Article

Isolation and Characterization of a Burkholderia sp. USM (JCM15050) Capable of Producing Polyhydroxyalkanoate (PHA) from Triglycerides, Fatty Acids and Glycerols

Journal of Polymers and the Environment (Impact Factor: 1.67). 12/2010; 18(4):584-592. DOI: 10.1007/s10924-010-0204-1

ABSTRACT

A consortium of microorganisms from oil polluted wastewater sample was cultivated to promote polyhydroxyalkanoate (PHA) accumulation
before subjecting the mixed cultures to sucrose density gradient ultracentrifugation. This resulted in the fractionation of
the bacterial cells according to their physical features such as size, morphology and/or densities. An isolate was identified
as Burkholderia sp. USM (JCM15050), which was capable of converting palm oil products [crude palm kernel oil (CPKO), palm olein (PO), palm
kernel acid oil (PKAO), palm stearin (PS), crude palm oil (CPO), palm acid oil (PAO) and palm fatty acid distillate (PFAD)],
fatty acids and various glycerol by-products into poly(3-hydroxybutyrate) [P(3HB)]. Up to 70 and 60wt% of P(3HB) could be
obtained when 0.5%(v/v) CPKO and glycerol was fed, respectively. Among the various fatty acids tested, lauric acid followed
by oleic acid and myristic acid gave the best cell growth and PHA accumulation. Compared to Cupriavidus necator H16, the present isolate showed better ability to grow on and produce PHA from various glycerol by-products generated by
the palm oil industry. This study demonstrated for the first time an isolate that has the potential to utilize palm oil and
glycerol derivatives for the biosynthesis of PHA.

KeywordsPHA-Ultracentrifugation-Palm oil-Glycerol-
Burkholderia sp.

Full-text

Available from: Jiun Yee Chee
ORIGINAL PAPER
Isolation and Characterization of a Burkholderia sp. USM
(JCM15050) Capable of Producing Polyhydroxyalkanoate (PHA)
from Triglycerides, Fatty Acids and Glycerols
Jiun-Yee Chee
Yifen Tan
Mohd-Razip Samian
Kumar Sudesh
Published online: 25 May 2010
Ó Springer Science+Business Media, LLC 2010
Abstract A consortium of microorganisms from oil
polluted wastewater sample was cultivated to promote
polyhydroxyalkanoate (PHA) accumulation before sub-
jecting the mixed cultures to sucrose density gradient
ultracentrifugation. This resulted in the fractionation of the
bacterial cells according to their physical features such as
size, morphology and/or densities. An isolate was identified
as Burkholderia sp. USM (JCM15050), which was capable
of converting palm oil products [crude palm kernel oil
(CPKO), palm olein (PO), palm kernel acid oil (PKAO),
palm stearin (PS), crude palm oil (CPO), palm acid oil
(PAO) and palm fatty acid distillate (PFAD)], fatty acids
and various glycerol by-products into poly(3-hydroxybu-
tyrate) [P(3HB)]. Up to 70 and 60 wt% of P(3HB) could be
obtained when 0.5%(v/v) CPKO and glycerol was fed,
respectively. Among the various fatty acids tested, lauric
acid followed by oleic acid and myristic acid gave the best
cell growth and PHA accumulation. Compared to Cupri-
avidus necator H16, the present isolate showed better ability
to grow on and produce PHA from various glycerol
by-products generated by the palm oil industry. This study
demonstrated for the first time an isolate that has the potential
to utilize palm oil and glycerol derivatives for the biosyn-
thesis of PHA.
Keywords PHA Ultracentrifugation Palm oil
Glycerol Burkholderia sp.
Introduction
Polyhydroxyalkanoate (PHA) is a family of bio-based and
biodegradable polymers synthesized by a wide variety of
naturally occurring microorganisms [1, 2]. PHA serves as
carbon and energy storage compound for these microor-
ganisms. Approximately 150 types of monomers have been
identified as PHA constituents which are produced via
various metabolic pathways using different carbon sources.
PHA containing short-chain-length monomers such as
3-hydroxybutyrate (4C) and/or 3-hydroxyvalerate (5C) are
termed SCL-PHA. The PHA made of medium chain-length
monomers such as 3-hydroxyhexanoate (6C), 3-hydrox-
yoctanoate (8C), 3-hydroxydecanoate (10C), 3-hydroxyd-
odecanoate (12C) and 3-hydroxytetradecanoate (14C) are
termed MCL-PHA. SCL and MCL-PHA are often syn-
thesized by different types of bacteria although some
bacteria are capable of synthesizing both types of PHA
[3, 4]. PHA is stored in the form of water insoluble gran-
ules in the cell cytoplasm. It is known that the density of
SCL-PHA granules is higher than that of MCL-PHA
granules due to the presence of longer side chains in MCL-
PHA structure that might cause loose packing of the bulky
MCL-PHA polymers in the granules [5].
There is much interest in PHA as a class of bio-based
and biodegradable polymers that can be produced using
renewable resources such as sugars and vegetable oils
[69]. In addition, the ability to produce PHA from
industrial and domestic wastes is also gaining much
importance as this approach can minimize waste disposal
problems while at the same time reduce the production cost
J.-Y. Chee Y. Tan M.-R. Samian K. Sudesh (&)
School of Biological Sciences, Universiti Sains Malaysia,
11800 Penang, Malaysia
e-mail: ksudesh@usm.my
123
J Polym Environ (2010) 18:584–592
DOI 10.1007/s10924-010-0204-1
Page 1
of PHA. Among the various fermentation feedstocks
studied for the production of PHA, vegetable oils gave the
best yield because of the high carbon content in fatty acids
[8]. Recently, because of the booming biofuel industry,
glycerol is also becoming an attractive feedstock for fer-
mentation. Bacteria that have the ability to use triglycerides
and glycerol for cell growth and PHA production would be
very useful.
In previous studies, glycerol and triacylglycerol have
been the subject of PHA production by Pseudomonas
oleovorans NRRL B-14682, Pseudomonas corrugata 388,
Pseudomonas resinovorans [1012] and Burkholderia
cepacia ATCC 17759 [13]. Here we report the identifica-
tion of a new potential bacterium, Burkholderia sp. USM
(JCM15050) with the ability to use vegetable oils, fatty
acids and glycerol as the sole carbon sources for growth
and PHA production.
Previous studies reported the application of ultracentri-
fugation to study the biological macromolecules such as
proteins [14], carbohydrates [1517], and nucleic acids [18,
19]. Kalscheuer et al. [20] reported the isolation of lipid
inclusions containing different specific densities using
glycerol density gradient centrifugation. This method was
also applied by Matsumoto et al. [21] as well as Loo and
Sudesh [5] in separating different types of PHA granules.
This idea has inspired the application of ultracentrifugation
in separating the mixture of PHA-producing microorgan-
isms. Conventional bacterial isolation involved several
steps of serial dilutions prior to selecting pure colonies.
This method has made the isolation procedure time-con-
suming. The idea of applying ultracentrifugation to par-
tially separate the microorganisms from the mixed cultures
arose from the basic concepts of the PHA densities that is
dependent on existence of the type of monomers and thus
affects the total densities of the cell biomass. However, to
date, no study was reported about the application of
ultracentrifugation in separating a mixture of microor-
ganisms.
In this study, the isolation procedure involved discrete
sucrose density gradient ultracentrifugation. The resulting
fractionation of microorganisms appeared as distinct bands
in the sucrose gradient with denser microorganisms
migrating furthest to the bottom of the tube. This is because
the SCL monomers contain not only shorter side chain but
also has been packed compactly in the PHA granules [5].
Conversely, MCL monomers possess longer side chain and
exhibit steric effects, thus causing the polymer chain to
arrange loosely in the PHA granules [5]. The difference in
the packing of SCL and MCL polymer chains in the PHA
granules is thought to contribute to their density difference.
By using this method, we have successfully isolated a
bacterium with the ability to convert various palm oil
derivatives into PHA.
Materials and Methods
Cultivation of Mixed Microbial Culture Samples
Oil polluted wastewater samples were collected from the
drainage system of Seberang Prai industrial area, Penang,
Malaysia. The natural microbial consortium in the samples
was directly inoculated into nitrogen-limiting mineral salts
medium with 1% (v/v) oleic acid as the sole carbon source.
The mineral salts medium was prepared according to the
method described previously [22]. A 1 mL of filter steril-
ized trace element and 0.25 g/L of MgSO
4
7H
2
O were
added aseptically into the mineral salts medium. The trace
element solution consists of (per liter) 0.22 g CoCl
2
6H
2
O,
9.7 g FeCl
3
, 7.8 g CaCl
2
, 0.12 g NiCl
2
6H
2
O, 0.11 g
CrCl
3
6H
2
O, 0.16 g CuSO
4
5H
2
O in 0.1 N HCl [9]. The
pH of the medium was adjusted to 7.0. The culture was
incubated at 30 °C, 200 rpm for 72 h to promote PHA
accumulation.
The wastewater sample was collected from the open
environment during daytime where temperature was
around 30 °C. Therefore, this temperature was applied
during screening of PHA-producing microorganisms. After
obtaining pure isolate, a series of bacterial identification
tests were done to characterize the isolate, and at that time
37 °C was found to be the most optimum temperature for
the growth of this isolate. Therefore, subsequent experi-
ments were carried out at this temperature.
Concentration of PHA-Containing Bacterial Strains
Using Sucrose Density Gradient Ultracentrifugation
Initially, the environmental sample was grown under PHA-
accumulation conditions to promote the biosynthesis of
PHA by the bacterial consortium in the sample. Subse-
quently, the cultivated cell suspension was centrifuged
(4 °C, 92009g, 10 min) and the supernatant discarded. The
cell pellet was then resuspended in 1 mL of mineral salts
medium. The suspension was then subjected to ultracen-
trifugation by placing the suspension on sucrose density
gradients. For this, an ultracentrifugation tube with 4 dif-
ferent sucrose concentrations, 1.0, 1.33, 1.67 and 2.0 M
was prepared (Fig. 1). The resulting layers were subjected
to ultracentrifugation at 4 °C, 280,0009g for 2 h [5].
After 2 h of ultracentrifugation, the fractionated bands
of cell suspension were carefully withdrawn by pipetting
out the layers of sucrose densities and cell suspension. The
cell suspensions were then serially diluted and spread onto
nutrient rich (NR) agar plate, containing (per liter) 10 g
meat extract, 10 g peptone, 2 g of yeast extract and 15% of
agar powder. The pH of the medium was adjusted to 7.0.
The agar plates were incubated at 30 °C for 24 h. The
resulting single colonies were picked and further purified
J Polym Environ (2010) 18:584–592 585
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by streaking on fresh NR plates until pure isolates were
obtained.
Identification of the Isolate
Identification of the isolate was performed based on mor-
phological observation, biochemical characterization, API
20NE and 16S rDNA analysis. Genomic DNA was
extracted using G-spin
TM
Genomic DNA Extraction Kit
(for Bacteria) (iNtRON Biotechnology, Inc., South Korea).
Amplification of the 16S rDNA fragment was performed by
PCR using universal primers 68F (5
0
-TNANACATGCAAG
TCGAKCG-3
0
) and 1392R (5
0
-ACGGGCGGTGTGTRC-3
0
)
[23]. The following PCR parameters were used: 94 °C for
2 min, 30 thermal cycles of 94 °C for 30 s, 58 °C for 30 s,
72 °C for 2 min; followed by 58 °C for 1 min and a final
extension step at 72 °C for 2 min. Plasmids extraction,
digestion of DNA with restriction endonucleases and
transformation of Escherichia coli JM109 were carried out
by standard procedures [24]. DNA sequencing analysis was
performed using ABI 3730xl DNA Analyzer (Applied
Biosystem Co., USA).
The similarity and identity of the sequence obtained
were compared to other sequences in the GenBank data-
base using nucleotide-nucleotide BLAST command [25]in
National Center for Biotechnology Information (NCBI).
Clustal X was used for alignment purposes [26].
Phylogenetic Analysis
Nineteen 16S rDNA genes were utilized for phylogenetic
analysis. These bacteria are PHA producing strains except
for Burkholderia sp. isolate CRE 7 and Burkholderia
cepacia strain B03. The PHA synthase protein sequences
of these two strains were not found in the NCBI database.
The 16S rDNA sequences (partial/full) were obtained from
NCBI. Alignments and Bootstrap consensus tree using
neighbor-joining program based on 500 replicates were
generated using MEGA version 4 [27].
Carbon Sources
Acidchem International Ltd. (Penang, Malaysia) and Uni-
tata Ltd. (Perak, Malaysia) kindly provided crude palm
kernel oil (CPKO), crude palm oil (CPO), palm olein (PO),
palm kernel acid oil (PKAO), palm acid oil (PAO), palm
stearin (PS), crude glycerol, glycerin pitch (GP), pure
glycerol and palm fatty acid distillate (PFAD).
Crude glycerol contained impurities including waste-
water, spent catalysts, salts after neutralization, fat soap,
free fatty acids (oleic acid and linoleic acid) as well as
methanol from transesterification of biodiesel refineries
[2830]. The purity of glycerol in crude glycerol reportedly
range from 65–85% [28]. Glycerine pitch is a waste gen-
erated from glycerol refining process, which contains
Before After
Cell
suspension
1.0 M
1.33 M
1.66 M
2.0 M
Fig. 1 Ultracentrifugation tube with discrete sucrose gradient and cell suspension before and after ultracentrifugation. Sucrose density gradient
ultracentrifugation was used to fractionate different types of microorganisms
586 J Polym Environ (2010) 18:584–592
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55–65% of glycerol and other impurities such as diglyc-
erol, fatty acids and inorganic salts [31, 32]. PFAD is
obtained as a by-product of palm oil refining, which con-
tains mainly unesterified fatty acids such as palmitic acid,
oleic acid, linoleic acid and stearic acid [33].
PHA Biosynthesis of the Isolate
The isolate was grown in NR medium for 24 h. Then,
1.5 mL of the resulting cell culture was transferred to
nitrogen-limiting mineral salts medium containing 0.5%
(v/v or w/v) of various carbon sources. The incubation was
carried out in a temperature-controlled shaker for 72 h at
37 °C and 200 rpm to enable cell growth and PHA accu-
mulation. Later the cells were harvested by centrifugation
(92009g, 10 min, 4 °C), washed once with hexane and
finally with distilled water. The cells were then lyophilized
before subjecting to methanolysis and gas chromatography
(GC) analysis.
Analytical Methods
Determination of PHA content and composition by GC
were carried out as described by Braunegg et al. [34].
Lipase Activity Assay
Lipase activity in the supernatant of the culture was
determined using p-nitrophenol laurate (pNPL) as the
substrate. Approximately 5 mmol/L of pNPL dissolved in
10 mL of dimethyl sulphoxide (DMSO) was emulsified in
90 mL of 100 mM phosphate buffer (pH 7.0) containing
mixtures of 0.1% polyvinyl alcohol (PVA) and 0.4% Tri-
ton-X 100. Approximately 25 lL of the cell-free superna-
tant was mixed with the emulsion solution to make 2 mL of
mixture and incubated for 10 min at 40 °C. The absorbance
was measured by spectrophotometer at 410 nm. One unit
of lipase activity was defined as the amount of enzyme
required to release 1 lmol of p-nitrophenol per min at
40 °C.
Results and Discussion
Isolation and Identification of the Bacterial Strains
Oil polluted wastewater samples were chosen as the source
of microorganisms in this study. We hypothesized that the
presence of oil would favor the growth and multiplication
of microbes that have the ability to metabolize oil, fatty
acids and/or glycerol. The wastewater samples were inoc-
ulated directly into mineral salts medium containing oleic
acid as the sole carbon source for growth and biosynthesis
of PHA. The isolate described here was enriched and
purified from cultures grown on oleic acid. Initially Nile
Blue A was applied to detect the accumulation of PHA
under fluorescence microscope.
Sucrose density gradient ultracentrifugation is a method
that is widely used to isolate PHA granules based on their
density [6, 21]. It is known that the increase in the side-
chain of PHA monomer from SCL to MCL would corre-
spond to a decrease in the PHA granules densities, thus
contributed to the differences in microbial cell densities.
Microbial cells with different types of PHA granules may
be fractionated and concentrated by this method.
Figure 1 shows the result that was obtained. Bacterial
cells containing P(3HB) granules were concentrated in the
band between 1.67 and 2.0 M sucrose solutions. While
other bands contained MCL-PHA-producing bacterial
cells. The concentrated bacterial cells in each band were
gently withdrawn and further purified to obtain pure cul-
tures. A total of four different strains were observed fre-
quently and isolated from the four bands. Upon obtaining
pure cultures, each bacterium was grown separately in oleic
acid as the sole carbon source under conditions that favor
PHA accumulation. GC analysis revealed that the isolates
from band one to band three produced MCL-PHA while
the isolate obtained from band four produced P(3HB)
(Table 1). The latter isolate was the subject of this study
due to its ability to convert palm oil derivatives into high
PHA content. The morphological and physiological char-
acteristics of the isolate are summarized in Table 2. The
Table 1 PHA contents and compositions produced by bacteria from band 1 to band 4 using oleic acid as the sole carbon source
Samples DCW PHA content (wt%) PHA compositions (mol%)
(g/L) SCL MCL
3HB 3HV 3HHx 3HO 3HD 3HDD: cis 3HDD 3HTD
Band 1 1.3 ± 0.3 8 ± 5 3 25 10 47 4 9 2
Band 2 1.4 ± 0.2 4 ± 2 65 60222 4 1
Band 3 1.0 ± 0.1 10 ± 4 38 10 37121 2
Band 4 0.7 ± 0.1 48 ± 5 100
3HB 3-hydroxybutyrate, 3HV 3-hydroxyvalerate, 3HHx 3-hydroxyhexanoate, 3HO 3-hydroxyoctanote, 3HD 3-hydroxydecanoate, 3HDD:cis
3-hydroxy-5-cis-dodecanoate, 3HDD 3-hydroxydodecanoate, 3HTD 3-hydroxytetradecanoate
J Polym Environ (2010) 18:584–592 587
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isolate was grown in various types of carbohydrates and
alcohols as the carbon source. It was found that this isolate
was able to utilize most of the carbon sources, with better
growth noticed in vegetable oils and glycerol.
A series of biochemical test and also API 20NE were
utilized to identify the isolate. The identification was then
further performed with 16S rDNA analysis. A partial 16S
rDNA sequence of 1349 bp was obtained by PCR. The
BLASTX analysis revealed a 99% identity to partial
sequence of 16S rRNA gene of Burkholderia sp. isolate CRE
7 (1347/1349) (accession no. U37340); followed by also a
99% identity to partial sequence of 16S rRNA gene of
Burkholderia cepacia strain B03 (1346/1349) (accession no.
DQ387437). The third closest identity was showed by
16S rRNA gene of Burkholderia vietnamiensis G4 with
99% identity (1346/1349) (accession no. CP000614). The
sequence was deposited in the GenBank database with the
accession number FJ667272. The isolate was found to be in
the Burkholderia sp. Linage according to the phylogenetic
tree generated (Fig. 2). The isolate was identified as a strain
of Burkholderia cepacia according to all of the identification
results. The isolate was then deposited in the Japanese Cul-
ture of Microorganisms under the code name Burkholderia
sp. USM (JCM15050).
Study of Growth Profile and P(3HB) Accumulation
The ability of Burkholderia sp. USM (JCM15050) to utilize
triglyceride, glycerol and fatty acids to produce PHA was
investigated. As shown in Fig. 3, the growth profile of the
isolate was studied in mineral medium containing CPKO as
the sole carbon source. The cells grew rapidly after 12 h of
adaptation and the cell biomass increased until about 36 h of
cultivation. During the period of exponential growth, accu-
mulation of P(3HB) increased gradually, amounting to
47 wt% of the dry cell weight at 24 h, 63 wt% at 36 h until it
reached a maximum of 70 wt% at 72 h.
Conversion of readily available carbon sources such as
vegetable oil and its by-products into PHA has been the
subject of studies in recent years. Burkholderia sp. is known
as one of the most metabolically versatile bacterium [36], is
able to secrete lipase to break down triglycerides [37] into
fatty acids and glycerol. Therefore, the production of lipase
by the isolate was also determined during the growth study.
In the first 12 h, the lipase was induced and secreted into the
culture medium. During the first 48 h, there was a steep
increase in lipase activity until it reached a maximum of 117
U/mL, even though the residual oil concentration showed
that CPKO was completely hydrolyzed at 24 h. Although the
depletion of triglyceride was detected at 24 h, the accumu-
lation of P(3HB) in the cells continued to increase until 36 h.
Biosynthesis of PHA from Fatty Acids and Glycerol
Derivatives by Burkholderia sp
Table 3 shows the relative % (w/w) of fatty acids com-
positions in the samples used in this study. Palm kernel
Table 2 Taxonomic characteristics of the isolate
Characteristics
Morphological characteristics
Shape Rod
Size (lm) (0.5–0.9) 9 (1.0–2.0)
Motility ?
Gram staining Negative
Features of colonies
Shape Circular
Opacity Opaque
Elevation Convex
Surface Smooth, glistening
Edge Entire
Emulsifiability Forms uniformly turbid
suspension in water
Color Beige
Physiological characteristics
Catalase ?
Oxidase Oxidative
O/F test ?
Urease test ?
Gelatin liquefaction -
Simmons citrate test ?
Arginine dihydrolase -
Reduction of nitrate ?
Growth temperatures 9–44 °C
Aerobic conditions ?
Nutritional characteristics
Glucose ?
Fructose ?
Sucrose ?
Lactose ?
D-Arabinose ?
Raffinose -
D-Xylose ?
Glycerol ?
Inositol ?
Mannitol ?
Sorbitol ?
D-Gluconate ?
Starch -
Vegetable oil ?
Morphological characteristics and features of the isolate were moni-
tored on NR agar plate. Growth on carbohydrates and triglyceride was
monitored on mineral salts medium. Other physiological character-
istics were determined according to Mac Faddin [35]
588 J Polym Environ (2010) 18:584–592
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extracts consisted of higher concentrations of lauric acid
(C12) and myristic acid (C14), whereas high concentration
of palmitic acid (C16) was found in mesocarp extracts. The
effect of different fatty acids on cell growth and PHA
production were determined by feeding the cells with
individual fatty acid as the sole carbon source. We also
determined the various concentrations of fatty acids to
investigate the level of tolerance of the fatty acids by the
isolate. Table 4 shows that neither caproic acid (C6) nor
caprylic acid (C8) was able to support the growth of the
isolate at the concentrations of more than 0.1% (v/v).
Capric acid (C10) could not at all support the cell growth
even at 0.1% (v/v). In contrast, with lauric acid and
myristic acid, Burkholderia sp. USM (JCM15050) pro-
duced relatively high amount of P(3HB) whereby 69 and
38 wt% respectively were produced at concentrations of
0.5% (w/v). Therefore, higher concentrations of lauric acid
and myristic acid in PKO, CPKO and PKAO were believed
to contribute to better cell growth and P(3HB) production.
Unsaturated fatty acid such as oleic acid (C18:1) contrib-
uted to the production of P(3HB) but had an adverse effect
on the cell biomass. Saturated stearic acid (C18:0) and
unsaturated oleic acid produced significantly different
amount of P(3HB), whereby the former resulted in the
production of less than 1 wt% while the latter produced
48 wt% P(3HB).
It is reported that the toxicity level of the fatty acid is
related to the concentration of the unionized form of the
fatty acid and the size of the carbon chain [38]. Indeed the
fatty acids with shorter n-alkyl chains exhibit higher tox-
icity than the longer chains [39]. On the other hand, long-
chain fatty acids were preferentially acted as the substrates
for phospholipids acylation reactions [40, 41] during high
rate of b-oxidation metabolism at exponential growth
phase.
Another lipase-hydrolyzed compound, glycerol could
also support both the cell growth and P(3HB) accumulation.
Glycerol derivatives which varied in the degree of purities
were used as the sole carbon source. The results showed that
the P(3HB) content was 54 wt% in 2.5 g/L of dry cell weight
when fed with pure glycerol (Table 5). However, other
by-products such as crude glycerol and glycerine pitch (GP),
only achieved 31 and 22 wt% of P(3HB) content respec-
tively. P(3HB) concentration obtained from glycerol
(1.35 g/L) was almost similar to those obtained from CPKO
(1.54 g/L) and lauric acid (1.17 g/L).
The P(3HB) contents obtained from crude glycerol,
glycerine pitch and PFAD were lower than that obtained
from pure glycerol. This is attributed to the fact that these
by-products contain different impurities of approximately
12–15% methanol and 23–25% soap as a product from
transesterification of biodiesel refineries [28]. Glycerol is
metabolized via glycolysis pathway. Initially, glycerol is
first converted into glycerol-3-phosphate with the aid of
ATP molecule to activate the reaction. Then it is followed
by the conversion to dihydroxyacetone phosphate before
entering the glycolysis pathway to generate acetyl-CoA
and followed by three step P(3HB) synthesis pathway.
Besides Burkholderia sp., glycerol derivatives were also
tested independently for their ability to support cell growth
and PHA production by Cupriavidus necator H16. Table 5
Fig. 2 Neighbour-joining tree of 16S rDNA gene sequence similarity
showing phylogenetic positions of Burkholderia sp. USM (JCM15050)
(FJ667272), Burkholderia sp. isolate CRE 7 (U37340), B. cepacia strain
B03 (DQ387437), PHA-producing bacteria: B. vietnamiensis G4
(CP000614), B. cenocepacia MC0-3 (CP000958), B. ambifaria MC40-
6 (CP001025), B. multivorans ATCC 17616 (AP009385), B. pseudo-
mallei K96243 (BX571966), B. mallei NCTC 10229 (CP000546),
R. eutropha H16 (AM260479), A. vinosum DSM 180 (FM178268),
A. vinelandii (AB175657), P. aeruginosa PAO1 (AE004091),
P. mendocina ymp (CP000680), P. oleovorans (D84018), P. corrugata
(AF348508), P. putida KT2440 (AE015451), P. fluorescens Pf0-1
(CP000094) and B. megaterium (AB334764). Numbers at nodes
represent the levels of bootstrap support based on analyses on 500
replicates. Bar: 0.01 substitutions per site
0
1
2
3
4
5
6
0 12 24 36 48 60 72 84 96
Time (h)
Dry cell weight (g/L)
P(3HB) concentration (g/L)
Residual oil concentration (g/L)
0
20
40
60
80
100
120
140
Lipase activity (U/mL)
Fig. 3 Time profiles of growth and lipase activity for growth-
associated P(3HB) synthesis by Burkholderia sp. grown in nitrogen-
limiting MM containing 0.5% (v/v) of CPKO and 0.5 g/L of NH
4
Cl at
37 °C. (filled diamond) Dried cell weight (DCW); (filled triangle)
P(3HB) concentration; (cross) Residual oil concentration; (filled
square) Lipase activity
J Polym Environ (2010) 18:584–592 589
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shows that palm kernel and palm mesocarp derivatives
could readily support the growth and P(3HB) accumulation
of C. necator H16. Approximately, 71 wt% P(3HB) of the
dry cell weight was obtained. Glycerol, which supported
the growth and P(3HB) production of Burkholderia sp.
USM (JCM15050), however, was less preferred by
C. necator H16, whereby, only 30 wt% of P(3HB) was
attained (Table 5).
Biosynthesis of PHA from Various Palm Oil Products
by Burkholderia sp
A total of nine types of palm oil products were tested for
the synthesis of PHA by this isolate. This strain was
capable of incorporating P(3HB) homopolymer regardless
of the type of triglycerides fed. Among the various tri-
glycerides tested, CPKO supported the best synthesis of
P(3HB), amounting to 70 wt% of the dry cell weight using
one-stage cultivation. Table 5 shows that products derived
from palm kernel such as CPKO and PKAO contributed
to higher P(3HB) accumulation rather than PO, PS, CPO
and PAO, which products derived from palm mesocarp.
Overall, the dry cell weight from all the carbon sources
screened was in the range of 1.7–2.6 g/L with P(3HB)
content ranging from 41–70 wt%.
In this study, although the P(3HB) contents gained from
glycerol derivatives were not as high as that from CPKO,
Burkholderia sp. USM (JCM15050) demonstrated good
versatility in the choice of palm oil derived carbon sources
for both cell growth and P(3HB) production.
Table 3 Relative % (w/w) fatty acid compositions of crude palm oil, palm stearin, palm olein, palm acid oil, crude palm kernel oil, palm kernel
oil and palm kernel acid oil
Fatty
acids
Saturated Unsaturated
Caproic
(6:0)
Caprylic
(8:0)
Capric
(10:0)
Lauric
(12:0)
Myristic
(14:0)
Palmitic
(16:0)
Stearic
(18:0)
Arachidic
(20:0)
Palmitoleic
(16:1)
Oleic
(18:1)
Linoleic
(18:2)
Linolenic
(18:3)
CPO 0.1 0.9 43.8 4.0 0.4 42.1 8.9 0.2
PS 1.1 58.9 4.4 27.7 8.1
PO 0.2 1.0 35.8 4.1 0.4 0.1 43.8 14.3 0.2
PAO 0.8 1.1 44.7 3.7 0.4 40.3 9.4
CPKO 3.0 3.5 48.5 16.2 7.5 2.6 15.7 2.1
PKO 0.5 4.0 3.6 48.3 15.5 8.0 2.1 15.4 2.6
PKAO 2.0 2.5 44.1 17.8 10.8 3.1 17.3 2.3
Source: Unitata Ltd
CPO crude palm oil, PS palm stearin, PO palm olein, PAO palm acid oil, CPKO crude palm kernel oil, PKO palm kernel oil, PKAO palm kernel
acid oil
Table 4 Biosynthesis of P(3HB) from various fatty acids as the sole carbon sources using one-stage cultivation nitrogen-limiting MM by the
isolate Burkholderia sp
a
Concentrations of fatty
acids
0.1% (v/v) or (w/v) 0.3% (v/v) or (w/v) 0.5% (v/v) or (w/v)
Dry cell weight
(g/L)
P(3HB) content
b
(wt%)
Dry cell weight
(g/L)
P(3HB) content
b
(wt%)
Dry cell weight
(g/L)
P(3HB) content
b
(wt%)
Caproic (6:0) 0.7 ± 0.3 Tr NG ND NG ND
Caprylic (8:0) 0.8 ± 0.1 Tr NG ND NG ND
Capric (10:0) NG ND NG ND NG ND
Lauric (12:0) 1.4 ± 0.2 8 ± 1 2.1 ± 0.1 49 ± 3 1.7 ± 0.1 69 ± 7
Myristic (14:0) 1.1 ± 0.3 1 ± 0.2 1.6 ± 0.1 49 ± 2 1.9 ± 0.2 38 ± 4
Palmitic (16:0) 0.6 ± 0.2 Tr 1.2 ± 0.1 3 ± 1 1.5 ± 0.2 9 ± 6
Stearic (18:0) 0.5 ± 0.1 1 ± 0.1 1.0 ± 0.3 1 ± 0.8 0.9 ± 0.3 Tr
Oleic (18:1) 1.0 ± 0.1 1 ± 0.2 1.4 ± 0.2 40 ± 2 0.7 ± 0.1 48 ± 5
a
Cultivation at 37 °C for 72 h, pH 7.0, 200 rpm
b
P(3HB) content in freeze-dried cells
Tr PHA content less than 1 wt%, NG no growth, ND not determined
590 J Polym Environ (2010) 18:584–592
123
Page 7
Acknowledgements This material is based upon work supported by
the Malaysia Toray Science Foundation. The authors would like to
thank Teoh Chai Sin and Chan Chin Keong for assisting with the
experimental works. Generous supply of palm oil products by Acid-
chem International Ltd. and Unitata Ltd. is gratefully acknowledged.
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Table 5 Biosynthesis of P(3HB) from various types of palm oil and glycerol as the sole carbon source using one-stage cultivation of nitrogen-
limiting MM by Burkholderia sp. USM (JCM15050)
a
and C. necator H16
Carbon sources (0.5%) (v/v) or (w/v) Burkholderia sp. USM (JCM15050) Cupriavidus necator H16
Dry cell weight
(g/L)
P(3HB) content
b
(% wt)
Dry cell weight
(g/L)
P(3HB) content
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(% wt)
Source
Palm kernel
CPKO
c
2.2 ± 0.4 70 ± 3 5.0 67 [42]
PKAO
c
1.9 ± 0.2 58 ± 4 4.7 ± 0.3 42 ± 2[43]
Palm mesocarp
PO
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2.4 ± 0.1 46 ± 6 5.2 70 [42]
PS
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PS
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CPO
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PAO
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1.8 ± 0.1 57 ± 1 4.5 ± 0.2 31 ± 2[43]
Glycerol derivatives
Glycerol
c
2.0 ± 0.4 60 ± 4 1.3 ± 0.3 33 ± 3 This study
Glycerol
d
2.5 ± 0.4 54 ± 4 1.8 ± 0.1 31 ± 3 This study
Crude glycerol 1.9 ± 0.2 31 ± 3 1.2 ± 0.2 34 ± 4 This study
GP 1.9 ± 0.1 22 ± 3 1.3 ± 0.2 18 ± 1 This study
Fatty acid derivative
PFAD 1.3 ± 0.3 43 ± 3 1.9 ± 0.5 47 ± 1 This study
a
Cultivation at 37 °C for 72 h, pH 7.0, 200 rpm
b
P(3HB) content in freeze-dried cells
c
Source from Unitata Ltd
d
Source from Acidchem Ltd
CPKO crude palm kernel oil, PO palm olein, PKAO palm kernel acid oil, PS palm stearin, CPO crude palm oil, PAO palm acid oil, Glycerol pure
glycerol, GP glycerine pitch, PFAD palm fatty acid distillate
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  • Source
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