Method for assessing the biodegradation of poly(lactic acid) in vitro (on agar plates): Application using PLA oligomers and Bacillus licheniformis vegetative cells or spores

This study focuses on an improvement of the clear zone method using agar plates, a widely used method for screening polymer degraders. The improvement consists in performing simple complementary analyses directly on the inoculated agar plates (isolated from Petri dishes) containing the polymer to be degraded in order to i) give the biodegradation kinetics of the entrapped water insoluble polymer; ii) assess the nature of the water soluble oligomeric/monomeric components after hydrolyze by the microorganisms. The technique was applied to poly (lactic acid), PLA. PLA oligomers (2000 g/mol) were chosen to model aged PLA, in order to accelerate the biodegradation kinetics, and Bacillus licheniformis (spores and vegetative cells) was used to validate the method. Therefore, complementary analyses focused on the chemical hydrolysis of ester bonds to convert residual polymeric/oligomeric components into monomers, which were then determined using a kit for assaying lactic acid. Thus, agar plates opacified with emulsified PLA oligomers were made (without organic solvent) and inoculated with either spores or vegetative cells (three loads 103, 105, 107 CFU/plate, at 23 and 37 °C). A remarkable result was the absence of free lactic acid (LA) monomers in all the clarified plates, due to their almost total consumption by the microorganisms. Moreover, the PLA oligomers biodegradation rate reached a remarkable 70 % by weight in 30 days, after direct analysis of the agar plates. These results demonstrated that the method is informative and quantitative, to select suitable strains able to degrade polyesters.