Enhancing Sustainability: Bioprospecting soil bacteria with cellulase production potential for industrial applications
Sunny Dhiman1, Anu Kumar1*, Gurpreet Singh1, Shabnum Shafi1,Bhanu Krishan2
1University Institute of Biotechnology, Chandigarh University, Gharuan, Mohali-140413, Punjab (India).
2Department of Biotechnology, Himachal Pradesh University, Summerhill, Shimla,
Himachal Pradesh, 171005.
*Corresponding Author E-mail: anu.uibt@cumail.in
ABSTRACT:
In this study, we investigated the local dump yard soil ecosystem in Khanpur (Kharar), Punjab, India, with the aim of identifying bacterial isolates exhibiting potential cellulase production. The primary screening involved the utilization of the CMC agar plate method and Congo red staining to authenticate and validate cellulase-producing microorganisms. To isolate cellulolytic bacteria, soil samples were added into enrichment liquid media containing 1% (w/v) Carboxy Methyl Cellulose (CMC) and incubated at 37°C for 72hours. Following incubation, enriched samples were plated on CMC agar plates. Among the 24 bacterial colonies observed on the CMC agar plates, only two displayed halo zone formation, a characteristic indicative of cellulase production, as confirmed by Congo Red staining. These two bacterial isolates, identified as SD2 and SR3, exhibited positive results for cellulase production. The results demonstrated that isolate SD2 and SR3 exhibited significant enzyme activities of 5.42U/ml and 9.71U/ml, respectively under submerged conditions. Further investigation into the morphological and microscopic features of bacterial isolate SR3 revealed its gram-negative, rod-shaped characteristics. The identification of bacterial isolate SR3 was conducted through a comprehensive analysis of morphological and biochemical tests. This research underscores the significance of bioprospecting soil bacteria as a sustainable approach towards meeting the demands of modern industrial processes.
KEYWORDS: Sustainable, Carboxymethyl cellulose, Fermentation, Cellulase, Enzyme activity.
INTRODUCTION:
In the realm of sustainable biotechnology, the bioprospecting of microorganisms has become a promising avenue for the discovery of valuable enzymes with industrial applications1. One such endeavour involves exploring the diverse microbial communities residing in unconventional habitats, such as local dump yards. The microbial diversity in soil ecosystems is relatively higher compare to other microbial ecosystems2.
Local dump yards, often considered as ecological eyesores, host a plethora of microorganisms uniquely adapted to thrive in complex and dynamic environments. Soil bacteria, in particular, play a vital role in the decomposition of organic matter, including cellulose-rich waste. A great majority of soil microorganisms remains unexplored as culturing dominant members using standard cultivation media has been a challenging enterprise. Consequently, the soil ecosystem is widely regarded as an immensely valuable source for discovering new microbial enzymes and bioactivities3. The diversity and adaptability of bacteria in these environments make them attractive candidates for biotechnological applications.
Cellulose is most bountiful biomass that serves as a key structural component of plants. It represents a renewable energy source in the biosphere4,5,6. Cellulases are responsible for the degradation of plant polysaccharide cellulose and is a commercially important enzyme that finds its application in many industries7. Cellulose degradation primarily occurs through the action of cellulase enzymes which are typically synthesized by bacteria and fungi8. These cellulases facilitate the efficient hydrolysis of cellulose into glucose units through their synergistic action of enzymes9. Cellulase is an enzyme complex crucial for the hydrolysis of cellulose into simpler sugars. This process, known as cellulolysis, has widespread applications in various industries, including biofuel production, textiles, food and beverage, and pharmaceuticals. Cellulases have garnered significant attention due to their wide-ranging applications across diverse industries such as textile, detergent, leather, food, feed, and paper10,11,12,13. Their exploitation in various industries necessitates the discovery of robust enzymes capable of functioning at high pH and elevated temperatures10. Cellulases are produced by large number of microorganisms14. Microorganisms such as fungi and bacteria are important producers of cellulases15. Traditionally, cellulase has been obtained from fungi, but recent research has shifted towards exploring bacterial sources due to their potential advantages, such as faster growth rates and ease of genetic manipulation16. Submerged fermentation, a widely employed bioprocessing technique, involves cultivating microorganisms in a liquid medium. This method provides a controlled environment for microbial growth and enzyme production.
This article delves into the fascinating world of bioprospecting soil bacteria in dump yard ecosystems specifically for cellulase production through submerged fermentation.
MATERIALS AND METHODS:
Chemicals:
All the chemicals used in this work were of pure grade, purchased from Himedia laboratories, India. All the glassware used was of Borosil.
Sample collection and enrichment:
Soil samples were aseptically collected in sterile plastic bags from a fruit waste dumping site near Khanpur, Kharar, Punjab (India). Collected soil samples were enriched with 100mL of enrichment media (Carboxy Methyl Cellulose Broth). Briefly One gram of each sample was suspended in 100ml of enrichment media. Incubation was done under shaking conditions at 37°C for 48hours.
Isolation and screening of cellulase producing bacteria:
The cultures exhibiting substantial growth were transferred to similar fresh medium. Upon three more transfers, 1mL of turbid broth was serially diluted up to 10-8 dilutions. The conventional serial dilution agar plating method was employed to isolate cellulase producer bacteria. The production medium contained 0.5 % peptone, 0.5% yeast extract, 0.5% NaCl, 1.0% carboxymethylcellulose (CMC), 0.1% KH2PO4 and 1% agar, at pH 7.0. Each serially diluted sample (0.1ml) was plated on CMC Agar plates. The plates were incubated at 37ºC for 48 hours. Bacteria were isolated according to their clear zone forming ability on CMC-Agar plates. The isolates exhibiting maximum zone of clearance were selected for further screening. Pure bacterial cultures were established by repeatedly streaking single colonies on nutrient agar plates with addition of an antifungal compound and were maintained on nutrient agar slants supplemented with 0.01% CMC. Regular sub culturing of the pure cultures was done after every 30 days. The purified colonies were preserved at 4oC. 2 best bacterial isolates designated as SD2 and SR3 exhibiting maximum halo zone around them were interpreted as positive for extracellular cellulase production. The pure culture of isolates SD2 and SR3 were individually streaked in a circular pattern at the centre of the CMC agar plates. Following 1-2 days of plate incubation, the plates were flooded with a 1% w/v Congo red solution for 15minutes. Subsequently, the plates were counterstained with a 1M NaCl solution for 10minutes. The presence of a transparent zone surrounding the bacterial isolates indicated hydrolysis of CMC. These bacterial cultures were individually preserved on CMC agar slants at 4°C.
Cellulase production by Submerged Fermentation Process:
Enzyme production was carried out by employing both cellulolytic bacterial isolates SD2 and SR3 in the cellulase production media under submerged conditions. To achieve this, a loopful of bacterial cells from the selected isolates were inoculated into 100ml of inoculum preparation media (pH 7-7.5) comprising 1g CMC, 0.5g yeast extract, 0.5g peptone, 0.5g NaCl, and 0.1g KH2PO4 respectively. The incubation was done at 37°C for 24hours. Fermentation media was prepared to contain (g/L) glucose 0.5gm, peptone 0.75gm, MgSO4 0.5gm, KH2PO4 0.5gm, and FeSO4 0.01gm. An inoculum level of 2% v/v (OD600=0.6–0.8) of each culture was added separately to 100ml production medium (pH 7-7.5) and incubated at 37oC for 48hrs at 150rpm. Samples were withdrawn from each flask every 12hours and centrifugation was done at 10000rpm for 10minutes at 4°C. Supernatant was used as enzyme source for the assay.
Enzyme assay:
To measure cellulolytic activity, Miller's method was employed17. A reaction mixture was prepared in a 10mL test tube, comprising 0.2mL of crude enzyme and 1.8 mL of 0.5% CMC in 50mM sodium phosphate buffer (pH=7). The reaction mixture was incubated in water bath at 37ºC for 30 minutes. Simultaneously, a Blank was prepared containing only 1.8mL of 0.5% CMC in 50mM sodium phosphate buffer. Subsequently, DNS (3ml) was added to both the test tubes containing the sample and the blank to terminate the reaction. Both the sample and blank mixtures were then incubated in a boiling water bath for 10 minutes to develop colour.
The absorbance of the sample was measured against the blank at 540nm. One unit of cellulase enzyme activity is defined as the amount of enzyme producing 1μmole of glucose in 1 min in experimental conditions.
Morphological Characterization:
The bacterial isolate SR3 with prominent cellulolytic activity was subjected to morphological, microscopic and biochemical characterization. Bergey’s manual of systematic bacteriology was used for morphological and microscopic characterization of the cellulase producing bacterial isolate SR3.
Biochemical characterization:
Various biochemical tests such as Methyl Red Test, Citrate utilization test, Voges–Proskauer test, Urease Test, Starch Hydrolysis Test, Catalase test, Indole Test, Oxidase test etc. were performed by standard methods for identification of the bacterial isolate SR318.