FEMS Microbiology Letters 161 (1998) 255^261
Bacterial isolates degrading aliphatic polycarbonates
Tetsushi Suyama *, Hiroyuki Hosoya, Yutaka Tokiwa
National Institute of Bioscience and Human Technology, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan
Received 13 January 1998; revised 6 February 1998; accepted 6 February 1998
Abstract
Bacteria that degrade an aliphatic polycarbonate, poly(hexamethylene carbonate), were isolated from river water in Ibaraki
Prefecture, Japan, after enrichment in liquid medium containing poly(hexamethylene carbonate) suspensions as carbon source,
and dilution to single cells. Four of the strains, 35L, WFF52, 61A and 61B2, degraded poly(hexamethylene carbonate) on agar
plate containing suspended poly(hexamethylene carbonate). Degradation of poly(hexamethylene carbonate) was confirmed by
gel permeation chromatography. Besides poly(hexamethylene carbonate), the strains were found to degrade poly(tetramethylene carbonate). The strains were characterized morphologically, physiologically, and by 16S rDNA sequence analysis. Strains
35L and WFF52 were tentatively identified as Pseudomonas sp. and Variovorax sp., respectively, while strains 61A and 61B2
constitute an unidentified branch within the L subclass of the Proteobacteria. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
Keywords : Aliphatic polycarbonate; Biodegradation; Lipolytic enzyme; Pseudomonas sp.; Variovorax sp.
1. Introduction
Development of biodegradable plastics is being extensively promoted as one of the solutions to plastic
waste disposal. Aliphatic polyesters are currently
considered the most promising materials for the production of biodegradable plastics. Strains of microbial degraders for biodegradable plastics were reported for: naturally occurring biodegradable
polymer, poly-L-hydroxybutyrate (PHB) [1,2]; and
arti¢cially synthesized polymers, poly(ethylene adipate) [3], poly(O-caprolactone) [1], poly(tetramethylene succinate) [4] and poly(lactic acid) [5].
Aliphatic polycarbonates are also biodegradable
* Corresponding author. Tel.: +81 (298) 54 6591;
Fax: +81 (298) 54-6587; E-mail: [email protected]
[6^9], and with their chemical property of resistance
to water are thought to provide a new possibility for
designing biodegradable plastics with unique properties, as raw materials of polyurethanes, copolymers,
and as matrix of polymer blends. We have tested the
populations of polycarbonate-degrading microorganisms using poly(ethylene carbonate) as a substrate
[8]. Of the colonies plated out, 0.2^5.7% showed activities of decreasing the turbidity of the suspended
poly(ethylene carbonate), con¢rming the distribution
of polycarbonate-degrading microorganisms in the
natural environment. However, a detailed characterization of the degrading microorganisms has not yet
been carried out. We previously examined the susceptibility of polycarbonates to commercial lipolytic
enzymes [9]. Enzymatic degradation of poly(tetramethylene carbonate) into low-molecular-mass com-
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 0 6 3 - 9
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ponents was demonstrated by lipoprotein lipase
(from Pseudomonas sp., Toyobo, Japan). However,
besides 1,4-butanediol and carbonic acid, diester was
detected as a dead-end product of enzymatic degradation. For practical use of biodegradable plastics in
place of non-degradable ones, it is necessary to prove
their degradation by activities present in the environment.
To investigate the degradation of aliphatic polycarbonates in the natural environment, we tried in
the present study to isolate environmental microorganisms that degrade aliphatic polycarbonates, and
also tried to characterize the isolates morphologically, physiologically, and phylogenetically.
2. Materials and methods
2.1. Isolation of polycarbonate-degrading
micro-organisms
The sample water was obtained from the Hanamuro river in Ibaraki Prefecture, Japan. The samples
were incubated in the liquid medium containing suspended poly(hexamethylene carbonate) (PHC, Fig.
1a) described below, at 30³C with vigorous shaking.
The achievement of degradation was monitored by
the decrease of turbidity in the medium due to degradation of PHC suspension. Pure strains of PHC
degrading microorganisms were obtained by repeated application of subcultures with dilution to
single cells. The degradation activity of the isolates
was checked by the formation of clear zones around
the colonies on agar plates prepared with the same
medium.
2.2. Culture media containing suspended polymers
The medium containing PHC (number-average
molecular mass: Mn = 2000; Toagosei Co., Japan)
was prepared by the protocol reported earlier [10]
with slight modi¢cations. PHC was suspended into
the medium as a substrate polymer at a ¢nal concentration of 0.1% (w/v). The medium also contained
(per liter), 1 g of (NH4 )2 SO4 , 0.1 g of yeast extract
(Difco, USA), 20 mg of CaCl2 W2H2 O, 10 mg of
NaCl, 10 mg of FeSO4 W7H2 O, 0.5 mg of
Na2 MoO4 W2H2 O, 0.5 mg of Na2 WO4 W2H2 O, 0.5 mg
of MnSO4 , 60 mg of surfactant (Plysurf A210G:
Daiichi Kogyo Seiyaku, Japan), 0.2 g of KH2 PO4
and 1.6 g of K2 HPO4 (pH 7.0). The liquid medium
was dispensed into test tubes in 10-ml aliquots and
then autoclaved. The agar plates were prepared by
addition of 1.5% agar and poured into petri dishes
after autoclaving.
The medium containing poly(tetramethylene carbonate) (PTC [9]; Mn = 2000; Asahi Chemical Industry Co., Japan) was prepared in the same way.
2.3. Analytical methods in the degradation of
polymers
Analysis of the bacterial degradation products was
performed as reported previously [9]. Strains were
precultured in the PTC/PHC medium at 30³C with
vigorous shaking and transferred into a fresh PTC/
PHC medium (100 Wl of cell suspension containing
2U103 cfu was inoculated into 10 ml medium). After
incubation the culture medium was dried with a rotary evaporator at 40³C and the degradation products from PTC/PHC were extracted with chloroform
(10 ml).
Chromatograms of gel permeation chromatography (GPC) were taken by an HLC-8020 GPC system
(Tosoh Co., Japan) with two wide-spectrum TSK gel
columns (GMHHR -M and GMHHR -L). Chloroform
was used as an eluent with a £ow rate of 1.0 ml
min31 and the measurements were performed at
40³C. NMR spectra and gas chromatograms were
taken by a model JNM-EX 270 FT-NMR spectrometer (JEOL, Japan) and a model GC-14B gas chromatograph (Shimadzu, Japan), respectively, in the
conditions reported previously [9].
Authentic 1,6-hexanediol and 1,4-butanediol were
purchased from Nacalai Tesque Co., Japan. Authentic diesters were prepared from enzymatic digestion
of PTC and PHC by the method previously described [9]. The production and the purity of diester
products were con¢rmed as follows.
Di(6-hydroxyhexyl) carbonate (formula given in
Fig. 1b): 1 H-NMR (N ppm: number of protons, coupling, assignment), 4.13 (4H, t, 1 and 1P-H), 3.63
(4H, t, 6 and 6P-H), 1.99 (2H, s, hydroxyl-H), 1.69
(4H, t-t, 2 and 2P-H), 1.58 (4H, t-t, 5 and 5P-H) and
1.40 (8H; m; 3, 3P, 4 and 4P-H); 13 C-NMR (Nc ppm),
155.5 (carbonyl-C), 67.9 (1 and 1P-C), 62.7 (6 and 6P-
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T. Suyama et al. / FEMS Microbiology Letters 161 (1998) 255^261
C), 32.5 (5 and 5P-C), 28.7 (2 and 2P-C), 25.5 (4 and
4P-C) and 25.4 (3 and 3P-C).
Di(4-hydroxybutyl) carbonate [9]: 1 H-NMR, 4.18
(4H, t, 1 and 1P-H), 3.68 (4H, t, 4 and 4P-H), 1.78
(4H, t-t, 2 and 2P-H), 1.66 (4H, t-t, 3 and 3P-H) and
1.63 (2H, s, hydroxyl-H); 13 C-NMR, 155.3 (carbonyl-C), 67.7 (1 and 1P-C), 62.3 (4 and 4P-C), 28.9 (3
and 3P-C) and 25.2 (2 and 2P-C).
2.4. Characterization of isolates
The accumulation of PHB was tested by Nile blue
A staining using the method of Ostle et al. [11]. Nile
blue A staining, Gram staining, morphology and
motility were observed with an Aristoplan phasecontrast microscope equipped with an Ortomat E
camera system (Ernst Leitz, Wetzlar, Germany).
Some standard physiological tests, e.g., oxidase and
catalase production, nitrate reduction, and denitri¢cation reaction, were carried out by conventional
methods [12]. Most biochemical properties, including
enzymatic activities and acid production from carbohydrates, were determined using substrate disks of a
commercial identi¢cation kit (Minitek; BBL Microbiology Systems, USA).
2.5. Phylogenetic study
Almost full-length 16S rRNA genes were ampli¢ed
by polymerase chain reaction (PCR) from chromosomal DNA extracted and puri¢ed by the method of
Marmur [13] using an AmpliTaq polymerase kit
(Perkin Elmer, USA) and a pair of primers targeted
for conserved regions of eubacteria homologous or
complementary to positions 8^27 and 1492^1511
(Escherichia coli numbers [14,15]). The PCR products were cloned using a pT7Blue T-Vector Kit (Novagen, USA) with E. coli JM109 (Takara, Japan).
All the procedures were performed according to the
standard protocols of the kits. The DNA inserts
were sequenced with an ABI 373A DNA sequencing
system (Applied Biosystems, USA). Sequence uniformity of 20^30 clones was determined.
The 16S rRNA gene sequences were compared
with previously published sequences in GenBankEMBL DNA sequence data libraries. Phylogenetic
analysis was performed with GENETYX 9.0 (Software Development Co., Japan) and CLUSTAL
257
W1.6 [16] programs. The nucleotide sequences determined in the present study have been deposited in
the DDBJ/EMBL/GenBank data libraries under accession numbers: AB003623, strain 61A clone group
1; AB003624, strain 61A clone group 2; AB003625,
strain 61B2 clone group 1; AB003626, strain 61B2
clone group 2; AB003627, strain WFF52; and
AB003628, strain 35L.
3. Results and discussion
3.1. The new strains
A total of 10 PHC-degrading strains with distinct
colony types were isolated from the water samples.
All of the strains had activity to reduce the turbidity
of opaque PHC suspension in liquid culture. Four of
the isolates had comparatively strong activity to
make clear zones on the PHC suspension plates.
The clear zones began to appear 2 days after inoculation. The four strains were also culturable on
agar plates of 0.08% nutrient broth (Difco) while
maintaining PHC-degrading activity, whereas the
other strains lost degrading activities during cultivation in the medium lacking PHC. The four isolates
were designated strains 35L, WFF52, 61A and
61B2.
The colonies of the four strains (Fig. 2) were surrounded by clear zones on the opaque plate of suspended PHC. Strain 35L formed a disk-shaped white
colony that was thin in the peripheral region. Strain
WFF52 spread a £at yellowish colony from a central
dot. Degradation of suspended PHC on the agar
plates was most visible in the colonies of strain
WFF52, by their rapid growth with high motility
and by the permeability of their degrading enzyme
into the medium. The other two strains, 61A and
61B2, initially formed white colonies which exhibited
pink in the course of the growth. The colonies of
strain 61A were smooth and soft. The transparency
of clear zones produced by strain 61A was the highest among those by the new isolates. It is likely that
the enzyme activity of PHC degradation produced
by strain 61A was the strongest. Strain 61B2 resembled strain 61A but the colonies grew more
slowly and larger. The colonies of strain 61B2 were
hard and less moist. Flocculation in liquid culture
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T. Suyama et al. / FEMS Microbiology Letters 161 (1998) 255^261
Fig. 1. Chemical structures of (a) PHC and (b) di(6-hydroxyhexyl) carbonate.
was often formed by this strain. These facts suggest
that the cells of strain 61B2 exhibit behavior causing
coagulation.
3.2. Con¢rmation of degradation of polymers by
strain 61A
The degradation process of PHC in the culture
medium incubated with the strains was con¢rmed
in detail using GPC. Fig. 3 shows the changes in
GPC chromatograms of PHC incubated with strain
61A. The main peak (retention time at around 14
min) was identi¢ed as the substrate polymer (PHC,
Mn = 2000), whose area was decreased by incubation
with strain 61A. Little degradation occurred during
12 h of incubation; the chromatogram of the sample
incubated for 12 h (Fig. 3a) was almost identical to
that of the sample before inoculation. The other minor peaks extracted from the culture medium were
unidenti¢ed. (The peaks were observed at the same
retention times in any extract from di¡erent kinds of
substrate polymer medium. It is possible that the
components of the minor peaks were complex products resultant from autoclaving of other substances
in the culture medium.) After 24 h of incubation, a
large peak at retention time 20 min appeared which
was identi¢ed as a peak of diester, i.e., di(6-hydroxyhexyl) carbonate (Fig. 3b, see Section 2). The peak
disappeared after an additional 24 h of incubation
(Fig. 3c).
Fig. 2. Colonies of PHC-degrading strains: (a) 35L; (b) WFF52 ; (c) 61A; (d) 61B2. The colonies were grown for 5 days on agar plates
with suspended PHC.
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T. Suyama et al. / FEMS Microbiology Letters 161 (1998) 255^261
259
Fig. 3. Changes in GPC chromatograms in the time course of
PHC (Mn = 2000) degradation after the inoculation of strain 61A
(2U103 cfu): (a) 12 h ; (b) 24 h ; (c) 48 h. The arrow shows the
peak of di(6-hydroxyhexyl) carbonate.
Fig. 4. Changes in GPC chromatograms in the time course of
PTC (Mn = 2000) degradation after the inoculation of strain 61A
(2U103 cfu): (a) 12 h; (b) 24 h; (c) 48 h. The arrow shows the
peak of di(4-hydroxybutyl) carbonate.
The four strains also made clear zones on the PTC
suspension plate. The degradation of PTC was also
determined in the same way. Observed changes in
time course of PTC degradation were almost identical with that of PHC degradation (Fig. 4a^c). Similar to the processes of PHC degradation, temporal
accumulation and disappearance of diester, di(4-hydroxybutyl) carbonate (Fig. 4b, a peak at retention
time 20.2 min, see Section 2) was observed.
The diesters (arrows in Figs. 3b and 4b) were
dead-end products of enzymatic degradation by lip-
oprotein lipase [9]. The temporal accumulation of the
diester products strongly suggests that the lipolytic
enzymes, similar to what we discovered previously,
are the key enzymes of PHC/PTC degradation also
in microbial processes.
In the case of microbial degradation, the diesters
were short-lived (Figs. 3c and 4c). This is due to the
secondary degradation pathways (esterases degrading the diesters) existing in the strains. Unlike the
enzymatic degradation [9], no monomeric diols
(1,6-hexanediol or 1,4-butanediol) were observed by
Table 1
Biochemical characteristics of new isolatesa
Characteristics
35L
WFF52
61A
61B2
Morphology
Straight rod
0.5U2^4 Wm
3
+
+
3
3
+
+
Straight rod
0.5U3^5 Wm
+
+
3
3
3
3
3
Rod
0.5U2 Wm
+
3
3
+
+
3
3
Curved rod
0.5U3^5 Wm
+
3
3
+
+
3
+
+
+
+
+
3
3
+
3
3
3
+
3
+
3
3
+
Motility
Catalase
Nitrate reduced to nitrite
Starch hydrolysis
L-Galactosidase (ONPG)
Arginine dihydrolase
Lysine decarboxylase
Acid production from
Glucose
Mannose
Arabinose
Xylose
a
+, positive reaction; 3, negative reaction. All four strains were negative for the following characteristics: Gram stain ; degradation of
extracellular PHB; denitri¢cation ; hydrolysis of esculin and salicin ; ornithine decarboxylase ; urease; indole production; acid production
from ra¤nose, maltose, trehalose, cellobiose, lactose, sorbitol, mannitol, rhamnose and glycerol ; and positive for the following characteristics: PHB accumulation, oxidase and utilization of citrate.
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Fig. 5. Phylogenetic positions of new isolates (nucleotide sequence accession numbers AB003623^AB003628) among Proteobacteria derived
from analysis of 16S rRNA genes by the method of neighbor-joining [17]. Positions with alignment gaps and unidenti¢ed bases were not
taken into consideration. The percentages of 1000 bootstrap trials that support each topological element are indicated beside the nodes.
GPC or gas chromatography in the entire course of
degradation. On the other hand, evolution of organic
acids that was not observed in the enzymatic degradation was observed. Adipic acid (PHC) or succinic
acid (PTC) and some other unidenti¢ed acids were
detected from the microbial degraded samples.
3.3. Characterization and phylogenetic analysis of
new strains
The isolates were characterized by the standard
tests listed in Table 1. As the strains did not grow
on bouillon media, they were cultured on nutrient
broth agar plates for the tests.
The phylogenetic tree implied from 16S rRNA
gene sequences (Fig. 5) indicates that the strains belong to the L or Q subclass of Proteobacteria. Strain
35L was classi¢ed in the Q subclass as a close relative
of Pseudomonas aureofaciens and P. chlororaphis
with similarities of 97.9% and 97.7%, respectively.
Other strains were classi¢ed in the L subclass. Strain
WFF52 was classi¢ed as a close relative of Variovorax paradoxus with a similarity of 99.0%. The sequences of strains 61A and 61B2 were almost identical,
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T. Suyama et al. / FEMS Microbiology Letters 161 (1998) 255^261
except for a single base, and made a distinct cluster
in the L subclass. They made no obvious cluster with
other speci¢ed genera (Fig. 5). A pair of slightly
di¡erent sequences were detected in both clones
from the two strains (shown as clone group 1 and
clone group 2). The detection was reproducible in at
least four distinct ampli¢cation experiments and the
strains are thought to have multicopies of the 16S
rRNA gene with slight mutation in their cells.
The new isolates were the ¢rst strains found and
isolated as degraders of aliphatic polycarbonates.
The speci¢city of the polycarbonate-degrading activity was not so strict, since all of the PHC-degrading
isolates also had PTC-degrading activity. Although
they require yeast extract or nutrient broth as cosubstrates for optimum growth, they have obvious activity to convert PHC/PTC into biomass and minerals under the conditions, and have a function to
restore the synthetic polymers into natural carbon
cycle. The phylogenetic study (Fig. 5) showed that
the four strains were classi¢ed into Proteobacteria
but all were not closely related to each other. This
result indicates that the polycarbonate-degrading activity is not a property limited to a speci¢ed organism of a restricted group.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
Acknowledgments
We wish to thank Akira Iwamoto (JSP Co.) and
Satoshi Hanada (RITE: Research Institute of Innovative Technology for the Earth) for technical advice.
[13]
[14]
[15]
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