Development of a process for the biotechnological large-scale production of 4-hydroxyvalerate-containing polyesters and characterization of their physical and mechanical properties

A process for the large-scale production of 4-hydroxyvalerate (4HV)-contain ing biopolyesters with a new monomer composition was developed by means of high-cell-density cultivation applying recombinant strains of Pseudomonas p utida and Ralstonia eutropha, harboring the PHA-biosynthesis genes phaC and phaE of Thiocapsa pfennigii. Cell densities of about 20 g/L revealing a PH A content of 52% (w/w) and a molar fraction of 4HV of up to 15.4 mol % were obtained by a two-stage fed-batch cultivation process at a 25-L scale usin g octanoic acid during the growth phase and levulinic acid for the accumula tion of 4HV-containing polyesters. Besides 4HV the polyester contained sign ificant amounts of both 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric ac id (3HV) and traces of 3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoic acid (3HO). With glucose or gluconic acid as the growth Substrate,the compo nents of the polyester could be reduced to mainly 3HV and 4HV with only a n egligible fraction of 3HB, resulting in a polyester with a new composition. Scale-up of the cultivation process to a 500-L scale was successfully perf ormed, resulting in the production of these polyesters at a pilot plant sca le. Short-term shifts in temperature and pH resulted in the formation of ce ll agglomerates of about 50-100 mum by which the effectiveness of the semic ontinuous centrifugation process was drastically increased. Washing of the freeze-dried cells with boiling methanol significantly shortened the extrac tion process and resulted in a polyester of higher purity. The physical and mechanical properties of these copolyesters were characterized by means of size exclusion chromatography, dynamic mechanical analysis, differential s canning calorimetry, stress-strain measurements, and measurements of the vi scosity of the solution. The copolyesters were cast into films, spun to fib ers, or processed into test bars by melt spinning and injection molding, re spectively. They revealed an almost entirely amorphous structure and conseq uently were sticky and lacked strength. However they showed high thermal st ability and an unusually high elongation at break of about 200%; the molecu lar weights (M-w) were between 2.0 x 10(5) and 3.3 x 10(5) g/mol. It was sh own that 4HV-containing polyesters belong to the class of thermoplastic ela stomeres.