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Optimizing acidic methanolysis of poly(3-hydroxyalkanoates) in gas chromatography analysis.

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ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING
Asia-Pac. J. Chem. Eng. 2009; 4: 487–494
Published online 20 February 2009 in Wiley InterScience
(www.interscience.wiley.com) DOI:10.1002/apj.260
Research Article
Optimizing acidic methanolysis of
poly(3-hydroxyalkanoates) in gas chromatography analysis
Chi-Wei Lo,1 Ho-Shing Wu1 * and Yu-Hong Wei2
1
2
Department of Chemical Engineering and Materials Science, Yuan Ze University, Chung Li, Taoyuan 32003, Taiwan
Graduate School of Biotechnology and Engineering, Yuan Ze University, Chung Li, Taoyuan 32003, Taiwan
Received 19 September 2008; Revised 26 November 2008; Accepted 2 December 2008
ABSTRACT: This work was undertaken to develop an improved gas chromatography (GC) analysis of poly (3hydroxyalkanoate) (PHA) quantification method based on acidic methanolysis. This is achieved by investigating the
kinetics of acidic hydrolysis of PHAs with sulfuric acid in the chloroform/aqueous solution to identify suitable hydrolytic
pretreatment conditions for quantitative analysis of PHAs. The target parameters included sulfuric acid concentration,
salt (NaCl) addition, kind of PHAs (commercial products of poly-3-hydroxybutyrate (PHB), poly(3-hydroxybutyrateco-3-hydroxyvalerate) (PHBV-8%) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx-3.8% and-10.5%),
as well as the type of PHA-producing microorganisms (Cupriavidus taiwanensis 184 and Burkholderia sp. PTU9).
These results show that esters would preferentially accumulate in the organic phase when NaCl was added in the twophase system, thereby enhancing the accuracy of GC analysis. Decomposition efficiency of different types of PHAs
was found to be dependent on sulfuric acid concentration, such as 1% H2 SO4 was favorable for PHB decomposition,
while 5 and 7% H2 SO4 should be used to decompose PHBV and PHBHHx.  2009 Curtin University of Technology
and John Wiley & Sons, Ltd.
KEYWORDS: poly (3-hydroxyalkanoates); PHAs; quantitative analysis; hydrolysis; gas chromatography
INTRODUCTION
Formation of polyesters by microorganisms has caught
more attention, due primarily to potential applications of the polyesters as bioplastics.[1 – 11] One of
these polyesters is called poly (3-hydroxyalkanoates)
(PHAs), which represent a whole family of different polyesters. Among various biodegradable polymer materials known, PHAs demonstrate excellent
and complete biodegradability, making them promising alternatives to petrochemical plastics. The properties of PHAs are very similar to those of polyethylene
(PE) and polypropylene (PP). Copolymers of PHAs,
such as poly (3-hydroxybutyrate-co-3-hydroxyvalerate)
(PHBV), are far less permeable to oxygen than PE
and PP. This character makes PHA copolymers to be
a better material for food packaging because there is
a reduced need for antioxidant addition. The lower
oxygen permeability of PHA copolymers results from
incorporation of a multitude of middle chain length
*Correspondence to: Ho-Shing Wu, Department of Chemical Engineering and Materials Science, Yuan Ze University, Chung Li,
Taoyuan 32003, Taiwan. E-mail: cehswu@saturn.yzu.edu.tw
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
(MCL) monomers into the polymers. PHAs are composed of monomers with 3-hydroxy, 4-hydroxy or 5hydroxy groups, different lengths of carbon backbones
(between 4 and 16 carbon atoms), and a broad range of
functional groups (e.g. halogens, phenoxy, cyano and
epoxy groups). To date, more than 120 different constituents of PHAs have been identified. Among them,
poly (3-hydroxybutyrate) (PHB), PHBV and poly(3hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx)
are the most famous members in the PHA groups.[12]
Several analytical methods are known for the determination of cellular PHA content in laboratory-scale
cultures. Chromatographic techniques are often used to
determine the hydrolytic products of the PHA polymer
for PHA quantification. High performance liquid chromatography (HPLC) was conducted to analyze the products from the digestion of PHA polymer with sulfuric
acid.[13] Gas chromatography (GC) was used to determine the methyl esters of the hydroxyalkanote acids
generated from the digestion of the bacterial pellets
with acidic methanolysis.[14] In addition, infrared (IR)
spectroscopy was used to quantify the polymer content
of a bacterial culture.[15] Nuclear magnetic resonance
(NMR) is also very useful in the analysis of all kinds
of specialized PHA, such as halogenated or acetylated
488
C.-W. LO, H.-S. WU AND Y.-H. WEI
PHA. It is essential to analyze epoxidized PHA resulting from acidic hydrolysis of the PHA as epoxy groups
will split into diols[16] for the study of the physical
and chemical properties and the metabolism of PHA
in intact cells.[17] However, among the aforementioned
four analytical methods for PHA, GC analysis is the
most convenient one and is frequently used to quantify
the PHA content.
The GC analysis method first described by Braunegg
et al .[18] is based on direct acidic methanolysis on the
cells followed by GC analysis of the resulting ester
products. Since extraction of the PHB is not required,
the GC analysis method possesses the advantages of
rapidity and reliability. However, using the Braunegg’s
protocol for PHB quantification, Jan et al .[19] found
that only 62% of PHB was detected in the chloroform
phase. They indicated that the PHB content could not
be accurately obtained for the hydrolysis of PHB under
Braunegg’s method. Riis and Mai[20] had modified
the Braunegg’s method and used hydrochloric acid in
propanol to reduce side products of the esterification
and to minimize losses due to the partitioning to the
aqueous phase.
For more effective depolymerization of PHA into
monomers, different sulfuric acid concentrations were
used to hydrolyze different types of PHAs. This
approach was used in the analysis of poly (3HB-co3HV) in Escherichia coli,[21] Rhodococcus rubber,[22]
and Ralstonia eutropha.[23] This method can be
upgraded to analyze MCL PHA, which was the starting
point of many researchers in this area.[24 – 29] However,
some other factors affecting the accuracy of PHA analysis, such as ester product distribution in the two phases,
sulfuric acid concentration in methanol from 3%[7,30 – 34]
to 10%,[35] and the reaction time from 3.5 h[32,36] to
6 h[30,31] to 20 h.[33 – 35,37] Oehmen et al .[34] optimized
the derivatization conditions (reaction time, acid concentration etc.) for PHB and PHBV using sulfuric acid.
Moreover, the purity of PHAs influences the processing of PHAs in industrial applications. Hence, how to
obtain higher purity of PHAs in the cultures is a crucial issue in industrial uses of PHAs. This study aims
to improve the GC analysis protocol for PHA quantification by investigating the kinetics of acidic hydrolysis
of PHA with sulfuric acid in the chloroform/aqueous
solution. Factors (e.g. sulfuric acid concentration, NaCl
content) influencing the effectiveness of hydrolytic pretreatment of PHA were examined. This study provides
innovative analytical concepts leading to more accurate
determination of PHAs.
Asia-Pacific Journal of Chemical Engineering
95%) were obtained from P&G and Aldrich (American). Methyl benzoate (99%) was obtained from
Riedel-deHaen (American). Chloroform (99%) was purchased from J. T. Baker (American). Methyl alcohol
(99%), sulfuric acid (98%) and sodium chloride (99%)
were obtained from Mallinckrodt (American), Osaka
(Japan) and WAKO (Germany), respectively. Both
methyl(R)-3-hydroxybutyrate (MHB) (C5 H10 O3 , 99%)
and methyl(R)-3-hydroxyvalerate (C6 H12 O3 , 99%) were
obtained from Fluka (Germany).
Bacteria strain and culture conditions
Cupriavidus taiwanensis 184 and Burkholderia sp.
PTU9 strains are provided by Prof. Wen-Ming Chen
from National Kaohsiung Marine University, Taiwan.
Luria-Bertani (LB) broth consisting of tryptone (10 g
l−1 ), yeast extract (5 g l−1 ) and NaCl (10 l−1 ) was
used for revival and preculture of both strains from
frozen stock. Overnight 24 h culture of the strain
was inoculated (2% inoculum) into a defined medium
consisting of gluconic acid (32 g l−1 ), NH4 Cl (1 g
l−1 ), Na2 HPO4 · 12H2 O (7 g l−1 ), KH2 PO4 · 2H2 O (3 g
l−1 ), CaCl2 · 7H2 O (0.01 mM), and MgSO4 (0.01 mM)
(working volume = 100 ml) in shake flasks. The batch
growth was carried out at 30 ◦ C under an agitation rate
of 200 rpm in a rotary incubator (Model LM570R, Yih
Der, Taipei, Taiwan). Samples were taken at designated
time intervals to determine cell concentration, PHA concentration and PHA content in the cell.
Pretreatment by acidic methanolysis for PHB
analysis
This method was modified from the method reported by
Braunegg et al .[18] The samples (pure PHAs or wetted
cell) were dissolved and acidified in 2 ml of methanol
with four concentrations [1, 3, 5 or 15% (v/v)] of
H2 SO4 , and 2 ml of chloroform in a screw-capped test
tube. The solution was then kept at 100 ◦ C for 6 h.
After cooling to ambient temperature, NaCl aqueous
solution (1 ml, 1 M) was added, and the sample was then
shaken for 10 min. After staying still for 6 h, the two
phases were permitted to separate. The organic phase
was withdrawn for GC analysis. The benzoate methyl
ester was used as the external standard compound. The
acidic methanolysis reactions are expressed as follows:
MATERIALS AND METHODS
O
H
CH3
C
C
C
H
H
Materials
O
PHB, 99%, PHBV, 8 wt%, PHBHHx, 3.8 wt%,
and 10.5 wt%, and (R) hydroxybutyric acid (HB
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
n
H
O
C
O
nH2O
H2SO4
n
H
CH3
C
C
H
H
(1)
OH
Asia-Pac. J. Chem. Eng. 2009; 4: 487–494
DOI: 10.1002/apj
Asia-Pacific Journal of Chemical Engineering
H
O
O
H
CH3
C
C
C
H
H
O
H3C
O
C
OH
H
CH3
C
C
H
H
OPTIMIZING ACIDIC METHANOLYSIS OF POLY(3-HYDROXYALKANOATES)
RESULTS AND DISCUSSION
CH3OH
(2)
OH
H2O
Analysis of PHB produced from different
bacterial strains
C. taiwanensis 184 and Burkholderia sp. PTU9 were
used to produce PHB products via flask fermentation. The fermentation conditions for C. taiwanensis
184 were modified medium, 37 ◦ C, 200 rpm and pH
7.0. The PHA was isolated from the mature cells by
NaOH method, e.g. 4%, 4-ml NaOH. The purified samples were used for PHA analysis by first applying the
improved acid methanolysis conditions cited above, second improving the partition of ester product, and third
quantifying the PHA by GC analysis. In order to obtain
the optimal methanolysis conditions, the experiments
were also conducted at different sulfuric acid concentrations (1–15%) and treatment times (3–12h). The
amount of by-products in the reaction system was fewer
for total product in GC analysis. It can be neglected for
the product analysis.
(a)
Effect of purification on the characteristics of
PHA
A variety of microorganisms is known to produce
PHA.[1 – 11] Commercial products of PHAs have been
available in the market offered by many companies (e.g.
Nodex, P&G, Metabolix etc.). However, the application of PHAs is restricted by its purity. Figure 1 shows
the conformations of three types of PHB by thermal
press process. The PHB products were obtained from
(a) C. taiwanensis 184 of purity > 99% and molecular weight (MW) = 1 200 000 from our laboratory,
(b) C. taiwanensis 184 of purity = 97% and MW =
1 100 000 from our laboratory, and (c) Aldrich (American) of purity = 99% and MW = 675 000. The fermentation conditions are shown in Experimental section.
According to samples shown in Fig. 1(a) and (c), PHB
membranes were easily made by thermal press process
at 178 ◦ C. On the basis of the thermogravimetric analyzer (TGA) result shown in Fig. 1(d), the polymer
backbones formed in a one-stage process where the
decomposing temperature range was 190–300 ◦ C. The
onset temperature of the samples obtained from Aldrich
of purity 99% and C. taiwanensis 184 (99, 97% pure)
was 242, 245 and 198 ◦ C, respectively, indicating that
the onset temperature slightly increased with increasing
(d)
(b)
(c)
Figure 1. Effect of thermal press for (a) 99% of Cupriavidus taiwanensis 184 and (b) 97%
of Cupriavidus taiwanensis 184, (c) Aldrich and (d) thermogravimetric analysis for–: 97%
of Cupriavidus taiwanensis 184, – – –: 99% of Cupriavidus taiwanensis 184, –.–: 98% of
Aldrich. This figure is available in colour online at www.apjChemEng.com.
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
Asia-Pac. J. Chem. Eng. 2009; 4: 487–494
DOI: 10.1002/apj
489
C.-W. LO, H.-S. WU AND Y.-H. WEI
purity of PHB. When the onset temperature is lower
than 220 ◦ C (i.e. PHB from C. taiwanensis 184), the
sample cannot be used to make a membrane (Fig. 1(b)).
The impurity in the sample would play an important
role in influencing the thermal press process and thermal properties. The impurity may be lipid, medium and
digestion agents. The char yield represents the residual amount of the tested samples at 600 ◦ C. The char
yield in TGA for samples from Aldrich, 99% pure and
C. taiwanensis 184 of 99 and 97% were 1.1, 1.29, and
0.93%, respectively. The inorganic impurities for three
cases are almost the same. Hence, the purity differences
among three cases are from the organic impurity in
the PHAs. Furthermore, how to determine the purity
of the sample plays a crucial role in using the material in industrial applications. In the past, the purity of
PHAs was determined by Braunegg’s method using GC.
However, the method has some problems that need to
be solved to improve the accuracy of PHA analysis. The
problems and the improving strategies are addressed in
the following text.
Quantitative analysis of standard PHB using
Braunegg’s method
In the method described by Braunegg et al ,[18] the
standard PHB were treated with 2 ml of chloroform
and 2 ml of methanol/3% H2 SO4 at 100 ◦ C for 3 h, and
then mixed with 1 ml of water to separate two phases.
The organic phase was withdrawn and injected for GC
analysis. They claimed this method could accurately
determine the purity of PHB. However, based on the
reactions indicated in Eqns (1) and (2), two reactions
are involved in this method. First, PHB is hydrolyzed
by sulfuric acid. The resulting 3-hydroxybutyric acid
is then reacted with methanol. According to theory of
chemistry, the two reactions are reversible (equilibrium
reaction). The final product, MHB, was measured by
GC in order to determine the purity of PHB. However,
the purity of PHB determined by GC using methyl
ester as the calibration standard was around 80%
which will be discussed below. If the concentration of
PHB increases, the yield of MHB determined slightly
increases (Fig. 2) because the reaction in Eqn (2) is
pushed to the right according to Le-Chatelier law.
This finding demonstrates that the accuracy of PHB
determined by GC was influenced by the amount of
PHB used for the analysis. In the past, the researchers
used one-point calibration of PHB to predict different
concentrations of PHB from microorganisms, resulting
in poor accuracy of PHB quantification. Hence, the
results of kinetic study (Fig. 2) suggest that if the PHB
(or PHA) is also used as the calibration standard, the
calibration curve must be generated using multipoint
calibration. The calibration curve may be a curviform
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
Asia-Pacific Journal of Chemical Engineering
100
80
Yield of MHB (%)
490
60
40
20
0
0
10
20
30
40
50
60
Weight of PHB (mg)
Figure 2. Relationship between of commercial PHB
(Aldrish) and MHB. Acidified methanol (2 ml, H2 SO4 ,
1%), chloroform (2 ml), 100 ◦ C, reaction time (6 h), NaCl
solution (1 M, 1 ml).
line and not linear line if the detective range of PHA
concentration is broad.
Effect of NaCl concentration on salting out
In addition, the Braunegg’s method[18] was conducted
in the two phases of chloroform and aqueous solution.
Adding water to organic phase will lead to the partition
of the ester between the organic and the aqueous phases.
As the MHB can be distributed into both phases, the
accuracy of PHB quantification is reduced, since only
organic phase was taken for GC analysis. Figure 3
shows the results of methanolysis in the presence and
absence of water. When 2 ml of water was added, only
48% of MHB was detected in the organic phase under
our experimental conditions (the H2 SO4 concentration
was 1%). Without water addition, 82% of the MHB
was detected. This observation is in agreement with the
results reported by Jan et al .[19] The foregoing results
clearly show that partition of MHB at aqueous phase
may cause severe error in Braunegg’s method.
In order to avoid the distribution of MHB in the
aqueous phase, the salting out method (i.e. addition
of NaCl) was conducted to avoid partition of ester
into aqueous phase, thereby increasing the accumulation
of MHB in the organic phase. Figure 4 shows the
relationship between MHB and NaCl addition for PHB
and PHBV-8%. With the addition of NaCl (0.5 M) or
higher, the yield of MHB determined increased from
44 to 85%. Therefore, adding NaCl into two-phase
system can increase the accuracy of PHA analysis.
Asia-Pac. J. Chem. Eng. 2009; 4: 487–494
DOI: 10.1002/apj
Yield of MHB (%)
Asia-Pacific Journal of Chemical Engineering
OPTIMIZING ACIDIC METHANOLYSIS OF POLY(3-HYDROXYALKANOATES)
100
120
80
100
80
Yield of MHB and MHHx (%)
60
40
20
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
H2SO4 (%)
60
(a)
40
120
100
80
Figure 3. Effect of water on H2 SO4 concentration for
PHB acidic methanolysis. PHB (20 mg), acidified methanol
(2 ml, H2 SO4 , 1%, chloroform (2 ml), 100 ◦ C, reaction
time: (6 h), : no added water, : added water.
60
(b)
°
40
0.0
0.5
1.0
1.5
2.0
2.5
3.0
NaCl (M)
100
Figure 5. Effect of NaCl for methyl (R)3-hydroxybutyrate
(MHB) and methyl(R)3-hydroxyhexanoate (MHHx) in
PHBHHx copolymer (PHHx fraction 3.8% (a) and 10.5%
(b)) (20 mg), acidified methanol (2 ml, H2 SO4 , 1%),
:
chloroform (2 ml), 100 ◦ C, reaction time (6 h). (a)
RMHB, : MHHx, (b) : RMHB, : MHHx.
Yield of MHB (%)
90
80
°
70
60
50
For short chain length PHA (i.e. PHB, PHB-co-PHV),
sodium chlorite solution could allow the monomer
to stay in organic phase by salting out effect. For
MCL PHA (PHB-co-PHHx), a higher concentration of
sulfuric acid and sodium chlorite would have the same
effect to enhance monomer to stay in the organic phase.
(a)
40
Yield of MHB and MHV (%)
°
100
90
80
70
Effect of H2 SO4 concentration
60
50
40
(b)
0
1
2
3
4
5
6
NaCl (M)
Figure 4. Effect of NaCl for methyl (R)3-hydroxybutyrate
(MHB) and methyl(R)3-hydroxyvalerate (MHV). PHB (or
PHBV-8%) (20 mg), acidified methanol (2 ml, H2 SO4 , 1%),
chloroform (2 ml), 100 ◦ C, reaction time (6 h). (a) : MHB,
(b) : MHB, : MHV.
°
However, the effect of salting out with NaCl may be
reduced while determining the MCL PHAs because the
methyl ester is of higher lipophilicity (Fig. 5). On the
other hand, the concentration of sulfuric acid would be
increased to treat higher MCL of PHA copolymer. The
suitable concentrations are 1, 5, 7% for PHB, PHBV,
PHBHHx. When the reaction completed, the monomer
of PHA would stay in both aqueous and organic phases.
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
The H2 SO4 concentrations used by Braunegg et al .[18]
and Lageveen et al .[24] were 3 and 15%, respectively. It
is thus of interest to identify which H2 SO4 concentration
is optimal. In this study, different sulfuric acid concentrations (1, 3, 5 and 15%) were used to prepare the
acidified methanol for the determination of PHB polymer. For PHB, Fig. 6(a)–(c) shows that the initial rate
of MHB formation increases when the H2 SO4 concentration increases. This result reveals that higher H2 SO4
concentration enables faster decomposition of polymers
to monomers and then reacted with methanol to form
MHB, as shown in Eqn (1). However, the equilibrium
yield of MHB decreases when the H2 SO4 concentration
increases because the ester reaction tends to shift left,
as shown in Eqn (2). Hence, the elevation of H2 SO4
concentration would increase the polymer decomposition but decrease the production of MHB. The difference in the yield of MHB for 1 and 15% of H2 SO4
concentration were over 20%. Figure 6(d) shows the
Asia-Pac. J. Chem. Eng. 2009; 4: 487–494
DOI: 10.1002/apj
491
C.-W. LO, H.-S. WU AND Y.-H. WEI
Asia-Pacific Journal of Chemical Engineering
100
80
60
40
Yield of MHB (%)
20
(a)
(b)
(c)
(d)
0
100
80
60
40
20
0
0
2
4
6
8
10 0
Time (h)
2
4
6
8
10
Figure 6. Effect of H2 SO4 concentration on reaction time for PHB and
HB at different temperatures. Acidified methanol (2 ml), chloroform
(2 ml), reaction time (6 h), NaCl solution (1 M, 1 ml). (a) PHB (20 mg),
90 ◦ C, : 1%, : 3%, : 5%, ◊: 15%. (b) PHB (20 mg), 100 ◦ C, : 1%,
: 3%, : 5%, ◊: 15%. (c) PHB (20 mg), 110 ◦ C, : 1%, : 3%, :
5%, ◊: 15%. (d) HB (20 mg), 100 ◦ C, : 1%, : 3%, : 5%, ◊: 15%.
°
acidic methanolysis of pure HB, as shown in Eqn (2).
The yield of MHB decreases from 85% with increasing sulfuric acid concentration. This finding shows that
higher sulfuric acid concentration allowed the reaction
to go left (Eqn (2)). In this case, increasing H2 SO4
concentration would reduce the detection accuracy of
PHB. Moreover, Fig. 6 also shows that when temperature increases, the reaction rate also increases and so
the reaction time required to reach equilibrium state
would become shorter. However, the reaction condition
of using a screw-capped test tube may explode when the
temperature is more than 100 ◦ C. Hence, more reaction
time at 100 ◦ C was needed to increase the degree of
hydrolysis and so as to have the equilibrium value of
MHB, and thus increase the accuracy of PHB measurement. Therefore, the suggested reaction time is more
than 6 h. If the reaction time is less than 6 h, the variation of GC accuracy will be increased. The results
correspond to the report of Oehmen et al .[34]
Figure 7 shows that using 1% sulfuric acid concentration could not decompose PHBV-8% copolymer completely. Increasing H2 SO4 concentration from 1 to 5%
led to get a better yield. In both cases, longer treatment
time of more 6 h was needed to complete the reaction
but the monomer concentration would decrease more
than 10 h.
Figures 8 and 9 also show that using 1% sulfuric acid
concentration could not decompose PHBHHx-3.8% and
PHBHHx-10.5% copolymer completely, too. Increasing
H2 SO4 concentration from 1 to 7% led to a better yield
of MHB and MHHx. However, when the concentration
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
°
°
°
100
90
Yield of MHB and MHV (%)
492
80
70
60
50
40
0
2
4
8
6
Time (h)
10
12
14
Figure 7. Effect of H2 SO4 concentration on reaction time
for PHBV-8%. PHBV-8% (20 mg), acidified methanol
(2 ml), chloroform (2 ml), reaction time (6 h), NaCl
solution (1 M, 1 ml)., 100 ◦ C. : 1% H2 SO4 (MHB): 1%
3% H2 SO4 (MHV)
H2 SO4 (MHV) : 3% H2 SO4 (MHB):
: 5% H2 SO4 (MHB):
5% H2 SO4 (MHV): ◊ 15%
15% H2 SO4 (MHV).
H2 SO4 (MHB):
°
of sulfuric acid increased to 12%, the yields of MHB
and MHHx will decrease slightly (Fig. 8). This is
because that higher concentration of sulfuric acid allow
Asia-Pac. J. Chem. Eng. 2009; 4: 487–494
DOI: 10.1002/apj
Asia-Pacific Journal of Chemical Engineering
OPTIMIZING ACIDIC METHANOLYSIS OF POLY(3-HYDROXYALKANOATES)
ester product to produce acid compound (Eqn (2)).
It also happens on longer treatment time. On the
basis of Figs 6–9, the yields of MHB and MHHx
will be reduced with using longer treatment time.
When increased contents of MCL-PHA copolymer
(Figs 7–9), it would be improved. Although higher
H2 SO4 concentration would decompose the MCL PHAs
into monomers, it also allows the reaction shift to left,
as shown in Eqn (2), or even decompose ester product
to small compound, reported by Oehmen et al .[34]
Yield of MHB (%)
120
100
80
60
40
20
(a)
0
Yield of MHHx (%)
120
100
PHB analysis from PHB-containing bacterial
cells
80
60
40
20
0
(b)
0
2
4
6
8
10
Time (h)
Figure 8. Effect of H2 SO4 concentration on reaction time for
PHBHHx-3.8%. PHBHHx-3.8% (20 mg), acidified methanol
(2 ml), chloroform (2 ml), reaction time (6 h), NaCl solution
(1 M, 1 ml), 100 ◦ C. : 1% H2 SO4 : 5% H2 SO4 : 7%
H2 SO4 .
°
Yield of MHB (%)
120
100
The PHB produced from different bacterial strains (i.e.
C. taiwanensis 184 and Burkholderia sp. PTU9) was
analyzed by the improved PHA quantification method
mentioned above. The optimal sulfuric acid concentration and reaction time for acidic methanolysis were
also identified. There was an optimal treatment time for
acidic methanolysis of wetted C. taiwanensis 184 cells,
which needed longer reaction time than pure polymers.
However, the trend for H2 SO4 concentration was the
same as treating the pure polymer (PHB). In contrast,
treating the sample with Burkholderia sp. PTU9 displayed a different trend, as the yield of MHB decreased
with increasing H2 SO4 concentration (Fig. 10). Therefore, a lower H2 SO4 concentration is preferred for
the acidic methanolysis-GC analysis method for that
sample.
80
60
80
40
20
(a)
0
60
Yield of MHB (%)
Yield of MHHx (%)
120
100
80
60
40
40
20
0
20
(b)
0
2
4
6
8
10
Time (h)
0
Figure 9. Effect of H2 SO4 concentration on reaction
time for PHBHHx-10.5%. PHBHHx-10.5% (20 mg), acidified
methanol (2 ml), chloroform (2 ml), reaction time (6 h), NaCl
solution (1 M, 1 ml), 100 ◦ C : 1% H2 SO4 , : 3% H2 SO4 , :
5% H2 SO4 , : 7% H2 SO4 , : 9% H2 SO4 , : 12% H2 SO4 .
°
0
2
4
6
8
10
12
14
16
Concentration of sulfuric acid (%)
Figure 10. Effect of kind of organism (Cupriavidus
taiwanensis 184 and Burkholderia sp. PTU9) for organism
(20 mg), acidified methanol (2 ml, H2 SO4 ), chloroform
(2 ml), NaCl solution (1 M, 1 ml), reaction time (6 h), :
Cupriavidus taiwanensis 184, : Burkholderia sp. PTU9.
°
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
Asia-Pac. J. Chem. Eng. 2009; 4: 487–494
DOI: 10.1002/apj
493
494
C.-W. LO, H.-S. WU AND Y.-H. WEI
CONCLUSION
PHB, PHB-co-PHV and PHB-co-PHHx were identified
with sulfuric acid and methanol. Adding 1-M NaCl
to aqueous phase can avoid the problem which just
sample from the organic phase will loss some ester
that still stay at aqueous phase. The optimal acid
methanolysis for PHB analysis shows 1% sulfuric acid
concentration and 6 h of reaction time. But treating
PHB-co-PHV-8%, PHB-co-PHHx-3.8% and PHB-coPHHx-10.5% must increase concentration of sulfuric
acid to 5 and 7%. This result in the study shows
the higher sulfuric acid concentration was used in this
analysis of PHB copolymer. The reaction time needs
long time (e.g. more 6 h) to complete the reaction.
If the reaction time was hoped to be reduced below
20 min, which is almost applied in chemical analysis,
the determining accuracy by GC was reduced when the
organism was directly reacted. Increasing accuracy of
GC will increase the purity of PHAs. Therefore, the new
method for methanolysis of PHAs must be developed
to reduce the reaction time and improve the accuracy
of GC in the future.
Acknowledgements
The authors would like to thank the financial support
from Oriental Union Chemical Corporation (Taipei,
Taiwan) and from National Science Council of Taiwan
(grant No 94-2622-E-155-009).
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DOI: 10.1002/apj
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