Journal of Environmental Biotechnology
Vol. 4, No. 1, 49–55, 2004

Original paper (regular paper)

Biosynthesis of Novel Poly(3-hydroxyalkanoates) Containing
Benzoyl Groups
TSUTOMU HONMA*, TAKESHI IMAMURA, TAKASHI KENMOKU, SHIN KOBAYASHI and TETSUYA YANO
Leading-Edge Technology Development Headquarters, Canon Inc., 5–1, Morinosato-Wakamiya,
Atsugi-shi, Kanagawa 243–0193, Japan
TEL: +81–46–247–2111 FAX: +81–46–248–0306
E-mail: homma.tsutomu@canon.co.jp(Received; 1 April, 2004/Accepted; 16 June, 2004)
We studied novel poly(3-hydroxyalkanoates) (PHAs) containing benzoyl groups. Production of PHAs by Pseudomonas
cichorii strain YN2 cultured with 4-benzoylbutyrate, 5-benzoylvalerate, 6-benzoylhexanoate, 7-benzoylheptanoate, or 8benzoyloctanoate was investigated. NMR and GC-MS analysis indicated that the polymer consisted of 3-hydroxybenzoylalkanoates as monomeric units. The polymer, which was biosynthesized from 5-benzoylvalerate, contained 3-hydroxy-5benzoylvalerate units. The average molecular number (Mn), the average molecular weight (Mw), the glass transition
temperature (Tg), and the melting point (Tm) of this polymer were higher (330,000, 1,300,000, 36°C, and 150°C, respectively)
than for other unusual-PHAs (PHAs having substituents other than alkyl groups introduced in the side chains) previously
reported. This polymer has potential for use to some purpose, to which existing biosynthetic PHAs cannot be used becauseof their thermal properties.
Key words: poly(3-hydroxyalkanoate), benzoyl group, Pseudomonas, thermal property

1.

Introduction

Many microorganisms produce and accumulate poly(3hydroxybutyrate) (PHB) or other poly(3-hydroxyalkanoates)(PHAs) in cells4,20). These polymers can be used to produce
various kinds of products by using melting processes, etc.,
like with other conventional plastics. PHAs have the advantage of being completely decomposed by microorganisms innature because of their biodegradability, and so they would
not remain in the natural environment to cause pollution
unlike many conventional synthetic polymers. They are also
excellent in biological compatibility and are expected to be
used in medicine, etc.
Such microbial PHAs may have a variety of compositions and structures depending on the types of microorganisms, compositions of culture media, culture conditions, etc.,
used for their production. Studies of PHAs have been done
so far on controlling such compositions and structures. For
example, Ralstonia eutropha can synthesize PHAs containing short-chain-length 3-hydroxyalkanoate units which havethree to five carbon atoms (scl-PHA), i.e., 3-hydroxypropionate unit, 3-hydroxybutyrate unit, 3-hydroxyvalerate unit
and 4-hydroxybutyrate unit4,19,20). PHAs containing mediumchain-length 3-hydroxyalkanoate units, which have 6 to 14
carbon atoms (mcl-PHAs), have been produced by Pseudomonas oleovorans4,19), etc.
However, if a wider range of applications using microbial

PHA, such as a functional polymer, is considered, PHAs
having substituents other than alkyl groups introduced in
the side chains (unusual-PHAs) are expected to be useful.
Examples of these substituents include those containing an
aromatic ring (such as a phenyl group9,12), a phenoxy
group9,13), a thiophenoxy group21)), a cyclohexyl group1,9), an
unsaturated hydrocarbon6), a cyano group16), a halogenatedhydrocarbon16), or an epoxide2,11).
In this study, we investigated PHA produced by Pseudomonas cichorii strain YN29,11) cultured with a benzoylalkanoate. The PHAs biosynthesized from benzoylalkanoates
contained monomer units having benzoyl groups. The
average molecular number (Mn), the average molecularweight (Mw), the glass transition temperature (Tg) and themelting point (Tm) of the PHA biosynthesized from 5benzoylvalerate (5-BzVA) were higher than for other unusual-PHAs previously reported.

2.
2.1.

Materials and Methods

Materials

4-Benzoylbutyrate (4-BzBA), 5-BzVA, 6-benzoylhexanoate (6-BzHxA), 7-benzoylheptanoate (7-BzHpA), and8-benzoyloctanoate (8-BzOA) were used as “unusual carbon sources” in this study. These chemicals were of thehighest purity grade available from Lancaster Synthesis Ltd.
(Manchester, UK), and were used without further purifica-HONMA et al.

50

tion.
5-Phenylvalerate (5-PVA), 5-phenoxyvalerate (5-PxVA)
and 4-cyclohexylbutyrate (4-CHBA) were purchased from
Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan), and nnonanoate was purchased from Kishida Chemical Co., Ltd.
(Osaka, Japan).
2.2.

Biosynthesis of polymers

The composition of M9 medium used in this study was (perliter): Na2HPO4, 6.2 g; KH2PO4, 3.0 g; NaCl, 0.5 g; NH4Cl,
1.0 g; and 3 ml of a trace element solution. The trace element solution contained (per liter): nitrilotriacetic acid, 1.5 g;
MgSO4, 3.0 g; MnSO4, 0.5 g; NaCl, 1.0 g; FeSO4, 0.1 g;
CaCl2, 0.1 g; CoCl2, 0.1 g; ZnSO4, 0.1 g; CuSO4, 0.1 g;
AlK(SO4)2, 0.1 g; H3BO3, 0.1 g; Na2MoO4, 0.1 g; NiCl2, 0.1 g.
The medium was sterilized by autoclaving at 121°C for
10 min.
Two-stage shake flask cultures of P. cichorii strain YN2
were done aerobically in 500 ml shaking flasks (Sakaguchi
flasks) containing 200 ml of the medium. P. cichorii strain
YN2 was inoculated into M9 medium containing 0.5%
(wt./vol.) D-glucose and 0.1% (wt./vol.) of an unusual carbon source and then was cultured with shaking at 125 rpm
and 30°C on a reciprocating shaker. After the culture for48 hours, the cells were harvested by centrifugation (9,000×g, 4°C, 10 min.), were resuspended in M9 medium containing 0.5% (wt./vol.) D-glucose and 0.1% (wt./vol.) of an
unusual carbon source but not containing a nitrogen source(NH4Cl), and were further cultured with shaking at 125 rpm
and 30°C on a reciprocating shaker. After the culture for48 hours, the cells were harvested by centrifugation (9,000×g, 4°C, 10 min.), were washed once with cold methanol andwere lyophilized.
The lyophilized cells were suspended in 20 ml of chloroform and was stirred at 60°C for 20 hours to extract thepolymer. The extract was filtered through a membrane filterof pore size 0.45 µm. The filtrate was concentrated by using
a rotary evaporator. The concentrate was reprecipitated incold methanol, and then only the precipitate was recovered
and was dried in vacuo to obtain the polymer.
Poly(3-hydroxy-5-phenylvalerate) (PHPV), poly(3hydroxy-5-phenoxyvalerate) (PHPxV), poly(3-hydroxy-4cyclohexylbutyrate) (PHCHB), mcl-PHA and PHB werebiosynthesized for comparative experiments in this study.
PHPV and PHPxV were biosynthesized from 5-PVA and
5-PxVA, respectively, by using the method reported previously by us10). PHCHB was biosynthesized by culturing P.
cichorii strain YN2 in M9 medium containing 4-CHBA andyeast extract (Bacto® Yeast Extract, Difco Laboratories, Detroit, USA) for 24 hours. Mcl-PHA was biosynthesized byusing the same method as described in this paper, excepts
for using n-nonanoate as a solo carbon source instead of
benzoylalkanoate and D-glucose. PHB was obtained byculturing Alcaligenes sp. strain TL2 with sodium lactate asa solo carbon source8).

2.3. Analytical methods
The molecular weight of the polymer was measured byusing gel permeation chromatography (GPC) at 30°C witha TOSOH HLC-8020 GPC system and a refractive index
detector with a PL gel MIXED-C column (5 µM, Polymer
Laboratory). Chloroform (HPLC grade) was used as an eluent. Polystyrene standards with low polydispersity (Polymer
Laboratory) were used to generate calibration curves.
To find the polymer composition, 5 mg of polymer sample was mixed with 2 ml of 3.0% (vol./vol.) sulfuric acid inmethanol and 2 ml of chloroform, and the mixture was refluxed for 3.5 hours at 100°C to convert monomer units totheir corresponding methyl ester. The mixture was cooled
and washed with 10 ml of distilled water. The organic phase
was collected, was dried over MgSO4, and was analyzed byusing a gas chromatography-mass spectrometer (GC-MS)(Shimadzu QP-5050A) with a J&W DB-WAXETR capillarycolumn (30 m×0.32 mm×0.5 µm; carrier gas, He, 2.2 ml/
min.; temperature program, 80°C for 5 min., then the temperature was increased at 5°C/min. to 200°C and remainedat this temperature).
Proton (1H) and carbon (13C) NMR spectra were recordedby using a Brucker FT-NMR DPX400 spectrometer at
400 MHz and 100 MHz, respectively, at the following experimental conditions: 0.5% (wt./vol.) polymer sample inchloroform-d, 298 K, tetramethylsilane was used as an internal reference.
Differential scanning calorimetry (DSC) was done by using Perkin Elmer Pyris 1. A polymer sample was tested
from –50 to 200°C in a nitrogen atmosphere at a heating
rate of 10°C/min. The Tg and the Tm were taken from theinflection point.
Thermogravimetry-differential thermalanalysis (TG-DTA)
was done by using MAC Science TG-DTA 2000S. A polymer sample was tested from room temperature to 300°C in
an air flow (100 ml/min.) at a heating rate of 10°C/min.

3.
3.1.

Results and Discussion

Biosynthesis of polymers

Table 1 lists the cell dry weights and polymer yields ofthe cultures of P. cichorii strain YN2 grown with different
unusual carbon sources. Cell growth and PHA production
depend on the used unusual carbon source. Table 1 showsthat the produced cell and polymer using 5-BzVA and
7-BzHpA were greater than those obtained by using
4-BzBA, 6-BzHxA and 8-BzOA.
The cell dry weight results of the cultures of P. cichorii
strain YN2 grown with 5-BzVA, 5-PVA and 5-PxVA were0.89 g/L, 1.30 g/L and 0.84 g/L, respectively. These results
show that benzoylalkanoate has not marked growth inhibitory effect compared with phenoxyalkanoate. The polymer
yields of the cultures of P. cichorii strain YN2 grown with
5-BzVA, 5-PVA and 5-PxVA were 0.30 g/L, 0.94 g/L and
0.40 g/L, respectively, and indicate that benzoylalkanoate

Biosynthesis of Novel Poly(3-hydroxyalkanoates) Containing Benzoyl Groups

51

Table 1. Production of PHAs by Pseudomonas cichorii strain YN2 in a two-stage cultivation in the media
containing benzoylalkanoate.

apolymer

Unusual carbon
sourceDry cell weight(g/l)

Polymer yield(g/l)

Polymer contenta(%wt.)

1
2
3
4
54-BzBA
5-BzVA
6-BzHxA
7-BzHpA
8-BzOA

0.31
0.89
0.71
1.44
0.71

0.01
0.30
0.18
0.59
0.17

3
34
25
41
24

PHPV
PHPxV
PHCHB
mcl-PHA
PHB

5-PVA
5-PxVA
4-CHBA

1.30
0.84
1.10
1.19
0.71

0.94
0.40
0.23
0.51
0.56

72
48
21
43
79

―
―

Percentage of polymer yield per dry cell weightand phenoxyalkanoate are comparatively hard to incorporate into polymer compared with phenylalkanoate.
3.2.

Characterization of polymers

The polymers obtained from P. cichorii strain YN2 weremethanolized to the methyl ester of the repeating units, andthe products were analyzed by using a GC-MS.
Some total ion chromatogram (TIC) peaks were detectedin the gas chromatogram of the polymer biosynthesizedfrom 5-BzVA (data not shown). TIC minor peaks wereidentified as the methyl ester from medium-chain-length
alkyl 3-hydroxyalkanoate units (3-hydroxyoctanoate methyl
ester, 3-hydroxydecanoate methyl ester, 3-hydroxydodecanoate methyl ester and 3-hydroxydodecenoate methyl ester).
But the main peak (retention time, R.T.=54.8 min.), which
was 80% of the total peak area, was previously unknown.
Unknown peaks were also detected in the chromatogramsof the analysis of the other polymer samples biosynthesizedfrom 4-BzBA (R.T.=52.6 min.), 6-BzHxA (R.T.=69.8 min.),
7-BzHpA (R.T.=54.8 and 192.5 min.) and 8-BzOA(R.T.=70.5 and approx. 550 min.; the latter was a broadpeak).
These unknown GC peaks were identified by mass spectroscopy measurements (Table 2). Characteristic m/z peaks
derived from the benzoyl structure (m/z=77, 105) were de-tected in both the unknown peaks. The m/z peaks of thefragment of the benzoyl structure and proximal methylenegroup (m/z=120, 121) were also detected in the unknown
peaks except for the R.T. 52.6 min. peak. Therefore these
unknown GC peaks were derived from methyl esters of monomeric units of PHAs that have benzoyl groups. Judging
from the retention times and the maximum m/z peaks ineach mass pattern, the unknown GC peaks were estimatedto derive from the structures shown in Table 2.
The polymers were characterized by NMR spectroscopy.
Fig. 1 and 2 show 1H- and 13C-NMR spectra of the polymer
biosynthesized from 5-BzVA. Chemical shifts and patternsof peaks corresponded to those expected from a PHA containing 3-hydroxy-5-benzoylvalerate units. Therefore thepolymer was found to be a PHA containing 3-hydroxy-5benzoylvalerate units. Similarly, the polymers biosynthesizedfrom 4-BzBA, 6-BzHxA, 7-BzHpA or 8-BzOA were judged
from their 1H-NMR spectra to be PHAs containing 3hydroxybenzoylalkanoate units (data not shown). We believethat PHAs containing benzoyl groups have not beenreported, and therefore this study may be the first report.
Table 3 shows monomer unit compositions (GC-MS, TIC
peak area proportion) of the polymers. The polymer
biosynthesized from 5-BzVA contained 3-hydroxy-5benzoylvalerate units and usual (alkyl) 3-hydroxyalkanoate

Table 2. Mass spectroscopy measurement of PHAs produced by Pseudomonas cichorii strain YN2 cultivated in the media containing benzoylalkanoate.

polymer

Retention timeof GC peak(min.)

1, 3, 5

52.6

2, 4

54.8

3, 5

68.8, 70.5

4

192.5

5

applox. 550

Main m/z peaks
51, 58, 63, 77, 95, 105, 115, 144, 172
41, 51, 59, 71, 75, 77, 85, 89, 105, 117, 120,
131, 144, 159,177, 187, 218
41, 51, 59, 71, 75, 77, 85, 105, 117, 120, 133,
147, 159, 173, 191, 200, 232
43, 51, 55, 71, 77, 91, 105, 120, 133, 145,
159, 173, 197, 215, 228, 246
41, 44, 57, 63, 69, 77, 91, 105, 121, 256

Structure

Molecular
weight3-hydroxy-4-benzoylbutyrate methyl ester

222

3-hydroxy-5-benzoylvalerate methyl ester236

3-hydroxy-6-benzoylhexanoate methyl ester

250

3-hydroxy-7-benzoylheptanoate methyl ester

264

3-hydroxy-8-benzoyloctanoate methyl ester

278

HONMA et al.

52

H-NMR spectrum of the polymer synthesized from 5-BzVA by Pseudomonas cichorii strain YN2.

Fig. 1.

1

Fig. 2.

13

C-NMR spectrum of the polymer synthesized from 5-BzVA by Pseudomonas cichorii strain YN2.

Table 3. Relative amount of repeating units in PHA produced by Pseudomonas cichorii strain YN2 with two-stage cultivation in the media
containing benzoylalkanoate.

aRelative amount of monomeric units in polymer (%)apolymer

Unusual carbon
source3HBzBb3HBzVc3HBzHxd

3HBzHpe

3HBzOfusual-3HAg

1
2
3
4
5

4-BzBA
5-BzVA
6-BzHxA
7-BzHpA
8-BzOA

8.3
—
—
—
—

—
79.8
—
30.8
0.2

—
—
69.8
—
37.8

—
—
—
34.2
—

—
—
—
—
49.0

91.7
20.2
30.2
35.0
13.0

GC-MS, peak area %, b 3-hydroxy-4-benzoylbutyrate, c 3-hydroxy-5-benzoylvalerate, d 3-hydroxy-6-benzoylhexanoate, e 3-hydroxy-7benzoylheptanoate, f 3-hydroxy-8-benzoyloctanoate, g alkyl 3-hydroxyalkanoate (C6–C14).

units at 79.8% and 20.2%, respectively. Similarly, the polymer biosynthesized from 6-BzHxA contained 3-hydroxy-6benzoylhexanoate units at 69.8%. However, the polymer
biosynthesized from 4-BzBA contained only 8.3% of 3-hydroxy-4-benzoylbutyrate units and the major part (91.7%)
consisted of usual 3-hydroxyalkanoate units. Because theyield of the polymer was also smaller than from otherbenzoylalkanoates, 4-BzBA may be hard to incorporate

Biosynthesis of Novel Poly(3-hydroxyalkanoates) Containing Benzoyl Groups

into PHA because of the shortage of its alkyl chain length.
The polymer biosynthesized from 7-BzHpA contained
3-hydroxy-7-benzoylheptanoate units and 3-hydroxy-5benzoylvalerate units (the chain length decreased by two-CH2-). The polymer biosynthesized from 8-BzOA contained 3-hydroxy-8-benzoyloctanoate units and 3-hydroxy-6benzoylhexanoate units (the chain length decreased by two-CH2-). These short length units may be produced by βoxidation reported by previous studies13,21).
In GC-MS experiments of PHPV, PHPxV, PHCHB, mclPHA and PHB, PHPV, PHPxV, PHCHB and PHB were
homopolymers consisting of 3-hydroxy-5-phenylvalerateunits, 3-hydroxy-5-phenoxyvalerate units, 3-hydroxy-4cyclohexylbutyrate units and 3-hydroxybutyrate units, respectively. The relative amount of monomeric units in mclPHA was (GC-MS, TIC peak area %): 3-hydroxyvalerate unit,
1%; 3-hydroxyheptanoate unit, 29%; 3-hydroxyoctanoate
unit, 3%; 3-hydroxynonanoate unit, 67%.
Table 4 show the molecular weights of the polymers measured by GPC experiments. The molecular weight of thepolymer biosynthesized from 5-BzVA (M n=330,000,
Mw=1,300,000) was markedly higher than for other polymer samples (Mn=18,000 to 41,000, Mw=42,000 to 93,000).
Incorporation of a 3-hydroxy-5-benzoylvalerate unit may
influence the molecular weight of a polymer, but this mechanism is unknown. 5-BzVA or 3-hydroxy-5-benzoylvalerateunits may inhibit the end of the biosynthesis of the polymer
chain, of which cause is unknown. But the comparatively
high Mw/Mn value of the polymer (Mw/Mn=3.9) suggests
the intermolecular binding is caused in some way by the
benzoyl groups.
The biodegradation property of the polymer biosynthesized in this study is of high interest to us, but it remains
unknown.
One of the expected characteristics of biosynthesized
PHAs would be their biodegradability. Some scl- and mclPHAs are degraded by microorganisms in the natural environment with relative ease, and the details have been stud-53

ied18,20), but information on biodegradation of unusual-PHAs
is limited.
We have not tested the biodegradability of the PHA containing benzoyl groups, but the unusual-PHA containing
similar aromatic side chains were degraded rapidly in compost (data not shown). PHAs containing 3-hydroxy-5phenylvalerate or 3-hydroxy-6-(4-cyanophenoxy)hexanoate
as unusual monomeric units were degraded by some Pseudomonas strains3,5,7). These results indicate a potential of
the PHA containing benzoyl groups to be degraded by natural microbes; further study is needed to show the biodegradation property of the PHA containing benzoyl groups.
3.3.

Thermal analysis of polymers

The texture of the polymer biosynthesized from 5-BzVA
is relatively hard at room temperature, which indicates that
this polymer has characteristic thermal properties.
To find if incorporation of benzoyl groups influences
thermal properties of the polymer, DSC and TG-DTA
analyses were done. As references, PHPV, PHPxV, PHCHB,
mcl-PHA, and PHB were also tested.
Table 5 shows the thermal properties of these PHAs, andFig. 3 shows the DSC thermogram of the polymer biosynthesized from 5-BzVA. The Tg value estimated by the resultof the second heating scan was 36°C, and the Tm value estimated by the result of the first heating scan was 150°C (Fig.
3). Tg and Tm values of the other unusual- and mcl-PHAs
tested were lower than these values. The thermal decomposition temperatures of the polymers were about the same,and a crystallization of the polymer was not clearly observed, except for PHB.
We believe such high Tg and Tm values of biosynthesizedunusual-PHAs have not been reported. So the thermal
properties of the polymer in this study stand out from thoseof other biosynthesized unusual-PHAs.
A high molecular weight can cause high Tg or Tm values
or both, but mcl-PHA (Mn=170,000, Mw=350,000) andPHB (Mn=1,800,000, Mw=2,600,000), which have relatively

Table 4. Molecular weight of PHA produced by Pseudomonas cichorii strain YN2 in a two-stage cultivation in
the media containing benzoylalkanoate.

apolymer

Unusual carbon
source

1
2
3
4
5
PHPV
PHPxV
PHCHB
mcl-PHA
PHB

Molecular weight of polymer
Mna

M wb

Mw/Mn

4-BzBA
5-BzVA
6-BzHxA
7-BzHpA
8-BzOA

18,000330,000
24,00023,00041,00042,0001,300,000
62,00053,00093,000

2.3
3.9
2.6
2.3
2.3

5-PVA
5-PxVA
4-CHBA——

69,00074,00049,000170,000
1,800,000

120,000
150,000
100,000
350,000
2,600,000

1.7
2.0
2.0
2.1
1.4

Average molecular number estimated by GPC, b Average molecular weight estimated by GPC

HONMA et al.

54

Table 5. Thermal properties of PHA.

apolymer

Unusual carbon
sourceTga (°C)

Tmb (°C)

Thermal decompositionc
(°C)

2
PHPV
PHPxV
PHCHB
mcl-PHAPHB

5-BzVA
5-PVA
5-PxVA
5-CHBA
—
—

36
19
23
23
–35
5

150
58
88
n.d.d
50
182

278
281
284
n.t.e
280
274

Glass transition temperature detected by DSC, b melting point detected by DSC, c detected by TG-DTA, d notdetected, and e not tested.

Fig. 3.

DSC thermogram of the polymer synthesized from 5-BzVA by Pseudomonas cichorii strain YN2.
(a) First heating scan of the polymer, (b) Second heating scan of the polymer.

high molecular weights, had no such high Tg or Tm values.
Therefore, incorporation of benzoyl groups may influence
the thermal properties of the polymer.
However, the appearances and the thermal properties ofthe polymers biosynthesized from 4-BzBA, 6-BzHxA,
7-BzHpA, and 8-BzOA were similar to those of the otherunusual-PHAs (data not shown). Therefore, in addition tomolecular weight and incorporation of benzoyl groups, thelength of the side chain containing a benzoyl group and thenumber of monomeric units can also influence thermal
properties of a polymer.
Incorporation of 3-hydroxy-5-benzoylvalerate units into a
PHA at a certain monomeric-unit-proportion may affectthe molecular weight and the thermal properties of the
PHA. The mechanism of this is unknown, but one hypothesis may be that the steric hindrance and the intermolecularbinding derived by the benzoyl group cause the molecularweight and the thermal properties of the polymer. If thesteric hindrance derived by the benzoyl group limits themolecular motion and the stereochemical configuration of
the polymer intra- or inter-molecularly or both, the Tg andTm values of the polymer will increase.
Usual-PHAs, consisting of scl- or mcl-3-hydroxyalkanoate

units (3-hydroxybutyrate unit, 3-hydroxyvalerate unit, 3hydroxyhexanoate unit etc.) or both, have been studied ingreat detail, for example, by Doi et al.4,17,20). But this study
differs from other studies in the chemical structure of
PHAs, which causes important differences in properties andfunctions of the polymer.
PHA containing 3-hydroxy-5-benzoylvalerate units shows
characteristic thermal properties. Especially, the Tg value ofthe polymer is much higher than for PHB, a representative
scl-PHA, and typical mcl-PHA. Unusual-PHAs containing
aromatic groups, such as benzoyl substituents, are estimatedto differ in the solubility in organic solvent from usualPHAs. These differences, which may be caused by thechemical structure, are important when considering industrial process and practical use.
The polymers in this study are classified as unusualPHAs, not usual-PHAs. Usual-PHAs are generally expected
to be ‘earth-friendly plastics’ or ‘biomaterials’ that are biodegradable, reproducible and biocompatible. Unusual-PHAs
are expected to be ‘functional polymers’ that have usefulfunctions derived from their functional groups, in addition
to environmental suitability and biocompatibility.
This study is basic research on novel PHAs containingBiosynthesis of Novel Poly(3-hydroxyalkanoates) Containing Benzoyl Groups

benzoyl groups, and its main purposes were: (1) to test if
monomeric units that have a benzoyl group can be incorporated into biosynthetic PHA, (2) to check if the unit can beeasily incorporated compared with other monomeric units,(3) to show some basic properties of PHAs containing benzoyl groups. Also examining the cost of using PHA in the
future is necessary.
A wide range of applications of PHAs have been hampered by high production cost of PHAs compared with conventional petrochemical-based plastics. Much effort has
been devoted to reduce the cost of PHA by increasing thecell density and polymer yield in the fermentation process,and positive results have been achieved in producing sclPHAs (PHB, etc.) and mcl-PHAs by microorganisms14,15).
For example, a mcl-PHA is produced by P. putida at a highcell density and high polymer yield (141 g/L and 72.6 g/L,
respectively) when using the high-cell-density fed-batch fermentation technique with phosphorus limitation15). Cell dry
weights (0.31 to 1.44 g/L) and polymer yields (0.01 to
0.59 g/L) in this study may not be sufficient for a wide use,
so using such a fermentation technique will be necessary toincrease cell dry weight and polymer yield.
We believe this is the first report on PHAs containing
benzoyl groups. Our results suggest that PHAs containing
benzoyl groups have potential for use, to which existing
unusual-PHAs cannot be used because of their thermal
properties. Especially, polymers that have a hard texture atroom temperature are useful. Our novel polymers widen therange of use of biodegradable polymers.

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