Process Biochemistry 46 (2011) 15721578

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Process Biochemistry
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Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymers using jatropha oil as the main carbon source
Ko-Sin Ng a , Yoke-Ming Wong a , Takeharu Tsuge b , Kumar Sudesh a,
a b

Ecobiomaterial Research Laboratory, School of Biological Science, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan

a r t i c l e

i n f o

a b s t r a c t
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-hydroxybutyrate-co-3hydroxyhexanoate) [P(3HB-co-3HHx)] copolymers were produced using jatropha oil as the carbon source. P(3HB-co-3HV) with a 3HV monomer as high as 42 mol% was produced by wild-type Cupriavidus necator H16 from a mixture of jatropha oil and sodium valerate, and P(3HB-co-3HHx) with a 3HHx monomer of 3 mol% was produced by transformant C. necator PHB 4/pBBREE32d13 harboring Aeromonas caviae PHA synthase, using jatropha oil as the sole carbon source. The results of differential scanning calorimetry, thermogravimetric analysis and gel permeation chromatography revealed that the copolymers produced from jatropha oil were essentially the same as those produced from other, more established carbon sources, such as sugars and other plant oils. This study demonstrates that jatropha oil is a potential renewable carbon source for the large-scale production of copolymers by C. necator and its transformant. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 8 December 2010 Received in revised form 4 April 2011 Accepted 26 April 2011 Keywords: Jatropha oil Physic nut Jatropha curcas Poly(3-hydroxybutyrate-co-3hydroxyvalerate) Poly(3-hydroxybutyrate-co-3hydroxyhexanoate) Bioplastics

1. Introduction Polyhydroxyalkanoate (PHA) is a polymer that is accumulated by bacteria as a carbon and energy storage material in the presence of an excess amount of carbon and a limited supply of another nutrient, which can be in the form of nitrogen, sulfur, phosphorus, magnesium or oxygen [1]. Among various known biodegradable polymeric materials, PHA is known to be a fully biodegradable alternative to conventional plastics [2]. In terms of its applications, PHA is a good material for bioplastics and implant biomaterials due to its biodegradability, biocompatibility, thermoprocessibility and sustainability [3]. Although PHA is a good substitute for petroleum-based plastics, the high cost of production makes PHA more expensive than conventional plastics [4]. In an effort to reduce the production cost of PHAs, various carbon sources, such as plant oils and sugars, have been explored for use in PHA production. To date, the use of various plant oils, including soybean oil [5], palm oil [6,7], coconut oil [6] and corn oil [8], has been reported to result in high yields of PHA.

Corresponding author. Tel.: +60 4 6534367; fax: +60 4 6565125. E-mail address: ksudesh@usm.my (K. Sudesh). 1359-5113/$ see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.04.012

However, the use of these edible oils as primary carbon sources for bioplastic production may not be sustainable [9]. Continuous conversion of edible oils to bioplastics may lead to a shortage in food supplies and cause ination in certain countries, especially in third world countries. Therefore, our study aims to evaluate the use of jatropha oil, which is a non-edible plant oil, for the production of PHA copolymers. In a previous study, we showed that jatropha oil can be used by Cupriavidus necator H16 to grow and synthesize poly(3-hydroxybutyrate) [P(3HB)] homopolymer [10]. However, it is not known if jatropha oil affects the incorporation of comonomer and the properties of PHA copolymers. Jatropha curcas, also known as physic nut, is a multipurpose shrub that can reach a height of 20 ft and has glabrous branchlets [11]. The life expectancy of J. curcas is almost 50 years, and this shrub is distributed natively in Central and South America, including in Mexico, Brazil and Argentina and can now also be found in many parts of Africa and Asia [12]. J. curcas is characterized as a hardy, highly adaptable, drought-resistant crop and consequently has high ecological adaptability. Furthermore, J. curcas is a disease-resistant plant because only a few insects or fungi can transmit their diseases to the plants [12]. Jatropha oil can be considered to be a biodiesel fuel because it has a low oil viscosity when compared to soybean, cottonseed and sunower oils [13]. The blackish J. curcas seeds contain ve main toxins, which are phorbol

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esters, phytates, trypsin inhibitors, lectins and curcin [14]. Besides, a variety of terpene alcohols and sterols were detected in jatropha oil [15,16]; thus, to some extent, jatropha oil is toxic and classied as a non-edible oil. Regarding its use as a carbon source for PHA biosynthesis, it is not known whether the toxins and/or any other unknown components in the jatropha oil have any effects on the various monomer supplying pathways involved in the synthesis of the PHA copolymers. In Malaysia, Jatropha has been put into consideration as a supplementary renewable resource in addition to palm oil to balance the renewable energy supply and exportation of biodiesel [17], and jatropha oil is a potential renewable feedstock for bioplastics. Recognizing the potential of Jatropha as a renewable energy source, many industrial companies are now investing in its research and cultivation. By the end of 2010, the cultivation area for Jatropha is expected to reach 1 million ha [17]. For the above reasons, this study extended the evaluation of jatropha oil for the production and characterization of PHA copolymers. The ability to biosynthesize PHA copolymers from the co-feeding of jatropha oil and precursors (sodium valerate and sodium propionate) was investigated using wild-type C. necator H16. In addition, copolymer biosynthesis from jatropha oil using transformant C. necator PHB 4/pBBREE32d13 was evaluated.

2.4. Residual oil measurement Approximately 2 mL of culture broth was collected at 48 h and centrifuged. The supernatant was mixed with 5 mL of hexane and vortexed for 1 min to dissolve the residual oil. Subsequently, 1 mL of the upper layer (hexane layer) was transferred to a pre-weighed plastic plate and left to dry in the fume hood until a constant weight was obtained. 2.5. Analytical procedures PHA content and composition were determined by gas chromatography (GC) analysis. Approximately 20 mg of lyophilized cells were subjected to methanolysis in the presence of 15% (v/v) sulfuric acid and 85% (v/v) methanol for 140 min at 100 C. The resulting hydroxyacyl methyl esters were then analyzed by GC [20]. To extract PHA from the lyophilized cells, approximately 3 g of freeze dried cells were reuxed in 300 mL of chloroform in a ratio of 1:100 for 4 h at 60 C. The reuxed solution was cooled to room temperature and ltered to remove the cell debris. The ltrate was then concentrated using a rotary evaporator before it was added, dropwise, into vigorously stirred, cool methanol. The precipitated and puried polymer was then collected and air dried in the fume hood. 2.6. Copolymer characterization The puried and dried extracted polymer was used for molecular weight determination. The molecular weight was determined at 40 C using a gel permeation chromatography (Agilent 1200 GPC) system equipped with a refractive index detector and SHODEX K-802 and K-806M columns. The samples were prepared by dissolving the extracted PHA in chloroform at a concentration of 1 mg/mL. Chloroform was used as the eluent at a ow rate of 0.8 mL/min. The weight-average molecular weight (Mw ), number-average molecular weight (Mn ), and polydispersity index (Mw /Mn ) were determined from the curve that was obtained. Calorimetric measurements (DSC) of the PHA were conducted using a Perkin Elmer Pyris 1 differential scanning calorimetry (DSC) thermal analysis system in the range of 30 C to 200 C at a heating rate of 20 C/min. The glass transition temperature (Tg ), crystalline melting point (Tm ) and enthalpy of fusion ( Hm ) were determined from the DSC thermogram of the second scan. Thermogravimetric analysis (TGA) was performed using a Mettler-Toledo TGA/SDTA 851 thermobalance with STARe thermal analysis software. TGA heating of 10 mg of PHA under a nitrogen atmosphere started at 30900 C at a heating rate of 20 C/min. The decomposition temperature (Td ) at 5% weight loss was determined.

2. Materials and methods 2.1. Bacterial strain and maintenance C. necator H16 (formerly known as Alcaligenes eutrophus, Ralstonia eutropha and Wautersia eutropha) and transformant C. necator PHB 4/pBBREE32d13 harboring the PHA synthase gene of Aeromonas caviae [18] were used throughout this study. For short-term maintenance, the bacterial strains were routinely streaked onto nutrient-rich (NR) agar plates with the following composition (per liter): 10 g peptone, 10 g meat extract and 2 g yeast extract [19]. Kanamycin at a concentration of 50 mg/L was added into the agar for maintenance of the transformant strain plasmid. For long-term storage, the bacteria were maintained in a 25% (v/v) glycerol stock solution. The glycerol stock was prepared by the addition of 12.5 mL of pure glycerol to an overnight culture of the bacterial cells in 50 mL of NR. The tubes were stored in aliquots of 1 mL at 20 C.

3. Results 3.1. Biosynthesis of P(3HB-co-3HV) copolymer from mixtures of jatropha oil and sodium valerate or sodium propionate In a previous study [10], jatropha oil was evaluated and found to be a suitable, sole carbon source for the biosynthesis of poly(3hydroxybutyrate) [P(3HB)] homopolymer. However, it is not yet known how jatropha oil will perform when added together with precursor carbon sources for the biosynthesis of PHA copolymers. In this study, the biosynthesis of P(3HB-co-3HV) copolymer was investigated for mixtures of jatropha oil and sodium valerate or sodium propionate (Table 1). The total carbon from jatropha oil and precursor for cell growth and PHA biosynthesis was xed at 9.51 g/L, which was equivalent to the carbon content of jatropha oil at the optimal concentration (12.5 g/L) used for the biosynthesis of P(3HB) in previous experiments. When the concentrations of the precursors were increased, the concentration of jatropha oil was decreased to result in a total carbon concentration of 9.51 g/L. The concentration of precursors mentioned in the text always refers to the carbon concentration. Here, sodium valerate or sodium propionate was co-fed with jatropha oil to produce P(3HB-co-3HV) copolymers with different 3HV monomer compositions. The 3HV precursors, such as propionic and valeric acids, have certain level of toxicity to the cells [21,22] and have to be fed in a timely and controlled manner. P(3HB-co-3HV) copolymers biosynthesized from late feeding of the precursors resulted in the formation of copolymer blends having different 3HV molar fractions [7]. In addition, the high content of readily accumulated P(3HB) in cells could reduce the utilization of precursors and incorporation of 3HV [22]. However, the feeding of precursors at an early stage would have stronger

2.2. Carbon source Jatropha oil (Sarawak, Malaysia) was used solely and in conjunction with 3HV precursors to produce P(3HB-co-3HHx) and P(3HB-co-3HV), respectively. The fatty acid composition of jatropha oil was described in the previous paper [10]. Jatropha oil was ltered with a hydrophobic PTFE membrane lter (0.2 m pore size) and subsequently autoclaved at 121 C for 15 min before the oil was added into mineral medium (MM) broth. Sodium valerate and sodium propionate were used as 3HV precursors. Stock solutions of both precursors at 20% (w/v) were prepared and autoclaved separately.

2.3. Cultivation and PHA synthesis One-stage batch cultivation in shake asks was conducted for PHA biosynthesis. The bacterial cells were rst grown in NR medium to enrich the cells. Two loops of bacteria, cultured for 1618 h, from the NR plate were grown for 6 h in 50 mL of NR medium at 30 C and 200 rpm. Approximately 3% (v/v) of the inoculum (OD600nm = 4.55) was transferred into 100 mL of MM broth and incubated for 48 h at 30 C and 200 rpm for PHA accumulation. The MM was prepared according to the following composition (per liter): 3.32 g Na2 HPO4 , 2.80 g KH2 PO4 , 0.54 g (NH2 )2 CO, 0.25 g MgSO4 7H2 O and 1 mL trace element solution [19]. The trace element solution consisted of 0.22 g CoCl2 6H2 O, 9.7 g FeCl3 , 7.8 g CaCl2 , 0.12 g NiCl2 6H2 O, 0.11 g CrCl3 6H2 O and 0.16 g CuSO4 5H2 O in 1 L of 0.1 N HCl [5]. In addition, 50 mg/L of kanamycin was added for cultures of the transformant strain. To induce the biosynthesis of P(3HB-co-3HV), 3HV precursors were added after 12 h of cultivation. The cells were harvested at the end of the 48-h cultivation period. Centrifugation at 8000 rpm and 4 C for 5 min using a KUBOTA 6500 was conducted to pellet the cells. Approximately 20 mL of hexane was added to the cell pellet followed by vortexing and centrifugation at 8000 rpm and 4 C for 3 min to remove the residual oil. The nal centrifugation (8000 rpm, 4 C for 5 min) was performed after adding 50 mL of distilled water to the pellet to remove the remaining hexane. The harvested cells were frozen at 20 C for about 24 h before freeze drying.

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Table 1 Biosynthesis of P(3HB-co-3HV) copolymer by C. necator H16 using the mixture of jatropha oil as the main carbon source and sodium valerate or sodium propionate as the precursor carbon sources.a Data shown are the means of triplicate. Mean data accompanied by different superscript alphabets are signicantly different (Tukeys HSD test, p < 0.05). Carbon concentration (g/L) CDW (g/L) PHA content (wt%)b Total PHA (g/L) PHA composition (mol%) Residual biomass (g/L) Conversion of precursors into 3HV monomer (%)c

Precursors

Jatropha oil 9.2d 7.5bc 7.3b 6.7b 6.7b 8.3cd 7.1b 6.8b 5.6a 5.6a 0.0 0.5 0.4 0.4 0.2 0.2 0.3 0.3 0.3 0.1 90d 74abc 75abc 70ab 76abc 81c 76abc 73abc 69a 77bc 1 2 1 1 3 3 3 3 2 3 8.3f 5.6d 5.4cd 4.7bc 5.1cd 6.6e 5.4cd 4.9bcd 3.8a 4.3ab 0.1 0.5 0.2 0.3 0.4 0.3 0.2 0.3 0.1 0.1

3HB 97g 91f 80de 68b 59a 98g 92fg 86e 77cd 73c 1 1 2 1 1 1 1 1 0 4

3HV 3a 9b 20cd 32f 41g 2a 8ab 14c 23de 27e 1 1 2 1 1 1 1 1 0 4 0.9a 1.7b 1.8b 2.0b 1.6ab 1.8b 1.7b 1.8b 1.7b 1.3ab 0.1 0.4 0.1 0.2 0.2 0.3 0.2 0.2 0.2 0.2 31 21 27 27 29 17 18 17 16 16

Sodium valerate 9.03 0.48 1.44 8.07 7.11 2.40 6.15 3.36 5.19 4.32 Sodium propionate 9.03 0.48 1.44 8.07 2.40 7.11 3.36 6.15 4.32 5.19

a Incubated for 48h at 30 C, initial pH 7, 200 rpm. Total carbon content of jatropha oil and precursors was set at 9.51 g/L. One gram of jatropha oil contains 0.76 g of carbon. Precursors were added separately at 12 h. b PHA content in freeze dried cells. c Conversion of precursor into 3HV (%) = carbon concentration of 3HV (g/L)/carbon concentration of precursors (g/L) 100%.

toxic effects on the cells due to the lower initial cell density that is less able to tolerate the higher concentration of precursors [23]. In this study, the cells were rst grown in jatropha oil for 12 h to achieve adequate cell density (about 2 g/L) with a P(3HB) content of 35 wt% to tolerate the toxic effects of the precursors that were fed later. Generally, the presence of higher concentrations of precursor in the culture medium resulted in increased 3HV accumulation but reduced cell dry weight (CDW) and PHA content (Table 1). When the concentrations of sodium valerate and sodium propionate were increased from 0.48 g/L to 1.44 g/L, a signicant drop in CDW from 9.2 g/L to 7.5 g/L and 8.3 g/L to 7.1 g/L were observed, respectively. When the concentrations of precursors were increased from 1.44 g/L to 4.32 g/L, the residual biomass did not show any signicant difference. The PHA content was as high as 90 wt% when 0.48 g/L of sodium valerate was fed, but PHA content was reduced to around 7076 wt% with increasing concentrations of sodium valerate. Generally, the copolymer concentrations were lower than that of the P(3HB) homopolymer produced in previous experiments [10]. The 3HV monomer composition was found to increase proportionally with the amount of precursors fed. To further examine the effects of the mixture of jatropha oil and sodium valerate or sodium propionate on the generation of 3HV for PHA biosynthesis, sodium valerate and sodium propionate were fed by standardizing the amount of carbon present in the precursors. This procedure enabled the calculation of the conversion percentage of carbon in the precursors into 3HV units. The calculation was based on the carbon content of the total 3HV units incorporated in P(3HB-co-3HV) and total carbon provided by precursors. In general, the conversion percentage of sodium valerate into 3HV units in the presence of jatropha oil was in the range of 2431% with an average of 28%, which was 1.6-fold higher than the average conversion percentage for sodium propionate (17%). Our results indicated that sodium valerate was more efciently utilized by the cells for biosynthesis of 3HV monomer when compared to sodium propionate because the conversion of sodium valerate to 3HV monomer (2131%) was higher than that of sodium propionate (1618%) (Table 1). The 3HV monomer composition in P(3HB-co-3HV) produced from sodium valerate addition was higher (342 mol%) compared to that produced from sodium pro-

pionate (227 mol%). The highest 3HV fraction of 42 mol% was obtained when 4.32 g/L of sodium valerate was added. However, only 27 mol% of 3HV was obtained with sodium propionate. 3.2. Biosynthesis of P(3HB-co-3HHx) using jatropha oil as sole carbon source The feasibility of jatropha oil as the sole carbon source to produce copolymer by transformant C. necator was also evaluated in this study. Different concentrations of jatropha oil were tested for the biosynthesis of P(3HB-co-3HHx) by C. necator PHB 4/pBBREE32d13 (Fig. 1A and B). Cell dry weight, total PHA, PHA content, PHA composition and residual oil were studied in this experiment. The CDW and total PHA increased with increasing concentrations of jatropha oil of up to 12.5 g/L, and these values decreased with concentrations of oil higher than 12.5 g/L. Meanwhile, the PHA content showed an increase with up to 10 g/L of jatropha oil fed, and this value remained constant in the range of 7884 wt% for higher concentrations of oil used. Conversely, the amount of residual oil was below 1 g/L when the amount of jatropha oil supplied ranged from 2.5 g/L to 10 g/L. The residual oil showed an increasing trend from 1.9 g/L to 7.3 g/L when the concentration of jatropha oil was increased from 12.5 g/L to 20 g/L. The most suitable concentration of jatropha oil for C. necator PHB 4/pBBREE32d13 was 10 g/L. When 10 g/L of jatropha oil was used, cell dry weight, total PHA and PHA content were 8.0 g/L, 6.7 g/L and 84 wt%, respectively, and PHA compositions of 97 mol% 3HB and 3 mol% 3HHx were determined. Based on this experiment, variations in the concentration of jatropha oil did not have a signicant effect on the 3HHx molar fraction. The monomer composition of 3HHx remained in the range of 34 mol% after 48 h of cultivation. After screening for the optimal concentration of jatropha oil (10 g/L), the time prole of P(3HB-co-3HHx) biosynthesis with supplementation of 10 g/L jatropha oil was performed. The purpose of this experiment was to study the trend in the 3HHx molar fraction and PHA production. For this experiment, cells were harvested at 12-h intervals up to 48 h of cultivation. Cell biomass and PHA production increased during the 48 h cultivation period (Fig. 2A and

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Fig. 1. Biosynthesis of P(3HB-co-3HHx) by Cupriavidus necator PHB 4/pBBREE32d13 with different concentrations of jatropha oil. (A) PHA content and PHA composition. (B) Total PHA, CDW and residual oil. Cells were cultivated in 100 mL of MM for 48 h at 30 C in a 500 mL ask. Data shown are the means of triplicate measurements. Mean data accompanied by different alphabet letters are signicantly different (Tukeys HSD test, p < 0.05).

Fig. 2. Time prole of P(3HB-co-3HHx) biosynthesis by Cupriavidus necator PHB 4/pBBREE32d13. (A) PHA content and PHA composition. (B) Total PHA and CDW. Cells were cultivated in 100 mL of MM with 10 g/L of jatropha oil for 48 h at 30 C and 200 rpm in a 500 mL ask. Mean data accompanied by different alphabet letters are signicantly different (Tukeys HSD test, p < 0.05).

B). The PHA content increased from 14 to 75 wt%, CDW increased from 1.2 to 8.7 g/L and total PHA increased from 0.2 to 6.5 g/L. The molar fraction of 3HHx was highest at 12 h (4 mol%), and this value then decreased slightly and remained at 2 mol% until the end of the incubation period. 3.3. Characterization of P(3HB-co-3HHx) and P(3HB-co-3HV) copolymers The P(3HB-co-3HHx) copolymer synthesized by C. necator PHB 4/pBBREE32d13 for different incubation periods was characterized to understand the changes in the properties of PHA produced. The P(3HB-co-3HHx) copolymer showed fairly high weight-average molecular weights, Mw , of 16.724.2 105 Da and broad polydispersities, Mw /Mn , of 3.14.8 (Table 2). Higher molecular weights were recorded at the early stages of cultivation. The thermal properties of P(3HB-co-2 mol% 3HHx) were similar to those of P(3HB-co-3HV) with 4 mol% of 3HV monomer (Table 3). GPC analysis revealed that the Mn and Mw values for P(3HB-co3HV) with 442 mol% of 3HV were in the range of 2.45.1 105 Da and 9.018.4 105 Da, respectively (Table 3). The polydispersity index remained in the range of 3.13.9. The highest Mw of 18.4 105 Da was determined for P(3HB-co-27 mol% 3HV). The Mw of P(3HB-co-3HV) and P(3HB-co-3HHx) produced in this study were moderately high. Increasing the 3HV composition did not result in relative effects on the molecular weight of the copolymer produced, and this result is also consistent with that of a previous study [24]. In this study, the thermal properties of moderately high-molecular-

weight P(3HB-co-3HV) and P(3HB-co-3HHx) copolymers produced from jatropha oil, either solely or cooperatively with precursor, were comparable to those of copolymers produced from other plant oils [6,7,18]. Multiple melting peaks were also observed for the P(3HB-co3HV) copolymers. The incorporation of comonomers, such as 3HV and 3HHx monomers, has been shown to improve the thermal and mechanical properties of P(3HB) [18,25]. The melting point of P(3HB) was reduced from 180 C when comonomer was incorporated into the P(3HB) homopolymer [1]. A similar observation was also made in this study (Table 3). P(3HB-co-2 mol% 3HHx) exhibited similar thermal properties to P(3HB-co-4 mol% 3HV). The incorporation of 3HHx and 3HV resulted in decreases in the Tg , Tm and Td to 1.8 C, 146150 C and 251252 C, respectively. Generally, Tm decreased from 150 C to a minimum of 131 C as the 3HV molar fraction was increased to 22 mol%, but Tm then increased back to 162 C as the 3HV molar fraction was further increased to 42 mol%. The trend of decreasing Tm followed by an increase as the molar fraction of 3HV is increased represents pseudoeutectic melting behavior of the isomorphism in the P(3HB-co-3HV) with the transition of crystal phases from the P(3HB) lattice to the P(3HB-co3HV) lattice [26]. According to several studies, the pseudoeutectic composition for the transition of the crystal phase ranged from approximately 30 to 56 mol% of 3HV [2729]. A lower pseudoeutectic composition at 25 mol% of 3HV and narrow changes of Tm for the second heating (154174 C) were reported [30]. Generally, the Tg and Hm values for P(3HB-co-3HV) dropped from 1.8 to 6.1 C and from 58.7 to 3.5 J/g, respectively, as the 3HV

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Table 2 Time prole changes in the molecular weights of P(3HB-co-3HHx) copolymers synthesized from 10 g/L jatropha oil as the sole carbon source. Data shown are the means of triplicate. Mean data accompanied by different superscript alphabets are signicantly different (Tukeys HSD test, p < 0.05). Harvesting time (h) 3HHx molar fraction (mol%) Molecular weight Mn (105 Da) 12 24 36 48 4 2 2 2 3.1 6.2a 4.9a 5.4a
a

Mw (105 Da) 24.2 21.1a 21.9a 16.7a

a

Mw /Mn 4.3a 3.4a 4.8a 3.1a 1.4 0.8 1.3 0.3

2.2 0.8 1.6 0.9

8.3 2.2 0.9 1.2

Table 3 Characteristics of P(3HB-co-3HV) and P(3HB-co-3HHx) copolymers biosynthesized by wild type and transformed strains of C. necator from jatropha oil as the main carbon source. Data shown are the means of triplicate. Mean data accompanied by different superscript alphabets are signicantly different (Tukeys HSD test, p < 0.05). PHA copolymers P(3HB-co-2 mol% 3HHx) P(3HB-co-4 mol% 3HV) P(3HB-co-10 mol% 3HV) P(3HB-co-22 mol% 3HV) P(3HB-co-27 mol% 3HV) P(3HB-co-42 mol% 3HV) Mn (105 ) 5.4 4.2bc 3.6b 2.4a 5.1c 4.6bc
c

Mw (105 ) 16.7 13.8b 11.1a 9.0a 18.4c 14.3b

c

Mw /Mn 3.1 3.3a 3.1a 3.9a 3.6a 3.1a

a

Tg ( C) 1.8 1.8 2.4 0.4 2.8 6.1

Tm ( C) 146, 158 150, 163 145, 157 131, 162 164 162

Hm (J/g) 50.2 58.7 47.5 24.2 1.4 3.5

Td ( C) 251 252 252 254 252 253

0.9 0.1 0.4 0.2 0.7 0.4

1.2 1.0 0.8 0.4 1.2 0.5

0.3 0.2 0.1 0.4 0.3 0.3

fraction increased from 4 to 42 mol%. The low Hm value obtained in this study, which was nearly zero, indicated that the P(3HBco-3HV) copolymer possessed low crystallinity and thus remained in the amorphous phase with no cocrystallization [31]. Quenched P(3HB-co-3HV) with intermediate compositions (3471 mol%) did not crystallize during the second heating [32]. This result explains why no melting peaks were observed at lower temperatures for P(3HB-co-3HV) with 3HV content as high as 27 mol% and 42 mol%. Since the crystallinity is so low, the higher melting temperature might not reect the true phase change of crystal but might indicate the coexistence of copolymers with a lower 3HV fractions [29] or rearrangement of crystal structure during the heating of DSC [33,34]. As reported in other studies, increased incorporation of the 3HV or 3HHx monomer into the copolymers decreased the Tg [25,35]. The Td values for all of the P(3HB-co-3HV) copolymers produced using mixtures of jatropha oil and 3HV precursors were in the range of 252254 C.

4. Discussion Jatropha oil is attracting signicant attention as a potential renewable resource for the production of biodiesel and the cost of production using jatropha oil is expected to be lower than that using rapeseed oil, soybean oil, palm oil and waste cooking oil [17]. In addition, unlike all of the other vegetable oils, jatropha oil is non-edible because of the presence of toxins. Consequently, jatropha oil has high potential as a feedstock for PHA biosynthesis by microorganisms. Jatropha oil was found to be a suitable feedstock for P(3HB) production in a previous study [10]. P(3HB) homopolymer is known to be brittle and stiff [2], and the incorporation of a second monomer into the homopolymer chain has been shown to improve the properties of the resulting polymer [36]. P(3HB-

co-3HV) and P(3HB-co-3HHx) are among the most well-studied copolymers. These copolymers are more exible and have lower melting temperatures, which yields a wider processing window [36]. The best proven strategy to produce P(3HB-co-3HV) with controlled composition is by co-feeding sugars with specic precursors of 3HV, such as propionic acid. Recent studies have shown that the co-feeding of 3HV precursors with triglycerides also produces P(3HB-co-3HV) with controlled composition [6]. Jatropha oil is rich in oleic acid (42%), which is known to improve the productivity of P(3HB-co-3HV) in C. necator by inducing cell growth, but this component simultaneously decreases the 3HV fraction [37]. In a previous study, the 3HV fraction increased when the ratio of the precursors and main carbon sources increased [38]. The composition of 3HV is generally inuenced by the concentration and the ratio of the precursors. Thus, in this experiment, the ratio of jatropha oil and the precursor was varied while xing the total amount of carbon in the culture medium. Addition of 3HV precursors was found to signicantly reduce the CDW and PHA accumulation (Table 1). A 40% reduction in CDW was observed, as compared to that obtained in the previous study [10] when jatropha oil was used as the sole carbon source. The PHA content was signicantly affected when the concentration of sodium valerate exceeded 0.48 g/L. In this study, a concentration of fatty acids of only 0.48 g/L was found to exhibit a certain level of inhibition towards cell growth and PHA synthesis. However, the salt forms of valeric acid and propionic acid used in this study were considered to be less toxic [39]. Table 1 shows that the incorporation of 3HV into the copolymer increased proportionally with the concentration of sodium valerate or sodium propionate added, and this result is similar to that observed in previous studies [23,40]. Sodium valerate can be converted via the -oxidation cycle into a 3-hydroxyvaleryl-CoA intermediate that can be incorporated directly into P(3HB-co-3HV) without catabolism [41]. The

Table 4 The yield and productivity of copolymer P(3HB-co-3HHx) in C. necator PHB 4/pBBREE32d13 and C. necator H16CAc supplemented with oils. Strains Carbon source CDW (g/L) 8.0 4.3 3.2 5.2 PHA content (wt%) 84 87 83 89 Total PHA (g/L) 6.7 3.7 2.7 4.6 Yield (%) (g-PHA/g-carbon source) 67 74 15 51 Productivity (PHA/L/h) 0.14 0.05 0.04 0.06 3HHx composition (mol%) 3 5 3.5 0.7 Reference

C. necator PHB 4/pBBREE32d13 C. necator PHB 4/pBBREE32d13 C. necator PHB 4/pBBREE32d13 C. necator H16CAc

Jatropha oil (10 g/L) CPKO (5 g/L) Soybean oil (18 g/L) Soybean oil (9 g/L)

This study [7] [16] [44]

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3HV molar fraction produced from sodium valerate was almost 2fold higher than that produced from sodium propionate, which is similar to results observed in a previous study [42]. The low conversion percentage of short-chain fatty acids is common and may be due to the accumulation of undissociated fatty acids in the cells, which inhibits substrate utilization [43]. However, the conversion percentages of sodium valerate and sodium propionate into 3HV monomer in this study are relatively higher than those reported in some studies [6,21]. The ability of jatropha oil to act as a sole carbon source for the biosynthesis of P(3HB-co-3HHx) by the transformant, C. necator PHB 4/pBBREE32d13, was also tested. Fig. 1 shows the effects of different concentrations of jatropha oil on the production of copolymer when the nitrogen concentration is xed at a constant value. A high C/N ratio in the range of 2050 is commonly used for PHA accumulation in bacterial cells [6]. In this study, a concentration of jatropha oil at 10 g/L (C/N ratio = 30) resulted in excellent cell growth and PHA accumulation. After the oil is hydrolyzed and broken down into glycerol and fatty acids by the lipase, the fatty acids enter the cell membrane and undergo -oxidation [44,45]. Based on the report of Loo et al. [7], a concentration of 5 g/L of palm kernel oil (PKO) fed to transformant C. necator PHB 4/pBBREE32d13 produced CDW and PHA contents of 4.3 g/L and 87 wt%, respectively, after 72 h of cultivation. Recent studies in our lab with crude palm kernel oil (CPKO) showed that the P(3HB-co-3HHx) copolymer content and the CDW can be further improved up to 87 wt% and 11.3 g/L, respectively (unpublished data). In comparison, 10 g/L of jatropha oil yielded 8 g/L and 84 wt% of CDW and P(3HB-co-3HHx) content, respectively, in 48 h of cultivation. Therefore, jatropha oil provides comparable yield and productivity (67% and 0.14 g-PHA/L/h, respectively) to CPKO. Furthermore, the yield and productivity of PHA from jatropha oil was higher than that reported by some studies [18,46] (Table 4). Palmitic acid, oleic acid, and linoleic acid were reported to yield good cell growth of C. necator [5], and these fatty acids are primary components of jatropha oil with a composition of 17.1%, 42% and 34.8% for palmitic acid, oleic acid and linoleic acid, respectively [10]. We were also interested in studying the trend of 3HHx incorporation into the copolymer, so we studied the time prole of the biosynthesis to determine the effect of jatropha oil on the 3HHx molar fraction. Loo [47] reported that the molar fraction of 3HHx at an earlier stage of cultivation was the highest (15 mol% at 12 h), but this fraction decreased and then remained constant at later stages of cultivation. In this study, the 3HHx molar fraction at 12 h was 4 mol% (Fig. 2), and it decreased to 2 mol% at 24 h and remained constant until the end of the cultivation period. Unlike PKO and CPKO, using jatropha oil resulted in a P(3HB-co-3HHx) copolymer with a lower 3HHx molar fraction at the earlier stage. The reason of the higher molar fraction of 3HHx at the beginning of the fermentation is still remained unknown. The time prole study also revealed the production amount of 3HHx comonomer for each incubation period; although the 3HHx fraction decreased with increasing cultivation period, the highest amount of 3HHx (0.13 g/L) was produced at 48 h. The productivity of 3HHx monomer was comparable to that reported in a previous study [18]. We have shown that jatropha oil in combination with precursor carbon sources is suitable for the biosynthesis of PHA copolymers by C. necator H16. In addition, when jatropha oil was fed as the sole carbon source to C. necator PHB 4/pBBREE32d13, P(3HB-co3HHx) copolymer was biosynthesized. We were also able to control the compositions of the P(3HB-co-3HV) copolymers by varying the concentration of the 3HV precursors. Jatropha oil supported both good cell growth and the biosynthesis of PHA copolymers by the wild-type and transformed strains of C. necator. The thermal properties and molecular weights of P(3HB-co-3HV) and P(3HBco-3HHx) copolymers produced from jatropha oil either solely or

cooperatively with precursor were similar to those of copolymers produced from other sugars and plant oils.

Acknowledgements This work was supported by a short-term research Grant from the Universiti Sains Malaysia. K.S. Ng thanks the USM Fellowship for nancial support. We are grateful to Dr. Ling Lay Pee for providing us with the jatropha oil used in this study.

References
[1] Doi Y. Microbial polyesters. New York: VCH; 1990. [2] Sudesh K, Abe H, Doi Y. Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog Polym Sci 2000;25:150355. [3] Chen G-Q, Wu Q. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials 2005;26:656578. [4] Steinbchel A, Fchtenbusch B. Bacterial and other biological systems for polyester production. Trends Biotechnol 1998;16:41927. [5] Kahar P, Tsuge T, Taguchi K, Doi Y. High yield production of polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant strain. Polym Degrad Stabil 2004;83:7986. [6] Lee W-H, Loo C-Y, Nomura CT, Sudesh K. Biosynthesis of polyhydroxyalkanoate copolymers from mixtures of plant oils and 3-hydroxyvalerate precursors. Bioresour Technol 2008;99:684451. [7] Loo C-Y, Lee W-H, Tsuge T, Doi Y, Sudesh K. Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from palm oil products in a Wautersia eutropha mutant. Biotechnol Lett 2005;27:140510. [8] Fukui T, Doi Y. Efcient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain. Appl Microbiol Biotechnol 1998;49:3336. [9] Sudesh K, Iwata T. Sustainability of biobased and biodegradable plastics. Clean 2008;36:43342. [10] Ng K-S, Ooi W-Y, Goh L-K, Shenbagarathai R, Sudesh K. Evaluation of jatropha oil to produce poly(3-hydroxybutyrate) by Cupriavidus necator H16. Polym Degrad Stabil 2010;95:13659. [11] Kumar A, Sharma S. An evaluation of multipurpose oil seed crop for industrial uses (Jatropha curcas L.): a review. Ind Crop Prod 2008;28:110. [12] Openshaw K. A review of Jatropha curcas: an oil plant of unfullled promise. Biomass Bioenergy 2000;19:115. [13] Kammann KP, Phillips AI. Sulfurized vegetable oil products as lubricant additives. J Am Oil Chem Soc 1985;62:91723. [14] Martnez-Herrera J, Siddhuraju P, Francis G, Dvila-Ortz G, Becker K. Chemical composition, toxic/antimetabolic constituents, and effects of different treatments on their levels, in four provenances of Jatropha curcas L. from Mexico. Food Chem 2006;96:809. [15] Adebowale KO, Adedire CO. Chemical composition and insecticidal properties of the underutilized Jatropha curcas seed oil. Afr J Biotechnol 2006;5:9016. [16] Akintayo ET. Characteristics and composition of Parkia biglobbossa and Jatropha curcas oils and cakes. Bioresour Technol 2004;92:30710. [17] Lim S, Teong LK. Recent trends, opportunities and challenges of biodiesel in Malaysia: an overview. Renew Sust Energ Rev 2010;14:93854. [18] Tsuge T, Saito Y, Kikkawa Y, Hiraishi T, Doi Y. Biosynthesis and compositional regulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) in recombinant Ralstonia eutropha expressing mutated polyhydroxyalkanoate synthase genes. Macromol Biosci 2004;4:23842. [19] Doi Y, Kitamura S, Abe H. Microbial synthesis and characterization of poly(3hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules 1995;28:48228. [20] Braunegg G, Sonnleitner B, Lafferty RM. A rapid gas chromatographic method for the determination of poly- -hydroxybutyric acid in microbial biomass. Appl Microbiol Biotechnol 1978;6:2937. [21] Du GC, Chen J, Yu J, Lun S. Feeding strategy of propionic acid for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with Ralstonia eutropha. Biochem Eng J 2001;8:10310. [22] Khanna S, Srivastava AK. Production of poly(3-hydroxybutyric-co-3hydroxyvaleric aicd) having a high hydroxyvalerate content with valeric acid feeding. J Ind Microbiol Biotechnol 2007;34:45761. [23] Shang L, Yim SC, Park HG, Chang HN. Sequential feeding of glucose and valerate in a fed-batch culture of Ralstonia eutropha for production of poly(hydroxybutyrate-co-hydroxyvalerate) with high 3-hydroxyvalerate fraction. Biotechnol Progr 2004;20:1404. [24] Volova TG, Kalacheva GS. The synthesis of hydroxybutyrate and hydroxyvalerate copolymers by the bacterium Ralstonia eutropha. Microbiology 2005;74:549. [25] Savenkova L, Gercberga Z, Bibers I, Kalnin M. Effect of 3-hydroxy valerate content on some physical and mechanical properties of polyhydroxyalkanoates produced by Azotobacter chroococcum. Process Biochem 2000;36:44550. [26] Yoshie N, Saito M, Inoue Y. Structural transition of lamella crystals in a isomorphous copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Macromolecules 2001;34:895360.

1578

K.-S. Ng et al. / Process Biochemistry 46 (2011) 15721578 [38] Choi J-C, Shin H-D, Lee Y-H. Modulation of 3-hydroxyvalerate molar fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) using Ralstonia eutropha transformant co-amplifying phbC and NADPH generation-related zwf genes. Enzyme Microb Technol 2003;32:17885. [39] Loo C-Y, Sudesh K. Biosynthesis and native granule characteristics of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Delftia acidovorans. Int J Biol Macromol 2007;40:46677. [40] Abdelhad HM, Hafez AMA, El-sayed AA, Khodair TA. Copolymer [P(HB-co-HV)] production as affected by strains and fermentation techniques. J Appl Sci Res 2009;5:34353. [41] Doi Y, Tamaki A, Kunioka M, Soga K. Production of copolyesters of 3hydroxybutyrate and 3-hydroxyvalerate by Alcaligenes eutrophus from butyric and pentanoic acids. Appl Microbiol Biotechnol 1988;28:3304. [42] Bhubalan K, Lee W-H, Loo C-Y, Yamamoto T, Tsuge T, Doi Y, Sudesh K. Controlled biosynthesis and characterization of poly(3-hydroxybutyrate-co3-hydroxyvalerate-co-3-hydroxyhexanoate) from mixtures of palm kernel oil and 3HV-precursors. Polym Degrad Stabil 2008;93:1723. [43] Wang J, Yue Z-B, Sheng G-P, Yu H-Q. Kinetic analysis on the production of polyhydroxyalkanoates from volatile fatty acids by Cupriavidus necator with a consideration of substrate inhibition, cell growth, maintenance, and product formation. Biochem Eng J 2010;49:4228. [44] Akiyama M, Tsuge T, Doi Y. Environmental life cycle comparison of polyhydroxyalkanoates produced from renewable carbon resources by bacterial fermentation. Polym Degrad Stabil 2003;80:18394. [45] Anderson AJ, Dawes EA. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 1990;54:45072. [46] Mifune J, Nakamura S, Fukui T. Engineering of pha operon on Cupriavidus necator chromosome for efcient biosynthesis of poly(3-hydroxybutyrate-co-3hydroxyhexanoate) from vegetable oil. Polym Degrad Stabil 2010;95:130512. [47] Loo CY. Synthesis and characterization of poly(3-hydroxybutyrate-co-3hydroxyhexanoate) from palm oil products by Ralstonia eutropha PHB 4 harboring the polyhydroxyalkanoate synthase gene of Aeromonas caviae. M.Sc. thesis. Malaysia: Universiti Sains Malaysia; 2007. p. 60.

[27] Bluhm TL, Hamer GK, Marchessault RH, Fyfe CA, Veregin RP. Isodimorphism in bacterial poly( -hydroxybutyrate-co- -hydroxyvalerate). Macromolecules 1986;19:28716. [28] Mitomo H, Morishita N, Doi Y. Composition range of crystal phase transition of isodimorphism in poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Macromolecules 1993;26:580911. [29] Mitomo H, Morishita N, Doi Y. Structural changes of poly(3-hydroxybutyrateco-3-hydroxyvalerate) fractionated with acetonewater solution. Polymer 1995;36:25738. [30] Keenan TM, Nakas JP, Tanenbaum SW. Polyhydroxyalkanoate copolymers from forest biomass. J Ind Microbiol Biotechnol 2006;33:61626. [31] Da Silva MG, Vargas H, Poley LH, Rodriguez RS, Baptista GB. Structural impact of hydroxyvalerate in polyhydroxyalkanoates (PHAscl ) dense lm monitored by XPS and photothermal methods. J Brazil Chem Soc 2005;16:7905. [32] Scandola M, Ceccorulli G, Doi Y. Viscoelastic relaxations and thermal properties of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate). Int J Biol Macromol 1990;12: 1127. [33] Saito M, Inoue Y, Yoshie N. Cocrystallization and phase segregation of blends of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Polymer 2001;42:557380. [34] Yoshie N, Menju H, Sato H, Inoue Y. Complex composition distribution of poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Macromolecules 1995;28:651621. [35] Keenan TM, Tanenbaum SW, Stipanovic AJ, Nakas JP. Production and characterization of poly- -hydroxyalkanoate copolymers from Burkholderia cepacia utilizing xylose and levulinic acid. Biotechnol Progr 2004;20:1697704. [36] Tsuge T. Metabolic improvements and use of inexpensive carbon sources in microbial production of polyhydroxyalkanoates. J Biosci Bioeng 2002;94:57984. [37] Marangoni C, Furigo A, Falco de Arago GM. Oleic acid improves poly(3hydroxybutyrate-co-3-hydroxyvalerate) production by Ralstonia eutropha in inverted sugar and propionic acid. Biotechnol Lett 2000;22:16358.