Isolation and Optimization of Lignocellulose Degrading Fungi

Today bioethanol has been recognized as the most common non-petroleum based renewable fuel as it provides unique environmental and economic strategic benefits and can be considered as a safe and cleanest liquid fuel alternative to fossil fuels. It has also been reported that total potential bioethanol production from lignocellulosic biomass (LB) is about 16 times higher than current ethanol production from sugars or starch biomass (Kim and Dale, 2004). Hence, LB is considered a future alternative for the agricultural products that are currently used as feedstock for bioethanol production, because it is more abundant and less expensive than food crops, especially when their wastes are used. Furthermore, the use of lignocellulosic biomass is more attractive in terms of energy balances and emissions (Kheshgi et al., 2000). Lignocellulosic biomass consists of three main components, i.e., carbohydrate polymers called cellulose and hemicellulose that can be converted to sugars, and a non-fermentable fraction called lignin (Hayn et al., 1993) that can be utilized for the production of electricity and/or heat. Typical bioethanol production processes using lignocellulosic feedstocks consist of four major steps: pretreatment, hydrolysis, fermentation, and separation (Joanne et al., 2010). The key element in bioconversion process of lignocellulose is the hydrolytic enzymes mainly cellulases. This enzyme is produced by several microorganisms, mainly by bacteria and fungi. Fungi are the main cellulase producing microorganisms, although a few bacteria and actinomycetes have also been reported to yield cellulase activity. Crude enzymes produced by these microorganisms are commercially available for agricultural and industrial uses. There are growing market for cellulases in the field of detergent industry and saccharification of agriculture wastes for bioethanol technology (Sukumaran et al., 2009). The ability of fungi to degrade lignocellulosic materials is due to their ability to secrete highly efficient enzymatic system and many studies have been done on cellulase enzymes complex consists of three major enzymes components that works synergically in cellulose degradation. Filamentous fungi, particularly Aspergillus and Trichoderma species, are well known as efficient producers of these cellulases. Wild type and mutant strains of Trichoderma spp. (T. viride, T. resei, T. longibrachiatum) have long been considered to be the most productive and powerful destroyers of crystalline cellulose (Gusakov et al., 2007). Carboxymethyl cellulase (CMCase), one of the members of cellulase complex, cleaves the internal glycosidic bonds of cellulosic chains and acts synergistically with exoglucanases and β-glucosidases during the solubilization of cellulosic materials. The study was aimed to screen the cellulolytic ability of fungi.

Soil samples were collected from different degrading sites of Rohtak (Haryana). Samples were collected from a depth of 4-6cm and were carried to laboratory in sterilized polythene bags. One gram of soil sample was suspended in 100mL sterilized normal saline and incubated at 30oC at 200rpm for one hour. Hundred microlitre of soil suspension from 10־¹ to 10-7 dilution were spread on the (PDA)

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medium plates and incubated at 30oC for 3-5 days. The petriplates were gently rotated clockwise and anticlockwise for uniform spreading of the soil suspensions. The PDA medium was supplemented with 60µg/mL of ampicillin as an antibacterial agent. The petriplates were then incubated at 30ºC in an incubator for 5 days. Isolated fungi were streaked extensively on PDA plates.

Potato peels - 200g Dextrose - 20g Agar - 15g pH - 7.0

For plate screening, carboxymethylcellulose-agar Mandles medium was used. This medium consisted of (g/L): (NH4)2SO4, 1.0; KH2PO4, 2.0; Urea, 0.3; MgSO4.7H2O, 0.3; CaCl2, 0.3; FeSO4, 5.0(mg/L); MnSO4, 1.6(mg/L); ZnCl2, 1.7(mg/L); Agar, 20 and CMC, 5.0. Fungal discs (dia 4mm) were inoculated on these plates in triplicates and incubated at 300C for 2-4 days to accelerate the action of extracellular cellulases and thus rapidly develop clear zone around the cellulose-producing colonies. For cellulolytic activity observations, plates were stained with 0.1% (w/v) Congo red dye for 15min followed by destaining with 1M NaCl solution for 15 to 20min. The zone of cellulose hydrolysis was apparent as a clear area in the otherwise congo red stain background and its diameter was measured. The strains that showed the most promising result was selected as potential cellulase producing fungi for further experiments.

To optimize the growth of fungi, the selected fungi were grown at different temperatures ranging from 20-45oC on PDA medium of varying pH range of 4.0-7.5 to observe the growth. The fungal growth was measured after definite time intervals.

The soil is always inhabitated by a large number of microorganisms like bacteria, actinomycetes, algae, fungi which form a major biotic component of soil. The PDA medium, meant for the growth of fungi, was utilised for isolating various degrading fungi from 20 soil samples collected from different sites of district Rohtak in Haryana (INDIA) by spread plate method through serial dilutions. Various fungi isolated from different soil samples of Rohtak were identified as follows: Aspergillus sp. R-1, Aspergillus sp. R-2, Aspergillus sp. R-3, Aspergillus sp. R -4, Rhizopus sp. R-1, Fusarium sp. R-1, Alternaria sp. R-1, Penicillium sp. R-1, Penicillium sp. R-2, Penicillium sp. R-3, Mucor sp. R-1, Trichoderma sp. R-1.

The cellulase producing colonies were identified by observing hydrolytic zone/s around colonies against dark background. The zone of hydrolysis was observed and its diameter was measured. Aspergillus sp. R-2 and Trichoderma sp. R-1 showed the maximum hydrolytic zone than fungus Penicillium sp. R-1. The hydrolytic zones of fungus Aspergillus sp. R-2 and Trichoderma sp. R-1 were also the highest among all the tested fungal isolates. Owing the higher cellulase enzymatic activity of Aspergillus sp. R-2, Trichoderma sp. R-1 and Penicillium sp. R-1 among all the tested fungal isolates, only these fungi were screened for further studies (Table 1). Table 1: Hydrolytic zone of various fungal isolates for their screening

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For optimizing the growth of all the screened fungi, different temperatures and pH were applied on various fungi and the diameter of hydrolytic zone was measured after a fixed interval of time as also shown in Table 2, 3 and 4. The Table 2 shows the effect of variable temperature and pH on the growth of fungus Aspergillus sp. R-2. The fungus Aspergillus sp. R-2 showed least growth rate at temperature 20oC and 45oC in presence of variable pH. Medium growth of the fungus was observed at temperature 25oC and 40oC and variable pH ranging from 4.0-6.5 and 5.0-7.0 respectively for different temperatures. More than 7.0 pH was found to be hindering for the growth of fungus Aspergillus sp. R-2 at all the temperatures. It was found that the maximum growth rate of fungi Aspergillus sp. R-2 was observed at temperature 30oC and pH 5.5.

Corroborative results have also been observed by Negi and Banerjee (2006) according to whom maximum amylase secretion was obtained at pH 5.5 and protease was found to be maximum at pH 4 and 7. They also found the temperature of 37oC as best for the production of both amylase and protease enzymes which was contradictory to the required temperature of 30oC for the Aspergillus sp. R-2 which was corroborative with the results of Ja’afaru and Fagade (2010). The impact of variable temperature and pH on the growth of fungus Penicillium sp. R-1 has been given in Table 3. It was clearly observed that maximum growth rate was observed at temperature 30oC-35oC and pH 4.0-5.5 while minimum growth rate was observed at temperature 45oC and all the pH ranging from 4.0-7.5. It was also noted that if the temperature remains constant at 20-25oC, minimum growth rate could occur at all the pH ranging from 4.0-7.5. Medium growth rate of the fungus could be observed at the temperatures 30-35oC at variable pH ranging from 6.0-7.5. Vamsi et al. (2009) also observed that the best results of Penicillium growth could be observed at 30ºC which was found to be corroborative with the present results. They also noted that the optimum pH for production of protease enzyme by Penicillium species was 6.6 which was found to be contradictory to the present results as the optimum pH for the growth of fungus Penicillium sp. R-1 has been found to be ranging from 4.0-5.5. Table 3: Effect of temperature and pH on the growth of fungus Penicillium sp. R-1 The effect of variable temperature and pH on the growth of fungus Trichoderma sp. R-1 has been given in Table 4.This Table shows that good growth rate of the fungus occurred at 35oC and in a pH range of pH 4.0-5.0.Temperatures 20-25oC and 45oC were found to show inhibitory effect on the growth of fungus Trichoderma sp. R-1 at all the pH ranging from 4.0-7.5 while medium growth rate could be observed at 30oC and 40oC at all the pH, i.e., 4.0-7.5. The effect of temperature 35oC and pH ranging from 5.5-7.5 also showed medium growth rate in fungus Trichoderma sp. R-1. Table 4: Effect of temperature and pH on the growth of Trichoderma sp. R-1 The results of Al-taweil and Osman (2009) indicated that an optimal medium for maximizing the production of biomass in batch cultures of Trichoderma viride contained temperature 30oC and pH 6.0 which was found to be contrary to the present results as the optimum temperature and pH required for the growth of Trichoderma sp. RTK -1 were found to be 35oC and 4.0-5.0 respectively. Contrary to the studies of Al-taweil and Osman (2009), Ahmed et al. (2009) reported the optimum fermentation temperature of 28oC for endoglucanase production from

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