- Email: [email protected]
Microbial synthesis and characterization of polyhydroxyalkanoates with fluorinated phenoxy side groups from Pseudomonas putida
European Polymer Journal 40 (2004) 1551–1557
EUROPEAN POLYMER JOURNAL www.elsevier.com/locate/europolj
Microbial synthesis and characterization of polyhydroxyalkanoates with fluorinated phenoxy side groups from Pseudomonas putida Yasuo Takagi a
a,*
, Ryou Yasuda a, Akira Maehara b, Tsuneo Yamane
b
Department of Environmental Materials, Nagoya Municipal Industrial Research Institute, 3-4-41 Rokuban, Atuta-ku, Nagoya 456-0058, Japan b Laboratory of Molecular Biotechnology, Department of Biological Mechanisms and Functions, Graduate School of Bio- and Agro-Science, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan Received 29 July 2003; received in revised form 22 January 2004; accepted 22 January 2004 Available online 16 March 2004
Abstract Microbial poly(3-hydroxyalkanoates) (PHAs) with fluorinated phenoxy side groups were produced by Pseudomonas putida when fluorophenoxyalkanoic acids were used as carbon sources. 11-(2-Fluorophenoxy)undecanoic acid (2FPUDA), 11-(3-fluorophenoxy)undecanoic acid (3FPUDA), 11-(4-fluorophenoxy)undecanoic acid (4FPUDA), 11(2,4-difluorophenoxy)undecanoic acid (2,4DFPUDA), 11-(2,4,6-trifluorophenoxy)undecanoic acid (2,4,6TFPUDA), and 11-(2,3,4,5,6-pentaflurophenoxy)undecanoic acid (2,3,4,5,6PFPUDA) were used as carbon sources in the present study. When cells were grown with 2,4DFPUDA, the production of homo poly(3-hydroxy-5-(2,4-difluorophenoxy)pentanoate) was confirmed by NMR and GC/MS analyses. Fluorine atoms inserted into the side chain of the PHA dramatically affected its physical properties. In marked contrast to medium chain length (MCL) PHA, this fluorinated PHA was opaque, cream colored, and possessed greater crystallinity and a higher melting point (100 C) than did the other MCL PHAs. Surface contact angle evaluation revealed that the PHA with two fluorine atoms possessed water-shedding properties. The number of substituted fluorine atoms in the carbon source affected cell growth and difluorine-substituted phenoxyalkanoic acids reduced cell growth, and polymer production compared to non-substituted phenoxyalkanoic acids. No polymeric materials were obtained using either 2,4,6TFPUDA or 2,3,4,5,6PFPUDA. 2004 Elsevier Ltd. All rights reserved. Keywords: Polyhydroxyalkanoate; Bacteria; Biodegradable plastic
1. Introduction Poly(3-hydroxyalkanoates) (PHAs) are bacterial storage compounds that have found use as biodegradable and biocompatible thermoplastic polymers. Microbial PHAs have a common structure containing chiral centers yielding polymers that are isotactic and
* Corresponding author. Tel.: +81-52-654-9890; fax: +81-52652-6776/654-9895. E-mail address: [email protected] (Y. Takagi).
optically active [1–3]. Several approaches to increase the utilization of PHAs have been investigated in the areas of agricultural, marine, and medical materials [4,5]. The physical and mechanical properties of PHAs are dependent on the compound’s monomeric composition, and, at present, more than 100 different PHAs have been produced and characterized [6]. Most of the PHAs with unusual groups have been produced from either Pseudomonas oleovorans or Pseudomonas putida when unusual carbon sources were supplied during fermentation. In particular, production of PHAs with unusual groups produced by P. oleovorans has been investigated extensively [7–10]. In addition to PHAs with long alkyl
0014-3057/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2004.01.030
1552
Y. Takagi et al. / European Polymer Journal 40 (2004) 1551–1557
groups, P. oleovorans has produced PHAs with functional groups such as an olefin [7–9], branched alkyl [11], halogen [10], phenyl [12], phenoxy [13–15], and cyclohexyl [16]. PHAs, with fluorinated side chains have been reported by several researchers. PHA containing 27 mol% of 3-hydroxy-9-fluorononanoate and 3-hydroxy-7fluoronoheptanoate was produced when P. oleovorans was cultivated on a mixture of nonane and 1-fluorononane [17]. Compared to PHA produced from nonanoic acid (a non-halogenated carbon source), the melting temperature (Tm) of this fluoro-PHA was increased from 47 to 61 C, and its glass transition temperature (Tg) increased from )44 to )39 C. Kim et al. [18] investigated PHA with fluorine atoms produced by P. oleovorans and P. putida grown with a mixture of multifluorinated alkanoic acid and nonanoic acid, which yielded copolymers containing up to 17.3 mol% nonafluoro 3-hydroxynonanoate. Fully or partially fluorinated polymers showed unique physical properties with respect to thermal, oxidative, and photochemical stability [19,20]. In addition, PHAs with monohalogenated side groups of fluorine, bromine, and chlorine have been produced in P. oleovorans from corresponding alkanes or alkanoic acids, resulting in copolymers containing a halogenated side group and an alkyl group due to the use of a mixture of halogenated carbon source and alkanoic acid [10,17,18,21]. A higher content of halogenated side groups significantly improves the physical properties of the PHA. To our knowledge, few reports have been published about PHAs with a high halogen content produced from bacteria. In this study, we produced PHAs with fluorinated side groups and investigated their properties.
2. Experimental 2.1. Materials The same general synthetic procedure was used for preparation of 11-phenoxyundecanoic acid (PUDA), 11-(4-fluorophenoxy)undecanoic acid (4FPUDA), 11(3-fluorophenoxy)undecanoic acid (3FPUDA), 11-(2fluorophenoxy)undecanoic acid (2FPUDA), 11-(2, 4-difluorophenoxy)undecanoic acid (2,4DFPUDA), 11(2,4,6-trifluorophenoxy)undecanoic acid (2,4,6TFPUDA), and 11-(2,3,4,5,6-pentaflurophenoxy)undecanoic acid (2,3,4,5,6PFPUDA). Potassium hydroxide (0.11 mol) was added to 0.07 mol of phenol (Aldrich Chemical Co., 99%), 4-flurophenol (Aldrich Chemical Co., 99%), 3-fluorophenol (Aldrich Chemical Co., 98%), 2-fluorophenol (Aldrich Chemical Co., 98%), 2,4-difluorophenol, (Aldrich Chemical Co., 99%), 2,4,6-trifluorophenol (Aldrich Chemical Co., 99%), or pentafluorophenol (Aldrich Chemical Co., 99%) in a 500 ml round bottom
flask containing 200 ml of ethanol. To this mixture, 0.06 mol of 11-bromoundecanoic acid (Aldrich Chemical Co., 99%) was added. The reaction was allowed to continue at the reflux temperature of ethanol for 24 h. The corresponding acids were precipitated by adding the reaction solution to 200 ml of 0.1 M aqueous HCl. This solution was then filtered, dried in vacuo, and purified by recrystallization from hot methanol. The yields of the purified acids ranged from 20% to 90%. The structures of the products were confirmed by NMR spectroscopy.
2.2. Cell growth A culture of P. putida 27N01 was used throughout the study. This strain was isolated from soil and identified by National Collections of Industrial and Marine Bacteria Limited (NCIMB) in Scotland. P. putida was precultivated under aerobic conditions at 30 C and pH 7.0 for 48 h on a reciprocating shaker in 10 ml of medium containing defined mineral salts and 20 mM octanoic acid (OA) as a carbon substrate. Composition of the mineral salt medium (per liter) was 1.5 g (NH4 )2 SO4 , 1.4 g KH2 PO4 , 1.4 g Na2 HPO4 , 0.5 g NaHCO3 , 0.3 g MgSO4 Æ 7H2 O, 0.3 g yeast extract (Difco), and 0.05 g ammonium iron (III) citrate. In addition, 0.1 ml of trace element solution was added to the medium. The trace element solution contained the following salts (per liter): 3.0 g H3 BO4 , 2.0 g CoCl2 Æ 6H2 O, 1.0 g ZnSO4 Æ 7H2 O, 0.3 g MnCl2 Æ 4H2 O, 0.2 g NaMoO4 Æ 2H2 O, 0.28 g NiSO4 Æ 7H2 O, and 0.1 g CuSO4 Æ 5H2 O. After precultivation, 1 ml of the cells was inoculated with modified medium containing OA, PUDA, 4FPUDA, 3FPUDA, 2FPUDA, 2,4DFPUDA, 2,4,6TFPUDA, or 2,3,4,5,6PFPUDA as the carbon source. The first cultivation was conducted in a 500 ml flask containing 100 ml of medium under the same conditions as the preculture for up to two weeks. Thereafter, the cells were transferred and grown for one week in a 5 l jar-fermenter (model KMJ-5C; Mitsuwa Bio Systems Co., Osaka, Japan) with a culture volume of 3 l, containing the same medium composition. In the fermenter, agitation speed, aeration rate, and temperature were set at 500 rpm, 0.7 vol vol1 min1 , and 30 C, respectively. The pH of the culture was adjusted by addition of 3 N KOH or 2 N HCl using a pH controller. Antifoam (Adecanol LG-109; Asahi Denka Kogyo Co., Tokyo, Japan) was added to the medium at an initial concentration of 0.03% (v/v). Direct monitoring of cell growth by measuring optical density was not possible due to emulsified liquid particles of undissolved carbon sources. Therefore, the mixture was filtered through cellulose filter paper (Toyo Roshi Kaisya, Ltd., porosity no. 2) to remove the particles and was then monitored daily at 660 nm to obtain cell growth curves.
Y. Takagi et al. / European Polymer Journal 40 (2004) 1551–1557
The cells were harvested by centrifugation, washed with 30% methanol solution in water, dried and weighed. The dried cells were suspended in 100 ml chloroform at room temperature for 24 h. The mixture was filtered through a cellulose filter paper, and the filtrate was concentrated by evaporation. The polymer was reprecipitated in methanol and dried in order to determine PHA yield. 2.4. Analyses To identify the composition of the polymer by GCMS, samples were prepared as described previously [15]. Methyl esters of PHA monomers were analyzed by a Hewlett Packard 5890 system equipped with a J&W SCIENTIFIC capillary column (30 mm · 0.25 mm) and a total ion detector. The 1 H and 13 C nuclear magnetic resonance (NMR) spectra of PHA samples were recorded on a JEOL JNM-EX 400 FT-NMR spectrometer. The 400 MHz 1 H-NMR and the 100 MHz 13 C-NMR spectra of the polymers were recorded at 25 C in CDCl3 . The average molecular mass and molecular mass distributions of the polymer were obtained at 30 C by gel permeation chromatography (Shodex GPC system 11; Showa Denko K.K., Tokyo, Japan) using AC-800P, AC-80M, and K-806M sample columns. Chloroform was used as an eluent at a flow rate of 1.0 ml min1 . Polystyrene standards having a low polydispersity rate were used to produce the calibration curve. Thermal characterization was carried out using a MacScience DSC-3100S differential scanning calorimeter (DSC) equipped with an MTC-1000S data station. The sample was sealed in an aluminum pan, and analysis was conducted while maintaining a dry nitrogen purge. The polymer sample was analyzed at a heating rate of 20 C/min from )100 to 150 C. X-ray diffraction analysis was carried out using a Routaflex RU-200 generator operating at 40 kV and 140 mA. Nickel-filtered Cu Ka radiation (k ¼ 1:54050 A) was used. An approximately 0.3 mm polymer sample was exposed to the X-rays. 2.5. Surface property To characterize the wettability of the PHA with fluorinated groups, the surface contact angle through the profile of a liquid drop (water) placed on a polymer film surface was measured by the observation-tangent method together with image analysis. Homopolymer of poly(3-hydroxyvalerate) was prepared by fermentation of Paracoccus denitrificans. Copolymer of poly(3hydroxyhexanoate) and poly(3-hydroxyoctanoate) was prepared using octanoic acid as a carbon source by P. putida. Polymer films were formed on the glass slides. Average surface contact angle values were obtained
from the duplicate measurements at 20 different sites on the polymer films.
3. Results and discussion 3.1. Cell growth Fig. 1 shows the growth curves of P. putida obtained by feeding 11-phenoxyundecanoic acid (PUDA), 11-(4fluorophenoxy)undecanoic acid (4FPUDA), 11-(2, 4-difluorophenoxy)undecanoic acid (2,4DFPUDA), 11(2,4,6-trifluorophenoxy)undecanoic acid (2,4,6TFPUDA) and 11-(2,3,4,5,6-pentaflurophenoxy)undecanoic acid (2,3,4,5,6PFPUDA), respectively. All growth curves show that the bacterium was capable of growing with unusual acids as a sole carbon source. Cells of P. putida grown with fluorine-substituted phenoxyalkanoic acids grew relatively slowly. Although cultivation at a higher carbon source concentration (3 mM/l) was attempted, cell growth was not increased due to the poor solubility of the carbon source in the medium. It was difficult to provide consistent feed to the bacteria, because carbon sources with more than two fluorine atoms floated on the medium and attached to the vessel wall during the fermentation process. The dry cell weight and polymer yield results from the P. putida grown with phenoxyundecanoic acid and various fluorinated phenoxyalkanoic acids are listed in Table 1, which shows that fluorine atoms on the aromatic ring had a negative effect on cell yields and polymer production. While the carbon source containing one or two fluorines decreased cell yield and polymer production compared to non-substituted phenoxyundecanoic acid, sources with more than 3
Optical Density at 660 nm
2.3. Polymer isolation
1553
2
1
0 0
10
20
30
40
50
60
70
Time (hr)
Fig. 1. Growth of P. putida on unusual carbon sources as determined by optical density measurement.; 11-phenoxyundecanoic acid (5 mM/l); 11-(4-fluorophenoxy)undecanoic acid (3 mM/l); O 11-(2,4-difluorophenoxy)undecanoic acid (3 mM/l); r 11-(2,4,6-trifluorophenoxy)undecanoic acid (3 mM/l); 11-(2,3,4,5,6-pentafluorophenoxy)undecanoic acid (3 mM/l).
1554
Y. Takagi et al. / European Polymer Journal 40 (2004) 1551–1557
Table 1 Cell growth and PHA production for P. putida grown with several carbon sources Carbon source
Dry cell (mg/l)
PHA (mg/l)
PHA content (%)
11-Phenoxyundecanoic acid 11-(2-Fluorophenoxy)undecanoic acid 11-(3-Fluorophenoxy)undecanoic acid 11-(4-Fluorophenoxy)undecanoic acid 11-(2,4-Difluorophenoxy)undecanoic acid 11-(2,4,6-Trifluorophenoxy)undecanoic acid 11-(2,3,4,5,6-Pentafluorophenoxy)undecanoic acid
230 46 53 62 59 53 41
32 3 4 6 5 n.d. n.d.
13.9 6.5 7.5 9.7 8.5 – –
three fluorines produced no polymeric materials when P. putida 27N01 was used for polymer production, suggesting that a limit of polymer production exists between two and three fluorine-substituted carbon sources. 3.2. Characterization of PHAs The 13 C-NMR spectrum of the polymer obtained from the 11-(3-fluorophenoxy)undecanoic acid (3FPUDA) is presented in Fig. 2(a). For a complete assignment of the NMR signals, this polymer was methanolized and the resulting methyl 3-hydroxyalkanoates were identified by GC-MS spectrometry [7]. Two peaks with different retention times in the gas chromatogram and characteristic m/z peaks in mass spectroscopy supported the suggestion that the polymer contained 3-hydroxy-7-(3fluorophenoxy)heptanoate (10.5%) and 3-hydroxy-5-(3fluoropheoxy)pentanoate (89.5%). Similarly, the peak in Fig. 2(b) was assigned to the polymer containing 3hydroxy-7-(4-fluorophenoxy)heptanoate (8.7%) and 3hydroxy-5-(4-fluoropheoxy)pentanoate (91.3%). When 2,4DFPUDA was used as the sole carbon source, poly(3-hydroxy-5-(2,4-difluorophenoxy)pentanoate) was produced. The 13 C-NMR spectrum of this polymer is shown in Fig. 2(c). The molecular weights of PHAs isolated from P. putida were measured by GPC, and their results are presented in Table 2. In the case of PHAs with fluorinated substituent groups, the molecular weights were about 10,000. The lower molecular weight compared to non-substituted PHAs can be explained by the reduced supply of monomer CoA for polymer synthase or by steric hindrance of the side groups during polymerization. 3.3. Thermal properties of PHA Generally, fluoropolymers can be expected to possess higher thermal resistance. The influence of fluorine atom incorporation on polymer thermal transitions was investigated in PHA from PUDA, 4FPUDA, and 2,4DFPUDA by DSC. As shown in Fig. 3, the DSC thermograms of the polymers showed different thermal behaviour. While PHA from PUDA did not show any clear crystalline melting peak (a), PHAs with a fluori-
nated side group exhibited melting point transitions at 52 C (monofluorinated) (b) and 102 C (difluorinated) (c). Incorporation of fluorine atoms into side group clearly contributes to the improvement of the thermal properties. To our knowledge, this is the first report of a 100 C melting point for a PHA with an unusual functional group. 3.4. X-ray analyses The PHA produced from 2,4DFPUDA was cream colored and brittle at room temperature. The characteristics of this PHA are quite different from those of the PHAs produced by Pseudomonas species capable of growing on unusual carbon sources. WAXS diffraction patterns of this PHA and the PHA from PUDA are shown in Fig. 4. The difluorinated PHA (a) produced sharp peaks, but non-fluorinated PHA (b) showed no peaks, indicating that fluorophenoxy PHA (a) was crystalline and phenoxy PHA (b) was nearly amorphous. These results are in agreement with those obtained by DSC, indicating that fluoro-PHA possesses crystalline melting behaviour. 3.5. Surface property Surface contact angles were measured using the tangent water drop method together with image analysis to investigate the surface properties of PHAs. PHAs with a phenoxy or alkyl group (3 and 5 carbons) in the side chain show surface contact angles of approximately 50, while those of the crystalline-like poly(3-hydroxyvalerate) P(3HV) film gave a surface contact angle of 92 and the difluorinated PHA gave an angle of 104. Therefore, fluorine incorporation into PHA influences the surface characteristics, suggesting that the chemical composition is related to the surface properties resulting in P(3HV) with slightly superior properties. The surface contact angle of commercial fluorine-containing plastics such as poly(tetrafluoroethylene) is 108. In general, a surface contact angle of over 100 is sufficient to allow utilization of materials as non-wetting materials. In fact, a drop of water on the surface of PHA produced from 2,4DFPUDA was actually repelled.
Y. Takagi et al. / European Polymer Journal 40 (2004) 1551–1557
1555
Fig. 2. 13 C-NMR spectra of the polymers produced by P. putida grown with different carbon sources. (a) Polymer from 11-(3-fluorophenoxy)undecanoic acid, (b) polymer from 11-(4-fluorophenoxy)undecanoic acid and (c) polymer from 11-(2,4-difluorophenoxy)undecanoic acid.
4. Conclusion P. putida 27N01 was able to accumulate PHAs containing fluorine atoms in all repeating units from carbon
sources of fluorine-substituted phenoxyalkanoic acids. According to previous investigations, PHAs with halogen atoms have been produced from a mixture of halogenated and non-halogenated carbon sources, but few
1556
Y. Takagi et al. / European Polymer Journal 40 (2004) 1551–1557
Table 2 Molecular weights of PHAs with substituted side groups obtained from P. putida Carbon source
Mn (g/mol)
Mw/Mn
11-Phenoxyundecanoic acid 11-(4-Fluorophenoxy) undecanoic acid 11-(2,4-Difluorophenoxy) undecanoic acid
104,000 11,000
1.6 1.9
13,000
2.8
PHAs from bacterial production using sole fluorinated carbon sources such as fluoroalkanoic acid were reported. The fluorinated aromatic group improved the proportion of the fluorinated PHAs in the cells, resulting in almost completely fluorinated PHAs. The molecular weights of these PHAs were lower than those of PHAs from fatty acids or phenoxyundecanoic acid. However, PHAs with fluorinated substituent groups possessed more ideal physical properties compared to those with non-substituted polyesters.
Acknowledgements The authors would like to thank Mr. M. Hashii and M. Asahi for their help in the analysis of polymer composition, Dr. M. Oda and Y. Ishigaki for their help with the NMR spectroscopy and gel permeation chromatography, and Dr. K. Ito for his help with the DSC measurements.
References Fig. 3. DSC thermograms of the polymers produced by P. putida grown with several carbon sources. (a) Polymer from 11-phenoxyundecanoic acid, (b) polymer from 11-(4-fluorophenoxy)undecanoic acid and (c) polymer from 11-(2,4-dirluorophenoxy)undecanoic acid.
Cu-Kα Intensity ( arb. unit )
Al plate
(a) (b) 10
20
30 40 2θ /deg
50
Fig. 4. X-ray diffraction patterns of PHAs. (a) Polymer from 11-(2,4-difluorophenoxy)undecanoic acid and (b) polymer from 11-phenoxyundecanoic acid.
[1] Akita S, Einaga Y, Miyaki Y, Fujita H. Macromolecules 1976;9:774. [2] Alper R, Lundgren DG, Marchessault RH, Cote WA. Biopolymers 1963;1:545. [3] Gross RA, Konrad G, Zhang Y, Lenz RW. Polym Prepr (Am Chem Soc, Div Polym Chem) 1987;28:373. [4] Holmes PA. Phys Technol 1985;16:32. [5] Byrom D. Trends Biotechnol 1987;5:246. [6] Steinb€ uchel A, Valentin HE. FEMS Microbiol Lett 1995; 128:219. [7] Lageveen RG, Huisman GW, Preusting H, Ketelaar P, Eggink G, Witholt B. Appl Environ Microbiol 1988;54: 2924. [8] Fritzsche K, Lenz RW, Fuller RC. Int J Biol Macromol 1990;12:85. [9] Kim YB, Lenz RW, Fuller RC. J Polym Sci Part A Polym Chem 1995;33:1367. [10] Doi Y, Abe C. Macromolecules 1990;23:3705. [11] Fritzsche K, Lenz RW, Fuller RC. Int J Biol Macromol 1990;12:92. [12] Fritzsche K, Lenz RW, Fuller RC. Macromol Chem 1990;191:1957. [13] Ritter H, Grafin Von Spee A. Macromol Chem Phys 1994; 195:1665. [14] Kim YB, Rhee YH, Han SH, Heo GS, Kim JS. Macromolecules 1996;29:5256. [15] Song JJ, Yoon SC. Appl Environ Microbiol 1996;62: 536. [16] Andujar M, Aponte MA, Diaz E, Schroder E. Macromolecules 1997;30:1611. [17] Abe C, Taima Y, Nakamura Y, Doi Y. Polym Commun 1990;31:404.
Y. Takagi et al. / European Polymer Journal 40 (2004) 1551–1557 [18] Kim O, Gross RA, Hammar WJ, Newmark RA. Macromolecules 1996;29:4572. [19] H€ opken J, Sheiko S, Czech J, M€ oller M. Polym Prepr (Am Chem Soc, Div Polym Chem) 1992;33:937.
1557
[20] Shimizu T, Tanaka Y, Kutsumizu S. Macromol Symp 1994;82:173. [21] Kim YB, Lenz RW, Fuller RC. Macromolecules 1992;25: 1852.
EUROPEAN POLYMER JOURNAL www.elsevier.com/locate/europolj
Microbial synthesis and characterization of polyhydroxyalkanoates with fluorinated phenoxy side groups from Pseudomonas putida Yasuo Takagi a
a,*
, Ryou Yasuda a, Akira Maehara b, Tsuneo Yamane
b
Department of Environmental Materials, Nagoya Municipal Industrial Research Institute, 3-4-41 Rokuban, Atuta-ku, Nagoya 456-0058, Japan b Laboratory of Molecular Biotechnology, Department of Biological Mechanisms and Functions, Graduate School of Bio- and Agro-Science, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan Received 29 July 2003; received in revised form 22 January 2004; accepted 22 January 2004 Available online 16 March 2004
Abstract Microbial poly(3-hydroxyalkanoates) (PHAs) with fluorinated phenoxy side groups were produced by Pseudomonas putida when fluorophenoxyalkanoic acids were used as carbon sources. 11-(2-Fluorophenoxy)undecanoic acid (2FPUDA), 11-(3-fluorophenoxy)undecanoic acid (3FPUDA), 11-(4-fluorophenoxy)undecanoic acid (4FPUDA), 11(2,4-difluorophenoxy)undecanoic acid (2,4DFPUDA), 11-(2,4,6-trifluorophenoxy)undecanoic acid (2,4,6TFPUDA), and 11-(2,3,4,5,6-pentaflurophenoxy)undecanoic acid (2,3,4,5,6PFPUDA) were used as carbon sources in the present study. When cells were grown with 2,4DFPUDA, the production of homo poly(3-hydroxy-5-(2,4-difluorophenoxy)pentanoate) was confirmed by NMR and GC/MS analyses. Fluorine atoms inserted into the side chain of the PHA dramatically affected its physical properties. In marked contrast to medium chain length (MCL) PHA, this fluorinated PHA was opaque, cream colored, and possessed greater crystallinity and a higher melting point (100 C) than did the other MCL PHAs. Surface contact angle evaluation revealed that the PHA with two fluorine atoms possessed water-shedding properties. The number of substituted fluorine atoms in the carbon source affected cell growth and difluorine-substituted phenoxyalkanoic acids reduced cell growth, and polymer production compared to non-substituted phenoxyalkanoic acids. No polymeric materials were obtained using either 2,4,6TFPUDA or 2,3,4,5,6PFPUDA. 2004 Elsevier Ltd. All rights reserved. Keywords: Polyhydroxyalkanoate; Bacteria; Biodegradable plastic
1. Introduction Poly(3-hydroxyalkanoates) (PHAs) are bacterial storage compounds that have found use as biodegradable and biocompatible thermoplastic polymers. Microbial PHAs have a common structure containing chiral centers yielding polymers that are isotactic and
* Corresponding author. Tel.: +81-52-654-9890; fax: +81-52652-6776/654-9895. E-mail address: [email protected] (Y. Takagi).
optically active [1–3]. Several approaches to increase the utilization of PHAs have been investigated in the areas of agricultural, marine, and medical materials [4,5]. The physical and mechanical properties of PHAs are dependent on the compound’s monomeric composition, and, at present, more than 100 different PHAs have been produced and characterized [6]. Most of the PHAs with unusual groups have been produced from either Pseudomonas oleovorans or Pseudomonas putida when unusual carbon sources were supplied during fermentation. In particular, production of PHAs with unusual groups produced by P. oleovorans has been investigated extensively [7–10]. In addition to PHAs with long alkyl
0014-3057/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2004.01.030
1552
Y. Takagi et al. / European Polymer Journal 40 (2004) 1551–1557
groups, P. oleovorans has produced PHAs with functional groups such as an olefin [7–9], branched alkyl [11], halogen [10], phenyl [12], phenoxy [13–15], and cyclohexyl [16]. PHAs, with fluorinated side chains have been reported by several researchers. PHA containing 27 mol% of 3-hydroxy-9-fluorononanoate and 3-hydroxy-7fluoronoheptanoate was produced when P. oleovorans was cultivated on a mixture of nonane and 1-fluorononane [17]. Compared to PHA produced from nonanoic acid (a non-halogenated carbon source), the melting temperature (Tm) of this fluoro-PHA was increased from 47 to 61 C, and its glass transition temperature (Tg) increased from )44 to )39 C. Kim et al. [18] investigated PHA with fluorine atoms produced by P. oleovorans and P. putida grown with a mixture of multifluorinated alkanoic acid and nonanoic acid, which yielded copolymers containing up to 17.3 mol% nonafluoro 3-hydroxynonanoate. Fully or partially fluorinated polymers showed unique physical properties with respect to thermal, oxidative, and photochemical stability [19,20]. In addition, PHAs with monohalogenated side groups of fluorine, bromine, and chlorine have been produced in P. oleovorans from corresponding alkanes or alkanoic acids, resulting in copolymers containing a halogenated side group and an alkyl group due to the use of a mixture of halogenated carbon source and alkanoic acid [10,17,18,21]. A higher content of halogenated side groups significantly improves the physical properties of the PHA. To our knowledge, few reports have been published about PHAs with a high halogen content produced from bacteria. In this study, we produced PHAs with fluorinated side groups and investigated their properties.
2. Experimental 2.1. Materials The same general synthetic procedure was used for preparation of 11-phenoxyundecanoic acid (PUDA), 11-(4-fluorophenoxy)undecanoic acid (4FPUDA), 11(3-fluorophenoxy)undecanoic acid (3FPUDA), 11-(2fluorophenoxy)undecanoic acid (2FPUDA), 11-(2, 4-difluorophenoxy)undecanoic acid (2,4DFPUDA), 11(2,4,6-trifluorophenoxy)undecanoic acid (2,4,6TFPUDA), and 11-(2,3,4,5,6-pentaflurophenoxy)undecanoic acid (2,3,4,5,6PFPUDA). Potassium hydroxide (0.11 mol) was added to 0.07 mol of phenol (Aldrich Chemical Co., 99%), 4-flurophenol (Aldrich Chemical Co., 99%), 3-fluorophenol (Aldrich Chemical Co., 98%), 2-fluorophenol (Aldrich Chemical Co., 98%), 2,4-difluorophenol, (Aldrich Chemical Co., 99%), 2,4,6-trifluorophenol (Aldrich Chemical Co., 99%), or pentafluorophenol (Aldrich Chemical Co., 99%) in a 500 ml round bottom
flask containing 200 ml of ethanol. To this mixture, 0.06 mol of 11-bromoundecanoic acid (Aldrich Chemical Co., 99%) was added. The reaction was allowed to continue at the reflux temperature of ethanol for 24 h. The corresponding acids were precipitated by adding the reaction solution to 200 ml of 0.1 M aqueous HCl. This solution was then filtered, dried in vacuo, and purified by recrystallization from hot methanol. The yields of the purified acids ranged from 20% to 90%. The structures of the products were confirmed by NMR spectroscopy.
2.2. Cell growth A culture of P. putida 27N01 was used throughout the study. This strain was isolated from soil and identified by National Collections of Industrial and Marine Bacteria Limited (NCIMB) in Scotland. P. putida was precultivated under aerobic conditions at 30 C and pH 7.0 for 48 h on a reciprocating shaker in 10 ml of medium containing defined mineral salts and 20 mM octanoic acid (OA) as a carbon substrate. Composition of the mineral salt medium (per liter) was 1.5 g (NH4 )2 SO4 , 1.4 g KH2 PO4 , 1.4 g Na2 HPO4 , 0.5 g NaHCO3 , 0.3 g MgSO4 Æ 7H2 O, 0.3 g yeast extract (Difco), and 0.05 g ammonium iron (III) citrate. In addition, 0.1 ml of trace element solution was added to the medium. The trace element solution contained the following salts (per liter): 3.0 g H3 BO4 , 2.0 g CoCl2 Æ 6H2 O, 1.0 g ZnSO4 Æ 7H2 O, 0.3 g MnCl2 Æ 4H2 O, 0.2 g NaMoO4 Æ 2H2 O, 0.28 g NiSO4 Æ 7H2 O, and 0.1 g CuSO4 Æ 5H2 O. After precultivation, 1 ml of the cells was inoculated with modified medium containing OA, PUDA, 4FPUDA, 3FPUDA, 2FPUDA, 2,4DFPUDA, 2,4,6TFPUDA, or 2,3,4,5,6PFPUDA as the carbon source. The first cultivation was conducted in a 500 ml flask containing 100 ml of medium under the same conditions as the preculture for up to two weeks. Thereafter, the cells were transferred and grown for one week in a 5 l jar-fermenter (model KMJ-5C; Mitsuwa Bio Systems Co., Osaka, Japan) with a culture volume of 3 l, containing the same medium composition. In the fermenter, agitation speed, aeration rate, and temperature were set at 500 rpm, 0.7 vol vol1 min1 , and 30 C, respectively. The pH of the culture was adjusted by addition of 3 N KOH or 2 N HCl using a pH controller. Antifoam (Adecanol LG-109; Asahi Denka Kogyo Co., Tokyo, Japan) was added to the medium at an initial concentration of 0.03% (v/v). Direct monitoring of cell growth by measuring optical density was not possible due to emulsified liquid particles of undissolved carbon sources. Therefore, the mixture was filtered through cellulose filter paper (Toyo Roshi Kaisya, Ltd., porosity no. 2) to remove the particles and was then monitored daily at 660 nm to obtain cell growth curves.
Y. Takagi et al. / European Polymer Journal 40 (2004) 1551–1557
The cells were harvested by centrifugation, washed with 30% methanol solution in water, dried and weighed. The dried cells were suspended in 100 ml chloroform at room temperature for 24 h. The mixture was filtered through a cellulose filter paper, and the filtrate was concentrated by evaporation. The polymer was reprecipitated in methanol and dried in order to determine PHA yield. 2.4. Analyses To identify the composition of the polymer by GCMS, samples were prepared as described previously [15]. Methyl esters of PHA monomers were analyzed by a Hewlett Packard 5890 system equipped with a J&W SCIENTIFIC capillary column (30 mm · 0.25 mm) and a total ion detector. The 1 H and 13 C nuclear magnetic resonance (NMR) spectra of PHA samples were recorded on a JEOL JNM-EX 400 FT-NMR spectrometer. The 400 MHz 1 H-NMR and the 100 MHz 13 C-NMR spectra of the polymers were recorded at 25 C in CDCl3 . The average molecular mass and molecular mass distributions of the polymer were obtained at 30 C by gel permeation chromatography (Shodex GPC system 11; Showa Denko K.K., Tokyo, Japan) using AC-800P, AC-80M, and K-806M sample columns. Chloroform was used as an eluent at a flow rate of 1.0 ml min1 . Polystyrene standards having a low polydispersity rate were used to produce the calibration curve. Thermal characterization was carried out using a MacScience DSC-3100S differential scanning calorimeter (DSC) equipped with an MTC-1000S data station. The sample was sealed in an aluminum pan, and analysis was conducted while maintaining a dry nitrogen purge. The polymer sample was analyzed at a heating rate of 20 C/min from )100 to 150 C. X-ray diffraction analysis was carried out using a Routaflex RU-200 generator operating at 40 kV and 140 mA. Nickel-filtered Cu Ka radiation (k ¼ 1:54050 A) was used. An approximately 0.3 mm polymer sample was exposed to the X-rays. 2.5. Surface property To characterize the wettability of the PHA with fluorinated groups, the surface contact angle through the profile of a liquid drop (water) placed on a polymer film surface was measured by the observation-tangent method together with image analysis. Homopolymer of poly(3-hydroxyvalerate) was prepared by fermentation of Paracoccus denitrificans. Copolymer of poly(3hydroxyhexanoate) and poly(3-hydroxyoctanoate) was prepared using octanoic acid as a carbon source by P. putida. Polymer films were formed on the glass slides. Average surface contact angle values were obtained
from the duplicate measurements at 20 different sites on the polymer films.
3. Results and discussion 3.1. Cell growth Fig. 1 shows the growth curves of P. putida obtained by feeding 11-phenoxyundecanoic acid (PUDA), 11-(4fluorophenoxy)undecanoic acid (4FPUDA), 11-(2, 4-difluorophenoxy)undecanoic acid (2,4DFPUDA), 11(2,4,6-trifluorophenoxy)undecanoic acid (2,4,6TFPUDA) and 11-(2,3,4,5,6-pentaflurophenoxy)undecanoic acid (2,3,4,5,6PFPUDA), respectively. All growth curves show that the bacterium was capable of growing with unusual acids as a sole carbon source. Cells of P. putida grown with fluorine-substituted phenoxyalkanoic acids grew relatively slowly. Although cultivation at a higher carbon source concentration (3 mM/l) was attempted, cell growth was not increased due to the poor solubility of the carbon source in the medium. It was difficult to provide consistent feed to the bacteria, because carbon sources with more than two fluorine atoms floated on the medium and attached to the vessel wall during the fermentation process. The dry cell weight and polymer yield results from the P. putida grown with phenoxyundecanoic acid and various fluorinated phenoxyalkanoic acids are listed in Table 1, which shows that fluorine atoms on the aromatic ring had a negative effect on cell yields and polymer production. While the carbon source containing one or two fluorines decreased cell yield and polymer production compared to non-substituted phenoxyundecanoic acid, sources with more than 3
Optical Density at 660 nm
2.3. Polymer isolation
1553
2
1
0 0
10
20
30
40
50
60
70
Time (hr)
Fig. 1. Growth of P. putida on unusual carbon sources as determined by optical density measurement.; 11-phenoxyundecanoic acid (5 mM/l); 11-(4-fluorophenoxy)undecanoic acid (3 mM/l); O 11-(2,4-difluorophenoxy)undecanoic acid (3 mM/l); r 11-(2,4,6-trifluorophenoxy)undecanoic acid (3 mM/l); 11-(2,3,4,5,6-pentafluorophenoxy)undecanoic acid (3 mM/l).
1554
Y. Takagi et al. / European Polymer Journal 40 (2004) 1551–1557
Table 1 Cell growth and PHA production for P. putida grown with several carbon sources Carbon source
Dry cell (mg/l)
PHA (mg/l)
PHA content (%)
11-Phenoxyundecanoic acid 11-(2-Fluorophenoxy)undecanoic acid 11-(3-Fluorophenoxy)undecanoic acid 11-(4-Fluorophenoxy)undecanoic acid 11-(2,4-Difluorophenoxy)undecanoic acid 11-(2,4,6-Trifluorophenoxy)undecanoic acid 11-(2,3,4,5,6-Pentafluorophenoxy)undecanoic acid
230 46 53 62 59 53 41
32 3 4 6 5 n.d. n.d.
13.9 6.5 7.5 9.7 8.5 – –
three fluorines produced no polymeric materials when P. putida 27N01 was used for polymer production, suggesting that a limit of polymer production exists between two and three fluorine-substituted carbon sources. 3.2. Characterization of PHAs The 13 C-NMR spectrum of the polymer obtained from the 11-(3-fluorophenoxy)undecanoic acid (3FPUDA) is presented in Fig. 2(a). For a complete assignment of the NMR signals, this polymer was methanolized and the resulting methyl 3-hydroxyalkanoates were identified by GC-MS spectrometry [7]. Two peaks with different retention times in the gas chromatogram and characteristic m/z peaks in mass spectroscopy supported the suggestion that the polymer contained 3-hydroxy-7-(3fluorophenoxy)heptanoate (10.5%) and 3-hydroxy-5-(3fluoropheoxy)pentanoate (89.5%). Similarly, the peak in Fig. 2(b) was assigned to the polymer containing 3hydroxy-7-(4-fluorophenoxy)heptanoate (8.7%) and 3hydroxy-5-(4-fluoropheoxy)pentanoate (91.3%). When 2,4DFPUDA was used as the sole carbon source, poly(3-hydroxy-5-(2,4-difluorophenoxy)pentanoate) was produced. The 13 C-NMR spectrum of this polymer is shown in Fig. 2(c). The molecular weights of PHAs isolated from P. putida were measured by GPC, and their results are presented in Table 2. In the case of PHAs with fluorinated substituent groups, the molecular weights were about 10,000. The lower molecular weight compared to non-substituted PHAs can be explained by the reduced supply of monomer CoA for polymer synthase or by steric hindrance of the side groups during polymerization. 3.3. Thermal properties of PHA Generally, fluoropolymers can be expected to possess higher thermal resistance. The influence of fluorine atom incorporation on polymer thermal transitions was investigated in PHA from PUDA, 4FPUDA, and 2,4DFPUDA by DSC. As shown in Fig. 3, the DSC thermograms of the polymers showed different thermal behaviour. While PHA from PUDA did not show any clear crystalline melting peak (a), PHAs with a fluori-
nated side group exhibited melting point transitions at 52 C (monofluorinated) (b) and 102 C (difluorinated) (c). Incorporation of fluorine atoms into side group clearly contributes to the improvement of the thermal properties. To our knowledge, this is the first report of a 100 C melting point for a PHA with an unusual functional group. 3.4. X-ray analyses The PHA produced from 2,4DFPUDA was cream colored and brittle at room temperature. The characteristics of this PHA are quite different from those of the PHAs produced by Pseudomonas species capable of growing on unusual carbon sources. WAXS diffraction patterns of this PHA and the PHA from PUDA are shown in Fig. 4. The difluorinated PHA (a) produced sharp peaks, but non-fluorinated PHA (b) showed no peaks, indicating that fluorophenoxy PHA (a) was crystalline and phenoxy PHA (b) was nearly amorphous. These results are in agreement with those obtained by DSC, indicating that fluoro-PHA possesses crystalline melting behaviour. 3.5. Surface property Surface contact angles were measured using the tangent water drop method together with image analysis to investigate the surface properties of PHAs. PHAs with a phenoxy or alkyl group (3 and 5 carbons) in the side chain show surface contact angles of approximately 50, while those of the crystalline-like poly(3-hydroxyvalerate) P(3HV) film gave a surface contact angle of 92 and the difluorinated PHA gave an angle of 104. Therefore, fluorine incorporation into PHA influences the surface characteristics, suggesting that the chemical composition is related to the surface properties resulting in P(3HV) with slightly superior properties. The surface contact angle of commercial fluorine-containing plastics such as poly(tetrafluoroethylene) is 108. In general, a surface contact angle of over 100 is sufficient to allow utilization of materials as non-wetting materials. In fact, a drop of water on the surface of PHA produced from 2,4DFPUDA was actually repelled.
Y. Takagi et al. / European Polymer Journal 40 (2004) 1551–1557
1555
Fig. 2. 13 C-NMR spectra of the polymers produced by P. putida grown with different carbon sources. (a) Polymer from 11-(3-fluorophenoxy)undecanoic acid, (b) polymer from 11-(4-fluorophenoxy)undecanoic acid and (c) polymer from 11-(2,4-difluorophenoxy)undecanoic acid.
4. Conclusion P. putida 27N01 was able to accumulate PHAs containing fluorine atoms in all repeating units from carbon
sources of fluorine-substituted phenoxyalkanoic acids. According to previous investigations, PHAs with halogen atoms have been produced from a mixture of halogenated and non-halogenated carbon sources, but few
1556
Y. Takagi et al. / European Polymer Journal 40 (2004) 1551–1557
Table 2 Molecular weights of PHAs with substituted side groups obtained from P. putida Carbon source
Mn (g/mol)
Mw/Mn
11-Phenoxyundecanoic acid 11-(4-Fluorophenoxy) undecanoic acid 11-(2,4-Difluorophenoxy) undecanoic acid
104,000 11,000
1.6 1.9
13,000
2.8
PHAs from bacterial production using sole fluorinated carbon sources such as fluoroalkanoic acid were reported. The fluorinated aromatic group improved the proportion of the fluorinated PHAs in the cells, resulting in almost completely fluorinated PHAs. The molecular weights of these PHAs were lower than those of PHAs from fatty acids or phenoxyundecanoic acid. However, PHAs with fluorinated substituent groups possessed more ideal physical properties compared to those with non-substituted polyesters.
Acknowledgements The authors would like to thank Mr. M. Hashii and M. Asahi for their help in the analysis of polymer composition, Dr. M. Oda and Y. Ishigaki for their help with the NMR spectroscopy and gel permeation chromatography, and Dr. K. Ito for his help with the DSC measurements.
References Fig. 3. DSC thermograms of the polymers produced by P. putida grown with several carbon sources. (a) Polymer from 11-phenoxyundecanoic acid, (b) polymer from 11-(4-fluorophenoxy)undecanoic acid and (c) polymer from 11-(2,4-dirluorophenoxy)undecanoic acid.
Cu-Kα Intensity ( arb. unit )
Al plate
(a) (b) 10
20
30 40 2θ /deg
50
Fig. 4. X-ray diffraction patterns of PHAs. (a) Polymer from 11-(2,4-difluorophenoxy)undecanoic acid and (b) polymer from 11-phenoxyundecanoic acid.
[1] Akita S, Einaga Y, Miyaki Y, Fujita H. Macromolecules 1976;9:774. [2] Alper R, Lundgren DG, Marchessault RH, Cote WA. Biopolymers 1963;1:545. [3] Gross RA, Konrad G, Zhang Y, Lenz RW. Polym Prepr (Am Chem Soc, Div Polym Chem) 1987;28:373. [4] Holmes PA. Phys Technol 1985;16:32. [5] Byrom D. Trends Biotechnol 1987;5:246. [6] Steinb€ uchel A, Valentin HE. FEMS Microbiol Lett 1995; 128:219. [7] Lageveen RG, Huisman GW, Preusting H, Ketelaar P, Eggink G, Witholt B. Appl Environ Microbiol 1988;54: 2924. [8] Fritzsche K, Lenz RW, Fuller RC. Int J Biol Macromol 1990;12:85. [9] Kim YB, Lenz RW, Fuller RC. J Polym Sci Part A Polym Chem 1995;33:1367. [10] Doi Y, Abe C. Macromolecules 1990;23:3705. [11] Fritzsche K, Lenz RW, Fuller RC. Int J Biol Macromol 1990;12:92. [12] Fritzsche K, Lenz RW, Fuller RC. Macromol Chem 1990;191:1957. [13] Ritter H, Grafin Von Spee A. Macromol Chem Phys 1994; 195:1665. [14] Kim YB, Rhee YH, Han SH, Heo GS, Kim JS. Macromolecules 1996;29:5256. [15] Song JJ, Yoon SC. Appl Environ Microbiol 1996;62: 536. [16] Andujar M, Aponte MA, Diaz E, Schroder E. Macromolecules 1997;30:1611. [17] Abe C, Taima Y, Nakamura Y, Doi Y. Polym Commun 1990;31:404.
Y. Takagi et al. / European Polymer Journal 40 (2004) 1551–1557 [18] Kim O, Gross RA, Hammar WJ, Newmark RA. Macromolecules 1996;29:4572. [19] H€ opken J, Sheiko S, Czech J, M€ oller M. Polym Prepr (Am Chem Soc, Div Polym Chem) 1992;33:937.
1557
[20] Shimizu T, Tanaka Y, Kutsumizu S. Macromol Symp 1994;82:173. [21] Kim YB, Lenz RW, Fuller RC. Macromolecules 1992;25: 1852.