Cellulase is a group of hydrolytic enzymes that accelerate the process of degrading cellulose, the most prevalent polysaccharide within the plant cell wall, into glucose or shorter oligosaccharides. This enzymatic transformation is an essential step in the global carbon cycle, enabling the breakdown of plant biomass and the recycling of organic matter (Lynd et al., 2002). Cellulases is of great interest both in bioprocessing facilities and in various other industrial applications, such as biofuel production (Singhania et al., 2013), textile, paper and pulp and animal feed, food processing and waste management (Kuhad et al., 2011). The need for sustainable and cost-effective enzymes has led to considerable investigation of cellulase-producing microorganisms, specifically bacteria, owing to their fast growth rate, straightforward genetic manipulation and relative hardiness against deviated environmental conditions (Sadhu and Maiti, 2013).
       
Bacteria have emerged as effective cellulase producers and their use presents advantages over the use of fungi, including increased stability of enzymes, greater production efficiency and robustness and adaptability to extreme environmental conditions (Bayer et al., 2006). Bacillus, Clostridium, Cellulomonas, Pseudomonas and Actinobacteria are among the bacterial genera found to be highly cellulase-generating (Sadhu and Maiti, 2013). Cellulolytic bacteria produce cellulases which break down cellulose into simple sugars that they use as their primary carbon and energy source. Three classes of enzymes make up the cellulolytic system in the majority of cellulolytic bacteria: endoglucanases, which can cleave internal β-1,4-glycosidic bonds in a cellulose chain; exoglucanases, also known as cellobiohydrolases, which release cellobiose units from the end of chains; and β-glucosidases, which convert cellobiose into glucose units (Singhania et al., 2013). Numerous factors, such as carbon and nitrogen supplies, temperature, pH, aeration and fermentation conditions, influence the synthesis of cellulase from bacteria (Sukumaran et al., 2005).
       
In order to make the industrial application more possible, the yield and activity of bacterial cellulase have to be improved. Various strategies like choice of host strain, metabolic engineering, media optimization and fermentation techniques (submerged and solid-state) have been implemented for enhanced bacterial cellulase production (Lynd et al., 1991). Moreover, both genetic engineering and recombinant DNA technology have facilitated the overexpression of cellulase genes in bacterial hosts, leading to increased productivity and stability of the enzymes (Singh et al., 2021).
       
Dozens of cellulolytic bacterial strains are found in natural environments, including soil, compost, decaying plant matter and extreme ecological niches (Bayer et al., 2006). The discovery and characterization of novel bacterial isolates with significant cellulose utilization capacity could have important ramifications for the development of sustainable biotechnological processes (Sadhu and Maiti, 2013). Also, omics technology, including genomics, transcrip-tomics and proteomics, has enabled the identification of important genes and regulatory mechanisms in bacterial cellulase biosynthesis (Kuhad et al., 2011).
 
Significance of enzymatic degradation in sustainable waste management
 
The increasing generation of cellulose-rich agro-industrial waste has intensified the search for eco-friendly technologies capable of converting waste materials into useful products. Among various approaches, enzymatic degradation has emerged as a key component of sustainable waste management, offering an efficient, green and biologically safe method to treat lignocellulosic residues (Howard et al., 2003; Bhat, 2000). Enzymatic degradation employs cellulases, hemicellulases and ligninases-produced by microorganisms such as bacteria, fungi and actinomycetes-to depolymerize complex polysaccharides into simpler monomeric sugars that can be further utilized in bioconversion processes (Lynd et al., 2002; Chavda et al., 2023).
 
Principles of enzymatic cellulose degradation
 
Three main groups of cellulases collaborate in the intricate and cooperative process of cellulose enzymatic degradation to transform cellulose into fermentable sugars. Endoglucanases (EC 3.2.1.4) randomly break internal β-1,4-glycosidic connections inside cellulose chains to produce new chain ends. Exoglucanases or cellobiohydrolases subsequently liberate cellobiose units from these chain ends (EC 3.2.1.91). β-glucosidases (EC 3.2.1.21) then hydrolyse the resulting cellobiose into glucose monomers (Zhang and Lynd, 2004; Tomme et al., 1995). This coordinated enzymatic activity enables the efficient conversion of insoluble cellulose into soluble sugars under mild environmental conditions, without generating harmful byproducts, thereby making the process both biocompatible and sustainable (Singhania et al., 2013).
 
Environmental advantages of enzymatic waste conversion
 
Enzymatic waste conversion presents several environmental advantages over conventional physical and chemical treatment methods. Unlike combustion or acid hydrolysis, enzymatic degradation does not release harmful greenhouse gases such as CO₂, SO₂, or NO_x, thereby contributing to the reduction of atmospheric pollution (Kuhad et al., 2016). Additionally, enzyme-mediated hydrolysis operates efficiently under moderate conditions-typically at temperatures between 30-50^oC and near-neutral pH-resulting in significantly lower energy consumption compared to traditional processes (Bhat and Bhat, 1997). The high biodegradability and substrate specificity of enzymes ensure selective action on target polymers without generating toxic intermediates, thus safeguarding soil and water quality (Bugg et al., 2011). Moreover, enzymatic degradation supports the concept of closed-loop sustainability by facilitating the conversion of agro-industrial waste into valuable bioproducts such as bioethanol, biogas and bioplastics, effectively aligning with circular economy principles (Sukumaran et al., 2010).
 
Microbial cellulases in waste bioconversion
 
Microorganisms such as Bacillus subtilis, Pseudomonas aeruginosa, Trichoderma reesei and Aspergillus niger are well-known producers of cellulases that effectively hydrolyse complex agro-wastes (Chavda et al., 2023; Immanuel et al., 2006). The integration of bacterial and fungal cellulases has been shown to enhance conversion efficiency due to the complementary action of extracellular enzymes (Sukumaran et al., 2010).
       
For instance, Enterobacter cloacae has demonstrated high cellulase activity (up to 344 IU/mL) under optimized conditions using rice husk as substrate, confirming the potential of microbial enzymes for large-scale bioconversion (Chavda et al., 2023). Such microbial degradation approaches not only recover energy and carbon but also reduce landfill accumulation and environmental pollution (Kumar et al., 2017).
 
Economic and industrial significance
 
The enzymatic approach holds significant industrial promise due to its ability to convert waste into economically valuable materials (Table 1). Enzyme-assisted degradation can transform cellulose into fermentable sugars for biofuel production, produce compost and organic fertilizers and assist in bioremediation of effluents from textile, paper and food industries (Klein-Marcuschamer et al., 2012). The global enzyme market for biomass conversion is projected to exceed USD 10 billion by 2030, reflecting growing industrial adoption of sustainable enzymatic technologies (Markets and Markets, 2023).


 
Significance and justification of the present study
 
The present investigation is designed to address existing research gaps by focusing on the isolation, characterization and application of efficient cellulase-producing bacteria derived from agro-industrial environments. The study emphasizes the utilization of inexpensive and abundantly available agro-wastes such as sugarcane bagasse, wheat bran, coconut shell and vegetable residues as substrates for enzyme production. Furthermore, it aims to characterize and optimize the activity of bacterial cellulases to enhance their suitability for potential industrial applications. In addition to enzyme production, this research contributes to the sustainable management of agro-industrial waste through microbial valorization, converting low-value residues into valuable bioproducts. By fulfilling these objectives, the study promotes the development of eco-sustainable bioprocesses that integrate waste reduction with cost-effective enzyme production, thereby advancing both environmental sustainability and industrial efficiency (Kuhad et al., 2016; Chavda et al., 2023).

Isolation of cellulase producing bacteria
 
The experiment was conducted during the 2025-2026 session  at the research lab Department of Allied Sciences, Institute for Technology and Management-ITM VocationalUniversity, Vadodara, Gujarat, Vadodara, Gujarat. Soil and cow dung samples were collected from Khanderao Vegetable market, Vadodara, Gujarat in sample collection containers. A 1g weighted sample was suspended in test tube containing 10ml sterile saline water and left for 2 hours to create a sample suspension. To obtain isolated colonies a ten-fold serial dilution of the sample suspensions upto 10^-10 were made. Aliquots of 0.1 ml of each dilution were inoculated on CMC Agar plate (g/l: CMC - 10 g, Tryptone - 2 g, Yeast Extract -10 g, KH₂PO₄- 4 g, Na₂HPO₄ - 4 g, MgSO₄7H₂O - 0.2 g, FeSO₄7H₂O - 0.004 g, CaCl₂2H₂O - 0.001 g, Agar - 1.5g, pH 7.0) and incubated at 37^oC for 48 Hours. Distinct well-isolated colonies were further streaked on CMC agar plates for screening of cellulase producing bacteria. The agar plates were kept at 37^oC for 48 Hours in incubator. Following incubation, agar plates were flooded with iodine solution and colonies producing cellulase were identified by the formation of clear halos surrounding the bacterial growth, indicating the hydrolysis of CMC.
 
Identification of cellulase producing bacteria
 
Gram staining was done to check the morphological charac-teristics of bacterial isolates. Biochemical characterization  was done according the standard protocol.
 
Production of cellulase enzyme
 
Cellulase production was carried out in 250 ml Erlenmeyer flask containing 50ml production media (g/l: CMC - 10 g, Glucose - 5 g, Tryptone - 2 g, Yeast Extract -10 g, KH₂PO₄ - 4 g, Na₂HPO₄ - 4 g, MgSO₄7H₂O - 0.2 g, FeSO₄7H₂O - 0.004 g, CaCl₂2H₂O - 0.001 g, pH 7.0) for 120 hrs. at 37^oC and 120 rpm. Broth was centrifuged at 10,000 × g at 4^oC to separate proteins and enzyme and the clear supernatant was used as crude enzyme extract source and stored at 4^oC for further use.
 
Partial purification of crude cellulase enzyme
 
Ammonium sulphate purification 0-80%
 
Solid ammonium sulphate was added to the crude supernatant until it reached 70% saturation at 20^oC. The precipitate was then left to develop at 4^oC for the entire night. After centrifuging the precipitated enzyme for 20 minutes at 10,000 × g, solid ammonium sulphate was added to the residual supernatant until it reached 80% saturation. The precipitate was collected by centrifugation at 10,000g for 20 minutes after being allowed to develop at 4^oC for the entire night. 50 mM Phosphate Buffer pH 7.0 was used to redissolve the precipitates.
 
Dialysis of cellulase
 
The Ammonium Sulphate 0-80% precipitated enzyme solution is placed in a dialysis bag and checked for the leakage of the sample in it. Before adding the sample, the dialysis tube is activated by soaking it in the phosphate buffer (pH 7.0) for 10 mins. This dialysis bag after the sample is filled is then weighed and suspended in a beaker containing the phosphate buffer (pH 7.0) solution and kept for 2 hours at room temperature. After incubation, the buffer is then discarded and the new buffer is filled in the beaker and the dialysis bag is again dipped in the buffer for the next 2 hours at room temperature. The process is once again repeated and the dialysis tube is placed in a new buffer and kept at 4^oC for overnight.
 
Enzyme activity
 
DNS was used to measure the pure cellulase’s activity. 2.5% CMC was dissolved in 0.1M citrate phosphate buffer to create the substrate. In a test tube, 0.5 ml of substrate and 0.5 ml of enzyme solution were combined. For ten minutes, the tubes were kept at 40^oC, the water bath temperature. To halt the enzyme reaction, 1.5 ml of DNS reagent was added to the test tubes following the incubation period. For ten minutes, the test tubes were submerged in a boiling water bath to allow the coloured complex to develop. The absorbance of the coloured complex at 540 nm was measured using a UV-visible spectrophotometer. The enzyme activity was computed as
 

Enzyme activity (U/ml) = ((Net O.D + Constant)/Slope) *1000*D.F.)/ (180*10*0.5)

 
Where,
D.F = Dilution factor.
0.5 = Vol. of enzyme.
10 = Inc. Time in min.
2.6 = Enzyme characterization.
       
The enzyme assay was conducted across a temperature range of 30-60^oC to identify the optimal temperature for enzymatic activity. 40^oC was used as standard. The optimum pH range for activity was determined under 5.0 to 9.0 of pH. Tests were carried out in duplicates.
 
Treatment of agriculture waste for its use as animal feed
 
Agriculture waste from wheat farm (WFAW) was taken and grinded into fine powder using a grinder.
       
Precisely weighed amount of 5 g WFAW was dissolved in 50 ml distilled water in a 250 ml flask.

5 ml dialysed enzyme solution was added to the flask containing WFAW, maintaining a solid-liquid ratio of 1:10 (w/v). The flasks were incubated in an incubator shaker for 2 hours at 50^oC.
       
After the incubation 1 ml hydrolysate sample was taken from the flask to estimate the concentration of simple sugar. 1 mL of sample was taken for DNS analysis before carrying out the enzymatic hydrolysis as control.
       
1.5 mL of DNS reagent was added in the test tube and kept in water bath at 100^oC for 10 minutes.
       
After 10 min, tubes were cooled and the absorbance of the sample was read at 540 nm. 1 mL of solution was taken from test tubes and diluted with 9ml distilled water to measure the absorbance.
 
Calculation
 
Glucose concentration was measured and the glucose released was calculated by:
 

Glucose (mg/g WFAW) = (Glucose concentration (mg/ml)* Volume of Hydrolysate (ml) Weight of WFAW

 
Treatment of Sugarcane Bagasse for its use as raw material in Bioethanol production
 
•   Precisely weighed amount of 10 g Sugarcane Bagasse (SGB) was taken and dissolved in 100 mL distilled water in a 500 mL flask.
•    Different concentrations of 5 mL, 10 mL and 15 mL dialysed enzyme solution was added to the flask containing Sugarcane Bagasse. The flasks were incubated in an incubator shaker at 50^oC for 24 h.
•    A 1-mL volume of hydrolysate sample was removed from the flask after incubation for determination of the concentration of simple sugar. A sample of 1 mL was taken for DNS analysis before the enzymatic hydrolysis to serve as control.
•    In the test tube, 1.5 mL of DNS reagent was added and boiled in the water bath for 10 min at 100^oC.

The tubes were cooled and after 10 min the absorbance was measured at 540 nm. From the test tubes, 1 mL aliquots of the solution were taken and diluted to 9 mL with the distilled water for absorbance reading.
 
Calculation
 
Glucose concentration was measured and the glucose released was calculated by:
 

Glucose (mg/g SGB) = (Glucose Concentration (mg/ml) * Volume of Hydrolysate (ml))/Weight of SGB

Isolation of Cellulase producing bacteria
 
Eight isolates from vegetable market soil, while four isolate from cow dung were obtained from the samples. Only three isolates from VM soil were cellulase producing bacteria, while one from cow dung sample was cellulase producing bacteria (Table 2.1, Fig 1). The cellulase producing isolates were purified and then maintained on agar slants for further analysis.




 
Identification of cellulase producing bacteria
 
All the positive isolates were gram positive and rod-shaped bacteria belonging to Bacillus species. Upon biochemical characterization, following results were obtained (Table 2.2).


 
Production of cellulase enzyme
 
The cellulase production by the isolated bacterial strain was assessed under submerged fermentation conditions. 100 µL of crude cellulase enzyme was pipetted in the wells of 1% CMC agar plate.  V4 isolate gave maximum zone of clearance on the plate having 0.66 cm, followed by V8 having 0.62 cm (Graph 1). These findings suggest that the selected bacterial strain exhibits promising cellulolytic enzyme production potential, which could be further optimized for industrial applications.


 
Enzyme assay
 
Ammonium sulphate precipitation was used to partially purify the crude cellulase enzyme obtained from isolate V4, which was then dialysed. Precipitation of ammonium sulphate was done at various saturation levels, with the 70-80% fraction showing the highest activity. Ammonium sulphate precipitation was carried out at different saturation levels, with maximum activity at the 70-80% fraction. The fraction was then dialyzed against buffer to get rid of excess salts which also resulted in a higher enzyme purity. Upon dialysis, enzyme activity increased, indicating that low molecular weight contaminants were removed while enzymatic activity remained intact. Cellulase activity was measured using the DNS (3,5-Dinitrosalicylic acid) method which quantifies reducing sugars liberating from the substrate. Enzyme activity of different purified cellulase enzymes is given in Table 2.3. The comparative enzyme activity after partial purification and dialysis is illustrated in Graph 2.




 
Enzyme characterization
 
The enzyme activity of purified cellulase enzyme was measured at pH range from 5.0 to 9.0. The enzyme showed maximum activity at pH 7 suggesting its preferred pH for catalytic action (Graph 3). At pH lower than, and higher than, optimum, a decrease of activity was detected, indicating that extreme conditions promote enzyme denaturation or decrease the affinity with the substrate. The dialyzed cellulase enzyme showed maximum activity at 50^oC, suggesting its optimum temperature for catalysis (Graph 4). The enzyme retained substantial activity at elevated temperatures, suggesting its thermostability. Analysis of the enzyme stability over a wide temp. range indicates the possible industrial applications that can use them for functioning in dynamic temperature scenarios.

In conclusion, this study isolated and screened cellulase producing bacteria from cow dung and vegetable market soil sample. The strain with the highest cellulase activity was selected and used for enzyme production. The enzymatic specific activity increased after ammonium sulphate precipitation and dialysis. Experimental characterization studies showed that the isolated enzyme demonstrated optimal activity at a pH of 7 and a temperature of 50^oC, suggesting that the purified cellulase could have potential industrial applications. Additional studies are needed on purification techniques and kinetic studies to optimize the enzyme’s efficiency and stability for commercial applications.

The present study was supported by Knowledge consortium of Gujarat (KCG).

Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided and do not accept any liability for any direct or indirect loss arising from the use of this content.

The authors declare that there is no conflict of interest regarding the publication of this article. No funding agency or sponsor influenced the study design, data collection, analysis, interpretation of data, or preparation of the manuscript.