Pak. J. Bot., 41(2): 907-916, 2009.

CELLULASE BIOSYNTHESIS BY SELECTED TRICHODERMA SPECIES

SHAZIA SHAFIQUE*, RUKHSANA BAJWA AND SOBIYA SHAFIQUE Institute of Mycology and Plant Pathology, University of the Punjab, Quaid-e-Azam Campus Lahore, Pakistan.
Abstract The enzyme cellulase, a multi enzyme complex made up of several proteins, catalyses the conversion of cellulose to glucose in an enzymatic hydrolysis. The indigenous fungi of Pakistan viz., Trichoderma viride Pers. ex Gray, T. reesei Rifai and T. harzianum Rifai were selected and analyzed on the basis of extent of hydrolyzing zones for the evaluation of their enzymatic activity in solid state fermentation on PDA and plate screening medium (PSM) at pH 4, 5 and 6. PSM at pH 4 depicted best results for all the strains tested. Strains FCBP-142 and FCBP-232 of T. viride, strains FCBP-271 and FCBP-364 of T. reesei and strains FCBP-210 and FCBP-325 of T. harzianum were considered best in their extent of hydrolyzing ability and were selected for evaluation of their cellulolytic activity through submerged fermentation using 3, 5-dinitrosalicylic acid (DNS) method. The maximum enzymatic activity was achieved after 72 hours of incubation at 30 2oC at initial pH 4.

Introduction Cellulose is the most abundant and renewable biopolymer on earth. Cellulose has been used by man for centuries, however, its enormous potential as a renewable source of energy was recognized only after cellulose degrading enzymes or cellulases had been identified (Bhat & Bhat, 1997). A cellulosic enzyme system consists of three major components: endo--glucanase (EC 3.2.1.4), exo--glucanase (EC 3.2.1.91) and -glucosidase (EC 3.2.1.21) (Knowles et al., 1987). The mode of action of each of these is as follows: 1. 2. 3. Endo--glucanase, 1,4Dglucan glucanohydrolase, CMCase, Cx: random scission of cellulose chains yielding glucose and cell-oligo saccharides. Exo--glucanase, 1,4-D-glucan cellobiohydrolase, Avicelase, C1: exo-attack on the non-reducing end of cellulase with cellobiose as the primary structure. -glucosidase, cellobiase: hydrolysis of cellobiose to glucose.

These components act synergistically in the conversion of cellulose to glucose (Eveleigh, 1987). Ali & Akhand (1992) worked on cellulase production by mesophilic Trichoderma isolate during growth on water hyacinth under optimized conditions. The cellulase complex of Trichoderma reesei has been most thoroughly studied. It is complete in that it can convert native cellulose as well as derived celluloses to glucose (King & Nessal 1969). Ahmad et al., 2003 worked on Trichoderma harzianum for cellulase enzyme production by using different carbon sources and reported that CMC is the best for substantial amount of enzyme production.
*

Corresponding author: [email protected]

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Since growth of fungi as well as the enzyme production depends on the composition of the growth media, water activity (aw), pH, temperature, light and the surrounding atmospheric gas mixture. The effect of environmental factors on the growth of fungi is generally less specific and restricted than the effect on secondary metabolite production. For example, the ranges of water activity, growth medium and pH within which formation of certain secondary metabolites occur, is narrower, than the range of conidial growth (Northolt & Bullerman, 1982). The work on cellulase enzyme production by Trichoderma species has been conducted all over the world. But the physiological responses of same organism or species may vary with ecological variations. Therefore, the present research work was aimed to evaluate the cellulase production potential by native Trichoderma strains, in particular by strains of Trichoderma viride, Trichoderma reesei and Trichoderma harzianum that were available in First Fungal Culture Bank of Pakistan (FCBP). Thus, the novelty of this work is that the work is concerned with commercialization of mycoflora of FCBP to meet industrial sector demands. Materials and Methods Experimental design: For Trichoderma reesei and Trichoderma viride a 332 factorial and for Trichoderma harzianum 632 factorial with 3 and 6 fungal strains respectively, 3 pH levels viz. 4, 5 and 6, were assessed on two growth media viz., Potato Dextrose Agar medium (PDA) and Plate Screening Medium (PSM), with 6 replicates each in a completely randomized design. Microorganism: The strains of Trichoderma viride (isolates FCBP-142, FCBP-167, FCBP-232), T. reesei (isolates FCBP-84, FCBP-271, FCBP-364) and T. harzianum (isolates FCBP-125, FCBP-139, FCBP-140, FCBP-193, FCBP-210, FCBP-325) were obtained from stock cultures of First Fungal Culture Bank of Pakistan, Institute of Mycology and Plant Pathology, University of the Punjab, Lahore. The cultures were maintained as direct stock cultures on Potato Dextrose Agar (PDA) plates at 30 2oC and stored at 4oC with regular sub culturing. Inoculum/spore suspension: The inoculum was prepared according to the method of Noomrio & Dahot, (1992). The stock suspension was serially diluted to prepare a conidial suspension of 5 x 105 conidia m L-1 with the help of haemacytometer (Neubauer Precidor HBG, Germany). This appropriate dilution was used as inoculum. Plate screening assay: The experiment was carried in two sets. In one set, the plate screening medium (PSM) contained Mendels mineral salt solution that is: Urea 0.3 g L-1, (NH4)2SO4 1.4 g L-1, KH2PO4 2.0 g L-1, CaCl2 0.3 g L-1, MgSO4 0.3 g L-1, yeast extract 0.25 g L-1 and proteose peptone 0.75 g L-1 with 10 g L-1 of cellulose and 17.5 g L-1 agar (Mandels, 1974) while the other set was composed of PDA. Three levels of pH: 4, 5 and 6 were adjusted for each set (pH was adjusted by either 0.5M NaOH or 0.5M HCl) before autoclaving. The suspensions were plated on both sets and were incubated at 30 2oC for five to seven days. After 7 days, the colonies with clear zones of cellulose digestion were selected and top two strains among each species were further assayed for cellulase activity.

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Fermentation conditions: Fermentation experiment was run according to Mendels method (Mandels, 1975). The mass concentration of nutrients in Mendels salt solution is: Urea 0.3 g L-1, (NH4)2SO4 1.4 g L-1, KH2PO4 2.0 g L-1, CaCl2 0.3 g L-1, MgSO4 0.3 g L-1, yeast extract 0.25 g L-1 and proteose peptone 0.75 g L-1 with 10 g L-1 of Carboxymethylcellulose (CMC) in 0.05M citrate buffer (pH 4). In 250 mL conical flasks, 50 mL of sterilized culture medium was taken and inoculated with 1.0 mL of conidial suspension (5 x 105 conidia mL-1) of each fungal species in triplicates. The flasks were incubated in orbital shaker incubator (100rpm) at 30 20C for 96 hours. After every 12 hours interval, the fermented broth was centrifuged at 3,000 rpm for 15 minutes and the supernatant (crude enzyme) was used for further analysis. Enzyme estimation assays: The overall cellulolytic activity in broth culture was determined by 3-5, dinitrosalicylic acid (DNS) method of Miller (1959). The reaction mixture contained 0.5 mL of 1% CMC in 0.05 M citrate buffer (pH 4) and 0.5 mL of enzyme solution. It was incubated at 50C for 15 min. Then 3 mL of DNS reagent was added and the mixture was boiled for 5 min. After cooling, the absorbance was measured at 600 nm using spectrophotometer. Sugar content was measured using a calibration curve of glucose. Enzymatic activity of cellulase complex was expressed as International Units/mL defined as the amount of enzyme which releases one micro mole of reducing sugar expressed as glucose per minute. Statistical analysis: Data regarding the hydrolyzing zones formed by different strains of three test species of Trichoderma as affected by pH and growth media were statistically analyzed using three way analysis of variance (ANOVA) followed by LSD method to demarcate mean differences. Data regarding cellulase activity of the selected strains of Trichoderma spp., were subjected to one way ANOVA followed by Duncans Multiple Range Test to separate the means (Steel & Torrie, 1980). Results The test strains of each Trichoderma species viz., Trichoderma viride, T. reesei and T. harzianum were analyzed on the basis of extent of hydrolyzing ability. Cellulose hydrolysis of all three strains of T. viride was immensely affected by varying pH and medium. The results indicated statistically significant interaction in all correlating factors of strain, growth medium and pH level in case of T. viride (Table 1). Cellulose digestion occurred in all three isolates between pH 46 but the maximum extension rate of approximately 53 1 mm was exhibited at pH 4 on PSM. The rate of hydrolysis was reduced tremendously as pH was increased from 46 (Fig. 1A-C). The three strains of T. reesei followed similar trends in response to changes in pH and medium (Fig. 2). Highest cellulose digestion, in terms of hydrolyzing zone, was observed at low pH (4) in PSM (Fig. 2A-C). There appeared reduction in hydrolyzing activity followed by further increase in pH. This effect was more pronounced and significant in strain FCBP-364 (Fig. 2C). Analysis of variance for Trichoderma harzianum (Table 3) depicted that increasing pH and substrates variation had a highly significant influence (p0.001) on hydrolyzing capabilities of different strains. The interaction between strain x growth medium had significant effect (p0.05) whereas the interactive effect between strain x pH x growth medium was highly significant (p0.001). All test strains showed the best hydrolyzing zone formation potential at pH 4, PSM further improved their activity significantly (p0.05) (Fig. 3). Cellulose digestion was inhibited by further increase in pH level.

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Table 1. ANOVA for hydrolyzing zones of different strains of Trichoderma viride under three pH levels and on two growth media. Sources of variation df SS MS F-values Treatments 17 2266 133 70*** Strains (S) 2 878 439 231*** pH 2 904 452 238*** Growth media (G) 1 334 334 176*** 4 65 16 8.5*** S pH 2 12 6 3.2* SG 2 48 24 12.7*** pH G 4 24 6 3.2* S pH G Error 90 171 1.9 Total 107 2437
Note: numbers represent F-values *** = p 0.001, * = p 0.05.

Table 2. ANOVA for hydrolyzing zones of different strains of Trichoderma reesei under three pH levels and on two growth media. Sources of variation df SS MS F-values Treatments 17 825 49 78*** Strains (S) 2 171 86 138*** pH 2 520 260 418*** Growth media (G) 1 82 82 131*** 4 30 7.6 12.2*** S pH 2 8 4 6.5** SG 2 6.3 3.2 5.1** pH G 4 6.4 1.6 2.5* S pH G Error 90 56 0.62 Total 107 881
Note: Numbers represent F-values *** = p 0.001, * = p 0.05.

Table 3. ANOVA for hydrolyzing zones of different strains of Trichoderma harzianum under three pH levels and on two growth media. Sources of variation df SS MS F-values Treatments 35 6659 190 149*** Strains (S) 5 4693 939 733*** pH 2 1317 658 514*** Growth media (G) 1 81 81 63*** 10 452 45 35*** S pH 5 15 3 2.3* SG 2 50 25 19*** pH G 10 51 5.1 4 *** S pH G Error 180 230 1.3 Total 215 6889
Note: Numbers represent F-values *** = p 0.001, * = p 0.05.

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P ot at o Dext rose Agar P lat e Screening Medium T. viride FC BP-142 LSD = 1.64 A

Hydrolyzing zone (mm)

55 50 45 40 35 pH 4

pH 5

pH 6

Hydrolyzing zone (mm)

55 50 45 40 35 pH 4

T. viride FC BP-167 LSD = 1.87 B

pH 5

pH 6

Hydrolyzing zone (mm)

55 50 45 40 35 pH 4

T. viride FC BP-232 LSD = 1.30 C

pH 5

pH 6

Fig. 1. Effect of pH and growth media on hydrolyzing zones of different strains of Trichoderma viride.

LSD at p0.05

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P ot at o Dext rose Agar P lat e Screening Medium

H ydrolyzing zone (m ) m

55 50 45 40 35 pH 4

T. reesei FC BP-84 LSD = 0.98 A

pH 5

pH 6

H ydrolyzing zone (m ) m

55 50 45 40 35 pH 4

T. reesei FC BP-271 LSD = 0.90 B

pH 5

pH 6

T. reesei FC BP-364 H ydrolyzing zone (m ) m 55 50 45 40 35 pH 4 pH 5 pH 6 LSD = 0.90 C

Fig. 2. Effect of pH and growth media on hydrolyzing zones of different strains of Trichoderma reesei.

LSD at p0.05

CELLULASE BIOSYNTHESIS BY TRICHODERMA SPECIES

Potato Dextrose Agar Plate Screening Medium 50 45 40 35 30 25 pH 4 pH 5 pH 6 T. harzianum FCBP-125 LSD = 1.89 A H y d ro ly z in g zo n e (m m ) T. harzianum FCBP-139 50 45 40 35 30 25 pH 4 pH 5 pH 6 LSD = 1.20 B

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H y d r o ly z in g z o n e ( m m )

H y d ro ly z in g zo n e (m m )

45 40 35 30 25 pH 4 pH 5

LSD = 0.93

H y d ro ly z in g z o n e ( m m )

50

T. harzianum FCBP-140

50 45 40 35 30 25 pH 4

T. harzianum FCBP-193 LSD = 1.66 D

pH 6

pH 5

pH 6

H y d r o ly zin g z o n e ( m m )

50 45 40 35 30 25

T. harzianum FCBP-210 LSD = 0.95 E H y d ro ly z in g zo n e (m m )

50 45 40 35 30 25

T. harzianum FCBP-325 LSD = 1.04 F

pH 4

pH 5

pH 6

pH 4

pH 5

pH 6

Fig. 3. Effect of pH and growth media on hydrolyzing zones of different strains of Trichoderma harzianum. LSD at p0.05

The selected fungal isolates were tested and compared for their cellulase activity by submerged fermentation. According to results obtained, it became obvious that enzyme activities of these fungi varied considerably even though they belong to the same genus. The comparison of Trichoderma species involving in the quantitative assay of cellulase activity has been presented in Fig. 4. The rate of saccharification of cellulases produced by Trichoderma species was carried out up to 96 hours. The maximum enzyme production was recorded up to 53.42 units mL-1 after incubation period of 72 hours at 30 2oC. All the strains exhibited over all good cellulolytic activity after 72 hours of incubation (Fig. 4A-C).

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0h
70 60 C ellulase activity (U nits m -1) L 50 40 30 20 10 0 S trai n FC BP-142

SHAZIA SHAFIQUE ET AL.,

12 h 24 h 36 h 48 h 60 h 72 h 84 h 96 h A

Trichoderma viride a a e f g h g
S trai n FC BP-232

b c e f d d

a b

Trichoderma reesei
70 60 C ellulase activity (U nits m -1) L 50 40 30 20 10 0

B a a b

a c f g h e d

b b f g h e

S trai n FC BP-271

S trai n FC BP-364

Trichoderma harzianum
70 60 C ellulase activity (U nits m -1) L 50 40 30 20 10 0 S trai n FC BP-210 S trai n FC BP-325

a a b f e g h g d c f e e

a d c

b b

Fig. 4. Cellulase activity of selected Trichoderma viride, T. reesei and T. harzianum strains. Each value is a mean average of three parallel replicates and Y-error bars indicate standard error among the replicates.

Of all the cultures tested, Trichoderma reesei strain FCBP-364 and Trichoderma viride strain FCBP-142 showed promising results by exhibiting 53.42 and 52.97 units mL-1 respectively. The strain FCBP-271 of T. reesei and strain FCBP-232 of T. viride though showed significant increase in enzymatic activity up to 72 hours but the level of their enzyme production was significantly lowered (47.755 and 48.333 units mL-1, respectively) than the other strains of same species. In case of T. harzianum strains FCBP-210 and FCBP-325 almost similar patterns of enzyme activity prevailed. However, the cellulase activity was found to be lowest i.e., 45.366 units mL-1 of strain FCBP-210 and 49.422 units mL-1 of strain FCBP-325, as compared to other two species of Trichderma. While among both strains of T. harzianum, the strain FCBP-325 was found to be more efficient as compared to other one.

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Discussion The pH of the medium has a direct influence on the growth of microorganisms carrying out fermentation process (Figs. 1-3). The rate of enzyme synthesis was favoured most at pH 4. Similar results were reported earlier by Suhr et al., (2002) while working on Penicillium caseifulvum who stated that low pH induces the production of secondary metabolites. Efficient colonization and utilization of lignocellulosic growth substrate depends upon the capacity of the organisms to produce the extracellular enzymes required to degrade the main polymers of the substrate, cellulose, hemicellulose and lignin. The physiology and extracellular lignocellulolytic enzyme production have been studied for some fungi from genus Pleurotus in submerged cultivation on synthetic media (Lee et al., 2000) and in solid-state fermentation of lignocellulosic substrates (Giardina et al., 2000). The results showed that all the selected isolates produced detectable quantities of cellulase on plate screening medium. Thus solid-state fermentation process was found to give fairly reliable indication of elevated cellulolytic activities. These findings are in line with the work conducted by Zaldivar et al., 2001. However, the selection of more efficient cellulolytic strains was made on biochemical basis (Elander, 1982). The ability of crude enzyme of Trichoderma species to hydrolyze cellulose presented remarkable variation in activity. Elisashvili et al., 2003, reported similar variations in enzymatic activity by different species of Pleurotus. Considerable variations in results have been observed among different strains of same species analyzed. These findings are correlated with the work on saccharifying activity of cellulases by T. harzianum (Haq et al., 2005). The temperature of the medium was kept at 30 2oC with shaking at 100 rpm, which is in accordance with the previous work conducted by Esterbaur et al., (1991) who found that optimum temperature for cellulase production from Trichoderma reesei was 15-28oC and optimal temperature for its growth was 30oC. Similarly, Smith & Wood (1991) have reported the optimum temperature of 30oC and 35oC for the production of extra cellular xylanases and xylosidase by Aspergillus awamori. The maximum activity was achieved after incubation period of 72 hours at 30 2oC. This is due to the fact that during this phase the microbes were in stationary phase. The results are in accordance with those of earlier work conducted on cellulolytic fungi by Aurangzeb et al., 1997. Further increase in the incubation period did not show any enhancement (Haq et al., 2005). Similarly glucose produced during fermentation may also be responsible for the feed back inhibition of the enzyme production as reported by Podukhe & Soman (1993). The data recommend that Trichoderma reesei strain FCBP-364 and Trichoderma viride strain FCBP-142 illustrated promising result by exhibiting 53.422 and 52.977units mL-1 respectively. The cellulolytic activities of these strains can be further enhanced by the mutagenic treatment (UV and chemical) of fungus. Further studies on these lines are in progress in our laboratory.
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