Fractionation and Characterization of Microbial Polyesters Containing 3-Hydroxybutyrate and 4-Hydroxybutyrate Repeat Units Fengying Shi, Richard D. Ashby, and Richard A. Gross* Department of Chemistry, University of Massachusetts Lowell, One University Avenue, Lowell, Massachusetts 01854 Received November 27, 1996 Revised Manuscript Received January 28, 1997 Introduction. Poly(hydroxyalkanoic acids) (PHAs) are a family of polyesters produced by a wide variety of microorganisms. 1-6 Most naturally occurring PHAs have -linked repeat units that are enantiopure ([R]- stereochemical configuration) and possess the general structure shown below where R is the pendant group. 2 The variability in both the number of carbons between ester group linkages and the side group structure for -linked PHAs provides a wide variety of microbial polyesters with diverse physical properties. 5 Research pioneered by Doi and co-workers reported that the bacteria Alcaligenes eutrophus, Alcaligenes latus, and Comamonas acidovorans produce copolymers of 3-hy- droxybutyrate (3HB) and 4-hydroxybutyrate (4HB, γ-linked) when presented with suitable carbon sources such as 4-hydroxybutyric acid, 4-chlorobutyric acid, γ-butyrolactone, and even-chain diols such as 1,4- butanediol and 1,6-hexanediol. 7-16 These copolymers, P(3HB-co-4HB), have been shown to have rather ex- traoridinary properties. For example, the product with 90 mol % 4HB had % elongation at break and tensile strength values of 1080% and 65 MPa, respectively. 15 It has generally been reported that P(3HB-co-4HB) copolyesters formed by both A. eutrophus (ATCC 17699) and A. latus (ATCC 29713) have statistically random sequence distributions of 3HB and 4HB units. 7-14 Abate et al. 17 cultured A. eutrophus (ATCC 17699) following biosynthetic methods described by Kunioka and Doi; however, a product fraction consisting of 97 mol % 4HB was isolated from the unfractionated product composed of 61 mol % 3HB and 39 mol % 4HB. Furthermore, Doi et al. 12 reported that, by feeding γ-butyrolactone and butyric acid as cosubstrates, A. eutrophus (ATCC 17699) produced a mixture of P(3HB- co-4HB) random copolyesters. Moreover, Saito and Doi 15 used the bacterium Comamonas acidovorans DS- 17 to produce mixtures of compositionally different random P(3HB-co-4HB) copolymers as well as products that deviated significantly from random Bernoullian statistics. These later 3HB/4HB products were not fractionated for further analysis. Since (i) polymer microstructure will strongly influ- ence the physicomechanical and biological properties of 3HB/4HB containing polymeric materials and (ii) previ- ous work either disregarded or only partially addressed the formation and characterization of P(3HB-co-4HB) mixtures, we performed a detailed investigation of 3HB/ 4HB product microstructure and heterogeneity. For this purpose, A. eutrophus (ATCC 17699) was used to form a 3HB/4HB-containing product using 4-hydroxy- butyric acid as the sole carbon source. Experimental Section. Fermentations for Poly- ester Production. Polyester production by A. eutro- phus was by two-stage fermentation conditions following a method by Doi and co-workers. 7,8 This involved growth (first stage) and polymer accumulation (second stage) in a nutrient-rich medium and a nitrogen-free defined medium, respectively. Details of the culture conditions and media compositions were described elsewhere (cultivation condition B was followed). 18 The carbon source used in the polymer-producing medium was the sodium salt of 4-hydroxybutyric acid (15.0 g/L). The cells were harvested by centrifugation and lyoph- ilized. 7,8 Polymer Purification. The intracellular PHAs were isolated by chloroform extraction at room temper- ature followed by precipitation into methanol, as was described previously. 18 For cells at the end of the second stage of cultivations, the % PHA in lyophilized cells and the volumetric PHA yield were 28% and 1.1 g/L, respectively. Isolation of polymer from cells at the end of the first stage showed that 3% of the cell dry weight contained P3HB (0.1 g/L). Fractionation of Polyesters. Acetone (10 volumes) was added slowly to a chloroform solution (0.1 g/mL) of the purified PHA, the mixture was stored at -10 °C overnight, and the resulting white precipitate formed was isolated by filtration and termed the acetone- insoluble (AIS) fraction. The solvent (acetone/chloro- form) was then removed by rotoevaporation, giving the acetone-soluble (AS) fraction. Further fractionation of the AS fraction was carried out by the addition of methanol (1.25 volumes) to an acetone solution (0.02 g/mL). The precipitate isolated by filtration was termed the AS methanol/acetone insoluble fraction (AS-MAIS); the solvent of the solution was removed by rotoevapo- ration and gave the corresponding AS methanol/acetone soluble fraction (AS-MAS). Residual solvents were removed from product fractions at room temperature in a vacuum desiccator (10 mmHg, 24 h). The samples were then allowed to age under ambient conditions for at least 1 week prior to thermal analysis (see below). Structural Analysis by 1 H and 13 C NMR. Proton ( 1 H) NMR spectra were recorded on a UNITY-250 NMR spectrometer at 250 MHz. Carbon ( 13 C) NMR spectra were recorded on a UNITY-250 NMR spectrometer at 63 MHz. Details on experimental parameters used were given elsewhere. 18 Molecular Weight Determinations. Molecular weight averages were measured by gel permeation chromatography (GPC). Polystyrene standards (Ald- rich) with low polydispersities were used to generate a calibration curve from which product molecular weights were determined with no further corrections. Ad- ditional details for the method used are described elsewhere. 18,19 Thermal Analysis by Differential Scanning Cal- orimetry. All thermal characterizations were carried out using a DuPont 2910 differential scanning calorim- eter (DSC) equipped with a TA 2000 data station at a heating rate of 10 °C/min and with a dry-nitrogen purge. Polyesters obtained either from solution precipitation or rotoevaporation of solvent (see the section on poly- ester fractionation, above) were dried in vacuo (50 °C, 24 h), sealed in aluminum pans, heated from 25 to 185 °C (first heat), rapidly quenched to -80 °C, and then * Corresponding author. O O H R 2521 Macromolecules 1997, 30, 2521-2523 S0024-9297(96)01737-8 CCC: $14.00 © 1997 American Chemical Society