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Screening, Isolation and Characterization of Cellulolytic Bacteria from Agroindustrial Soils of Bangladesh and Their Optimization for Cellulase Enzyme Production

Md. Ahasun Habib
Department of Microbiology, International Institute of Applied Science and Technology, Rangpur 5400, Bangladesh and Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Fahmida Khatun
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Sabina Yasmin
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Arrafy Rahman
Department of Microbiology, International Institute of Applied Science and Technology, Rangpur 5400, Bangladesh

Abstract/ Executive Summary:

Cellulase enzymes are capable of degrading cellulose-containing industrial pollutants. Nowadays, they have a broad range of applications in different industries including textile, detergent, food and fermentation industries. In the present study, soil samples were collected from different agro-industrial regions of Bangladesh. Eighteen cellulolytic bacteria were isolated from the diluted soil samples. These bacterial isolates were preliminarily screened on Bushnell Haas medium (BHM) agar plate with the help of 0.3% congo red for the qualitative determination of cellulase activity where six isolates made the highest clearing zone around the bacterial colony. The highest clear zone forming bacterial isolate represented the highest cellulase activity. Different morphological (gram staining) and biochemical tests (catalase, indole, urease, oxidase, methyl red, Voges-Proskauer, gelatin hydrolysis, hydrogen sulfide, antibiotic sensitivity and carbohydrate including glucose, sucrose, lactose, xylose, sorbitol, mannitol and myo-inositol utilization tests) were performed to identify cellulolytic bacteria. Isolate 4, 6, 13 and 17 were identified as Streptococcus sp., Xanthomonas sp., Brucella sp. and Bacillus sp., respectively while isolate 1 and 9 were identified as Staphylococcus sp. In the present study, order (the highest to the lowest) of cellulase activity (U/mL) of bacterial isolate 13, 17, 6, 4, 9, 1 was (1.85±0.05) > (1.56±0.07) > (1.26±0.06), (0.84±0.05) > (0.56±0.04) > (0.52±0.03) at their respective optimum temperature and pH. The findings of this study will help to identify other cellulolytic bacteria that can degrade cellulose containing soil pollutants.

Keywords: Cellulase production, Cellulolytic bacteria, Cellulose utilization, Screening, Soil Pollutants

Location: In Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Bacteria

Objectives:

To screen and identify cellulose-degrading bacteria from different agro-industrial soil samples of Bangladesh and also to measure the cellulase production capability of the bacterial isolates by the submerged fermentation process.

Methodology:

Sample collection: The industrial soil samples were collected from different regions of Bangladesh (Osman Agro Industries, Mohadevpur, Naogaon; Ifad Agro-complex Ltd, Bhaluka, Mymensingh; Chittagong Meridian Agro Industries Ltd., Chittagong; Mazim Agro Industries Ltd., Gazipur, Dhaka). Soil samples were collected from 10-15cm depth of soil. Pre-sterilized spatula and plastic bags were used for sample collection and before bacterial isolation the samples were stored at 4°C in the icebox. Isolation of cellulolytic bacteria: Ten-fold serial dilutions of each soil sample were prepared in sterilized distilled water. One gram of soil sample was suspended in 9 ml of sterile distilled water. After serial dilution (10-1 to 10-6 times), an aliquot of 200 ????L of each dilution was taken and spread plated onto Bushnell Haas medium (BHM) (Bushnell and Haas, 1941). Each agar plate contains the following components (g/L): Carboxymethylcellulose (CMC) (10.0), NH₄NO₃ (1.0), KH₂PO₄ (1.0), K₂HPO₄ (1.0), CaCl₂ (0.02), MgSO₄⋅7H₂O (0.2), FeCl₃⋅6H₂O (0.05) and agar (20.0). Then plates were incubated at 37°C for 48hr. Qualitative screening of cellulolytic bacteria: Morphologically distinct colonies were selected from different dilution plates and streaked on separate BHM agar plate and incubated at 37°C for 72 hr. To perform staining the replica plates were also prepared individually (Ruijssenaars and Hartmans, 2001). The 0.3% congo red was added to the replica plates for 12-15 min. Then congo red solution was poured off and each agar plate was washed with 1M NaCl (Teather and Wood, 1982). The isolates showing a clear zone around the colonies were selected and taken from the master plate and further used for the enzyme production in a liquid medium. The selected bacterial isolates were cultured on BHM nutrient broth containing the following components: CMC (10.0 g/L), NH₄NO₃ (1.0 g/L), KH₂PO₄ (1.0 g/L), K₂HPO₄ (1.0 g/L), MgSO₄⋅7H₂O (0.2 g/L), CaCl₂ (0.02 g/L) and FeCl₃⋅6H₂O (0.05 g/L). The culture slants were stored at 4°C as inoculum and sub-cultured every 10–15 days. Morphological characterization: Gram staining is a powerful technique to identify bacteria and it was conducted according to the procedure of Cappuccino and Sherman (2005). Statistical analysis: All analyses were performed in three replicates and the experimental data were statistically analyzed using the Statistical Package for Social Sciences (SPSS, 2007) database, version 16.0, submitting the data to a simple one-factor Analysis of Variance (ANOVA). ANOVA was performed and mean separation was done by SPSS database (p<0.05).

Source of Information: J Bangladesh Agril Univ 19(2): 198–205, 2021; ISSN 1810-3030 (Print) 2408-8684 (Online)

Article Link (Online Journal): https://doi.org/10.5455/JBAU.63173

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Cellulolytic bacteria played an important role for the decomposition of cellulose and hemicellulose present in soil. We performed screening and isolation of bacteria showing potential cellulolytic activity from soil samples collected from different agro-industrial regions. Different cellulolytic (cellulase producing) bacteria including Streptococcus sp., Staphylococcus sp., Xanthomonas sp. and Brucella sp. were identified based on their morphological and biochemical characteristics. Production of cellulase enzymes was observed and a comparison of enzyme activities of the isolates was also compared. The findings of this study will play an important role for textile, paper, food and agricultural industries as most of these industries import cellulase from other countries. Cellulase degrading bacteria can also be utilized for the degradation of cellulose-rich pollutants dumped in soil near agro-industrial regions of Bangladesh.

Knowledge Management: Journal