Graft Copolymerisation of Methyl Methacrylate onto a
Bacterial Polyester Containing Unsaturated Side Chains
Songun Ilter,1Baki Hazer,*1, 2Mehlika Borcakli,1Oya Atici31TUBITAK-Marmara Research Center, Food Science and Technology Research Institute, Gebze 41470 Kocaeli, Turkey
Fax: 0372 323 86 93; E-mail: bhazer@karaelmas.edu.tr
2Zonguldak Karaelmas University, Department of Chemistry, 67100 Zonguldak, Turkey 3Istanbul Technical University, Department of Chemistry, 80626 Maslak, Turkey
Introduction
Poly(3-hydroxy alkanoate)s, PHAs, are biodegradable and natural aliphatic polyesters, and exist as intracellular energy and carbon storage materials, produced by some micro-organisms.[1 – 3] Pseudomonas oleovorans produce
medium chain-length PHA random copolymers of repeat-ing units containrepeat-ing 6 to 12 carbon atoms. This bacterium is very versatile for PHA production because it can pro-duce polyester from a wide variety of carbon substrates, including alkanes, alcohols, alkanoic acid, alkenes and its derivatives.[4 – 9]
Unsaturated PHAs were produced from the substrates of edible oily acids obtained from hazelnut, sesame, but-ter, olive, coconut and anchovy oil.[10, 11] Bacterial
poly-ester synthesized from soybean oily acids was a sticky, waxy and soft material. To improve the mechanical and viscoelastic properties of biodegradable PHAs, several attempts have been reported.[12 – 17]Grafting is considered
an important technique for modifying physical and
chem-ical properties of polymers.[18 – 22]
Hazer[12]
has reported grafting reactions of Poly(3-hydroxynonanoate) (PHN) with polystyrene and poly(methyl methacrylate) by per-oxidic initiators. c-Irradiation of the mixture of PHN and methyl methacrylate (MMA) also yielded PHN-g-PMMA graft copolymers.[14]Lenz et al.[15 – 17]obtained PHAs with
double bonds from P. oleovorans co-fed with 10-undece-noic acid and nona10-undece-noic or octa10-undece-noic acid. They performed the modification of the biopolyesters, containing double bonds, via irradiation,[15]
epoxidation[23]
and hydroxyla-tion.[24]Poly(ethylene glycol) was thermally grafted onto
the PHAs containing double bonds by polyazoester.[25]
Crosslinking properties of the unsaturated PHAs were also studied.[26]
The goal of this study is to combine the advantages of natural and synthetic polymers to obtain new composite materials via grafting reactions of MMA on PHA-soy-bean.
Full Paper:Poly(3-hydroxy alkanoate) containing unsat-urated side chains was produced by feeding Pseudomonas oleovorans with soybean-oil acid (PHA-soybean). The composition of PHA-soybean were found to be 10 mol-% of unsaturated side chains with the saturated hexanoate, octanoate and decanoate units. Methyl methacrylate (MMA) was thermally grafted on PHA-soybean in the presence of benzoyl peroxide. Fractional precipitation was used to isolate the graft copolymer from related homopolymers. PHA content in copolymer samples was between 15 to 30 mol-%. Graft copolymer samples were kept in hydroquinone to prevent post polymerization lead-ing to crosslinklead-ing in a day under laboratory atmospheric conditions. Copolymer characterization was performed using GPC and NMR techniques. Thermal analysis of the graft copolymer indicated the decomposition and glass transition temperatures of the PHA and PMMA segments of the graft copolymers. They also showed elongation at break in the range 10 to 21 related to the plasticizer effect of the biopolyester segments.
Macromol. Chem. Phys. 2001, 202, No. 11 iWILEY-VCH Verlag GmbH, D-69451 Weinheim 2001 1022-1352/2001/1107–2281$17.50+.50/0 Thermogravimetric trace of graft copolymer SI-13.
Experimental Part
Materials
Methyl methacrylate (MMA), CHCl3, benzoyl peroxide and
CH3OH were purchased from Merck and used without
further purification. Substrate
Soybean oil was a commercial product obtained from soy-bean grown in Turkey. Soysoy-bean oil was hydrolyzed in a 10% solution of KOH in ethanol, after which the solution was neutralized with a 10 wt.-% solution of sulfuric acid in water to obtain the carboxylic acid substrates.[6]The acid obtained
from the hydrolysis of soybean oil included both saturated and unsaturated acids in different percentage weights. Unsat-urated acids, including oleic, linoleic, linolenic and palmito-leic acids, comprised of the acids from soybean.[27]Saturated
acids, including both palmitic and stearic acids, were approximately 18%. Enes, dienes and trienes as unsaturated acids were about 18–26, 50–55 and 7–10 wt.-% respec-tively.
PHA-Soybean Biosynthesis
Stock cultures of P. oleovorans (ATCC 29347) were used in growth and polymer production experiments. Bacterial polyester containing olefinic groups in side chains was pre-pared by feeding P. oleovorans with soybean oily acids as reported elsewhere[10, 24] and the resulting polymer was
extracted with chloroform.[1, 4]
Grafting Procedure
For the grafting reaction 0.5 g of PHA-soybean, 2 mL of MMA and 0.01 g benzoyl peroxide were mixed in a Pyrex tube. Nitrogen was introduced through a needle into the tube for about 5 min. The tightly capped tube was kept at 70– 80 8C for a given polymerization time. The polymer product was taken out by dissolving in chloroform and was coagu-lated into methanol. The polymer sample was dried over-night under vacuum. The polymerization conditions and the results are given in Table 1.
Fractional Precipitation of PHA-Soybean-graft-PMMA The graft copolymer was isolated from the homopolymer of MMA and unreacted PHA-soybean by a fractional precipita-tion method. The vacuum-dried graft copolymer sample was dissolved in 10 mL of CHCl3. While the solution was slowly
stirred, MeOH was added dropwise until completion of the first precipitation. After decanting, MeOH was added to the upper solvent for precipitation of the second fraction. The same procedure was applied until precipitation ended. c-Values were calculated as the ratio of the total volume of MeOH used for each fraction to the volume of CHCl3 in
which the graft copolymer was dissolved. The fractionated polymer was dried under vacuum. Table 1 includes results of the fractional precipitation.
Polymer Characterization
1H NMR and13C NMR Analysis
1H NMR and 13C NMR spectra were recorded in CDCl 3 at
17 8C with a TMS internal standard using a Bruker 200 MHz NMR AC 200L. The signals in13C NMR spectra of
PHA-soybean and copolymer are listed in Table 2. GPC Analysis
Molecular weights were determined by gel permeation chro-matography (GPC) with a Waters model 6000A solvent delivery system with model 401 refractive-index detector, and a mode 730 data module with two Ultrastyragel linear columns in series. THF was used as the eluent at a flow rate of 1.0 mL/min. A calibration curve was generated from six polystyrene standards.
Methanolysis and GC-MS Analysis
The methanolysis reaction was carried out in CHCl3/MeOH/
H2SO4 (1 mL/1.85 mL/0.15 mL) at 100 8C for 140 min
fol-lowing a procedure identical with that described pre-viously.[5] The methyl esters obtained were assayed by gas
chromatography and mass spectroscopy (GC-MS) using a Perkin Elmer instrument with He as the carrier gas. After injection the column was maintained at 60 8C for 4 min and
Table 1. Results and conditions of the free radical polymerization of MMA in the presence of PHA-soybean. Run No. PHA-soybean
g a) MMA ml Benzoyl peroxide g Reaction time h Polymer yield g
Block copolymer fractionation
c wt.-% SI-7 1 3 0.05 1.15 1.46 crosslinked SI-13 0.5 3 0.05 1.15 1.15 crosslinked SI-15 0.3 3 0.01 1.15 1.04 crosslinked SI-24 0.5 2 0.01 2 1.93 2.7–3.2 80 SI-30 0.5 2 0.01 2 1.94 2.5–3.1 84 SI-33 0.5 1 0.02 2 1.42 2.9–3.1 90 SI-34 0.5 0.5 0.02 2 0.68 PMMA – 3 0.01 1 1.42 3.2–3.4 80 PHA-soybean 0.5 – – – – 1.4–2.0 80
a) PHA-soybean was obtained by 24 h of fermentation in 17.8 g of dry cell; the polymer yield was 3.6 gL–1. GPC results of the
PHA-soybean gave M—w: 1.3 6 10–5, M —
then heated at 10 8C/min to 270 8C. A temperature program was used which efficiently separated the different methyl 3-hydroxy alka(e)noates. Each peak in the chromatogram was analyzed with a mass spectrometer.
Thermal Analysis
Differential scanning calorimetry (DSC) and Thermogravi-metric Analysis (TGA) of the graft copolymers were per-formed on a DuPont 2910 instrument to determine the glass transition temperatures (Tg), melting transition temperatures
(Tm) and thermal degradation. For DSC analysis, samples
were heated from –100 to +200 8C in a nitrogen atmosphere at a rate of 10 8C/min and heated a second time using the same range and heating rate.
Stress-Strain Measurements
The mechanical properties of the copolymer films were measured on a Tensilon (UTM II) tester at room temperature with a crosshead speed of 10 mm/min. The copolymer films were prepared by solvent casting from chloroform solution. The mechanical test specimens were punched from the well dried film. These measurements were made for 3 samples of each copolymer samples and the results are shown in Table 3.
Results and Discussion
PHA containing unsaturation in the side chains was obtained in a 10 L glass fermentation by feeding Pseudo-monas oleovorans with 20 mm of soybean oily acids. After 24 h fermentation time, 19 g of dry cell and 2.9 g of polyester were obtained. M—w, M
—
n and molecular weight
distribution (MWD) were found to be 1.3 6 10–5,
0.7 6 10–5
and 1.8 respectively. Typical double bond sig-nals of PHA were observed at 2.0 and 5.4 ppm in the
1H NMR spectrum. GC-MS analysis was also used for the
chemical structure characterization. The methanolysis product of soybean was analyzed by GC-MS. PHA-copolymer content was found to be saturated hexanoate (3 mol-%), octanoate (59 mol-%) and decanoate units (10 mol-%) with the total unsaturation (10 mol-% of -ene, -diene and -triene). The biopolyester obtained was a sticky, waxy and soft material. Thermal analysis of the PHA-soybean was performed using DSC. The glass tran-sition temperature (Tg) of PHA was around –50 8C, the
melting temperature (Tm) was not detected, while
satu-rated biopolyester, e. g., poly(3-hydroxy octanoate)[7, 8]
has a melting transition at 66 8C and a glass transition at –35 8C. Presumably, unsaturation in side chains and a Table 2. 13C NMR chemical shift data (ppm) of PHA-soybean and PHA-soybean-graft-PMMA samples: SI-30.
Samples 1CH3 1CH2 (side chains) b-Carbon of PHA 1CH2CO2 1CH2CH1 1C2O 1O1CH3 1CH2C(CH3) (COOCH3)1 PHA-soybean 13.1 14 22.5, 24.7 25, 25.6 26.6, 27.2 29.3, 31.7 33.7 70.8 39.1 130.7 124 169.4 SI-30 14 16.4 18.7 22.5, 24.6, 29.3 29.1, 31.1 70.8 39.1 169.4 177.7 52.4 44.6 44.7
Table 3. Results of copolymer characterization. Run no: Molecular weights PHA
in copolymer
Thermal analysis Mechanical properties
M—n6 10–5 MWD mol-% Glass transition
8C Decomposition 8C Strain at break Tensile strength (F) Tg1 Tg2 Tg3 Td1 Td2 Td3 e % N mm2 SI-24 3.2 3.1 15 –50 112 290 (30)a) 390 21 10.5 SI-30 2.3 2.9 13 –50 –40 112 285 (25) 385 13 5.5 SI-33 0.9 4.2 30 12 105 270 (30) 345 (50) 405 10 7.5 SI-7 crosslink –32 88 285 (25) 405 SI-15 crosslink 285 (15) 390 SI-13 crosslink 285 (25) 400 PMMA 2.5 3.5 110 275 (45) 350 (55) 5 6.8 PHA-soybean 0.7 1.8 –50 270 –
higher C-number in repeating units lowered the glass transition temperature (Tg) of PHA.
Methyl methacrylate was grafted on PHA-soybean via a free radical mechanism in the presence of benzoyl per-oxide. Growing PMMA radicals can attach to the double bonds of the biopolyester to form a graft copolymer (Scheme 1). Meanwhile, phenyl radicals can also attach to the double bonds of the biopolyester leading to poly-ester radicals, which can initiate MMA polymerization. Table 1 includes the results and conditions of the grafting procedure at 80 8C. Copolymer samples were obtained as a thin film (U = 10 cm) by solvent casting in an alumi-nium dish. Post-polymerization produced the crosslinked samples (SI-7, SI-13 and SI-15 in Table 1), under labora-tory atmospheric conditions. Therefore, 2 mg of hydro-quinone was added to the copolymer samples in the sol-vent casting procedure to presol-vent post-polymerization (SI-24, SI-30 and SI-33 are not crosslinked copolymer samples). The crosslinked and non-crosslinked copoly-mer films were all highly transparent and brittle.
Fractional precipitation is a useful separation method of block/graft copolymers from the related homopoly-mers. Graft copolymer, PHA-soybean and PMMA were performed by means of precipitation with methanol as a non-solvent from their chloroform solution. c-Values were calculated from the ratio of the total volume of MeOH used for each fraction to the volume of CHCl3in
which graft copolymer was dissolved. Results of frac-tional precipitation are shown Table 1. The c-value of PHA-soybean and that of PMMA were 1.4–2.0 and 3.2– 3.4, respectively. c-Values of copolymer samples were between those of the related homopolymers (1.4–2.0 for PHA and 3.2–3.4 for PMMA), e. g., 2.5–3.2, which con-firms the graft copolymer structure. In this manner, GPC traces of the pure graft copolymers were all unimodal. Additionally, after extraction with a suitable solvent, crosslinked copolymer films did not give a considerable
soluble part which could be the homopolymer.1H NMR
spectra of the copolymer samples contained characteristic peaks (d ppm): –COOCH3of methyl methacrylate at 3.5
and (1O1CH1) of polyester at 5.1. Figure 1 shows a typical1H NMR spectrum of the graft copolymer SI-33.
When compared, the peaks areas at 3.5 and 5.1 ppm in
1
H NMR spectra of SI-24, SI-30 and SI-33 copolymer samples were found to include 15, 13, and 30 mol-% of biopolyester, respectively.
13C NMR spectra of the graft copolymer SI-30 can be
seen in Figure 2, and the signals of PHA-soybean and graft copolymer are listed in Table 2. When compared with the copolymer spectrum, the olefinic signals of the biopolyester at 124–130.7 ppm disappeared, and PMMA characteristic peaks appeared at 16.4, 44.7, 52.4 and 177.7 ppm for methyl, tertiary carbon, methoxy and car-bonyl bands, respectively.
GPC was used to determined the molecular weight and polydispersity of graft copolymers, PMMA and PHA-soybean (Table 3). Unimodal GPC chromatograms observed of the graft copolymer samples isolated by frac-tional precipitation can also be attributed to proper separation of the graft copolymer.
Scheme 1.
Figure 1. 1
H NMR spectrum of the graft copolymer sample, SI-33.
Figure 2. 13C NMR spectra of the graft copolymer sample,
Thermal analysis of graft copolymers was performed by DSC and TGA. The results of the thermal analysis are collected in Table 3. DSC results revealed the immiscibil-ity of the segments. The degradation of PHA-g-PMMA graft copolymer started above 270 8C (Figure 3 a). The degradation appeared to be a two-stage process, i. e., from 285 8C to 385 8C. The maximum weight loss occurred at around 400 8C (Td2). However, sample SI-33 has the
high-est amount of PHA (30 wt.-%). In the case of this sample, the degradation appeared to be three stage i. e. 270 8C, 345 8C and 405 8C (Figure 3 b). The maximum weight loss occurred at 345 8C. The grafting of PHA lowers the initial decomposition temperature because PHA chains degrade at approximately 270 8C.
The strain at break and tensile strength values of PHA-g-PMMA graft copolymers are shown in Table 3. The strain at break of the copolymer samples were changing in range between 10 to 21% while that of PMMA was 5%. Increase in elongation of the copolymer samples can be attributable to the plasticizer effect of the soft biopo-lyester segments son the copolymer.
Conclusion
Double bonds are open to further functionalization of bio-polyesters to improve mechanical and viscoelastic prop-erties. Grafting vinyl monomers on PHAs obtained from renewable sources can be carried out by a free radical mechanism. The viscous, sticky bacterial polyester can be handle in the form of copolymeric material. PMMA-g-PHA graft copolymers can also be used as compatibili-zers in polymer blends of PHA and PMMA.
Acknowledgement: This work was financially supported by an Eureka E2004 ‘Micropols’ grant.
Received: June 26, 2000 Revised: January 4, 2001
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