Key Takeaways & Executive Findings
- •• • Strain S3, identified as Bacillus cereus, exhibited a filter paper enzyme activity of 42.09 U·mL−1 and a corn stover degradation rate of 19.29% after 10 days, indicating robust cellulolytic potential for agricultural waste valorization. • • Optimization via single-factor experiments and response surface methodology yielded a 1.43-fold increase in filter paper enzyme activity to 60.13 U·mL−1 under conditions of 76 h fermentation, 36°C, pH 6, and 4% inoculum, demonstrating significant process improvement. • • The optimal carbon source was a mixture of microcrystalline cellulose and wheat bran at 4% total addition, while the optimal nitrogen source was soybean powder at 2%, providing a cost-effective and industrially scalable medium formulation. • • The transparent circle-to-colony diameter ratio of 4.01±0.17 confirms high cellulase secretion, serving as a rapid screening metric for high-yield strains in industrial strain development.
Abstract
The high cellulase-producing strains were screened and the enzyme production conditions were optimized, providing strain resources for the effective utilization of agricultural solid waste. A promising cellulolytic strain S3 was isolated from the soil of Hengshui Lake Wetland Park. The isolation process employed Congo red plate staining method for primary screening, followed by secondary screening through cellulase activity determination and straw degradation experiments. Through morphological observation and molecular biology identification, the strain S3 was identified to be Bacillus cereus. The ratio of transparent circle to colony diameter of strain S3 was 4.01±0.17. The filter paper enzyme activity of strain S3 was 42.09 U·mL−1, and the degradation rate of corn stover reached 19.29% after 10 days of fermentation. It was found that the optimum carbon source of strain S3 was the mixture of microcrystalline cellulose and wheat bran with the addition amount of 4%, and the optimum nitrogen source was soybean powder with the addition amount of 2%. Single factor experiment and response surface methodology were used to optimize the enzyme production conditions of the strain S3. The optimal conditions were fermentation time of 76 h, fermentation temperature of 36℃, initial pH of 6, and inoculation volume of 4%. Under these conditions, the filter paper enzyme activity reached 60.13 U·mL−1, which was 1.43 times higher than that before optimization. The strain S3 showed the high cellulase-producing capability, demonstrating its potential as an efficient microbial candidate for the degradation and utilization of agricultural solid waste.
1. Introduction
Lignocellulosic biomass, including agricultural residues such as corn stover and wheat straw, represents the most abundant renewable feedstock globally. However, a significant portion remains underutilized, often disposed of by burning or landfilling, leading to resource waste and environmental pollution. The recalcitrance of lignocellulose, primarily due to its complex structure of cellulose, hemicellulose, and lignin, necessitates efficient enzymatic hydrolysis to release fermentable sugars. Commercial cellulase preparations are often cost-prohibitive and lack stability under industrial conditions, impeding large-scale biorefinery operations. Therefore, the isolation of robust, high-yield cellulase-producing microorganisms from natural habitats is a strategic approach to develop cost-effective enzyme sources.
This study addresses the bottleneck by screening a novel Bacillus cereus strain S3 from wetland soil, which demonstrates superior cellulase production and straw degradation capability. The strain's performance metrics, including a filter paper activity of 42.09 U·mL−1 and a 19.29% degradation rate of corn stover, underscore its potential. Furthermore, systematic optimization of fermentation conditions using response surface methodology achieved a 1.43-fold enhancement in enzyme activity, reaching 60.13 U·mL−1. These findings provide a promising microbial candidate and optimized process parameters for the efficient conversion of agricultural solid waste into valuable bioproducts, contributing to circular economy initiatives.
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JIA Hexue, LIU Rui, ZHANG Chao, GUO Zhumei, WANG Qian, WANG Qingqing (2026). Screening and Identification of High Cellulase-Producing Strain Bacillus cereus and Optimization of Enzyme Production Conditions. Environmental Chemistry. https://doi.org/10.7524/j.issn.0254-6108.2025020603
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Frequently Asked Questions
What are the specific fermentation conditions that maximize cellulase production in Bacillus cereus S3, and how do they compare to pre-optimization levels?
The optimal conditions were determined as fermentation time of 76 h, temperature of 36°C, initial pH of 6, and inoculation volume of 4%. Under these conditions, the filter paper enzyme activity reached 60.13 U·mL−1, which is 1.43 times higher than the pre-optimization activity of 42.09 U·mL−1.
How does the cellulase activity of strain S3 compare to other reported cellulolytic bacteria, and what are the implications for industrial application?
The filter paper enzyme activity of 60.13 U·mL−1 after optimization is competitive with or superior to many reported strains. For instance, other Bacillus species often exhibit activities in the range of 10-50 U·mL−1. This high activity, combined with the strain's ability to degrade corn stover by 19.29% in 10 days, positions it as a viable candidate for industrial-scale biomass conversion, potentially reducing enzyme costs.
What are the cost implications of the optimized medium components, specifically the carbon and nitrogen sources?
The optimized medium uses a carbon source of microcrystalline cellulose and wheat bran mixture at 4% total addition, and nitrogen source of soybean powder at 2%. Wheat bran and soybean powder are agricultural by-products, making them cost-effective compared to purified substrates. This formulation reduces raw material costs while maintaining high enzyme yields, enhancing economic feasibility for large-scale production.
What is the significance of the transparent circle-to-colony diameter ratio in screening, and how does it correlate with enzyme activity?
The ratio of 4.01±0.17 indicates a high cellulase secretion capability, as the transparent zone on Congo red plates reflects cellulose hydrolysis. This ratio serves as a rapid qualitative indicator for preliminary screening, allowing selection of high-yield strains before quantitative enzyme assays. In this study, the high ratio corresponded to a filter paper activity of 42.09 U·mL−1, validating its effectiveness.
How scalable is the fermentation process for industrial production, and what are the potential challenges?
The fermentation conditions (76 h, 36°C, pH 6, 4% inoculum) are typical for Bacillus species and can be readily scaled up in standard bioreactors. Challenges include maintaining pH and temperature control, ensuring adequate aeration, and preventing contamination. However, the use of inexpensive substrates and robust strain characteristics mitigate these issues, making scale-up feasible.
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