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Process Biochemistry 46 (2011) 1572–1578

Contents lists available at ScienceDirect

Process Biochemistry

jo u rn al hom epa ge: www .e lsev ier .com/ locate /procbio

iosynthesis and characterization ofoly(3-hydroxybutyrate-co-3-hydroxyvalerate) andoly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymers using jatropha oils the main carbon source

o-Sin Nga, Yoke-Ming Wonga, Takeharu Tsugeb, Kumar Sudesha,∗

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

r t i c l e i n f o

rticle history:eceived 8 December 2010eceived in revised form 4 April 2011ccepted 26 April 2011

eywords:atropha oil

a b s t r a c t

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [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 necatorH16 from a mixture of jatropha oil and sodium valerate, and P(3HB-co-3HHx) with a 3HHx monomer of3 mol% was produced by transformant C. necator PHB−4/pBBREE32d13 harboring Aeromonas caviae PHAsynthase, 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

hysic nutatropha curcasoly(3-hydroxybutyrate-co-3-ydroxyvalerate)oly(3-hydroxybutyrate-co-3-ydroxyhexanoate)

from jatropha oil were essentially the same as those produced from other, more established carbonsources, such as sugars and other plant oils. This study demonstrates that jatropha oil is a potentialrenewable carbon source for the large-scale production of copolymers by C. necator and its transformant.

© 2011 Elsevier Ltd. All rights reserved.

ioplastics

. Introduction

Polyhydroxyalkanoate (PHA) is a polymer that is accumulatedy bacteria as a carbon and energy storage material in the pres-nce of an excess amount of carbon and a limited supply of anotherutrient, which can be in the form of nitrogen, sulfur, phosphorus,agnesium or oxygen [1]. Among various known biodegradable

olymeric materials, PHA is known to be a fully biodegradablelternative to conventional plastics [2]. In terms of its applications,HA is a good material for bioplastics and implant biomaterials dueo its biodegradability, biocompatibility, thermoprocessibility andustainability [3].

Although PHA is a good substitute for petroleum-based plastics,he high cost of production makes PHA more expensive than con-entional plastics [4]. In an effort to reduce the production cost ofHAs, various carbon sources, such as plant oils and sugars, have

een explored for use in PHA production. To date, the use of variouslant oils, including soybean oil [5], palm oil [6,7], coconut oil [6]nd 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).

359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.procbio.2011.04.012

However, the use of these edible oils as primary carbon sourcesfor bioplastic production may not be sustainable [9]. Continuousconversion of edible oils to bioplastics may lead to a shortage infood supplies and cause inflation in certain countries, especially inthird world countries. Therefore, our study aims to evaluate the useof jatropha oil, which is a non-edible plant oil, for the productionof PHA copolymers. In a previous study, we showed that jatrophaoil can be used by Cupriavidus necator H16 to grow and synthesizepoly(3-hydroxybutyrate) [P(3HB)] homopolymer [10]. However, itis not known if jatropha oil affects the incorporation of comonomerand the properties of PHA copolymers. Jatropha curcas, also knownas physic nut, is a multipurpose shrub that can reach a height of 20 ftand has glabrous branchlets [11]. The life expectancy of J. curcas isalmost 50 years, and this shrub is distributed natively in Central andSouth America, including in Mexico, Brazil and Argentina and cannow also be found in many parts of Africa and Asia [12]. J. curcas ischaracterized as a hardy, highly adaptable, drought-resistant cropand 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 canbe considered to be a biodiesel fuel because it has a low oil viscositywhen compared to soybean, cottonseed and sunflower oils [13]. Theblackish J. curcas seeds contain five main toxins, which are phorbol

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sters, phytates, trypsin inhibitors, lectins and curcin [14]. Besides, variety of terpene alcohols and sterols were detected in jatrophail [15,16]; thus, to some extent, jatropha oil is toxic and classifieds a non-edible oil. Regarding its use as a carbon source for PHAiosynthesis, it is not known whether the toxins and/or any othernknown components in the jatropha oil have any effects on thearious monomer supplying pathways involved in the synthesis ofhe PHA copolymers.

In Malaysia, Jatropha has been put into consideration as a sup-lementary renewable resource in addition to palm oil to balancehe renewable energy supply and exportation of biodiesel [17], andatropha oil is a potential renewable feedstock for bioplastics. Rec-gnizing the potential of Jatropha as a renewable energy source,any industrial companies are now investing in its research and

ultivation. By the end of 2010, the cultivation area for Jatrophas expected to reach 1 million ha [17]. For the above reasons, thistudy extended the evaluation of jatropha oil for the production andharacterization of PHA copolymers. The ability to biosynthesizeHA copolymers from the co-feeding of jatropha oil and precursorssodium valerate and sodium propionate) was investigated usingild-type C. necator H16. In addition, copolymer biosynthesis from

atropha oil using transformant C. necator PHB−4/pBBREE32d13as evaluated.

. Materials and methods

.1. Bacterial strain and maintenance

C. necator H16 (formerly known as Alcaligenes eutrophus, Ralstonia eutropha andautersia eutropha) and transformant C. necator PHB−4/pBBREE32d13 harboring

he PHA synthase gene of Aeromonas caviae [18] were used throughout this study.or short-term maintenance, the bacterial strains were routinely streaked ontoutrient-rich (NR) agar plates with the following composition (per liter): 10 g pep-one, 10 g meat extract and 2 g yeast extract [19]. Kanamycin at a concentration of0 mg/L was added into the agar for maintenance of the transformant strain plas-id. For long-term storage, the bacteria were maintained in a 25% (v/v) glycerol

tock solution. The glycerol stock was prepared by the addition of 12.5 mL of purelycerol to an overnight culture of the bacterial cells in 50 mL of NR. The tubes weretored in aliquots of 1 mL at −20 ◦C.

.2. Carbon source

Jatropha oil (Sarawak, Malaysia) was used solely and in conjunction with 3HVrecursors to produce P(3HB-co-3HHx) and P(3HB-co-3HV), respectively. The fattycid composition of jatropha oil was described in the previous paper [10]. Jatrophail was filtered with a hydrophobic PTFE membrane filter (0.2 �m pore size) andubsequently autoclaved at 121 ◦C for 15 min before the oil was added into min-ral medium (MM) broth. Sodium valerate and sodium propionate were used asHV precursors. Stock solutions of both precursors at 20% (w/v) were prepared andutoclaved separately.

.3. Cultivation and PHA synthesis

One-stage batch cultivation in shake flasks was conducted for PHA biosynthe-is. The bacterial cells were first grown in NR medium to enrich the cells. Twooops of bacteria, cultured for 16–18 h, from the NR plate were grown for 6 h in0 mL of NR medium at 30 ◦C and 200 rpm. Approximately 3% (v/v) of the inoculumOD600nm = 4.5–5) was transferred into 100 mL of MM broth and incubated for 48 ht 30 ◦C and 200 rpm for PHA accumulation. The MM was prepared according to theollowing composition (per liter): 3.32 g Na2HPO4, 2.80 g KH2PO4, 0.54 g (NH2)2CO,.25 g MgSO4·7H2O and 1 mL trace element solution [19]. The trace element solu-ion consisted of 0.22 g CoCl2·6H2O, 9.7 g FeCl3, 7.8 g CaCl2, 0.12 g NiCl2·6H2O, 0.11 grCl3·6H2O and 0.16 g CuSO4·5H2O in 1 L of 0.1 N HCl [5]. In addition, 50 mg/L ofanamycin was added for cultures of the transformant strain. To induce the biosyn-hesis of P(3HB-co-3HV), 3HV precursors were added after 12 h of cultivation. Theells were harvested at the end of the 48-h cultivation period. Centrifugation at000 rpm and 4 ◦C for 5 min using a KUBOTA 6500 was conducted to pellet the cells.

pproximately 20 mL of hexane was added to the cell pellet followed by vortexingnd centrifugation at 8000 rpm and 4 ◦C for 3 min to remove the residual oil. Thenal centrifugation (8000 rpm, 4 ◦C for 5 min) was performed after adding 50 mL ofistilled water to the pellet to remove the remaining hexane. The harvested cellsere frozen at −20 ◦C for about 24 h before freeze drying.

try 46 (2011) 1572–1578 1573

2.4. Residual oil measurement

Approximately 2 mL of culture broth was collected at 48 h and centrifuged. Thesupernatant was mixed with 5 mL of hexane and vortexed for 1 min to dissolve theresidual oil. Subsequently, 1 mL of the upper layer (hexane layer) was transferred toa pre-weighed plastic plate and left to dry in the fume hood until a constant weightwas 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 methanolysisin the presence of 15% (v/v) sulfuric acid and 85% (v/v) methanol for 140 min at100 ◦C. The resulting hydroxyacyl methyl esters were then analyzed by GC [20]. Toextract PHA from the lyophilized cells, approximately 3 g of freeze dried cells wererefluxed in 300 mL of chloroform in a ratio of 1:100 for 4 h at 60 ◦C. The refluxedsolution was cooled to room temperature and filtered to remove the cell debris. Thefiltrate was then concentrated using a rotary evaporator before it was added, drop-wise, into vigorously stirred, cool methanol. The precipitated and purified polymerwas then collected and air dried in the fume hood.

2.6. Copolymer characterization

The purified and dried extracted polymer was used for molecular weight deter-mination. The molecular weight was determined at 40 ◦C using a gel permeationchromatography (Agilent 1200 GPC) system equipped with a refractive index detec-tor and SHODEX K-802 and K-806M columns. The samples were prepared bydissolving the extracted PHA in chloroform at a concentration of 1 mg/mL. Chlo-roform was used as the eluent at a flow rate of 0.8 mL/min. The weight-averagemolecular weight (Mw), number-average molecular weight (Mn), and polydisper-sity index (Mw/Mn) were determined from the curve that was obtained. Calorimetricmeasurements (DSC) of the PHA were conducted using a Perkin Elmer Pyris 1 dif-ferential scanning calorimetry (DSC) thermal analysis system in the range of −30 ◦Cto 200 ◦C at a heating rate of 20 ◦C/min. The glass transition temperature (Tg), crys-talline melting point (Tm) and enthalpy of fusion (�Hm) were determined from theDSC thermogram of the second scan. Thermogravimetric analysis (TGA) was per-formed using a Mettler-Toledo TGA/SDTA 851 thermobalance with STARe thermalanalysis software. TGA heating of 10 mg of PHA under a nitrogen atmosphere startedat 30–900 ◦C at a heating rate of 20 ◦C/min. The decomposition temperature (Td) at5% weight loss was determined.

3. Results

3.1. Biosynthesis of P(3HB-co-3HV) copolymer from mixtures ofjatropha oil and sodium valerate or sodium propionate

In a previous study [10], jatropha oil was evaluated and foundto be a suitable, sole carbon source for the biosynthesis of poly(3-hydroxybutyrate) [P(3HB)] homopolymer. However, it is not yetknown how jatropha oil will perform when added together withprecursor carbon sources for the biosynthesis of PHA copolymers.In this study, the biosynthesis of P(3HB-co-3HV) copolymer wasinvestigated for mixtures of jatropha oil and sodium valerate orsodium propionate (Table 1). The total carbon from jatropha oiland precursor for cell growth and PHA biosynthesis was fixed at9.51 g/L, which was equivalent to the carbon content of jatrophaoil at the optimal concentration (12.5 g/L) used for the biosynthe-sis of P(3HB) in previous experiments. When the concentrationsof the precursors were increased, the concentration of jatropha oilwas decreased to result in a total carbon concentration of 9.51 g/L.The concentration of precursors mentioned in the text always refersto the carbon concentration. Here, sodium valerate or sodium pro-pionate was co-fed with jatropha oil to produce P(3HB-co-3HV)copolymers with different 3HV monomer compositions. The 3HVprecursors, such as propionic and valeric acids, have certain level oftoxicity to the cells [21,22] and have to be fed in a timely and con-trolled manner. P(3HB-co-3HV) copolymers biosynthesized fromlate feeding of the precursors resulted in the formation of copoly-

mer blends having different 3HV molar fractions [7]. In addition, thehigh content of readily accumulated P(3HB) in cells could reducethe utilization of precursors and incorporation of 3HV [22]. How-ever, the feeding of precursors at an early stage would have stronger

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Table 1Biosynthesis 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 theprecursor carbon sources.a Data shown are the means of triplicate. Mean data accompanied by different superscript alphabets are significantly different (Tukey’s 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 ofprecursors into3HV monomer(%)c

Precursors Jatropha oil 3HB 3HV

Sodium valerate0.48 9.03 9.2d ± 0.0 90d ± 1 8.3f ± 0.1 97g ± 1 3a ± 1 0.9a ± 0.1 311.44 8.07 7.5bc ± 0.5 74abc ± 2 5.6d ± 0.5 91f ± 1 9b ± 1 1.7b ± 0.4 212.40 7.11 7.3b ± 0.4 75abc ± 1 5.4cd ± 0.2 80de ± 2 20cd ± 2 1.8b ± 0.1 273.36 6.15 6.7b ± 0.4 70ab ± 1 4.7bc ± 0.3 68b ± 1 32f ± 1 2.0b ± 0.2 274.32 5.19 6.7b ± 0.2 76abc ± 3 5.1cd ± 0.4 59a ± 1 41g ± 1 1.6ab ± 0.2 29

Sodium propionate0.48 9.03 8.3cd ± 0.2 81c ± 3 6.6e ± 0.3 98g ± 1 2a ± 1 1.8b ± 0.3 171.44 8.07 7.1b ± 0.3 76abc ± 3 5.4cd ± 0.2 92fg ± 1 8ab ± 1 1.7b ± 0.2 182.40 7.11 6.8b ± 0.3 73abc ± 3 4.9bcd ± 0.3 86e ± 1 14c ± 1 1.8b ± 0.2 173.36 6.15 5.6a ± 0.3 69a ± 2 3.8a ± 0.1 77cd ± 0 23de ± 0 1.7b ± 0.2 164.32 5.19 5.6a ± 0.1 77bc ± 3 4.3ab ± 0.1 73c ± 4 27e ± 4 1.3ab ± 0.2 16

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.

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oxic effects on the cells due to the lower initial cell density that isess able to tolerate the higher concentration of precursors [23].n this study, the cells were first grown in jatropha oil for 12 h tochieve adequate cell density (about 2 g/L) with a P(3HB) contentf 35 wt% to tolerate the toxic effects of the precursors that wereed later.

Generally, the presence of higher concentrations of precursor inhe culture medium resulted in increased 3HV accumulation buteduced cell dry weight (CDW) and PHA content (Table 1). Whenhe concentrations of sodium valerate and sodium propionate werencreased from 0.48 g/L to 1.44 g/L, a significant drop in CDW from.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.44 g/L to 4.32 g/L, the residual biomass did not show any sig-ificant difference. The PHA content was as high as 90 wt% when.48 g/L of sodium valerate was fed, but PHA content was reduced toround 70–76 wt% with increasing concentrations of sodium valer-te. Generally, the copolymer concentrations were lower than thatf the P(3HB) homopolymer produced in previous experiments10]. The 3HV monomer composition was found to increase pro-ortionally with the amount of precursors fed.

To further examine the effects of the mixture of jatropha oilnd sodium valerate or sodium propionate on the generation ofHV for PHA biosynthesis, sodium valerate and sodium propionateere fed by standardizing the amount of carbon present in the pre-

ursors. This procedure enabled the calculation of the conversionercentage of carbon in the precursors into 3HV units. The cal-ulation was based on the carbon content of the total 3HV unitsncorporated in P(3HB-co-3HV) and total carbon provided by pre-ursors. In general, the conversion percentage of sodium valeratento 3HV units in the presence of jatropha oil was in the range of4–31% with an average of 28%, which was 1.6-fold higher thanhe average conversion percentage for sodium propionate (17%).ur results indicated that sodium valerate was more efficiently uti-

ized by the cells for biosynthesis of 3HV monomer when comparedo sodium propionate because the conversion of sodium valer-

te to 3HV monomer (21–31%) was higher than that of sodiumropionate (16–18%) (Table 1). The 3HV monomer composition

n P(3HB-co-3HV) produced from sodium valerate addition wasigher (3–42 mol%) compared to that produced from sodium pro-

centration of precursors (g/L) × 100%.

pionate (2–27 mol%). The highest 3HV fraction of 42 mol% wasobtained 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 solecarbon source

The feasibility of jatropha oil as the sole carbon source toproduce copolymer by transformant C. necator was also eval-uated in this study. Different concentrations of jatropha oilwere tested for the biosynthesis of P(3HB-co-3HHx) by C. neca-tor PHB−4/pBBREE32d13 (Fig. 1A and B). Cell dry weight, totalPHA, PHA content, PHA composition and residual oil were stud-ied in this experiment. The CDW and total PHA increased withincreasing concentrations of jatropha oil of up to 12.5 g/L, andthese values decreased with concentrations of oil higher than12.5 g/L. Meanwhile, the PHA content showed an increase withup to 10 g/L of jatropha oil fed, and this value remained con-stant in the range of 78–84 wt% for higher concentrations of oilused.

Conversely, the amount of residual oil was below 1 g/L whenthe 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/Lwhen the concentration of jatropha oil was increased from 12.5 g/Lto 20 g/L. The most suitable concentration of jatropha oil for C. neca-tor PHB−4/pBBREE32d13 was 10 g/L. When 10 g/L of jatropha oilwas 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 signifi-cant effect on the 3HHx molar fraction. The monomer compositionof 3HHx remained in the range of 3–4 mol% after 48 h of cultiva-tion.

After screening for the optimal concentration of jatropha oil(10 g/L), the time profile of P(3HB-co-3HHx) biosynthesis with sup-plementation of 10 g/L jatropha oil was performed. The purpose of

this experiment was to study the trend in the 3HHx molar fractionand PHA production. For this experiment, cells were harvested at12-h intervals up to 48 h of cultivation. Cell biomass and PHA pro-duction increased during the 48 h cultivation period (Fig. 2A and

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K.-S. Ng et al. / Process Biochemistry 46 (2011) 1572–1578 1575

Fig. 1. Biosynthesis of P(3HB-co-3HHx) by Cupriavidus necatorPHB−4/pBBREE32d13 with different concentrations of jatropha oil. (A) PHAcontent and PHA composition. (B) Total PHA, CDW and residual oil. Cells werecml

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ultivated in 100 mL of MM for 48 h at 30 ◦C in a 500 mL flask. Data shown are the

eans of triplicate measurements. Mean data accompanied by different alphabetetters are significantly different (Tukey’s HSD test, p < 0.05).

). The PHA content increased from 14 to 75 wt%, CDW increasedrom 1.2 to 8.7 g/L and total PHA increased from 0.2 to 6.5 g/L. The

olar fraction of 3HHx was highest at 12 h (4 mol%), and this valuehen decreased slightly and remained at 2 mol% until the end of thencubation period.

.3. Characterization of P(3HB-co-3HHx) and P(3HB-co-3HV)opolymers

The P(3HB-co-3HHx) copolymer synthesized by C. necatorHB−4/pBBREE32d13 for different incubation periods was char-cterized to understand the changes in the properties of PHAroduced. The P(3HB-co-3HHx) copolymer showed fairly higheight-average molecular weights, Mw, of 16.7–24.2 × 105 Da and

road polydispersities, Mw/Mn, of 3.1–4.8 (Table 2). Higher molec-lar weights were recorded at the early stages of cultivation.he thermal properties of P(3HB-co-2 mol% 3HHx) were simi-ar to those of P(3HB-co-3HV) with 4 mol% of 3HV monomerTable 3).

GPC analysis revealed that the Mn and Mw values for P(3HB-co-HV) with 4–42 mol% of 3HV were in the range of 2.4–5.1 × 105 Dand 9.0–18.4 × 105 Da, respectively (Table 3). The polydisper-ity index remained in the range of 3.1–3.9. The highest Mw of8.4 × 105 Da was determined for P(3HB-co-27 mol% 3HV). The Mw

f P(3HB-co-3HV) and P(3HB-co-3HHx) produced in this study were

oderately high. Increasing the 3HV composition did not result in

elative effects on the molecular weight of the copolymer produced,nd this result is also consistent with that of a previous study [24].n this study, the thermal properties of moderately high-molecular-

CDW. Cells were cultivated in 100 mL of MM with 10 g/L of jatropha oil for 48 h at30 ◦C and 200 rpm in a 500 mL flask. Mean data accompanied by different alphabetletters are significantly different (Tukey’s HSD test, p < 0.05).

weight P(3HB-co-3HV) and P(3HB-co-3HHx) copolymers producedfrom jatropha oil, either solely or cooperatively with precursor,were comparable to those of copolymers produced from other plantoils [6,7,18].

Multiple melting peaks were also observed for the P(3HB-co-3HV) copolymers. The incorporation of comonomers, such as 3HVand 3HHx monomers, has been shown to improve the thermaland mechanical properties of P(3HB) [18,25]. The melting point ofP(3HB) was reduced from 180 ◦C when comonomer was incorpo-rated into the P(3HB) homopolymer [1]. A similar observation wasalso made in this study (Table 3). P(3HB-co-2 mol% 3HHx) exhibitedsimilar thermal properties to P(3HB-co-4 mol% 3HV). The incorpo-ration of 3HHx and 3HV resulted in decreases in the Tg, Tm andTd to 1.8 ◦C, 146–150 ◦C and 251–252 ◦C, respectively. Generally,Tm decreased from 150 ◦C to a minimum of 131 ◦C as the 3HVmolar fraction was increased to 22 mol%, but Tm then increasedback to 162 ◦C as the 3HV molar fraction was further increased to42 mol%. The trend of decreasing Tm followed by an increase as themolar fraction of 3HV is increased represents pseudoeutectic melt-ing behavior of the isomorphism in the P(3HB-co-3HV) with thetransition of crystal phases from the P(3HB) lattice to the P(3HB-co-3HV) lattice [26]. According to several studies, the pseudoeutecticcomposition for the transition of the crystal phase ranged fromapproximately 30 to 56 mol% of 3HV [27–29]. A lower pseudoeu-

tectic composition at 25 mol% of 3HV and narrow changes of Tm forthe second heating (154–174 ◦C) were reported [30].

Generally, the Tg and �Hm values for P(3HB-co-3HV) droppedfrom 1.8 to −6.1 ◦C and from 58.7 to 3.5 J/g, respectively, as the 3HV

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1576 K.-S. Ng et al. / Process Biochemistry 46 (2011) 1572–1578

Table 2Time profile 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 oftriplicate. Mean data accompanied by different superscript alphabets are significantly different (Tukey’s HSD test, p < 0.05).

Harvesting time (h) 3HHx molar fraction (mol%) Molecular weight

Mn (×105 Da) Mw (×105 Da) Mw/Mn

12 4 3.1a ± 2.2 24.2a ± 8.3 4.3a ± 1.424 2 6.2a ± 0.8 21.1a ± 2.2 3.4a ± 0.836 2 4.9a ± 1.6 21.9a ± 0.9 4.8a ± 1.348 2 5.4a ± 0.9 16.7a ± 1.2 3.1a ± 0.3

Table 3Characteristics 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 carbonsource. Data shown are the means of triplicate. Mean data accompanied by different superscript alphabets are significantly different (Tukey’s HSD test, p < 0.05).

PHA copolymers Mn (×105) Mw (×105) Mw/Mn Tg (◦C) Tm (◦C) �Hm (J/g) Td (◦C)

P(3HB-co-2 mol% 3HHx) 5.4c ± 0.9 16.7c ± 1.2 3.1a ± 0.3 1.8 146, 158 50.2 251P(3HB-co-4 mol% 3HV) 4.2bc ± 0.1 13.8b ± 1.0 3.3a ± 0.2 1.8 150, 163 58.7 252P(3HB-co-10 mol% 3HV) 3.6b ± 0.4 11.1a ± 0.8 3.1a ± 0.1 2.4 145, 157 47.5 252P(3HB-co-22 mol% 3HV) 2.4a ± 0.2 9.0a ± 0.4 3.9a ± 0.4 0.4 131, 162 24.2 254

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P(3HB-co-27 mol% 3HV) 5.1c ± 0.7 18.4c ± 1.2 3.6P(3HB-co-42 mol% 3HV) 4.6bc ± 0.4 14.3b ± 0.5 3.1

raction increased from 4 to 42 mol%. The low �Hm value obtainedn this study, which was nearly zero, indicated that the P(3HB-o-3HV) copolymer possessed low crystallinity and thus remainedn the amorphous phase with no cocrystallization [31]. Quenched(3HB-co-3HV) with intermediate compositions (34–71 mol%) didot crystallize during the second heating [32]. This result explainshy no melting peaks were observed at lower temperatures for

(3HB-co-3HV) with 3HV content as high as 27 mol% and 42 mol%.ince the crystallinity is so low, the higher melting temperatureight not reflect the true phase change of crystal but might indi-

ate the coexistence of copolymers with a lower 3HV fractions [29]r rearrangement of crystal structure during the heating of DSC33,34]. As reported in other studies, increased incorporation ofhe 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

sing mixtures of jatropha oil and 3HV precursors were in the rangef 252–254 ◦C.

. Discussion

Jatropha oil is attracting significant attention as a potentialenewable resource for the production of biodiesel and the costf production using jatropha oil is expected to be lower than thatsing rapeseed oil, soybean oil, palm oil and waste cooking oil [17].

n addition, unlike all of the other vegetable oils, jatropha oil ison-edible because of the presence of toxins. Consequently, jat-opha oil has high potential as a feedstock for PHA biosynthesis byicroorganisms. Jatropha oil was found to be a suitable feedstock

or P(3HB) production in a previous study [10]. P(3HB) homopoly-er is known to be brittle and stiff [2], and the incorporation of

second monomer into the homopolymer chain has been showno improve the properties of the resulting polymer [36]. P(3HB-

able 4he yield and productivity of copolymer P(3HB-co-3HHx) in C. necator PHB−4/pBBREE32d

Strains Carbon source CDW(g/L)

PHA content(wt%)

Tota(g/L)

C. necator PHB−4/pBBREE32d13 Jatropha oil (10 g/L) 8.0 84 6.7

C. necator PHB−4/pBBREE32d13 CPKO (5 g/L) 4.3 87 3.7

C. necator PHB−4/pBBREE32d13 Soybean oil (18 g/L) 3.2 83 2.7

C. necator H16CAc Soybean oil (9 g/L) 5.2 89 4.6

.3 −2.8 164 1.4 252

.3 −6.1 162 3.5 253

co-3HV) and P(3HB-co-3HHx) are among the most well-studiedcopolymers. These copolymers are more flexible and have lowermelting temperatures, which yields a wider processing window[36].

The best proven strategy to produce P(3HB-co-3HV) with con-trolled composition is by co-feeding sugars with specific precursorsof 3HV, such as propionic acid. Recent studies have shown thatthe co-feeding of 3HV precursors with triglycerides also producesP(3HB-co-3HV) with controlled composition [6]. Jatropha oil is richin oleic acid (42%), which is known to improve the productivityof P(3HB-co-3HV) in C. necator by inducing cell growth, but thiscomponent simultaneously decreases the 3HV fraction [37]. In aprevious study, the 3HV fraction increased when the ratio of theprecursors and main carbon sources increased [38]. The composi-tion of 3HV is generally influenced by the concentration and theratio of the precursors. Thus, in this experiment, the ratio of jat-ropha oil and the precursor was varied while fixing the total amountof carbon in the culture medium.

Addition of 3HV precursors was found to significantly reducethe CDW and PHA accumulation (Table 1). A 40% reduction in CDWwas observed, as compared to that obtained in the previous study[10] when jatropha oil was used as the sole carbon source. ThePHA content was significantly affected when the concentration ofsodium valerate exceeded 0.48 g/L. In this study, a concentrationof fatty acids of only 0.48 g/L was found to exhibit a certain levelof inhibition towards cell growth and PHA synthesis. However, thesalt forms of valeric acid and propionic acid used in this study wereconsidered to be less toxic [39]. Table 1 shows that the incorpora-tion of 3HV into the copolymer increased proportionally with theconcentration 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 cycleinto a 3-hydroxyvaleryl-CoA intermediate that can be incorpo-rated directly into P(3HB-co-3HV) without catabolism [41]. The

13 and C. necator H16CAc supplemented with oils.

l PHA Yield (%)(g-PHA/g-carbonsource)

Productivity(PHA/L/h)

3HHx composition(mol%)

Reference

67 0.14 3 This study74 0.05 5 [7]15 0.04 3.5 [16]51 0.06 0.7 [44]

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HV molar fraction produced from sodium valerate was almost 2-old higher than that produced from sodium propionate, which isimilar to results observed in a previous study [42]. The low con-ersion percentage of short-chain fatty acids is common and maye due to the accumulation of undissociated fatty acids in the cells,hich inhibits substrate utilization [43]. However, the conversionercentages of sodium valerate and sodium propionate into 3HVonomer in this study are relatively higher than those reported in

ome studies [6,21].The ability of jatropha oil to act as a sole carbon source for the

iosynthesis of P(3HB-co-3HHx) by the transformant, C. necatorHB−4/pBBREE32d13, was also tested. Fig. 1 shows the effects ofifferent concentrations of jatropha oil on the production of copoly-er when the nitrogen concentration is fixed at a constant value.

high C/N ratio in the range of 20–50 is commonly used for PHAccumulation in bacterial cells [6]. In this study, a concentrationf jatropha oil at 10 g/L (C/N ratio = ∼30) resulted in excellent cellrowth and PHA accumulation. After the oil is hydrolyzed and bro-en down into glycerol and fatty acids by the lipase, the fatty acidsnter the cell membrane and undergo �-oxidation [44,45]. Basedn the report of Loo et al. [7], a concentration of 5 g/L of palm ker-el oil (PKO) fed to transformant C. necator PHB−4/pBBREE32d13roduced CDW and PHA contents of 4.3 g/L and 87 wt%, respec-ively, after 72 h of cultivation. Recent studies in our lab withrude palm kernel oil (CPKO) showed that the P(3HB-co-3HHx)opolymer content and the CDW can be further improved up to7 wt% and 11.3 g/L, respectively (unpublished data). In compar-

son, 10 g/L of jatropha oil yielded 8 g/L and 84 wt% of CDW and(3HB-co-3HHx) content, respectively, in 48 h of cultivation. There-ore, jatropha oil provides comparable yield and productivity (67%nd 0.14 g-PHA/L/h, respectively) to CPKO. Furthermore, the yieldnd productivity of PHA from jatropha oil was higher than thateported by some studies [18,46] (Table 4). Palmitic acid, oleic acid,nd linoleic acid were reported to yield good cell growth of C. neca-or [5], and these fatty acids are primary components of jatropha oilith a composition of 17.1%, 42% and 34.8% for palmitic acid, oleic

cid and linoleic acid, respectively [10].We were also interested in studying the trend of 3HHx incor-

oration into the copolymer, so we studied the time profile of theiosynthesis to determine the effect of jatropha oil on the 3HHxolar fraction. Loo [47] reported that the molar fraction of 3HHx at

n earlier stage of cultivation was the highest (15 mol% at 12 h), buthis fraction decreased and then remained constant at later stagesf cultivation. In this study, the 3HHx molar fraction at 12 h was

mol% (Fig. 2), and it decreased to 2 mol% at 24 h and remainedonstant until the end of the cultivation period. Unlike PKO andPKO, using jatropha oil resulted in a P(3HB-co-3HHx) copolymerith a lower 3HHx molar fraction at the earlier stage. The reason of

he higher molar fraction of 3HHx at the beginning of the fermenta-ion is still remained unknown. The time profile study also revealedhe production amount of 3HHx comonomer for each incubationeriod; although the 3HHx fraction decreased with increasing culti-ation period, the highest amount of 3HHx (0.13 g/L) was producedt 48 h. The productivity of 3HHx monomer was comparable to thateported in a previous study [18].

We have shown that jatropha oil in combination with precursorarbon sources is suitable for the biosynthesis of PHA copolymersy C. necator H16. In addition, when jatropha oil was fed as theole carbon source to C. necator PHB−4/pBBREE32d13, P(3HB-co-HHx) copolymer was biosynthesized. We were also able to controlhe compositions of the P(3HB-co-3HV) copolymers by varyinghe concentration of the 3HV precursors. Jatropha oil supported

oth good cell growth and the biosynthesis of PHA copolymers byhe wild-type and transformed strains of C. necator. The thermalroperties and molecular weights of P(3HB-co-3HV) and P(3HB-o-3HHx) copolymers produced from jatropha oil either solely or

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try 46 (2011) 1572–1578 1577

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

Acknowledgements

This work was supported by a short-term research Grant fromthe Universiti Sains Malaysia. K.S. Ng thanks the USM Fellowship forfinancial support. We are grateful to Dr. Ling Lay Pee for providingus 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 polyhydroxyalka-

noates: biological polyesters. Prog Polym Sci 2000;25:1503–55.[3] Chen G-Q, Wu Q. The application of polyhydroxyalkanoates as tissue engineer-

ing materials. Biomaterials 2005;26:6565–78.[4] Steinbüchel A, Füchtenbusch B. Bacterial and other biological systems for

polyester production. Trends Biotechnol 1998;16:419–27.[5] Kahar P, Tsuge T, Taguchi K, Doi Y. High yield production of polyhydroxyalka-

noates from soybean oil by Ralstonia eutropha and its recombinant strain. PolymDegrad Stabil 2004;83:79–86.

[6] Lee W-H, Loo C-Y, Nomura CT, Sudesh K. Biosynthesis of polyhydroxyalkanoatecopolymers from mixtures of plant oils and 3-hydroxyvalerate precursors.Bioresour Technol 2008;99:6844–51.

[7] Loo C-Y, Lee W-H, Tsuge T, Doi Y, Sudesh K. Biosynthesis and characterizationof poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from palm oil products ina Wautersia eutropha mutant. Biotechnol Lett 2005;27:1405–10.

[8] Fukui T, Doi Y. Efficient production of polyhydroxyalkanoates from plant oilsby Alcaligenes eutrophus and its recombinant strain. Appl Microbiol Biotechnol1998;49:333–6.

[9] Sudesh K, Iwata T. Sustainability of biobased and biodegradable plastics. Clean2008;36:433–42.

10] Ng K-S, Ooi W-Y, Goh L-K, Shenbagarathai R, Sudesh K. Evaluation of jatropha oilto produce poly(3-hydroxybutyrate) by Cupriavidus necator H16. Polym DegradStabil 2010;95:1365–9.

11] Kumar A, Sharma S. An evaluation of multipurpose oil seed crop for industrialuses (Jatropha curcas L.): a review. Ind Crop Prod 2008;28:1–10.

12] Openshaw K. A review of Jatropha curcas: an oil plant of unfulfilled promise.Biomass Bioenergy 2000;19:1–15.

13] Kammann KP, Phillips AI. Sulfurized vegetable oil products as lubricant addi-tives. J Am Oil Chem Soc 1985;62:917–23.

14] Martínez-Herrera J, Siddhuraju P, Francis G, Dávila-Ortíz G, Becker K. Chemicalcomposition, toxic/antimetabolic constituents, and effects of different treat-ments on their levels, in four provenances of Jatropha curcas L. from Mexico.Food Chem 2006;96:80–9.

15] Adebowale KO, Adedire CO. Chemical composition and insecticidal propertiesof the underutilized Jatropha curcas seed oil. Afr J Biotechnol 2006;5:901–6.

16] Akintayo ET. Characteristics and composition of Parkia biglobbossa and Jatrophacurcas oils and cakes. Bioresour Technol 2004;92:307–10.

17] Lim S, Teong LK. Recent trends, opportunities and challenges of biodiesel inMalaysia: an overview. Renew Sust Energ Rev 2010;14:938–54.

18] Tsuge T, Saito Y, Kikkawa Y, Hiraishi T, Doi Y. Biosynthesis and compositionalregulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) in recombi-nant Ralstonia eutropha expressing mutated polyhydroxyalkanoate synthasegenes. Macromol Biosci 2004;4:238–42.

19] Doi Y, Kitamura S, Abe H. Microbial synthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules 1995;28:4822–8.

20] Braunegg G, Sonnleitner B, Lafferty RM. A rapid gas chromatographic methodfor the determination of poly-�-hydroxybutyric acid in microbial biomass. ApplMicrobiol Biotechnol 1978;6:29–37.

21] Du GC, Chen J, Yu J, Lun S. Feeding strategy of propionic acid for productionof poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with Ralstonia eutropha.Biochem Eng J 2001;8:103–10.

22] Khanna S, Srivastava AK. Production of poly(3-hydroxybutyric-co-3-hydroxyvaleric aicd) having a high hydroxyvalerate content with valeric acidfeeding. J Ind Microbiol Biotechnol 2007;34:457–61.

23] Shang L, Yim SC, Park HG, Chang HN. Sequential feeding of glucose andvalerate in a fed-batch culture of Ralstonia eutropha for production ofpoly(hydroxybutyrate-co-hydroxyvalerate) with high 3-hydroxyvalerate frac-tion. Biotechnol Progr 2004;20:140–4.

24] Volova TG, Kalacheva GS. The synthesis of hydroxybutyrate and hydrox-yvalerate copolymers by the bacterium Ralstonia eutropha. Microbiology2005;74:54–9.

25] Savenkova L, Gercberga Z, Bibers I, Kalnin M. Effect of 3-hydroxy valerate con-

tent on some physical and mechanical properties of polyhydroxyalkanoatesproduced by Azotobacter chroococcum. Process Biochem 2000;36:445–50.

26] Yoshie N, Saito M, Inoue Y. Structural transition of lamella crystals ina isomorphous copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate).Macromolecules 2001;34:8953–60.

Journal Identification = PRBI Article Identification = 9229 Date: June 27, 2011 Time: 8:16 am

1 chemi

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

578 K.-S. Ng et al. / Process Bio

27] Bluhm TL, Hamer GK, Marchessault RH, Fyfe CA, Veregin RP. Isodimorphismin bacterial poly(�-hydroxybutyrate-co-�-hydroxyvalerate). Macromolecules1986;19:2871–6.

28] Mitomo H, Morishita N, Doi Y. Composition range of crystal phase transitionof isodimorphism in poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Macro-molecules 1993;26:5809–11.

29] Mitomo H, Morishita N, Doi Y. Structural changes of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fractionated with acetone–water solution. Polymer1995;36:2573–8.

30] Keenan TM, Nakas JP, Tanenbaum SW. Polyhydroxyalkanoate copolymers fromforest biomass. J Ind Microbiol Biotechnol 2006;33:616–26.

31] Da Silva MG, Vargas H, Poley LH, Rodriguez RS, Baptista GB. Structural impactof hydroxyvalerate in polyhydroxyalkanoates (PHAscl) dense film monitoredby XPS and photothermal methods. J Brazil Chem Soc 2005;16:790–5.

32] Scandola M, Ceccorulli G, Doi Y. Viscoelastic relaxations and thermalproperties of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) andpoly(3-hydroxybutyrate-co-4-hydroxybutyrate). Int J Biol Macromol 1990;12:112–7.

33] Saito M, Inoue Y, Yoshie N. Cocrystallization and phase segregation of blends ofpoly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate).Polymer 2001;42:5573–80.

34] Yoshie N, Menju H, Sato H, Inoue Y. Complex composition distri-bution of poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Macromolecules1995;28:6516–21.

35] Keenan TM, Tanenbaum SW, Stipanovic AJ, Nakas JP. Production and charac-terization of poly-�-hydroxyalkanoate copolymers from Burkholderia cepaciautilizing xylose and levulinic acid. Biotechnol Progr 2004;20:1697–704.

36] Tsuge T. Metabolic improvements and use of inexpensive carbon sources

in microbial production of polyhydroxyalkanoates. J Biosci Bioeng2002;94:579–84.

37] Marangoni C, Furigo A, Falcão de Aragão GM. Oleic acid improves poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Ralstonia eutropha ininverted sugar and propionic acid. Biotechnol Lett 2000;22:1635–8.

[

stry 46 (2011) 1572–1578

38] Choi J-C, Shin H-D, Lee Y-H. Modulation of 3-hydroxyvalerate molar fractionin poly(3-hydroxybutyrate-co-3-hydroxyvalerate) using Ralstonia eutrophatransformant co-amplifying phbC and NADPH generation-related zwf genes.Enzyme Microb Technol 2003;32:178–85.

39] Loo C-Y, Sudesh K. Biosynthesis and native granule characteristics ofpoly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Delftia acidovorans. Int J BiolMacromol 2007;40:466–77.

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 Res2009;5:343–53.

41] Doi Y, Tamaki A, Kunioka M, Soga K. Production of copolyesters of 3-hydroxybutyrate and 3-hydroxyvalerate by Alcaligenes eutrophus from butyricand pentanoic acids. Appl Microbiol Biotechnol 1988;28:330–4.

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-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) from mixtures of palm kernel oiland 3HV-precursors. Polym Degrad Stabil 2008;93:17–23.

43] Wang J, Yue Z-B, Sheng G-P, Yu H-Q. Kinetic analysis on the production ofpolyhydroxyalkanoates from volatile fatty acids by Cupriavidus necator with aconsideration of substrate inhibition, cell growth, maintenance, and productformation. Biochem Eng J 2010;49:422–8.

44] Akiyama M, Tsuge T, Doi Y. Environmental life cycle comparison of poly-hydroxyalkanoates produced from renewable carbon resources by bacterialfermentation. Polym Degrad Stabil 2003;80:183–94.

45] Anderson AJ, Dawes EA. Occurrence, metabolism, metabolic role, and industrialuses of bacterial polyhydroxyalkanoates. Microbiol Rev 1990;54:450–72.

46] Mifune J, Nakamura S, Fukui T. Engineering of pha operon on Cupriavidus necatorchromosome for efficient biosynthesis of poly(3-hydroxybutyrate-co-3-

hydroxyhexanoate) from vegetable oil. Polym Degrad Stabil 2010;95:1305–12.

47] Loo CY. Synthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from palm oil products by Ralstonia eutropha PHB−4harboring the polyhydroxyalkanoate synthase gene of Aeromonas caviae. M.Sc.thesis. Malaysia: Universiti Sains Malaysia; 2007. p. 60.