Introduction

A large amount of plant waste is being continuously accumulated on the surface of earth. A part of the waste gets into the soil due to the biodegradation process of soil microbes including microfungi. Intensity of decay and degradation process depends on different environmental factors such as species of plant, sort of soil, moisture, temperature, the type of soil microflora and their metabolic processes. In order to increase the efficiency of these metabolic processes, the scientific society is making strong efforts in this area of research. Moreover, the aim of the latest researchers are oriented to intensify plant remnants decay and degradation and modify metabolites enriching the soil by useful biologically active substances and finding materials able to enrich forage and food by important biologically valuable additions (Reid, 1989; Kelley, 1992; Varnaite, 2001).

Different substrata basically consisting of cellulose, hemicellulose and lignin are the main nutrient source of microorganisms. Degradation process of plant waste is stimulated by various enzymes based on the microorganisms activity (Yoshida et al., 1996; del Pilar Castillo et al., 1997; Ortega et al., 2001; Bridziuviene and Lugauskas, 2003; Tanaka et al., 2009).

Fungi play an important role in the biodegradation of various substances rich in cellulose and lignin (Babickaja, 1994; Daljit, 1995; Petre et al., 2005). Cellulose of plant material is degraded by fungal cellulolytic enzyme cellulase. It is capable of degrading crystalline forms of cellulose. The enzyme composed of three enzyme species: endo-b-1,4 glucanase, exo-b-1,4-glucanase (cellobiohydrolases) and b-glucosidase (cellobiases). The three enzyme groups work synergistically to hydrolyse crystalline cellulose. The net effect is the release of carbondioxide and it is used as an index to find out decomposition of cellulose (Paul, 1992).

The aim of this study is to isolate fungi from virgin soil of Ooty forest ecosystem and to estimate the cellulose degradation potentials of Acacia sp. leaf litter by these fungi through CO₂ releasing method.

Materials and Methods

Isolation and identification of microfungi from leaf litter degrading virgin soils

The surface of the soil profile was cleaned and vertical samples were taken from 10 cm depth with a disinfected spatula. The spatula was applied perpendicular to the vertical surface of the soil profile. The samples were stored in a sterilized cooled bottle until they reached the laboratory. The samples were processed in an isolation process using the soil dilution plate (Waksman, 1922) in Czapek’s sucrose-nitrate-agar medium (Booth, 1971) in the inoculation chamber in Petri dishes and then incubated at 28 ± 1° C. Further subcultures were made and the pure cultures were maintained in the laboratory for further studies. The identification of fungi was performed following Domsch et al. (1980). The 40 fungal species isolated and identified are listed below:

Acremonium murorum (Corda) W. Gams (Moniliaceae)

Amorphotheca resinae Parbery (Amorphothecaceae)

Aspergillus alutaceus Berk and Curt. (Moniliaceae)

Aspergillus candidus Link ex Link (Moniliaceae)

Aspergillus erythrocephalus Berk. and Curt. (Moniliaceae)

Aspergillus flavus Link ex. Gray (Moniliaceae)

Aspergillus fumigatus Fres. (Moniliaceae)

Aspergillus niger van Tieghem (Moniliaceae)

Aspergillus oryzae (Ahlburg) Cohn (Moniliaceae)

Aspergillus restrictus G. Sm. (Moniliaceae)

Aspergillus terreus Thom. (Moniliaceae)

Aspergillus ustus (Bain) Thom and Church (Moniliaceae)

Aspergillus versicolor (Vuill.) Tiraboschi (Moniliaceae)

Botrytis cinera Pers. ex Nocca and Balb (Moniliaceae)

Chaetomium crispatum (Fuckel) Fuckel (Melanosporaceae)

Chaetomium funicola Cooke (Melanosporaceae)

Cladosporium herbarum (Pers) Link ex Gray (Dematiaceae)

Curvularia lunata (Wakker) Boediju (Dematiaceae)

Fusarium chlamydosporum Wollenw and Reink (Tuberculariaceae)

Fusarium oxysporum Schlect emend. Sny. and Hans. (Tuberculariaceae)

Fusarium poae (Peck) Wollenw (Tuberculariaceae)

Fusarium solani (Mart) Sacc (Tuberculariaceae)

Helminthosporium solani (Pleosporaceae)

Humicola fuscoatra Traaen (Deuteromycotina)

Memnoniella echinata (Riv) Galloway (Deuteromycotina)

Mortierella alpina Peyronel (Deuteromycotina)

Mucor mucedo Mich. ex St-Am (Mucoraceae)

Mucor racemosus Fres. (Mucoraceae)

Paecilomyces carneus (Duche and Heim) A. H. S. Brown and G. Sm. (Deuteromycotina)

Penicillium chrysogennm Thom. (Moniliaceae)

Penicillium funiculosum Thom. (Moniliaceae)

Penicillium janthinellum Biourge (Moniliaceae)

Penicillium verrucosum Dierckx (Moniliaceae)

Periconia prolifica Lefebvre and A. G. Johnson (Dematiaceae)

Pleospora herbarum (Fr ex Fr) Rabenh (Pleosporaceae)

Rhizoctonia solani Kuhn (Ceratobasidiaceae)

Rhizopus oryzae Went and Prinsen Geerligs (Mucoraceae)

Trichoderma viride Pers ex Gray (Tuberculariaceae)

Trichosporiella cerebriformis (de Vries and Kleine-Natrop W.Gams) (Deuteromycotina)

Trichothecium roseum (Pers) Link ex Gray (Moniliaceae)

Fungal cultures for biodegradation studies

The pure cultures of all the forty fungal species were tested for cellulose biodegradation studies. From the pure culture, a loopful of fungal spores along with mycelia were inoculated in 250 ml conical flask under sterile condition in 50 ml Czapek’s-Sucrose-Nitrate liquid medium in an inoculation chamber. After inoculation the culture bottles were incubated at 28 ± 1° C. The growth of fungus appeared after 24 hours of inoculation.

Determination of CO₂ release during cellulose biodegradation

To the conical flask with 24 hr of fungal inoculum, 500 mg of dried leaf material as substrate was added in small pieces. Vials containing 5 ml of 5 N sodium hydroxide solution was suspended with the help of a thread. The conical flask was closed with stopper and sealed with parafilm membrane to ensure air-tight condition and incubated at 28° ± 1° C. The fungus colonizes the dried Acacia dealbata leaf material degrading cellulose material with the release of CO₂. It was absorbed by sodium hydroxide in the vials. During each estimation the content of the vials was quantitatively transferred to a flask followed by the addition of 5 ml of saturated solution of barium chloride to precipitate the CO₂ as barium carbonate. Two drops of phenolphthalein was added. The residual amount of sodium hydroxide in the flask was measured by titrating against 0.1 N hydrochloric acid. The end point is the disappearance of pink colour (Gaur et al., 1971).

Calculation

1 ml of 0.1 N hydrochloric acid = 1 ml of 0.1 N sodium hydroxide

= 2.2 mg of carbondioxide evolved

Results and Discussion

Forty fungal species which colonize the forest ecosystem of Ooty have been isolated and their cellulose biodegradation potentials were estimated through CO₂ release method on 10^th, 20^th and 30^th days of degradation process and the results are presented in Table-1.

On the 10^th day of cellulose biodegradation, CO₂ release by Humicola fuscoatra showed a maximum of 72 mg followed by 68 mg by Paecilomyces carneus, 66 mg by Memnoniella echinata and Rhizotonia solani, 62 mg by Acremonium murorum,  60 mg by Fusarium oxysporum and F. solani, the minimum of 28 mg by Aspergillus fumigatus and Penicillium verrucosum. On the 20^th day of cellulose biodegradation CO₂ release by H. fuscoatra showed a maximum of 96 mg followed by 92 mg by  A. murorum and M. echinata, 86 mg by P. carneus, 82 mg by R. solani, 80 mg by  F. solani and 72 mg by A. versicolor, Cladosporium herbarum and F. oxysporum, the minimum of 38 mg by Helminthosporium solani and P. chrysogenum. On the 30^th day of cellulose biodegradation CO₂ release by H. fuscoatra showed a maximum of 64 mg followed by 58 mg by P. carneus, 56 mg by A. murorum, C. herbarum and P. carneus, 54 mg by R. solani and Trichoderma viride, 52 mg by Trichothecium roseum, the minimum of 24 mg by Aspergillus candidus, A. flavus and Mucor racemosus.

Table 1: CO₂ release (mg) during Acacia dealbata leaf litter cellulose biodegradation of fungi isolated from forest ecosystem of Ooty.

At the end of 30^th day of cellulose biodegradation the maximum total amount of CO₂ release of 232 mg was found in H. fuscoatra, followed by 220 mg by P. carneus, 216 mg by M. echinata, 210 mg by A. murorum, 202 mg by R. solani, 186 mg by F. solani, the minimum of 92 mg by A. restrictus.

From this result it can be grouped that A. murorum, H. fuscoatra, M. echinata, P. carneus and R. solani are the fast cellulose degrading fungi. Amorphotheca resinae, Aspergillus alutaceus, A. erythrocephalus, A. niger, A. oryzae, A. terreus,  A. versicolor, Botrytis cinera, Chaetomium funicola, C. herbarum, Curvularia lunata, F. chlamydosponum, F. oxysporum, F. solani, Rhizopus oryzae, Trichoderma viride and Trichothecium roseum are moderate cellulose degrading fungi, whereas Aspergillus candidus, A. flavus, A. fumigatus, A. restrictus, A. ustus, Chaetomium crispatum, Fusarium poae, Helminthosporium solani, Mortierella alpina, Mucor mucedo, M. racemosus, Penicillium chrysogenum, P. funiculosum, P. janthinellum,  P. verrucosum, Periconia prolifica, Pleospora herbarum and Trichosporiella cerebriformis are the slow cellulose degrading fungi.

The various levels of biodegradation are due to the activities of fungal enzymes. These fungi are capable of producing cellulose enzymes such as endoglucanase, exoglucanase and b-glucosidase in their system (Fahnrich et al., 1981; Schuabel, 1981; Kannan et al., 1990). Cellulose enzyme is capable of degrading crystalline forms of cellulose of endo b-1, 4-glucanases, exo-b-1,4-glucanases (Cellobiohydrolases) and b-glucosidases (Cellobiases). The net effect of these three enzymes is to rapidly decrease the polymer length with a slow increase in reducing group (Moore and Landecker, 1972; Coughlan, 1989). The fast cellulose degrading fungi in this study namely A. murorum, H. fuscoatra, M. echinata, P. carneus and R. solani have such high potential of producing cellulose enzymes in their system so as to degrade the cellulose in leaf litter substrate more effectively and the result is in accordance with the study conducted by Abubacker et al. (2001) with respect to cellulose degradation of commercial paper as substrate. Cellulolytic enzymes play an important role in natural biodegradation process in which plant lignocellulose materials are efficiently degraded by fungi (Varnaite et al., 2011). Many fungi probably produce b-glucosidase, a large enzyme with a molecular mass of 32 KDa, active with glucose and cello oligosaccharides (Goodell, 2003). b-glucosidase enzyme responsible for most of the glucosidase activity is optimum in certain fungi which are capable of degrading cellulose. The cellulolytic and hemicellulolytic enzymes systems cannot be separated, since several enzymes show activity against more than one substrate (Cohen et al., 2005). However the measurement of carbondioxide release during the biodegradation process is used as an index of cellulose decomposition (Gaur et al., 1971). From this study it is to be concluded that the fungi like A. murorum, H. fuscoatra, M. echinata, P. carneus and R. solani are capable of producing cellulose degrading enzymes at a faster rate to decompose the leaf litter substrate cellulose and released more CO₂ and hence these potential fungi can effectively add more nutrients by way of degradation in forest ecosystem.

Acknowledgement

The authors thank DST-FIST, Government of India, New Delhi for providing the infrastructure facilities for the Department of Botany, National College, Tiruchirappalli. The authors also thank Shri. K. Ragunathan, Secretary and  Dr. K. Anbarasu, Principal, National College, Tiruchirappalli for their encouragement.

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