Microplate-Based Quantification of Poly-γ-Glutamic Acid (γ-PGA) levels in Biofilm Samples

Poly-γ-glutamic acid (PGA) is a commercially useful biopolymer produced by many Bacillus species. PGA has a diverse range of applications across medicine and industry generating significant interest in optimising PGA production and enhancing yields. One approach to improve PGA recovery involves identification of high-yield PGA producer strains and determining optimal production conditions, both require an appropriate screening method. Here we present a sensitive and reproducible assay for quantifying PGA from B. subtilis biofilms, whereby the spectral profile of methylene blue changes when bound to PGA. PGA was purified and lyophilised from NCIB 3610 ΔtasA liquid cultures grown at 50°C, allowing production of protein-free PGA for use as a standard at known concentrations. Standard curves were generated from methylene blue absorbance readings at 564 nm and 664 nm, enabling subsequent quantification of PGA from biofilm extracts. We validated the quantification protocol and determined the treatment steps required to minimise interference. The assay has a 96-well plate format, enabling quantification of many samples at low sample volume, while minimising waste of laboratory consumables. Overall, our method offers a sensitive, reproducible approach for PGA quantification in biofilm research and should facilitate comparative analyses across strains, treatments, or environmental conditions.