title: Development of a process for the biotechnological large-scale production of 4-hydroxyvalerate-containing polyesters and characterization of their physical and mechanical properties.
abstract: A process for the large-scale production of 4-hydroxyvalerate (4HV)-containing biopolyesters with a new monomer composition was developed by means of high-cell-density cultivation applying recombinant strains of Pseudomonas putida and Ralstonia eutropha, harboring the PHA-biosynthesis genes phaC and phaE of Thiocapsa pfennigii. Cell densities of about 20 g/L revealing a PHA 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 using octanoic acid during the growth phase and levulinic acid for the accumulation of 4HV-containing polyesters. Besides 4HV the polyester contained significant amounts of both 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) and traces of 3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoic acid (3HO). With glucose or gluconic acid as the growth substrate, the components of the polyester could be reduced to mainly 3HV and 4HV with only a negligible fraction of 3HB, resulting in a polyester with a new composition. Scale-up of the cultivation process to a 500-L scale was successfully performed, resulting in the production of these polyesters at a pilot plant scale. Short-term shifts in temperature and pH resulted in the formation of cell agglomerates of about 50-100 microm by which the effectiveness of the semicontinuous centrifugation process was drastically increased. Washing of the freeze-dried cells with boiling methanol significantly shortened the extraction 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 scanning calorimetry, stress-strain measurements, and measurements of the viscosity of the solution. The copolyesters were cast into films, spun to fibers, or processed into test bars by melt spinning and injection molding, respectively. They revealed an almost entirely amorphous structure and consequently were sticky and lacked strength. However they showed high thermal stability and an unusually high elongation at break of about 200%; the molecular weights (M(w)) were between 2.0 x 10(5) and 3.3 x 10(5) g/mol. It was shown that 4HV-containing polyesters belong to the class of thermoplastic elastomeres.
authors.affiliation: Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, D-48149 Münster, Germany.
authors.forename: V
authors.initials: V
authors.lastname: Gorenflo
authors.forename: G
authors.initials: G
authors.lastname: Schmack
authors.forename: R
authors.initials: R
authors.lastname: Vogel
authors.forename: A
authors.initials: A
authors.lastname: Steinbüchel
mesh_terms: D000332:Aerobiosis; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D002152:Calorimetry, Differential Scanning; D002210:Caprylates / Q000378:metabolism; D002845:Chromatography; D003470:Culture Media; D020118:Cupriavidus necator / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D005942:Gluconates / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007982:Levulinic Acids / Q000378:metabolism; D011091:Polyesters / Q000737:chemistry* / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D013997:Time Factors; D014631:Valerates / Q000378:metabolism; D014783:Viscosity
chemical_list: D002210:Caprylates; D003470:Culture Media; D005942:Gluconates; D007982:Levulinic Acids; D011091:Polyesters; D012996:Solutions; D014631:Valerates; C469548:4-hydroxyvaleric acid; D005947:Glucose; C031492:octanoic acid; C030691:gluconic acid; C032246:levulinic acid