°C.

Molecular identification of Trichoderma sp.
Isolation of genomic DNA from Trichoderma sp. was done using the QIAamp® DNA Mini Kit following proto col suggested by Qiagen (Qiagen 2020). After that poly merase chain reaction (PCR) was conducted using ITS primer. The reaction was performed in a 25 µL final vol ume containing 0.1 µg of genomic DNA, 10 pM of each primer (ITS4 and ITS5), 1 × Taq polymerase buffer, 1.5 mM MgCl 2 , 0.2 mM dNTPs, and 1 U of Taq DNA poly merase. PCR thermal cycle parameters used were 94°C for 3 min followed by 35 cycles of 30 s at 94°C, 40 s at 55°C and 35 s at 72°C and a final extension at 72°C for 7 min (Lu et al. 2012). The PCR products were later subjected to purification using QIAquick PCR Purification Kit (Qiagen 2020). The purified PCR products were later sent for sequencing at Apical Scientific Sdn. Bhd., Se langor. The results obtained were then compared with the databases from National Center for Biotechnology Infor mation (NCBI).

Experimental design
The best cellulase producer of Trichoderma sp. was cho sen for SSF process. The effect of Trichoderma sp. on each of the following parameters was conducted by chang ing one factor at one time. All experiments were con ducted in a triplicate manner and the means of the results were compared with oneway ANOVA and considered sig nificant when p < 0.05.

Preparation of Trichoderma sp. spores
The Trichoderma sp. was grown on the petri dish for 7 days. After 7 days the plate was flooded with sterile dis tilled water (dH 2 O) and spores were harvested using a ster ile glass scraper. The harvested spores were then mea sured for their concentration using a hemocytometer and adjusted using dH 2 O to obtain a concentration of 10 7 .

Solid extraction of cellulase
The weight of the fermented solid substrate was deter mined and dH 2 O was added into the flasks, approximately five times of the solid substrate weight. The mixture was then stirred with a magnetic stirrer for 30 min at 700 rpm. After that, the mixture was centrifuged at 8000×g for 15 min (Sigma 4K10, B. Braun, Germany) and the super natant separated was taken as the enzyme extract and used in FPase and CMCase tests.

Effect of different agriculture waste on cellulase production
Three agricultural wastes were used in this study (wheat bran, rice bran and rice husk). Five millilitter of a mixture containing 10 7 of Trichoderma sp. spores was inoculated into a conical flask containing 20 g of each agriculture waste while moisture content and temperature were main tained at 50% and 28°C respectively. Results were ob served every day for 12 days and the cellulases (FPase and CMCase) activity was measured using the DNS method.

Effect of different moisture content on cellulase production
Trichoderma sp. spores concentration of 10 7 was inoc ulated into a conical flask containing 20 g of each agri culture waste according to the moisture content desired (20%-80%) and the temperature was maintained at 28±2°C . Results were observed for 12 days and the cellulases (FPase and CMCase) activity was measured using the DNS method.

Effect of different temperatures on cellulase production
Five mililitter of Trichoderma sp. spore of 10 7 was inoc ulated into a conical flask containing 20 g of each agricul ture waste. Moisture content was set at 50% while tem perature was set at 28°C, 35°C, 45°C and 55°C. Results were observed for 12 days and the cellulases (FPase and CMCase) activity was measured using the DNS method.

Determination of cellulase production using optimized condition
Trichoderma sp. spores of 10 7 was inoculated into a con ical flask containing 20 g optimized substrate and incu bated under optimized moisture content and temperature for 7 days. Cellulases (FPase and CMCase) activity was measured using the DNS method.

Isolation, cellulase screening and identification of Trichoderma sp.
A total of 30 Trichoderma sp. were isolated from the soil samples. All the Trichoderma sp. was screened for their ability to secrete FPase and CMCase activity which will gave a representative on the total cellulase activity se creted by Trichoderma sp. Out of the 30 isolates of Tri choderma sp. isolated, only five isolates of the Tricho derma sp. showed high FPase and CMCase activity of 7.4 U/mL-9.5 U/mL and 7.5 U/mL-10.9 U/mL respectively at day 6 of incubation (Table 1). Based on the results of the five potential isolates of Trichoderma sp. that we ob tained (Table 2), Trichoderma harzanium TF2 was chosen for further investigation due to its highest cellulases pro

Effect of different agriculture waste on cellulase production
From the obtained data, it was observed that wheat bran showed a better source for the secretion of enzyme cel lulase compared to rice bran and rice husk (Figure 1). The use of wheat bran showed an increase of 23.6% and 64.4%, respectively ,in the cellulases activity production (FPase and CMCase activity) by T. harzianum TF2 when compared to rice bran and rice husk. These results were in accordance to a study conducted by Haq et al. (2006), whereby they observed that T. harzanium KM07 produced 16 U/g of total cellulases in wheat bran, but the amount de creases when they are using rice bran (13 U/g) and rice husk (12 U/g). According to Nochur et al. (1993), the amount of nutrients in wheat bran, such as protein 1.32%, carbohydrate 69.0%, fats 1.9%, fiber 2.6%, ash 1.8%, Ca 0.05%, P 0.35%, Mg 0.17%, S 0.12% and K 0.45% might be one of the factors that influence the fungal growth and subsequently better cellulases production when wheat bran was used as substrate in the fermentation. This state ment was further supported by Brijwani et al. (2010) and Kittanan et al. (2018) when these researchers stated that an increased in the secretion of cellulase by Trichoderma sp. was due to the presence of wheat bran which con tained high protein and starch. This was noted by Kit tanan et al. (2018), when wheat bran was added to copra waste, the cellulase activity produced by Trichoderma ree sei increased from 0.31 FPU/g-5.23 FPU/ g dry substrate at day 2 and 0.42U/g-5.18 U/ g dry substrate at day 6 of solid state fermentation respectively. Triwahyuni et al. (2018) also showed that Trichoderma sp. T004 isolated from Indonesian soil produces highest cellulase activity (0.52 FPU/mL) when wheat bran was used. This further established the reason why wheat bran was the best sub strate for cellulase production by Trichoderma sp.

Effect of different moisture content on cellulase production
According to Liu and Yang (2007), the optimal moisture content for solid state fermentation should be 40%-60% (v/w). In this study, it was observed that at 60% of mois ture content (Figure 2c), the production of cellulases activ ity by Trichoderma harzianum TF2 was induced from 23.8 U/g to 33.3 U/g when moisture content increased from 40% to 60% (v/w) (Figure 2b and Figure 2c). According to Liu and Yang (2007), an increase moisture content at a certain level will caused the cellulases enzyme production to decrease. This is because the moisture content will re duce the surface area of the substrate and this will affect the accessibility of the air to the substrate, thus affecting the growth and metabolism of the microbes (Liu and Yang 2007). Irfan et al. (2014), concurred with the statement made by Liu and Yang (2007), when they observed that at the ratio of 11:10 (mL:g) Trichoderma virideIR05 pro duces 64.3 U/g of xylanase activity, but when the ratio of water was increased to 13:10 (mL:g) the activity was re duced to 55 U/g. In this study, it was also observed that the cellulase activity reduced from 33.3 U/g to 29.6 U/g in wheat bran when the moisture content was further in creased from 60%-80% (v/w) (Figure 2c and Figure 2d). At 20% (v/w) moisture content it was observed that T. harzanium TF2 required 9 days to colonize the substrate and to obtain optimum cellulase activity of 19.6 U/g in wheat bran (Figure 2a). This is much lower when we com pared to cellulase activity at 60% (v/w), which is 33.3 U/g in 7 days of fermentation (Figure 2c). Another research done by Sachdev et al. (2018) noted that Trichoderma lixii growth at optimum when 68.87% of moisture content was used. This further justified that Trichoderma sp. grows well in moisture content of approximately 60%.

Effect of different temperature on cellulase production
The optimum temperature for the highest cellulase pro duction was when Trichoderma sp. was grown at 28°C (Figure 3). It was observed that, at 28°C (Figure 3a), Trichoderma harzianum TF2 produced 32.6 U/g of cellu lases activity (total of CMCase and FPase activity) when wheat bran was used however, when the temperature was increased to 45°C ( Figure 3c) the cellulases activity was drastically decreased to 16.1 U/g for the same substrate. Darabzadeh et al. (2019), reported that at 30°C produc tion of cellulase was 1.16 U/g but decreased to 0.85 U/g when the temperature was increased to 35°C. This indi cates that the optimum temperature for the enzymatic re action to take place should be around 2530°C. Our find ings fit well in this region. According to Ali et al. (2017), fungi grow best at 25-30°C. However, this differs from the genus, species and strain of the fungus. Singh et al. (2014), in their study, observed that T. harzianum pro duces the highest biomass at 25°C -30°C. According to work done by Iqbal et al. (2010), CMCase activity in creases until the temperature reached 35°C and decreased as the temperature increased further. In our study, it was observed that CMCase activity do increase until 35°C but the incremental was just 0.1U/g. However, beyond 35°C all cellulases activity (CMCase and FPase) reduced and this concurred with Iqbal et al. (2010).

Cellulase production using optimized conditions
Trichoderma harzianum TF2 showed an optimum cellu lase activity (CMCase and FPase) of 38.5 U/g when SSF was conducted under the combined optimized condition (Figure 4). This showed an incremental of approximately 19% compared to cellulases activity produced when op timization was done for each parameter only. Sari et al. (2013), reported that the combination of T. reesei with rice straw as substrate only gives a reading of 1.80 IU/mL of cellulase activity which much lower than the results showed by T. harzanium TF2 in this study. Haq et al. (2006), indicated that their formulated condition was only able to give an incremental of 11% in cellulase produc tion for T. harzanium KM07 used. In a study conducted by Rahnama et al. (2013), T. harzanium SNRS3 produces 117.56 U/g of cellulase activity (6.25 U/g of FPase and 111.31 U/g of CMCase), the results obtained were high compared to the results we obtained in this study. How ever, it was noticed that the FPase activity obtained was low compared to 17.7 U/g obtained in this study. The abil ity of different isolates of T. harzanium to react to their op timized condition will produce different increment rates in their cellulases activity. The current conditions used for T. harzianum TF2 indicated that these were the most suitable conditions for T. harzanium TF2 to produce cellu lases activity at this moment. There might be other param eters that will increase the cellulase activity of T. harza nium TF2 however, those parameters were not yet tested in this study. From all the test conducted it was noted that cellulase production increase as the biomass of T. harza nium TF2 increased. Darabzadeh et al. (2019), stated that T. reesei biomass production correlates with the enzyme production.

Conclusions
Trichoderma harzanium TF2, isolated from banana rhi zosphere, produced a high cellulases activity of 38.5 U/g when wheat bran was used as the substrate, incubated at 28±2 ºC and moisture was kept at 60% (v/w).