Eng. Life Sci. 2009, 9, No.

6, 454461

454

Monica Lady Fiorese1

Filomena Freitas2

Research Article

Joana Pais2
Ana Maria Ramos2
Glaucia M. F. de Aragao1

Recovery of polyhydroxybutyrate (PHB) from

Cupriavidus necator biomass by solvent
extraction with 1,2-propylene carbonate

Maria A. M. Reis2
1

Universidade Federal de
Santa Catarina/
UFSCCentro Tecnologico/
CTC, Florianopolis, Santa
Catarina, Brazil

CQFB/REQUIMTE,
Chemistry Department,
FCT/Universidade Nova de
Lisboa, Caparica, Portugal

An integrated procedure for the recovery of polyhydroxybutyrate (PHB) produced by

Cupriavidus necator based on the extraction with 1,2-propylene carbonate was evaluated. The effect of temperature (1001451C) and contact time (1545 min), precipitation period, and biomass pretreatments (pH shock and/or thermal treatments) on
PHB extraction efficiency and polymer properties was evaluated. The highest yield
(95%) and purity (84%) were obtained with the combination of a temperature of
1301C and a contact time of 30 min, with a precipitation period of 48 h. Under these
conditions, PHB had a molecular weight of 7.4  105, which was the highest value
obtained. Lower values (2.2  105) were obtained for higher temperatures (1451C),
while lower temperatures resulted in incomplete extraction yields (4554%). No
further yield improvement was achieved with the pH/heat pretreatments, but the
polymers molecular weight was increased to 1.3  106. The PHB physical properties
were not significantly affected by any of the tested procedures, as shown by the
narrow ranges obtained for the glass transition temperature (4.85.01C), melting
temperature (170.1180.11C), melting enthalpy (77.888.5 J/g) and crystallinity
(5562%). 1,2-Propylene carbonate was shown to be an efficient solvent for the
extraction of PHB from biomass. The precipitation procedure was found to highly
influence the polymer recovery and its molecular weight. Although polymer molecular weight and purity were improved by applying pH/heat pretreatment to the
biomass, the procedure involves the use of large amounts of chemicals, which
increases the recovery costs and makes the process environmentally unfriendly.
Keywords: 1,2-Propylene carbonate / Cupriavidus necator / Polyhydroxybutyrate
Received: April 28, 2009; revised: October 14, 2009; accepted: October 27, 2009
DOI: 10.1002/elsc.200900034

Introduction

Plastics, owing to their thermoplastic properties, versatility,

and cost, have conquered a very important place in our
everyday life, replacing glass, wood, and metal in many
industrial, domestic, and environmental applications. There is
a great concern about the plastics final disposal and reuse
because they are not biodegradable, accumulating in the
environment, where they have a huge deleterious impact. An
ecological alternative is the development of biodegradable
plastics with identical physical and chemical properties, such as
polyhydroxyalkanoates (PHA) [1, 2]. PHA are biopolymers
accumulated in intracellular granules, as carbon and energy
reserves, by a great number of prokaryotes [3]. Usually, PHA
Correspondence: Dr. Maria A. M. Reis (amr@dq.fct.unl.pt), CQFB/
REQUIMTE, Chemistry Department, FCT/Universidade Nova de
Lisboa, 2829-516 Caparica, Portugal.

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

are accumulated under conditions of unbalanced growth due

to nutrient limitation (e.g. nitrogen, phosphorus, sulfur, or
magnesium) [4]. Among the PHA-producing microorganisms,
Cupriavidus necator (formerly known as Ralstonia eutropha) is
the most widely studied and used for its industrial production.
This bacterium is able to accumulate intracellular reserves of
polyhydroxybutyrate (PHB), which accounts for up to 80% of
its cell dry weight (CDW) [1]. The molecular weight of PHB
produced by wild-type bacteria is usually in the range of
1.0  104 to 3.0  106, with a polydispersity of about 2.0 [5]. It
is a highly crystalline thermoplastic, with physical properties
comparable to some petrochemical plastics like polypropylene,
polyethylene, or polyvinylchloride. Nevertheless, so far it has
not been able to replace conventional plastics on a large scale,
mainly because of its high cost.
PHB recovery from biomass is estimated to represent up to
50% of the polymers production costs [6]. The major step of
the separation process is the extraction of PHB granules, which

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Eng. Life Sci. 2009, 9, No. 6, 454461

involves the disruption of the cells and the subsequent

separation of the polymer from the non-PHB cell material [7].
To improve cell disruption, a pretreatment step may be used,
prior to the extraction, employing chemical (e.g. treatments
with alkali and/or acids) and/or physical methods (e.g. heat
treatment) [8]. The extraction process usually involves the use
of organic solvents that first modify the cell membrane
permeability and then dissolve the polymer. The separation of
PHB from the solvent is performed by solvent evaporation or
precipitation in a non-solvent [8]. The most commonly used
solvents are chlorinated hydrocarbons, such as chloroform,
methylene chloride, and 1,2-dichloroethane [9, 10]. Usually,
the extracted polymer solutions are very viscous, making it
necessary to use large solvent volumes to ease cell debris
removal. Consequently, unless some solvent recycling is
performed, the extraction process becomes too expensive [8].
The objective of this work was to carry out the recovery of
PHB produced by C. necator, by solvent extraction with 1,2propylene carbonate. The starting point was a US patent that
describes the use of cyclic carbonates, such as propylene
carbonate and ethylene carbonate [11]. To our knowledge, this
patent document and a publication by the same authors [12]
are the only references to this subject reported in the literature.
These authors have studied the effect of temperature and
contact time on yield, intrinsic viscosity, and molecular weight
of the extracted polymer. They have shown that propylene
carbonate was more effective than ethylene carbonate in the
recovery of PHB from dry cells of Hydrogenomonas eutropha
(currently renamed as C. necator), Azotobacter chroococcum,
and Bacillus megatherium, and that their extraction procedure
was suitable for the treatment of dry and wet biomass.
In the present study, PHB was extracted from C. necator
biomass using 1,2-propylene carbonate as solvent, based on the
method described by Lafferty and Heinzle [11, 12]. Several
temperatures and contact times were applied. Some modifications were introduced to the Lafferty and Heinzle method,
namely, different precipitation procedures for polymer
separation from the solvent. Additionally, the best conditions
of polymer extraction and precipitation were tested for the
recovery of PHB from biomass subjected to different
pretreatments. These pretreatment procedures (heat and/or pH
shock) were adapted from Kanani et al. [13].
The purpose of this study was the optimization, in a
systematic way, of PHB extraction from biomass, involving the
integration of three different methodologies: biomass
pretreatment, extraction with 1,2-propylene carbonate, and
optimized precipitation. The effect of all these parameters on
the recovered polymers was evaluated with regard to yield,
purity, average molecular weight, and polydispersity, and
thermal characteristics (glass transition and melting temperatures).

Materials and methods

2.1

Polymer production

C. necator DSM 545 was used for the production of PHB.

Cultivations were carried out using a mineral medium with the

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Recovery of polyhydroxyalkanoate

455

following composition (g/L) [14, 15]: nitrilotriacetic acid, 0.19;

Na2HPO4  7H2O, 6.7; KH2PO4, 1.5; (NH4)2SO4, 5.0;
MgSO4  7H2O, 0.5; ferrous ammonium citrate, 0.06;
CaCl2  2H2O, 0.01; 1 mL trace element solution. Each liter of
trace element solution contained 0.3 g H3BO3, 0.2 g
CoCl2  6H2O, 0.1 g ZnSO4  7H2O, 30 mg MnCl2  4H2O, 30 mg
NaMoO4  2H2O, 20 mg NiCl2  6H2O, and 10 mg CuSO4  5H2O.
Glucose and fructose (1:1) were used as carbon sources in a total
concentration of 30 g/L. Cells were incubated without any
nutrient limitation, at 301C for 24 h, and used as inoculum for
bioreactor cultivation.
Polymer production was performed in a 5L bioreactor
(BIOFLO 110; New Brunswick Scientific, CO, USA), with a
working volume of 4 L. The temperature was controlled at
32.51C. The pH was controlled at 7.0 by the automatic addition of 1 M NaOH and 1 M H2SO4. The dissolved oxygen
concentration was controlled above 40% of the saturation
concentration by gradually increasing the stirring rate
(200900 rpm) and the air flow rate (0.1250.9 vvm). Samples
(2030 mL) were withdrawn from the bioreactor every 30 min
and centrifuged (18 000  g, 10 min) for cell separation. The
cell-free supernatant was stored at 201C for the determination of nitrogen and substrate. The cell pellet was also stored at
201C for the quantification of the intracellular PHB content.
The CDW was determined by gravimetry [16]. Glucose and
fructose concentrations in the cell-free supernatant were
determined by high-performance liquid chromatography [17].
The ammonium concentration was determined using a
potentiometric sensor (Orion 9512; Thermo Electron, Massachusetts, USA). The PHB content of the biomass was determined by GC [1820]. Determinations of PHB content, CDW,
glucose, fructose, and ammonium concentrations were
performed in replicate experiments, and the associated error
was in the range of 15%.

2.2

Biomass pretreatments

Biomass pretreatments were adapted from a US ICI patent

[13]. At the end of the cultivation, the standard procedure
consisted in the recovery of the biomass by centrifugation of
the culture broth (21 000  g, 15 min). The biomass was
washed twice with deionized water and stored at 201C. The
tested pretreatment procedures consisted in subjecting the
culture broth to:
(i) thermal treatment at a temperature of 601C for 5 min;
(ii) pH treatment by sequentially raising the pH to 9.0
(addition of 1 M NH4OH), stirring for 5 min, and
lowering the pH to 4.0 (addition of 1 M HCl);
(iii) thermal/pH treatment by sequentially raising the pH to
9.0 (addition of 1 M NH4OH), heating to a temperature
of 601C for 5 min, and finally lowering the pH to 4.0
(addition of 1 M HCl).
Following each procedure, the treated biomass was
recovered from the broth by centrifugation (21 000  g,
15 min), washed twice with deionized water, and stored at
201C.

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456

2.3

Polymer extraction with chloroform

Extraction of PHB from the biomass with chloroform

was performed as described by Dalcanton [21] and used
as the standard extraction technique. Briefly, the wet biomass
(2 g) was heated in chloroform (100 mL) to a temperature
of 601C, under stirring, for 2 h. Later, the cell debris was
separated by centrifugation (3000  g, 15 min, at room
temperature). The polymer was recovered by solvent
evaporation.

2.4

Polymer extraction with 1,2-propylene carbonate

PHB in the biomass was extracted as described by

Lafferty and Heinzle [11, 12], with some modifications.
About 11.5 g of wet biomass was suspended in 150 mL
1,2-propylene carbonate (Fluka, Missouri, USA), in a flask
equipped with a condenser and magnetic stirring, immersed
in a thermostatic oil bath. The suspension was homogenized
and heated at different temperatures: 100, 115, 130 and
1451C; the highest temperature tested was the solvent
boiling point. Once the suspension had reached the set
temperature, it was maintained at that temperature to test
different contact times: 15, 30 and 45 min. Later, the hot
mixture was filtered under vacuum with hydrophilic
polypropylene membrane filters of 0.45 mm porosity.
The retentate, mainly constituted by cell debris, was
washed with 150 mL hot 1,2-propylene carbonate (1001C).
All the filtrate fractions were collected for testing the
precipitation procedures described in the following
section.

2.5

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M. L. Fiorese et al.

Polymer precipitation

The hot polymer solution in 1,2-propylene carbonate,

obtained by filtration, was submitted to three different procedures:
(i) Centrifugation, while still hot (14 000  g, 15 min). The
precipitated polymer, which had a yellowish coloration,
was washed several times with acetone until it presented a
white coloration indicative of the absence of 1,2propylene carbonate. The polymer was suspended in
acetone and filtered under vacuum with membrane
filters, being further washed with acetone during the
filtration (150 mL). Finally, the polymer was dried at
room temperature.
(ii) Cooling to room temperature for 24 h. The precipitated
polymer was separated from the solvent by filtration,
washed with acetone, and dried.
(iii) Precipitation for 48 h. The polymer solution in 1,2propylene carbonate was left to stand for 24 h at room
temperature. Then, two volumes of acetone were added
to the mixture and left at room temperature for another
24 h, after which the precipitated polymer was separated
from the solvent by filtration, washed with acetone, and
dried.

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.6

Polymer characterization

The polymer obtained with each of the procedures tested was

analyzed by GC for the determination of its purity. About 2 mg
of polymer was subjected to methanolysis, as described in
Section 2.1. Purity (%) was calculated as:
purity

mPHP
 100
mfilm

where mPHB is the mass of polymer as quantified by GC and

mfilm is the total mass of sample used for the GC analysis.
The yield of PHB was calculated by a gravimetric method
where the polymer obtained after each extraction/purification/
pretreatment was dried at 601C for 24 h and weighed. The yield
Y (%) was expressed as:
Y

mfilm  purity
 100
mcell  PHBcell

where mfilm (g) is the mass of the polymer film obtained after
extraction from a cell mass mcell (g) with a PHBcell (%)
polymer content.
Number (Mn) and weight average molecular weights (Mw),
as well as the polydispersity index (MwMn), named PD, were
obtained by size exclusion chromatography in a Waters
apparatus equipped with a series of three Waters Ultrastyragel
columns, with porosities of 103, 104 and 105 A. The analysis
was performed at 301C, with chloroform as eluent, at a flow
rate of 1.0 mL/min. PHB was analyzed at a concentration of
1 g/L. Sample injection volumes of 150 mL were used. Absolute
values of Mw and Mn were determined. Universal calibration
was performed and the calibration curve was generated with
monodisperse polystyrene (PS) standards (in the range of
2  103 to 4  106; Waters, Minneapolis, USA and Polymer
Laboratories, Shropshire, UK). The calibration curve was
correlated with PHB using the Mark-Houwink-Sakurada
relationship, [Z] 5 K M a, where [Z] is the viscosity number
limit and K and a are the Mark-Houwink constants for each
polymer/solvent/temperature system [22]. The values of these
constants used for the pairs PHB/chloroform and PS/chloroform were K 5 0.0118 mL/g, a 5 0.78, and K 5 0.0049 mL/g,
a 5 0.78, respectively [23]. Sample injection volumes of 150 mL
were used.
Thermal analysis was performed by differential scanning
calorimetry with a Setaram equipment model DSC 131
(France). The temperature can range from 150 to 7001C,
with heating and cooling velocities between 0.01 and 99.991C/
min, and the flow signal is comprised between 100 and 1
100 mV, with a resolution of 70.2 mW. The samples were
subjected to increasing temperature of up to 3001C (101C/
min). The polymers crystallinity was obtained considering a
melting enthalpy of 142 J/g for 100% PHB [24].

Results and discussion

3.1

PHB production by C. necator

C. necator cultivation runs for PHB production are usually

performed at 301C [5]. Nevertheless, based on previous studies

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Eng. Life Sci. 2009, 9, No. 6, 454461

Recovery of polyhydroxyalkanoate

0.8
10
0.6

8
6

0.4

Nitrogen(g/L)

Total Biomass (g/L); Residual Biomass (g/L)

P(3HB) (g/L)

(data not shown), a temperature of 32.51C was chosen since it

had resulted in the highest cell growth and PHB synthesis. The
average values for biomass, PHB, and residual nutrients
obtained in the fermentation of glucose and fructose by
C. necator are shown in Fig. 1. There are two clear phases: (i) a
growth phase that lasted for about 10 h, where the biomass
concentration increased to 4.6 g/L and (ii) a production phase
that started when nitrogen-limiting conditions occurred,

0
2

10 12 14 16 18 20 22 24 26 28
Cultivation time (h)

Figure 1. Batch fermentation kinetics by C. necator. Total

Biomass (m), PHB (d), residual biomass (D), and residual
nitrogen (&). The vertical line indicates the moment of nitrogen
limitation that induced the beginning of PHB production.

100
Purity (%)

where the total biomass concentration increased to 10.3 g/L

within 27 h of cultivation, mainly due to the increase of PHA
cell content. In this period, 0.49 g/L nitrogen and approximately 20 g/L of fructose and glucose were metabolized.
Although a small amount of PHB was produced during the
growth phase, increased synthesis was observed under nitrogen-limiting conditions. According to Ramsay et al. [14], a
nitrogen concentration below 0.2 g/L allows for PHB accumulation under non-limiting growth conditions. The final
PHB concentration obtained at the end of the fermentation
was 7.3 g/L, corresponding to a PHB biomass content of 71%.
The yield of PHB from fructose and glucose was 0.38 g/g and
the productivity was 0.33 g/L.h.

3.2

Characterization of the polymer extracted with

chloroform

0.2
2

80
60

Yield (%)

457

40
20
0
chloroform 100/15

100/45

115/45

130/15

130/30

145/15

145/30

145/45

Figure 2. Yield and purity of PHB obtained by solvent extraction

with chloroform and with 1,2-propylene carbonate at different
temperatures and contact times.

Chloroform extraction was used as the reference method for

comparison with the extraction with 1,2-propylene carbonate.
Chloroform extraction of the polymer produced by C. necator
yielded 96% of the PHB contained in the cells, with a purity of
95% (Fig. 2). Similar values have been reported for solvent
extraction with chloroform, namely, a yield of 94% and a
purity of 98% [21]. The impurities were probably cell
components that were solubilized by the solvent during the
extraction process.
The polymer extracted with chloroform had a molecular
weight of 1.0  106 and a polydispersity of 3.2 (Fig. 3). The
molecular weight was in the range of the values reported in the
literature (1.0  104 to 4.0  106), but the polydispersity was
higher than the usual value for PHB extracted with chloroform
[5, 25]. The molecular weight and the polydispersity are
important parameters that will determine the suitability of the
polymer for specific applications [25]. The company ZENECA
considers a molecular weight of Mw 5 6  105 (absolute value)
already acceptable for its thermoplastic BIOPOLs [26]. The
polydispersity reflects the degree of heterogeneity of the
polymers chain lengths. Thus, the PHB obtained by chloroform extraction has a good molecular weight, but it is just a
little more heterogeneous.
The physical properties (glass transition temperature, Tg;
melting temperature, Tm; melting enthalpy, DHm; crystallinity, wc)

3,5

3,0

1,2x10

1,0x10

MW

2,0
5

6,0x10

1,5
5

4,0x10

Polydispersity (%)

2,5
5

8,0x10

1,0

2,0x10

0,5

0,0

0,0
chloroform

100/15

100/45

115/45

130/15

130/30

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

145/15

145/30

145/45

Figure 3. Molecular weight and polydispersity of PHB obtained by solvent

extraction with chloroform and with
1,2-propylene carbonate at different
temperatures and contact times.

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M. L. Fiorese et al.

Table 1. Physical characteristics of PHB obtained by solvent

extraction with chloroform and with 1,2-propylene carbonate at
different temperatures and contact times.a)
Temperature (1C)
Standard
chloroform
extraction
145
145
145
130
130
115
100
100

Contact
time (min)

45
30
15
30
15
45
45
15

Tg
(1C)

Tm
(1C)

DHm
(J/g)

wc (%)

4.9

173.5

80.0

56

4.8
5.0
5.0
4.9
4.9
4.9
4.9
4.8

170.1
180.1
177.5
175.0
173.5
175.1
175.8
171.7

77.8
85.0
88.5
85.0
87.8
87.0
84.5
80.5

55
60
62
60
62
61
60
57

a) Tg, Glass transition temperature; Tm, melting temperature; DHm,

melting enthalpy; wc, crystallinity.

of PHB obtained by chloroform extraction are presented

in Table 1. Similar values were reported for chloroform
extraction, namely, glass transition temperatures varying
in the range of -5 to 51C, melting temperatures of 1741801C,
and crystallinities of 5580% [21, 27]. PHB having crystallinity
values ranging between 60 and 80% indicates that the
polymer is a rigid and brittle material, which limits its
use in some final applications that need a high impact resistance [5].

3.3

Polymer extraction with 1,2-propylene carbonate

The processes of extraction and purification of biopolymers

from biomass that utilize halogenated solvents are totally
forbidden nowadays, since they are highly aggressive to the
environment and to human health. To the best of our
knowledge, PHB extraction with 1,2-propylene carbonate has
been first proposed by Lafferty and Heinzle [11, 12] as an
alternative to chlorinated solvents. The advantages of using
1,2-propylene carbonate are its high boiling point, its reusability for several times without purification, and its low toxicity.
PHB produced by C. necator was extracted with 1,2propylene carbonate. Different temperatures (100, 115, 130
and 1451C) and contact times (15, 30 and 45 min) were tested
in order to determine the most appropriate conditions for the
extraction. The efficiency of the process at each temperature/
contact time was evaluated considering the polymer extraction
yield and purity obtained, its molecular weight, and some
thermophysical properties, namely, glass transition temperature, melting temperature, melting enthalpy, and crystallinity
(Table 1). Chloroform extraction was used as the reference
method for comparison of the results obtained.
At low temperatures (100 and 1151C), the extraction was
incomplete, as shown by the low yield obtained (4554%)
(Fig. 2). This result is in accordance with Lafferty and Heinzle
[11, 12] who reported incomplete PHB extraction with

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1,2-propylene carbonate at temperatures below 1201C. In their

studies, the increase of the contact time from 1 to 10 min, for
the extraction at 1101C, slightly improved the yield from 30 to
37%, but longer contact times (1530 min) resulted in a
negligible improvement (up to 38%) [11]. In our study,
increasing the contact time from 15 to 45 min, for the
extraction at 1001C, also did not result in any significant yield
improvement (Fig. 2), but the polymer purity increased from 7
to 43%. Probably, a higher contact time promoted an increase
of the solubility of the impurities. The best performance was
obtained with the combination of a temperature of 1301C and
a contact time of 30 min, with a yield of 95% and a polymer
purity of 84% (Fig. 2). With a lower contact time (15 min),
extraction at 1301C was not complete, resulting in a yield of
66%. The highest yield obtained by Lafferty and Heinzle
[11, 12] (7687%), using 1,2-propylene carbonate as the
extraction solvent, was observed at temperatures of 1201401C
and contact times of 2030 min, but the purity of the polymer
was not evaluated.
The effect of temperature and contact time on the molecular weight was also evaluated. The molecular weights of PHB
obtained for all temperatures/contact times tested were lower
than the molecular weight of the chloroform-extracted polymer (Fig. 3). The highest values obtained were 6.5  105 and
7.4  105, with polydispersity indexes of 2.5 and 3.1, respectively. These values correspond to the extractions performed
with 1,2-propylene carbonate at 1301C with contact times of
15 and 30 min, respectively. The PHB molecular weight is
highly influenced by the temperature and the length of the
thermal treatment applied during the extraction procedure
[28, 29]. A reduction of the polymers molecular weight with
increasing temperature (from 110 to 1401C) and contact time
(from 1 to 30 min) has been reported by Lafferty and Heinzle
for extraction of PHB with propylene carbonate [11, 12]. By
increasing the extraction temperature, the solubility of the
longer PHB chains is enhanced. Consequently, the polymers
molecular weight is also higher for higher temperatures.
Nevertheless, there is a maximum value for temperature that
can be used at which the solvent starts to degrade the polymer,
thus reducing its molecular weight. Interestingly, the polymer
extracted with 1,2-propylene carbonate is more homogeneous
(polydispersity between 2.1 and 3.1) than the one extracted
with chloroform (polydispersity of 3.2), except for the
extraction performed at 1451C with a contact time of 15 min
(polydispersity of 3.3) (Fig. 3).
For the highest temperature tested (1451C) neither yield
nor purity were improved (Fig. 2). In fact, both parameters
showed a decrease for all contact times tested as the
temperature was raised from 130 to 1451C. Moreover, there
was a reduction of the molecular weight, which was more
pronounced as the contact time was increased (Fig. 3). The
same behavior has been observed by Lafferty and Heinzle [11,
12]. The molecular weight of the polymer extracted at 1451C/
30 min contact time was 70% lower than the polymer obtained
at 1301C/30 min. Figure 3 is very clear with regard to the effect
of solvent on polymer degradation for temperatures above
1301C, and of increasing contact time at that temperature (the
boiling point of the solvent, which is 1401501C). Moreover,
the decrease in polydispersity means that the scission

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Recovery of polyhydroxyalkanoate

459

Table 2. Effect of the different procedures used for the precipitation of PHB after extraction with 1,2-propylene carbonate (temperature
and contact time of 1301C and 30 min, respectively) on polymer yield and purity.a)
Procedure
Hot centrifugation
Cooling to room temperature
Precipitation for 48 h

Yield (%)
63
73
95

Purity (%)
72
81
84

Mw
4

2.0  10
6.6  105
7.4  105

PD

Tg (1C)

Tm (1C)

DHm (J/g)

wc (%)

1.6
3.0
3.1

4.9
4.9
4.9

175
174.5
175

83
83.5
85

58
59
60

a) Thermophysical characteristics of the extracted polymer in terms of molecular weight (Mw), glass transition temperature (Tg), melting temperature
(Tm), melting enthalpy (DHm), and crystallinity (wc).

mechanism due to degradation creates very small chains with

similar length.
The polymer obtained by 1,2-propylene carbonate extraction at different temperatures and contact times had melting
temperatures in the range of 170.1180.11C, melting enthalpies
of 77.888.51C, and crystallinities of 5562% (Table 1). These
results show that the physical properties of the polymer were
similar to the PHB extracted with chloroform and within the
ranges reported in the literature for each characteristic parameter evaluated.

3.4

Comparison of precipitation procedures

In this work, three different precipitating procedures

were tested, namely, centrifugation of the hot polymer solution, precipitation by cooling to room temperature, and
precipitation during 48 h. The different precipitating
procedures were performed in order to test the influence
of the contact time between the solvent and the polymer
on the obtained polymer yield and their characteristics.
The improvement of the polymer separation and the homogeneity of the polymeric chains, using the three alternative
methods not tested in the patent, was also an achievement of
these tests.
The results obtained are presented in Table 2. It is clear that
the yield of the extraction with 1,2-propylene carbonate was
increased with the precipitation. In the hot centrifugation
procedure, there was a short precipitation time, resulting in an
incomplete polymer recovery (63%). The molecular weight
was 2.0  104, which was much lower than that of the polymer
obtained by chloroform extraction (Table 1). This result
indicates that the precipitation process was not complete and
only a small fraction of polymer chains was recovered. The
lower value of Mw obtained, differing by two orders of
magnitude from the chloroform extraction, suggests that
degradation phenomena promoting chain scission have
occurred. On the other hand, the polymer obtained by hot
centrifugation was more homogeneous, as shown by its low
polydispersity of 1.6 (Table 2).
In the precipitation by cooling to room temperature, the
time allowed for the precipitation of PHB was increased to 4 h.
With this procedure, 73% of the polymer was recovered with a
higher purity (81%) (Table 2). Furthermore, the molecular
weight (6.6  105) was considerably higher than that of the
polymer obtained by hot centrifugation, but less homogeneous. These results show that longer PHB chains were
recovered from the solution, which could be related to the

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

longer precipitation period and/or the lower temperature of

the precipitation.
In the last precipitation procedure tested, the polymer was
allowed to settle for a much longer period (48 h). The yield was
considerably improved, with 95% of the PHB being recovered
(Table 2), thus confirming that extending the precipitation
period enhances polymer recovery. The polymer obtained by
this procedure had a slightly higher purity and molecular
weight than the polymer obtained by cooling the solution to
room temperature.
The results obtained show that the precipitation procedure
highly influences the polymer recovery and its molecular
weight. As expected, the physical properties were not significantly affected by the precipitation period and the polymer
had identical glass transition and melting temperatures,
melting enthalpies, and crystallinities (4.91C, 1741751C, and
5860%, respectively) (Table 2). These values are within the
literature ranges referred for chloroform extraction, namely,
glass transition temperatures varying in the range of5 to 51C,
melting temperatures of 1741801C, and crystallinities of
5580% [21, 27].

3.5

Biomass pretreatments

Due to its intracellular nature, it is necessary to break the cells

in order to facilitate the release of PHB. Gram-negative
bacteria such as C. necator have a very resistant peptidoglycan
layer in their cell wall [30]. Biomass pretreatments are intended
to make the biomass more amenable to the subsequent solvent
extraction process, thus improving polymer purity and
recovery.
Heat treatment has an impact on the cell solidity, as it
denatures genetic material and proteins and destabilizes the
outer membrane [8]. Additionally, in the case of C. necator,
heat pretreatment also denatures the PHB depolymerase, thus
preventing polymer degradation [2]. Temperatures in the
range of 501401C and durations of 160 min have been
studied by several authors [30, 31]. There are also several
reports on the use of acid or alkaline pretreatments to disrupt
the cells prior to solvent extraction [32, 33]. Acid/alkaline
treatments in combination with heat treatment were first
proposed as processes for biomass flocculation and cell
hydrolysis [13, 34]. The procedure in those treatments
involved raising the pH up to 811 and/or heating to
temperatures in the range of 502001C, followed by lowering
the pH to 25. This strategy has been used as a pretreatment of
PHA-containing cells, prior to the solvent extraction process,

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Eng. Life Sci. 2009, 9, No. 6, 454461

M. L. Fiorese et al.

Table 3. Comparison between the different biomass pretreatments used for the disruption of Cupriavidus necator, followed by extraction
with 1,2-propylene carbonate (temperature and contact time of 1301C and 30 min, respectively), on polymer yield and purity.a)
Biomass pretreatment
None
Heat
pH
Heat/pH

Yield (%)
95
94
94
95

Purity (%)
84
83
86
88

Mw
5

7.4  10
8.0  105
9.5  105
1.3  106

PD

Tg (1C)

Tm (1C)

DHm (J/g)

wc (%)

3.1
3.2
3.4
3.5

4.9
5.0
5.0
4.9

175
177
177
175

85
88
84
82

60
62
59
58

a) Thermophysical characteristics of the extracted polymer in terms of molecular weight (Mw), glass transition temperature (Tg), melting temperature
(Tm), melting enthalpy (DHm), and crystallinity (wc).

with the aim of destabilizing the bacterial cell wall and, hence,
increasing polymer extraction [31].
In this work, the standard procedure consisted in the
recovery of the biomass by centrifugation of the culture broth,
followed by washing with deionized water. The biomass
pretreatment procedures tested were based on the treatment of
the culture broth with high temperature (601C), pH variation
(raising to pH 9, followed by lowering to pH 4), and a
combination of heat and pH variation. The treated biomass
was recovered from the broth by centrifugation and washed
with deionized water, similar to the standard procedure. The
biomass samples obtained with each of the three different
pretreatments were next extracted with 1,2-propylene carbonate under the best conditions selected (temperature and
contact time of 1301C and 30 min, respectively), followed by
polymer precipitation for 48 h.
The results obtained are presented in Table 3. Heat treatment
alone did not improve the yield or the purity of the polymer, but
there was a slight increase of molecular weight. On the other
hand, pH treatment alone did not improve the yield, but the
polymers purity was slightly increased. The main improvement
achieved by the pH treatment was an increase of 22% on
molecular weight, by comparison with the standard procedure.
Even when the combined heat/pH treatment was used, the
yield was not improved. Nevertheless, the polymer had a
higher purity (Table 3). The best performance, in terms of
polymer molecular weight, was observed for the combination
of thermal and pH treatments (1.3  106). In fact, there was an
increase of 43% in comparison to the standard procedure. The
value obtained was also 23% higher than that of the PHB
obtained with chloroform extraction (Table 1).
The biomass pretreatments tested did not significantly
change the PHB physical properties (Table 3). In fact, glass
transition temperature (4.91C), melting temperature
(1751771C), melting enthalpy (8288 J/g), and crystallinity
(5862%) were similar to the standard procedure (4.91C,
1751C, 85 J/g and 60%, respectively). However, there was a
reduction of the degree of crystallinity when pH treatments
were used, being more pronounced for the combined heat/pH
treatment. Also, there was an increase of the polydispersity
indexes for all the pretreatments tested in comparison to the
standard procedure where no pretreatment was applied.
Therefore, pH pretreatments, involving the use of large
amounts of chemicals, are highly disadvantageous for the cost
of the recovery process and pose environmental issues. The
analysis of the polymers characteristics tested enables the
option not to perform these treatments because they are very

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

similar to the ones obtained for the standard polymer. Unless a

very specific chemical or physical property related with a
higher molecular weight will be required, this option shall not
be a reasonable choice.

Concluding remarks

In this study, a procedure for the recovery of PHB from C.

necator biomass was evaluated, involving the integration of
three different methodologies, namely, biomass pretreatment,
extraction with 1,2-propylene carbonate, and optimized
precipitation. The results obtained in this study show that 1,2propylene carbonate can be efficiently used for solvent
extraction of PHB from biomass. Due to its low toxicity and
high boiling point, which enables it to be reused several times
without purification, it can be considered as an alternative to
the highly aggressive chlorinated solvents commonly used. The
best performance was obtained with the combination of a
temperature of 1301C and a contact time of 30 min, with a
polymer yield of 95% and a purity of 84%. The polymer thus
obtained was characterized by a molecular weight of 7.4  105
and a polydispersity of 3.1. The precipitation procedure was
shown to highly influence the polymer recovery and its
molecular weight, but did not significantly affect the PHB
physical properties. Although the polymer yield did not
improve with the heat/pH treatment, this treatment increased
the PHB molecular weight and purity. In order to evaluate the
potential interest of this treatment, the polymer quality
increase must be balanced against the need of using larger
amounts of chemicals, which would increase the overall
polymer recovery costs and require an additional process step
to treat the acid/alkaline stream resulting from this treatment.
Abbreviations : CDW, cell dry weight; PHA, polyhydroxyalkanoate;
PHB, polyhydroxybutyrate

Acknowledgements
M.L.F. acknowledges the Brazilian Government for financing a
CAPES fellowship (Coordenac- ao de Aperfeic- oamento de
Pessoal de Nvel Superior).

Conflict of interest
The authors have declared no conflict of interest.

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Eng. Life Sci. 2009, 9, No. 6, 454461

Recovery of polyhydroxyalkanoate

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