Separation and Purication Technology 102 (2013) 111117

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Separation and Purication Technology

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Effect of different recovery strategies of P(3HB-co-3HHx) copolymer from

Cupriavidus necator recombinant harboring the PHA synthase of Chromobacterium
sp. USM2
Siti Nor Syairah Anis a, Nurhezreen Md Iqbal a, Sudesh Kumar a, Al-Ashraf Amirul a,b,
a
b

School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
Malaysian Institute of Pharmaceuticals and Nutraceuticals, MOSTI, Malaysia

a r t i c l e

i n f o

Article history:
Received 6 January 2012
Received in revised form 18 September 2012
Accepted 27 September 2012
Available online 10 October 2012
Keywords:
Biodegradable polymers
Alkaline digestion
P(3HB-co-3HHx)
PHA recovery
Cupriavidus necator
Industrial application

a b s t r a c t
Five strategies (Experiment A, B, C, D and E) for recovery of PHA were carried out in 10 L bioreactor using
cells which contained 68 wt% PHA and 3 mol% 3HHx. NaCl (8 g/L), NaOH (0.1 M) and ethanol (20% v/v)
were used in the treatments. Separated treatments of NaCl and NaOH with ethanol (Experiment D)
was found as the best strategy with high PHA recovery (95 wt%) and purity (90 wt%). Although, combined
treatments (Experiment B) obtained the highest PHA purity (97 wt%) severe degradation of polymer was
detected. The molecular weight (Mw) was reduced to almost half (2.8 105 Da) compared to polymer
extracted using chloroform (4.6  105 Da). In addition, pre-treatment using NaCl was found to increase
the PHA recovery and purity to 97.7 wt% and 97.5 wt%, respectively. Also, no signicant changes in the
PHA recovery and purity were observed when lyophilized cell or wet cell was used. Further characterizations of recovered polymers were carried out using GPC, DSC, TGA and tensile machine.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Nowadays, plastics are the most important things in our daily
life because many things are created from plastic including in the
household, industrial, food sector and also in medical applications.
Consequently, collection and waste of plastics can threat the environment because synthetic plastics are toxic and non-biodegradable [1]. The petrochemical-based plastics or conventional
plastics persist for many years after disposal [2]. Therefore, many
researches were done to nd out solution to replace the nondegradable plastic to biodegradable plastics or polymers. Polyhydoxyalkanoates (PHAs) are produced by various microorganisms
under stress condition through fermentation. Currently, over 150
different monomers of PHAs have been identied with molecular
masses ranging from 50,000 to 1,000,000 Da [3]. Different monomers of PHA can be manipulated using different types of bacteria
(Gram-positive or Gram-negative), fermentation conditions, fermentation modes, substrates used, recombinant gene expression
and protein engineering of PHA biosynthetic [3,4].

Corresponding author at: School of Biological Sciences, Universiti Sains

Malaysia, 11800 Minden, Penang, Malaysia. Tel.: +604 6533888x4013; fax: +604
6565125.
E-mail address: amirul@usm.my (A.A. Amirul).
1383-5866/$ - see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.seppur.2012.09.036

PHAs are intracellular products and are required to be extracted

from the interior of the bacterial cells. After appropriate cultivation, the bacterial cells containing PHA granules are harvested by
centrifugation and freeze dried. The next step is to recover the
PHA via isolating, purifying and extracting out the produced polymers from the bacterial cells [5]. The extracted PHAs whether
insoluble or granules associated forms are then puried from other
impurities such as nucleic acids, lipids, phospholipids, peptidoglycan and proteinaceous materials [6,7]. The purity and recovery
yield of PHA should be in the deliberation before and after the
extraction and purication processes. Recovery of polymers contributes signicantly to the overall cost of PHA production. Therefore, development of recovery process that allows the simple and
efcient extraction will be well rewarded [8]. There are many
methods developed by researchers for the extraction, purication
and recovery of PHA but the methods are still in consideration because of the high cost, complex process and non-environmental
friendly aspects [7].
For large scale production, PHA extraction cost should be as low
as possible with high PHA recovery yield at the end of this process.
The efcient recovery process for PHA production depends on
several factors. The main factor is that the chemicals or solvents
used for recovery process should be a green chemical or a green
solvent. It will be more economical if the chemical or solvent is
an inexpensive material. Besides, a simple operation during the

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recovery of PHA with low energy input and short time will greatly
reduce the cost and this should be taken into consideration especially in the large scale recovery process. In addition, the percentage of recovery and purity must be determined after the recovery
process. A high recovery and purity of recovered polymer with no
reduction of molecular weight represents the efciency of the
recovery process. Furthermore, the thermal and mechanical properties of the recovered polymer must be determined to ensure that
the polymer does not undergo any deterioration after recovery
process.
The aim of this study is to develop a simple, economical and
environmental friendly procedure for recovering biodegradable
polymer from bacterial cells using NaOH digestion. NaOH in alkaline conditions causes saponication of the lipid layer in the cell
wall and increases the permeability of cell membrane that helps
release the nonpolymeric protein material [5,7,9]. Recombinant
bacterium, Cupriavidus necator harboring pBBR1MCS-C2 plasmid
polyhydroxyalkanoate (PHA) synthase gene was used for the production of copolymer P(3HB-co-3HHx) from crude palm kernel
oil (CPKO). For large volume recovery, different strategies were
studied to develop economical large volume recovery process. Several centrifugation steps could be avoided in order to reduce the
energy consumption. The most efcient procedure was selected
based on the improvement condition with high quality product.
2. Materials and methods
2.1. Microorganism
Recombinant bacterium,
C. necator PHB4 harboring
pBBR1MCS-C2 plasmid/ Chromobacterium sp. PHA synthase gene
was used for the production of copolymer P(3HB-co-3HHx). The recombinant C. necator used in this study was kindly provided by
Prof. Sudesh Kumar from Eco-biomaterial laboratory, Universiti
Sains Malaysia.
2.2. Culture media
Nutrient-rich medium composed of (g/L): meat extract, 10;
peptone, 10; yeast extract, 2; was used for enrichment of recombinant C. necator. The medium for PHA production was a mineral salt
medium (MSM) composed of (g/L): KH2PO4, 2.8; Na2HPO4.12H2O,
3.32; urea, 0.54. Medium components were dissolved in distilled
water and autoclaved at 121 C for 20 min. CPKO was autoclaved
separately and added to medium giving an initial concentration
of 20 g/L. 1 mL/L of trace elements solution and 1.4 g/L of MgSO47H2O were added to the medium after sterile ltration. Composition of the trace elements solution was (g/L in 0.1 N HCl):
CoCl26H2O, 0.22; FeCl3, 9.7; CaCl2, 7.8; NiCl26H2O, 0.12; CrCl3
6H2O, 0.11; CuSO45H2O, 0.16. Kanamycin was added to the
medium giving a concentration of 50 mg/L for maintenance of
the plasmid in strain PHB-4/ pBBR1MCS-C2.
2.3. Production of P(3HB-co-3HHx) in 150 L fermenter
Approximately, two loopfuls of C. necator cells from NR agar
were transferred into the NR medium. The preculture was incubated for 12 h at 30 C and then transferred to 500 mL ask containing 100 mL of NR medium for another 18 h. After cultivation,
10% (v/v) of inoculums from NR were transferred into 7 L of NR
medium as preparation for seed culture and was further incubated
for 12 h. PHA biosynthesis was carried out in a 150 L fermenter
(BIOSTAT D, B-Braun) with the initial working volume set at 70 L.
The cultures were incubated for 72 h at 30 C. The agitation speed
was xed at 250 rpm with air ow rate of 1 vvm. During fermenta-

tion, 20 g/L of CPKO was fed at 12, 24 and 36 h time interval. Additional magnesium sulfate, 1.4 g/L; trace elements, 1 mL/L and urea;
2 g/L were also added at the 24 h time interval. The cells were harvested by centrifugation and washed with hexane to remove excess oils and nally rinsed with distilled water. The bacterial
cells obtained from this fermentation were used throughout the
recovery experiments.
2.4. Recovery of P(3HB-co-3HHx) in bioreactor
The recovery process using total volume of 10 L was carried out
using improved parameters established in previous experiments
(data not shown). The bacterial cells obtained from the 70 L fermentation were adjusted with distilled water until the cell concentration of 10 g/L was obtained. Approximately, 4 g/L of NaOH
(0.1 M) was added into the bacterial suspension and stirred at
350 rpm for 1 h. Then, the suspensions were centrifuged, washed
and polished using 20% (v/v) of ethanol for 3 h. The recovered polymers were freeze-dried and analyzed using GC.
2.5. Recovery of P(3HB-co-3HHx) using different cells conditions
The recovery of P(3HB-co-3HHx) using NaOH was carried out
using three cells conditions; lyophilized cells, wet cells (without
culture medium) and wet cells with the culture medium (without
centrifugation). The cell concentrations were xed at 10 g/L and
treated with 0.1 M of NaOH at 30 C for 1 h. The recovered polymers were centrifuged, washed and polished using 20% (v/v) of
ethanol for 3 h before freeze-dried.
2.6. Recovery of P(3HB-co-3HHx) with NaCl pre-treatment
The pre-treatment of NaCl was carried out before treatment
using NaOH. The cells concentration was xed at 10 g/L while
the concentrations of NaCl were varied from 2 to 10 g/L and incubated at 30 C for 3 h. The recovered polymers were centrifuged,
washed and polished using 20% (v/v) of ethanol for 3 h before
freeze-dried.
2.7. Recovery of P(3HB-co-3HHx) in bioreactor using different
strategies
The bacterial cells obtained through the 70 L fermentation was
harvested and separated from the culture broth by centrifugation.
Only the cell pellets were used in this experiment. Several strategies for the recovery of P(3HB-co-3HHx) were developed (Fig. 1)
and tested. The pure polymers obtained from each recovery process were further characterized using GPC, DSC, TGA and tensile
machine.
2.8. Analytical procedures
2.8.1. Gas chromatography
The PHA concentration, composition, recovery and purity were
determined using gas chromatography (GC) (Shimadzu GC-2014)
based on method by [10]. Approximately 58 mg of extracted polymer were subjected to methanolysis in the presence of chloroform
with methanol and sulfuric acid [85%:15% (v/v)]. The reaction mixture was incubated at 100 C for 3 h. The organic layer containing
the reaction products was separated, dried over Na2SO4, and analyzed by GC. Caprylic methyl ester (CME) 1:500 was used as the
internal standard.
2.8.2. Determination of molecular weight
The molecular mass data for the polyesters were obtained by
gel permeation chromatography (GPC) machine using a Waters

S.N.S. Anis et al. / Separation and Purication Technology 102 (2013) 111117

113

Cell pellets containing P(3HB-co-3HHx) obtained from

70 L fermentation

Pretreatment using NaCl at

30 C for 3 hours, 350 rpm
Centrifugation

Treatment with NaOH (0.1 M)

at 30 C for 1 hour, 350 rpm
Centrifugation
Polish using 20 % ethanol (v/v)
at 30 C for 1 hour, 350 rpm

Centrifugation

Freeze-drying

Experiment A
Experiment B
Experiment C
Experiment D
Experiment E
Fig. 1. The ow of recovery process using various strategies.

Model 600E (Multi Solvent Delivery System), Waters 410 refractive-index detector with Waters 746-Data Module PLgel Mixed C
column (Polymer Laboratories, Ltd., UK). The polymer samples
were dissolved in chloroform to a nal concentration of 2.0 mg/
mL and ltered through 0.45 lm PTFE membranes (Millipore,
USA). The ow rate was at 1.0 mL/min. Polystyrene standards with
low polydispersity (1.1) (Polymer Laboratories, Ltd., UK) were
used to plot the standard curve.
2.8.3. Tensile test
The mechanical properties of the P(3HB-co-3HHx) were characterized using tensile machine (GOTECH A1-3000) to determine the
tensile strength, Youngs modulus and elongation to break. Tensile
test pieces with 4 mm width, 45 mm gauge length were cut from
polymer lms using steel ASTM regulation punches. The crosshead speed used is 10 mm/min. A minimum of ve replicates were
tested for each polymer sample.

2.8.4. Thermal properties of P(3HB-co-3HHx)

Differential scanning calorimeter (DSC) (Pyris DSC, PerkinElmer) was used to determine the thermal properties of the polymer
sample. The pure polymer (5 mg) were encapsulated in aluminum
pans and heated from 50 to 200 C at a heating rate of 20 C/min
(rst heating scan). The melt samples were then maintained at
200 C for 2 min and followed by rapid quenching to 50 C at rate
of 5 C/min. They were heated from 50 to 200 C at a heating rate
of 20 C/min (second heating scan). The glass transition temperature (Tg) was taken as the midpoint of the heat capacity change.
The melting temperature (Tm) and the enthalpy of fusion (DHm)
were determined from the DSC endotherm. Thermal decomposition of P(3HB-co-3HHx) was determined using Mettler Toledo,
TGA/SDTA 851e. Approximately, 1015 mg of samples was heated
with nitrogen ow at rate of 10 C/min in order to ramp the initial
temperature to 800 C. Three replicates were prepared for each
sample.

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2.9. Calculations
The purity and recovery of P(3HB-co-3HHx) were calculated
according to the following equations:

phag
 100 w=w%
biomassg
phag
%Recovery P
 100 w=w%
PHAg

%Purity

%NPCM 100%  %Purity

For PHA purity, pha(g) represent the amount of PHA in biomass(g) of cells, calculated based on the GC analysis. The PHA
recovery was determined by dividing the amount of recovered
P
PHA, pha(g) with a known amount of PHA in the biomass, PHA(g)
obtained using chloroform extraction method.
3. Results and discussions
3.1. Effect of cell conditions and NaCl pre-treatment
In small scale laboratory processes, the bacterial cells were usually harvested by centrifugation and freeze-dried. However, centrifugation and drying procedure require high energy consumption
and are costly to be carried out in industrial scale [11]. In order
to reduce the production cost, direct recovery of PHA from the culture broth was to be expected. Therefore, the recovery of copolymer P(3HB-co-3HHx) carried out using lyophilized cells, wet cells
and cells with culture broth (without centrifugation). Recombinant
bacterium C. necator PHB4 harboring phaC of Chromobacterium sp.
USM2 was used to synthesize P(3HB-co-3HHx) using CPKO as sole
carbon source. After 72 h of cultivation, 42 g/L of cell biomass with
68 wt% PHA and 3 mol% of 3HHx was obtained. The PHA purity and
recovery of lyophilized cells and wet cells are 80.5 wt% purity with
88.6 wt% recovery and 83.9 wt% purity with 91.1 wt% recovery,
respectively. The results were as shown in Table 1. There was no
signicant difference in lyophilized cells or wet cells using this
treatment. On the other hand, the recovery of cells with culture
broth yields low PHA purity (76.4 wt%) and recovery (68.9 wt%).
Earlier work by [12] has concluded that the drier the bacterial cells,
the easier the recovery process. In addition, [11] also reported that
the purity of recovered polymer would decrease compared to the
recovered polymer using lyophilized cells. Direct recovery from
fermentation also has a drawback due to the high viscosity of the
fermentation broth during the recovery process. Besides that, the
residual oil from the fermentation also decreases the NaOH concentration and lessens its efciency to digest NPCM. It was suggested that in direct recovery using chemical digestion method
must involve shaking, heat treatment and washing [13]. However,
as heating is a crucial step, it requires high energy, thus it is not
suitable for large volume recovery. In our study, both lyophilized
cells and wet cells produce desirable results for PHA purity and
recovery yield. In the subsequent experiment, wet cells were used
for PHA recovery.
Pre-treatment of bacterial cells before NaOH treatment is another method to increase the PHA purity and recovery as it is done

Table 1
Recovery of copolymer P(3HB-co-3HHx) using different type of cells.

Table 2
Effect sodium chloride (NaCl) pre-treatment on the recovery of copolymer P(3HB-co3HHx).

Type of cells

Purity
(wt%)a

Recovery
(wt%)a

Residual NPCM
(wt%)a

Lyophilized cell
Wet cell
Wet cell with broth

80.5 4.4
83.9 3.0
76.4 2.2

88.6 4.3
91.1 2.7
68.9 2.1

19.5 4.4
16.1 3.0
23.6 2.2

Calculated from GC analysis. Values are means of three replications SD.

NaCl
concentration (g/L)

Purity
(wt%)a

Recovery
(wt%)a

Residual
NPCM (wt%)a

Controlb
2
4
6
8
10

82.2 3.7
87.8 2.2
93.1 2.8
93.0 4.3
97.7 1.6
94.8 0.7

83.9 1.2
82.3 2.6
84.0 4.9
86.2 3.3
97.5 2.0
97.1 1.8

17.8 3.7
12.2 2.2
6.9 2.8
7.0 4.3
2.3 1.6
5.2 0.7

Calculated from GC analysis. Values are means of three replications SD.

Recovery treatment using NaOH without NaCl pre-treatment was used as a
control.
b

to deliberately make the bacterial cells more amenable during

digestion treatment [14]. The bacterial cells were pre-treated with
NaCl before treatment using NaOH. The PHA purity and recovery
increased from 87.8 wt% and 82.3 wt% to 97.7 wt% and 97.5 wt%,
respectively, as the NaCl concentration increased from 2 mg/mL
to 8 mg/mL (Table 2). Pre-treatment with NaCl causes imbalanced
in the cell membrane, as a result the cell wall is easier to disrupt
when treated with NaOH. High concentrations of NaCl will create
an osmotic pressure to the bacterial cells that leads the cells to
dehydrate and shrivel making the release of PHA granules easier
when treated with NaOH. Based on the results, 8 g/L of NaCl was
found to be a suitable concentration for the pre-treatment process.
NaCl pre-treatment was found to improve the recovery of PHA despite via mechanical approach using bead mill disruption method
[15].
Suzuki and co-workers had also discovered that by using pretreatments of H2O2 and SDS before enzymatic digestion, it increases the PHA purity and recovery [16]. Besides using chemical
as pre-treatment, heat pre-treatment [17] and freezing pre-treatment [18] also showed impact on the cell sturdiness. Even so, these
operations require high energy and are costly to be considered for
large scale recovery process.

3.2. Recovery of P(3HB-co-3HHx) using various strategies

In large scale production, it is important to have a recovery process that is inexpensive with a high PHA purity and recovery yield.
Two factors have been considered to ensure an efcient recovery
process. First, the chemical used should not be hazardous to the
environment. Second, the recovery process must be as economical
as possible. Thus, in our study we had focused on using green
chemicals or solvents viz. NaCl, NaOH and ethanol for the recovery
process. In addition, these strategies were developed in order to
limit the use of energy by omitting several centrifugation processes
as shown in Fig. 1.
The highest PHA purity was obtained using strategies in experiment B and C with 97 wt% and 95 wt%, respectively (Table 3). The
PHA purity for others were as followed, experiment D (90.8 wt%), E
(87.5 wt%) and A (82.7 wt%). However, experiment A and D obtained highest PHA recovery yield with 94.4 wt% and 95.3 wt%,
respectively. These were followed by B (86.4 wt%), C (79.5 wt%)
and E (70.3 wt%). In addition, the molecular weights (Mw) of these
recovered PHA were determined using GPC analysis to compare
the overall effects of each strategy. The control is the recovered
polymer (4.63 kDa) using chloroform extraction as it is reported
to cause negligible degradation of PHA [19]. It was observed that
there were negligible degradation of PHA occurred in recovered
polymers obtained using A (4.93 kDa), D (4.90 kDa) and E
(4.95 kDa). On the other hand, recovered polymers using experi-

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Table 3
Recovery of copolymer P(3HB-co-3HHx) in bioreactor using different strategies.
Experiment

Control
A
B
C
D
E

Purity (wt%)a

ND
82.7 2.5
97.0 1.1
95.1 2.7
90.8 2.6
87.5 2.4

Recovery (wt%)a

ND
94.4 2.8
86.4 1.0
79.5 2.3
95.3 1.2
70.3 1.9

Residual NPCM (wt%)a

ND
17.3 2.5
3.0 1.1
4.9 2.7
9.2 2.6
12.5 2.4

Molecular Weight
Mw ( 105)b(Da)

Pdc (Mw/ Mn)

4.6 0.1
4.9 0.5
2.8 0.0
3.2 0.1
4.9 0.1
4.9 0.0

3.2 0.2
3.1 0.5
2.3 0.2
2.8 0.1
3.0 0.1
3.5 0.1

ND: Not determined.

a
Calculated from GC analysis.
b
Molecular weight.
c
Polydispersity index.
d
Molecular weight was determined using chloroform extraction as a control.

ment B and C showed considerable decrease in the molecular

weights with 2.84 kDa and 3.17 kDa, respectively.
Experiment A was carried out without NaCl pre-treatment in
the recovery process. This method yields high PHA recovery and
negligible degradation of polymers but has the lowest PHA purity.
This shows that NaCl pre-treatment is an essential step in the
recovery process as it increases the efciency of NaOH to digest
the cell membrane. Meanwhile, the recovered polymers using B
and C have the highest PHA purity but a lower PHA recovery yield
with severe degradation of polymers. This was due to the formation of sodium ethoxide or alkoxide when ethanol was mixed with
the NaOH in the mixture reacts. Sodium ethoxide is just like sodium hydroxide, except that the hydrogen has been replaced by
an ethyl group. Sodium hydroxide contains OH ions; sodium ethoxide contains CH3CH2O ions [20]. This indirectly causes an increase of basicity of the mixture. High concentration of NaOH or
alkaline would not only inuence the PHA purity and recovery
but also increase the risk of polymers degradation [19]. Therefore,
the recovered polymer granules after the NaOH treatment must be
separated and washed before further treatment using ethanol to
avoid degradation of polymers. In experiment D and E, the recovered polymers after NaOH treatment were separated and washed
before further treatment. Recovered polymers using experiment
D resulted in higher PHA purity, recovery with negligible degradation of molecular weights. It was suggested that using combined
salt, NaCl and NaOH treatment for PHA recovery would increase
the effectiveness of cells disruption [21].
Additional studies were carried out using scanning electron
microscopy (SEM) to examine any degradation effects on the surface of the PHA granules caused by treatments during the recovery process. Fig. 2a showed the bacterial cells morphology before
treatments and Fig. 2b and c showed the PHA granules obtained
after treatments. Fig. 2b showed the surface of PHA granules obtained by using experiment B. Single PHA granule is visible
although the granules are small and misshapen. The surfaces of
the granules are also very rough due to erosion caused by the
strong base used during treatment. This correlates with the result
obtained that polymers recovered using experiment B, which
gave a high PHA purity but have low molecular weights. Fig. 2c
showed the effect of experiment D on the PHA granules. Single
PHA granule is noticeably larger and the surface of the granules
was much smoother compared to the granules obtained in experiment B. In addition, cross section of the bacterial cells before
and after treatment was observed using transmission electron
microscopy (TEM) (Fig. 3). From Fig. 3a and b, it showed that
the treatments will only affect the bacterial cell wall leaving
the PHA granule intact. Experiment D was chosen as the best
strategy for PHA recovery in bioreactor as it gives a high PHA
purity, recovery with negligible degradation and similar molecu-

Fig. 2. Scanning electron micrograph of bacterial cells 9 (a) before treatment and
recovered polymers after (b) experiment B and (c) experiment D. Observed under
Scanning Electron Microscope (SEM) (10,000  magnication).

lar weight with the polymer recovered using chloroform

extraction.

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Fig. 3. Transmission electron micrograph of bacterial cells (a) before treatment and
(b) recovered polymers after treatment using recovery process by experiment D.
Observed under Transmission Electron Microscope (TEM) (10,000  magnication).

3.3. Thermal and mechanical properties of recovered P(3HB-co-3HHx)

Based on the results, it is evident that the recovery of copolymer
P(3HB-co-3HHx) from a recombinant bacterium, C. necator harboring pBBR1MCS-C2 plasmid through NaOH digestion treatments
have good comparability with chloroform extraction. When compared, recovery using NaOH has a percentage purity of 90.8 wt%
with recovery of 95.3 wt% while the chloroform extraction results
in 98.2 wt% of purity and 92.1 wt% of recovery. Characterization of
polymer properties by both NaOH recovery and chloroform extraction method showed almost similar results and negligible change
in the polymer properties (for experiment A, D and E).
To evaluate the effect of the recovery process on the thermal
property of the recovered P(3HB-co-3HHx), the melting temperature, the enthalpy of fusion and the decomposition temperature
of recovered polymers using experiment A, B, C, D and E were

determined with DSC and TGA (Table 4). The melting temperature
(Tm) of polymers recovered by experiment D (146 C) is similar to
polymers recovered by chloroform extraction (149 C). However,
the enthalpy of fusion of polymers recovered by using experiment
D (49.6 J/g) was slightly higher than by using chloroform extraction (46.7 J/g). This indicates that the crystallinity of this polymer
was slightly higher than by using chloroform extraction. In fact,
polymer extracted through chloroform extraction will dissolve in
the solvent and later is re-crystallized through non solvent precipitation. On the other hand, chemical digestion polymer is recovered
as crude granule from cell cytoplasm, thus have no chance for recrystallization [22]. Decomposition temperatures (Td) of recovered
polymers for each experiment were determined using TGA. Experiment D and E gave Td of 265 C and 260 C, respectively. These
decomposition temperatures were almost similar with the control
(278.3 C). Whereas, Td below than 245 C was obtained for experiment A, B, and C and they showed signicant reduction compared
to the control.
The mechanical properties of the recovered polymers such as
tensile strength (MPa), Youngs modulus (MPa), and elongation to
break (%) were determined using tensile machine (Table 4). The
values could only be obtained for experiment D and E. The results
were compared with recovered polymers obtained by chloroform
extraction. However, the recovered polymers from experiment D
and E showed a slight decrease in tensile strength (MPa), Youngs
modulus (MPa), and elongation to break (%) compared to polymers
extracted using chloroform. This was perceived to be due to the
impurities in the chemically recovered polymer, such as CPKO
and cell debris that can affect on the tensile, as compared to the
solvent-extraction PHA [18,23]. Since the percentage of these
impurities is low, further chloroform extraction is not necessary
for certain applications, such as for building materials, binders,
paints and paper coating [23]. However, for biomedical purpose
these polymers must be pure as most biomedical application involves contact in human body and the purity of the material is
an important criterion [23,24]. The polymers recovered using
NaOH treatment can be further treated using other solvents, since
only a small amount of solvents would be required to increase the
PHA purity to as high as 99 wt% [25].

4. Conclusion
Industrialized recovery and purication of copolymer P(3HB-co3HHx) from recombinant bacterium strain of C. necator PHB4
using the PHA synthase of Chromobacterium sp. USM2 are possible
using a low cost and environmental friendly recovery method.
High PHA purity and recovery of polymers was achieved using
the simple, inexpensive and efcient recovery process using re-

Table 4
Thermal and mechanical properties of recovered copolymer P(3HB-co-3HHx) in bioreactor using different strategies.
Experiment

Thermal properties
Tg (C)

Control
A
B
C
D
E

1.7 0.7
17.9 0.7
22.8 0.3
18.6 2.0
0.5 0.1
1.1 0.4

Tm (C)

Mechanical properties
b

149.0 0.3
123.3 0.6
116.9 0.2
120.2 2.7
146.0 0.1
143.7 0.3

ND: Not detected.

a
Glass transition temperature.
b
Melting temperature.
c
Heat of fusion.
d
Decomposition temperature.
e
Determined using tensile machine.

DHm (J/g)
46.7 2.5
17.6 2.1
19.1 0.8
16.9 3.0
49.6 0.1
51.1 1.0

Td (C)

Tensile strength (MPa)e

Youngs modulus (MPa)e

Elongation (%)e

278.3 16.1
245.0 0.0
240.0 0.0
240.0 0.0
265.0 0.0
260.0 0.0

16.0 1.4
ND
ND
ND
8.4 1.6
6.1 1.5

51.4 5.1
ND
ND
ND
23.0 4.8
16.0 5.2

3.3 0.4
ND
ND
ND
0.9 0.2
0.8 0.2

S.N.S. Anis et al. / Separation and Purication Technology 102 (2013) 111117

combinant bacterium, C. necator. Besides, the use of inexpensive

carbon source, chemicals and solvents for the biosynthesis and
recovery process is also an added advantage for PHA production
at an industrial scale.

[11]

[12]

Acknowledgements
[13]

The authors acknowledged the research grants provided by

Ministry of Science Technology and Innovations (MOSTI), Malaysia
(304/PBIOLOGI/650464/S105), Universiti Sains Malaysia (304/PBIOLOGI/639015) and National Science Fellowship. Also, we greatly
appreciate the suggestions given from Prof. Anthony J. Sinskey
(Department of Biology, Massachusetts Institute of Technology)
in the development of this project.
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