Poly(ethylene glycol) of Mn 200 g/mol was added to cultivation media of Alcaligines eutrophus during polymer formation, and the carbon source used was 4-hydroxybutyrate. The addition of PEG-200 from 0 to 2 wt % resulted in increased 4-hydroxybutyrate (4HB) incorporation from 66 to 86 mol % into the poly(3-hydroxyalkanoate) (PHA), formed. When 4% PEG was used, the mole percent of 4HB in PHA decreased to 64 mol %. Thus, PEG in cultivation media resulted in polyester compositional changes. An unexpectedly large amount of the dry cell weight (∼10%) was found to be PEG-200 for cultivation media amended with 4% PEG-200. Analysis of this product by gel permeation chromatography (GPC) showed it to be complex, having multiple peaks. The repeat unit sequence distribution determined by 75 MHz 13 C NMR spectroscopy indicated that the addition of PEG-200 to media resulted in the formation of a product with predominantly 3-hydroxybutyrate (3HB)-3HB and 4HB-4HB diads instead of random copolyesters which are formed in the absence of PEG addition to media. Two dimensional homonuclear ( 1 H) correlated NMR analysis along with a number of control experiments indicates that PEG was covalently linked at the carboxy terminal position of PHA chains, forming natural-synthetic diblocks. Fractionation of this product into acetone soluble (AS, 84 mol % 4HB, Mn ) 37 400 g/mol) and insoluble (AIS, 95 mol % 3HB, Mn ) 130 000 g/mol) fractions was carried out which proved that the product formed is a mixture of polyesters differing in composition and molecular weight. Analysis of the unfractionated product as well as AS and AIS fractions by differential scanning calorimetry supported the above conclusions.

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Novel functional poly(P-hydroxyalkanoates), PHAs, with hyperpolarizable side groups were prepared. Pseudomonas oleovorans ATCC 29347 and Pseudomonas putida KT 2442 were selected as biocatalysts. These organisms were first grown on citrate (40 mM) before polymer formation was studied in a second stage. The carbon sources in second-stage cultivations were mixtures (total of 15 mM) of octanoate (OA) with either 7.5 or 10 mMpara-cyanophenoxyhexanoate (CPH), para-cyanophenoxyvalerate (CPV), para-cyanophenoxybutyrate (CPB), or para-nitrophenoxyhexanoate (NPH). For both organisms, only small deviations in the number of colony-forming units per millilitre were observed when second-stage cultivations containing only 15 rnM octanoate were compared with those using mixtures of OA with either CPB, CPV, CPH, or NPH. PHA volumetric yields as a function of organism, carbon source mixture, and culture time are reported. The percent incorporations of substituted phenoxy side groups in the polymer products were determined by 'H NMR spectroscopy. Pseudomonasputida formed PHAs with up to 24.2% 3-hydroxy-6-(para-cyanophenoxy)hexanoate side groups when 5 mM OA and 10 mM CPH were used. A dramatic decrease in the percent incorporation of para-cyanophenoxy (CP) by P . putida was observed when the relatively shorter CP-substituted n-alkanoate chain CPV and CPB cosubstrates were used. Use of NPH in place of CPH had deleterious effects on both polymer formation and percent incorporation of substituted phenoxy side groups in P. putida. Pseudomonas oleovorans formed PHAs with only up to -2% CP side chains when a combination of OA and CPH was used and little to no CP side groups when a combination of OA and CPV or CPB was used. Substitution of NPH in place of CPH led to a modest increase (up to -5%) in substituted phenoxy side groups. Thus, a new route to first-generation chiral polymer structures for nonlinear optical applications was demonstrated.

Pseudomonas putida KT 2442 was utilized as biocatalyst to form optoactive poly(/?-hydroxya1kanoate)s (PHAs) from a cosubstrate mixture of octanoate and the achiral polarizable carbon source 6(4-cyanophenoxy)hexanoate, CPH. COSY and heteronuclear multiplet quantum correlation experiments were used to assign 'H and 13C NMR signals of 3-hydroxy-6(4cyanophenoxy)hexanoate (3HCPH) repeat units. The methine carbon of 3HCPH repeat units was sensitive to repeat unit sequence effects, indicating that a substantial fraction of 3HCPH centered triad sequences in the product contain neighboring 3-hydroxyoctanoate and 3-hydroxhexanoate repeat units. Comparing the thermal properties of 0 and 19.6mol% 3HCPH samples by differential scanning calorimetry shows that 3HCPH incorporation results in melting at temperatures > 64°C (not seen for the 0 mol% sample), more rapid crystallization and a new Tg transition at --21°C. These characteristics indicate that chains and/or chain segments are formed that are enriched in 3HCPH which phaseseparate and form a unique crystal structure. Measurements of second harmonic generation (SHG) intensities carried out using in-situ corona-poled samples showed weak SHG signals that increased by a factor of 8 for an increase in the 3HCPH content from 26 to 34mol%. Comparatively higher SHG intensities (5 times) were found for PHAs which contained 5.1 mol% 3-hydroxy-6(4-nitrophen0xy)hexanoate (3HNPH) repeat units relative to a PHA with 17 mol% 3HCPH. In-vivo biodegradation studies of microbial polyesters prepared with and without 3HCPH repeat units showed that PHA chains with 3HCPH degraded to lesser extents (weight loss of -20 and 50% over 72 h incubations). The large increase in polydispersity from 1.9 to 4.3 observed during in-vivo biodegradation of microbial polyesters containing 3HCPH repeat units was attributed to the existence of chains with highly variable contents of 3HCPH repeat units.

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