An enhanced degradation of polyhydroxyalkanoates (PHAs) using adaptive laboratory methods: A sustainable approach alternates to genetic engineering

Polyhydroxybutyrate (PHB) is a biodegradable plastic synthesized by several bacterial species, regarded as a sustainable alternative to petroleum-derived plastics owing to its biodegradability, biocompatibility, and renewable production processes. Although PHB exhibits inherent biodegradability, its natural degradation can be slow and inefficient in real-world conditions. This study employed the Adaptive Laboratory Evolution (ALE) method to enhance the degradation of PHB in natural environments. This study started with an examination of the degradation processes of PHB and poly (butylene adipate-co-terephthalate) (PBAT) mulch films in soil using two bacterial strains, Ralstonia insidiosa C1 and Comamonas sp. K5. A two stage ALE methodology achieved 70-80 % degradation of PHB, while a four-stage approach resulted 100 % degradation. R. insidiosa C1 degraded 100 % PHB within 144 h at 1.5 % (w/v), whereas Comamonas sp. K5 within 120 h at 1.0 % (w/v). Further, the gene expression during ALE showed that a 1 % (w/v) PHB significantly increased the gene expression levels at 1.7 times than the baseline. Conversely, the 2 % (w/v) PHB resulted in lower gene expressions indicating a correlation between ALE's degradation activity enhancement and phaZ gene expression levels. The identification of 3-hydroxybutyrate (3-HB) and acetoacetate via NMR analysis substantiates that Ralstonia sp. C1 effectively degrades PHB into 3-HB as a principal metabolite resulting from the hydrolysis of PHB's ester bonds. Finally, strain C1's PHB production was examined in the context of ALE; a reduction in PHB production compared to non-ALE, suggesting that ALE may compromise PHB production. The study advocates further research into ALE's effectiveness for synthetic and bioplastic degradation in real-world scenarios, particularly under varying environmental conditions.