MICROBIOLOGY Screening of cellulolytic activity of some rhizospheric soil fungi isolated from different sources

ABSTRACT


Lignocellulosic
material is primarily composed of three different polymers: lignin, hemicellulose, and cellulose that are linked together.Cellulose is the most common organic compound in the world and the primary component of plant cell walls.It is composed of nearly 8,000 to 12,000 Dglucose residues linked together by β-1,4 bonds (Aro et al., 2005).Cellulose chains have a flat structure that is stabilized by internal hydrogen bonds.

(Festucci-Buselli et al., 2007).
Hemicellulose is a complex carbohydrate structure composed of various polymers such as pentoses (arabinose and xylose), hexoses (glucose, mannose, and galactose), and sugar acids.In hardwood and agricultural plants such as grasses and straw, xylan is the most abundant component of hemicellulose, whereas glucomannan is the most abundant component in softwood (Fengel and Wegener, 1984;Saha, 2003).Lignin is an amorphous heteropolymer composed of three different phenylpropane units (coniferyl, p-coumaryl and sinapyl alcohol) held together by various linkages.Lignin's major function is to provide impermeability, structural support, and resistance to microbial attack and oxidative stress to plants.The amorphous heteropolymer is also nonwater soluble and optically inactive, making lignin breakdown extremely difficult (Fengel and Wegener, 1984).
Lignocellulosic biomass can be transformed into fuels and other compounds by using a multienzyme system that hydrolyzes biomass to glucose.Hydrolytic efficiency is well established to be the result of the synergistic actions of a multi-component system containing at least three major groups of enzymes: Endoglucanases hydrolyze the cellulose polymer internally, while exoglucanases or cellobiohydrolases act on the reducing and non-reducing ends, releasing cellobiose and cellooligosaccharides; and β-glucosidases which cleaves cellobiose and releases two molecules of glucose (Gottschalk et al The present work aimed to isolate, identify, and screen the cellulolytic activity of some rhizospheric soil fungi.

Sample collection
Six samples were collected from Al-Gharbia governorate, Egypt.The soil samples were from various plant rhizospheres (rice, wheat, bean, sugar beet, and pea).
Soil samples were collected from Al-Gharbia governorate at a depth of 5 cm from the top and sieved using a two mm sieve.The samples were collected in bags and transported to the lab.The soil sample was air-dried before being used in vitro to isolate the fungi (Johnson et al., 1959).

Isolation of fungi from the collected soil samples
The dilution plate method was used to isolate soil fungi from the collected soil samples (Johnson et al., 1959).One gram of each soil sample was dissolved in 9 ml of sterilized distilled water in a sterile 100 ml conical flask.The flask containing the soil suspension was shaken vigorously in a shaker for 30 min at 160 rpm.One ml of the suspension was pipetted into a sterilized 100 ml Erlenmeyer flask containing 9 ml of sterile distilled water and shaked for a few minutes.Consecutive dilutions were made in the same way until a 10 -4 dilution was found to be suitable for plating, 0.1ml of 10 -4 dilution was transferred to a plate containing Czapek's -Dox agar medium with rose Bengal 25 µg/ml and spread over the surface.Three replicas of agar plates were prepared and incubated at 28 o C for 5 days.The fungal colonies were purified, identified, and kept as stock cultures in sterile Czapek's Dox agar slants at 4 ± 1 o C.

Identification of isolated fungi
The pure isolates were identified based on cultural, morphological features such as colony colour and growth, spore coloration, and microscopic features of hyphae and spore structures described in standard mycology books (Booth, 1971;Raper and Fennell, 1977;Moubasher, 1978;Domsch et al., 1980;Kitch and Pitt, 1992).

Screening for cellulolytic activity based on clear zones using solid medium
Using Czapek's agar medium with carboxymethyl cellulose (CMC) as the only carbon source rather than sucrose, the diameter of clear zones surrounding growing fungal colonies was used to evaluate the cellulolytic activity of fungal isolates (Hasanin et al., 2018).The medium was prepared and sterilized at 121°C for 15 min before being poured into the sterilized Petri dishes.Separate discs of fungal isolates (5mm) were inoculated after solidification and incubated at 30±2°C for 5 days.Following incubation, 10 ml of Congo red (0.1 % w/v) was added to each dish for 30 minutes before being washed with 1M NaCl for 20 min.After that, dishes were treated with 5% acetic acid for 5min before being washed with distilled water (Darwesh et al., 2020).The clear zone is an indicator of cellulose degradation.

Carboxymethyl cellulase (CMCase) assay
Carboxy-methyl cellulase (CMCase) activity was determined as described by (Mandels et al., 1976).In this method 0.5 ml of fungal filtrate was added to 1% CMC dissolved in 0.05M Na-citrate buffer (pH 4.8) and incubated for 45 minutes at 45°C, then 1ml of dinitrosalicylic acid reagent was added to each tube and immersed in a boiling water bath at 95-100°C for 10 minutes before cooling after that the amount of reducing sugar was measured at 540 nm.This assay was performed on each of three fungal suspension replicas.

β-glucosidase (βGase) assay
β-glucosidase (βGase) activity was determined using the method described by (Mandels et al., 1976).In this method 0.5 ml of fungal filtrate was added to 1% D-salicin dissolved in 0.05M of Na-citrate buffer (pH 4.8) and incubated for 45 min of incubation at 45°C then 1ml dinitrosalicylic acid of reagent was added to each tube and immersed in a boiling water bath at 95-100°C for 10 min after cooling the amount of reducing sugar was measured at 540 nm.This assay was performed on each of three fungal suspension replicas.

Filter-paper cellulase (FPase) assay
Filter-paper cellulase (FPase) activity was determined as described by (Mandels et al., 1976).In this method 0.5 ml of fungal filtrate was added to 50 mg of filter paper (Whatman No. 1) in 1 ml of 0.05M Na-citrate buffer (pH 4.8) and incubated for 45 min at 45°C, then1ml of dinitrosalicylic acid reagent was added to each tube and immersed in a boiling water bath for 10 min after cooling the amount of reducing sugar was measured at 540 nm.This assay was performed on each of three fungal suspension replica.The amount of reducing sugar (glucose) was measured at 540 nm after cooling.This assay was performed on each of three fungal suspension replicas.

Statistical analysis
The data were subjected to one way analysis of variance (ANOVA) using the SPSS 19.0 software program, and the significance of the mean difference was determined using (Duncan, 1955).Values are very highly significant when p < 0.001, highly significant when p < 0.01 and significant when p ≤ 0.05.Results were reported as mean value ± SD.

Isolation of Fungi from different collected samples
Twenty fungal species were isolated from different places in Al-Gharbia governorate.These fungal species were found to belong to seven different genera "Trichoderma, Asperagillus, Rhizopus, Cephalosporium, Fusarium, Penicillium, Mucor".

Identification of isolated fungi
The fungal isolates were purified and identified using the morphological and microscopic features of their hyphae and spore's structures as indicated by consult keys in standard mycology books (Table 1).Screening for cellulolytic activity using solid culture Among 20 fungi, 17 were found to have cellulolytic activity as indicated by the clear zones, ranging from 1.2 to 9.3 cm in diameter as shown in Fig. 1.The highest clear zone (9.3 cm) was recorded by Trichoderma virdi (Rs-4).While the lowest clear zone diameter (1.2 cm) was recorded by Aspergillus nidulans (Ps-1) as shown in Table 2   Table (2

Screening for cellulase activity using liquid culture
Data represented in Table 3 indicated that the maximum carboxymethyl cellulase (CMCase) activity was recorded in Trichoderma virdi (Rs-4) (27.7 U/ml) followed by Aspergillus candidus (Ws-1) and Aspergillus niger (Bs-1) with 24.0 and 22.7 U/ml respectively.The activities of other remaining isolates ranged from 22.7 to 10.0 U/ml.Also, the maximum β glucosidase (βGase) activity was recorded in Trichoderma virdi (RS-1) (30.1 U/ml) and the remaining isolates ranged from 22.4 to 10.0 U/ml.Filterpaper cellulase (FPase) activity was the highest when Aspergillus niger (Ps-1) was applied and recorded (11.9 U/ml) and the remaining isolates ranged from 9.4 to 3.4 U/ml.Also, Table 3 indicated that the total cellulolytic activity was recorded in Trichoderma virdi (Rs-4), (67.2 U/ml), and the remaining isolates ranged from 52.1 to 26.8 U/ml.Rs: Rice soil, Ws: Wheat soil, Bs: Bean soil, Sbs: Suger beet soil, Ps: Pea soil, Ms: Mixed soil All data represented means of 3 replica ± standard Deviation (SD).The P-value is < 0.001.Results nonsignificant = non-significant difference at P > 0.05, *significant at P ≤ 0.05, **highly significant at P ≤ 0.01, ***very highly significant at P ≤ 0. 001.Different capital letters indicate significant differences at p ≤ 0.05 using Duncan's test.The results of a screening of cellulase activities by isolated fungi using a liquid medium indicated that Trichoderma viride Rs-4 had the highest carboxymethyl cellulase (CMCase) activity, followed by Asperagillus candidus (Ws-1) and Asperagillus niger (Ps-1).Also, the maximum β glucosidase (βGase) activity was recorded by Trichoderma viride (Rs-4).Filter-paper cellulase (FPase) activity recorded with Asperagillus niger (Ms-2) was the highest.The highest total cellulolytic activity was recorded by Trichoderma viride (Rs-4).

Conclusion
This study revealed that Trichoderma viride and Aspergillus niger are promising fungi that have cellulolytic activity.Therefore, we recommend the use of these promising isolates in further study to improve the production of cellulase enzyme for use in saccharification and the use of the resulting reducing sugars in the production of bioethanol and biohydrogen.

Table ( 1
): List of fungal species isolated from different sources Rs: Rice soil, Ws: Wheat soil, Bs: Bean soil, Sbs: Suger beet soil, Ps: Pea soil, Ms: Mixed soil

Table ( 3
): Screening for the cellulolytic activity of tested fungi in liquid culture.

) and Kuczek- Turpeinen et al., (2005) obtained
similar results, reporting that cellulolytic activity of tested Trichoderma and Asperagillus species was relatively higher.According to Lynd