Influence of glycerine pitch on the metabolite profiling and enzyme activity in the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) biosynthesis by Cupriavidus malaysiensis USMAHM13

Aims: Glycerine pitch is an industrial waste by-product generated mainly from oleochemical industries and has received attention as a potential carbon substrate.Cupriavidus malaysianensis USMAHM13 possesses the capability to produce poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer using glycerine pitch and 1,4-butanediol.This study was aimed to investigate the metabolic changes and enzyme activities during different bacterial growth phases to gain deeper insights into the influence of glycerine pitch in the P(3HB-co-4HB) copolymer synthesis.Methodology and results: It was found that glycerine pitch enhanced bacterial growth and 4HB accumulation compared to pure glycerine via one-stage and two-stage cultivation.The highest PHA concentration (4.45 g/L) and 4HB monomer concentration (1.18 g/L) were achieved via one-stage cultivation using glycerine pitch (10 g/L) and 1,4butanediol (5 g/L).Metabolite profiling studies revealed that lysine, proline and threonine amino acids were only detected when glycerine pitch was used as a carbon source in both P(3HB) homopolymer and P(3HB-co-4HB) copolymer synthesis.Enzymatic assay of P(3HB-co-4HB) synthesis revealed that phaA activity diminished at 36 h onwards, whereas phaB and phaC activity increased to 48 or 60 h.The level of hydroxybutyric acid and PHA content was high at 72 h for both P(3HB) and P(3HB-co-4HB) polymer production using glycerine pitch, which indicated a positive influence of glycerine pitch on the PHA synthase enzyme. Conclusion, significance and impact of study:The present study could potentially unveil an exciting prospect for controlling the composition of 4HB monomers using glycerine pitch, which can be customised to suit specific applications.