Kumar Sudesh, Ecobiomaterial Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia. E-mail: email@example.com
Aims: Burkholderia sp. USM (JCM15050) isolated from oil-polluted wastewater is capable of utilizing palm oil products and glycerol to synthesize poly(3-hydroxybutyrate) [P(3HB)]. To confer the ability to produce polymer containing 3-hydroxyhexanoate (3HHx), plasmid (pBBREE32d13) harbouring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae (phaCAc) was transformed into this strain.
Methods and Results: The resulting transformant incorporated approximately 1 ± 0·3 mol% of 3HHx in the polymer when crude palm kernel oil (CPKO) or palm kernel acid oil was used as the sole carbon source. In addition, when the transformed strain was cultivated in the mixtures of CPKO and sodium valerate, PHA containing 69 mol% 3HB, 30 mol% 3-hydroxyvalerate and 1 mol% 3HHx monomers was produced. Batch feeding of carbon sources with 0·5% (v/v) CPKO at 0 h and 0·25% (w/v) sodium valerate at 36 h yielded 6 mol% of 3HHx monomer by controlled-feeding strategies.
Conclusions: Burkholderia sp. USM (JCM15050) has the metabolic pathways to supply both the short-chain length (SCL) and medium-chain length (MCL) PHA monomers. By transforming the strain with the Aer. caviae PHA synthase with broader substrate specificity, SCL-MCL PHA was produced.
Significance and Impact of the Study: This is the first study demonstrating the ability of transformant Burkholderia to produce P(3HB-co-3HHx) from a single carbon source.
Rapid consumption of natural resources throughout our daily activities has led to overcrowding of wastes in our environment. Among the myriad wastes accumulated, synthetic plastics, which are derived from petrochemicals, have been defined as compounds that may contribute to global warming from incineration (Kim and Dale 2005). Polyhydroxyalkanoate (PHA) has received much attention as a potential substitute for synthetic and nonbiodegradable plastics.
To date, more than 150 different PHA constituents have been identified (Steinbüchel 2001). They are composed of monomers with carbon atom numbers ranging from 3 to 14 carbon atoms. Physical properties of PHA polymers range from highly crystalline material to rubber-like elastomers with increasing carbon chain length (Sudesh and Doi 2000). The type of PHA synthesized by bacteria depends on the carbon substrate and the substrate specificity of the PHA synthase in the bacterium (Ashby et al. 2001). Poly(3-hydroxybuyrate) [P(3HB)] homopolymer is the most common type of PHA synthesized. However, it is known to be a highly crystalline and brittle material (Doi 1990). To improve the mechanical properties, addition of different monomers such as 3-hydroxypropionate (Andreeβen and Steinbüchel 2010), 4-hydroxybutyrate, 3-hydroxyvalerate (3HV), 3-hydroxyhexanoate (3HHx) (Fukui and Doi 1997; Sudesh and Doi 2000; Steinbüchel and Lütke-Eversloh 2003; Bhubalan et al. 2010), polylactate (Petersen et al. 2001), poly(β-hydroxybutyrate-co-ε-caprolactone) or poly(β-hydroxybutyrate-co-lactide) (Reeve et al. 1993) has been practiced in different ways such as feeding with structurally related carbon sources, employing genetically engineered bacterial strains or by chemical modifications.
Burkholderia sp. USM (JCM15050), isolated from oil-polluted wastewater, is capable of accumulating P(3HB) homopolymer from plant oils (Chee et al. 2010b). The ability to utilize triglycerides has made this bacterium a good candidate for producing PHA. In this study, heterologous expression of the phaCAc gene of Aer. caviae was performed in the wild-type Burkholderia sp. USM (JCM15050). The resulting Burkholderia sp. transformant harboured both the inserted phaCAc and its natural PHA synthase gene (phaCBsp). This transformant serves as a host to determine the ability of this strain in supplying 3HHx monomer for PHA biosynthesis. In addition, ultrastructural studies of this transformant revealed the effect of heterologous PHA synthase gene dosage on PHA granule formation.
Materials and methods
Wild-type Burkholderia sp. USM (JCM15050) was previously isolated from oil-polluted wastewater (Chee et al. 2010b). The transformed strain of Burkholderia sp. USM (JCM15050) was obtained by transconjugation of pBBREE32d13 plasmid harbouring the PHA synthase gene of Aer. caviae (phaCAc). Escherichia coli S17-1 was used as the donor, and transconjugation was carried out according to methods described by Friedrich et al. (1981). Construction of the plasmid is described in detail elsewhere (Tsuge et al. 2004). Bacterial strains were routinely streaked on nutrient rich (NR) agar consisting of 10 g l−1 peptone, 10 g l−1 meat extract, 2 g l−1 yeast extract, 15 g l−1 agar powder (pH 7·0). For long-term storage, the strain was kept at −20°C in 25% glycerol.
Crude palm kernel oil (CPKO), palm kernel acid oil (PKAO) and glycerol were used as carbon sources throughout this study. Precursors such as sodium valerate and sodium propionate were used to generate 3HV monomer. For preparation of sodium valerate, 40 g sodium hydroxide was dissolved in 1 l of absolute ethanol by vigorous stirring. Approximately 102·14 ml of valeric acid was then slowly added into the sodium hydroxide solution that was being stirred gently. Upon completion, sodium valerate was obtained in the form of precipitant. It was later placed in an oven at 55°C for approximately 12 h to dry the excess ethanol. Sodium valerate, sodium propionate and palm oil products were sterilized separately by autoclaving at 121°C for 15 min under 15 psi.
PHA biosynthesis media and growth conditions
PHA biosynthesis was carried out using one-stage and two-stage cultivation methods. For one-stage cultivation, the strains were grown in NR broth medium for 24 h. Next, 3% (v/v) of the inoculum was transferred into nitrogen-limiting mineral salt medium (MM) with 0·5% (v/v) of the carbon sources. The MM was prepared according to Doi et al. (1995). Ammonium chloride (0·5 g l−1) was used as the nitrogen source. The medium formulation of trace element solution was obtained from Kahar et al. (2004). Carbon substrates were added aseptically into the MM medium to promote PHA synthesis. Incubation was carried out under aerobic condition, in an incubator shaker for 72 h at 37°C and 200 rev min−1 to enable cell growth and PHA accumulation. The cells were subsequently harvested, washed with hexane and finally with distilled water. The cells were then lyophilized before subjecting to gas chromatography (GC) analysis.
For two-stage cultivation, 3% (v/v) of the culture grown on NR medium was inoculated into a fresh NR medium and incubated for another 24 h. The cells were then harvested by centrifugation and washed once with sterile distilled water before aseptically transferring them into nitrogen-free MM (in the absence of ammonium chloride). Carbon substrates were added aseptically into the MM medium to promote PHA synthesis. The cells were then incubated further for 48 h, 200 rev min−1 at 37°C before harvesting.
Determination of PHA content and monomer composition in the lyophilized cells were carried out using GC analysis as described by Braunegg et al. (1978). Approximately 5 mg of lyophilized dry cells was subjected to methanolysis in 2·0 ml solution consisting of 85% (v/v) methanol and 15% (v/v) concentrated sulphuric acid and 2·0 ml chloroform at 100°C for 140 min. The reaction converts the monomer constituents to their hydroxyacyl methyl esters. After completion, the reaction mixture was allowed to cool down to room temperature before 1·0 ml of distilled water was added and then was vortexed vigorously to induce phase separation. A 0·5 ml volume of the lower chloroform layer was then mixed with 0·5 ml of caprylate methyl ester as internal standard to the ratio of 1 : 1 before proceeded to GC analysis.
Transmission electron microscope (TEM)
The harvested cells were prefixed in McDowell–Trump fixative as described by McDowell and Trump (1976) and postfixed by 1% OsO4. After a series of dehydration processes using different grades of ethanol, the cells were embedded in Spurr’s low viscosity resin (Spurr 1969). The ultrathin sections were cut and later placed onto copper grids, immersed in uranyl acetate and lead citrate solution prior to electron microscope examination (Phillip CM 12/STEM and JLM-2000FX11) at an acceleration voltage of 80 kV.
PHA biosynthesis by wild-type and transformed Burkholderia sp. USM (JCM15050) using different carbon sources in one-stage and two-stage cultivations
In our previous work, Burkholderia sp. USM (JCM15050) was found to possess the ability to utilize palm oil products for the synthesis of P(3HB) homopolymer (Chee et al. 2010b). PHA accumulation of up to 70 wt% and 58 wt% was obtained from CPKO and PKAO, respectively (Table 1). Besides, glycerol was also identified as a potential carbon source for P(3HB) production. To investigate the ability of this strain to synthesize monomers other than 3HB, it was cultivated with sodium valerate or sodium propionate as the sole or as co-precursor substrates. Co-polymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), was produced by the wild-type strain using two-stage cultivation. As shown in Table 1, when either sodium valerate or sodium propionate was fed as the sole carbon source in one-stage cultivation the cell growth was very poor, and therefore, the PHA content and composition could not be determined. In contrast, when cultivated in two-stage cultivation, the cells were able to produce P(3HB-co-3HV) from both sodium valerate and sodium propionate (Table 2). The co-polymers contained up to 78 mol% and 24 mol% of 3HV monomer, respectively.
Table 1. Biosynthesis of polyhydroxyalkanoate (PHA) by Burkholderia sp. USM (JCM15050) and transformed strain using palm oil derivatives as the sole carbon source
Table 2. Biosynthesis of polyhydroxyalkanoate (PHA) by Burkholderia sp. USM (JCM15050) and transformed strain using 3HV precursors in two-stage cultivation
Carbon sources 0·5% (w/v)
Dry cell weight (g l−1)
PHA content* (wt%)
PHA concentration (g l−1)
PHA compositions (mol%)
Tr, trace; 3HV, 3-hydroxyvalerate.
*PHA content in freeze-dried cells.
1·6 ± 0·2
16 ± 2
0·3 ± 0·02
1·3 ± 0·1
1 ± 0·2
2·0 ± 0·2
33 ± 3
0·7 ± 0·1
1·9 ± 0·2
7 ± 1
0·1 ± 0·03
Aeromonas caviae is known to produce a random co-polymer of PHA containing 3HHx monomer when grown in oil-based products (Fukui and Doi 1998) and alkanoic acids (Doi et al. 1995). Loo et al. (2005) revealed that the heterologous expression of phaCAc in C. necator H16 PHB−4 enabled the production of co-polymers containing 3HHx monomer from palm oil products. Insertion of the phaCAc into Burkholderia sp. USM (JCM15050) enabled the production of a second monomer besides 3HB, when triglycerides were fed as the sole carbon source.
Incorporation of 1 mol% of 3HHx monomer was detected in the transformed Burkholderia sp. cultivated on CPKO and PKAO (Table 1). These results demonstrated that the PhaCAc was able to incorporate 3HHx upon expression in Burkholderia sp. USM (JCM15050). As both CPKO and PKAO are rich in lauric acid (C12) and myristic acid (C14) (Chee et al. 2010b), these two fatty acids were tested separately as the sole carbon source to investigate their contribution to the synthesis of 3HHx monomer. Results showed that only lauric acid (0·5% w/v) contributed to the production of 3HHx monomer (1 mol%) (data not shown). Table 1 also shows that the utilization of glycerol by both the wild-type and transformed strain resulted in the synthesis of P(3HB) homopolymer. The cell biomass attained from glycerol was better than those from triglycerides (data not shown).
In two-stage cultivation, the PHA content increased almost 1·5 times in culture supplemented with sodium valerate in the second stage, and high 3HV monomer composition of up to 84 mol% was obtained (Table 2). Feeding of sodium valerate together with glycerol resulted in the synthesis of P(3HB-co-3HV) by the transformed strain. The 3HV monomer composition in the co-polymer was 44 mol% (Table 3). Substitution of glycerol with CPKO and the co-feeding with sodium valerate reduced the PHA content and 3HV monomer fraction to 38 wt% and 30 mol%, respectively. Approximately 1 mol% of 3HHx was incorporated into the PHA synthesized by the transformed strain when cultivated in the mixture of CPKO and sodium valerate. However, under identical culture conditions, the wild-type strain was only capable of accumulating 31 wt % of PHA with 2 mol% of 3HV composition.
Table 3. Biosynthesis of polyhydroxyalkanoate (PHA) by Burkholderia sp. USM (JCM15050) and transformed strain using mixtures of palm oil derivatives with 3HV precursors in one-stage cultivation
†0·25% (v/v) of sodium valerate or sodium propionate was added into nitrogen-limiting mineral salt medium (MM) together with 0·25% (v/v) of CPKO or glycerol at 0 h.
‡0·25% (v/v) of sodium valerate or sodium propionate was added at 36-h growth in nitrogen-limiting MM with 0·5% (v/v) of CPKO.
CPKO + sodium valerate†
1·6 ± 0·1
31 ± 3
0·5 ± 0·1
CPKO + sodium propionate†
0·9 ± 0·2
1 ± 0·1
CPKO + sodium valerate‡
2·3 ± 0·4
72 ± 3
1·7 ± 0·3
CPKO + sodium propionate‡
2·2 ± 0·3
65 ± 4
1·4 ± 0·2
Glycerol + sodium valerate†
2·0 ± 0·3
42 ± 4
0·8 ± 0·1
Glycerol + sodium propionate†
1·6 ± 0·1
47 ± 5
0·8 ± 0·01
CPKO + sodium valerate†
1·6 ± 0·2
38 ± 4
0·6 ± 0·1
CPKO + sodium propionate†
1·1 ± 0·1
43 ± 2
0·5 ± 0·1
CPKO + sodium valerate‡
1·8 ± 0·1
86 ± 1
1·5 ± 0·1
CPKO + sodium propionate‡
1·5 ± 0·6
76 ± 2
1·1 ± 0·4
To reduce the toxicity of 3HV precursors towards cell growth and PHA production, the feeding time of 3HV precursor was delayed from 0 to 36 h after the addition of CPKO. Interestingly, the changes in feeding time resulted in an increase in 3HHx monomer composition from 1 mol% to approximately 6 mol% (Table 3). Although there was an increment in 3HHx monomer compositions, the 3HV monomer composition in the transformed strain was not affected.
Under similar culture conditions, the transformed strain produced higher PHA content than the wild-type strain; 86 wt% using CPKO and sodium valerate and 76 wt% using CPKO and sodium propionate. Nevertheless, there was no variation in 3HV composition although 3HV precursors feeding time was delayed to 36 h. The molar fraction of 3HV produced using sodium valerate was approximately 34 mol%. However, only 1 mol% of 3HV was obtained using with sodium propionate.
Analysis of PHA granules formation in wild-type and transformed Burkholderia sp.
Further work was undertaken to observe the ultrastructure of PHA granules produced by the wild-type and transformed Burkholderia sp. using TEM analysis. The formation and morphology of the granules were observed every 24 h over the cultivation period of 72 h in 0·5% (v/v) CPKO. At the early stage (0 h), PHA accumulation was not observed in the cells. The TEM images revealed that some of the cells were still dividing after 24 h of cultivation in NR medium (Fig. 1a). Formation of septum could be observed when the cells began to divide. The cell cytoplasm was separated from the cell wall while a dark-stained element was found at the centre of the cells. This element was believed to be the nucleoid region. As shown in Fig. 1a, the cells underwent cell division and the so-called nucleoid region was divided into two daughter cells. At this moment, 3% (v/v) of the inoculum was transferred from NR medium into nitrogen-limiting MM.
At 24 h cultivation, a substantial amount of P(3HB) granules were accumulated intracellularly and have almost occupied the entire cells (Fig. 1b,c). These intracellular granules were closely packed because of space limitation in the cell cytoplasm. The TEM micrographs also showed that the granules were surrounded by a thin boundary layer, which separated the granules from one another. At this stage, the granule boundaries were still noticeable, as compared to the later stages. Nevertheless, the nucleoid region was obscured at 24 h. Initially, the cytoplasm was found to be distanced from the inner layer of cell wall membrane. When the bacterial cells started to accumulate P(3HB), the cytoplasm was apparently pushed towards the cell wall to maximize the cell volume for storage of these compounds.
Figure 1d–g show that the granule boundary was vague as the granules became larger after prolonged cultivation of 48 and 72 h. The granules seemed to interact with the neighbouring granules, and their boundary region was not visible. This may result in the coalescences of neighbouring granules to form one large granule. Gerngross et al. (1993) and Tian et al. (2005) reported that the increasing granular size would gradually be forced to coalesce together. Figures 1e,g show that the granules were in contact with one another. When the cells were grown for 24 h or longer, another type of black-mediated pigment was found (Figs 1b–g and 2a–d). These black-mediated pigments were located beside the granules.
TEM study showed that the granules in wild-type Burkholderia sp. occupied almost the entire cell volume and were spherical. Approximately 2–7 P(3HB) granules of variable sizes were observed in each cell, with the average number of four granules per cell. Insertion of the plasmid pBBREE32d13 harbouring phaCAc gene resulted in a transformed strain containing two different types of PHA synthases. Figure 3a–d clearly shows that the number of granules increased when the phaCAc gene was expressed in the cells. The number of granules were around 5–13 granules per cell. In some cells, there were even more than 13 granules. However, the sizes of these granules were smaller than those found in the wild-type strain.
Aeromonas caviae is a bacterium capable of synthesizing PHA containing 3HHx monomer when cultured in fatty acids or oil-based carbon sources (Kobayashi et al. 1994; Tsuge et al. 2007). However, Aer. caviae cannot utilize triglycerides efficiently (Fukui and Doi 1998). Burkholderia sp. is capable of utilizing plant oils to produce P(3HB) (Rodrigues et al. 1995; Silva et al. 2000; Zazali and Tan 2005). Because of the poor material properties of P(3HB) (Doi et al. 1995; Ashby et al. 2001), it is desirable to produce PHA co-polymers. The incorporation of 3HHx monomers into the P(3HB) polymer chain is known to produce co-polymers with improved properties (Tsuge et al. 2007). To elucidate the monomer supplying pathway in Burkholderia sp. and at the same time improve the properties of the polymer produced, the phaCAc was co-expressed in Burkholderia sp. along with its native PHA synthase. This attempt conferred Burkholderia sp. USM (JCM15050) with the ability to produce PHA consisting of 3HB, 3HV and 3HHx monomers.
The synthesis of P(3HB-co-3HHx) by Burkholderia sp. USM (JCM15050) indicated that the β-oxidation pathway of this bacterium was able to provide enoyl-CoA intermediates for the production of 3HHx monomers, which were subsequently polymerized by the PhaCAc. The molar fractions of 3HHx in the co-polymer produced by the transformed strain from CPKO and PKAO were constant at 1 mol%. The feeding of 3HV precursors enhanced the incorporation of co-monomers. The timing of 3HV precursor addition into the culture medium affected the PHA content and 3HHx composition. Up to 7 mol% of 3HHx was incorporated in addition to 3HV following this strategy.
Bhubalan et al. (2008) reported that the incorporation of 3HV monomer was better from sodium valerate than from the same amount of sodium propionate. This statement is in accordance with our results which revealed that sodium valerate was a more suitable substrate for 3HV monomer production than sodium propionate in terms of 3HV composition. Reducing the concentration of sodium valerate and co-feeding of CPKO increased both cell biomass and PHA content. In other cases, addition of sodium valerate in two-stage cultivation resulted in higher 3HV fraction, up to 78 mol%. Based on the results obtained, it can be postulated that 3HV monomers are generated more efficiently when the bacterial cells are not growing or have reached the stationary phase. Bhubalan et al. (2008) made similar observations when the transformed strain of C. necator PHB−4-harbouring plasmid pBBREE32d13 was cultured in mineral medium containing palm kernel oil and 3HV precursors.
Bhubalan et al. (2008) reported a terpolymer, P(3HB-co-39 mol% 3HV-co-3 mol% 3HHx), that exhibited elastomeric behaviour and did not show any melting peak (Tm). They also found that the feeding time of 3HV precursors affected the molecular weights of the terpolymer.
Higher magnification of Figs 1g and 2b,d (arrows) shows that this black-mediated pigment may serve as the granule initiation site. Upon initiation of P(3HB) synthesis by the black-mediated pigment, the granules were layered with a thin membrane. Tian et al. (2005) reported that black-mediated pigment served as scaffolds, which offered a site to initiate granule formation by the PHA synthase.
When the cells were incubated for a longer period, the granule boundary region was less visible. Adjacent granules coalesced with one another and became one large granule inside the cells. This may suggest that the cells undertake self-adjustment metabolism to degrade the membrane layer with the purpose to increase the space for loading of more granules. Kelley and Srienc (1999) claimed that P(3HB) granules were more difficult to coalesce when compared to P(3HB-co-3HV) granules because of higher crystallinity of P(3HB) homopolymer. However, in this study, it was observed that the fusion of P(3HB) granules inside Burkholderia sp. led to the formation of one large granule (Fig. 1f,g). Barnard and Sanders (1988, 1989) reported that the polymer chains inside the granules exist in an amorphous state. Thus, flowing and interchanging of the fluid polymer was believed to occur between the fused granules.
Although TEM micrographs revealed that cells were packed with PHA granules, GC results revealed that the PHA content of the transformant that was fed with CPKO and sodium valerate was only 38 wt% (Table 3). On the other hand, the wild-type strain produced 70 wt% of P(3HB) after 72 h of cultivation. The illusion of transformed cells that were apparently full of PHA granules may be due to the 3HV and 3HHx monomers that were composed of longer side chain, thus exhibited ‘looser’ structure and causing the polymer to occupy a larger space. Consequently, the polymer chains were not closely packed, resulting in large but loose granules. Similar observation was also made in a study that used Delftia acidovorans to synthesize various types of PHA co-polymers (Loo and Sudesh 2007). They reported that the granules of P(3HB-co-3HV) occupied more space in the cell cytoplasm and had a lower density compared with P(3HB) or poly(4-hydroxybutyrate) granules.
Finally, it was observed that the transformed strain accumulated numerous smaller granules compared with the wild type. Similar observations were reported before in cases where the phasin proteins were manipulated. Overexpression of phasin genes resulted in smaller and numerous granules (Jossek et al. 1998). On the other hand, the deletion of phasin genes resulted in single or a few large granules (Steinbüchel et al. 1997; Fukui et al. 1999; Maehara et al. 1999). Pieper-Fürst and Steinbüchel (2000) showed that the existence of phasin gene was important in determining the size and number of PHA granules. However, in the present study, the phasin genes were not manipulated. Only an additional PHA synthase gene was introduced, which resulted in similar effect as phasin overexpression. Therefore, it can be suggested that higher concentration of PHA synthase results in smaller and numerous granules and vice versa.
In conclusion, we have successfully inserted pBBREE32d13 plasmid harbouring phaCAc into wild-type Burkholderia sp. USM (JCM15050). The resulting transformant possessed the ability to utilize CPKO and PKAO to synthesize PHA containing 3HHx monomer. TEM study demonstrated that heterologous expression of an additional synthase gene resulted in smaller but increased number of PHA granules inside the cells.
This material is based upon work supported by the Malaysia Toray Science Foundation. The authors would like to thank Saw Siew May, Kesaven Bhubalan and Tan Yifen for their assistance while carrying out the experiments. Generous supply of palm oil products by Acidchem International Ltd and Unitata Ltd is gratefully acknowledged.