ACS PHA graphene composites 11908

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/251565474

Synthesis of poly-(R)-3-hydroxyalkanoic acids (PHA) and their graphene nanocomposites ARTICLE in PAPERS PRESENTED AT THE ... MEETING. AMERICAN CHEMICAL SOCIETY. DIVISION OF POLYMER CHEMISTRY · JANUARY 2013

READS

163

4 AUTHORS, INCLUDING: Ahmed A. Abdala

Muhammad Zafar Iqbal

Qatar Environment and Energy Research Inst…

Colorado School of Mines

56 PUBLICATIONS 4,863 CITATIONS

9 PUBLICATIONS 42 CITATIONS

SEE PROFILE

SEE PROFILE

Available from: Ahmed A. Abdala Retrieved on: 11 January 2016

Synthesis of Poly-(R)-3 hydroxyalkanoic acids (PHA) and their Graphene Nanocomposites Ahmed Abdala1, Muhammad Iqbal1, John Barrett2, and Friedrich Srienc2 1

Department of Chemical Engineering, The Petroleum Institute, Abu Dhabi, UAE 2 Department of Chemical Engineering and Materials Science, and BioTechnology Institute, University of Minnesota, Minneapolis/St.Paul, MN

Introduction Polyhydrxyalkanotes (PHAs) have become an important class of biopolymers due to their renewable sources, biodegradation and applications in tissue engineering because of their biocompatbility1. However, their thermal stability and mechanical properties limit their utilization in other applications. Therefore, Nanocomposites of biopolymers with nano-fillers such as carbon nanotubes or clay, offer significant potential for their increased utilization, as a result of the improvements in mechanical and thermal properties. Poly 3-hydroxybutyrate (PHB) is the most widely studied biopolymer in the PHA family. PHB are polyesters that can be biologically synthesized by microbial cultivation or in other biological systems 2. There are a few reports on nanocomposites of PHB with nanofillers such as wood flour3, clay4, layered silicate5 and carbon 6 nanotubes . These nanofillers have been reported to affect the thermal, crystallization and mechanical properties of PHB. For example, incorporation of 10 wt.% of CNT into PHB matrix doubles the tensile modulus and further improved the hardness of the composite. The crystal nucleation was also found to increase with a loading of 1 wt.% of CNT. The discovery of graphene in 2004 has introduced a new class of polymer graphene nanocomposite as a result of the extraordinary mechanical, thermal, and electrical properties of graphene and the ability to disperse in polymer matrices7. Therefore, graphene-based biopolymer composites have drawn attention due to the improved electric conductivity that can further improve the rate of proliferation, an advantage in stimulation of bone repair8, and benefit PHB fibers used as neutral scaffolds1. Moreover, dispersion of graphene into PHB matrix is expected to enhance the thermal, mechanical and electrical conductivity properties of PHB. However, to the best of our knowledge, no report has been made on graphene/PHB composites. In this study, we present the development of PHB-graphene nanocomposite using melt and solution mixing. The effect of the graphene loading and processing method on the dispersion of graphene into the matrix and on the mechanical and thermal properties is discussed. Experimental Materials used in PHB synthesis: D-Fructose (Aldrich); Boric Acid, Cupric Sulfate Pentahydrate (Mallinckrodt); Cobalt Chloride Hexahydrate, Zinc Sulfate Heptahydrate, Sodium Phosphate Dibasic Heptahydrate (Fisher); Potassium Phosphate Dibasic, Ammonium Sulfate, Magnesium Sulfate Heptahydrate, Ferric Ammonium Citrate, Calcium Chloride Dihydrate, Manganese Chloride Tetrahydrate, Nickel Chloride Hexahydrate, Sodium Molybdate Dihydrate, Sodium Hydroxide, Methanol (Chromasolve) (Sigma); Chloroform (ACS) (Alfa Aesar). Materials used in graphene production. Natural flake graphite (-10 mesh, 99.9%, Alfa Aesar), Sulfuric Acid (95-97%, J.T. Bakers), Hydrochloric Acid (37%, Reidel- deHaen), Hydrogen Peroxide (30% solution, BDH), Potassium Permanganate and Sodium Nitrate (Fisher Scientific). All reagents were used without further purification. Synthesis of Poly (3-hydroxybutyrate). Poly-3-hydroxybutyrate (PHB) was prepared by biosynthesis using the organism Ralstonia eutropha9. Cells were cultivated in minimal salts media containing fructose, 20 g/L. During cultivation additional fructose was

added to sustain growth. The biosynthesis was performed in a 10-L bioreactor (Sartorius, Germany) containing 5 L of media. Temperature was controlled at 30C and pH is maintained at 7.0 by automatic addition of 6 M sodium hydroxide. Agitation was set at 400 RPM and airflow was adjusted to maintain the dissolved oxygen above 50% saturation. Following biosynthesis for 48 hr, the cell broth was centrifuged, washed in one volume of deionized water, and centrifuged again. The resulting cell pellet was lyophilized for ~12 hrs to dry the cells. PHB 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 was dry. Graphene production. Thermally reduced graphene (TRG) is produced following the thermal exfoliation method10. In this method, graphite is oxidized using Staudenmaier method11 as follows: graphite (5 g) is placed in ice-cooled flask contains a mixture of H2SO4 (90 ml) and HNO3 (45 ml). Potassium chlorate (55 g) is added slowly to the cold reaction mixture. The reaction is stopped after 96 h by pouring the reaction mixture into water (4 L). HCl solution (5%) is used to wash the produced graphite oxide (GO). The mixture is then washed with water till no chloride ions are detected. GO is dried in a vacuum overnight. GO was exfoliated by rapid heating at 1000 oC in a tube furnace (Barnstead Thermolyne) under flow of nitrogen for 30 s. Nanocomposite Fabrication. TRG/PHB nanocomposites are prepared by melt and solution blending. In melt blending, different TRG loadings (0.1-3 wt.%) are extruded using microextruded (HAAKE MiniLab II microcompounder ) operating at 100 RPM screw and 175o C for 5 minutes. Test samples for mechanical and electrical measurement are made by injection molding the extrudate using mini-injection molding (HAAKE MiniCTW) with cylinder temperature and mould temperature of 200 and 50o C and injection and post injection pressure of 600 and 200 bar. Pure PHB is also extruded under the same conditions. In solvent blending, TRG is dispersed in chloroform by sonication then mixed with PHB dissolved in chloroform at 60o C for 4 hours. The mixture is pulse sonicated for additional 5 minutes. The composite solution is solvent cast and the dry sample is hot pressed to make test samples. PHB Characterization. Purified PHB was derivatized to its propyl ester by the method of propanolyis12 before analysis by gas chromatography with FID (GC-17A, Shimadzu) using a DB-WAX column (ID 0.32 mm, 0.5 m film thickness) (Agilent Technologies). Quantitative determination of PHB was made by comparison to a commercial standard (Polysciences, Inc.). Graphene Characterization. X-Ray diffraction and Transmission Electron Microscopy, TEM (FEI Tecnai G20) was used to study the exfoliation of graphite oxide. Differential scanning calorimeter (DSC) (Netzsch 204 F1 Phoenix) and Thermogravimetric analyzer (TGA) (Netzsch STA 409 PC) were employed to investigate thermal properties of PHB and its composites. Tensile testing machine (Testometric M350-10CT) and Dynamic Mechanical Analyzer (TA RSAIII) are used to study the mechanical and dynamic thermo-mechanical properties of composites. Results and Discussion Biosynthesis of PHB. PHB is produced naturally by numerous species of microorganisms, of which Ralstonia eutropha is widely studied. Fructose is converted to PHB by way of acetyl-CoA, a central metabolic intermediate common in most microorganisms. In R. eutropha, acetyl-CoA is converted to PHB by the action of three key enzymes: PhbA, acetoacetyl-CoA synthase, PhbB, acetoacetyl-CoA reductase ,and PhbC, PHB synthase. The extent of intracellular accumulation and the composition of PHB and other PHAs were

quantitatively determined using GC as described in the Materials section. The bacterial synthesis pathway to PHB is shown in Fig. 1.

The sheets are very thin with dark area representing the edges or overlay of multiple sheets as the elastic corrugations and the scrolled or folded edges often result in different brightness in the surface of TRG.

Characterization of TRG. Thermal, mechanical, and electrical properties of PHB and the composites. The oxidation of graphite leads to the appearance of polar oxygen functionalities on the surface of GO and change in the hyperdization from all sp2 carbon to a mixture of sp2 and sp3 carbon. The presence of these functionalities in addition to the adsorbed water intercalation lead to expansion of the interlayer inter spacing from the graphite 3.35 Å (002 peak at 2 = 26.5) to 7.8 Å (2 = 11.4) as indicated in the XRD patterns for graphite and GO, Figure 2. In contrast to graphite and GO diffraction patterns which indicate the presence of ordered layered

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. The effect of TRG on crystallization and melting characteristics of PHB is examined using DSC. The pure and nanocomposite samples were initially heated to 200o C at a rate of 30o C/min in order to erase any previous thermal history present in PHB, and cooled to 35o C at 10o C/min. Figure 5 shows DSC cooling traces of the pure PHB, 0.5 and 1% nanocomposite samples cooled from 200 to 35° C at a rate of 10o C/min. For pure PHB, the crystallization onset and the peak are observed at 107.2o and 96.5o C, whereas for 0.5% TRG/PHB composites are observed at 112.1o and 103o C, respectively. The increase in the onset and peak temperatures suggests that TRG play a role in non-isothermal crystallization of PHB due to heterogeneous nucleation in the composites. However, the onset and peak temperatures of crystallization for 1%TRG/PHB composites appear to be lower than those for 0.5% TRG/PHB sample but still higher than those for the pure sample. This may be attributed to the agglomeration of TRG sheets during melt blending resulted in ineffectiveness in crystallization extent than 0.5% TRG/PHB composites.

Figure 1. Biosynthesis route of PHB in Ralstonia eutropha structure, TRG diffraction pattern shows no noticeable diffraction peaks confirming the complete exfoliation of GO and production of TRG.

-0.3 -0.5 -0.7

Exo

DSC Signal (mW/mg)

-0.1

-0.9 -1.1 Pure PHB 0.5% TRG/PHB 1% TRG/PHB

-1.3 -1.5 50

60

70

80

90

100 110 120 130 140 150

Temperature

Figure 5. DSC cooling traces for pure PHB and TRG/PHB nanocomposites.

Figure 2. XRD patterns of pure graphite, GO and TRG TEM image of TRG (Figure 2) shows very large (micron size) graphene sheets with wrinkled structure due to the chemical nature of TRG.

On the other hand, the melting endotherms of pure PHB and its nancomposites did not show any significant differences in the onset and peak values. The characteristic values for melting of pure PHB and TRG/PHB nanocomposites are provided in Table 1. The area under the melting peak is used to calculated total enthalpy of melting and the %crystallinity using the theoretical enthalpy of fusion of a 100% crystalline PHB of 146 J/g 13. As shown in table 1, there is a slight increase in the %crystallinity with TRG loading . These results are consistent with the crystallization resuls which suggest that incorporation of TRG increases the rate of nucleation in PHB. Therefore, the size and nature of the crystal structure are examined with optical microscope and will be discussed in detailed during the oral presentation. Table 1 Effect of TRG loading on thermal properties

Pure PHB 0.5% TRG/PHB 1% TRG/PHB

Figure 3. TEM micrograph of TRG

Onset (oC) 167.4 167.8 167.0

Peak (oC) 174.8 174.3 172.7

Melting Area (J/g) 66.27 69.19 72.15

% Crystallinity 45.4 47.4 49.4

The effect of TRG on the thermal stability of PHB is investigated using DSC and TGA. It has been also observed a significant increase in the degradation onset temperatures of PHB and the composites. The onset for pure PHB (264.8o C) has increase to 277.2° C and 272.5° C with incorporation of 0.5 and 1 wt.% TRG, respectively. A difference of around 12.4o C in degradation onset starting temperature indicates higher stability of the composites as compared to that of pure PHB. Moreover, area under degradation peak reflects energy required for degradation. Although, there is slight increase on the onset of degradation, the overall degradation behavior does not show any significant difference between the pure and the composite samples.

Figure 5 shows the TGA thermographs of PHB, 0.5 and 1 wt.% TRG composite samples. The samples were heated from 50 to 350° C temperature at a rate of 5° C/min under inert atmosphere of nitrogen. The TGA results are consistent with the DSC observation. The composite samples show slightly enhanced thermal stability compared to the pure PHB. The mechanical properties of pure and nanocomposites have been determined by a tensile machine. We observe that TRG increase the Young’s modulus of PHB. For example, addition of 0.5 wt.% of TRG leads to about 10% increase in modulus while 1 wt.% results in 25% increase in the modulus for melt blended samples. Solvent blending leads to a more significant increase in modulus. In addition to the modulus, incorporation of TRG also affects the ultimate strength and the elongation at break.

7.

8.

9.

10.

11. 12.

13. Figure 5. TGA thermograms for pure PHB and 0.5% TRG/PHB composite The electrical properties of the Melt blended composite samples with TRG loading of 0.5 and 1 wt.% did not improve as the samples remains nonconductive at this loading. Solution blending and/or higher TRG loading are used to make the sample electrically conductive. Conclusions Thermally reduced graphene is successfully synthesized using thermal exfoliation of GO. The results from XRD and TEM measurements indicate a complete exfoliation of GO to TRG. Nanocomposites of PHB with TRG at different loadings are prepared by melt blending using microcompounder and by solvent blending using chloroform. Our results indicate that TRG affected the rate of crystallization and the increases the %crystallinity. On the other hand, no significant changes in melting properties are observed for the melt blended samples. A slight increase in mechanical properties of PHB is observed with increasing TRG loading. The thermal stability of PHB is enhanced by the addition of TRG as the onset degradation temperature increases with TRG loading.

References 1. 2.

3.

4.

5.

6.

Chen, G. Q.; Wu, Q., The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials 2005, 26 (33), 6565-6578. Jackson, J. K.; Srienc, F., Effects of recombinant modulation of the phbCAB operon copy number on PHB synthesis rates in Ralstonia eutropha. Journal of Biotechnology 1999, 68 (1), 49-60. Fernandes, E. G.; Pietrini, M.; Chiellini, E., Bio-based polymeric composites comprising wood flour as filler. Biomacromolecules 2004, 5 (4), 1200-1205. Botana, A.; Mollo, M.; Eisenberg, P.; Torres Sanchez, R. M., Effect of modified montmorillonite on biodegradable PHB nanocomposites. Applied Clay Science 2010, 47 (3-4), 263-270. Maiti, P.; Batt, C. A.; Giannelis, E. P., New biodegradable polyhydroxybutyrate/layered silicate nanocomposites. Biomacromolecules 2007, 8 (11), 3393-3400. (a) Xu, C.; Qiu, Z., Crystallization behavior and thermal property of biodegradable poly (3‐hydroxybutyrate)/multi‐walled carbon nanotubes nanocomposite. Polymers for Advanced Technologies 2011, 22 (5), 538544; (b) Yun, S. I.; Gadd, G. E.; Latella, B. A.; Lo, V.; Russell, R. A.; Holden, P. J., Mechanical properties of biodegradable polyhydroxyalkanoates/single wall carbon nanotube nanocomposite films. Polymer Bulletin 2008, 61 (2), 267-275.

Kim, H.; Abdala, A.; Macosko, C. W., Graphene/Polymer Nanocomposites. Macromolecules (Washington, DC, United States) 2010, 43 (16), 6515-6530. Supronowicz, P.; Ajayan, P.; Ullmann, K.; Arulanandam, B.; Metzger, D.; Bizios, R., Novel current‐conducting composite substrates for exposing osteoblasts to alternating current stimulation. Journal of biomedical materials research 2002, 59 (3), 499-506. Ramsay, B. A.; Lomaliza, K.; Chavarie, C.; Dube, B.; Bataille, P.; Ramsay, J. A., Production of poly-(β-hydroxybutyric-co-βhydroxyvaleric) acids. Applied and Environmental Microbiology 1990, 56 (7), 2093-2098. (a) Prud'homme, R. K.; Aksay, I. A.; Adamson, D.; Abdala, A. Thermally exfoliated graphite oxide. 7,658,901, 2010; (b) McAllister, M. J.; Li, J.L.; Adamson, D. H.; Schniepp, H. C.; Abdala, A. A.; Liu, J.; HerreraAlonso, M.; Milius, D. L.; Car, R.; Prud'homme, R. K.; Aksay, I. A., Single Sheet Functionalized Graphene by Oxidation and Thermal Expansion of Graphite. Chemistry of Materials 2007, 19 (18), 43964404. Staudenmaier, L., Method for the preparation of graphitic acid. Ber. Dtsch. chem. Ges. 1898, 31, 1481-87. Riis, V.; Mai, W., Gas chromatographic determination of poly-βhydroxybutyric acid in microbial biomass after hydrochloric acid propanolysis. Journal of Chromatography A 1988, 445 (C), 285-289. Hahn, S. K.; Chang, Y. K.; Lee, S. Y., Recovery and characterization of poly (3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli. Applied and environmental microbiology 1995, 61 (1), 34-39.