Synthesis of Poly-(R)-3 Hydroxyoctanoate (PHO) and Its Graphene

Chapter 14

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Synthesis of Poly-(R)-3 Hydroxyoctanoate (PHO) and Its Graphene Nanocomposites Ahmed Abdala,*,1 John Barrett,2 and Friedrich Srienc2 1Department

of Chemical Engineering, The Petroleum Institute, Abu Dhabi, United Arab Emirates Permanent Address: Department of Chemical Engineering and Petroleum Refining, Faculty of Petroleum and Mining Engineering, Suez University, Suez, Egypt 2Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455 and BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108 *E-mail: [email protected]

Polyhydroxyalkanoates are a popular class of bioplastics valued for their rapid biodegradadion, biocompatibility, and renewable feedstocks. While there are already a few commercial applications for these biopolymers, a greater diversity of properties is needed to compete with petroleum based polymers. In this chapter, we report the synthesis and characterization of polyhdroxyoctanoate and its nanocomposite with thermally reduced graphene. The results indicate the incorporation of graphene into the PHO matrix leads to a small upshift in the glass transition, enhance the thermal stability, and ~600% increase in modulus. Electrical percolation between 0.5 and 1 vol.% TRG was obtained.

© 2013 American Chemical Society In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Introduction Polyhydrxyalkanotes (PHAs) are polyesters that can be biologically synthesized by microbial cultivation or in other biological systems (1). They become an important class of biopolymers due to their renewable sources, biodegradation and applications in tissue engineering because of their biocompatbility (2). Structurally, the PHA backbone is comprised of 3-carbon repeat units with oxo-ester linkages. The attachment of various aliphatic and aromatic moieties stemming from the 3-carbon postion of each monomer imparts a range of material properties. Polyhydroxybutyrate (PHB), which possesses a single methyl group at the 3-carbon postion, is a stiff thermoplastic with a high melting temperature and is by far the most commonly used form of PHA. In contrast, medium chain-length PHA, (PHAmcl) contains longer aliphatic appendages (3-11 carbons) at the 3-carbon position which enhance elasticity but reduce polymer strength and melting temperature (3). Among PHAmcl, poly(3-hydroxyoctanoate) (PHO), which is a heteropolymer composed of C6, C8, and C10 monomers, is significantly more amorphous and flexible than PHB (4, 5). Thus, new methods to increase the strength and melting temperature of PHAmcl have significant potential for stimulating commercial proliferation of these materials and encouraging development of the larger natural products industry. Therefore, Nanocomposites of biopolymers with nano-fillers such as carbon nanotubes or clay, offer a significant potential for their increased utilization, as a result of the improvements in mechanical and thermal properties. There are a few publications that reports the production and characterization nanocomposites of PHB with nanofillers such as clay/layered silicate (6, 7) and carbon nanotubes (8, 9). Two new carbon allotropes, carbon nanotubes (10) and graphene (11), have received much attention as nanofillers because of their extraordinary mechanical, thermal, and electrical properties. With Young’s modulus of 1 TPa and ultimate strength of 130 GPa, graphene is the stiffest and strongest material ever measured (12). The incorporation of graphene into polymer matrices is expected to result in significant enhancement of the thermal, mechanical, electrical, and barrier properties (13). In this study, we develop a series of PHO-graphene nanocomposites with different graphene loading using solvent mixing in chloroform. Graphene dispersion in the PHO matrix is examined using TEM and the effect of graphene loading on the mechanical, thermal, and electrical properties are discussed.

Experimental PHO Synthesis PHO was produced via a fed-batch biosynthesis using the wild-type organism, Pseudomonas oleovorans . Initially, one fresh colony was selected and inoculated into a test tube containing 5-mL of LB medium (10 g Tryptone, 5 g Yeast Extract Powder, and 5 g NaCl in 1L of water) and grown overnight at 30°C. The culture was then transferred into a 2-L baffled flask with 500-mL of LB medium + 1% (v/v) 200 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


alkane for an additional 16 hours at 30°C with shaking at 250 RPM. This 500-mL culture was used to inoculate a 10-L bioreactor containing 5-L of E medium + 2% (v/v) alkane. E medium consisted of 1.1 g (NH4)2HPO4, 5.8 g K2HPO4, 3.7 g KHPO4, 0.25 g MgSO4•7H2O, and 1mL of trace metals in 1 L of water. Trace metals consisted of 2.78 g FeSO4•7H2O, 1.98 g MnCl2•4H2O, 2.81 g CoSO4•7H2O, 0.17 g CuCl2•2H2O, 0.29 g ZnSO4•7H2O, 1.67 g CaCl2•2H2O, 1M 1 mL in 1 L of water. Airflow and agitation were adjusted to maintain dissolved oxygen in the culture above 40%. During biosynthesis carbon dioxide evolution rate (CER) of the culture was monitored via mass spectroscopy. A sharp decline in CER indicated depletion of the carbon source, at which time more alkane was added to maintain growth. Batches were harvested at 50 hrs. The resulting cell pellet was lyophilized to dry the cells. PHO within the pellet was extracted in boiling chloroform using a Soxhlet apparatus for 16 hrs. Dissolved polymer was then precipitated with excess methanol, 8:1 v/v. The solvent mixture was decanted and residual solvent was evaporated under ambient conditions until the polymer is dry. Purified PHO were analyzed via gas chromatography fitted with a flame ionization detector (GC-17A, Shimadzu) using a DB-WAX column (ID 0.32 mm, 0.5 µm film thickness) (Agilent Technologies). Prior to injection, polyhydroxyalkanoic acids were converted to 3-hydroxyalkanoic propyl esters by the method of propanolyis (14). Quantitative determination the of different PHAs was made by comparison to standards synthesized from purified 3-hydroxyalkanoic acids (Sigma) Graphene Production and Characterization Thermally reduced graphene (TRG) is produced following the thermal exfoliation method (15, 16). In this method, natural flake graphite (-10 mesh, 99.9%, Alfa Aesar) is oxidized using Staudenmaier method (17) using a mixture of H2SO4 (95-97%, J.T. Bakers) and HNO3 (68%, J.T. Bakers), and Potassium chlorate (Fisher Scientific). The produced graphite oxide (GO) is washed with 5% HCl (37%, Reidel-de Haen), until no sulfate ions are detected then it repeatedly washed with water till no chloride ions are detected and dried in a vacuum overnight. GO was exfoliated by rapid heating at 1000 °C in a tube furnace (Barnstead Thermolyne) under flow of nitrogen for 30 s. XRD (X’Pert PRO MPD diffractometer, PANalytical) was used to test the oxidation of graphite and the complete exfoliation of graphite oxide. XRD scan between 5-35° was conducted at a scan rate of 0.02°/sec with instrument parameters of 40 kV voltage, 20 A intensity and 1.5406 Å CuKα radiation. TEM images were obtained using FEI Tecnai G20 TEM. Fabrication and Characterization of the Nanocomposites PHO/graphene nanocomposites with 1, 2, 5 wt.% (0.5, 1, and 2.5 vol.%) graphene were prepared by the following procedure. First, 1 g of polymer was dissolved in 20 mL of chloroform using a vortex tube mixer. Periodic incubation of the mixture in an 80° water bath was used to promote dissolution. Second, graphene powder was dispersed in chloroform at a concentration of 0.5 mg/L. To 201 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


promote the dispersion of graphene sheets, sonication was applied to the mixture using a electrode sonicator (Misonix 3000) at a power density of 1.5-3 W/mL. The graphene dispersion was added to PHO solution and stirrer for 1 hr. This mixture was then poured into a petri dish and evaporated on a hotplate at 55°C. Films were dried overnight to remove excess chloroform. For mechanical, rheological, and surface resistance measurements specimens were prepared from the dry composite samples by hot press (Tetrahedron, MTP-10) at 100°C and 1.0 MPa for 5 minutes. 5 cm x 5 cm square with a thickness of 0.5 mm was prepared and cut into rectangles (5 cm x 5 cm x 0.5 mm) for (1.25 cm x 0.5 mm) disks for electrical conductivity. All samples were aged at room temperature for more than 72 hrs prior to testing. Differential scanning calorimeter (DSC) (Netzsch 204 F1 Phoenix) and thermogravimetric analyzer (TGA) (Netzsch STA 409 PC) were employed to investigate thermal properties of PHO and its graphene nanocomposite. The mechanical properties of the pure and composite samples were measured using dynamic mechanical analyzer (TA Instruments, RSAIII) operating in a tension mode at an extension rate of 5 mm/min. Surface resistance measurements were taken from circular disks, 10 x 0.5 mm, using an 11-point probe (Prostat Corp., PRF-914B probe with PRS-801 meter). For each sample, 4 readings were collected, two from each side of the film. Graphene morphology and graphene dispersion into the PHO matrix is analyzed with TEM (FEI Tecnai G20 TEM). 80-100 nm thick composite films for TEM imaging were prepared at -80°C using an ultramicrotome (Leica, EM UC6).

Results and Discussion Preparation of Purified PHO The biosynthesis route for PHO in Pseudomonas oleovorans is shown in Figure 1. Briefly, octane in the media is consumed by the microorganism while undergoing a string of enzymatic conversions to produce a biologically active fatty acyl-CoA molecule. This intermediate molecule is degraded via successive fatty acid β-oxidation cycle in which two carbons are removed to produce the central metabolite, acetyl-CoA, and the corresponding n-2 fatty acyl-CoA. In Pseudomonas oleovorans, one of the intermediates of β -oxidation, trans-2-enoyl-CoA is converted via a 3-R-enoyl-CoA hydratase, PhaJ, to the monomer species, 3-hydoxyalkanoyl-CoA. The cyclical nature of β-oxidation results in a distribution of different monomers. This variation in the monomer pool combined with the promiscuous nature of the PhaC polymerase results in a random co-polymer comprised of several unique but structurally similar monomers. The synthesized sample is a random polymer of 3-hydroxyoctanoate (3HB), 3-hydroxyhexanoate (3HH), 3-hydroxydecanoate (3HD), and 3-hydroxybutyrate (3HB) with composition of 91.4, 7, 1.2, and 0.4 mol%, respectively.

202 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Figure 1. Metabolic pathway for PHO synthesis in Pseudomonas oleovorans.

Production and Characterization of TRG Oxidation of graphite leads to the introduction of polar oxygen functionalities on the surface of GO and change in carbon hyperdization to a mixture of sp2 and sp3 carbon. This leads to the expansion of the interlayer inter spacing from the graphite 3.35 Å (002 peak at 2θ = 26.5) to 7.8 Å (2θ = 11.4) as indicated by the XRD patterns for graphite and GO, Figure 2-a. In contrast, TRG diffraction pattern shows no noticeable diffraction peaks confirming the complete exfoliation of GO and production of TRG. TEM image of TRG (Figure 1-b) shows very thin and large (micron size) graphene sheets with wrinkled structure. The dark areas on the TEM micrograph represent the edges of folded or overlapped sheets.



Figure 2. a) XRD patterns of pure graphite, GO and TRG. b) TEM of TRG.

Morphology PHO-TRG Nanocomposites The dispersion of TRG in PHO is examined using TEM. As shown in Figure 3, although TRG is homogeneously distributed in the PHO matrix, it is not very well dispersed into the matrix as evidence by the presence of dark areas that represent the edges of stacked graphene layers. The high resolution image in the right indicates that TRG maintained its wrinkled structure while imbedded into the matrix.

Figure 3. TEM images of PHO-TRG composite with 1% TRG. Thermal Properties The nonoxidative thermal degradation of PHO and its TRG nanocomposites is studied using TGA and the results are shown in Figure 4. The pure and composite samples are stable up to 275°C as they show no significant weight loss below that temperature. Above 275°C, the pure and composite sample undergoes a slow degradation up to 295°C. Above 295°C , the pure samples rapidly degrade to 204 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

almost zero weight over a narrow range of about 30°C . The degradation rate of the composite samples is lower than that of the pure sample suggesting TRG inhibits the nonoxidative thermal degradation of PHO.


Table 1. Effect of TRG loading on thermal transitions of PHO TRG (vol%)

Tg (°C)

Tm (°C)

ΔHm (J/g)

T90% (°C)

T50% (°C)

0

-41.9

53.7

15.5

297.1

309.8

0.5

-38.1

55.7

13.9

299.3

311.7

1

-38.6

55.5

9.0

300.5

315.6

2.5

-39.0

54.1

12.2

299.5

314.5

The effects of TRG loading on the thermal transitions of PHO are also provided in Table 1. The addition of TRG increases the glass transition of PHO by a few degrees. The increase in the glass transition can be attributed to restraining the motion of PHO chains by the TRG sheets.

Figure 4. TGA thermograms of PHO and its TRG nanocomposites with 0, 0.5, 1, and 2.5 vol.% TRG. The crystallization behavior of biodegradable polymers is an important parameter because it significantly affects not only the crystalline structure and morphology but also the final physical properties and biodegradability of the polymer. Table 1 provides the melt temperature and heat of melting for the pure PHO and the composite samples. The melt temperature increases by 2°C with the addition of 0.5 vol.% TRG. A further increase in the TRG loading does not increase the melt temperature. In contrary, the melt temperature decreases when TRG loading increases from 1 to 2.5 vol.%. This behavior could be attributed to 205 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

the agglomeration of TRG sheets at higher loading. On the other hand, the heat of melting and consequently the %crystallinity decreases with the addition of TRG. This decrease in crystallinity suggests that the incorporation of TRG decreases the nucleation rate of PHO.


Mechanical Properties The mechanical properties of pure PHO and its TRG nanocomposite have been studied using DMA. The addition of TRG significantly increases the stiffness of PHO, reduces the elongation at break, and have no significant effect of the ultimate strength as shown in Figure 5 and table 2.

Figure 5. Stress-strain curves of PHO and its TRG nanocomposites with different TRG loading, vol.%.

Table 2. Mechanical properties of PHO-TRG nanocomposites TRG (vol%)

Modulus (MPa)

Strength (MPa)

Elongation at break (%)

0

4.5

6.4

425

0.5

7.2

7.0

346

1

10.9

5.6

205

2.5

31.0

6.7

105



The addition of TRG resulted in a very significant increase in the modulus (600% at 2.5 vol.%). This is even more impressive if we recall that the composite samples has a lower crystallinity compared to the pure sample as discussed earlier. This level of enhancement is significantly higher than the reported for graphene and graphene oxide composite with glassy polymers such as PA (18), PMMA (19), and PCL (20) but similar to that of elastomeric polymers such as natural rubber (21), PDMS (21), and TPU (22). Although, Figure 5 shows no appreciable change in the ultimate strength, we claim that TRG enhances the strength of PHO just enough to overcome the decrease in strength due to the lower crystallinity of the composite samples. The main drawback of the addition of TRG is the reduction in the elongation at break. Nevertheless, the composite samples remain highly flexible with a minimum elongation at break of over 100%.

Electrical Properties The greatest advantage of carbon nanofillers and graphene in particular versus other nanofillers is the ability to increase the electrical conductivity of nonconductive polymers and the measured electrical resistivity of PHO and the nanocomposite samples provides an example of such ability. The required TRG loading to produce electrically conductive PHO, the electrical percolation, is slightly above 0.5 vol.%. This low percolation limit is an indication of a good dispersion of TRG into the matrix. The observed percolation limit is similar to that solution processed polyethylene-TRG (23) but lower than that of polar polymer-TRG composites (22). A further increase in the loading of TRG greatly increases the electrical conductivity (decreases resistivity) as shown in Figure 6. Compared to the resistivity of the pure polymer of 1.3x103 MΩ.m, a resistivity of less than 5 Ω.m is obtained with 2.5 vol.% loading of TRG.

Figure 6. Effect of TRG loading on the electrical resistance of PHO-TRG nanocomposites. 207 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Conclusions We have successfully produced PHO, TRG, and their nanocomposites. The solvent blended PHO-TRG nanocomposite with TRG loading of 0.5, 1, and 2.5 vol.% exhibited enhanced thermal, mechanical, and electrical properties compared to the pure PHO. The addition of TRG resulted in a slight upshift in the glass transition, increase in the melt temperature, and decrease in crystallinty. Regardless of this decrease in crystallinity, the mechanical properties of the composite sample revealed a striking 600% increase in modulus with 2.5 vol.% TRG while maintaining elongation at break above 100%. Electrically conductive PHO samples can be made with the addition of slightly more than 0.5 vol% TRG and a very low resistivity of less than 5 Ω.m is obtained with 2.5 vol.% TRG. These promising results would increase the applications of medium chain PHAs.

Acknowledgments Financial support from the Abu Dhabi-Minnesota Institute for Research Excellence (ADMIRE) is acknowledged. The authors also thank Dr. Marios Katsiotis at the Department of Chemical Engineering, the Petroleum Institute for the TEM work and Mr. Daniel Rouse, the university of Minnesota, for his role in the synthesis of PHO.

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Synthesis of Poly-(R)-3 Hydroxyoctanoate (PHO) and Its Graphene

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