A resource-efficient agar segment method for qualitative cellulase screening: reducing processing time, waste generation, and environmental impact.

Sustainable and cost-effective screening methods are essential for identifying microorganisms with cellulolytic activity. Traditional plate-based assays require substantial volumes of dye solutions (typically 30-40 mL per plate for flooding protocols) and generate contaminated liquid waste, including residual Congo red and NaCl solutions, as well as spore-contaminated liquid waste when working with sporulating microorganisms. Here, we present an Agar Segment Method (ASM) for the qualitative detection of carboxymethylcellulase (CMCase) activity, involving excision of small agar segments from pre-grown CMC plates followed by staining in minimal volumes of Congo red and NaCl. Compared to the conventional flooding technique, ASM reduced reagent consumption and liquid waste by 86.7%, solid waste by 95.4%, and processing time by 80%. A complementary semi-quantitative assessment showed strong correlation between halo measurements obtained with ASM and conventional flooding (r = 0.95), with an equivalent detection limit of 0.05 U/mL. The method showed improved reproducibility (CV = 8-9%) and lower estimated CO₂-equivalent emissions (78% reduction) and water consumption (86% reduction) per sample. The method was further evaluated using representative fungal and bacterial strains, including actinomycetes. This miniaturized protocol offers a simple, low-cost, and environmentally friendly alternative suitable for high-throughput screening in resource-constrained laboratories, with potential applications in enzyme discovery and strain selection programs.