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Screening of Cellulolytic Fungi and Evaluation of Biodegradation Potentialities of a Selected Strain on Organic Solid Waste

Kamrun Naher
Department of Botany, University of Rajshahi, Rajshahi- 6205, Bangladesh

Md. Showkat Hossain
Department of Botany, University of Rajshahi, Rajshahi- 6205, Bangladesh

Nargis Tanjila
Department of Botany, University of Rajshahi, Rajshahi- 6205, Bangladesh

Most. Ferdousi Begum
Department of Botany, University of Rajshahi, Rajshahi- 6205, Bangladesh

Abstract/ Executive Summary:

This study focused on the screening of cellulolytic fungi isolated from different sources including soil, humus, agricultural wastes and kitchen wastes. Fungal isolates were isolated from different samples on selective medium and 32 isolates were tested on congo red plate for primary screening. Eight fungal strains have good cellulase potential and showed considerable amount of clear zone. These isolates are identified as Trichoderma virens, T. pseudokoningii, T. harzianum Aspergillus ficuum, A. niger, A. tubingensis, Rhizopus sp. and Fusarium sp. Out of eight isolates T. harzianum had more cellulose hydrolyzing capability and showed better performance on TLC plate. The maximum reducing sugar (365 µg/ml), protein (355 µg/ml) and biomass production (711 mg) was observed in T. harzianum. The maximum CMCase and FPase were also recorded in T. harzianum. This potential cellulolytic strain was further used for biodegradation of OSW with different treatments. The highest volume loss (43.59%) and weight loss (20.31%) was observed in T₁ treatment using kitchen waste and T. harzianum which was greater than 87.01 % and 79.71 %, respectively to control. It indicates T. harzinum is a promising fungus and can be apply in enhancing of organic solid waste biodegradation process to compost.

Keywords: Cellulolytic fungi, Congo red plate, TLC, CMCase, FPase, Biodegradation

Location: Department of Botany, University of Rajshahi

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Non-commodity: Performance

Objectives:

To screen out a potential cellulolytic fungus through conventional method and its bioconversion potentialities were evaluated on organic solid waste to prepare compost.

Methodology:

Sample collection - For isolation of cellulolytic fungi a total of twenty six samples were collected from different types of cellulosic wastes such as soil (usually where the cellulosic materials were rotten), humus, agricultural and kitchen wastes. The samples were kept in sterile polythene bags and preserved in refrigerator at 4º C for further study. Isolation of cellulolytic fungi - Agar plate method of Muskett and dilution plate method of Brierley et al was followed for isolation of fungi. Fungi, those grew upon on selective medium (PDA with 1% CMC) were considered as cellulolytic fungi and selected for primary screening. Primary screening of cellulolytic fungi - The selected fungal isolates were screened for their ability to produce cellulases complex following the congo red plate screening method. The isolates were grown on PDA medium supplemented with 1% CMC and incubated at 27ºC for 5 days. After incubation the Petri plates were flooded with congo red solution (0.1%), and after 15 min the congo red solution was discarded, and the plates were washed with 1M NaOH solution allowed to stand for 15–20 minutes. The clear zone was observed around the colony when the enzyme had utilized the cellulose. Identification of selected fungi - Identification of selected strains was made by macroscopic and microscopic characteristics of the isolates. The generic identity of each colony was recorded and identification up to species level was tried wherever possible, with the help of standard mycological books and manuals. Preparation of culture extract - The selected organism was inoculated in 100 ml PDB medium with 1% CMC and allowed to grow at 28⁰C with manual shaking for 7 days. When the organism was grown profusely, the culture medium was filtered and the filtrate was centrifuged at 10,000 rpm for 10 minutes. Then the supernatant was used as the crude enzyme solution for the experimental purpose. Measurement of reducing sugar The amount of reducing sugar in culture filtrate was measured by using dinitro-salicylic acid (DNS) reagent and measuring the absorbance at 550 nm in a spectrophotometer (Spectronic 21) ) using glucose monohydrate as the standard. The reducing sugar produced in the reaction mixture was expressed by the amount of reducing sugar released/ml. Estimation of protein - Protein was estimated by measuring the absorbance at 660 nm in a spectrophotometer (Spectronic 21) using bovin serum albumin as the standard. Confirmation of cellulase activity by TLC - Cellulase is the enzyme which release sugar from cellulosic substrate and the released sugar were identified by thin layer chromatography (TLC). Biomass yield - Biomass produced by fungi in stationary method was taken. The filter paper containing biomass residue was dried in oven at 80⁰C for a constant weight and amount of biomass was calculated by subtracting the weight of filter paper. Carboxymethyl cellulase activity (CMCase) - Endo-glucanase activity (CMCase) was determined by using 1% CMC as the substrate in 0.02M acetated buffer pH 5.2. The reaction mixture containing 2 ml of 1% CMC and 2 ml of enzyme extract were incubated for 2 hours at 45⁰C, after which the release of reducing sugar was measured using DNS reagent. One unit (IU) of enzyme activity was defined as the amount of enzyme released reducing sugar/ml/min. Filter paper activity (FPase) - For assay of FP-ase activity, 2 ml of enzyme extract was added to 1 ml of 0.02M acetate buffer, pH 5.2 belong with 50 mg filter paper strip (Whatman no. 1, 1x6 cm) in a test tube and after incubation at 50⁰C for 1 hrs, the reducing sugar released was estimated using DNS reagent. One unit of filter paper (FPU) activity was defined as the amount of released reducing sugar/ ml/min. Biodegradation of organic solid waste (OSW) - Organic solid as kitchen waste and garden waste were used for biodegradation process. Kitchen wastes were collected from different garbage centre of RU Campus and garden wastes were from garden of RU with polythene bag and cut into 2-3 mm in size and weighed into 50 g and kept in 150 ml quantity bottle and tightened with cotton plug. The bottles with OSW were then autoclaved at 121⁰C and 1.05 kg/cm2 pressure for 15 minutes. The selected strain was cultured on PDA medium at 30⁰C for 48 hours. Then culture disc (5 mm diam.) was cut with cork borer and applied @ 6 culture discs / bottle. Control treatments were performed with no inoculation. Sterile thermometer was also pushed through cotton plug for each bottle. Then the bottles were sealed with para film and labeled. All the process was completed inside the running laminar airflow. The changes of odor, pH, temperature, volume loss (%) and weight loss (%) of decomposed organic solid waste were observed after 10 days interval up to 30 days. For measurement of volume loss (%) and weight loss (%) the following formula was used: Volume loss (%) = [(V - V₁)/ V] x 100, where V is initial volume and V₁ is final volume. Weight loss (%) = [(W - W1)/ W x 100], where W is initial weight and W₁ is final weight. Statistical analysis of the data - The experiment was conducted by using completely randomized design with three replications. Analysis of the variance (ANOVA) was done accordingly and significant differences among the treatments mean were identified by least significant differences (LSD) test at 5% level.

Source of Information: International Journal of Biosciences | IJB |; ISSN: 2220-6655 (Print), 2222-5234 (Online); Vol. 7, No. 3, p. 1-10, 2015

Article Link (Online Journal): http://www.innspub.net; http://dx.doi.org/10.12692/ijb/7.3.1-10

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The results showed that the maximum pH of treatments reached to approximately 6.01 to 8.48 in T₁treatment where kitchen wastes meet the compost regulations of pH 5.0–8.0 for the US, and pH 5.5–8.0 for the Council of European Communities (CEC). The maximum volume loss and weight loss was also observed in same treatment which was 87.01% and 79.71 % greater than control.

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