International Journal of Biological Macromolecules 35 (2005) 127–133
Bacterial synthesis of polyhydroxyalkanoates containing aromatic and
aliphatic monomers by Pseudomonas putida CA-3
Patrick G. Ward, Kevin E. O’Connor ∗
Department of Industrial Microbiology, Centre for Synthesis and Chemical Biology, Conway Institute for Biomolecular and Biomedical Research,
National University of Ireland, University College Dublin, Belfield, Dublin 4, Ireland
Received 13 April 2004; received in revised form 7 January 2005; accepted 7 January 2005
Abstract
Pseudomonas putida CA-3 has the ability to accumulate to high levels unique polyhydroxyalkanoate (PHA) heteropolymers composed of
aromatic and aliphatic monomers. The majority of monomers are aromatic making up 98% of the polymer. (R)-3-hydroxyphenylvalerate and
(R)-3-hydroxyphenylhexanoate are the most abundant monomers found in polymers accumulated from phenylalkanoic acids with an uneven
and even number of carbons on the acyl side chain respectively. PHAs accumulated from phenylvaleric and phenylhexanoic acid were partially
crystalline while all other PHAs were amorphous. Significant differences in the yield and PHA content of the cells occurred when different
phenylalkanoic acids were supplied as growth substrates. Increasing the initial concentration of the growth substrate increased both the PHA
content of the cells and the overall yield (g PHA/g carbon supplied) of PHA accumulated by P. putida CA-3 cells. The highest PHA content (%
cell dry wt.) from an aromatic carbon source was 59% when 15 mM phenylvaleric acid was supplied as the sole source of carbon and energy.
This corresponded to a maximum PHA yield of 0.42 g PHA/g carbon supplied. In and attempt to increase the level of PHA accumulated from
related growth substrates acrylic acid was added to the growth medium. However, the addition of various concentrations of acrylic acid to the
growth medium had either no effect or decreased the PHA content of the cell accumulated from phenylalkanoic acids by P. putida CA-3.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Polyhydroxyalkanoate; Aromatic; Pseudomonas putida
1. Introduction
Polyhydroxyalkanoates are polyesters produced by bacteria as intracellular storage materials in response to a variety
of nutritional and environmental conditions, such as nitrogen
limitation [1,2]. The monomer composition of PHA determines the physical and chemical properties of the polymer.
The alteration of monomer composition of PHA could lead
to altered and desirable polymer properties. PHA monomer
composition in turn depends on the carbon source, the
metabolic route to PHA and the specificity of the enzyme
system synthesising PHA [1,3].
␤-Oxidation is the main pathway involved in providing
substrates for PHA biosynthesis in pseudomonads when
∗
Corresponding author. Tel.: +353 1 706 1307; fax: +353 1 706 1183.
E-mail address: [email protected] (K.E. O’Connor).
0141-8130/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijbiomac.2005.01.001
related substrates such as alkanoic acids (Aks) and phenylalkanoic acids (PhAks) are used as the carbon source [2,4].
The accumulation of PHA during growth on non-related
carbon sources, such as glucose, acetate, and ethanol,
proceeds through acetyl-CoA and fatty acid de novo biosynthesis [5,6]. Although many different organisms can utilise
aromatic hydrocarbons as a carbon and energy source, only
a limited number of bacteria such as Pseudomonas putida
U, P. putida BM01 and P. oleovorans, have the ability to
produce PHA containing aromatic monomers [4,7–10]. This
may reflect the metabolic route through which these aromatic
compounds are metabolised but also the substrate range of
the PHA polymerase [4,11]. To date aromatic substrates
supplied as sole carbon sources for PHA accumulation
have resulted in moderate growth yields [9,12]. Even under
optimum conditions cell dry weights of between 0.33 and
0.4 g/l were obtained when P. oleovorans and P. putida
128
P.G. Ward, K.E. O’Connor / International Journal of Biological Macromolecules 35 (2005) 127–133
BMO1 were grown on 10 and 15 mM phenylvaleric acid
respectively as the sole carbon source [9,12].
PHA polymers containing phenylalkanoate monomers
have been identified as having possible uses as drug vehicles
with dual properties [13]. This is due to the fact that
the degradation of PHA may allow the slow release of
pharmacological substances from PHA implants. In addition
the degradation of aromatic PHA may also result in the
generation of intermediates such as phenylacetic acid and
phenylbutyric acid that have been identified as potential
anti-tumor, analgesic and chemopreventive compounds
[13–16].
In this study, the yield, monomer composition, and polymer properties of PHA accumulated by P. putida CA-3 when
grown on a variety of PhAks as a sole source of carbon and
energy is described. A comparison of the yield and composition of PHA accumulated from aromatic and aliphatic carbon
sources by the same bacterial strain is also described. Finally,
the effect of metabolic pathway inhibitors on PHA accumulation in P. putida CA-3 is reported.
2. Materials and methods
2.1. Strain, media and growth conditions
P. putida CA-3 (NCIMB 41162) is one of three cultures
isolated from a bioreactor containing styrene. P. putida CA-3
has previously been shown to have the ability to accumulate
PHA from styrene and phenylacetic acid [17]. Cultures
were grown in modified E2 medium (pH 7.1) at 30 ◦ C, with
shaking at 200 rpm. E2 medium was prepared as previously
described [18]. For PHA production, P. putida CA-3 was
grown in batch culture, under nitrogen limiting conditions.
Sodium ammonium phosphate (SAP) was supplied at a
concentration of 1.0 g/l (67 mg nitrogen/l) as the nitrogen
source in the E2 growth medium. Sterilised solutions of
all growth substrates were added directly to the media,
post-autoclaving, at the required concentrations. Cells were
harvested after 48 h for PHA quantification and monomer
determination.
For experiments investigating the effect of acrylic acid
on PHA accumulation, P. putida CA-3 cells were grown for
24 h in non-PHA accumulating E2 medium containing 3.5 g
SAP/l and the relevant growth substrate at a concentration of
5 mM. Cells were then washed and transferred to E2 medium
containing 0.125 g/l SAP, in order to induce PHA accumulation, a further 5 mM of the growth substrate and the relevant
concentration of acrylic acid (0–20 mM). PHA accumulation
was measured after a further 24 h of incubation.
2.2. Analysis of nitrogen source in the growth medium
The concentration of sodium ammonium phosphate in the
growth medium was determined by measuring the formation
of indophenol from phenol as previously described [19].
2.3. PHA monomer determination
PHA monomers composition was determined using the
method previously described [2]. Samples were analysed on
a Fisons GC-8000 series gas chromatograph. For peak identification, PHA standards from P. oleovorans (aliphatic) and P.
putida U (aromatic) were used. PHA monomer composition
was confirmed by gas chromatography–mass spectrometry
(GC–MS). The composition of aromatic PHA was further
confirmed by 13 C and 1 H NMR analysis of the polymer.
2.4. Isolation and analysis of PHA polymers
PHA polymers were isolated using the method previously
described [2]. 13 C nuclear magnetic resonance (NMR) spectra was performed in a Bruker advance 400 MHz NMR spectrometer operating at 111 MHz for 13 C measurements at room
temperature. NMR analysis was obtained from 25% (w/v)
chloroform solutions, and a delay time between pulses of 2.0 s
was applied. Differential scanning calorimetry (DSC) experiments were carried out in a Perkin-Elmer Pyris Diamond
system. Two scans were performed by using a 20 ◦ C/min
heating rate and a 200 ◦ C/min cooling rate (quenching) between runs. Thermograms were obtained in the range −60 to
+75 ◦ C under helium purge. The glass transition temperature
(Tg ) values were taken at the inflection point on the transition
of second scans. Thermogravimetric analysis investigation
was carried out using a Perkin-Elmer Pyris 1 TGA, over a
temperature range of 30–600 ◦ C and a rate was 10 ◦ C/min.
Average molecular weights, molecular weight distribution
and polydispersity index were determined using a Waters gel
permeation chromatograph equipped with a refractive index
detector series 410. A set of 104 and 105 , and 500 Å PLgel columns conditioned at 30 ◦ C were used to elute samples
(1 mg/ml) and 1 ml/min HPLC grade tetrahydrofuran flow
rate. Ten samples of polystyrene standards having molecular weights ranging from 1340 to 950,000 Da were used for
calibration.
3. Results and discussion
3.1. PHA accumulation from aromatic substrates
P. putida CA-3 is capable of accumulating PHA from a
number of PhAks with five carbons or more in the acyl side
chain (PhAksn≥5 ) (Table 1). However, when supplied with
phenylbutyric acid as the sole source of carbon and energy,
P. putida CA-3 was incapable of growth after 48 h of incubation (Table 1). In keeping with previous experiments with
P. putida CA-3 the concentration of nitrogen in the growth
medium was not detectable after 10–16 h of growth (depending on the growth substrate) [17]. The depletion of nitrogen
in the growth medium coincided with PHA accumulation.
In addition, any further increase in the cell dry weight after
the total depletion of nitrogen was due to the accumulation
P.G. Ward, K.E. O’Connor / International Journal of Biological Macromolecules 35 (2005) 127–133
129
Table 1
Data for P. putida CA-3 cells grown on a range of phenylalkanoic acids at concentrations where maximum yield (g PHA/g carbon supplied) was observed
Substrate
Initial substrate
concentration (mM)
Carbon supplied
(g carbon/l)
% PHAa
Cell dry
wt. (g/l)
Total PHA (g/l)
PHAb yield
(g/g carbon)
Phenylbutyric acid
Phenylvaleric acid
Phenylhexanoic acid
Phenylheptanoic acid
Phenyloctanoic acid
Phenyldecanoic acid
10
15
20
15
15
15
1.20
1.96
2.88
2.34
2.52
2.70
ND
59 ± 3
45 ± 2
50 ± 3
52 ± 2
53 ± 3
NG
1.39
1.07
1.12
1.16
1.19
ND
0.82
0.48
0.56
0.60
0.63
ND
0.42
0.17
0.24
0.24
0.23
ND: not determined; NG: no growth on this substrate.
a All experiments were carried out in 50 ml of E2 medium with a sodium ammonium phosphate (SAP) concentration of 1.0 g/l, over a 48 h period. PHA is
expressed as a percentage of cell dry weight.
b PHA yield (g/g carbon) = the grams of PHA accumulated per gram of carbon supplied.
of PHA (data not shown). When concentrations of 10 mM
PhAks were supplied to the growth media the carbon source
was no longer detectable after 48 h of growth. When 15 mM
PhAks were supplied, up to 3 mM of the carbon sources remained in the media after 48 h of growth (data not shown).
Increasing the initial concentration of the substrate supplied to the growth medium increased both the % cell dry
wt. of PHA and the PHA yield (g PHA/g carbon) accumulated by P. putida CA-3 cells for all aromatic substrates tested
(Fig. 1). The highest level of PHA (% cell dry wt.) accumulated by P. putida CA-3 cells grown on aromatic substrates
was 59%, when grown on 15 mM phenylvaleric acid (Fig. 1).
This corresponded to a PHA yield of 0.42 g PHA/g carbon
(Table 1, Fig. 1), and was the highest yield recorded for an
aromatic growth substrate at any of the concentrations tested.
The highest PHA contents of cells grown on phenylheptanoic
acid (50%), phenyloctanoic acid (52%) and phenyldecanoic
acid (52%) were also at a 15 mM concentration of these substrates (Fig. 1). This corresponded to PHA yields (g PHA/g
carbon) of 0.24, 0.24 and 0.23, respectively, which were also
the highest yields recorded for these substrates (Table 1). A
Fig. 1. PHA accumulation by P. putida CA-3 cells grown on phenylvaleric
acid (䊉), phenylhexanoic acid (), phenylheptanoic acid (), phenyloctanoic acid () and phenyldecanoic acid (×) at varying initial concentrations.
further increase in the initial concentration to 20 mM resulted
in a decrease in PHA accumulation for all but one substrate
namely, phenylhexanoic acid (Fig. 1).
The highest PHA content of 59%, achieved by P. putida
CA-3 cells grown on PhAksn≥5 is roughly equivalent to that
achieved by P. putida BM01 (56%) when grown on 20 mM
phenylvaleric acid and supplemented with 40 mM butyric
acid [9]. The highest PHA content of wild type P. putida
U was 28% from 15 mM phenylvaleric acid [4], a value 2.08
fold lower than P. putida CA-3.
GC–MS analysis of the monomer composition of the
PHA accumulated from these PhAksn≥5 revealed that the
majority of the monomers were aromatic (Table 2). All
aromatic monomers produced by P. putida CA-3 from
PhAksn≥5 had an acyl moiety either equal in length to the
growth substrate or shorter by multiples of two carbon units,
suggesting aromatic PHA intermediates are being generated through the ␤-oxidation pathway [5], i.e. when grown
on phenylheptanoic acid, the aromatic monomers incorporated into the PHA were (R)-3-hydroxyphenylheptanoate
(3HPHp) and (R)-3-hydroxyphenylvaleroate (3HPV). (R)3-hydroxyphenylbutyrate (3HPB) and 3HPV are the smallest aromatic monomers observed in PHA accumulated by P.
putida CA-3 cells grown on substrates with an even and uneven number of carbons in the acyl side chain respectively
(Table 2). Thus, while P. putida CA-3 is not capable of growth
with phenylbutyric acid (R)-3-hydroxyphenylbutyryl-CoA is
a substrate for PHA synthesis suggesting enzymes early in
the phenylalkanoic acid catabolic pathway leading to the
PHA biosynthetic pathway do not recognise phenylbutyric
acid as a substrate. (R)-3-hydroxyphenylhexanoate (3HPH)
is the predominant monomer in PHA accumulated from
PhAksn≥5 , with an even number of carbons on the acyl side
chain, while 3HPV is the predominant monomer in PHA
accumulated from PhAksn≥5 , with an uneven number of
carbons on the acyl side chain (Table 2). This is in keeping with observations made for aromatic PHA accumulation in P. putida U and P. putida BM01 [4,20]. However,
P. putida U does not accumulate PHA containing 3HPB
suggesting a difference in the affinity of the PHA polymerase or ␤-oxidation enzymes in P. putida U and P. putida
CA-3.
130
P.G. Ward, K.E. O’Connor / International Journal of Biological Macromolecules 35 (2005) 127–133
Table 2
PHA monomer distribution of the polymers synthesised by P. putida CA-3 grown on various PhAksn≥5 as the sole source of carbon and energy
Growth substratea
% Monomer composition of PHAb
Phenylvaleric acid
Phenylhexanoic acid
Phenylheptanoic acid
Phenyloctanoic acid
Phenyldecanoic acid
3HH
3HO
3HD
0.2
0.2
0.2
0.2
0.2
0.5
0.3
0.3
0.2
0.2
1.3
1.5
1.5
1.6
1.6
3HPB
–
15
–
7
6
3HPV
3HPH
3HPHp
3HPO
3HPD
98
–
85
–
–
–
83
–
61
57
–
–
13
–
–
–
–
–
30
26
–
–
–
–
9
All values are the mean of three independent determinations.
a All substrate were supplied at the concentrations resulting in maximum yield (g PHA/g carbon supplied) to the growth media. All experiments were carried
out in 50 ml of E2 medium with a sodium ammonium phosphate (SAP) concentration of 1.0 g/l, over a 48 h period.
b Calculated as the percentage of total PHA produced. 3HH: 3-hydroxyhexanoate; 3HO: 3-hydroxyoctanoate; 3HD: 3-hydroxydecanoate; 3HPB:
3-hydroxyphenylbutyroate; 3HPV: 3-hydroxyphenylvaleroate; 3HPH: 3-hydroxyphenylhexanoate; 3HPHp: 3-hydroxyphenylheptanoate; 3HPO: 3hydroxyphenyloctanoate; 3HPD: 3-hydroxyphenyldecanoate.
A small proportion of (R)-3-hydroxyhexanoate (3HH),
(R)-3-hydroxyoctanoate (3HO) and (R)-3-hydroxydecanoate
(3HD) monomers were present in PHAs accumulated from
PhAksn≥5 (Table 2). To the best of our knowledge no other
strain produces a heteropolymer containing both aromatic
and aliphatic monomers from a single substrate [4,8,9,21].
P. putida U is also incapable of PHA production from unrelated growth substrates such as glucose, suggesting an absent or unexpressed ACP:CoA transacylase gene (phaG),
known to transfer fatty acid intermediates to PHA synthesis. Conversely a transacylase (PhaG) is likely to be involved
in the transfer of aliphatic intermediates to PHA synthesis in
P. putida CA-3. To further test this theory, P. putida CA-3
cells were incubated with phenylheptanoic acid in the presence of 15 mM 2-bromooctanoic acid, a known inhibitor of
PhaG [22]. Interestingly, a much higher concentration of 2bromooctanoic acid was required to inhibit PHA accumulation from unrelated carbon sources in P. putida CA-3 (up
to 15 mM) than in P. fluorescens BM07 (1mM) [22]. In the
presence of 15 mM 2-bromooctanoic acid a 20% and 97%
decrease in the percentage of PHA accumulated from phenylheptanoate and glucose was observed respectively. However,
the presence of 2-bromooctanoic acid failed to significantly
decrease the proportion of the 3HH, 3HO and 3HD aliphatic
monomers, measured by GC–MS, in PHA accumulated from
phenylheptanoic acid. Thus, a phaG gene knockout mutant is required to conclusively determine the role of fatty
acid metabolism in PHA accumulation from PhAksn≥5 in P.
putida CA-3.
3.2. PHA accumulation from aliphatic substrates
As a comparative measure of the ability to accumulate
PHA from aromatic carbon substrates PHA accumulation by
P. putida CA-3 from aliphatic alkanoic acids (Aks) was also
determined. The total percentage PHA accumulated from
Aks varied significantly depending on the length of the acyl
chain (Table 3). The PHA content of the cell and the PHA
yield (g PHA/g carbon) for butyric acid and valeric acid
grown cells is significantly lower than the other aliphatic
growth substrates (Fig. 2, Table 3).
The highest level of PHA (% cell dry wt.) accumulated by
P. putida CA-3 cells grown on aliphatic substrates was 64%
with heptanoic acid supplied at an initial concentration of
20 mM in the growth medium (Fig. 2, Table 3). Cells grown
on octanoic (63%), nonanoic (60%) and decanoic acid (61%)
accumulated similarly high levels of PHA at initial concentrations of 20 mM in the growth medium. A further increase
in the initial concentration of these substrates did not increase
the PHA content of the cells further (Fig. 2). The highest PHA
yields (g PHA/g carbon) recorded for the aliphatic growth
substrates tested were for cells grown on an initial concentration of 15 mM heptanoic acid (0.64), octanoic acid (0.57),
nonanoic acid (0.43) and decanoic acid (0.41) respectively.
Table 3
Data for P. putida CA-3 cells grown on a range of alkanoic acids at concentrations where maximum yield (g PHA/g carbon supplied) was observed
Substrate
Initial substrate
concentration (mM)
Carbon supplied
(g carbon/l)
% PHAa
Cell dry wt. (g/l)
Total PHA (g/l)
PHAb yield
(g/g carbon)
Butyric acid
Valeric acid
Hexanoic acid
Heptanoic acid
Octanoic acid
Nonanoic acid
Decanoic acid
20
20
20
15
15
15
15
0.96
1.20
1.44
1.26
1.44
1.62
1.80
15 ± 1
17 ± 1
37 ± 2
59 ± 4
59 ± 3
55 ± 4
57 ± 4
0.66
0.68
0.93
1.36
1.39
1.26
1.31
0.10
0.12
0.35
0.80
0.82
0.69
0.75
0.10
0.10
0.24
0.64
0.57
0.43
0.41
a
All experiments were carried out in 50 ml of E2 medium with a sodium ammonium phosphate concentration of 1.0 g/l, over a 48 h period. PHA is expressed
as a percentage of cell dry weight.
b PHA yield (g/g carbon) = the grams of PHA accumulated per gram of carbon supplied.
P.G. Ward, K.E. O’Connor / International Journal of Biological Macromolecules 35 (2005) 127–133
Fig. 2. PHA accumulation by P. putida CA-3 cells grown on butyric acid (),
valeric acid (), hexanoic acid (×), heptanoic acid (), octanoic acid (),
nonanoic acid () and decanoic acid (♦) at varying initial concentrations.
Thus, P. putida CA-3 is capable of accumulating aromatic
PHA at levels equivalent to aliphatic PHA from Aks with
seven carbons or more. However, the yield of PHA is significantly lower from PhAks compared to Aks with seven
carbons or more.
In keeping with observations made for PhAks the majority
of PHA monomers produced from hexanoic, heptanoic,
octanoic, nonanoic, and decanoic acids have an acyl chain
length either equal in length to the growth substrate or
shorter by multiples of two carbon units, suggesting the
majority of PHA intermediates are being generated through
the ␤-oxidation pathway (Table 4). Fatty acid synthesis is
most likely responsible for the generation of even chain
monomers from heptanoic acid and nonanoic acid (Table 4).
Furthermore, the same pathway is most likely responsible
Table 4
PHA accumulation and monomer distribution of the polymers synthesised
by P. putida CA-3 cells grown on glucose and various alkanoic acids as the
sole source of carbon and energy
Growth substratea
% Monomer composition of PHAb
3HV
3HH
Glucose
Butyric acid
Valeric acid
Hexanoic acid
Heptanoic acid
Octanoic acid
Nonanoic acid
Decanoic acid
–
–
8
–
2.5
–
3
–
2
2
2
72
0.5
9
0.5
7
3HHp
3HO
3HN
3HD
–
–
–
90
–
27
–
23
23
20
6
2
88
2
55
–
–
–
–
–
–
65.5
–
75
75
70
22
5
3
2
38
All values are the mean of three independent determinations.
a All substrate were supplied at the concentrations resulting in maximum
yield (g PHA/g carbon supplied) to the growth media. All experiments were
carried out in 50 ml of E2 medium with a sodium ammonium phosphate
(SAP) concentration of 1.0 g/l, over a 48 h period.
b 3HV: 3-hydroxyvaleroate; 3HH: 3-hydroxyhexanoate; 3HHp: 3hydroxyheptanoate; 3HO: 3-hydroxyoctanoate; 3HN: 3-hydroxynonanoate;
3HD: 3-hydroxydecanoate.
131
for generating monomers that are longer than the growth
substrates octanoic acid and hexanoic acid (Table 4).
The monomer composition of PHA accumulated from
cells grown on butyric acid and valeric acid is similar to that
accumulated by cells grown on glucose (Table 4). However,
PHA accumulated by valeric acid grown cells contains a low
level of 3-hydroxyvalerate (8%). Thus (R)-3-hydroxyvalerylCoA appeared to be the smallest substrate for the PHA polymerising system in P. putida CA-3.
The monomer composition of aromatic PHA suggests that
the PHA polymerising system of P. putida CA-3 has a higher
affinity for hydroxylated aromatic monomers with acyl side
chains of five and six carbons (Table 2). PHA accumulated
by P. putida CA-3 grown on Aks suggest a higher affinity
for hydroxylated aliphatic substrates with acyl chain lengths
between seven and nine carbons (Table 4). This would suggest
that it is not solely acyl chain length that affects monomer
composition. This opens up the possibility that there may
be two different PHA polymerases, one with an affinity for
PhAks and one with an affinity for Aks. However, studies
on the PhaC1 and PhaC2 polymerases in P. putida U have
shown that the two polymerases act on both Aks and PhAks
[4]. Future work will involve attempting to clone the PHA
operon encoding the PHA polymerase (phaC) genes in P.
putida CA-3 and look at gene knockouts.
3.3. The effect of acrylic acid on PHA accumulation
It has been reported that the addition of low levels of the ␤oxidation inhibitor acrylic acid can increase the level of PHA
accumulated from related growth substrates by increasing the
intracellular concentration of PHA intermediates [23,24]. We
attempted to increase aromatic and aliphatic PHA accumulation, in P. putida CA-3, by adding various concentrations
of acrylic acid. The addition of acrylic acid did not increase
the level of PHA accumulated from any of the PhAks or Aks
tested (data not shown). Phenyloctanoic acid and octanoic
acid are illustrated as representative examples of the effect of
acrylic acid on PHA accumulation in P. putida CA-3 (Fig. 3).
At concentrations of up to 1 mM, acrylic acid had little effect
on PHA accumulation from phenyloctanoic acid and octanoic
acid, respectively. The addition of acrylic acid at concentrations above 1 mM strongly inhibited PHA accumulation from
phenyloctanoic acid and octanoic acid respectively (Fig. 3).
3.4. Polymer properties of aromatic PHA
The properties of the PHA accumulated by P. putida
CA-3 varied significantly depending on the growth substrate
supplied (Table 5). The polymers accumulated from the
aromatic growth substrates became increasing glue-like as
the acyl chain length of the growth substrate was increased.
The decomposition temperature of the polymers decreased
as the length of the acyl side chain of the aromatic growth
substrate was increased. The highest and lowest decomposition temperatures were recorded for PHA accumulated from
132
P.G. Ward, K.E. O’Connor / International Journal of Biological Macromolecules 35 (2005) 127–133
Table 5
Polymer properties of PHA polymers accumulated from PhAksn≥5
Substrate
Mw a (Da)
Mn (Da)
Q (Mw /Mn )
Tg (◦ C)
Tm (◦ C)
DT (◦ C)
Phenylvaleric acid
Phenylhexanoic acid
Phenylheptanoic acid
Phenyloctanoic acid
Phenyldecanoic acid
77000
316000
210000
292000
167000
25000
91000
67000
72000
57000
3.1
3.5
3.1
4.1
2.9
13.2
3.9
9.6
−14.3
−8.7
51.5
52.1
ND
ND
ND
283
274
259
257
254
ND: not detected.
a M : molecular weight, M , molecular number; Q (M /M ): polydispersity; T : glass transition temperature; T : melting temperature; DT: decomposition
w
n
w
n
g
m
temperature.
phenylvaleric acid and phenyldecanoic acid grown cells,
respectively (Table 5). Whether the polymers are amorphous
or partially crystalline can be determined by the presence of a
melting peak on a DSC thermogram. Completely amorphous
polymers (polymers that do not crystallise) will have a glass
transition temperature (Tg ) but not a melting temperature
(Tm ). Partially crystalline polymers have both a Tg and a
Tm . The PHAs accumulated from phenylvaleric acid and
phenylhexanoic acid were partially crystalline with Tm ,
at 51.5 and 52.1 ◦ C, respectively (Table 5). The polymers
accumulated from phenylheptanoic acid, phenyloctanoic
acid and phenyldecanoic acid had no Tm indicating that
these polymers were totally amorphous (Table 5). The
Tg of the polymers varied between 13.2 ◦ C for the PHA
accumulated from phenylvaleric acid to −14.3 ◦ C for the
PHA accumulated from phenyloctanoic acid (Table 5).
The unique monomer composition of the PHAs accumulated by P. putida CA-3 contributes significantly to the different polymer properties. The aromatic polymers accumulated by P. putida CA-3 have low crystallinity compared
to aliphatic PHA polymers [25]. Furthermore PHA accumulated by P. putida U from the same PhAks substrates
has different aromatic monomer ratios, does not contain
aliphatic monomers and is totally amorphous [13]. The Tg
of all the polymers accumulated by P. putida CA-3 from
these aromatic substrates was significantly higher than that
of medium chain length aliphatic PHAs previously reported
(Table 5) [17,25,26]. The Tg of aliphatic PHA accumulated
from styrene by P. putida CA-3 was previously reported as
−41.7 ◦ C [17]. In addition the Tg values of aromatic PHA accumulated from PhAks by P. putida U are different from the
polymers accumulated by P. putida CA-3. The Tg of aromatic
PHAs accumulated by P. putida U from phenylhexanoic and
phenylheptanoic acid were −1.3 and −11.2 ◦ C, respectively
[13].
The molecular weight of the PHAs accumulated from
phenylhexanoic acid, phenylheptanoic acid, phenyloctanoic
acid and phenyldecanoic acid were between 2.2- and 4.1-fold
higher than that of the PHA accumulated from phenylvaleric
acid (Table 5). The polydispersity of the polymers was high
relative to aliphatic and aromatic PHA previously reported
and varied between 2.92 and 4.06 [13,25].
4. Conclusions
P. putida CA-3 is one of a limited number of organisms
with the ability to produce PHA with aromatic monomers
[4,8,9,21]. To the best of our knowledge it is the only strain
to date to produce a heteropolymer of aromatic and aliphatic
monomers from a single substrate. The starting concentration of substrate has a dramatic effect on PHA content and
yield (Fig. 1). The monomer composition and the ratio of the
monomers present in the polymer are unique to P. putida CA3. However, subtle differences in the monomer composition
of PHA accumulated by P. putida CA-3 and other strains do
not appear to significantly change the polymer properties, i.e.
while PHA accumulated by P. putida CA-3 from phenylvaleric acid contains aromatic (98%) and aliphatic monomers
(2%) the aromatic homopolymer accumulated from the same
carbon source by P. oleovorans has an almost identical Tg
(13 ◦ C.) [12]. An increase in the proportion of aliphatic to
aromatic monomers is most likely required before a noticeable change in polymer properties occurs. Such an increase
may offer potential in the generation of PHA polymers with
new properties and possible biotechnological applications.
Acknowledgements
Fig. 3. The effect of acrylic acid on the level of PHA accumulated by P.
putida CA-3 cells grown on 10 mM octanoic acid (䊉) and 10 mM phenyloctanoic acid ().
Patrick Ward is the recipient of a PhD scholarship from
the Environmental Protection Agency (EPA) Ireland and
P.G. Ward, K.E. O’Connor / International Journal of Biological Macromolecules 35 (2005) 127–133
Enterprise Ireland. We thank Keith Fortune, Materials
Ireland, Trinity College Dublin, for polymer analysis, and
Guy de Roo ETH, Zurich, for GCMS analysis.
References
[1] B.H.A. Rehm, A. Steinbüchel, Int. J. Biol. Macromol. 25 (1999) 3.
[2] R.G. Lageveen, G.W. Huisman, H. Preusting, P. Ketelaar, G. Eggink,
B. Wiltolt, Appl. Environ. Microbiol. 54 (1988) 2924.
[3] A. Steinbuchel, B. Fuchtenbusch, Trends Biotechnol. 16 (1998)
419.
[4] B. Garcia, E.R. Olivera, B. Minambres, M. Fernandez-valverde, L.M.
Canedo, M.A. Prieto, J.L. Garcia, M. Martinez, J.M. Luengo, J. Biol.
Chem. 274 (1999) 29228.
[5] G.N.M. Huijberts, T.C. de Rijk, R. de Waard, G. Eggink, J. Bacteriol.
176 (1994) 1661.
[6] B.H.A. Rehm, N. Kruger, A. Steinbuchel, J. Biol. Chem. 273 (1998)
24044.
[7] G.A. Abraham, A. Gallardo, J. San Roman, E.R. Olivera, R. Jodra, B.
Garcia, B. Minambres, J.L. Garcia, J.M. Luengo, Biomacromolecules
2 (2001) 562.
[8] Y.B. Kim, D.Y. Kim, Y.H. Rhee, Macromol 32 (1999) 6058.
[9] J.J. Song, M.H. Choi, S.C. Yoon, N.E. Huh, J. Microbiol. Biotechnol.
11 (2001) 435.
[10] D.M. Chung, M.H. Choi, J.J. Song, S.C. Yoon, I.K. Kang, N.E. Huh,
Int. J. Biol. Macromol. 19 (2001) 243.
133
[11] B. Kessler, B. Witholt, J. Biotechnol. 86 (2001) 97.
[12] K. Fritzsche, R.W. Lenz, R.C. Fuller, Makromol. Chem. 191 (1990)
1957.
[13] E.R. Olivera, D. Carnicero, R. Jodra, B. Minambres, B. Garcia, G.A.
Abraham, A. Gallardo, J.S. Roman, J.L. Garcia, G. Naharro, J.M.
Luengo, Environ. Microbiol. 3 (2001) 612.
[14] Y.L. Chun, Y.H. Lee, S.H. Yen, K.H. Chi, Clin. Cancer Res. 6 (2000)
1452.
[15] E. Kebebew, M.G. Wong, A.E. Siperstein, Q.Y. Duh, O.H. Clark, J.
Clin. Endocrinol. Metab. 84 (1999) 2840.
[16] T.E. Witzig, M. Timm, M. Stenson, P.A. Svingen, S.H. Kaufmann,
Clin. Cancer Res. 6 (2000) 681.
[17] P.G. Ward, G. de Roo, K.E. O’Connor, Appl. Environ. Microbiol.
71 (4) (2005).
[18] H.J. Vogel, D.M. Bonner, J. Biol. Chem. 218 (1956) 97.
[19] D. Scheiner, Water Res. 10 (1976) 31.
[20] J.J. Song, S.C. Yoon, Appl. Environ. Microbiol. 62 (1996) 536.
[21] A. Steinbuchel, H.E. Valentin, FEMS Microbiol. Lett. 128 (1995)
219.
[22] H.J. Lee, M.H. Choi, T.U. Kim, S.C. Yoon, Appl. Environ. Microbiol. 67 (2001) 4963.
[23] Q. Qi, A. Steinbuchel, B.H.A. Rehm, FEMS Microbiol. Lett. 167
(1998) 89.
[24] K. Zhao, G. Tian, Z. Zheng, J.C. Chen, G.Q. Chen, FEMS Microbiol.
Lett. 218 (2003) 59.
[25] H. Preusting, A. Nijenhuis, B. Witholt, Macromol 23 (1990) 4220.
[26] L.L. Madison, G.W. Huisman, Microbiol. Mol. Biol. Rev. 63 (1999)
21.