Development of a high-throughput growth assay for bacteria or yeasts using an agar-based insoluble carbon source immobilization method

Polysaccharide degradation by microorganisms is essential in driving global nutrient cycling, developing renewable fuels or chemicals, and promoting human gut health. However, complex polysaccharides are often insoluble, making it challenging to study degradation with soluble enzymes or to measure microbial growth. Several protocols exist to address this challenge, including 3D-printed biomass containment devices or agar capture systems to facilitate the study of insoluble carbohydrate degradation. While these methods are functional, they are constrained by variable substrate loading and time-consuming preparation. To address these shortcomings, a 3D-printed pipette was designed and tested as part of a newly developed agar immobilization method to quantitatively monitor the degradation of insoluble polysaccharides for a 96-well microtiter assay. The pipette and method were validated via two mechanisms. First, growth analyses of Cellvibrio japonicus wild-type or bgl2A, cbp2D, and cbp2E single deletion mutant strains were conducted using glucose, barley β-glucan, starch, cellulose, chitin, pectin, galactan, or intact yeast biomass as a sole carbon source to benchmark the new method against previously described methods. Second, the agar pipette was used to screen the growth of bacteria or yeasts capable of utilizing insoluble polysaccharides as the sole carbon source. The 3D-printed pipette and agar immobilization method enabled a faster, more consistent, and cost-effective way for high-throughput screening of bacterial or yeast growth using insoluble carbohydrates, suggesting that it can be a useful tool for environmental and applied microbial research.