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Biosynthesis of polyhydroxyalkanoate copolyester containing cyclohexyl groups by Pseudomonas oleovorans
International Journal of Biological Macromolecules 29 (2001) 145– 150 www.elsevier.com/locate/ijbiomac
Biosynthesis of polyhydroxyalkanoate copolyester containing cyclohexyl groups by Pseudomonas oleo6orans Do Young Kim a, Saet Byel Jung a, Gang Guk Choi a, Young Baek Kim b, Young Ha Rhee a,* a
Department of Microbiology, Chungnam National Uni6ersity, Daejon 305 -764, South Korea b Department of Polymer Engineering, PaiChai Uni6ersity, Daejon 302 -735, South Korea Received 11 August 2000; accepted 8 April 2001
Abstract Production of polyhydroxyalkanoates (PHAs) substituted with cyclohexyl groups by Pseudomonas oleo6orans grown with 4-cyclohexylbutyric acid (4-CHB) and its mixtures with nonanoic acid (NA) was investigated. Addition of NA to medium gave rise to an increase in the total concentration of 3-hydroxy-4-cyclohexylbutyrate repeating unit in the PHAs, indicating that the bioconversion rate of 4-CHB to polyester was significantly improved by the cometabolic effect. Increasing the proportion of NA from 1.0 to 7.5 mM at a concentration of 10 mM total carbon substrate also accelerated the uptake speed of 4-CHB by the organism and resulted in an increase of the ratio of 3-hydroxynonanoate to 3-hydroxyheptanoate from 1.28 to 2.05. Differential scanning calorimetric analysis of the PHAs bearing the corresponding functional groups showed one melting transition and one glass transition temperature varying according to the composition. These results indicated that random copolyesters were obtained from the carbon substrates used in this study. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Copolyester; Cyclohexyl substituent; Polyhydroxyalkanoate; Pseudomonas oleo6orans
1. Introduction Medium-chain-length polyhydroxyalkanoates (MCLPHAs) that consist of constituents having a chain length of typically C6 – C12 are produced by a wide variety of Gram-negative bacteria, mainly pseudomonads. Until now, more than 100 different hydroxyalkanoates have been incorporated into MCL-PHAs as constituents of biosynthetic PHA [1,2]. Of the microorganisms capable of producing MCL-PHAs Pseudomonas oleo6orans has been investigated most extensively. This organism synthesizes MCL-PHAs containing various functional groups in the side chains when grown with carbon substrates bearing corresponding chemical structures [3 – 13]. Although P. putida has been also reported to be useful for the production of functional MCL-PHAs [9,11,13,14], recent results in our laboratory have demonstrated that * Corresponding author. Tel.: + 82-42-8216413; fax: +82-428227367. E-mail address: [email protected] (Y.H. Rhee).
P. putida cannot produce all of the PHA containing functional substituents produced by P. oleo6orans [15]. PHAs containing functional groups are of great interest, because these groups can improve the physical properties of MCL-PHAs. Moreover, some functional substituents can be modified by chemical reactions in order to obtain more useful groups that can extend the potential application of PHAs as environmentally biodegradable polymers and functional biomaterials for use in biomedical applications [16,17]. When P. oleo6orans is grown on mixtures of octanoic acid (OA) or nonanoic acid (NA) with alkanoic acids containing non-aromatic functional groups such as bromine [5], fluorine [9], olefin [6], or carbon –carbon triple bonds [11], the bacterium produces random copolyesters containing monomer units derived from both substrates. On the other hand, P. oleo6orans grown on a mixture of OA or NA with alkanoic acid containing aromatic groups such as phenyl [4], methylphenyl [8], nitrophenyl [12], or methylphenoxy group [13] produces mixtures of more than one copolyester with significantly different compositions. The mixtures in general
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are composed of copolyesters containing a larger concentration of aromatic repeating units and copolyesters containing a small amount of aromatic repeating units. Recently, polyestyers containing cyclohexyl groups were synthesized by P. oleo6orans when it was grown with mixtures of either 5-cyclohexylvaleric acid (5CHV) or 4-cyclohexylbutyric acid (4-CHB) and NA [10]. Interestingly, thermal analysis revealed that the polyesters were not copolyesters, but mixtures of two PHAs with significantly different compositions: one containing more cyclohexyl groups than the other. These results were rather unusual because the formation of mixtures of different copolyesters by P. oleo6orans has only been observed in co-feeding experiments using mixtures of OA or NA with aromatic alkanoic acids. In the present study, to understand the PHA biosynthesis patterns of P. oleo6orans grown with mixtures of 4-CHB and NA, the growth behavior of the organism on the substrates and the fermentation kinetics for PHA production were examined. Of considerable interest was to determine whether PHAs so produced were random copolyesters or mixtures of PHAs with different compositions.
2. Materials and methods
extracted crude PHA was purified by repeated precipitation by dropping into vigorously stirred cold methanol to obtain the fine product and this precipitation procedure was carried out at least three times.
2.3. Analytical methods Using gas chromatography (GC, Hewlett-Packard 5890 gas chromatograph equipped with a flame ionization detector and a HP-1 capillary column), the relative concentration of the PHA monomeric units synthesized were calculated by integrating the methyl-ester peaks from methanolyzed samples. The oven temperature was initially maintained at 80°C for 4 min and then raised at a rate of 10°C/min to 230°C. Identification of the PHA monomeric units was carried out by either gas chromatography/mass spectroscopy (GC/MS) or 1H nuclear magnetic resonance (NMR) spectroscopy. GC/ MS analysis was carried out using a Hewlett-Packard 5988 GC/MS system and NMR spectroscopy was performed using a Bruker 500 NMR spectrometer as described previously [5]. Differential scanning calorimetry (DSC) analysis was carried out using a Perkin-Elmer DSC 7. The temperature was scanned from −100 to 150°C at a ramp 20°C/min. The analysis of residual carbon substrates in the medium was done as previously described [11].
2.1. Culture of bacterium 3. Results and discussion P. oleo6orans ATCC 29347 was grown on a basal medium as described elsewhere [6]. The concentration of two carbon substrates (4-CHB and NA) was changed to evaluate their effect on growth and PHA biosynthesis. The total concentration of carboxylic acids in the medium was 10 mM. Shake flask cultures were carried out aerobically in 500 ml Erlenmeyer flasks containing 100 ml of the basal medium. Batch fermentations were conducted in a 5 l jar fermentor (Korea Fermentor Co. Ltd.) with a working volume of 3 l. The medium was inoculated with a 3% (v/v) inoculum of an overnight culture in the basal medium containing 10 mM NA as the sole carbon source. The temperature and pH were automatically controlled at optimal values of 30°C and 7.0, respectively. The airflow rate was 0.25 vvm and agitation speed was 250 rpm. Cell growth was monitored spectrophotometrically at 660 nm. Cell cultivation was stopped approximately 2 h after the growth reached the stationary phase. Cells were harvested by centrifugation followed by lyophilization.
2.2. Preparation of PHAs PHA was isolated from lyophilized cells by extraction with hot chloroform using a Soxhlet apparatus. The
3.1. Biosynthesis of PHAs bearing cyclohexyl groups The growth curves for P. oleo6orans grown with various molar mixtures of 4-CHB and NA are plotted in Fig. 1. When the bacterium was grown on a medium in which the concentration of NA was over 5 mM at a concentration of 10 mM total carbon substrate, this organism grew very well with a very short lag period. However, increasing the concentration of 4-CHB over 5 mM in given mixtures resulted in the poor growth of P. oleo6orans with longer lag time. In particular, the bacterium grown with 4-CHB as a sole carbon source required an induction period of approximately 5 days for growth and the resulting biomass was only approximately 0.21 g/l after 320 h cultivation. Fermentation results for P. oleo6orans grown with various mixtures of 4-CHB and NA and compositions of the produced PHAs were listed in Table 1. The bacterium was capable of utilizing 4-CHB as the sole carbon substrate for the growth and production of PHA bearing corresponding functional groups. However, the total amount of PHA accumulated in the cells was only about 2% of dry cell weight. These results correspond to those reported in another study where 4-CHB was classified as a carbon substrate that could
4-CHB
NA
g/l
mM
mM
1.70 1.53 1.28 0.85 0.42 –
10 9 7.5 5 2.5 –
– 1 2.5 5 7.5 10
a
Culture time (h)
320 47 25 17 15 14
Dry cell weight (g/l)
0.21 0.25 0.41 0.52 0.83 1.02
PHA content (%wt)
2 3 8 11 13 31
Weight of cyclohexyl unit (mg/l)
4.2 5.8 19.8 20.5 14.2 –
GC area%. 3HHp, 3-hydroxyheptanoate; 3HN, 3-hydroxynonanoate; 3HCHB, 3-hydroxy-4-cyclohexylbutyrate.
Yield of cyclohexyl unit (%)
0.2 0.4 1.54 2.40 3.40 –
Relative amount of monomeric units in PHAs (%)a 3HHp
3HN
3HCHB
– 9.8 14.2 22.8 28.4 26.5
– 12.6 25.5 41.2 58.4 73.5
100 77.6 60.3 36.0 13.2 –
D.Y. Kim et al. / International Journal of Biological Macromolecules 29 (2001) 145–150
Table 1 Fermentation results for P. oleo6orans grown with various mixtures of 4-CHB and NA and compositions of produced PHAs
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support both bacterial growth and PHA production [18]. The gas chromatographic analysis of the methanolyzed sample of the PHA biosynthesized from 4-CHB showed that this PHA contained only 3-hydroxy-4-cyclohexylbutyrate (3HCHB) repeating unit. P. oleo6orans cultivated with various mixtures of 4-CHB and NA, synthesized polyesters consisting of 3HCHB as well as 3-hydroxyalkanoates derived from NA. The PHAs produced were transparent at room temperature. Table 1 shows that the ratio of 3-hydroxynonanoate (3HN) to 3-hydroxyheptanoate (3HHp) in the PHAs produced from mixtures of these two carbon substrates was significantly changed from 1.28 to 2.05 depending on the concentration of 4-CHB in the carbon substrate mixtures. This result suggests that the change of compositions of two 3-hydroxyalkanoates in the PHAs synthesized from different molar mixtures was markedly affected by the addition of 4-CHB. These results were very similar to those previously obtained from PHAs synthesized by P. oleo6orans from various molar mixtures of NA and 10-undecynoic acid [11]. In that case, the ratio of 3HN to 3HHp of the PHAs produced by this bacterium was significantly changed from 2.9 to 1.1 when the fraction of 10-undecynoic acid was increased from 25 mol% to 100 mol% in the carbon substrate mixture.
3.2. Effects of NA on biocon6ersion of 4 -CHB to PHA Fig. 2 shows the total amounts of 3HCHB repeating unit in the PHAs obtained from P. oleo6orans cells harvested in each growth stage during cultivation with various mixtures of 4-CHB and NA. In the early
Fig. 2. Total amount of 3HCHB repeating units in the PHAs obtained from P. oleo6orans cells harvested in each growth stage during cultivation with various mixtures of 4-CHB and NA: ( ) 7.5 mM NA+2.5 mM 4-CHB; () 5 mM NA + 5 mM 4-CHB; () 2.5 mM NA+7.5 mM 4-CHB.
growth stage, increasing the concentration of NA in the carbon substrate mixture elevated the bioconversion speed of 4-CHB to polyester. Additionally, Table 1 and Fig. 2 clearly indicate that the conversion rate of 4CHB to PHA was significantly increased by raising the fraction of NA in the mixed carbon sources. The present result suggests that the production of PHAs bearing cyclohexyl groups by P. oleo6orans from various molar mixtures of 4-CHB and NA would be increased through cometabolism. This cometabolic biosynthesis is expected to offer the opportunity to design new functional polyesters and other unusual polyesters that have unique physico–chemical properties. The enhanced incorporation of corresponding functional groups into polyester by the co-feed of NA was also observed in P. putida grown with various mixtures of 10-undecylenic acid or 10-undecynoic acid with NA [11,15].
3.3. Relationship between carbon substrate consumption and PHA production
Fig. 1. Growth curves for P. oleo6orans grown with various molar mixtures of 4-CHB and NA: ( ) 10 mM NA; () 7.5 mM NA+ 2.5 mM 4-CHB; () 5 mM NA+ 5 mM 4-CHB; () 2.5 mM NA+7.5 mM 4-CHB; ( ) 1 mM NA+ 9 mM 4-CHB; ( ) 10 mM 4-CHB.
Fig. 3 shows the fermentation kinetics of PHA production by P. oleo6orans grown with an equimolar mixture of 4-CHB and NA. The early bacterial growth occurred by consumption of only NA and 4-CHB started to be consumed for the growth and PHA production after approximately 6 h cultivation. Most of NA in culture medium was depleted after 11 h while only about 50% of the given 4-CHB was used up throughout the cultivation. These results were very similar to those obtained by P. oleo6orans cultivated in an equimolar mixture of 5-phenylvaleric acid (5-PV)
D.Y. Kim et al. / International Journal of Biological Macromolecules 29 (2001) 145–150
Fig. 3. Fermentation kinetics of PHA production by P. oleo6orans grown with an equimolar mixture of 4-CHB and NA: ( ) dry cell weight; () PHA content; ( ) residual amount of NA; () residual amount of 4-CHB.
and NA [4]. In that case, 5-PV always maintained a concentration of approximately 25% of the carbon substrate supplemented in the culture broth, even after fermentation had stopped. However, the simultaneous utilization of NA and 4-CHB in this study during the exponential growth stage of cells was very comparable to the results of Andu´ jar et al. [10] who demonstrated that utilization of 5-CHV only after the complete consumption of NA when P. oleo6orans was cultivated in flasks with a medium containing a mixture of NA and 5-CHV as the carbon sources.
149
It is known that different culture conditions can result in significantly different physiological cell states, even though the cells are nominally in the same growth stage. In general, when cells are aerobically cultivated in a fermentor in which the temperature and pH are automatically controlled at optimal values, they adapt more rapidly to a given environment than when cultivated in a flask, and as a result, the cells show a relatively higher growth rate due to an enhanced physiological state in terms of the utilization of carbon substrates [19]. Therefore, it was presumed that the obvious discrepancy between flask culture of Andu´ jar et al. [10] and fermentor culture of the present study might be due to differences in the physiological state of the cells, due to different culture conditions, as the cultivation period for the highest dry cell weight was approximately 50 h in the shake flask culture [10] and 17 h in the fermentor used in the present study (Fig. 1).
3.4. Thermal analysis of the PHAs containing cyclohexyl groups DSC thermograms of the polyesters produced from mixtures of 4-CHB and NA by P. oleo6orans are shown in Fig. 4. The polyester produced from NA was referred to polyhydroxynonanoate (PHN) and used in this study as a control. The PHAs bearing terminal cyclohexyl groups in the side chains possessed higher glass transition temperatures (Tg ) and lower melting transition temperatures (Tm ) than those of PHN. Increasing the concentration of 3HCHB units in the PHAs resulted in an increase of Tg from − 40.13 to −28.26°C and a decrease of Tm from 50.85 to 42.66°C. These were very unusual phenomena because the in-
Fig. 4. DSC thermograms of copolyesters synthesized by P. oleo6orans; a PHA produced from NA (a), a PHA produced from a mixture of 2.5 mM 4-CHB and 7.5 mM NA (b), and a PHA produced from a mixture of 5 mM 4-CHB and 5 mM NA (c).
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crease of Tg of a polymer generally accompanies the rise of Tm [20]. DSC thermograms clearly showed that the PHAs synthesized from two different molar mixtures of 4-CHB and NA by P. oleo6orans had only one Tg and Tm. The experimental results were confirmed by three repeated experiments. The present results suggest that the PHAs synthesized in this study were most likely random copolyesters rather than physical mixtures of significantly different copolyesters. The results are comparable to those of Andu´ jar et al. [10], showing that P. oleo6orans grown with a mixture of 80% 5-CHV and 20% NA produced a physical mixture of two PHAs with significantly different compositions. It has been reported that the carbon availability of P. oleo6orans for aromatic and non-aromatic alkanoic acids is significantly different. When grown on mixtures of NA with an aromatic alkanoic acid, such as 5-PV [4,7] and methylphenoxyalkanoates [15], P. oleo6orans often utilizes aromatic alkanoic acids following the consumption of significant amounts of NA in the growth medium and produces a physical mixture of the two polyesters with significantly different compositions. Recently, an effort to produce a random copolyester with corresponding repeating units by P. oleo6orans grown on a mixture of NA and 5-PV was performed by Curley et al. [7]. However, in that case, P. oleo6orans synthesized a mixture of two polyesters due to the different uptake rates of the two carbon substrates, and it was concluded that this difference in utilization time of the carbon substrates might have been responsible for the production of the two different polyesters. Therefore, it is proposed that the formation of a random copolyester from various mixtures of NA and 4-CHB by P. oleo6orans is due to the simultaneous consumption of the carbon substrates.
4. Conclusions P. oleo6orans synthesized PHAs bearing cyclohexyl groups in the side chains when it was grown with 4-CHB alone or in mixtures with NA. Production of the PHAs bearing cyclohexyl substituents by this organism was significantly elevated by addition of NA, indicating that the incorporation of cyclohexyl units to PHA was mainly performed through cometabolism. The PHAs produced in this research were most likely
random copolyesters as determined by DSC analysis. These results suggested that the biosynthetic patterns of PHAs by P. oleo6orans from various mixtures of 4CHB and NA were accorded to those of PHAs from alkanoic acids containing non-aromatic functional groups rather than those of polyesters bearing aromatic functional substituents. It is also suggested that the simultaneous consumption of 4-CHB and NA by P. oleo6orans is probably responsible for the production of copolyesters containing cyclohexyl groups.
Acknowledgements This work was supported by a research grant from the interdisciplinary research program of the Korea Science and Engineering Foundation (Grant No. 19992-201-006-4).
References [1] Steinbu¨ chel A, Valentin HE. FEMS Microbiol Lett 1995;128:219. [2] Witholt B, Kessler B. Curr Opin Biotechnol 1999;10:279. [3] Fritzsche K, Lenz RW, Fuller RC. Int J Biol Macromol 1990;2:85. [4] Kim YB, Lenz RW, Fuller RC. Macromolecules 1991;24:5256. [5] Kim YB, Lenz RW, Fuller RC. Macromolecules 1992;25:1852. [6] Kim YB, Lenz RW, Fuller RC. J Polym Sci (Part A: polymer chemistry) 1995;33:1367. [7] Curley JM, Lenz RW, Fuller RC. Int J Biol Macromol 1996;19:29. [8] Hazer B, Lenz RW, Fuller RC. Polymer 1996;37:5951. [9] Kim OY, Gross RA, Hammer WJ, Newmark RA. Macromolecules 1996;29:4572. [10] Andu´ jar M, Aponte MA, Dı´az E, Schro¨ der E. Macromolecules 1997;30:1611. [11] Kim DY, Kim YB, Rhee YH. Macromolecules 1998;31:4760. [12] Aro´ stegui SM, Aponte MA, Dı´az E, Schro¨ der E. Macromolecules 1999;32:2889. [13] Kim YB, Kim DY, Rhee YH. Macromolecules 1999;32:6058. [14] Song JJ, Yoon SC. Appl Environ Microbiol 1996;62:536. [15] Kim DY, Kim YB, Rhee YH. Int J Biol Macromol 2000;28:23. [16] de Koning GJM, van Bilsen HMM, Lemstra PJ, Hazenberg W, Witholt B, Preusting H, van der Galie¨ n JG, Schirmer A, Jendrossek D. Polymer 1994;35:2090. [17] Madison LL, Huisman GW. Microbiol Mol Biol Rev 1999;63:21. [18] Lenz RW, Kim YB, Fuller RC. FEMS Microbiol Rev 1992;103:207. [19] Zeng AP, Deckwer WD. Biotechnol Prog 1995;11:71. [20] Doi Y. Microbial Polyesters. New York: VCH Publishers, Inc., 1990.
Biosynthesis of polyhydroxyalkanoate copolyester containing cyclohexyl groups by Pseudomonas oleo6orans Do Young Kim a, Saet Byel Jung a, Gang Guk Choi a, Young Baek Kim b, Young Ha Rhee a,* a
Department of Microbiology, Chungnam National Uni6ersity, Daejon 305 -764, South Korea b Department of Polymer Engineering, PaiChai Uni6ersity, Daejon 302 -735, South Korea Received 11 August 2000; accepted 8 April 2001
Abstract Production of polyhydroxyalkanoates (PHAs) substituted with cyclohexyl groups by Pseudomonas oleo6orans grown with 4-cyclohexylbutyric acid (4-CHB) and its mixtures with nonanoic acid (NA) was investigated. Addition of NA to medium gave rise to an increase in the total concentration of 3-hydroxy-4-cyclohexylbutyrate repeating unit in the PHAs, indicating that the bioconversion rate of 4-CHB to polyester was significantly improved by the cometabolic effect. Increasing the proportion of NA from 1.0 to 7.5 mM at a concentration of 10 mM total carbon substrate also accelerated the uptake speed of 4-CHB by the organism and resulted in an increase of the ratio of 3-hydroxynonanoate to 3-hydroxyheptanoate from 1.28 to 2.05. Differential scanning calorimetric analysis of the PHAs bearing the corresponding functional groups showed one melting transition and one glass transition temperature varying according to the composition. These results indicated that random copolyesters were obtained from the carbon substrates used in this study. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Copolyester; Cyclohexyl substituent; Polyhydroxyalkanoate; Pseudomonas oleo6orans
1. Introduction Medium-chain-length polyhydroxyalkanoates (MCLPHAs) that consist of constituents having a chain length of typically C6 – C12 are produced by a wide variety of Gram-negative bacteria, mainly pseudomonads. Until now, more than 100 different hydroxyalkanoates have been incorporated into MCL-PHAs as constituents of biosynthetic PHA [1,2]. Of the microorganisms capable of producing MCL-PHAs Pseudomonas oleo6orans has been investigated most extensively. This organism synthesizes MCL-PHAs containing various functional groups in the side chains when grown with carbon substrates bearing corresponding chemical structures [3 – 13]. Although P. putida has been also reported to be useful for the production of functional MCL-PHAs [9,11,13,14], recent results in our laboratory have demonstrated that * Corresponding author. Tel.: + 82-42-8216413; fax: +82-428227367. E-mail address: [email protected] (Y.H. Rhee).
P. putida cannot produce all of the PHA containing functional substituents produced by P. oleo6orans [15]. PHAs containing functional groups are of great interest, because these groups can improve the physical properties of MCL-PHAs. Moreover, some functional substituents can be modified by chemical reactions in order to obtain more useful groups that can extend the potential application of PHAs as environmentally biodegradable polymers and functional biomaterials for use in biomedical applications [16,17]. When P. oleo6orans is grown on mixtures of octanoic acid (OA) or nonanoic acid (NA) with alkanoic acids containing non-aromatic functional groups such as bromine [5], fluorine [9], olefin [6], or carbon –carbon triple bonds [11], the bacterium produces random copolyesters containing monomer units derived from both substrates. On the other hand, P. oleo6orans grown on a mixture of OA or NA with alkanoic acid containing aromatic groups such as phenyl [4], methylphenyl [8], nitrophenyl [12], or methylphenoxy group [13] produces mixtures of more than one copolyester with significantly different compositions. The mixtures in general
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are composed of copolyesters containing a larger concentration of aromatic repeating units and copolyesters containing a small amount of aromatic repeating units. Recently, polyestyers containing cyclohexyl groups were synthesized by P. oleo6orans when it was grown with mixtures of either 5-cyclohexylvaleric acid (5CHV) or 4-cyclohexylbutyric acid (4-CHB) and NA [10]. Interestingly, thermal analysis revealed that the polyesters were not copolyesters, but mixtures of two PHAs with significantly different compositions: one containing more cyclohexyl groups than the other. These results were rather unusual because the formation of mixtures of different copolyesters by P. oleo6orans has only been observed in co-feeding experiments using mixtures of OA or NA with aromatic alkanoic acids. In the present study, to understand the PHA biosynthesis patterns of P. oleo6orans grown with mixtures of 4-CHB and NA, the growth behavior of the organism on the substrates and the fermentation kinetics for PHA production were examined. Of considerable interest was to determine whether PHAs so produced were random copolyesters or mixtures of PHAs with different compositions.
2. Materials and methods
extracted crude PHA was purified by repeated precipitation by dropping into vigorously stirred cold methanol to obtain the fine product and this precipitation procedure was carried out at least three times.
2.3. Analytical methods Using gas chromatography (GC, Hewlett-Packard 5890 gas chromatograph equipped with a flame ionization detector and a HP-1 capillary column), the relative concentration of the PHA monomeric units synthesized were calculated by integrating the methyl-ester peaks from methanolyzed samples. The oven temperature was initially maintained at 80°C for 4 min and then raised at a rate of 10°C/min to 230°C. Identification of the PHA monomeric units was carried out by either gas chromatography/mass spectroscopy (GC/MS) or 1H nuclear magnetic resonance (NMR) spectroscopy. GC/ MS analysis was carried out using a Hewlett-Packard 5988 GC/MS system and NMR spectroscopy was performed using a Bruker 500 NMR spectrometer as described previously [5]. Differential scanning calorimetry (DSC) analysis was carried out using a Perkin-Elmer DSC 7. The temperature was scanned from −100 to 150°C at a ramp 20°C/min. The analysis of residual carbon substrates in the medium was done as previously described [11].
2.1. Culture of bacterium 3. Results and discussion P. oleo6orans ATCC 29347 was grown on a basal medium as described elsewhere [6]. The concentration of two carbon substrates (4-CHB and NA) was changed to evaluate their effect on growth and PHA biosynthesis. The total concentration of carboxylic acids in the medium was 10 mM. Shake flask cultures were carried out aerobically in 500 ml Erlenmeyer flasks containing 100 ml of the basal medium. Batch fermentations were conducted in a 5 l jar fermentor (Korea Fermentor Co. Ltd.) with a working volume of 3 l. The medium was inoculated with a 3% (v/v) inoculum of an overnight culture in the basal medium containing 10 mM NA as the sole carbon source. The temperature and pH were automatically controlled at optimal values of 30°C and 7.0, respectively. The airflow rate was 0.25 vvm and agitation speed was 250 rpm. Cell growth was monitored spectrophotometrically at 660 nm. Cell cultivation was stopped approximately 2 h after the growth reached the stationary phase. Cells were harvested by centrifugation followed by lyophilization.
2.2. Preparation of PHAs PHA was isolated from lyophilized cells by extraction with hot chloroform using a Soxhlet apparatus. The
3.1. Biosynthesis of PHAs bearing cyclohexyl groups The growth curves for P. oleo6orans grown with various molar mixtures of 4-CHB and NA are plotted in Fig. 1. When the bacterium was grown on a medium in which the concentration of NA was over 5 mM at a concentration of 10 mM total carbon substrate, this organism grew very well with a very short lag period. However, increasing the concentration of 4-CHB over 5 mM in given mixtures resulted in the poor growth of P. oleo6orans with longer lag time. In particular, the bacterium grown with 4-CHB as a sole carbon source required an induction period of approximately 5 days for growth and the resulting biomass was only approximately 0.21 g/l after 320 h cultivation. Fermentation results for P. oleo6orans grown with various mixtures of 4-CHB and NA and compositions of the produced PHAs were listed in Table 1. The bacterium was capable of utilizing 4-CHB as the sole carbon substrate for the growth and production of PHA bearing corresponding functional groups. However, the total amount of PHA accumulated in the cells was only about 2% of dry cell weight. These results correspond to those reported in another study where 4-CHB was classified as a carbon substrate that could
4-CHB
NA
g/l
mM
mM
1.70 1.53 1.28 0.85 0.42 –
10 9 7.5 5 2.5 –
– 1 2.5 5 7.5 10
a
Culture time (h)
320 47 25 17 15 14
Dry cell weight (g/l)
0.21 0.25 0.41 0.52 0.83 1.02
PHA content (%wt)
2 3 8 11 13 31
Weight of cyclohexyl unit (mg/l)
4.2 5.8 19.8 20.5 14.2 –
GC area%. 3HHp, 3-hydroxyheptanoate; 3HN, 3-hydroxynonanoate; 3HCHB, 3-hydroxy-4-cyclohexylbutyrate.
Yield of cyclohexyl unit (%)
0.2 0.4 1.54 2.40 3.40 –
Relative amount of monomeric units in PHAs (%)a 3HHp
3HN
3HCHB
– 9.8 14.2 22.8 28.4 26.5
– 12.6 25.5 41.2 58.4 73.5
100 77.6 60.3 36.0 13.2 –
D.Y. Kim et al. / International Journal of Biological Macromolecules 29 (2001) 145–150
Table 1 Fermentation results for P. oleo6orans grown with various mixtures of 4-CHB and NA and compositions of produced PHAs
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D.Y. Kim et al. / International Journal of Biological Macromolecules 29 (2001) 145–150
support both bacterial growth and PHA production [18]. The gas chromatographic analysis of the methanolyzed sample of the PHA biosynthesized from 4-CHB showed that this PHA contained only 3-hydroxy-4-cyclohexylbutyrate (3HCHB) repeating unit. P. oleo6orans cultivated with various mixtures of 4-CHB and NA, synthesized polyesters consisting of 3HCHB as well as 3-hydroxyalkanoates derived from NA. The PHAs produced were transparent at room temperature. Table 1 shows that the ratio of 3-hydroxynonanoate (3HN) to 3-hydroxyheptanoate (3HHp) in the PHAs produced from mixtures of these two carbon substrates was significantly changed from 1.28 to 2.05 depending on the concentration of 4-CHB in the carbon substrate mixtures. This result suggests that the change of compositions of two 3-hydroxyalkanoates in the PHAs synthesized from different molar mixtures was markedly affected by the addition of 4-CHB. These results were very similar to those previously obtained from PHAs synthesized by P. oleo6orans from various molar mixtures of NA and 10-undecynoic acid [11]. In that case, the ratio of 3HN to 3HHp of the PHAs produced by this bacterium was significantly changed from 2.9 to 1.1 when the fraction of 10-undecynoic acid was increased from 25 mol% to 100 mol% in the carbon substrate mixture.
3.2. Effects of NA on biocon6ersion of 4 -CHB to PHA Fig. 2 shows the total amounts of 3HCHB repeating unit in the PHAs obtained from P. oleo6orans cells harvested in each growth stage during cultivation with various mixtures of 4-CHB and NA. In the early
Fig. 2. Total amount of 3HCHB repeating units in the PHAs obtained from P. oleo6orans cells harvested in each growth stage during cultivation with various mixtures of 4-CHB and NA: ( ) 7.5 mM NA+2.5 mM 4-CHB; () 5 mM NA + 5 mM 4-CHB; () 2.5 mM NA+7.5 mM 4-CHB.
growth stage, increasing the concentration of NA in the carbon substrate mixture elevated the bioconversion speed of 4-CHB to polyester. Additionally, Table 1 and Fig. 2 clearly indicate that the conversion rate of 4CHB to PHA was significantly increased by raising the fraction of NA in the mixed carbon sources. The present result suggests that the production of PHAs bearing cyclohexyl groups by P. oleo6orans from various molar mixtures of 4-CHB and NA would be increased through cometabolism. This cometabolic biosynthesis is expected to offer the opportunity to design new functional polyesters and other unusual polyesters that have unique physico–chemical properties. The enhanced incorporation of corresponding functional groups into polyester by the co-feed of NA was also observed in P. putida grown with various mixtures of 10-undecylenic acid or 10-undecynoic acid with NA [11,15].
3.3. Relationship between carbon substrate consumption and PHA production
Fig. 1. Growth curves for P. oleo6orans grown with various molar mixtures of 4-CHB and NA: ( ) 10 mM NA; () 7.5 mM NA+ 2.5 mM 4-CHB; () 5 mM NA+ 5 mM 4-CHB; () 2.5 mM NA+7.5 mM 4-CHB; ( ) 1 mM NA+ 9 mM 4-CHB; ( ) 10 mM 4-CHB.
Fig. 3 shows the fermentation kinetics of PHA production by P. oleo6orans grown with an equimolar mixture of 4-CHB and NA. The early bacterial growth occurred by consumption of only NA and 4-CHB started to be consumed for the growth and PHA production after approximately 6 h cultivation. Most of NA in culture medium was depleted after 11 h while only about 50% of the given 4-CHB was used up throughout the cultivation. These results were very similar to those obtained by P. oleo6orans cultivated in an equimolar mixture of 5-phenylvaleric acid (5-PV)
D.Y. Kim et al. / International Journal of Biological Macromolecules 29 (2001) 145–150
Fig. 3. Fermentation kinetics of PHA production by P. oleo6orans grown with an equimolar mixture of 4-CHB and NA: ( ) dry cell weight; () PHA content; ( ) residual amount of NA; () residual amount of 4-CHB.
and NA [4]. In that case, 5-PV always maintained a concentration of approximately 25% of the carbon substrate supplemented in the culture broth, even after fermentation had stopped. However, the simultaneous utilization of NA and 4-CHB in this study during the exponential growth stage of cells was very comparable to the results of Andu´ jar et al. [10] who demonstrated that utilization of 5-CHV only after the complete consumption of NA when P. oleo6orans was cultivated in flasks with a medium containing a mixture of NA and 5-CHV as the carbon sources.
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It is known that different culture conditions can result in significantly different physiological cell states, even though the cells are nominally in the same growth stage. In general, when cells are aerobically cultivated in a fermentor in which the temperature and pH are automatically controlled at optimal values, they adapt more rapidly to a given environment than when cultivated in a flask, and as a result, the cells show a relatively higher growth rate due to an enhanced physiological state in terms of the utilization of carbon substrates [19]. Therefore, it was presumed that the obvious discrepancy between flask culture of Andu´ jar et al. [10] and fermentor culture of the present study might be due to differences in the physiological state of the cells, due to different culture conditions, as the cultivation period for the highest dry cell weight was approximately 50 h in the shake flask culture [10] and 17 h in the fermentor used in the present study (Fig. 1).
3.4. Thermal analysis of the PHAs containing cyclohexyl groups DSC thermograms of the polyesters produced from mixtures of 4-CHB and NA by P. oleo6orans are shown in Fig. 4. The polyester produced from NA was referred to polyhydroxynonanoate (PHN) and used in this study as a control. The PHAs bearing terminal cyclohexyl groups in the side chains possessed higher glass transition temperatures (Tg ) and lower melting transition temperatures (Tm ) than those of PHN. Increasing the concentration of 3HCHB units in the PHAs resulted in an increase of Tg from − 40.13 to −28.26°C and a decrease of Tm from 50.85 to 42.66°C. These were very unusual phenomena because the in-
Fig. 4. DSC thermograms of copolyesters synthesized by P. oleo6orans; a PHA produced from NA (a), a PHA produced from a mixture of 2.5 mM 4-CHB and 7.5 mM NA (b), and a PHA produced from a mixture of 5 mM 4-CHB and 5 mM NA (c).
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crease of Tg of a polymer generally accompanies the rise of Tm [20]. DSC thermograms clearly showed that the PHAs synthesized from two different molar mixtures of 4-CHB and NA by P. oleo6orans had only one Tg and Tm. The experimental results were confirmed by three repeated experiments. The present results suggest that the PHAs synthesized in this study were most likely random copolyesters rather than physical mixtures of significantly different copolyesters. The results are comparable to those of Andu´ jar et al. [10], showing that P. oleo6orans grown with a mixture of 80% 5-CHV and 20% NA produced a physical mixture of two PHAs with significantly different compositions. It has been reported that the carbon availability of P. oleo6orans for aromatic and non-aromatic alkanoic acids is significantly different. When grown on mixtures of NA with an aromatic alkanoic acid, such as 5-PV [4,7] and methylphenoxyalkanoates [15], P. oleo6orans often utilizes aromatic alkanoic acids following the consumption of significant amounts of NA in the growth medium and produces a physical mixture of the two polyesters with significantly different compositions. Recently, an effort to produce a random copolyester with corresponding repeating units by P. oleo6orans grown on a mixture of NA and 5-PV was performed by Curley et al. [7]. However, in that case, P. oleo6orans synthesized a mixture of two polyesters due to the different uptake rates of the two carbon substrates, and it was concluded that this difference in utilization time of the carbon substrates might have been responsible for the production of the two different polyesters. Therefore, it is proposed that the formation of a random copolyester from various mixtures of NA and 4-CHB by P. oleo6orans is due to the simultaneous consumption of the carbon substrates.
4. Conclusions P. oleo6orans synthesized PHAs bearing cyclohexyl groups in the side chains when it was grown with 4-CHB alone or in mixtures with NA. Production of the PHAs bearing cyclohexyl substituents by this organism was significantly elevated by addition of NA, indicating that the incorporation of cyclohexyl units to PHA was mainly performed through cometabolism. The PHAs produced in this research were most likely
random copolyesters as determined by DSC analysis. These results suggested that the biosynthetic patterns of PHAs by P. oleo6orans from various mixtures of 4CHB and NA were accorded to those of PHAs from alkanoic acids containing non-aromatic functional groups rather than those of polyesters bearing aromatic functional substituents. It is also suggested that the simultaneous consumption of 4-CHB and NA by P. oleo6orans is probably responsible for the production of copolyesters containing cyclohexyl groups.
Acknowledgements This work was supported by a research grant from the interdisciplinary research program of the Korea Science and Engineering Foundation (Grant No. 19992-201-006-4).
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