Biosynthesis and Properties of Medium-Chain-Length

Chapter 3

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Biosynthesis and Properties of Medium-Chain -Length Polyhydroxyalkanoates from Pseudomonas resinovorans Richard D . Ashby, Daniel Κ. Y . Solaiman, and Thomas A . Foglia Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, P A 19038

In an effort to create new polymers, and provide additional outlets for animal fat and vegetable oil commodities, several triacylglycerols (TAGs) were studied as substrates for the bacterial production of medium-chain-length poly(hydroxy -alkanoate) polymers (mcl-PHA) by Pseudomonas resinovorans. Polymer yields ranged from 40% to 60% of the cell dry weight depending on the substrate. Each of the PHA polymers was characterized with respect to molar mass (gel permeation chromatography), thermal properties (calorimetry), and repeat -unit composition (gas chromatography/mass spectrometry). Repeat-unit chain lengths ranged from C4 to C14 with varying amounts of side-chain unsaturation (double bonds). For example, PHA from coconut oil consisted almost entirely of saturated monomers, whereas PHA from soybean oil contained higher levels of unsaturated side-chains. Variation in repeat -unit composition resulted in polymers with properties ranging from elastomeric to adhesive-like. The presence of olefinic groups in the polymer side-chains allowed for radiation crosslinking which enhanced the tensile strength, flexibility, and Young's modulus of the polymer films.

Poly(hydroxyalkanoates) (PHA) are a class of naturally occurring, optically active polyesters that accumulate in numerous bacteria as carbon and energy storage U.S. government work. Published 2000 American Chemical Society Scholz and Gross; Polymers from Renewable Resources ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

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26 materials (1-3). In most cases the polymers contain β-linked repeat units and are compositionally distinct based on side-chain structure (4). However, some exceptions exist where the linkages are through the δ position of the monomers (e.g., 4-hydroxybutyrate, 4HB, 4-hydroxyvalerate, 4HV, 4-hydroxyhexanoate, 4HH) (5-7), or through the γ position (5-hydroxyvalerate, 5HV) (8). To date, approximately 100 different PHA structural analogs exist based on the large variety of PHA producers and the broad substrate specificity of the polymerization systems. Because they are viewed as "environmentally friendly," many PHAs are being studied as potential replacements for synthetic plastics in several applications. Triacylglycerols (TAGs) are non-traditional feedstocks for md-PHA production. By using agricultural products like fats and oils, new polymers may result with unusual repeat unit compositions and properties. It is known that several bacterial species produce medium-chain-length poly(hydroxyalkanoate) (md-PHA) polymers from fatty acids (9-11). Recently intact TAGs have also been considered as substrates for PHA production (10,1214). Organisms that metabolize TAGs to PHA polymer presumably have to meet two requirements: (i) production of a lipase enzyme that hydrolyzes the TAG to liberate the long-chain fatty acids and (ii) production of PHA from long-chain fatty acids. Lipase production is a common feature among Pseudomonas species (15,16), and likewise many pseudomonads produce md-PHA from a variety of carbon sources including long-chain fatty acids (11,17,18). Therefore, under certain growth conditions, selected bacterial strains have the ability to produce PHA polymers from TAGs. In addition, PHA biosynthesis from TAGs may provide new polymers with repeat-unit compositions containing saturated and unsaturated side-chains resulting in materials with elastomeric properties. One way to increase the strength of a polymer film is to crosslink the matrix. This can be accomplished by the use of chemical additives (peroxides, sulfur vulcanization) (19,20) or physical treatments (radiation) (21). Generally, the crosslink density is dependent upon the method used, and the number of functional groups present. Radiation provides a quick, and relatively clean method of crosslinking without the addition or formation of contaminants in the polymer matrix. The interaction of radiation with polymeric materials generally results in three-dimensional network structures with improved tensile properties (22). Because of this, three of the md-PHAs were selected (based on the number of olefinic groups present in their side-chains) for irradiation studies to determine the effects of olefinic content on radiation induced crosslink efficiency, and tensile properties. PHA films produced from coconut oil (PHA-C; low olefinic concentration), tallow (PHA-T; intermediate olefinic concentration), and soybean oil (PHA-So; high olefinic concentration) were solution cast and irradiated using a γ-emitter (cesium-137). The films were characterized by tensile testing before and after radiation treatment to compare the effect of γ-irradiation on the polymer tensile properties.

Scholz and Gross; Polymers from Renewable Resources ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

27 Experimental Materials All simple salts were obtained from Sigma Chemical Company (St. Louis, MO) and used as received. Each TAG was obtained as a commodity material as follows: coconut oil (C) and olive oil (O) (Sigma Chemical Company), tallow (T) (Miniat Inc., Chicago, IL), lard (L) (Holsum Foods, Waukesha, WI), butter oil (B) (ERRC dairy pilot processing plant), high oleic acid sunflower oil (Su) (SVO Enterprises, Eastlake, OH), and soybean oil (So) (purchased from the supermarket under the trade name Wesson oil). A l l organic solvents were HPLC grade (Burdick and Jackson, Muskegon, MI).

Strain Information and Polymer Synthesis Pseudomonas resinovorans NRRL B-2649 was obtained from the National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, Peoria, IL, and used as the producer strain in all fermentations. Stock cultures were prepared and stored as described previously (14). Shake flask experiments were conducted at 500 mL volumes (in 1 liter Erlenmeyer flasks) under batch culture conditions in Medium E* (for medium composition see reference 9). Each TAG substrate was heated at 60°C for 15 minutes and added to sterile Medium E* at a concentration of 1% (w/v). The flasks were inoculated with a 0.1% inoculum from a thawed cryovial. Bacterial growth and polymer production were carried out at 30°C with shaking at 250 rpm, for 72 hours in an orbital shaker-incubator. At 72 hours the cells were pelleted and washed twice in deionized water by centrifiigation (8000 χ g, 20 minutes, 4°C). Unused fats (solids) were removed from the cultures prior to centrifiigation by filtration through cheesecloth. Unused oils concentrated at the air/liquid interface upon centrifiigation and were removed either with the supernatant or by wiping the walls of the centrifuge bottles with paper towels. The cell pellets were then lyophilized (-24 hours) to a constant weight.

Electron Microscopy Scanning Electron Microscopy (SEM) A solution of 1% glutaraldehyde-0.1 M imidazole-HCl buffer (pH 6.8) was flooded onto the surface of a 48 hour medium E* agar plate inoculated with P. resinovorans and containing emulsified olive oil as the carbon substrate (the medium was modified from that described in reference 23). The cultures were incubated at room temperature for 2 hours and stored in sealed containers at 4°C.


28 Selected areas of the cultures were excised, washed by immersion in imidazole buffer for 30 minutes, and immersed in 2% osmium tetroxide-0.1 M imidazole buffer for two hours. Samples were then washed in distilled water, dehydrated in a graded series of ethanol solutions and critical point dried from liquid carbon dioxide. Finally, the dried samples were mounted on aluminum specimen stubs with colloidal silver adhesive and coated with a thin layer of gold by DC sputtering at low voltage (Model LVC-76, Plasms Sciences, Lorton, VA). Photographic images of the surfaces of sample cultures were made using a JSM840A scanning electron microscope (JEOL USA, Peabody, MA) operated in the secondary electron imaging mode. Transmission Electron Microscopy (TEM) One hundred microliters of 10% glutaraldehyde was added directly to 900 uL of a 72 hour broth culture of P. resinovorans and rapidly mixed in a 1.5 mL tube. The mixture was then spun in a Model 5413 Eppendorf centrifuge for 5 minutes. After standing at room temperature for 2 hours, the cell pellets were washed with 0.1 M imidazole buffer (pH 6.8) for 30 minutes and immersed in 2% osmium tetroxide-0.1 M imidazole buffer for 2 hours. Pellets were washed in distilled water, dehydrated in a series of ethanol solutions, soaked in propylene oxide and infiltrated with a 1:1 (v/v) mixture of propylene oxide and an epoxy resin mixture overnight. Pellets were next embedded infreshepoxy resin mixture and cured for 48 hours at 55°C. Thin sections of the embedded samples were cut with diamond knives and stained with solutions of 2% uranyl acetate and lead citrate. Photographic images were made using a Model C M 12 scanning-transmission electron microscope (Philips Electronics, Mahwah, NJ) operated in the bright field imaging mode.

PHA Purification and Isolation Supercritical fluid extraction (SFE) was used to remove residual TAG material prior to PHA isolation. The dried bacterial cells (5 g per extraction) were packed tightly into a 24 mL stainless steel extraction vessel (rated at 10,000 psi). Polypropylene wool was packed in front and behind the bacterial cells to protect the vessel frits (2 μιη). The extraction vessel was placed in the SFE apparatus oven (Applied Separations, Allentown, PA). The oven was adjusted to 60°C and the restrictor valve temperature was set at 100°C. When the vessel temperature reached 60°C carbon dioxide at 5000 psi was allowed to flow through the extraction vessel at 1.5 L/minute for 1 hour. The pressure was then raised to 7000 psi and carbon dioxide flow continued for 1 hour at 1.5 L/minute In the final hour, carbon dioxide flowed at 1.5 L/minute at a pressure of 9000 psi. At the end of 3 hours, the vessel temperature and pressure were reduced to room temperature and atmospheric pressure and the bacterial cells removed from the vessel for polymer isolation.


29 PHA isolation was accomplished by stirring SFE extracted cells (approximately 1 g) for 24 hours in chloroform (100 mL) at 30°C. The insoluble cellular material was removed by vacuum filtration through Whatman #1 filter paper (Whatman International Ltd., Maidstone, England) and the solvent was concentrated (to approximately 5 mL) in a rotary evaporator. Each md-PHA was precipitated by dropwise addition of the polymer concentrate into excess cold methanol and the precipitate dried in vacuo for 24 hours at 25°C and 1.0 mm Hg.

Instrumental Procedures PHA repeat unit compositions were determined by gas chromatography (GC) and GC/mass spectrometry (GC/MS) of the β-hydroxymethyl esters (β-HMEs). Samples were prepared according to the procedures of Brandi et al. (9) and analyzed by GC and GC/MS using conditions reported previously (10). Molar mass averages were determined by gel permeation chromatography (GPC) after the procedure of Cromwick et al. (10). Polystyrene standards (Polysciences Corp., Warrington, PA) were used to generate a calibration curve from which PHA molar masses were determined without further corrections. Chloroform was used as the eluent at a flow rate of 1 mL/min. The sample concentration and injection volume were 0.5% (w/v) and 200 uL, respectively. The thermal properties and the carbon ( C) nuclear magnetic resonance (NMR) spectra of each md-PHA were obtained as described elsewhere (14). 13

Film Preparation and Irradiation Films were cast from solutions of PHA-T, PHA-C, or PHA-So. Solutions were prepared by dissolving 1.5 g of purified PHA in 15 mL of chloroform. Films were cast in glass petri dishes (100 mm χ 15 mm), and the solvent evaporated under a nitrogen atmosphere. The resulting films were approximately 0.1 mm thick, and were stored desiccated in the dark under a nitrogen purge prior to irradiation. The films in the glass petri dishes were irradiated with 50 kGy of radiation (energy was based on an 8.5 h exposure) using a cesium-137 source at 20°C in a nitrogen atmosphere. The irradiator and dosimetry were described previously (24).

Sol/Gel Analysis Sol/gel tests were performed on both non-irradiated and irradiated film samples according to the method described previously (25).


30 Tensile Property Measurement Tensile properties of the PHA films were measured at 23°C and 50% relative humidity with a gauge length of 25 mm. An Instron tensile testing machine, model 1122 (Canton, MA), was used throughout this work. The cross-head was maintained at a constant speed of 50 mm/minute Measurements included tensile strength, elongation to break and Young's modulus. Tensile strength and elongation to break were defined as the ultimate stress and strain, respectively. Young's modulus is a physical quantity representing the stiffness of a material. It was determined by measuring the slope of a line tangent to the initial stress-strain curve. All the data were calculated and collected through the use of Instron series IX automated materials testing system version V.

Results and Discussion md-PHA Production and Characterization While many species of Pseudomonas have the ability to produce md-PHA from fatty acids, only two (P. resinovorans and P. aeruginosa) have been documented to produce md-PHA from TAGs (10, 13). Cromwick et al. (10) explored the reason why P. resinovorans grew and produced md-PHA from TAGs while the other known PHA producers tested did not. By using a fluorescent assay developed by Moreau (26), it was determined that P. resinovorans was the only organism tested that exhibited extracellular esterase (lipase) activity in the presence of TAG substrates. This enzyme activity liberates the fatty acids from the glycerol backbone of the TAG, thus providing a substrate that could be used in md-PHA synthesis. The inability of the other PHA-producing pseudomonads to grow and produce PHA from TAGs suggests that the esterase (lipase) activity is necessary for PHA biosynthesis by P. resinovorans. Each animal fat and vegetable oil is unique in its fatty acid composition. For this reason, seven different TAGs (tallow, lard, butter oil, olive oil, high oleic acid sunflower oil, coconut oil, and soybean oil) were screened as potential substrates for md-PHA production. Scanning electron microscopy (SEM) showed that P. resinovorans attaches itself to the fat or oil droplets, thus providing maximal access to the substrate (Fig. la). In each case, P. resinovorans produced an mdPHA polymer, which was evident by the presence of one or more PHA granules per bacterial cell (Fig. lb). The cells were harvested by centrifiigation and the cellular biomass and polymer yield determined gravimetrically. The PHA content was calculated as a percentage of the cell dry weight (CDW) (Table I).


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Figure L Electron micrographs showing growth and polymer production by P. resinovorans on TAG substrates: a) SEM of bacterial cells attached to a droplet of olive oil (the length of the average bacterial cell is approximately 1 μτη). b) TEM o/P. resinovorans grown on tallow showing granule production within the cells (scale: 22 mm = 1 μτη).


32 Table L Cell Dry Weights (CDW) and md-PHA Polymer Content of P. resinovorans Grown on TAG Substrates Substrate PHA yield PHA content Crude cell yield (% CDW) (R/L) (R/L) Animal fats Tallow 1.0 2.4 43 2.1 Lard 3.8 55 Butter oil 2.0 3.7 53 Vegetable oils Olive 2.3 4.0 58 Sunflower (HO)* 4.0 2.3 57 2.4 Soybean 4.1 59 Coconut 4.1 2.5 61 PHA yield is calculated by multiplying the cell yield (g/L) by the PHA content (% dry weight) of the cells. *HO = "high oleic." 3

a

The ability of P. resinovorans to grow and produce equal cell masses and polymer yields on coconut oil (highly saturated TAG) and soybean oil (highly unsaturated TAG) indicates that the concentration of cis double bonds has little effect on substrate metabolism. This suggests that the discrepancy between cell growth and PHA yields on animal fats vs. vegetable oils is primarily due to the physical state of the substrate at 30°C (incubation temperature) rather than fatty acid composition. The titer (melting range) of each vegetable oil tested is less than 25°C. Because of this, these oils are easily dispersed throughout the culture media by simple agitation at 30°C. In contrast, lard and butter oil have titers near 33°C, only slightly higher than the incubation temperature, while the titer of tallow is greater than 40°C. The result is that some of the animal fat TAGs remain solid throughout the fermentation, which retards both bacterial growth and polymer yield. In addition to TAGs, animal fats and vegetable oils contain small amounts of sterols (approx. 0.1%-0.5%) and tocopherols (primarily α and γ). Though the utilization of these compounds for cell growth and polymer production cannot be eliminated, high cell and polymer yields (along with polymer composition) suggest that TAGs were the primary carbon sources used by the bacterium. The composition of md-PHAs from long chain fatty acids is controlled by the specificity of the PHA-synthesizing system, the structures of the fatty acids, and the degradation pathway for long chain fatty acids (11). It has been reported that P. oleovorans grown on alkanoic acids produces md-PHAs that reflect the chain length of the substrate (27). For example, when grown on hexanoate or octanoate the md-PHA was composed predominantly of 3-hydroxyhexanoate and 3-hydroxyoctanoate monomers, respectively. When grown on alkanoates with chain lengths greater than Qo, it was observed that C and C i were the 8

0


33 Table Π. Repeat-Unit Composition of Poly(Hydroxyalkanoate) Polymers Isolated from P. resinovorans Cultures Grown on TAG Substrates Isolate β-hydroxymethyl ester (%) C4:0 Q:0 ^8:0 Cio.o Cl2:0 Ci2:l Cl4:0 Cl4:l Cl4:2 Cl4:3 n.d. n.d. PHA-T Tr* 4 6 9 3 15 17 46 n.d. 4 8 3 PHA-L Tr 7 4 26 34 14 4 5 n.d. n.d. PHA-B Tr Tr 9 31 15 35 n.d. 10 1 PHA-O 1 1 3 8 29 33 14 3 n.d. PHA-Su 2 3 13 5 22 14 3 35 Tr Tr 14 4 9 10 PHA-So 4 18 8 32 n.d. Tr Tr n.d. PHA-C 7 33 1 3 40 16 Average relative percent (n=5) as determined by GC of the β-HMEs obtained by acid hydrolysis and methylation of each md-PHA polymer. *Tr (trace) =

M - H 0 - C H O H = 152 2

3

Ν® s

0-

1 52 m/z Figwre 2. Electron-impact mass spectrum of β-hydroxydecanoate methyl ester. The presence of an ion fragment with m/z = 103 is indicative of a β-hydroxyalkanoate methyl ester. (SOURCE: Reproduced with permission from ref. 14.)


35 portion of the tallow rather than the more insoluble fraction. PHA-C contained very low levels of unsaturated monomers. This was undoubtedly due to the low levels of unsaturation in the substrate itself (oleic acid, 4.6%; linoleic acid, 0.9%). The PHA-So repeat-unit composition was more complex than the other PHAs. It contained a much larger fraction of di-unsaturated Q 4 monomers (and a small Ci4;3 fraction) and, with the exception of PHA-Su, had more than twice as many unsaturated side chains as the other md-PHAs. The PHAs also were studied by C-NMR (50 MHz). Figure 3 shows typical spectra of md-PHAs, in particular PHA-C, PHA-T, and PHA-So. The chemical shift assignments are based on those reported previously (18, 28, 29). The chemical shifts of the peaks in the 0 - 120 ppm region are identical apart from slight differences in their relative intensities. From the chemical shift intensities between 120 and 140 ppm (olefinic carbons) it was confirmed that PHA-C contained very few unsaturated side chains, whereas the side chains from PHA-So were highly unsaturated. As the concentration of olefinic carbons increased, the polymers became more amorphous. This was verified by measurement of the thermal properties of each polymer by calorimetry (Table III). With the exception of PHA-Su and PHA-So, each md-PHA was elastomeric at room temperature. PHA-C had the highest glass transition temperature (T ), melting temperature (T ), and enthalpy of fusion (AH ) of the polymers. While it seems likely that these increases were primarily due to a more ordered packing arrangement as the degree of side-chain unsaturation decreased, the increased molar masses of the md-PHAs containing more saturated monomers also may have helped to increase the T and T of the polymers. 13

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Table ΠΙ. Molar Masses and Thermal Properties of TAG-Derived md-PHA Polymers Produced by P. resinovorans Isolate T (°C) T rc) M AH (J/g) M» MJMn (xl(f) (xlO ) PHA-T 93 -46 42 11.7 269 2.89 PHA-L 84 -46 42 9.8 220 2.62 PHA-B 101 -43 43 12.0 298 2.95 PHA-0 82 214 42 10.1 -46 2.61 PHA-Su* 61 149 2.44 -47 PHA-So* 57 121 -47 2.13 PHA-C 15.4 133 -40 45 449 3.38 All molar masses were determined by gel-permeation chromatography. *The md-PHA polymers from high oleic sunflower oil and soybean oil were amorphous and showed no melting transition. w

g

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fl


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36

V

r

β

α

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[R]

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140

120

100

80

60

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ppm Figure 3. The C-NMR spectrum of mcl-PHA polymers isolated from P. resinovorans grown on a) coconut oil (PHA-C), b) tallow (PHA-T), and c) soybean oil (PHA-So). (SOURCE: Reproduced with permissionfromref. 14.) J3


37 At the other extreme, PHA-So and PHA-Su had T 's of -47°C and TVs and AH 's that were not measurable. This indicated that these polymers were completely amorphous and, in fact, they were almost liquid-like at room temperature. In contrast to PHA-C, for example, this was probably due to the smaller molar masses and higher concentrations of double bonds in the side-chains, which resulted in a lack of packing uniformity in the polymer. In addition to composition, molar mass also influences polymer properties. The molar masses of the PHA polymers are given in Table ΠΙ. It can be seen that an increased molar mass was associated with an increased concentration of saturated monomer side-chains. This suggests that an increased concentration of unsaturated fatty acids in the substrate interrupts the polymerization efficiency of the system. Because equal polymer yields were obtained from both coconut oil and soybean oil, it seems likely that the presence of unsaturated fatty acids, while not inhibiting polymerization, causes a more efficient termination of polymerization and results in a larger number of smaller molar mass PHA-So chains. In addition, unsaturated free fatty acids require additional isomerase and epimerase enzymes for oxidation to produce the CoA intermediates required for polymerization. The increased energy requirement involved in the production of two additional enzymes may slow the polymerization process and result in polymers with smaller relative molar masses. Whatever the predominant mechanism, it may be that both chemistries exist, resulting in a molar mass reduction in md-PHAs containing higher concentrations of unsaturated sidechains. g

m

Radiation Effects Radiation affects the properties of polymeric materials according to the type and source of radiation, the nature of the polymer structure, and the mechanism of reaction. It has been well established that crosslinking of a polymeric material requires the presence of functional groups (30). Olefinic groups are used frequently to this end since they can be crosslinked by both chemical (19, 20) and irradiation methods (21, 31, 32, 33). As seen from the md-PHA compositions (Table II), many of the double bonds from the TAG substrate were preserved by the bacterial PHA polymerization process. These groups provide the functionality necessary for crosslinking. Gamma-irradiation of a polymeric material generates radicals that lead to crosslink formation. However, these radicals also cause chain scission, the extent of which is based on the chemical composition of the material. Formally, the two processes, Le., crosslinking and chain scission, occur simultaneously, making it necessary to achieve some balance between the two processes to enhance the tensile properties of each material. In the present study three different md-PHAs were chosen based on their differing olefinic concentrations and γ-irradiated to determine the effects of side-chain unsaturation on radiation induced enhancement of polymer tensile


38 properties. After irradiation, each of the polymer films was subjected to a sol/gel analysis to determine the extent of crosslinking using non-irradiated films as controls. For non-irradiated films, the gelfractionamounted to between 1.0% and 1.4% of the total film weight tested. This indicated that prior to irradiation the films were relatively free of crosslinks. After irradiation, the gel fractions increased to 8%, 60%, and 91% for PHA-C, PHA-T, and PHA-So, respectively. Accordingly, the extent of crosslinking was proportional to the number of olefinic groups present in each mc/-PHA polymer side-chain. Tensile properties were measured immediately after irradiation to limit auto-oxidation. In each case, the tensile properties were enhanced by irradiation (Table IV). The most interesting result occurred with PHA-So films. While the tensile properties of the PHA-T film and PHA-C film increased, the PHA-So films were stiffened from a liquid-like texture to a solid film. This allowed the measurement of the tensile properties of PHA-So, which could not be done prior to irradiation. The tensile strength and percent elongation of the irradiated PHA-So film were 0.7 MPa and 25%, respectively, showing it to be a weaker film than the PHA-T and PHA-C crosslinked films. Young's modulus, which is a measure of the stiffness of a material, also increased in each polymer film after irradiation. Interestingly, PHA-So had the highest modulus after irradiation. These results indicated that irradiation had the greatest effect on the PHA-So films, which can be directly related to the high concentration of olefinic groups present in the PHASo polymer. Gel permeation chromatography (GPC) of the soluble (sol)fractionsfromthe sol/gel analyses of the irradiated polymers (Table V) revealed that the molar

Table IV. Tensile Properties of Select Irradiated and Non-Irradiated mcl PHA Polymer Films Young's Radiation Tensile Modulus Isotote Dose Strength % Elongation (MPa) (kGyf (MPa) 2.0 PHA-C 0 242 2.5 2.6 50 360 5.1 1.7 PHA-T 320 0 3.0 3.0 50 360 4.9 PHA-So* 0 3.1 50 25 0.7 Radiation source was cesium-137. In the absence of radiation PHA-So was amorphous and resulted in a noncoherent film. (SOURCE: Reproduced with permissionfromref. 33.)

a

b


39 Table V. Sol Fraction" Molar Masses of Select Irradiated mcl-VRk Polymer Films Radiation Molar mass (sol fraction) Isolate (film) Dose (kGy) M (x 10 ) M (x 10 ) M /M„ 59 205 3.47 PHA-C 50 48 194 4.04 PHA-T 50 33 102 3.09 PHA-So 50 Sol fractions were obtained from the chloroform soluble portions of the sol/gel analysis after film irradiation. (SOURCE: Reproduced with permission from ref. 33.) 3

n

3

w

w

a

masses had M and M values that were smaller and more polydisperse than the non-irradiated sol controls (Table III). This indicated that, in addition to crosslinking, random chain scission also occurred during the irradiation process. The formation of small quantities of large molar mass polymers helped increase polydispersity (M /M ). Specifically, the total amount of large molar mass fraction material varied from 3% (PHA-So) to 18% (PHA-C) with average M values well above 6.0 χ 10 g/mol. Because irradiation is a balance between crosslinking and chain scission, it is entirely possible that the large molar massfractionwas the result of the radiation induced formation of microgels that are small enough to pass through a 0.45 \im filter prior to GPC analysis. PHA-C is composed of the fewest olefinic carbons. This results in a limited number of crosslink sites compared to chain scission sites. Because of this, a larger concentration of microgels may be produced by PHA-C when compared to PHA-So during irradiation. This combination of microgel formation and chain scission resulted in increased polydispersities for the sol fractions of the irradiated films. In conclusion, it has been demonstrated that P. resinovorans produces mdPHA polymers from T A G substrates. Each md-PHA synthesized from T A G substrates reflects the fatty acid make-up of the TAG. Because of this, different polymers can be produced from various TAGs ranging from highly saturated (PHA-C) to highly unsaturated (PHA-So). The presence of olefinic groups in the md-PHA side-chains allows the manipulation of polymer properties by irradiation. Radiation generally results in polymer crosslinking; some chain scission, however, does occur. A positive balance between crosslinking and chain scission provides a relatively clean method of improving tensile properties of the TAG-derived md-PHAs. Lastly, optimization of md-PHA production from TAGs may provide farmers an additional outlet for fat and oil commodities, or provide a method to recycle used oils and greases. n

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40 Acknowledgement The authors thank Rob DiCiccio and Bina Christy for their technical assistance, as well as Dr. Alberto Nunez for performing the GC/MS analyses, Dr. Donald Thayer for performing the polymer irradiation, Dr. Peter Cooke for the electron microscopy, Ms. Janine Brouillette for the C-NMR, Dr. Jim Hampson for the supercritical fluid extraction, and Dr. Cheng-Kung Liu for the tensile testing. 13

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