Selection and Adaptive Laboratory Evolution (ALE) of Environment-Derived Microorganisms to Enhance Glycol Removal from Waste Coolants

Coolants containing glycols, corrosion inhibitors and anti-foaming additives pose a significant environmental risk due to their limited susceptibility to natural degradation. At the same time, glycols are valuable raw materials widely used across industrial sectors, and global consumption of ethylene glycol, the principal component of most coolants, has been steadily increasing. Microbial biodegradation offers an economically and environmentally attractive route for treating spent coolants. In this study, fourteen environment-derived strains from a culture collection, thirteen bacteria and one yeast, were screened for the ability to grow on two chemically distinct industrial coolant matrices: an unused, ethylene-glycol-based product (Petrygo Q NEW) and a propylene-glycol-dominated spent fluid drained from an engine cooling system. Six strains (AC2/2, B3J, 5A, B9F, B16B and 17E) showing the highest growth potential were subjected to adaptive laboratory evolution (ALE) by serial passaging for ten transfers in a minimal medium containing 1.5 g L−1 yeast extract, in which the coolant provided the principal carbon and energy source. Growth dynamics of evolved and ancestral strains were compared by microplate cultivation, glycol depletion was quantified by high-performance liquid chromatography (HPLC) in shake-flask cultures, and tolerance was assessed by spot assays. Adaptation was strain-specific and was generally more pronounced on the spent, propylene-glycol-dominated fluid than on the unused, ethylene-glycol-based product. After selection, removal of ethylene glycol from the spent fluid within 96 h increased from 33.3% to 79.7% for Wickerhamomyces anomalus B16B and from 24.8% to 60.1% for Bacillus sp. B9F, while Pseudomonas sp. AC2/2 depleted 64.3% of the propylene glycol present that is 25.6 g L−1 and the largest mass removed from either matrix. In the unused, ethylene-glycol-based product, removal also increased after ALE, reaching 10.9 g L−1 for AC2/2 from an initial load of about 50 g L−1, but none of these differences were significant. To the best of our knowledge, this is the first application of ALE to an intact commercial coolant and to a spent fluid drained from an engine cooling system, and it demonstrates that non-modified environmental isolates can be improved for the treatment of this hazardous waste stream without genetic modification.