WO1997007153A1 - Methods of controlling microbial polyester structure - Google Patents

Methods of controlling microbial polyester structure Download PDF

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WO1997007153A1
WO1997007153A1 PCT/US1995/010396 US9510396W WO9707153A1 WO 1997007153 A1 WO1997007153 A1 WO 1997007153A1 US 9510396 W US9510396 W US 9510396W WO 9707153 A1 WO9707153 A1 WO 9707153A1
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pha
peg
microorganism
polymer
percent
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PCT/US1995/010396
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French (fr)
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Richard A. Gross
Fengying Shi
Richard D. Ashby
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University Of Massachusetts Medical Center
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Priority to PCT/US1995/010396 priority Critical patent/WO1997007153A1/en
Priority to AU34064/95A priority patent/AU3406495A/en
Publication of WO1997007153A1 publication Critical patent/WO1997007153A1/en

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    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08GMACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/66Polyesters containing oxygen in the form of ether groups
    • C08G63/664Polyesters containing oxygen in the form of ether groups derived from hydroxy carboxylic acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/62Carboxylic acid esters
    • C12P7/625Polyesters of hydroxy carboxylic acids

Definitions

  • This invention relates to the control of the structure of microbially-produced polyester compositions such as polyhydroxyalkanoates.
  • Polyhydroxyalkanoates are a series of optically active, thermoplastic, water insoluble polyesters of alkanoic acids produced by various microorganisms, since natural microbial PHAs are synthesized in aqueous media from renewable resources to form biodegradable thermoplastics, this process for polymer synthesis is an "environmentally friendly" preparative route. The microbial synthesis also avoids the use of organic solvents and toxic chemicals required for the chemical synthesis of PHAs. Also, since these microbial polyesters are biodegradable, they can be disposed of as part of the biowaste fraction of municipal solid waste.
  • the first member of the PHA family to be identified was poly(3-hydroxybutyrate) , also known as "P3HB.” See, e.g., Lemoigne, Ann . Inst . Pasteur (Paris) , 21:144, 1925; Lemoigne, Bull . Soc . Chim . Biol . , 8.:770, 1926; and Lemoigne, Ann. Inst . Pasteur (Paris) , 4_1:148, 1927.
  • a problem associated with P3HB is that melt- crystallized and solution-cast films of P3HB show brittle behavior which increases upon aging at room temperature. PHAs with improved physico-mechanical properties have been created by incorporating different structural repeat units into PHAs.
  • Selected examples include poly[3HB-3- hydroxyvalerate-co-3-hydroxyhexanoate] (also referred to as "P[3HB-3HV-co-3HH”) , described in Brandl et al.. Int . J. Biol . Macromol . , 11:49 (1989); P[3HB-co-3HH] described in Shiotani et al., Japanese Pat. Appl. 93049 (1993), and Shimamura et al., Macromolecules, 21_:878-880 (1994); and P3HV described in Steinb ⁇ chel et al., Appl . Microbiol . Biotechnol . , .39:443-449 (1993).
  • 3-Hydroxyalkanoates that contain n-alkyl side groups with lengths generally from propyl to nonyl have also been produced, for example with functional side chain substituents such as phenyl and cyanophenoxy groups.
  • a number of PHAs have also been reported that contain 4-hydroxbutyrate (4HB) repeat units, such as P[3HB-co-4HB] described in Kunioka et al., Polym . Commun . , 2 ⁇ :174 (1988) and Kunioka et al., Appl . Microbiol . Biotechnol .
  • Control of composition for copolyesters of 3HB and 4HB is normally achieved by variation in the carbon sources used or by alteration of other physiological parameters such as the incubation time and nitrogen concentration.
  • other physiological parameters such as the incubation time and nitrogen concentration.
  • PHAs can be chemically tailored to exhibit the desired physical-mechanical properties, crystallization rates, optical clarity, rheological properties, and biodegradation rates.
  • the invention is based on the discovery that when polyethylene glycol (PEG) of a known molecular weight is added to the culture medium of the bacterium Alcaligenes eutrophus or Alcaligenes latu ⁇ , the structure of the resulting product can be controlled.
  • PEG polyethylene glycol
  • A. eutrophus is placed in 4.0% PEG-200 supplemented media under polymer producing conditions
  • PEG-200 interacts with enzyme systems involved in PHA biosynthesis to cause dramatic product structural modulation.
  • the cells respond to the PEG external stimulus by accumulating large quantities of oligomeric PEG that has a number average molecular weight (M n ) closely resembling that of the PEG added to cultivation media.
  • M n number average molecular weight
  • PEG-200 added to culture media resulted in the following: (1) the controlled decrease in PHA molecular weight, which decreases the melt viscosity and bioresorption time; (2) the modulation of the repeat unit composition of the PHA products containing 3HB, 3HV, and 4HB, which provides polymers with varied physical properties; (3) the alteration of PHA repeat unit sequence distribution so that complex polymeric mixtures are obtained in place of random copolymers; and (4) the formation of PHA-PEG diblock copolymers where the carboxylate terminus of PHA chains are covalently linked by an ester bond to PEG chain segments. This is an example of the cellular production of a naturally synthesized block copolymer.
  • the invention features a method for producing a PHA having a controlled, e.g., decreased, molecular weight. by culturing a PHA-producing microorganism, e.g., Alcaligenes , in a polymer production medium under conditions that allow the microorganism to produce a PHA, and adding PEG to the polymer production medium in an amount sufficient for the microorganism to produce a PHA having a molecular weight that is decreased relative to the molecular weight of a PHA produced by the same microorganism under the same growth conditions without PEG.
  • a PHA-producing microorganism e.g., Alcaligenes
  • a "polymer production medium” is used, e.g., for the second-stage fermentation, and includes a desired carbon source, but has a deficiency in one or more nutrients, e.g., nitrogen, oxygen, sulfur, or phosphate, that induces the microorganism to produce PHAs.
  • nutrients e.g., nitrogen, oxygen, sulfur, or phosphate
  • the PEG can be added to the polymer production medium at a concentration of, e.g., 0.25 to 10.0 percent (weight/volume).
  • the PEG can be added to the polymer production medium at a concentration of, e.g., up to 6.0 percent (weight/ volume) .
  • the invention features a method for incorporating 3-hydoxyvalerate (3HV) repeat units into a PHA using a non-3HV carbon source, e.g.
  • the invention further features a method for producing a PHA comprising a copolyester blend of at least two component polymers wherein each component polymer represents at least 30 percent by weight of the total blend, each component polymer is composed of at least 70 percent of a specific repeat unit structure, and the major repeat unit structure in each component polymer is different.
  • This method is carried out by culturing a PHA-producing microorganism in a polymer production medium containing a carbon source, e.g., 4- hydroxybutyrate, under conditions that allow the microorganism to produce a PHA, and adding PEG to the polymer production medium in an amount sufficient for the microorganism to produce a PHA comprising a copolyester blend, e.g. , at a concentration of 4 percent (weight/volume) .
  • a carbon source e.g., 4- hydroxybutyrate
  • the invention also features a method for producing a polyhydroxyalkanoate-polyethylene glycol (PHA-PEG) diblock copolymer in which the carboxyl terminus of a PHA chain segment is covalently linked by an ester bond to a PEG chain segment by culturing a PHA-producing microorganism in a polymer production medium under conditions that allow the microorganism to produce a PHA, and adding PEG to the polymer production medium in an amount sufficient for the microorganism to produce a PHA- PEG diblock copolymer.
  • PHA-PEG polyhydroxyalkanoate-polyethylene glycol
  • the polymer production medium can include glucose as the carbon source, and the microorganism can be A. latus . Then PEG can be added at a concentration of up to 6 percent (weight/volume) . This method can be used to produce a PHA chain segment containing only P3HB repeat units.
  • the polymer production medium can include 4-hydroxybutyric acid as the carbon source, and the microorganism can be A. eutrophus . Then the PEG can be added at a concentration of, e.g., 4 percent (weight/volume). This method can be used to produce a diblock copolymer comprising a majority of 4HB repeat units.
  • the invention features a method for increasing the 4-hydroxybutyrate (4HB) mol percent in a PHA by culturing a PHA-producing microorganism in a polymer production medium containing 4-hydroxybu yric acid as a carbon source under conditions that allow the microorganism to produce a PHA, and adding PEG to the polymer production medium in an amount sufficient for the microorganism to produce a PHA of increased 4HB mol percent, e.g., 1 or 2 percent (weight/volume) .
  • the invention features a PHA copolyester blend including first and second polymers each comprising at least 30 percent by weight of the blend, wherein the first polymer comprises at least 70 mol percent of a first repeat unit structure, the second polymer comprises at least 70 mol percent of a second repeat unit structure, and wherein the first and second repeat unit structures are different.
  • the first repeat unit structure can be 3-hydroxybutyric acid
  • the first polymer can comprise at least 90 mol percent of a first repeat unit structure
  • the second repeat unit structure can be 4-hydroxybutyrate
  • the second polymer can comprise at least 80 mol percent of a second repeat unit structure.
  • the invention also features a polyhydroxyalkanoate-polyethylene glycol diblock (PHA- PEG) copolymer including a first chain of PHA repeat units and a second chain of PEG repeat units, wherein the second chain of PEG repeat units is covalently bound via an ester bond to a carboxy terminal end of the first chain of PHA repeat units.
  • PHA- PEG polyhydroxyalkanoate-polyethylene glycol diblock
  • the first chain can be poly(3-hydroxyb tyrate)
  • the second chain can have an average of 5 PEG repeat units, in which case the first chain can comprise an average of 220 PHA repeat units, or the first chain can comprise at least 80 mol percent of 4-hydroxybutyrate, and the second chain can have an average of 5 PEG repeat units, in which case the first chain can have an average of 435 PHA repeat units.
  • Fig. 1 is a 500 MHz E NMR Spectrum of purified PHA (A. eutrophus , carbon source ( S.) 4- hydroxybutyrate, 4% PEG-200) .
  • Fig. 2 is an expansion of a two dimensional homonuclear ( ⁇ E) correlated (COSY) spectrum of PHA (A. eutrophus , CS. 4-hydroxybutyrate, 4% PEG-200) .
  • Figs. 3a to 3c are a series of gel permeation chromatography (GPC) traces of products formed (3a:A. eutrophus , CS. 4-hydroxybutyrate, 0% PEG-200, crude; 3b:A. eutrophus , CS. 4-hydroxybutyrate, 4% PEG-200, crude; and 3c:A. eutrophus , CS. 4-hydroxybutyrate, 4% PEG-200, one time precipitated) .
  • GPC gel permeation chromatography
  • Fig. 4 is a 125 MHz 13 C NMR spectrum of PHA (A. eutrophus , CS. 4-hydroxybutyrate, 4% PEG-200) .
  • Figs. 5a to 5c are a series of expanded 75 MHz 13 C NMR spectra for carbonyl resonances of PHA (5a:A. eutrophus , CS. 4-hydroxybutyrate, 4% PEG-200; 5b:Acetone soluble (AS) fraction of sample 5a; and 5c:Acetone insoluble (AIS) fraction of sample 5a) .
  • Figs. 6a to 6c are a series of differential scanning calorimetry (DSC) thermograms of PHAs (First Heating) (6a:A. eutrophus , CS. 4-hydroxybutyrate, 4% PEG- 200; 6b:Acetone soluble fraction of sample 6a; and 6c:Acetone insoluble fraction of sample 6a) .
  • DSC differential scanning calorimetry
  • Figs. 7a to 7c are a series of DSC thermograms of PHAs (Second Heating) (7a:A. eutrophus , CS. 4- hydroxybutyrate, 4% PEG-200; 7b:Acetone soluble fraction of sample 7a; and 7c:Acetone insoluble fraction of sample 7a) .
  • PHAs Synchronization Agent
  • Fig. 8 is a graph showing the effects of PEG-200 media concentration on the number average molecular weights (M n ) of the resulting PHAs from A. eutrophus and A. latus. Detailed Description Use of PEG to Control PHA Structure
  • PEG with M n of about 200 g/mol was added in an amount up to 10% (w/v) to cultivations of A. eutrophus and 6% to cultivations of A. latus , either initially or during the polymer production stage, e.g., the second stage of a two stage fermentation, to study: (1) the effect of PEG-200 on the conversion by A. eutrophus and A. latus of the carbon source 4- hydroxybutyrate (4HB) to polyester, (2) changes in product molecular weight, and (3) the incorporation of PEG chain segments that are covalently linked to microbial polyester products.
  • 4- hydroxybutyrate (4HB) 4- hydroxybutyrate
  • GPC Gel permeation chromatography
  • PEG-200 (200 g/mol) used in these studies was purchased from Aldrich.
  • the PEG number average molecular weight (M n ) was confirmed by using ⁇ -H NMR end group analysis and was found to be 194 g/mol.
  • Alcaligenes eutrophus (ATCC 17699) was used in this study. This strain was first grown under aerobic conditions as described in Ervine, Chap. 2 in Fermentation. A Practical Approach (McNeil et al. (eds.), IRL Press, 1990) at 30°C for 14 hours, the culture was then diluted with 2 parts of 20% glycerol and transferred into 1 L cryogenic vials. The vial contents were frozen in a dry ice-ethanol bath and then stored in liquid nitrogen. The cells contained in the vials were used as the inoculum for the two-stage fermentation reactions described below.
  • Alcaligenes latus (DMS 1122) was also used in the methods of the invention.
  • Cultivation Condition A A nutrient rich medium (100 mL, as described in Kunoika et al. , Appl . Microbiol . Biotech . , 3_0_:569, 1989) was prepared, autoclaved to sterilize, and inoculated with 0.1 L cells from a thawed cryovial. A. eutrophus was grown in 500 mL baffled Erlenmeyer flasks in a shaker incubator at 30°C, 250 RPM, for 24 hours.
  • the cells were harvested by centrifugation (4°C, 8,000 rpm for 20 minutes) and washed with a sterile Na 2 HP0 4 -NaH 2 P0 4 buffer solution at pH 7.0. Typically, the cell dry weight of these first stage cultivations was 0.5 g/L.
  • the washed cells were then transferred under aseptic conditions into 100 mL of a sterile filtered nitrogen-free medium which contained 1.51 g/L Na 2 HP0 4 , 2.65 g/L KH 2 P0 4 , 0.2 g/L MgS0 4 , 1.0 mL/L Microelement solution (Kunioka et al., Appl . Microbiol . Biotechnol .
  • Cultivation Condition B Increased PHA from media amended with 4% PEG needed for fractionation and subsequent analysis (see below) was obtained as described above by the two-stage method but using 2800 mL Erlenmeyer flasks and 500 mL cultivation volumes.
  • A. latus was grown on 1.0% glucose (w/v) in a one- stage fermentation where PEG-200 was added to the initial growth media as described in Hiramitsu, et al. , Biotechnol . Letters 15:461 (1993). It was anticipated that under these conditions, the PHA formed by A. latus would be P3HB.
  • A. latus grew and divided initially in the presence of up to 3% (w/v) PEG-200, but showed no bacterial growth, and hence no PHA production at 4% PEG- 200.
  • Increasing the media concentration of PEG-200 from 0 to 1% caused little change in the cell and polymer yields. However, at a media concentration of 3% PEG-200, the biomass and polymer yield productivity dropped precipitously.
  • Alcaligenes species were extracted from cells by stirring a suspension of lyophilized cells (about 0.5 g) for 48 hours in chloroform (80 L) at room temperature. The insoluble cellular material was removed by filtration, and the solvent was then evaporated to obtain what is termed herein the "crude product.” Precipitated products were isolated by concentrating the chloroform crude product solution to a total volume of ⁇ 4 mL and precipitation of the polymer in 30 mL of methanol. The resulting precipitate was washed with methanol and ether and then dried in vacuo . Unless otherwise specified, the isolated products were obtained using one precipitation/washing cycle.
  • the solvent was evaporated from the acetone-chloroform solution which gave the acetone soluble (AS) fraction. Removal of residual solvents from the AS and AIS fractions was carried out in a vacuum dessicator (10 mm Hg, 24 hours) and the samples were then allowed to age for at least one week at ambient temperature prior to carrying out thermal analyses.
  • composition for copolyesters of 3HB and 4HB is normally achieved by variation in the carbon sources used or by alteration of other physiological parameters such as the incubation time and nitrogen concentration.
  • PEG-200 was added to A. eutrophus cultivations in concentrations up to 4% (w/v) during the second or polymer producing stage of the fermentation where 4HB served as the carbon source.
  • the mol fractions of repeat units for PHAs isolated by one precipitation/washing cycle were analyzed by 1 H NMR spectral integration of well resolved signal regions (see Fig. 1) as has been previously described, e.g., in Nakamura et al., Macromolecules, 25:4237-4241 (1992).
  • polym cult cell polym.
  • the 1 H NMR spectrum of the PHA isolated by one precipitation/washing cycle for a cultivation containing 4% PEG-200 (culture condition B, see Table I) is shown in Fig. 1.
  • Weak 2 H NMR signals at -3.7 ppm were observed that correspond to protons (a,c,d,e) of ethylene glycol (EG) repeat units.
  • P(3HB-co-4HB) formed in the absence of PEG does not show any 1 H NMR signals in the 3.6 to 3.8 ppm spectral region.
  • a COSY spectrum of this product was recorded and the specific spectral regions of interest are shown in Fig. 2.
  • the signals in the 3.68 to 3.80 and 4.20 to 4.50 ppm regions were assigned to protons a and b, respectively, of esterified PEG chain segments. Correlation of the signals with peaks at 3.62 and 3.73 suggest that they are due to protons e and d of terminal free hydroxyl EG units (see Fig. 2) . Assuming that the contribution of the overlapping signals in the 3.6 to 3.8 region can be estimated by Bernoullian curve fitting, the area under peaks was measured by cutting and weighing. The integration results showed that the ratio of protons a + d to c to 2x e was 3:6:2. Using the ratio c to a + d and c to 2x e gives values of n (internal EG units of 2 and 3, respectively) .
  • the average chain length of PEG segments in the diblock copolymer is between 4 and 5 which corresponds to molecular weights of ⁇ 180 and 220 g/mol, respectively.
  • the above results are consistent with the formation of PHA chains that are covalently linked at the carboxylate chain terminus to PEG chain segments, which indicates that PHA-PEG diblock copolymers were formed (see Figs. 1 and 2) .
  • the average PEG chain length in the product is almost identical to that which was provided in the cultivation media.
  • Such PHA-PEG diblock copolymers include a long PHA chain segment (average of 430 4HB repeat units) that is covalently linked with an ester bond at its carboxy terminal end to a relatively short PEG chain segment (average of 5 repeat units) .
  • These PHA-PEG diblock copolymers provide unique characteristics compared to PHA products currently available.
  • the diblock copolymers include terminal ethylene glycol (EG) hydroxyl functionalities that allow the formation of chemical linkages with drugs, they have amphipathic characteristics, and they can be used in blends as compatibilizing agents.
  • the invention also provides a unique method to incorporate PEG into PHA formulations such that the PEG will leach out of the PHA into an aqueous media at a much slower rate than formulations in which PHAs and PEG are merely mixed together.
  • PEG-200 (0.3% w/w) was mixed with a PHA-PEG product (0.7% w/w) obtained after 3 precipitation/ washing cycles from 4% PEG amended cultivations.
  • PHA-PEG product (0.7% w/w) obtained after 3 precipitation/ washing cycles from 4% PEG amended cultivations.
  • the relative signal intensities of the 3.6-3.8 ppm signal region to PHA protons was identical to that of the PHA-PEG product prior to mixing with PEG-200.
  • repeated precipitation/washing (up to three times) of one-time- precipitated samples did not result in a change in the EG mol percentage. Therefore, non-covalently linked PEG-200 is indeed removed efficiently from the isolated products by one precipitation/washing cycle.
  • PHAs isolated from A. eutrophus cultivations in which 4-hydroxybutyric acid served as a carbon source and PEG-200 was not added to the media have sequence distributions of 3HB and 4HB repeat units that are approximately random. See, e.g., Nakamura et al., Macromolecules , 5:4237- 241, 1992.
  • the GPC trace of the crude polymer product obtained from cultivation media without PEG-200 shows only a uni odal peak (see Fig. 3a) .
  • the GPC trace of the crude product has a component peak with an elution volume which corresponds exactly with that of PEG-200 (Fig. 3b, peak at 200 g/mol) . This is further evidence that PEG-200 does indeed accumulate in the cells, and that this occurs without notable cellular selectivity as a function of PEG chain length.
  • the PHA product was fractionated based on its solubility in acetone. Fractionation resulted in an acetone soluble fraction (AS) representing 57% (w/w) of the total product, and an acetone insoluble fraction (AIS) representing 43% (w/w) of the total product.
  • AS acetone soluble fraction
  • AIS acetone insoluble fraction
  • the AS and AIS fractions had M n (M- ⁇ /M..) values of 37,400 (2.52) and 130,000 (3.42), respectively (see Table I). The fact that the PHA product could be fractionated provides additional evidence that the product is a mixture or blend of polyesters as opposed to a block copolymer.
  • PEG chain segments are found primarily in the AS high 4HB fraction (see Table I) . This is evidence that for A. eutrophus , linkages between PEG and PHA segments occur primarily between 4HB and EG repeat units.
  • each column shows the experimental values (exp) , determined by measuring the relative peak areas for the carbonyl carbon 13 C NMR signals assigned (see Figs. 5a to 5c) to the four dyad sequences, and the calculated value (calcd) , determined from equations 1 to 3, assuming a Bernoullian or random statistical process and that the contribution of 3HV and EG repeat units can be neglected.
  • the indicated percent PEG was added to the cultivation medium during the second or polymer producing stage.
  • the non-fractionated sample was obtained from one precipitation/washing cycle from a cultivation carried out using 500 mL of media in a 2.8 L shake flask.
  • the AS and AIS fractions of the 4% PEG product represent 57% and 43% (w/w) of the product, respectively.
  • Table II shows that the PHA produced with 0% PEG approximates a random copolyester.
  • the addition of 4% PEG to cultivations resulted in a novel product that has predominantly 3HB*-3HB and 4HB*-4HB dyads (see also, Table I rows 4-AS and 4-AIS, and Fig. 5a) .
  • the addition of PEG to the growth medium provides a new microbial polymerization process in which polymer blends are made directly by a single fermentation reaction.
  • this new process is much more efficient than prior methods to produce blends of polymers in which one component polymer has a high (greater than 70%, and preferably greater than 90%) 3HB content, and the other component polymer has a high (greater than 70%, and preferably greater than 80%) 4HB content. Furthermore, each of the two component polymers represents at least 30% of the total weight of the blend.
  • Table III shows the results of thermal analysis obtained by DSC measurements at a scanning rate of 10°C/min.
  • the percent PEG added to the cultivation medium was added during the second or polymer producing stage.
  • the non-fractionated samples were obtained from one precipitation/washing cycle.
  • 4-AS and 4-AIS are the acetone soluble (57% w/w) and insoluble (43%) fractions of the 4% PEG sample.
  • T g represents glass transition temperatures taken as the midpoint of the heat capacity change and measured during the second heating scan after rapidly quenching by liquid nitrogen at -70°C from the melt.
  • T m represents the peak melting temperatures for each endothermic melting transition determined during the first heating scan.
  • ⁇ H f (cal/g) represents the heat of fusion value measured for each melting endothermic transition. Cultivations were carried out using 500 mL of media in a 2.8 L shake flask (culture conditions B) .
  • the DSC thermograms of the 4% PEG product during a first heating scan showed two distinct T m values at 55° and 170°C (see also Fig. 6a) which closely approximate reported T m values for P3HB and P4HB (177° and 54° C, respectively) .
  • the DSC thermogram of this product recorded during a second heating scan after rapidly quenching from the melt showed T g values at -45° and -29°C (see also Fig. 7a).
  • the T g at -45°C closely approximates that reported for P4HB (-50°C) while the T g at -29°C is intermediate to those reported for P3HB (-4°C) and P4HB.
  • the dyad sequence distribution of the AS fraction determined experimentally (see Fig. 5b) , and calculated using equations 1 to 3, above, suggests that the product formed approximates that of a high 4HB content random copolyester (see Table II) . Further study of this fraction by DSC indicated product heterogeneity. Specifically, the AS fraction had multiple Tg (-15,-42°C) transitions and a broad melting region (see Table III, Figs. 6b and 6b) .
  • Table I also depicts the effects of PEG-200 on volumetric yield and product molecular weight for the series of fermentations of A. eutrophus carried out under culture conditions A.
  • the volumetric yield of the PHAs continued to decrease with increased PEG media concentration so that for 2 and 4% PEG-200 addition the yields were approximately 59% and 49%, respectively, of that for PEG deficient media.
  • the M n and M w /M n values measured by GPC of the products formed from cultivations with 0, 1, 2, and 4% PEG are also shown in Table I. The GPC traces of these products were unimodal.
  • PEG can be used to form PHAs that contain 4HB repeat units and have reduced molecular weights compared to PHAs produced without PEG.
  • a decrease in molecular weight affects the polymer characteristics, e.g., decreases the melt viscosity, and is useful to form sustained release compositions and biomaterials that require relatively shorter bioresorption times.
  • Table IV shows the effects of PEG-200 on bacterial growth, polymer production, and polymer composition from 1 and 2 stage cultivations of A. latus grown on glucose (designated by the letter L) , and 2 stage cultivations of A. eutrophus grown on fructose (designated by the letter E) .
  • R-CDW is "residual cell dry weight" which corresponds to the non-polymer weight of the cells which may be considered the residual biomass.
  • the molecular weights of the isolated polymer products formed by A. latus were analyzed by gel permeation chromatography (GPC) . Unless otherwise specified, the isolated products were obtained using 2 precipitation/ washing cycles.
  • A. eutrophus As the microbial production system where variable quantities of PEG-200 were added to cultivation media (see Table IV) .
  • Cellular growth and polymer production for all A. eutrophus cultures were carried out using a two-stage batch culture process as described above for cultivation conditions A, using fructose as the carbon source for the second stage cultivations.
  • A. eutrophus showed only small decreases in cell and product yield with the addition of up to 2% PEG-200.
  • the methods described above allow the modification of microbial polyester products by simply changing the concentration of PEG added to the cultivation medium.
  • concentration of PEG added By controlling the amount of PEG added, one can control the molecular weight, repeat unit composition and distribution, and produce specified copolyester blends as opposed to random copolyester chains.
  • such diblock copolymers and copolyester blends of the invention can be used to make biodegradable plastic articles and coatings, e.g., for paper, that are manufactured by standard thermal processing methods.
  • These new copolyesters can also be used for microencapsulation, e.g., of cells or drugs, to produce cell growth matrices, and to produce biomedical materials such as sutures, implants, and drug delivery vehicles.
  • component polymer repeat unit structures of 3- hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxy-4- pentenoate, and 3-hydroxypropionate can be prepared using the corresponding acids, e.g., 3-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxy-4-pentenoic acid, and 3-hydroxypropionic acid, as carbon sources.
  • carbon sources can be used to form 3HB and 4HB repeat unit structures.
  • 1,4-butane diol and 1,6-hexane diol can be used as carbon sources to produce 4HB repeat unit structures.
  • PEG polymer production media containing these carbon sources will also cause an increase in the 4HB mol percentage compared to fermentation without the added PEG.

Abstract

The addition of PEG to culture media of Alcaligenes eutrophus and A. latus resulted in the following: (1) the controlled decrease in polyhydroxyalkanoate (PHA) molecular weight, which decreases the melt viscosity and bioresorption time; (2) the modulation of the repeat unit composition of the PHA products containing 3-hydroxybutyrate, 3-hydroxyvalerate, and 4-hydroxybutyrate, which provides polymers with varied physical properties; (3) the alteration of PHA repeat unit sequence distribution so that complex polymeric mixtures are obtained in place of random copolymers; and (4) the formation of PHA-PEG diblock copolymers where the carboxylate terminus of PHA chains are covalently linked by an ester bond to PEG chain segments. This is an example of the cellular production of a naturally synthesized diblock copolymer. The invention features new diblock copolymers, copolyester blends, and methods of preparation.

Description

Methods of Controlling Microbial Polyester Structure
Statement as to Federally Sponsored Research This invention was made in part with Government support under grant No. DMR-9057233, awarded by the
National Science Foundation. The Government has certain rights in the invention.
Background of the Invention This invention relates to the control of the structure of microbially-produced polyester compositions such as polyhydroxyalkanoates.
Polyhydroxyalkanoates (PHAs) are a series of optically active, thermoplastic, water insoluble polyesters of alkanoic acids produced by various microorganisms, since natural microbial PHAs are synthesized in aqueous media from renewable resources to form biodegradable thermoplastics, this process for polymer synthesis is an "environmentally friendly" preparative route. The microbial synthesis also avoids the use of organic solvents and toxic chemicals required for the chemical synthesis of PHAs. Also, since these microbial polyesters are biodegradable, they can be disposed of as part of the biowaste fraction of municipal solid waste. The first member of the PHA family to be identified was poly(3-hydroxybutyrate) , also known as "P3HB." See, e.g., Lemoigne, Ann . Inst . Pasteur (Paris) , 21:144, 1925; Lemoigne, Bull . Soc . Chim . Biol . , 8.:770, 1926; and Lemoigne, Ann. Inst . Pasteur (Paris) , 4_1:148, 1927. A problem associated with P3HB is that melt- crystallized and solution-cast films of P3HB show brittle behavior which increases upon aging at room temperature. PHAs with improved physico-mechanical properties have been created by incorporating different structural repeat units into PHAs. Over 50 different structural repeat unit types have been incorporated into PHAs to produce a large range of homo- and copolyesters. This structural diversity has been achieved by using different microbial production systems and by varying media carbon sources. These carbon sources are metabolized into hydroxyalkanoate repeat units having variable pendant group structures and number of carbon atoms between ester linkages.
Selected examples include poly[3HB-3- hydroxyvalerate-co-3-hydroxyhexanoate] (also referred to as "P[3HB-3HV-co-3HH") , described in Brandl et al.. Int . J. Biol . Macromol . , 11:49 (1989); P[3HB-co-3HH] described in Shiotani et al., Japanese Pat. Appl. 93049 (1993), and Shimamura et al., Macromolecules, 21_:878-880 (1994); and P3HV described in Steinbϋchel et al., Appl . Microbiol . Biotechnol . , .39:443-449 (1993). 3-Hydroxyalkanoates that contain n-alkyl side groups with lengths generally from propyl to nonyl have also been produced, for example with functional side chain substituents such as phenyl and cyanophenoxy groups. A number of PHAs have also been reported that contain 4-hydroxbutyrate (4HB) repeat units, such as P[3HB-co-4HB] described in Kunioka et al., Polym . Commun . , 2^:174 (1988) and Kunioka et al., Appl . Microbiol . Biotechnol . , J30., 569 (1989), and terpolyesters of 3HB, 3HV, and 4HB, described in Kimura et al., Biotechnol . Lett . , 14(6) :445-450 (1992). In addition, 3- hydroxy-propionate (3HP) and 4HB repeat units have been found in PHAs produced by the bacterium Alcaligenes eutrophus (see, Kunioka et al., Polym. Commun . , 29:174- 76, 1988, and Nakamura et al., Macromol . Reports , A28CSUPP1.1. :15. 1991). Control of composition for copolyesters of 3HB and 4HB is normally achieved by variation in the carbon sources used or by alteration of other physiological parameters such as the incubation time and nitrogen concentration. For example, see. Nakamura et al., Macromolecules , 2J5, 4237-4241 (1992), and Doi, Y., Microbial Polyesters, VCH, New York (1990) . In this way, PHAs can be chemically tailored to exhibit the desired physical-mechanical properties, crystallization rates, optical clarity, rheological properties, and biodegradation rates.
Thus, a variety of carbon source substrates have been used to form novel PHAs. However, known microbial synthesis methods provide no rational strategies to control polymer molecular weight or end group structure during the microbial polymerization.
Summary of the Invention The invention is based on the discovery that when polyethylene glycol (PEG) of a known molecular weight is added to the culture medium of the bacterium Alcaligenes eutrophus or Alcaligenes latuε, the structure of the resulting product can be controlled. For example, when A. eutrophus is placed in 4.0% PEG-200 supplemented media under polymer producing conditions, PEG-200 interacts with enzyme systems involved in PHA biosynthesis to cause dramatic product structural modulation. The cells respond to the PEG external stimulus by accumulating large quantities of oligomeric PEG that has a number average molecular weight (Mn) closely resembling that of the PEG added to cultivation media.
Specifically, addition of PEG-200 to culture media resulted in the following: (1) the controlled decrease in PHA molecular weight, which decreases the melt viscosity and bioresorption time; (2) the modulation of the repeat unit composition of the PHA products containing 3HB, 3HV, and 4HB, which provides polymers with varied physical properties; (3) the alteration of PHA repeat unit sequence distribution so that complex polymeric mixtures are obtained in place of random copolymers; and (4) the formation of PHA-PEG diblock copolymers where the carboxylate terminus of PHA chains are covalently linked by an ester bond to PEG chain segments. This is an example of the cellular production of a naturally synthesized block copolymer.
In general, the invention features a method for producing a PHA having a controlled, e.g., decreased, molecular weight. by culturing a PHA-producing microorganism, e.g., Alcaligenes , in a polymer production medium under conditions that allow the microorganism to produce a PHA, and adding PEG to the polymer production medium in an amount sufficient for the microorganism to produce a PHA having a molecular weight that is decreased relative to the molecular weight of a PHA produced by the same microorganism under the same growth conditions without PEG.
As used herein, a "polymer production medium" is used, e.g., for the second-stage fermentation, and includes a desired carbon source, but has a deficiency in one or more nutrients, e.g., nitrogen, oxygen, sulfur, or phosphate, that induces the microorganism to produce PHAs. These media are well known in the fermentation arts.
When A. eutrophus is used, the PEG can be added to the polymer production medium at a concentration of, e.g., 0.25 to 10.0 percent (weight/volume). When A. latus is used, the PEG can be added to the polymer production medium at a concentration of, e.g., up to 6.0 percent (weight/ volume) . In another embodiment, the invention features a method for incorporating 3-hydoxyvalerate (3HV) repeat units into a PHA using a non-3HV carbon source, e.g. , 4- hydroxybutyric acid or 4-hydroxybutyrate, by culturing a PHA-producing microorganism in a polymer production medium containing a non-3HV carbon source under conditions that allow the microorganism to produce a PHA, and adding PEG to the polymer production medium in an amount sufficient for the microorganism to produce a PHA comprising 3HV, e.g., at a concentration of from 1 to 4 percent (weight/volume) .
The invention further features a method for producing a PHA comprising a copolyester blend of at least two component polymers wherein each component polymer represents at least 30 percent by weight of the total blend, each component polymer is composed of at least 70 percent of a specific repeat unit structure, and the major repeat unit structure in each component polymer is different. This method is carried out by culturing a PHA-producing microorganism in a polymer production medium containing a carbon source, e.g., 4- hydroxybutyrate, under conditions that allow the microorganism to produce a PHA, and adding PEG to the polymer production medium in an amount sufficient for the microorganism to produce a PHA comprising a copolyester blend, e.g. , at a concentration of 4 percent (weight/volume) .
The invention also features a method for producing a polyhydroxyalkanoate-polyethylene glycol (PHA-PEG) diblock copolymer in which the carboxyl terminus of a PHA chain segment is covalently linked by an ester bond to a PEG chain segment by culturing a PHA-producing microorganism in a polymer production medium under conditions that allow the microorganism to produce a PHA, and adding PEG to the polymer production medium in an amount sufficient for the microorganism to produce a PHA- PEG diblock copolymer.
For example, the polymer production medium can include glucose as the carbon source, and the microorganism can be A. latus . Then PEG can be added at a concentration of up to 6 percent (weight/volume) . This method can be used to produce a PHA chain segment containing only P3HB repeat units. In addition, the polymer production medium can include 4-hydroxybutyric acid as the carbon source, and the microorganism can be A. eutrophus . Then the PEG can be added at a concentration of, e.g., 4 percent (weight/volume). This method can be used to produce a diblock copolymer comprising a majority of 4HB repeat units. In another embodiment, the invention features a method for increasing the 4-hydroxybutyrate (4HB) mol percent in a PHA by culturing a PHA-producing microorganism in a polymer production medium containing 4-hydroxybu yric acid as a carbon source under conditions that allow the microorganism to produce a PHA, and adding PEG to the polymer production medium in an amount sufficient for the microorganism to produce a PHA of increased 4HB mol percent, e.g., 1 or 2 percent (weight/volume) . In another aspect, the invention features a PHA copolyester blend including first and second polymers each comprising at least 30 percent by weight of the blend, wherein the first polymer comprises at least 70 mol percent of a first repeat unit structure, the second polymer comprises at least 70 mol percent of a second repeat unit structure, and wherein the first and second repeat unit structures are different. For example, the first repeat unit structure can be 3-hydroxybutyric acid, the first polymer can comprise at least 90 mol percent of a first repeat unit structure, the second repeat unit structure can be 4-hydroxybutyrate, the second polymer can comprise at least 80 mol percent of a second repeat unit structure.
The invention also features a polyhydroxyalkanoate-polyethylene glycol diblock (PHA- PEG) copolymer including a first chain of PHA repeat units and a second chain of PEG repeat units, wherein the second chain of PEG repeat units is covalently bound via an ester bond to a carboxy terminal end of the first chain of PHA repeat units. In particular examples, the first chain can be poly(3-hydroxyb tyrate) , and the second chain can have an average of 5 PEG repeat units, in which case the first chain can comprise an average of 220 PHA repeat units, or the first chain can comprise at least 80 mol percent of 4-hydroxybutyrate, and the second chain can have an average of 5 PEG repeat units, in which case the first chain can have an average of 435 PHA repeat units.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the detailed description, and from the claims. Brief Description of the Drawings Fig. 1 is a 500 MHz E NMR Spectrum of purified PHA (A. eutrophus , carbon source ( S.) 4- hydroxybutyrate, 4% PEG-200) . Fig. 2 is an expansion of a two dimensional homonuclear (λE) correlated (COSY) spectrum of PHA (A. eutrophus , CS. 4-hydroxybutyrate, 4% PEG-200) .
Figs. 3a to 3c are a series of gel permeation chromatography (GPC) traces of products formed (3a:A. eutrophus , CS. 4-hydroxybutyrate, 0% PEG-200, crude; 3b:A. eutrophus , CS. 4-hydroxybutyrate, 4% PEG-200, crude; and 3c:A. eutrophus , CS. 4-hydroxybutyrate, 4% PEG-200, one time precipitated) .
Fig. 4 is a 125 MHz 13C NMR spectrum of PHA (A. eutrophus , CS. 4-hydroxybutyrate, 4% PEG-200) .
Figs. 5a to 5c are a series of expanded 75 MHz 13C NMR spectra for carbonyl resonances of PHA (5a:A. eutrophus , CS. 4-hydroxybutyrate, 4% PEG-200; 5b:Acetone soluble (AS) fraction of sample 5a; and 5c:Acetone insoluble (AIS) fraction of sample 5a) .
Figs. 6a to 6c are a series of differential scanning calorimetry (DSC) thermograms of PHAs (First Heating) (6a:A. eutrophus , CS. 4-hydroxybutyrate, 4% PEG- 200; 6b:Acetone soluble fraction of sample 6a; and 6c:Acetone insoluble fraction of sample 6a) .
Figs. 7a to 7c are a series of DSC thermograms of PHAs (Second Heating) (7a:A. eutrophus , CS. 4- hydroxybutyrate, 4% PEG-200; 7b:Acetone soluble fraction of sample 7a; and 7c:Acetone insoluble fraction of sample 7a) .
Fig. 8 is a graph showing the effects of PEG-200 media concentration on the number average molecular weights (Mn) of the resulting PHAs from A. eutrophus and A. latus. Detailed Description Use of PEG to Control PHA Structure
PEG with Mn of about 200 g/mol (PEG-200) was added in an amount up to 10% (w/v) to cultivations of A. eutrophus and 6% to cultivations of A. latus , either initially or during the polymer production stage, e.g., the second stage of a two stage fermentation, to study: (1) the effect of PEG-200 on the conversion by A. eutrophus and A. latus of the carbon source 4- hydroxybutyrate (4HB) to polyester, (2) changes in product molecular weight, and (3) the incorporation of PEG chain segments that are covalently linked to microbial polyester products.
Gel permeation chromatography (GPC) was used to investigate product molecular weight averages and dispersity. One- and two- dimensional 1H nuclear magnetic resonance (NMR) spectroscopy were used to study the repeat unit composition and incorporation of PEG-200 in various product fractions. 13C NMR spectroscopy was used to analyze polymer repeat unit sequence distribution. Fractionation by differential solubilities in acetone was used to investigate product heterogeneity. In addition, differential scanning calorimetry (DSC) was used to obtain information on thermal transitions of products and product fractions. Polyethylene Glycol
PEG-200 (200 g/mol) used in these studies was purchased from Aldrich. The PEG number average molecular weight (Mn) was confirmed by using ^-H NMR end group analysis and was found to be 194 g/mol.
Bacterial Preservation and Inoculum Preparation Alcaligenes eutrophus (ATCC 17699) was used in this study. This strain was first grown under aerobic conditions as described in Ervine, Chap. 2 in Fermentation. A Practical Approach (McNeil et al. (eds.), IRL Press, 1990) at 30°C for 14 hours, the culture was then diluted with 2 parts of 20% glycerol and transferred into 1 L cryogenic vials. The vial contents were frozen in a dry ice-ethanol bath and then stored in liquid nitrogen. The cells contained in the vials were used as the inoculum for the two-stage fermentation reactions described below.
Alcaligenes latus (DMS 1122) was also used in the methods of the invention. A. eutrophus Fermentation Conditions
100 mL Cultivations (Cultivation Condition A) : A nutrient rich medium (100 mL, as described in Kunoika et al. , Appl . Microbiol . Biotech . , 3_0_:569, 1989) was prepared, autoclaved to sterilize, and inoculated with 0.1 L cells from a thawed cryovial. A. eutrophus was grown in 500 mL baffled Erlenmeyer flasks in a shaker incubator at 30°C, 250 RPM, for 24 hours. The cells were harvested by centrifugation (4°C, 8,000 rpm for 20 minutes) and washed with a sterile Na2HP04-NaH2P04 buffer solution at pH 7.0. Typically, the cell dry weight of these first stage cultivations was 0.5 g/L. The washed cells were then transferred under aseptic conditions into 100 mL of a sterile filtered nitrogen-free medium which contained 1.51 g/L Na2HP04, 2.65 g/L KH2P04, 0.2 g/L MgS04, 1.0 mL/L Microelement solution (Kunioka et al., Appl . Microbiol . Biotechnol . , .10, 569, 1989), 4- hydroxybutyric acid (1.5 g) or fructose (1.5 g) , and either 0, 1, 2, or 4% (wt/vol) PEG-200. Polymer production was then carried out by cultivation of A. eutrophus in the above media using a 500 mL Erlenmeyer flask at 30°C, 250 RPM, for 48 hours. The cells were then separated by centrifugation, washed with about 10 L of water per gram of wet cells, and lyophilized.
500 mL Cultivations (Cultivation Condition B) : Increased PHA from media amended with 4% PEG needed for fractionation and subsequent analysis (see below) was obtained as described above by the two-stage method but using 2800 mL Erlenmeyer flasks and 500 mL cultivation volumes. A. latus Fermentation Conditions
A. latus was grown on 1.0% glucose (w/v) in a one- stage fermentation where PEG-200 was added to the initial growth media as described in Hiramitsu, et al. , Biotechnol . Letters 15:461 (1993). It was anticipated that under these conditions, the PHA formed by A. latus would be P3HB. A. latus grew and divided initially in the presence of up to 3% (w/v) PEG-200, but showed no bacterial growth, and hence no PHA production at 4% PEG- 200. Increasing the media concentration of PEG-200 from 0 to 1% caused little change in the cell and polymer yields. However, at a media concentration of 3% PEG-200, the biomass and polymer yield productivity dropped precipitously.
Polymer Isolation The intracellular PHAs formed from each
Alcaligenes species were extracted from cells by stirring a suspension of lyophilized cells (about 0.5 g) for 48 hours in chloroform (80 L) at room temperature. The insoluble cellular material was removed by filtration, and the solvent was then evaporated to obtain what is termed herein the "crude product." Precipitated products were isolated by concentrating the chloroform crude product solution to a total volume of ~4 mL and precipitation of the polymer in 30 mL of methanol. The resulting precipitate was washed with methanol and ether and then dried in vacuo . Unless otherwise specified, the isolated products were obtained using one precipitation/washing cycle.
The PHA formed from 4-hydroxybutyrate in the medium with 4% PEG (cultivation condition B, see above) and isolated by one precipitation/washing cycle was dissolved in chloroform (0.1 g/mL) . Acetone (10 volumes) was slowly added to the chloroform solution. The white cotton-like precipitate which resulted from acetone addition was isolated by filtration giving the acetone insoluble (AIS) fraction. The solvent was evaporated from the acetone-chloroform solution which gave the acetone soluble (AS) fraction. Removal of residual solvents from the AS and AIS fractions was carried out in a vacuum dessicator (10 mm Hg, 24 hours) and the samples were then allowed to age for at least one week at ambient temperature prior to carrying out thermal analyses. Polymer Characterization A UNITY-500 NMR Spectrometer was used for 1 and 2- D proton NMR experiments described below. Proton (1H) NMR were recorded at 500 MHz. Chemical shifts in parts per million (ppm) were reported downfield from 0.00 ppm using tetramethylsilane (TMS) as an internal reference. The experimental parameters were as follows: 0.5% w/v polymer in chloroform-d, temperature 298°K, 2.4 μsec
(14°) pulse width, 3 second acquisition time, and 6,000 Hz spectral width. Carbon (13C) NMR spectra were recorded using a Varian XL-300 at 75.4 MHz and the following parameters: 2.0% w/v polymer in chloroform-d, 298°K, 9.7 μsec pulse width, 1 second acquisition time and 2 second pulse delay, 16502 spectral width, 33024 data points, and 14400-19600 accumulations. The observed 13C NMR chemical shifts in ppm were referenced relative to chloroform-d at 76.91 ppm. For the COSY experiment (0.5% w/v polymer in chloroform-d) the data were collected in a 1024 x 256 data matrix and zero-filled to 1024 x 1024 using 8 scans per increment, a 4260 Hz sweep width, and a 1.1 second delay between transients. The data was processed using sinebell weighting. The polymers produced by A. latus were analyzed by l-D^H NMR (500 MHz) and COSY (500 MHz) spectroscopy. Spectra of PHA samples were recorded on a Varian XU-500 spectrometer. Parameters for the 1-D-1H and COSY polymer spectra were as follows. 1.0% (w/v) polymer in CDC13, temperature 298°K, 2.4 and 14.5 μsec pulse widths, 8000 and 2710 spectral widths, 3.0 and 0.189 second acquisition times, 0 and 1.0 second delay times, and 45 and 8 transients, respectively. The molecular weights of polyesters were determined by GPC studies using a Waters HPLC system with 500-, IO3-, IO4-, and 105-A Ultrastyragel columns placed in series. Chloroform (HPLC grade) was used as the eluent at a flow rate of 1.0 mL/min, sample concentrations were typically 3 to 10 mg/mL and the injection volume was 100 μL. Detection was by refractive index (Waters Model 410) . Polystyrene standards (Aldrich) with low polydispersities were used to generate a calibration curve from which product molecular weights were determined with no further corrections.
All thermal characterizations were carried out using a DuPont 2910 differential scanning calorimetry (DSC) equipped with a TA 2000 data station, using between 5.0 to 6.0 mg of sample sealed in aluminum pans and a dry nitrogen purge. The polymer samples were heated at a rate of 10 °C/min from room temperature to 200°C, rapidly quenched from the melt and then were analyzed during second heating scans from -80°C to 200°C Data reported for the melting temperature(s) , Tm, and enthalpy of fusion(s) , ΔHf, were taken from the first heating scan. The reported glass transition temperature (Tg) values were the midpoint values measured during the second heating scans. EXAMPLES Effect of PEG on PHA Repeat Unit Composition
Control of composition for copolyesters of 3HB and 4HB is normally achieved by variation in the carbon sources used or by alteration of other physiological parameters such as the incubation time and nitrogen concentration. In this study, PEG-200 was added to A. eutrophus cultivations in concentrations up to 4% (w/v) during the second or polymer producing stage of the fermentation where 4HB served as the carbon source. The mol fractions of repeat units for PHAs isolated by one precipitation/washing cycle were analyzed by 1H NMR spectral integration of well resolved signal regions (see Fig. 1) as has been previously described, e.g., in Nakamura et al., Macromolecules, 25:4237-4241 (1992). When A. eutrophus is grown on 4HB without PEG added, the resulting PHA closely approximates a random copolyester of 3HB and 4HB repeat units of high molecular weight, with no 3HV or EG repeat units (see Table I, below) . Upon the addition of PEG-200 in culture conditions A, dramatic shifts in the repeat unit composition were achieved. Table I below shows the effects of PEG-200 on the production and compositions of microbial polyesters formed by A. eutrophus using 4HB as carbon source. In particular, the mol% of 4HB in the product changed from 66% with 0% PEG-200 added, to 86% with 2% PEG-200 added. Upon further addition of PEG-200 from 2 to 4%, the mol% of 4HB decreased. Furthermore, the addition of PEG-200 resulted in products containing low level incorporation of 3HV repeat units (see Table I). Table I
polym, cult cell polym. PHA repeat units found in PHA Mn, Mw cond yield, content yield, mol % g/mol /Mn %PEG, g/L of g/L X IO"3 cells, %
3HB 4HB 3HV EG
5 O A 3.7 21 0.76 34 66 0 0 222.1 2.76
1 A 3.5 16 0.56 20 79 1.1 0 178.6 1.89
2 A 3.1 14 0.45 11 86 2.8 0.28 153.0 2.05
4 A 2.6 14 0.37 30 64 5.0 0.93 112.2 2.51
0 B 3.9 27 1.1 70 30 0 0 198.6 2.87
) 4 B 3.7 26 0.97 41 53 5.4 1.1 77.2 3.95
4-AS B 13 84 2.1 1.6 37.4 2.52
4-AIS B 95 3 2.0 0.1 130.0 3.42
In Table I, PEG was added to the cultivation medium during the second, polymer producing stage. Non- fractionated samples were obtained from one precipitation/ washing cycle. As described in further detail below, 4-AS and 4-AIS are the acetone soluble (57% w/w) and insoluble (43%) fractions of the 4% PEG product obtained using cultivation condition B. The cell yield is the quantity of harvested cells after they were washed with nanopure water and lyophilized. The polymeric content of the cells is expressed as the percent of the cellular dry weight which contains PHA. These values were obtained gravimetrically from the isolated product from chloroform extraction and one precipitation/washing cycle. The PHA yield is the (cell yield) X (fraction of the cellular dry weight which is PHA) . The Mn and Mw/Mn were determined by GPC The cultivation conditions are either condition A or B as indicated. This experiment was repeated and the identical trends were observed.
As shown in Table I, small quantities of PEG-200 added to fermentation media caused important product As shown in Table I, small quantities of PEG-200 added to fermentation media caused important product compositional changes. The addition of PEG to the media increased both 4HB and 3HV contents, while decreasing the 3HB contents. It is known, e.g., as described in Doi et al., Microbial Polyesters (VCH Publishers, N.Y. 1990), that by decreasing the mol percentage of 3HB while increasing the relative contents of 4HB or 3HV in polymers, products are formed that have a relatively higher flexibility and elongation at break.
Formation of PHA-PEG Diblock Copolymers
The 1H NMR spectrum of the PHA isolated by one precipitation/washing cycle for a cultivation containing 4% PEG-200 (culture condition B, see Table I) is shown in Fig. 1. Weak 2H NMR signals at -3.7 ppm were observed that correspond to protons (a,c,d,e) of ethylene glycol (EG) repeat units. In contrast, P(3HB-co-4HB) formed in the absence of PEG (not shown) does not show any 1H NMR signals in the 3.6 to 3.8 ppm spectral region. A COSY spectrum of this product was recorded and the specific spectral regions of interest are shown in Fig. 2. Three E signals at 4.25, 4.35, and 4.46 ppm were observed that have correlations (coupling between neighboring 1H nuclei) with signals at 3.70, 3.73 and 3.77 ppm, respectively. The signal at 4.25 ppm also has a contribution from a satellite peak of protons 8. (4.1 ppm) (in Fig. 1) due to 13C-1H coupling. Based on chemical shift parameters documented for model compounds, it is expected that esterification of a terminal PEG-CH2- OH will lead to a downfield shift from -3.7 to -4.25 ppm. Considering these results and data, the signals in the 3.68 to 3.80 and 4.20 to 4.50 ppm regions were assigned to protons a and b, respectively, of esterified PEG chain segments. Correlation of the signals with peaks at 3.62 and 3.73 suggest that they are due to protons e and d of terminal free hydroxyl EG units (see Fig. 2) . Assuming that the contribution of the overlapping signals in the 3.6 to 3.8 region can be estimated by Bernoullian curve fitting, the area under peaks was measured by cutting and weighing. The integration results showed that the ratio of protons a + d to c to 2x e was 3:6:2. Using the ratio c to a + d and c to 2x e gives values of n (internal EG units of 2 and 3, respectively) .
Thus, the average chain length of PEG segments in the diblock copolymer is between 4 and 5 which corresponds to molecular weights of ~180 and 220 g/mol, respectively. The above results are consistent with the formation of PHA chains that are covalently linked at the carboxylate chain terminus to PEG chain segments, which indicates that PHA-PEG diblock copolymers were formed (see Figs. 1 and 2) . Furthermore, the average PEG chain length in the product is almost identical to that which was provided in the cultivation media.
Such PHA-PEG diblock copolymers include a long PHA chain segment (average of 430 4HB repeat units) that is covalently linked with an ester bond at its carboxy terminal end to a relatively short PEG chain segment (average of 5 repeat units) . These PHA-PEG diblock copolymers provide unique characteristics compared to PHA products currently available. For example, the diblock copolymers include terminal ethylene glycol (EG) hydroxyl functionalities that allow the formation of chemical linkages with drugs, they have amphipathic characteristics, and they can be used in blends as compatibilizing agents.
The invention also provides a unique method to incorporate PEG into PHA formulations such that the PEG will leach out of the PHA into an aqueous media at a much slower rate than formulations in which PHAs and PEG are merely mixed together.
The following experiments were performed to provide further evidence that 1H NMR signals observed in the 3.6-3.8 ppm region for one-time-precipitated products were not due in part to residual PEG-200. P(3HB-co-30% 4HB) (produced by a cultivation of A. eutrophus with no added PEG-200) and PEG-200 (286 and 218 mg, respectively) were dissolved in chloroform and cast to form a film. This film contained 43% by weight PEG-200, which exceeds by a factor of ~2 times the quantity of PEG-200 found in the corresponding crude product (non-precipitated- solution extracted material, see discussion below) . The film was then purified by one precipitation/washing cycle using identical conditions as was used for isolated products. The resulting isolate contained 0 mol % PEG based on *H NMR analysis. Therefore, no residual PEG exists in the one-time-precipitated product.
In addition, PEG-200 (0.3% w/w) was mixed with a PHA-PEG product (0.7% w/w) obtained after 3 precipitation/ washing cycles from 4% PEG amended cultivations. Once again, after only one precipitation/washing cycle, the relative signal intensities of the 3.6-3.8 ppm signal region to PHA protons was identical to that of the PHA-PEG product prior to mixing with PEG-200. Moreover, repeated precipitation/washing (up to three times) of one-time- precipitated samples did not result in a change in the EG mol percentage. Therefore, non-covalently linked PEG-200 is indeed removed efficiently from the isolated products by one precipitation/washing cycle.
Formation of Copolyester Blends
PHAs isolated from A. eutrophus cultivations in which 4-hydroxybutyric acid served as a carbon source and PEG-200 was not added to the media have sequence distributions of 3HB and 4HB repeat units that are approximately random. See, e.g., Nakamura et al., Macromolecules , 5:4237- 241, 1992. A GPC trace of the extracted crude material from A. eutrophus cultivations (culture condition B using 4- hydroxybutyrate as the carbon source, see Table I) containing 4% PEG was quite complex indicating that it is a mixture or blend of polymers having very different molecular weight averages (see Fig. 3b) . The mixture has unique physical and biological properties. In contrast, the GPC trace of the crude polymer product obtained from cultivation media without PEG-200 shows only a uni odal peak (see Fig. 3a) . Also, the GPC trace of the crude product has a component peak with an elution volume which corresponds exactly with that of PEG-200 (Fig. 3b, peak at 200 g/mol) . This is further evidence that PEG-200 does indeed accumulate in the cells, and that this occurs without notable cellular selectivity as a function of PEG chain length.
Consistent with the studies above in which PEG-200 was mixed with PHAs and removed by one precipitation/washing cycle, the GPC trace of the one¬ time-precipitated product (Fig. 3c) shows no trace of residual PEG-200, but still shows multiple component peaks.
Fractionation of PEG Cultivation Products The PHA product was fractionated based on its solubility in acetone. Fractionation resulted in an acetone soluble fraction (AS) representing 57% (w/w) of the total product, and an acetone insoluble fraction (AIS) representing 43% (w/w) of the total product. The AS and AIS fractions had Mn (M-^/M..) values of 37,400 (2.52) and 130,000 (3.42), respectively (see Table I). The fact that the PHA product could be fractionated provides additional evidence that the product is a mixture or blend of polyesters as opposed to a block copolymer.
Analysis of the repeat unit composition of these fractions by 1H NMR spectral integration showed that the mol fractions of 3HB, 4HB, 3HV, and EG repeat units are 13, 84, 2.1, 1.6 and 95, 3, 2, 0.1, respectively (see Table I) . Thus, the addition of PEG-200 to cultivation media results in the formation of a new product that is a blend of polyesters in contrast to the random copolymers formed in the absence of PEG-200.
Also, the PEG chain segments are found primarily in the AS high 4HB fraction (see Table I) . This is evidence that for A. eutrophus , linkages between PEG and PHA segments occur primarily between 4HB and EG repeat units.
Using a model in which it is assumed that PEG segments are at all carboxyl terminal positions of PHA chains, the Mn calculated molecular weight based on H NMR spectral integration is 24,000 g/mol, whereas the experimentally determined value from GPC is 37,400 g/mol. From this analysis, the results are consistent with this model.
NMR Analysis of the Copolyester Blend Fractionations
The effects of PEG-200 on the repeat unit sequence distribution were studied using the 13C NMR spectra for the one time precipitated/washed products obtained from fermentations with 0 and 4% PEG-200 (culture condition B) . The 13C NMR spectrum for the latter product is shown in Fig. 4 and expansions of the carbonyl regions of the unfractionated product, acetone soluble (AS) fraction, and acetone insoluble (AIS) fraction are shown in Figs. 5a, 5b, and 5c, respectively. The assignments of the observed signals, including those in the carbonyl region which are sensitive to effects of repeat unit sequence distribution, were made as described in, e.g., Nakamura et al., Macromolecules , 25:4237-4241 (1992).
To simplify the repeat unit sequence analysis below, the small contributions from 3HV and EG repeat units were neglected so that the products were assumed to consist of only 3HB and 4HB repeat units. The relative mol fractions of 3HB*-3HB (3*3), 3HB*-4HB (3*4), 4HB*-3HB (4*3) and 4HB*-4HB (4*4) dyads (see Figs. 5a to 5c) were determined by spectrometer integration and are given in Table II below, which shows the experimental and calculated comonomer dyad fractions for PHAs and product fractions formed in cultivations with and without PEG- 200. Experimental values were compared to those calculated assuming a Bernoullian or random statistical process for microbial catalyzed copolymerization using the following relationships (equations 1-3) where F3 is the mole fraction of 3HB units in the polymer as described, e.g., in Doi et al., Macromolecules , 21:2722 (1988) :
[3*3] = F3 2 (1)
[ 3*4 ] = [4*3 ] = F3 ( l-F3) (2 ) [ 4*4 ] = ( I-F3) 2 (3 )
Table II
polym Dyad Sequence % PEG, 3HB-3HB 3HB-4HB 4HB-3HB 4HB-4HB exp (calcd) exp (calcd) exp (calcd) exp (calcd)
0 0.60 (0.49) 0.14 (0.21) 0.10 (0.21) 0.16 (0.096)
4 0.57 (0.32) 0 (0.25) 0 (0.25) 0.43 (0.18)
4-AS 0.07 (0.03) 0.14 (0.15) 0.14 (0.15) 0.66 (0.67)
4-AIS 1.0 (0.85) 0 (0.07) 0 (0.07) 0 (0.01)
In Table II, each column shows the experimental values (exp) , determined by measuring the relative peak areas for the carbonyl carbon 13C NMR signals assigned (see Figs. 5a to 5c) to the four dyad sequences, and the calculated value (calcd) , determined from equations 1 to 3, assuming a Bernoullian or random statistical process and that the contribution of 3HV and EG repeat units can be neglected. In the first column, the indicated percent PEG was added to the cultivation medium during the second or polymer producing stage. The non-fractionated sample was obtained from one precipitation/washing cycle from a cultivation carried out using 500 mL of media in a 2.8 L shake flask. The AS and AIS fractions of the 4% PEG product represent 57% and 43% (w/w) of the product, respectively.
Table II shows that the PHA produced with 0% PEG approximates a random copolyester. In contrast, the addition of 4% PEG to cultivations resulted in a novel product that has predominantly 3HB*-3HB and 4HB*-4HB dyads (see also, Table I rows 4-AS and 4-AIS, and Fig. 5a) . Thus, the addition of PEG to the growth medium provides a new microbial polymerization process in which polymer blends are made directly by a single fermentation reaction. Therefore, this new process is much more efficient than prior methods to produce blends of polymers in which one component polymer has a high (greater than 70%, and preferably greater than 90%) 3HB content, and the other component polymer has a high (greater than 70%, and preferably greater than 80%) 4HB content. Furthermore, each of the two component polymers represents at least 30% of the total weight of the blend.
Thermal Analysis of the Copolyester Blend Fractionations Table III, below, shows the results of thermal analysis obtained by DSC measurements at a scanning rate of 10°C/min. The percent PEG added to the cultivation medium was added during the second or polymer producing stage. The non-fractionated samples were obtained from one precipitation/washing cycle. Again, 4-AS and 4-AIS are the acetone soluble (57% w/w) and insoluble (43%) fractions of the 4% PEG sample. In Table III, Tg represents glass transition temperatures taken as the midpoint of the heat capacity change and measured during the second heating scan after rapidly quenching by liquid nitrogen at -70°C from the melt. Tm represents the peak melting temperatures for each endothermic melting transition determined during the first heating scan. ΔHf (cal/g) represents the heat of fusion value measured for each melting endothermic transition. Cultivations were carried out using 500 mL of media in a 2.8 L shake flask (culture conditions B) .
Table III
Figure imgf000026_0001
As shown in Table III, the DSC thermograms of the 4% PEG product during a first heating scan showed two distinct Tm values at 55° and 170°C (see also Fig. 6a) which closely approximate reported Tm values for P3HB and P4HB (177° and 54° C, respectively) . The DSC thermogram of this product recorded during a second heating scan after rapidly quenching from the melt showed Tg values at -45° and -29°C (see also Fig. 7a). The Tg at -45°C closely approximates that reported for P4HB (-50°C) while the Tg at -29°C is intermediate to those reported for P3HB (-4°C) and P4HB. This indicates the formation of a copolymer blend of predominantly P3HB and P4HB. The observed Tg at -29°C may result from the formation of a small product fraction that consists of random 3HB/4HB copolyester chains having the corresponding Tg value. In contrast, the product obtained from cultivations with no added PEG had Tm and Tg values of 165 and 3°C, respectively, which is consistent with the formation of a random copolyester.
13C NMR and DSC measurements of the AS and AIS fractions were made to further characterize the individual component polymers of the copolyester blend formed in 4% PEG amended media. Expansions of the 13C NMR carbonyl spectral regions for these fractions are shown in Figs. 5b and 5c, respectively. DSC thermograms of the first and second heating scans are shown in Figs. 6a to 6c and 7a to 7c, respectively. If the Tg, Tm, and ΔHf values for solution precipitated P3HB are taken as 4°C, 20.8 cal/g, and 177°C, respectively, comparison of these data to those obtained for the AIS fraction (see Table III) indicates that this fraction contains primarily P3HB homopolymer, as opposed to a random copolyester such as P(3HB-co-6 mol% 4HB) that has been shown to have Tm and ΔHf values of 162°C and 13.5 cal/g, respectively (Nakamura et al., Macromolecules , 25:4237-4241, 1992). This is further supported by the 13C NMR spectrum of the AIS fraction which shows only 3HB*-3HB dyads (see Fig. 5c) .
The dyad sequence distribution of the AS fraction determined experimentally (see Fig. 5b) , and calculated using equations 1 to 3, above, suggests that the product formed approximates that of a high 4HB content random copolyester (see Table II) . Further study of this fraction by DSC indicated product heterogeneity. Specifically, the AS fraction had multiple Tg (-15,-42°C) transitions and a broad melting region (see Table III, Figs. 6b and 6b) . A comparison of the thermal transitions of this product fraction with those previously reported for 3HB/4HB random copolyesters (Nakamura, 1992, supra) indicates that the AS fraction is composed of P(3HB-co-90% 4HB) and P(3HB-co-28% 4HB) random copolyesters (Tg values of -44 and -15°C, respectively) (Nakamura, 1992, supra) .
This analysis, assuming that the components are immiscible, indicates that the AS fraction is a mixture of random copolyesters with relatively high and low 4HB contents (-90-94 and -30 mol%, respectively) with weight fractions of -86 and 14%, respectively. Thus, it appears that the unfractionated product from media containing 4% PEG is indeed complex as was originally indicated by the GPC trace (see Figs. 3b and 3c, above) , and is composed of at least three different component polymers of different repeat unit composition.
Effect of PEG on PHA Molecular Weight and Yield Results of Studies on A. eutrophus (CS. 4- Hydroxybutyric Acid)
Table I also depicts the effects of PEG-200 on volumetric yield and product molecular weight for the series of fermentations of A. eutrophus carried out under culture conditions A. The volumetric yield of the PHAs continued to decrease with increased PEG media concentration so that for 2 and 4% PEG-200 addition the yields were approximately 59% and 49%, respectively, of that for PEG deficient media. The Mn and Mw/Mn values measured by GPC of the products formed from cultivations with 0, 1, 2, and 4% PEG are also shown in Table I. The GPC traces of these products were unimodal. An increase in the PEG concentration from 0 to 4 percent resulted in a decrease in product molecular weight (Mn) from 222,100 g/mol to 112,200 g/mol, e.g., about 50 percent. Thus, PEG can be used to form PHAs that contain 4HB repeat units and have reduced molecular weights compared to PHAs produced without PEG. A decrease in molecular weight affects the polymer characteristics, e.g., decreases the melt viscosity, and is useful to form sustained release compositions and biomaterials that require relatively shorter bioresorption times.
Comparison of PEG Effects on P3HB Molecular Weight in A . latus and A. eutrophus PEG-200 was added to the fermentation media of each organism either initially, or at the beginning of the second stage of a two stage fermentation. Table IV shows the effects of PEG-200 on bacterial growth, polymer production, and polymer composition from 1 and 2 stage cultivations of A. latus grown on glucose (designated by the letter L) , and 2 stage cultivations of A. eutrophus grown on fructose (designated by the letter E) . In Table IV, R-CDW is "residual cell dry weight" which corresponds to the non-polymer weight of the cells which may be considered the residual biomass.
Figure imgf000030_0001
Results in A . latus
As shown in Table IV, increasing the media concentration of PEG-200 from 0 to 1% caused little change in the cell and polymer yields for A. latus . However, at a media concentration of 3% PEG-200, the biomass and polymer yield productivity dropped precipitously. These results are likely due to the increased osmotic stress caused by increasing media PEG- 200 concentrations. Further, the cellular productivity of A. latus was constant between 0 and 2% PEG-200 concentrations, but at 3% PEG-200, cellular productivity was reduced by 80% to 0.3 mg/mg R-CDW. This reduction indicated that above 2% PEG 200, the decrease in product yield was due not only to poorer cell yields, but was also the result of a less efficient production system.
The molecular weights of the isolated polymer products formed by A. latus were analyzed by gel permeation chromatography (GPC) . Unless otherwise specified, the isolated products were obtained using 2 precipitation/ washing cycles.
The addition of only 1% PEG-200 to A. latus cultivation media resulted in a decrease in the Mn by 85% from 238,000 g/mol to about 35,000 g/mol (see Fig. 8). Further increases in the media PEG-200 concentration from 1 to 3% resulted in little to no further molecular weight change. Thus, low PEG-200 media concentrations (1%) can be used to modify product molecular weight.
For two-stage cultivations of A. latus , polymers were formed in production medium containing up to 6% PEG- 200, where 2, 3, and 4% PEG were added to cultivations containing 2% PEG-200 after a first-stage 24 hour cultivation. This two-stage approach resulted in polymers with Mn values as low as 19,000 g/mol. Furthermore, products L-2 to L-7, when analyzed by 1- and 2-D proton NMR as described above for products derived from 4-hydroxybutyric acid, were shown to contain diblock copolymers of a P3HB chain segment covalently linked at its carboxyl terminal end to a PEG chain segment. Results in A. eutrophus
For comparative purposes, a series of two-stage cultivations were carried out using A. eutrophus as the microbial production system where variable quantities of PEG-200 were added to cultivation media (see Table IV) . Cellular growth and polymer production for all A. eutrophus cultures were carried out using a two-stage batch culture process as described above for cultivation conditions A, using fructose as the carbon source for the second stage cultivations. A. eutrophus showed only small decreases in cell and product yield with the addition of up to 2% PEG-200. Also, the cellular productivity calculated using the non- polymer or residual cell dry weight (R-CDW, see Table IV) remained almost unchanged (-1.0 mg/mg R-CDW) for media containing up to 5% PEG-200, but decreased to 0.13 mg/mg non-polymer CDW when the PEG-200 media concentration was increased to 10%. Thus, A. eutrophus showed an excellent tolerance to the osmotic stress imposed by the solute PEG-200. Fig. 8 shows that the molecular weight of product polyesters was decreased by increasing the media PEG-200 concentration. In fact, there was a regular decrease in product molecular weight as the PEG-200 concentration was increased from 0 to 1% (Mn values of 650,000 g/mol and 104,000 g/mol, respectively). As was observed for A. latus , further increases in the media PEG-200 concentration from l to 5% resulted in substantially less molecular weight reduction per added PEG increment (see Fig. 8) . Thus, sensitive control of product molecular weight was achieved by variation of the media PEG-200 concentration from 0 to 1% for both A. latus and A. eutrophus .
USE The methods described above allow the modification of microbial polyester products by simply changing the concentration of PEG added to the cultivation medium. By controlling the amount of PEG added, one can control the molecular weight, repeat unit composition and distribution, and produce specified copolyester blends as opposed to random copolyester chains.
In particular, such diblock copolymers and copolyester blends of the invention can be used to make biodegradable plastic articles and coatings, e.g., for paper, that are manufactured by standard thermal processing methods. These new copolyesters can also be used for microencapsulation, e.g., of cells or drugs, to produce cell growth matrices, and to produce biomedical materials such as sutures, implants, and drug delivery vehicles.
Other Embodiments
As described in Shi et al. , Polymer Preprints, Am . Chem Soc , 36(1) .430-432 (April 1995), A. eutrophus grown on fructose resulted in the formation of P3HB with a high Mn. When PEG-200 was added to the culture media, the P3HB Mn decreased significantly. For example, the addition of 0.2% PEG decreased P3HB Mn by a factor of 2. Further increases in PEG concentration up to 10% resulted in decreased P3HB molecular weight by a factor of about 10. Copolyester blends including component polymers having repeat unit structures other than 3HB and 4HB can also be made according to the invention. For example, component polymer repeat unit structures of 3- hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxy-4- pentenoate, and 3-hydroxypropionate, can be prepared using the corresponding acids, e.g., 3-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxy-4-pentenoic acid, and 3-hydroxypropionic acid, as carbon sources.
In addition, other carbon sources can be used to form 3HB and 4HB repeat unit structures. For example, 1,4-butane diol and 1,6-hexane diol can be used as carbon sources to produce 4HB repeat unit structures. Thus, the addition of PEG to polymer production media containing these carbon sources will also cause an increase in the 4HB mol percentage compared to fermentation without the added PEG.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Claims
1. A method for producing a polyhydroxyalkanoate (PHA) having a controlled molecular weight, said method comprising culturing a PHA-producing microorganism in a polymer production medium under conditions that allow the microorganism to produce a PHA, and adding polyethylene glycol (PEG) to the polymer production medium in an amount sufficient for the microorganism to produce a PHA having a molecular weight that is decreased relative to the molecular weight of a PHA produced by the same microorganism under the same growth conditions without PEG.
2. A method of claim 1, wherein the PHA-producing microorganism is an Alcaligenes bacterium.
3. A method of claim 2, wherein the PHA-producing microorganism is Alcaligenes eutrophus , and wherein PEG is added to the polymer production medium at a concentration of 0.25 to 10.0 percent (weight/volume) .
4. A method of claim 2, wherein the PHA-producing microorganism is Alcaligenes latus , and wherein PEG is added to the polymer production medium at a concentration of up to 6.0 percent (weight/volume) .
5. A method for incorporating 3-hydoxyvalerate (3HV) repeat units into a polyhydroxyalkanoate (PHA) using a non-3HV carbon source, said method comprising culturing a PHA-producing microorganism in a polymer production medium containing a non-3HV carbon source under conditions that allow the microorganism to produce a PHA, and adding polyethylene glycol (PEG) to the polymer production medium in an amount sufficient for the microorganism to produce a PHA comprising 3HV.
6. A method of claim 5, wherein the non-3HV carbon source is 4-hydroxybutyric acid or 4- hydroxybutyrate.
7. A method of claim 5, wherein PEG is added to the polymer production medium at a concentration of from 1 to 4 percent (weight/volume) .
8. A method for producing a polyhydroxyalkanoate (PHA) comprising a copolyester blend of at least two component polymers wherein each polymer represents at least 30 percent by weight of the total blend, each component polymer is composed of at least 70 percent of a specific repeat unit structure, and the major repeat unit structure in each component polymer is different, said method comprising culturing a PHA-producing microorganism in a polymer production medium containing a carbon source under conditions that allow the microorganism to produce a PHA, and adding polyethylene glycol (PEG) to the polymer production medium in an amount sufficient for the microorganism to produce a PHA comprising a copolyester blend.
9. A method of claim 8, wherein PEG is added at a concentration of 4 percent (weight/volume) .
10. A method of claim 8, wherein the carbon source is 4-hydroxybutyrate.
11. A method for producing a polyhydroxyalkanoate-polyethylene glycol (PHA-PEG) diblock copolymer in which the carboxyl terminus of a PHA chain segment is covalently linked by an ester bond to a PEG chain segment, said method comprising culturing a PHA-producing microorganism in a polymer production medium under conditions that allow the microorganism to produce a PHA, and adding polyethylene glycol (PEG) to the polymer production medium in an amount sufficient for the microorganism to produce a PHA-PEG diblock copolymer.
12. A method of claim 11, wherein the polymer production medium comprises glucose as the carbon source, and wherein the microorganism is Alcaligenes latus .
13. A method of claim 12, wherein PEG is added at a concentration of up to 6 percent (weight/volume) .
14. A method of claim 12, wherein the PHA chain segment contains only P3HB repeat units.
15. A method of claim 11, wherein the polymer production medium comprises 4-hydroxybutyric acid as the carbon source, and wherein the microorganism is Alcaligenes eutrophus.
16. A method of claim 15, wherein PEG is added at a concentration of 4 percent (weight/volume) .
17. A method of claim 15, wherein the diblock copolymer comprises a majority of 4HB repeat units.
18. A method for increasing the 4-hydroxybutyrate (4HB) mol percent in a polyhydroxyalkanoate (PHA) , said method comprising culturing a PHA-producing microorganism in a polymer production medium containing 4-hydroxybutyric acid as a carbon source under conditions that allow the microorganism to produce a PHA, and adding polyethylene glycol (PEG) to the polymer production medium in an amount sufficient for the microorganism to produce a PHA of increased 4HB mol percent.
19. A method of claim 18, wherein PEG is added to the polymer production medium at a concentration of 1 percent (weight/volume) .
20. A method of claim 18, wherein PEG is added to the polymer production medium at a concentration of 2 percent (weight/volume) .
21. A polyhydroxyalkanoate (PHA) copolyester blend comprising first and second polymers each comprising at least 30 percent by weight of said blend, wherein said first polymer comprises at least 70 mol percent of a first repeat unit structure, said second polymer comprises at least 70 mol percent of a second repeat unit structure, and wherein said first and second repeat unit structures are different.
22. A copolyester blend of claim 21, wherein said first repeat unit structure is 3-hydroxybutyric acid.
23. A copolyester blend of claim 21, wherein said first polymer comprises at least 90 mol percent of a first repeat unit structure.
24. A copolyester blend of claim 21, wherein said second repeat unit structure is 4-hydroxybutyrate.
25. A copolyester blend of claim 21, wherein said second polymer comprises at least 80 mol percent of a second repeat unit structure.
26. A polyhydroxyalkanoate-polyethylene glycol diblock (PHA-PEG) copolymer comprising a first chain of PHA repeat units and a second chain of PEG repeat units, wherein the second chain of PEG repeat units is covalently bound via an ester bond to a carboxy terminal end of the first chain of PHA repeat units.
27. A PHA-PEG diblock copolymer of claim 26, wherein said first chain comprises poly-3- hydroxybutyrate, and said second chain comprises an average of 5 PEG repeat units.
28. A PHA-PEG diblock copolymer of claim 27, wherein said first chain comprises an average of 220 PHA repeat units.
29. A PHA-PEG diblock copolymer of claim 26, wherein said first chain comprises at least 80 mol percent of 4-hydroxybutyrate, and said second chain comprises an average of 5 PEG repeat units.
30. A PHA-PEG diblock copolymer of claim 29, wherein said first chain comprises an average of 435 PHA repeat units.
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