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.

<i>Ralstonia eutropha</i> Strain H16 as Model Organism for PHA Metabolism and for Biotechnological Production of Technically Interesting Biopolymers

“…An engineered strain of R. eutropha , in which the 2-methyl-cis -aconitate hydratase (acnM) gene was disrupted by directed insertion of an additional copy of a 2-methylcitrate synthase gene (prpC) , could be utilized for production of high concentrations of 2-methylcitrate in the medium when cultivated with levulinic acid plus succinate as carbon sources [Ewering et al, 2006b]. Levulinic acid is in addition also a suitable carbon source to produce 4-hydroxyvalerate containing PHAs [Gorenflo et al, 2001]. Another low-priced carbon source, which can be utilized for PHA production, is the residual oil from production of rhamnose [Füchtenbusch et al, 2000].…”

Large-Scale Production of Poly(3-Hydroxyoctanoic Acid) by Pseudomonas putida GPo1 and a Simplified Downstream Process

“…Different separation processes have been described before, starting from filtration (10), froth flotation (70), and continuous centrifugation (20). Continuous separation, also with continuous cell release from the separator, as used in this study, provided a fast and efficient method for separation of cultivation broth up to 1,000 liter/h, as it is possible with a CSA-8 Westfalia separator.…”

Generation and Utilization of Microbial Biomass Hydrolysates in Recovery and Production of Poly(3-hydroxybutyrate)

“…This was confirmed with the spectrum of cell mass extract in acetone. Acetone is a common solvent used to remove hydrophobic lipids, steroids and pigments from PHB-containing cell mass [17]. It does not dissolve and extract PHB and proteins.…”

“…Two technologies, based-on solvent extraction or biomass dissolution, are usually adopted in PHA recovery. With solvent extraction, the PHA granules are dissolved in appropriate organic solvents, leaving cells or residual biomass intact [17,18]. With cell mass dissolution, the PHA granules are left intact while the non-PHA cell mass is decomposed and dissolved in aqueous solutions with help of biological and/or chemical agents [19,20].…”