Development of a Process for the Biotechnological Large-Scale

Biomacromolecules 2001, 2, 45-57

45

Development of a Process for the Biotechnological Large-Scale Production of 4-Hydroxyvalerate-Containing Polyesters and Characterization of Their Physical and Mechanical Properties V. Gorenflo,† G. Schmack,‡ R. Vogel,‡ and A. Steinbu¨chel*,† Institut fu¨r Mikrobiologie, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Corrensstrasse 3, D-48149 Mu¨nster, Germany, and Institut fu¨r Polymerforschung Dresden e.V., Hohe Strasse 6, D-01005 Dresden, Germany Received September 27, 2000; Revised Manuscript Received November 16, 2000

A process for the large-scale production of 4-hydroxyvalerate (4HV)-containing biopolyesters with a new monomer composition was developed by means of high-cell-density cultivation applying recombinant strains of Pseudomonas putida and Ralstonia eutropha, harboring the PHA-biosynthesis genes phaC and phaE of Thiocapsa pfennigii. Cell densities of about 20 g/L revealing a PHA content of 52% (w/w) and a molar fraction of 4HV of up to 15.4 mol % were obtained by a two-stage fed-batch cultivation process at a 25-L scale using octanoic acid during the growth phase and levulinic acid for the accumulation of 4HV-containing polyesters. Besides 4HV the polyester contained significant amounts of both 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) and traces of 3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoic acid (3HO). With glucose or gluconic acid as the growth substrate, the components of the polyester could be reduced to mainly 3HV and 4HV with only a negligible fraction of 3HB, resulting in a polyester with a new composition. Scale-up of the cultivation process to a 500-L scale was successfully performed, resulting in the production of these polyesters at a pilot plant scale. Short-term shifts in temperature and pH resulted in the formation of cell agglomerates of about 50-100 µm by which the effectiveness of the semicontinuous centrifugation process was drastically increased. Washing of the freeze-dried cells with boiling methanol significantly shortened the extraction process and resulted in a polyester of higher purity. The physical and mechanical properties of these copolyesters were characterized by means of size exclusion chromatography, dynamic mechanical analysis, differential scanning calorimetry, stress-strain measurements, and measurements of the viscosity of the solution. The copolyesters were cast into films, spun to fibers, or processed into test bars by melt spinning and injection molding, respectively. They revealed an almost entirely amorphous structure and consequently were sticky and lacked strength. However they showed high thermal stability and an unusually high elongation at break of about 200%; the molecular weights (Mw) were between 2.0 × 105 and 3.3 × 105 g/mol. It was shown that 4HV-containing polyesters belong to the class of thermoplastic elastomeres. Introduction Aliphatic polyesters of hydroxyalkanoic acids (PHAs) are known as storage compounds since the discovery of poly(3-hydroxybutyric acid), poly(3HB), in Bacillus megaterium.1 They are mainly synthesized by various bacteria if a carbon source is provided in excess and if bacterial growth is impaired by the lack of at least one nutrient.2,3 Detailed knowledge about the synthesizing organisms and the physiological and genetic basis of polyester biosynthesis has been revealed.2-5 By means of genetic and metabolic engineering and by the control of the cultivation conditions in combination with the feeding of suitable precursor substrates, more than 100 hydroxyalkanoic acids have been detected as constituents of biosynthetic PHA.6,7 The composition of the PHAs produced and thus their physical and material properties depend both on the microorganism and carbon source † ‡

Institut fu¨r Mikrobiologie, Westfa¨lische Wilhelms-Universita¨t Mu¨nster. Institut fu¨r Polymerforschung Dresden e.V.

used.8,9 Various polyesters have been investigated with respect to their material properties revealing that they belong to the class of thermoplastic elastomeres which represent one of the most interesting developments in modern material research.8-14 The material properties of these biologically produced polyesters depend strongly on their monomer composition and can be varied in a wide range without losing biodegradability.14 Important developments concerning research and application prospects of mcl-PHAs were recently outlined.15 To enable thorough characterization of the properties of these novel materials, and to develop applications, a flexible process is needed to produce large amounts of these polyesters. Scale-up of the cultivation process to an industrial scale was developed successfully for very few polyesters. These are mainly poly(3HB) and the copolyester poly(3HBco-3HV), which are considered for several medical and technical applications.12,16 It would also be desirable to reach high production effectiveness in a large-scale process starting

10.1021/bm0000992 CCC: $20.00 © 2001 American Chemical Society Published on Web 01/11/2001

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Biomacromolecules, Vol. 2, No. 1, 2001

with cheap raw materials for other polyesters. High cell densities are a prerequisite for high productivity, and some bacteria can be grown to densities of more than 100 g/L.17 However these fermentation processes are often limited to small volumes or need particular cultivation strategies which are not applicable for economical large-scale production. Downstream processing must be an integral part of the production process. A number of solvent extraction processes for the recovery of poly(3-hydroxybutyric acid) (poly(3HB)) have been suggested based on chlorinated solvents such as chloroform, methylene chloride, or 1,2-dichlorethane.18 These methods lead to high extraction rates but are comparatively expensive and reveal a high potential for ecological pollution. One alternative has been to solubilize all cell material apart from poly(3HB), for instance, by employing alkaline sodium hypochlorite.19,20 This method appeared to be rather effective but led to a drastic reduction of the molecular weight of the polyester. As an alternative, an enzymatic digestion method was developed by ZENECA to recover poly(3HB) from Ralstonia eutropha.21 This process includes thermal treatment of poly(3HB)-containing biomass, enzymatic treatment, and washing with an anionic surfactant to dissolve non-poly(3HB) cell material. Cells of Pseudomonas putida could be broken by the combination of heat shock and treatment of the cells with alcalase, sodium dodecyl sulfate (SDS), and ethylenediaminetetraacetate without attacking the PHA granules. In this process SDS formed a major contaminant.22 Choi and Lee23 solubilized non-PHA material from Escherichia coli by treatment with a solution of sodium hydroxide. It recently was shown that polyesters containing 4-hydroxyvaleric acid (4HV) were synthesized and accumulated by several strains of Pseudomonas and Alcaligenes.24-26 4HV contributed up to 30 mol % of the hydroxyalkanoate constituents when 4-hydroxyvaleric acid was fed as the sole carbon source. In addition, these polyesters regularly contained 3HB and 3HV in considerable amounts. Since 4-hydroxyvaleric acid is not commercially available, levulinic acid (4-ketovaleric acid) was used as a precursor substrate for the accumulation of 4HV-containing polyesters. Levulinic acid can easily be produced from renewable resources such as the hydrolysis of fructose with HCl,27 making it a suitable carbon source for large-scale production processes. The aim of this study was to develop a system for the large-scale production of 4HV-containing polyesters with a high content of 4HV in order to facilitate the investigation of their material properties and the testing of the thermoplastic processing. This process should include the economical and ecological production of the polyesters by bacteria as well as the investigation of suitable downstream processing. In addition, the physical properties, such as the molecular weight and the thermal, rheological, and mechanical properties of the produced polyesters, were characterized and compared with those of a homopolyester of 3HV13 and copolyesters consisting of 4HV, 3HV, and traces of medium-chain-length hydroxyalkanoic acids.8 Materials and Methods Bacterial Strains and Plasmids. Bacterial strains used in this study are the PHA-negative mutants Ralstonia

Gorenflo et al.

eutropha H16 PHB-428 and Pseudomonas putida GPp10429 harboring the PHA synthase genes phaC and phaE of Thiocapsa pfennigii.30 Media. A mineral salts medium (MM) described by Schlegel et al.31 was used for PHA-accumulation experiments. Stock solutions of all media components, the carbon sources octanoic and levulinic acids and tetracycline or streptomycin, were sterilized separately and added to the basic medium as indicated in the text. Cultivation of bacteria. CultiVation in Erlenmeyer flasks was done in 500-mL Erlenmeyer flasks at 30 °C under aerobic conditions on a rotary shaker with 150 strokes/min (Pilotshake RC-4/6-W, Ku¨hner AG, Birsfelden, Switzerland) containing 10% (v/v) of MM. The pH of the supernatant was measured regularly and, if necessary, was maintained at 7.0 by the addition of the corresponding fatty acid or HCl (2 N), respectively. Cells were harvested by centrifugation at 8000 rpm (Sorvall RC-5B, Du Pont Instruments, Newton, MA). CultiVation of cells at a 25-L scale was done in a stirred (500-1000 rpm) and aerated (2-25 L of air min-1) 25-L stainless steel fermentor (Apparate- und Beha¨ltertechnik Harrislee GmbH, Germany), which was regulated by a control unit (EPSS, Industrie Technik GmbH, Flensburg, Germany). CultiVation of cells at a 500-L scale was done in a stirred (100-300 rpm) and aerated (100-300 L of air min-1) 650-L stainless steel fermentor (Biostat D 650, B. Braun, Biotech International, Melsungen, Germany) with the pH adjusted to 7.0. For inoculation, 25 L of a 24-h batch cultivation were transferred under axenic conditions from a 30-L stainless steel fermentor (Biostat UD 30, B. Braun, Biotech International, Melsungen). Octanoic acid, gluconic acid, or glucose was added as the sole carbon source for growth, and levulinic acid was added during the accumulation phase. Because octanoic acid showed an inhibitory effect at higher concentrations, it was fed as sodium salt in portions of 0.05-0.2% (w/v), whereas gluconic acid and glucose were added in portions of 0.12.0% (w/v). A solution of NH4Cl or NH4OH was provided as the nitrogen source. MgSO4, CaCl2, and Fe(III)-NH4citrate were added according to Schlegel et al.31 each time when the optical density (OD at 600 nm) of the cultivation broth had increased by 10 units. Concentrations of carbon sources and nitrogen sources in the supernatant were measured by high-performance liquid chromatography (HPLC) or test strips, respectively, and were supplemented as indicated in the text. Solutions of NaOH (2 N), NH4OH (32%, w/v), HCl (2 N), or levulinic acid (500 g L-1) were used to adjust the pH. Tetracycline (50 µg mL-1) was used for plasmid maintenance. Foam was destroyed by a FundaFoam foam centrifuge (B. Braun, Biotech International, Melsungen, Germany) or by adding Wacker Silicon Antischaum Emulsion SLE antifoam agent (Darwin Vertriebs GmbH, Ottobrunn, Germany). Accumulation of PHA within the Fermentor. PHA accumulation experiments were carried out as two-stage fedbatch fermentation processes. As a first step, cells were grown to high cell densities as described above. At the end of the growth phase, when the carbon and nitrogen source were exhausted, temperature, pH, aeration, and agitation were

Large-Scale Production of Biopolyesters

adjusted to the values indicated in the text. Levulinic acid was used both as carbon source and to adjust the pH during the accumulation phase. At the end of the accumulation phase, the cells were flocculated, harvested, and subjected to PHA analysis and extraction (see below). Automation of the Fed-Batch Fermentation Process. During fed-batch cultivations with glucose as the growth substrate, an aqueous solution of NH4OH (32%, w/v) was used to adjust the pH and to supplement consumed nitrogen automatically.32 Glucose was fed automatically by using the DO-stat. method. Supplements (MgSO4, CaCl2, and Fe(III)NH4-citrate) were added to the glucose stock solution. During cultivations with either gluconic acid or octanoic acid, NH4OH was added to the carbon stock solution. Monitoring of Cell Growth. Growth of the cultures was monitored by following the optical density by using either a Klett-Summerson filter photometer at 520-580 nm (filter No. 54) or a UV/VIS spectrophotometer at 600 nm (Ultrospec 2000, Pharmacia Biotech, Cambridge, England). Determination of the cellular dry matter was another method to monitor cell growth. Synthesis of Substrates. 4-HydroxyValeric acid (4HV) was obtained by alkaline saponification from the corresponding lactone. NaOH (10 N) was added dropwise to a chilled aqueous solution of 50% (w/v) γ-valerolactone, until the pH of the solution had increased to 13. The solution was stirred for 1 h and then neutralized through the addition of 1 N HCl.24 Sodium octanoate and leVulinate solutions were obtained by neutralizing an aqueous suspension of octanoic acid (30%, w/v) or a chilled aqueous solution of levulinic acid (50%, w/v), respectively, with NaOH microprills. Solutions of carbon sources containing ammonia were obtained by mixing an aqueous solution of NH4OH (32%, w/v) and either gluconic acid (50%, w/v) or octanoic acid (30%, w/v), until the pH of the solutions was 7.0. Concentrations of NH4OH, gluconic acid, or octanoic acid were adjusted by adding solutions of gluconic acid sodium salt or octanoic acid sodium salt, respectively. Simultaneous Detection of Glucose, Gluconic Acid, Levulinic Acid, and Metabolites. Concentrations of glucose, gluconic acid, 2-keto-gluconic acid, levulinic acid, and 4-hydroxyvaleric acid in the supernatants were determined by ion exchange chromatography using an HPLC apparatus (Knauer GmbH, Berlin, Germany). Supernatants were mixed (1:1, v/v) with sulfuric acid (5%, v/v), incubated at 70 °C for 3 h, and centrifuged at 13 000 rpm (Biofuge A, Heraeus, Osterode, Germany) for 20 min before subjecting 10 µL to HPLC analysis. Degassed 6.5 mM sulfuric acid at a flow rate of 0.5 mL/min (L-6200 Gradienten Pumpe Lichrograph, Merk, Darmstadt, Germany) was used as the mobile phase. Separation was done in an Aminex ion-exclusion column (HPX-87H, 300 × 7.8 mm, Biorad, Richmond, U.S.A.) at 65 °C (T-6300 column oven, Merck, Darmstadt, Germany). Glucose and gluconic acid could not be separated and were detected simultaneously. Detection was done using an RI detector (RI-71, Merck, Darmstadt, Germany) and evaluation of data by an integrator (Chromatopac C-R3A, Shimadzu, Duisburg, Germany).

Biomacromolecules, Vol. 2, No. 1, 2001 47

Simultaneous Detection of Octanoic Acid and Levulinic Acid. Simultaneous detection was done by reversed-phase chromatography using an HPLC apparatus (Knauer GmbH, Berlin, Germany). Substances were separated at room temperature using a Nucleosil-100 C18 column (7 µm, 250 × 4 mm) as a stationary phase, and a mixture of 47% (v/v) acetonitrile and 53% (v/v) potassium phosphate buffer (0.01 M in H2Obidest, pH 7.1) as mobile phase at a flow rate of 1.0 mL/min. Eluted substances were detected by a diode array detector (J&M, Analytische Mess- und Regeltechnik, Aachen, Germany), and signals were evaluated by using the Eurochrom 2000-Software. Ammonia and Glucose Quick Test. Concentration of ammonia in the supernatant was determined by test strips (Ammonium-Test, Merck, Darmstadt, Germany). Quick determination of glucose concentration within the supernatant was done by glucose test strips (Diaben Test 5000, Boeringer, Mannheim, Germany). Cell-Flocculation and Separation. Flocculation Process. Cells were flocculated within the fermentor to improve sedimentation (according to Walker et al.,33 modified). The pH of the cultivation broth was increased to 8.5 by the addition of NaOH (50%, w/v), and the temperature was adjusted to 70 °C and held for 15 min. After the broth was cooled to 25 °C, the pH was adjusted to 4.0 by the addition of HCl (37%, w/v). During this process, the suspension was stirred with 50 rpm for cultivations in a 500-L scale or 200 rpm for cultivations in a 25-L scale, respectively. Cell separation was then performed by a continuous centrifugation in Z41 or Z61 CEPA centrifuges (Carl Padberg, Lahr, Germany) for cultivations at a 25-L scale or a 500-L scale. The recovered biomass was frozen at -30 °C and then lyophilized (Beta 1-16, Christ, Osterode, Germany). Isolation of Polyhydroxyalkanoates. Washing. Lyophilized and crushed cells were washed in boiling methanol (50%, w/v) for 30 min to separate non-PHA fatty acids from the cells, and to increase cell permeability for the following PHA extraction step. Digestion of non-PHA Material by NaOH. Lyophilized cells were resuspended in 0.5 N NaOH to 50% (w/v) and stirred for 1 h at 30 °C. After the suspension was neutralized to pH 7.0 with HCl (37%, w/v) and cooled to room temperature, cells were collected by centrifugation (20 min, 13 000 rpm), washed twice with saline (0.9% NaCl in H2O), and then freeze-dried. Soxhlet Extraction. Lyophilized cells were extracted in a 0.3-0.5-L Soxhlet apparatus using thimbles (Schleicher & Schu¨ll, Dassel, Germany) under reflux with either chloroform or acetone.34 Precipitation and Purification of the Polyester. The polyester was precipitated from the chloroform solution by the addition to 10 vol of ethanol. The precipitate was separated from the solvents by filtration. Remaining solvents were removed by exposure of the polyester to a stream of air. Precipitation was repeated up to three times to obtain highly purified PHA samples. Filtration of the DissolVed Polyester. Insoluble particles remaining from the extraction process, and impending further investigation of the material properties of the polyester, were

48


removed by filtration of the polyester solution with a stainless steel fine-filter (SIKA-FIL 3, Krebso¨ge GmbH, Radevormwald, Germany). The filter enabling the separation of particles exceeding a size of 3 µm was fitted in a sterile filtration apparatus, and the solution was filtered by applying a pressure of about 50 mbar. Polyester Film Castings. Filtrated solutions of polyester in chloroform were put into glass or stainless steel bowls with different diameters to get casted films with a thickness of 1-5 mm. The remaining solvent was removed by exposing the solutions to a stream of air. Polyester Analysis. Polyester Content and Composition. The polyester content of the bacteria was determined by methanolysis of 3-5 mg of lyophilized cells in the presence of 15% (v/v) sulfuric acid.35 The resulting methyl esters of the constituent hydroxyalkanoic acids were characterized by gas chromatography.36,37 For identification of the methyl esters gas chromatography/mass spectrometry was also performed using a HP 6890 gas chromatograph equipped with a model 5973 mass selective detector (Hewlet Packard, Waldbronn, Germany). Molecular Mass Analysis. The molecular mass of the purified and filtered (Minisart SRP4, Sartorius, Go¨ttingen, Germany) PHA was determined by gel permeation chromatography (GPC) relative to polystyrene standards (Polymer Standards Service, Mainz, Germany). Analysis was performed by injecting 20-120 µL (717plus Autosampler, Rheodyne 7725i) of the PHA solution in chloroform on four Styragel columns (HR3, HR4, HR5, HR6) connected in line in a GPC apparatus (Waters, Milford, MA). Samples were eluted with chloroform at a flow rate of 1.0 mL/min (515 HPLC-pump) and at 35 °C (Jetstream 2 column oven), and the eluted compounds were monitored by a model 410 differential refractometer. Polydispersity and the number average (Mn) and weight average (Mw) molar masses were calculated by using the Millenium Chromatography-Manager GPC software (Waters). Processing of the Polyesters. After the isolation process the water content of the samples was reduced from about 0.2% to 0.06% (w/w) by 24 h of drying under vacuum at a temperature of 20 °C. The dried polyester was subsequently stored in a desiccator and was used for all experiments. The melt spinning experiments were performed with a selfconstructed spinning device,8 with a spinning velocity of up to 50 m/min. The injection molding experiments were carried out with a plunger injection molding machine (Minimixer, Musashino Kikai Co., Ltd., Japan). The polyester material was introduced into a preheated cylinder and was compressed by a piston under a nitrogen atmosphere. To homogenize the material, it was sheared with 10 rpm by shearing elements attached to the piston during the preheating of 5 min. When the melt reached 140 °C, it was pressed into the molding tool by a nozzle. For better demolding, the tool was cooled in liquid nitrogen for 30 s. Six to seven test bars were formed out of 3 g of polyester material. Characterization of the polyesters. The water content and the relatiVe Viscosity of the samples were determined as described elsewhere.8

Gorenflo et al.

Size exclusion chromatography (SEC) was performed by HPLC relative to polystyrene molecular weight standards.8 Differential Scanning Calorimetry (DSC). The glass transition temperature (Tg), the melting temperature (Tm), the melting enthalpy (∆Hm), and the heat capacity (cp) of the polyesters were determined by means of DSC.8 The DSC measurements were carried out in the temperature range of 30-150 °C under a nitrogen atmosphere, with a heating rate of 20 K/min. Dynamic Mechanical Analysis (DMA). The measurements were done on dried samples by means of a rotational rheometer (ARES, Rheometric Scientific, Inc., USA) under nitrogen at 140 °C in a shear frequency range between 10-1 and 102 rad‚s-1. The geometry chosen for these measurements was a plate-plate arrangement (25 mm plate diameter, 2 mm gap width). In addition, a temperature sweep was recorded beginning at 140 °C, at a mean shear frequency of 1 rad‚s-1 and at a cooling rate of 5.0 K‚min-1. The storage modulus G′ and the loss modulus G′′ were measured as a function of the shear frequency or the temperature. The complex melt viscosity (η*) and the tangent of the phase shift angle δ, tan δ ) G′′/G′, were calculated. The glasstransition temperature (Tg) was determined from the temperature sweeps. The mechanical properties of the original film castings (sample size 40 × 10 × 1 mm3) were determined by means of a stress-strain universal tester (TFCM, Instron, Offenbach, Germany) according to ISO 527. The mean value of the tensile test data of 10 specimens is reported. The physical break stress (σ) was calculated from the maximum strength (RH), and the elongation at break (H) according to the relation σ ) RH(1 + H/100). Results and Discussion Fermentative Production of the Polyester. Accumulation of PHA from Levulinic Acid by Strains of Pseudomonas putida and Ralstonia eutropha. Recently, it was found that cells of P. putida GPp104 (pHP1014::E156), harboring the PHA-biosynthesis genes from T. pfennigii, accumulated copolyesters consisting of 3HB, 3HV, and 4HV acid when cultivated in mineral salts medium with levulinic acid as the sole carbon source.7,38 The accumulation of 4HV-containing PHAs from levulinic acid as sole carbon source by the wild types and recombinant strains of P. putida and R. eutropha was investigated in detail (Table 1). The PHAs accumulated by the recombinant strains of P. putida revealed a significantly higher fraction of 4HV than those accumulated by the wild type or the strains of R. eutropha. For example, P. putida GPp104 (pHP1014::E156) accumulated a terpolyester with a 4HV content of 30 mol %. P. putida GPp104 (pHP1014::B28RV), which also harbors the PHA-biosynthesis genes from T. pfennigii, but on a shorter fragment, revealed a higher polyester content, but with less 4HV. The PHAs accumulated by the recombinant strains of P. putida consisted mainly of 3HV and 4HV and contained 3HB at only a minor fraction. It can be concluded that the drastic increase of the molar contents of 4HV and 3HV and the reduction in monomer types by the recombinant strains of P. putida are due to the recombinant PHA-synthase, which



Table 1. Composition of PHAs Accumulated by Recombinant Strains of R. Eutropha HF39 or P. Putida KT2440 after Growth on Levulinic Acida strain

Ralstonia eutropha HF 39 H16 PHB-4 (pHP1014::E156) H16 PHB-4 (pHP1014::B28) Pseudomonas putida KT2440 GPp104 (pHP1014::E156) GPp 104 (pHP1014::B28RV)

composition of PHA (mol %)

PHA content (% CDM)

3HB

3HV

4HV

3HHx

3HO

3HD

3HDD

66.2 60.3 57.5

46.4 73.6 85.5

51.5 22.6 13.0

2.1 3.8 1.5

n.d. n.d. n.d.

n.d. n.d. n.d.

n.d. n.d. n.d.

n.d. n.d. n.d.

22.3 41.8 54.7

3.2 0.8 0.7

22.7 69.2 80.4

0.8 30.0 18.9

10.7 n.d. n.d.

23.2 n.d. n.d.

36.4 n.d. n.d.

3.0 n.d. n.d.

a Cells were cultivated at 30 °C and 150 rpm in 50-mL Erlenmeyer flasks containing MM and levulinic acid as sole carbon source (1.2%, w/v in total, provided in proportions of 0.1 to 0.4%, w/v). PHA content and composition were determined after 72 h of cultivation. Abbreviations: 3HB, 3-hydroxybutyric acid; 3HV, 3-hydroxyvaleric acid; 4HV, 4-hydroxyvaleric acid; 3HHx, 3-hydroxyhexanoic acid; 3HO, 3-hydroxyoctanoic acid; 3HD, 3-hydroxydecanoic acid; 3HDD, 3-hydroxydodecanoic acid; CDM, cellular dry matter; n.d., not detectable.

exhibits a distinct substrate specificity, and the lack of its own PHA-synthase. Former studies on the PHA accumulation in P. putida GPp104 (pHP1014::E156) with octanoic acid as carbon source revealed a new type of PHA consisting of both short- and medium-chain-length 3-hydroxyalkanoic acids.30 This study revealed that this and other recombinant strains of P. putida GPp104 are suitable strains for the production of PHAs containing a high molar content of 4HV. Two-Stage Fed-Batch Cultivation with Octanoic Acid as Growth Substrate. To increase the cell density, a twostage fed-batch cultivation process with P. putida GPp104 (pHP1014::E156) was developed on the basis of an optimized 25 L scale two-stage batch process with octanoic acid as substrate for cell growth and levulinic acid as carbon source for the accumulation of 4HV-containing polyester. During the growth phase the pH was adjusted to 7.8 by adding 2 N HCl in order to minimize the accumulation of undesired constituents deriving from the catabolism of octanoic acid. Octanoate (300 g/L) was fed in small portions ensuring that the concentration within the culture did not exceed 0.1% (w/ v) in order to prevent excess synthesis of PHAs from octanoic acid. When the concentration of ammonia in the supernatant became less than 0.2% (w/v), ammonium chloride (21.3 g/L) was added. Accumulation of PHAs was induced after 46 h of cultivation by nitrogen starvation and by continuously supplying an excess of levulinic acid. Nitrogen starvation was revealed as the best strategy to enhance PHA accumulation in P. putida.32 At the beginning of the accumulation phase the pH was shifted to 7.1 and kept at this value by the addition of levulinic acid; the temperature was kept at 35 °C. At the end of the cultivation process, after 128 h, the cell density was about 20 g/L, and the cells revealed a PHA content of 52% (w/w). During growth on octanoic acid (up to 40 h) a copolyester consisting of 3HB, 3HHx, and 3HO was accumulated by the cells, contributing up to 30% (w/ w) to the cellular dry matter, whereas from the beginning of the accumulation phase onward, coinciding with the addition of levulinic acid, 3HV and 4HV were also incorporated into the polyester, with 4HV finally amounting to 15.4 mol %. Despite the choice of favorable cultivation conditions concerning pH, temperature, and a low proportion of carbon to nitrogen source by means of an automated feeding regime, it was not possible to prevent the incorporation of high amounts of octanoic acid-derived constituents such as 3HB,

Table 2. PHA Accumulation of P. Putida GPp104 (pHP1014::B28RV) with Gluconic Acid as Growth Substrate and Levulinic Acid as Accumulation Substratea

carbon source levulinic acid levulinic acid + glucose

PHA content levulinic acid (% of CDM) yield (%) 31.6 50.8

16.9 27.3

composition of PHA (mol %) 3HB

3HV

4HV

3.3 1.6

64.7 70.6

32.0 27.8

a Cells were cultivated at 30 °C and 150 rpm in 50-mL Erlenmeyer flasks containing MM and gluconic acid (0.5%, w/v) as growth substrate. Tetracycline (50 µg/mL) was used for plasmid maintenance. After 24 h of incubation, cells were collected, washed with saline, and resuspended in nitrogen-free MM. Levulinic acid (3 × 0.3%, w/v), or levulinic acid (3 × 0.3%, w/v) plus glucose (0.3 + 0.3 + 1.0%, w/v), was added as carbon source. PHA content and composition were determined after 72 h of cultivation. Abbreviations: See legend to Table 1.

3HHx, and traces of 3HO into PHA during the growth phase. Former investigations of PHA accumulation by P. putida GPp104 (pHP1014::E156) on octanoic acid as carbon source led also to the accumulation of a polyester consisting of 48.5 mol % 3HB, 47.3 mol % 3HHx, and 4.2 mol % 3HO.30 Two-Stage Fed-Batch Cultivation with Gluconic Acid or Glucose as Growth Substrate. Accumulation experiments of P. putida GPp104 harboring pHP1014::E156 or pHP1014::B28RV with gluconic acid or glucose in Erlenmeyer flasks revealed very low contents of poly(3HB) contributing to a maximum of 2% (w/w) or less of the cellular dry matter (data not shown). Since glucose and gluconic acid are less expensive substrates than octanoic acid and can be produced from renewable resources, and since glucose offers the advantage of an independently automated feeding regime of the carbon source and of ammonia, they might be more suitable carbon sources for the growth phase. Cells of P. putida GPp104 (pHP1014::B28RV) grown in MM containing gluconic acid in the first and levulinic acid in the second cultivation period accumulated a polyester consisting mainly of 3HV and 4HV with a minor fraction of 3HB (Table 2). In addition, the content of 4HV was higher than that in cells cultivated on octanoic acid. Since the wild type PHA-synthase, which preferentially uses 3-hydroxyacylCoA thioesters of C6 or higher carbon atoms, is not active in the mutant GPp104, and since the T. pfennigii PHAsynthase preferentially uses CoA thioesters of C4 to C6 hydroxy fatty acids, the recombinant strains of P. putida

50


employed in this study produced only minor amounts of PHAs from gluconic acid. When glucose was fed during the accumulation phase in addition to levulinic acid, the polyester content of the cellular dry matter increased from 31.6 to 50.8% (w/w), while the proportion of 4HV and 3HB decreased only slightly in favor of 3HV (Table 2). Simultaneously, the yield of PHA from levulinic acid increased significantly from 16.9 to 27.3%. This effect could be due to the observation that the catabolism of long-chain intermediates via the β-oxidation pathway is diminished by catabolic repression of enzymes involved in the β-oxidation by glucose as was shown for Escherichia coli39 and Caulobacter crescentus.40 An inhibitory effect of glucose on the β-oxidation pathway could also lead to the accumulation of intermediates of levulinic acid catabolism in the cells thereby providing substrates for PHAsynthase in excess and explaining the observed positive effect of the cosubstrate glucose during PHA accumulation from levulinic acid. This effect would also explain the enhanced yield for the transformation of levulinic acid into PHA because degradation of levulinic acid via the β-oxidation pathway was partially inhibited. The positive effect of an inhibition of β-oxidation by acrylic acid on the accumulation of PHA from fatty acids was shown recently for cells of recombinant E. coli.41 Another explanation for the increased accumulation and yield of PHA from levulinic acid in the presence of glucose or gluconic acid could be that more ATP is available for the activation of levulinic acid by a thiokinase. This strategy was applied to the cultivation of P. putida GPp104 (pHP1014::B28RV) at the 25-L scale using gluconic acid as the carbon source for cell growth. The use of gluconic acid as carbon source for cell growth in all precultures reduced the lag phase to less than 10 h. During the first 20 h the cells showed exponential growth behavior. Despite intensive feeding of gluconic acid, ammonia, and supplements when these nutrients were consumed, the increase of the cellular dry matter declined after 35 h of cultivation and stopped at a concentration of 9.4 g/L after 65 h. During the cultivation, 2-ketogluconic acid was formed up to a concentration of about 50 g/L, meaning that about 50% of the carbon source was converted to 2-ketogluconic acid and only 6.6% to PHA-free cellular dry matter. Presumably, the high concentration of 2-ketogluconic acid inhibited further cell growth. The formation of 2-ketogluconic acid during the cultivation of P. aeruginosa on gluconic acid, as it is known for most pseudomonads,42 and also for Klebsiella pneumoniae strain NCTC418,43 has been observed.44 In a fully automated two-stage fed-batch process, glucose instead of gluconic acid was fed in small proportions (below 9 g/L) in order to prevent excess formation of 2-ketogluconic acid. By this measure the formation of 2-ketogluconic acid during the exponential growth phase could be limited to 3-5 g/L (Figure 1C) and the cell density increased to 16 g/L during the growth phase and further to 25 g/L at the end of the accumulation phase (Figure 1A). To adapt the cells for the utilization of levulinic acid, small amounts of this carbon source (0.05-0.1%, w/v) were already provided during the growth phase from 23 h onward, immediately resulting in the accumulation of PHA. Further accumulation of PHA was

Gorenflo et al.

Figure 1. Time course of an automated two-stage fed-batch cultivation of P. putida GPp104 (pHP1014::B28RV) with glucose as carbon source for cell growth and levulinic acid as carbon source for the accumulation of 4HV-containing PHA and gluconic acid as cosubstrate. The fermentor was filled with 14 L of MM, containing glucose (1.2%, w/v), ammonium chloride (0.1%, w/v), and tetracycline (50 mg/ L), and was inoculated with 1 L of a preculture grown on gluconic acid. Growth conditions: temperature, 30 °C; pH, 7.0; stirrer speed, 500-700 rpm; aeration, 1-1.5 vvm. After 55 h of cultivation (see dotted line) the temperature was adjusted to 35 °C, the pH to 7.0, the stirrer speed to 500 rpm, and the aeration to 0.75 vvm. Abbreviations: MM, mineral salts medium; rpm, rounds per minute; 3HB, 3-hydroxybutyric acid; 3HV, 3-hydroxyvaleric acid; 4HV 4-hydroxyvaleric acid. (A) Cellular dry matter, polyester content, and concentration of levulinic acid. (B) Molar fraction of PHA constituents. (C) Concentration of ammonia, glucose/gluconic acid, and 2-ketogluconic acid within the supernatant.

stimulated in the accumulation phase by nitrogen starvation (Figure 1C) and by continuously supplying an excess of levulinic acid by means of the pH-regulation unit after a cultivation period of 35 h (Figure 1A). Furthermore, the temperature was shifted to 35 °C, and gluconic acid was used as cosubstrate in the accumulation phase in order to enhance PHA-accumulation from levulinic acid. This resulted in a sharp increase of the PHA content to about 50% (w/w) during the accumulation phase. During the first 17 h from the addition of levulinic acid onward, only 3HV and 4HV were incorporated into PHA (Figure 1B), whereas later 3HB also occurred; then the composition of the PHA remained constant until the end of the accumulation phase (15.1 mol % 4HV, 82.3 mol % 3HV, and 2.6 mol % 3HB).


These results show that the development of a fully automated cultivation with glucose as carbon source for cell growth and levulinic acid as carbon source for the accumulation of 4HV-containing PHA led to a reduction of the number of constituents of the polyester from five to two major hydroxy fatty acids, 3HV and 4HV, and to a significant increase of the molar 4HV content of the accumulated PHA. Scale-up of the Cultivation Process to a 500-L Scale. The above developed fermentation process, employing P. putida GPp104 (pHP1014::B28RV) for the production of 4HV-containing PHAs, was scaled to a 650-L fermentor. Gluconic acid and ammonia as carbon and nitrogen sources, respectively, were used during the growth phase, with levulinic acid, gluconic acid, and NaOH during the accumulation phase for the synthesis of PHA and the regulation of the pH to 7.0, respectively. During the growth phase, the cell density increased from 0.04 to 13.6 g/L with a PHA content of less than 0.5% (w/w) (Figure 2A). The concentration of 2-ketogluconic acid was low, and only for a short period of time a concentration of 2 g/L was detected. After a cultivation period of 40.5 h, temperature was maintained at 35 °C, levulinic acid was supplied at 3.0 g/L (Figure 2C), and accumulation conditions were induced by nitrogen starvation. During the following accumulation phase, the cell density reached 19.7 g/L, and the PHA content of the cells increased up to 38% (w/w) within the first 20 h and finally reached 50% (w/w). After 134 h of cultivation, 6.0 kg of cellular dry matter was collected which contained 3.0 kg of PHA composed of 34.6 mol % 4HV, 63.7 mol % 3HV, and 1.7 mol % 3HB (Figure 2B). In total, 9.6 kg of levulinic acid was consumed by the bacteria during the process, yielding 31%. In addition, 4HV could be detected in the medium after 63 h of cultivation as revealed by HPLC and GC analysis of cell free supernatants, amounting to 3.5 g/L at the end of the accumulation phase (Figure 2C). In contrast, 3HV and 3HB were detected up to concentrations of only 0.18 and 0.02 g/L, respectively. The total amount of these three fatty acids in the supernatant was 1.3 kg, meaning that 13.5% of the levulinic acid used as carbon source was converted to these free fatty acids. Therefore, 44.5% of the metabolized levulinic acid was converted by the cells to monomeric or polymeric 3HB, 3HV, and 4HV, whereas 55.5% was presumably metabolized to CO2, cell mass, and other products. The scale-up of the cultivation process, developed in the 25-L scale to a 500-L scale, was successfully achieved. During a 5-L fed-batch cultivation of the wild-type strain P. putida BM01 on glucose, a maximum cellular dry matter of 100 g/L was obtained, using pure oxygen for aeration,45 which is the highest value reported in the literature for a P. putida strain.17 Considering that the above presented cultivation process, which reached a maximum cellular dry matter of 25 g/L, was performed by using air instead of oxygen, it can be presumed that a significant increase of the cell density can hardly be expected, especially when the data revealed that the final attainable cell density was oxygen-limited (data not shown).


Figure 2. Time course of a 500-L fed-batch cultivation of P. putida GPp104 (pHP1014::B28RV) with gluconic acid as carbon source for cell growth and levulinic acid as carbon source for the accumulation of 4HV-containing PHA and gluconic acid as cosubstrate. The fermentor was filled with 350 L of MM containing gluconic acid (0.3%, w/v), ammonium chloride (0.12%, w/v), and tetracycline (50 mg/L) and was inoculated with 30 L of a preculture grown on gluconic acid. Growth conditions: temperature, 30 °C; pH, 7.0; stirrer speed, 100300 rpm; aeration, 0.25-0.75 vvm. After 40.5 h of cultivation (see dotted line) the temperature was adjusted to 35 °C, the pH to 7.0, the stirrer speed to 180-250 rpm, and the aeration to 0.25-0.75 vvm. Abbreviations: MM, mineral salts medium; rpm, rounds per minute; 3HB, 3-hydroxybutyric acid; 3HV, 3-hydroxyvaleric acid; 4HV 4-hydroxyvaleric acid. (A) Cellular dry matter and polyester content. (B) Molar fraction of PHA constituents. (C) Concentration of 4HV, gluconic acid, and levulinic acid within the supernatant. The arrows indicate a manual addition of levulinic acid.

The formation of comparatively high amounts of extracellular 4HV during the accumulation phase may be due to a membrane-bound reductase; 4HV is then taken up by the cells and incorporated into the polyester or catabolized to 3HV and other metabolites. Assuming that the uptake of 4HV into the cells is the limiting step, the accumulation of high amounts of 4HV in the medium can be explained. Alternatively, it is possible that levulinic acid is first transported into the cells and then reduced to 4HV. Assuming that the PHA-synthase exhibits a lower specificity for the thioester of 4HV than for those of 3HV and 3HB, 4HV may be produced in excess and excreted into the medium, while 3HV and 3HB are incorporated into the PHA.

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Figure 3. Extraction of 4HV-containing polyesters from cells of P. putida GPp104 (pHP1014::B28RV) with chloroform or acetone after different treatments of the cells. The cells of P. putida GPp104 (pHP1014::B28RV) were cultivated in a 500-L scale with gluconic acid during the growth phase and levulinic acid during the accumulation phase as described in the section above and treated as indicated. The PHAs were extracted in a Soxhlet apparatus, precipitated in ethanol, and analyzed by GC analysis as described in Materials and Methods.

Downstream Processing The above-described large-scale production of PHA by recombinant strains of P. putida led to cell suspensions exhibiting densities of 10-30 g/L usually containing 3050% (w/w) PHA. The next objective was to separate the intracellular PHA from all non-PHA material of the cells in order to obtain highly purified PHA for the investigation of its material properties. For all investigations, cells of P. putida GPp104 (pHP1014::B28RV) cultivated on gluconic acid and levulinic acid as carbon source for cell growth or accumulation of 4HV-containing PHAs were used. Optimization of the Solid-Liquid Separation by the Application of a Flocculation Process. The first step after the cultivation process is the separation of the PHAcontaining cells from the medium by continuous centrifugation. Despite a high residence time of the cells in the centrifuge, only 70% of the cell mass could be harvested. This significant loss resulted from poor sedimentation of the cells due to their low buoyant density.46 To increase the separation efficiency, a flocculation process was developed as described in Materials and Methods. Microscopic investigation of the flocculated cell suspension revealed cell agglomerates of about 50-100 µm in diameter which then sedimented much better. Applying a volumetric flow of 3.0 L/min, the residence time of the cells in the rotor was about 2.0 min. Analysis of the supernatant revealed that the loss of cell mass was less than 10%. Extraction of the Polyester by Solvents Applying a Soxhlet Apparatus. It was shown that the polyester derived from levulinic acid was insoluble in methanol, opening the possibility of using this solvent for lipid extraction without

Figure 4. Extraction of 4HV-containing polyester from cells of P. putida GPp104 (pHP1014::B28RV) with chloroform after flocculation and treatment with boiling methanol. The cells of P. putida GPp104 (pHP1014::B28RV) were cultivated with gluconic acid during the growth phase and levulinic acid during the accumulation phase as described in the section above. The PHAs were extracted in a Soxhlet apparatus, precipitated in ethanol, and analyzed by GC analysis as described in Materials and Methods. (A) Time course of the extraction efficiency. (B) Time course of the polyester extraction adopted to a model for mass transport.

dissolving the polyester. Therefore, aliquots of 250 g of lyophilized cells treated with either cold or boiling methanol were extracted in a 1.5-L Soxhlet apparatus under reflux with chloroform or acetone for 9, 33, 81, and 129 h. The extracted polyesters were then precipitated in cold ethanol, dried, and subjected to GC and GPC analysis in order to determine the composition and molar mass of the extracted PHA, respectively. As shown in Figure 3, pretreatment of the cells either with cold or, in particular, with boiling methanol increased the efficiency of extraction with chloroform, drastically reducing the time required for extraction. In addition, pretreatment with hot methanol yielded PHAs with a much higher purity; therefore, the precipitation of the polyester must not be repeated. Extraction of the cells with acetone was, however, not superior compared to chloroform. The extraction with chloroform of flocculated and freeze-dried cells was studied in detail. After 33 h, about 98% of PHA was extracted from the cells with the extraction rate decreasing continuously during the extraction process (Figure 4A). The time course of the extraction of the polyester should obey the general equation of convective mass transfer.47 In combination with a mass balance for the polyester, the time



course of the extraction of the polyester can be described by the following exponential equation (for the derivation of this expression see Appendix): mPol,ext(t) ) 1 - e(-k*t) mPol,tot The resolution of this equation to k*t, and the plotting of the expression -ln(1 - mPol,ext(t)/mPol,tot versus the extraction time, should give a straight line with the slope as k*. The application of this model shows a good correlation to the experimental data (Figure 4B), demonstrating the reliability of eq 9 to predict the time course of the Soxhlet extraction process when the coefficient is k*, which is 0.12 h-1 for this example and was experimentally determined before. Influence of the Extraction Process on the Molecular Weight and the Composition of the Polyester. The molecular weight and the composition of the polyester accumulated by P. putida GPp104 (pHP1014::B28RV) grown on gluconic acid and levulinic acid was investigated by GC and GPC during the extraction with acetone in a Soxhlet apparatus. During the time course of the extraction process over 129 h, no significant changes of the molar composition of the polyester were detected (3HB, 2.9 ( 0.8 mol %; 3HV, 72.0 ( 0.9 mol %; 4HV, 25.1 ( 0.4 mol %). The molecular weight (Mw, (528 ( 26) × 103) and the polydispersity index (Mw/Mn, 3.1 ( 0.2) varied up to 10%, which was within the range of measuring accuracy. Cells, which were not flocculated before treatment with cold methanol and extraction with cold chloroform, exhibited the same molecular weight and molar composition of the polyester. This implied that the polyester is a copolyester, presumably with random distribution of the constituents within the polyester backbone. If the polyester was not a copolyester, different fractions with variable compositions or molecular weights would presumably be extracted during the time course of the experiment according to their solubility in the solvent. The data also indicated that the polyester did not degrade during this process. Solubilization of the non-PHA Biomass by Treatment with NaOH. The solubilization of non-PHA material was investigated by treatment with NaOH (0.5 N) as described above. Freeze-dried cells were subjected to methanolysis and GC analysis. The untreated cells contained 50% (w/w) polyester consisting of 1.7 mol % 3HB, 63.7 mol % 3HV, and 34.6 mol % 4HV. After treatment of the cells with 0.5 N NaOH, the polyester content increased to 75.3% (w/w), while the molar composition of the polyester remained constant. This means that 67.2% of the non-PHA cellmaterial was solubilized during the treatment with NaOH. In comparison with this, poly(3HB) was isolated from recombinant cells of E. coli, originally containing 77% (w/ w) of polyester by treatment with only 0.2 N NaOH under the same conditions resulting in a 98.5% pure polyester.23 Extraction by Boiling in Chloroform. An aliquot of 360 g of freeze-dried cells of P. putida GPp104 (pHP1014:: B28RV) containing 44% (w/w) of polyester (molar composition: 1.7 mol % 3HB, 63.7 mol % 3HV, and 34.6 mol % 4HV) was resuspended in 2 L of chloroform and boiled under

Table 3. Composition of 4HV-Containing Polyesters Obtained from Levulinic Acid from Cells of the Recombinant Strain of P. putidaa

sample F-11b F-13 F-14 F-15 F-16

C source composition of PHA processed (growth) (mol %) mass polyester type (g) (g) 3HB 3HV 4HV 3HHx 3HO octanoate gluconate glucose glucose glucose

555 2000 1080 1055 2000

101 71 50 100 58

20.0 2.4 7.0 1.5 3.7

47.0 50.3 58.4 84.0 71.3

16.0 15.0 47.3 n.d. 34.6 n.d. 14.5 n.d. 25.0 n.d.

2.0 n.d. n.d. n.d. n.d.

a Strain: F-11 to F-14, P. putida GPp104 (pHP1014::E156); F-15 and F-16, P. putida GPp104 (pHP1014::B28RV). Experimental conditions: cultivation scale, 25 L; growth phase, T 30 °C; aeration 1 vvm; tetracyclin 50 µg/mL; F-11, pH 8.0; F-13 to F-16, pH 7.0; accumulation phase, T 35 °C; pH 7.0; aeration, 1 vvm; except F-11, pH 6.5; aeration, 0.75 vvm. Abbreviations: see legend to Table 1. bProduced as a reference according to Schmack et al., 1998.8

reflux conditions. After 0.5 and 6.5 h, the extracted and precipitated polyester as well as the cells were analyzed by GC: 30 min of boiling led to the extraction of 91.2 g of polyester, corresponding to 57.6% of the total polyester accumulated by the cells. Further 6 h extraction yielded 61.0 g of polyester, leaving 3.0% (w/w) of PHA in the cells. The Soxhlet extraction of quantities of cells exceeding 300 g and exhibiting a relatively high polyester content (ca. 50%, w/w) was less efficient and required an extended extraction period. This effect is presumably due to a partial clogging of the pores of the extraction thimbles by the highly viscous polyester solution, leading to a drastic decrease in the replacement of the polyester containing solvent by fresh solvent. The extraction of freeze-dried cells with boiling chloroform in a round-bottom flask, instead of a Soxhlet apparatus, decreased the time required for the extraction of 95% of the polyester to less than 30%. The main reason for the significantly decreased extraction time may be explained by the direct contact of the cells with the solvent and the improved mixing during the extraction process. Characterization of the Physical and Mechanical Properties of the Polyester Four different polyester samples were obtained from four independent cultivations of P. putida GPp104 (pHP1014:: B28RV) or P. putida GPp104 (pHP1014::E156) on gluconic acid or on glucose as carbon source, during the growth phase and on levulinic acid as carbon source for the accumulation of 4HV-containing polyesters during the accumulation phase as described above. The accumulated polyesters were isolated by solvent extraction. All these polyesters consisted of 3HV as the main constituent and revealed a 4HV-content of between 14.5 and 47.3 mol %; 3HB occurred only as a minor constituent (Table 3). A copolyester with 3-HHx and traces of 3-HO8 and poly(3HV)13 was produced as a reference to evaluate the properties of the new polyesters. Processing of the Polyester. Injection Molding. To determine the mechanical properties of the polyesters and to test the processing of the polyester by a piston extrusion device, the samples were processed to test bars. To demold the samples, the tool had to be cooled in liquid nitrogen for

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Table 4. Differential Scanning Calorimetry Analysis of Unprocessed 4HV-Containing Polyesters and Poly(3HV)a

sample

Tg (°C)

melting range (°C)

F-11 F-13 F-14 F-15 F-16 poly(3HV)

-15.0 -11.9 -11.5 -11.2 -12.9 -15.4

30-150 41-75 44-100 42-90 41-95 50-135

Table 5. Molecular Weights of the Unprocessed and of the Processed Polyestersa

Tm (°C)

Tm (°C)

Tm (°C)

Tm (°C)

∆Hm (J/g)

48.0 49.2 48.7 49.1* 53.2*

70.0 60.0 75.2 71.9 79.2

84.0

110.0

25.2 2.0 6.1 13.2 41.9 107.7

118.5

a Abbreviations and symbols: T , glass transition temperature; T , g m melting temperature; ∆Hm, melting enthalpy; *, Shoulder; poly(3HV), poly(3-hydroxyvaleric acid).

30 s. A clear shrinking process of the samples, dependent on the residence time of the melt within the cylinder, was revealed. Even after the test-bars were tempered at 10 °C for 6 days, no dimension stability could be reached. The bad demolding properties of the specimens and their relaxation could again be explained by the low crystallization rate (see section below, Table 4). Melt Spinning Process. The dried polyester samples were processed by melt spinning. The polyester melt leaving the spinning die exhibited periodically fluctuating diameters and a helical structure. This effect can even be observed without drawing the extrudates. These flow irregularities are known as “melt-rupture” or “elastic turbulence” and are generated while the melt is entering or passing the manifold. The reason for such flow irregularities is very high viscosity of the viscoelastic fluid and a very high elastic material function at the existing flow conditions within the die. The low glass transition temperature of about -12 °C, as revealed by DMA (see section below, Table 4), and the low tendency to crystallize explain the stickiness of the fibers. In addition to that, the orientations, which were generated in the melt spinning process, relaxed after a few minutes so that the fibers offered a low tenacity of beneath 10 MPa. Thermal Analysis by Differential Scanning Calorimetry (DSC). The thermal properties of the virgin samples of the new copolyesters were determined by DSC. The samples exhibited a broad melting range between 30 and 150 °C with several peaks occurring. Therefore, the melting enthalpy (∆Hm), which is a measure for the crystalline proportion, could only be calculated in total (Table 4). Their low values indicate a very small crystalline proportion. In addition, the differences between the samples were substantial, which can be attributed to their different molecular composition. The rate of crystallization of the copolyester was in the range of the cooling velocity of 20 K/min, already so low that the samples were quenched to a noncrystalline, amorphous state. To facilitate postcrystallization, the test bars were conditioned for 6 days at 10 °C and 60% humidity before being investigated by DSC. Only test bars of the higher crystalline sample F-16 postcrystallized; the test bars of the sample F-15 showed no crystallization at all. The heat capacity (cp) of the copolyesters showed values of about 0.3 J/K‚g, which were significantly reduced in comparison to poly(3HV) with 0.57 J/K‚g. Molecular Weight of Unprocessed and Processed Polyesters. The average values of the molecular parameters,

sample F-11 F-13 F-14 F-15 F-16 poly(3HV)

Mn Mw MZ (103 g/ (103 g/ (103 g/ Mw/ ηint η0 mol) mol) mol) Mn (dL/g) (103 Pa s) 165 154 98 149 144 60

unprocessed samples 410 870 2.5 2.7 278 446 1.8 2.0 198 321 2.0 1.4 275 447 1.8 2.0 325 630 2.3 2.2 155 280 2.6 1.0

80.5 19.2 3.5 28.0 39.4 1.0

DI -

fibers (F-11, poly(3HV)), or test bars (F-14 to F-16), respectively F-11 120 235 400 2.0 1.1 0.38 F-13 - F-14 91 193 317 2.1 0.08 F-15 134 240 369 1.8 0.11 F-16 139 338 672 2.4 0.04 poly(3HV) 70 160 285 2.3 1.0 -0.14 a The testbars (F-14 to F-16) remained in the cylinder (thermal strain) for 30 min. Abbreviations: Mn, number-average molar mass; Mw, weightaverage molar mass, MZ, Z-average of the molar mass; ηint, intrinsic viscosity; η0, zero viscosity; DI, dispersity index; -, no data available.

the number-average molar mass (Mn), the weight-average molar mass (Mw), the Z-average molar mass (MZ), and the molar mass distribution (Mw/Mn) of the virgin samples and of the test bars were determined by means of SEC using calibrations with polystyrene standards (Table 5). The values illustrate the influence of the thermal processing, especially the process of injection molding, on the change of the molar mass, of the molar mass distribution, and the extent of degradation. The degradation index DI ) (Mn0/Mn) - 1 is equal to the average number of chain scissions per number average of the initial macromolecules during the injection molding process.48 Despite the above-mentioned limitations concerning the relative character of the values, it can be concluded that the unprocessed samples of the new copolyesters (F-13 to F-16) exhibit relatively narrow polydispersity indexes (Mw/Mn) between 1.8 and 2.5. A comparison of the molecular weights of the unprocessed samples with the test bars revealed no significant changes during the processing, except for sample F-11 which showed a drastic decrease of the molecular weight to about one-half. Consequently, sample F-11, with low proportions of the monomers 3HHx and 3HO, reveals a significantly higher degradation index (DI) than the samples F-13 to F-16 or poly(3HV), which did not contain 3HHx or 3HO, respectively. These data confirm an improved thermal stability of the new copolyesters (F-13 to F-16) in comparison to the polyesters which contain 3HHx or 3HO. The intrinsic viscosities of the unprocessed polyesters F-13 to F-16 were significantly higher than that of the poly(3HV) sample but lower than that of F-11 (Table 5). Dynamic Mechanical Analysis (DMA). The melt viscosity function (η*) was calculated from the storage modulus (G′) and the loss modulus (G′′). These parameters provide a rheological characterization which is sensitive to differences of the structure, the monomer composition, and the molecular weight of the new copolyesters. Figure 5A shows the melt viscosity of the samples as a function of the shear frequency



Figure 6. Temperature dependence of the melt viscosity (η*) of the virgin samples.

Figure 5. Dynamic mechanical analysis (DMA) of 4HV-containing polyesters and poly(3HV): (A) melt viscosity (η*); (B) shear storage modulus (G’); (C) shear loss modulus (G’’) during the frequency (ω) shift.

(ω). The melt viscosities of the new copolyester are about 1 order in magnitude higher than the melt viscosity of poly(3HV) but in the same range as the copolyester F-11, except sample F-14 which showed relatively low values. The polyesters show a non-Newtonian, pseudoplastic behavior in the measured range. The distinct decrease of the melt viscosity with increasing frequency indicates that the relaxation time for the copolyesters is much longer than that for the homopolyester poly(3HV), most probably because of the stronger entanglement of the high molecular chains. The

viscosity function (η*) was fitted to the shear frequency (ω) with an expression by Ellis containing three parameters.8 One of these fit parameters corresponds to the zero shear viscosity (η0) (Table 5), which is independent of the molecular weight distribution, but depends on the weight average molecular weight and the molar composition of the polyester. It increases with increasing molecular mass to the power 3.4. Table 5 shows the values of the zero shear viscosity, the intrinsic viscosity, and the molecular weight estimated by means of SEC measurements of the virgin samples and the processed samples (test bars and the fibers), respectively. The values correlate in tendency; differences can be the result of the different molecular composition of the respective polyester. The shear storage modulus (G′) and the shear loss modulus (G′′) of the samples were approximately 1 order in magnitude higher than those of poly(3HV) (Figure 5B,C, at low frequencies) but lower than the values of the sample F-11. These results emphasize the completely different dynamicmechanical behavior of the new copolyesters in comparison to the brittle homopolyester poly(3HV), but they do not reach the high elastomeric character of the copolyester F-11 with low content of medium chain length hydroxyalkanoic acids.8 By means of a temperature sweep of the viscosity at an average shear frequency of 1 rad/s, tan δ ) G′′/G′ and the glass-transition temperature (Tg) of the polyesters were calculated revealing Tg values of about -12 °C (Figure 6, Table 4), being in the range of those for poly(3HV) and 4HVcontaining polyesters with a low content of medium-chainlength hydroxyalkanoic acids.8,13 Stress-Strain Measurements. Test samples were cut from the original film castings, and their mechanical properties were calculated from the stress-strain curves. A marked difference exists between the three types of the investigated polyesters: the storage modulus (G′) already indicated differences in the elastic behavior, the copolyester F-11 revealed an extremely high elongation at break of about 1000%, and the corresponding value for the brittle homopolyester poly(3HV) was only 1.4%.13 The values of the new copolyesters (F-15 and F-16) were in the middle range (F-16, 28%; F-15, 208%). The differences between these two curves

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Gorenflo et al.

can be explained by the different crystalline proportion of the samples F-15 and F-16. The level of the tensile strength was very low for all investigated polyesters and in the same order of magnitude as reported for poly(3HB) film castings.49 The experiments revealed a tensile strength at break for F-15 and F-16 of 4.1 and 7.0 MPa, respectively, in the same range as for poly(3HV).13 Conclusions The presented process for the biotechnological production of 4HV-containing PHAs by recombinant strains of P. putida and the combined use of glucose/gluconic acid and levulinic acid are shown to be suitable for the effective large-scale production of PHAs with high molar fraction of 4HV. The pretreatment of the cells prior to the extraction process revealed a high potential for reducing the costs of downstream processing. The presented investigation should facilitate the development of a high-performance strain for the industrial production of 4HV-containing polyesters. It was shown that poly(3HB-co-3HV-co-4HV) belongs to the class of thermoplastic elastomeres. The presented results revealed, when compared to the mechanical properties of poly(3HB) and poly(3HV), that the elastomeric behavior of the new polyester is due to the constituent 4HV. The low proportions of 3HHx and 3HO within the 4HV-containing polyesters, investigated in an earlier study, are not solely responsible for their elastomeric properties. A broad melting range in a low temperature field, low crystallization rate, and low crystallinity are characteristic for these new polyesters and are the reason for stickiness and the difficulty of the thermoplastic processing, although they exhibit a very high thermal stability in comparison to the polyesters containing 3HHx and 3HO. The viscosity functions of the new polyesters show a non-Newtonian, pseudoplastic behavior because of a stronger entanglement of the high molecular chains. The storage moduli of the new polyester confirm their elastomeric character. It may conveniently be handled as a latex like other mcl-PHAs.50 The high elasticity of the new polyesters makes them suitable candidates for coatings, which needs to be tested soon. Appendix

(2)

The mass balance for the polyester being extracted out of the cells mPol,ext is calculated according to eq 3, with mPol,tot as the total amount of the polyester. mPol,tot ) mPol,Cells + mPol,ext

(3)

Due to the construction of a Soxhlet apparatus, the polyester containing chloroform is continuously replaced by fresh chloroform with a high volumetric flow allowing the polyester concentration in the solvent to be neglected in comparison to the concentration in the filter cake, resulting in eq 4. cSolution(t) ≈ 0

(4)

The combination of eq 1 with (4) in (2) leads to eq 5. mPol,Cells(t) dmPol,Cells ) -βACell(cPol,Cells(t) - 0) ) -βACells dt VCells (5) Assuming that the total volume and the surface of the cells do not change significantly during the extraction process, eq 5 and the expression k* ) βACells/VCells lead to eqs 6 and 7.

∫mm

Pol,Cells(t)

Pol,tot

1

mPol,Cells(t)

dmPol,Cells ) -

∫0tk* dt

mPol,Cells(t) ) cPol,tot e(-k*t)

(6) (7)

Equations 7 and 3 can be combined to eq 8 or 9, respectively. mPol,ext(t) ) mPol,tot(1 - e(-k*t))

(8)

mPol,ext(t)/mPol,tot ) 1 - e(-k*t)

(9)

Acknowledgment. The authors thank D. Voigt and H. Kunath (IPF Dresden) for their support by characterizing and processing of the copolyesters. This study was partially supported by a grant provided by the European Commission (FAIR program, CT96-1780 “PHAstics”). References and Notes

The time course of the extraction of the polyester should obey eq 1 according to the general equation of convective mass transfer,47 with m˘ Pol,Cells(t) representing the mass change of the polyester within the cells, β the masstransfer coefficient, ACells the outer surface of the cells, cPol,Cells(t) the concentration of the polyester within the cells, and cPol,Solution(t) the concentration of the polyester within the solvent. m˘ Pol,Cells(t) ) βACells(cPol,Cells(t) - cPol,Solution(t))

dmPol,Cells/dt ) -m˘ Pol,Cells(t)

(1)

Assuming the limiting case of a polyester concentration within the solvent and the filter cake independent of place, the mass balance for the polyester within the cells m˘ Pol,Cells is calculated according to eq 2.

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BM0000992

Development of a Process for the Biotechnological Large-Scale

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