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									Soil Science and Plant Nutrition (2007) 53, 568–574                                                   doi: 10.1111/j.1747-0765.2007.00169.x

Blackwell Publishing Ltd

Fungal communities on biodegradable plastics

Molecular analysis of fungal communities of biodegradable
plastics in two Japanese soils
Masahiro KAMIYA, Susumu ASAKAWA and Makoto KIMURA
Laboratory of Soil Biology and Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University,
Nagoya 464-8601, Japan

                      This study aimed to elucidate the microbial communities responsible for the decomposition of poly-
                      (ε-caprolactone) (PCL), poly-(butylene succinate) (PBS), poly-(butylene succinate and adipate) (PBSA) and
                      poly-lactide (PLA) in two soils using a culture-independent, polymerase chain reaction-denaturing gradient
                      gel electrophoresis (PCR-DGGE) method with subsequent sequencing of the main DGGE bands. The PCL,
                      PBS and PBSA films were considerably degraded within 50 days at 25°C under upland dark conditions in
                      one soil, while the PLA film was not degraded at all after 120 days in the soil. In the other soil, with less soil
                      organic matter content, only the PBSA films showed any discernible degradation in the 50-day incubation.
                      Many fungal hyphae and hollows along fungal hyphae were observed on the surface of those PCL, PBS and
                      PBSA films. The PCR-DGGE patterns of fungal DNA that were extracted from the degrading plastic films
                      were similar between the soils, with a few different bands, irrespective of the type of plastic film. All four
                      sequenced DGGE bands belonged to Chaetothyriales or Ascomycota incertaesedis in Ascomycota. All the
                      fungal isolates, a total of 60 colonies, formed either white or yellow colonies on Rose Bengal agar medium
                      with similar appearance, and four representative isolates, two white and two yellow isolates from PCL and
                      PBSA films, showed the same mobility on DGGE gel to the mobility of a common band of DNA extracts.
                      Their closest relatives were Penicillium spp.
                      Key words: biodegradable plastics, DGGE, poly-(butylene succinate), poly-(butylene succinate and
                      adipate), poly-(ε-caprolactone), Penicillium.

INTRODUCTION                                                                     and many types of biodegradable plastics are in the
                                                                                 marketplace: for example, poly-(3-hydroxy butyrate)
The production of synthetic plastics amounted to 224                             (PHB), poly-(3-hydroxy butyrate and varelate) (PHB /V),
million tons in the world in 2004 (Plastics Europe;                              poly-(ε-caprolactone) (PCL), poly-(butylene succinate) These plastics with                              (PBS), poly-(butylene succinate and adipate) (PBSA) and
high performance and stability bring about serious                               poly-lactide (PLA). These plastics are decomposable in
problems in waste treatment because they remain unde-                            soils, and the rate of decomposition depends on climate
composed in landfills and produce toxic substances in                            (temperature and rainfall) and soil properties (Hoshino
incineration. To solve the serious problems of plastics                          et al. 2001). Their biodegradability has been evidenced
in the waste stage, biodegradable plastics are attracting                        by bacterial and fungal isolates, such as Alcaligenes
special attention from the public as “the plastics of the                        feacalis (Tanio et al. 1982), Pseudomonas lemoignei
21st century”.                                                                   (Nakayama et al. 1985) and Mucor sp. (Nishide et al.
   The production of biodegradable plastics amounted                             1999) for PHB, Cryptococcus laurentti (Benedict et al.
to approximately 3.0 × 105 tons in the world in 2004,                            1983) and Paecilomyces sp. (Nishide et al. 1999) for
                                                                                 PCL, Bacillus stearothermophilus (Tomita et al. 2000)
                                                                                 and Cunninghamella sp. (Nishide et al. 1999) for PBSA,
Correspondence: M. KAMIYA, Laboratory of Soil Biology
and Chemistry, Graduate School of Bioagricultural Sciences,                      and Fusarium moniliforme for PLA (Torres et al. 1996).
Nagoya University, Furocho, Chikusa, Nagoya 464-8601,                               The biodegradability of these plastics is also ascertained
Japan. Email:                                         using enzymes from fungi and bacteria: for example,
Received 31 January 2007.                                                        PHB/V by Nishide et al. (1999) and PCL by Tokiwa
Accepted for publication 7 May 2007.                                             and Suzuki (1977) and Tokiwa et al. (1986). Hoshino

                                                                                   © 2007 Japanese Society of Soil Science and Plant Nutrition
                                                                      Fungal communities on biodegradable plastics   569

and Isono (2002) observed the biodegradability of PCL,        specimens were placed on the soil and more soil (3 cm
PBS, PBSA and PLA by commercially available lipases.          thickness) was placed on the specimens. The container
   Although these studies clearly demonstrate biode-          was closed with a lid and kept in a room at 25°C under
gradability in the environment, the microorganisms            dark conditions. The lid was opened periodically to
responsible for plastic decomposition in respective envi-     maintain an inner aerobic atmosphere. The period of
ronments have not been well documented, especially in         incubation in the Anjo soil was 50 days for PCL,
the soil environment. As only a few microorganisms in         30 days for PBS, 24 days for PBSA and 120 days for
soil are detectable using culture methods on one hand,        PLA, while it was 50 days for every plastic film in the
and not a single but a group of microorganisms are            university soil.
estimated to contribute to plastics decomposition in             The plastic specimens were recovered four times at
soils on the other hand, this study aims to elucidate the     appropriate intervals and subjected to a determination
microbial communities responsible for the decom-              of decomposition rate, microscopic observation with
position of PCL, PBS, PBSA and PLA in two soils using         an optical microscope or a scanning electron micro-
a culture-independent, polymerase chain reaction-             scope (SEM) and PCR-DGGE analysis with subsequent
denaturing gradient gel electrophoresis (PCR-DGGE)            sequencing of the DGGE bands. Every determination
method with subsequent sequencing of the main DGGE            and observation was conducted in triplicate. Every
bands. As Nishide et al. (1999) observed that PCL, PBS        plastic specimen for the determination of decomposition
and PBSA were not degraded at 30ºC or 52ºC under              rate was weighed at the time of placement in the soil.
anaerobic conditions for 50 days, the experiments                In the determination of the decomposition rate of the
were conducted under aerobic conditions. In this study,       plastics, recovered specimens were gently rinsed with
we isolated several microorganisms from decomposing           distilled water to dislodge the soil and air-dried in a
plastic specimens with the same mobility to the dominant      desiccator with silica gel for 24 h. After weighing, the
community DNA bands on the DGGE gel. In addition,             specimens were incinerated overnight in an electric
we examined the commonality and dissimilarity of              furnace (Electric Furnace TMF-3000, Nisshin EM,
the responsible decomposer communities between the            Tokyo, Japan) at 550°C and weighed again to deter-
two soils.                                                    mine the organic matter content of the specimens.
                                                              The decomposition rate of the specimens was estimated
                                                              from the weight loss along with the incubation period.
                                                              Scanning electron microscope observation
Biodegradable plastics                                        Recovered specimens were gently rinsed with distilled
Four types of biodegradable plastics were used in the         water to dislodge the soil and dried for more than 12 h
present study. They were PCL (H7, Daicel Chemical             in a freeze-dryer (Freeze Dryer FDU-540, Eyela, Tokyo,
Industries, Tokyo, Japan), PBS (1001, Showa High              Japan). Dried specimens were mounted on a holder
Polymer Company, Tokyo, Japan), PBSA (3001, Showa             (Type-QM, Nisshin EM) and sputter-coated with
High Polymer Company) and PLA (Unitika, Osaka, Japan).        platinum palladium (Ion Sputter E-1030, Hitachi Co.,
All test specimens were in the form of a thin film (2 cm      Tokyo, Japan) for examining their decomposition with
× 2 cm with approximately 0.01 mm thickness) with a           a SEM (S-2300, Hitachi, Tokyo, Japan) at ×400 to
large area per weight to accelerate biodegradability.         ×8,000 magnification.

Degradation of plastics in soils                              DNA extraction
Two types of soil samples were used for estimating            The DNA extraction from the plastic films and the
microbial communities of plastics degradation. One            Anjo soil was carried out according to the procedure
was collected from a paddy field in the Aichi-ken Anjo        described by Zhou et al. (1996) with small modifications.
Research and Extension Center, Aichi, Japan (Oxyaquic         The plastic films were washed moderately in sterile
Dystrudept; total C = 13 g kg−1, total N = 1.1 g kg−1,        mili-Q water first. Then, three pieces of the film were
pH(H2O) = 6.3), and the other soil sample was from            put in a sterile 15-mL centrifuge tube, to which 10 mL
an upland field in the Nagoya University Farm, Aichi,         of DNA extraction buffer (100 mmol L–1 Tris-HCl
Japan (Hapludults; total C = 9.4 g kg−1, total N = 1.0        [pH 8.0], 100 mmol L–1 ethylenediaminetetraacetic acid
g kg−1, pH(H2O) = 5.2). The soils were passed through         [EDTA], 1.5 mol L–1 NaCl and 10 g L−1 cetyltrimethyl-
a 2-mm mesh screen and packed in a container (32 cm ×         ammonium bromide [CTAB]) and 100 μL of 10 mg mL−1
23 cm with a 10 cm depth) to become 3 cm in thickness         proteinase K (Promega, Madison, WI, USA) was added
after adjusting the moisture content to 50% of the            and incubated for 30 min at 37°C in a water bath. After
maximum water holding capacity. Then, the test plastic        1.5 mL of 100 g L−1 sodium dodecyl sulfate (SDS) was added

© 2007 Japanese Society of Soil Science and Plant Nutrition
570 M. Kamiya et al.

to the mixture, the mixture was incubated at 65°C for       10% formamide and 1.75 mol L–1 urea) to 45% (8%
30 min, frozen at −80°C for 20 min and thawed at 65°C       [w/v] acrylamide/bisacrilamide [37.5:1], 18% forma-
for 30 min three times. DNA extraction from the soil        mide and 3.15 mol L–1 urea). The electrophoresis was
was according to the method of Watanabe et al. (2004).      carried out using an electrophoresis cell D-code System
  Then, 1 mL of phenol–chloroform–isoamylalcohol            (Bio-Rad Laboratories, Hercules, CA, USA) in 1× TAE
(PCI) (25:24:1, v/v/v) was added to the mixture and it      buffer at 60°C and 100 V for 14 h. Visualization of
was centrifuged at 17,000 g for 5 min. The superna-         DGGE bands was achieved by staining with SYBR
tant phase was transferred to a 2-mL Eppendorf tube         Green Ι nucleic acid gel stain (BMA, Rockland, ME,
and 1 mL of chloroform–isoamylalcohol (24:1, v/v)           USA) for 30 min and photographing under UV light.
was added, followed by centrifugation at 17,000 g for
5 min. After transferring the upper phase of the solution   Direct sequencing
to a new Eppendorf tube, the DNA contained in this          All DGGE bands were excised from DGGE gels and
phase was precipitated with 1 mL of isopropanol at          put in 1.5-mL Eppendorf tubes. One hundred micro-
4°C and centrifuged at 17,000 g for 20 min. The super-      liters of TE buffer was added to the tube and kept at
natant was discarded and the pellet of crude DNA was        4°C overnight to diffuse DNA from the gel strip. One
washed with 700 mL L−1 cold ethanol and thereafter with     microliter of eluted DNA was used as a template to
100% cold ethanol. After drying on a heat block at 37°C,    amplify DNA from the excised DGGE band. The
the DNA was dissolved in 30 μL of TE buffer (10 mmol L–1    primer set and the PCR program were the same as those
Tris-HCL (pH 8.0), 1 mmol L–1 EDTA) and stored at           described above. The resulting PCR products were
4°C for immediate use or at −20°C for storage.              checked by DGGE for the same mobility with that of
                                                            the excised band in the original DGGE pattern. The PCR
PCR-DGGE analysis                                           products that matched the position were sequenced
As the SEM observations suggested that fungi were           using the Dynamic ET terminator Cycle Sequencing Kit
the predominant decomposers of every plastic film, the      (Amersham, Piscataway, NJ, USA) according to the
PCR-DGGE analysis targeted fungal communities. The          instructions, by using the set of two primers, EF4f and
18S rDNA was amplified with PCR using the fungal            fung5r (no GC clamp) with the 373S DNA sequencer
specific primer set of the forward primer EF4 (5′-GGA       (Applied Biosystems Japan, Chiba, Japan).
AGG G [G/A]T GTA TTT ATA G-3′) and the reverse
primer fung5r with GC-rich clamp (5′-CGC CCG CCG            Phylogenetic analysis
CGC GCG GCG GCG GGC GGG GCG GGG GCA                         Sequences of DGGE bands were compared with 18S
CGG GGT AAA GTC CTG GTC CCC-3′; the underlined              rDNA sequences obtained using the BLAST search from
sequence corresponded to the GC-rich clamp.) (Smit          the database of the National Centre of Biotechnology
et al. 1999). The PCR was carried out in a total volume     Information (NCBI) website (
of 20 μL in a 200-μL microtube, which contained 0.2 μL      The phylogenetic tree was constructed using 1,000-fold
of each primer (50 pmol each), 2 μL of 2.5 mmol L–1         bootstrap analysis using the neighbor-joining method
dNTP mixture, 2 μL of 10× Ex Taq buffer (20 mmol L–1        with nj plot software (Perriere and Gouy, 1996). The
Mg2+; TaKaRa, Ohtsu, Shiga, Japan), 0.1 μL of 5 units       18S rDNA partial sequences obtained in this study are
μL−1 Ex Taq DNA polymerase (TaKaRa), 1 μL DNA               available in the DNA Data Bank of Japan (DDBJ) database
template (approximately 15 ng) and 8.5 μL milli-Q           under the accession numbers AB292044–AB292052.
water. Cycle conditions for the amplification were as
follows: an initial denaturation at 94°C for 5 min,         Isolation of fungal degraders of plastic films
followed by 35 cycles of denaturation at 94°C for           Plastic films for fungal isolation were those recovered
1 min, annealing at 50°C for 1 min, extension at 72°C       on the last sampling date. Degraded plastic films were
for 2 min, and a final extension at 72°C for 8 min          washed in 10 mL sterile milli-Q water with ultrasonica-
with TaKaRa PCR Thermal Cycler (Model TP 240;               tion for 3 min (38 kHz, 80 W), and two methods were
TaKaRa). The PCR product was analyzed on 20 g L−1           applied for isolating fungal decomposers. Washed
agarose gels containing 20 g L−1 of 50× TAE buffer          plastic films were inoculated onto Rose Bengal agar
(40 mmol L–1 Tris-acetate, 1 mmol L–1 EDTA) by apply-       medium (KH2PO4 1 g, MgSO4·7H2O 0.5 g, peptone 5 g,
ing ethidium bromide (10 mg mL−1) staining. The gel         glucose 10 g, Rose Bengal 0.033 g, streptomycin 0.03 g
was photographed under ultraviolet (UV) light to            and agar 15 g per liter, pH 6.8) and incubated for 5–14
ascertain the successful amplification.                     days at 25°C. In addition, the milli-Q water used for
   The DNA fragments of the PCR products were sepa-         cleaning the films was diluted to the appropriate con-
rated on a polyacrylamide gel with a denaturing gradient    centration, inoculated to Rose Bengal agar medium and
from 25% (8% [w/v] acrylamide/bisacrilamide [37.5:1],       incubated for 5–14 days at 25°C. Developed fungi were

                                                              © 2007 Japanese Society of Soil Science and Plant Nutrition
                                                                          Fungal communities on biodegradable plastics       571

isolated with a sterile needle to a new Rose Bengal agar
plate and purified.
   To ensure their ability to degrade plastics, isolated fungi
were inoculated into autoclaved soil (121°C for 60 min)
with respective plastic films and incubated at 25°C.
Plastic degradation was examined using SEM observa-
tion. PCR-DGGE was also carried out to check the
mobility of the PCR product from fungal isolates. For
the DNA extraction from the isolates, approximately
1 g of fungal cells of isolate was suspended in 4 mL of
DNA extraction buffer in a sterile 15-mL centrifuge
tube and shaken vigorously. Two hundred microliters
of 10 mg mL−1 proteinase K was added to the tube, and            Figure 1 Degradation of biodegradable plastics in Anjo soil.
it was incubated for 2 h at 55°C followed by overnight                 poly-(ε-caprolactone) (PCL),      poly-(butylene succinate)
incubation at 37°C. The following procedures for DNA             (PBS),       poly-(butylene succinate and adipate) (PBSA), and
                                                                      poly-lactide (PLA).
extraction, PCR amplification and DGGE analysis were
the same as the procedures described earlier.

Identification of fungal isolates                                   In contrast, no degradation was observed for the PLA
Fungal isolates were identified based on 18S rDNA                films during the 120-day incubation. Although actino-
sequence and morphology on Rose Bengal agar plate.               mycete threads were observed accidentally on a PLA film,
The 18S rDNA sequence was determined for the PCR                 there was no evidence of decomposition around the threads
product with EF4f and fung5r primers (no GC-clamp).              and the surface of the PLA after 120-day incubation was
Dynamic ET terminator Cycle Sequencing Kit (Amersham)            as clean as that before placement in soil (data not shown).
was used for sequence determination with the 373S                Hoshino et al. (2001) summarized a 3-year field test of
DNA sequencer (Applied Biosystems Japan) or 310                  PLA degradation in soil at 19 locations in Japan that
Genetic Analyzer (Applied Biosystems Japan). The                 was conducted by the Biodegradable Plastics Society of
fungi grown on the medium were picked up with a                  Japan and concluded no discernible degradation of PLA
sterilized toothpick onto a slide glass and stained with         sheet during the first 3 months at any location.
lactphenol cotton blue (cotton blue 10 mg in water
20 mL, phenol 20 g, lactic acid 20 g and glycerol 40 g)          PCR-DGGE patterns of fungal communities
for 15 min to observe conidia and conidiophores with             on degrading plastic films
an optical microscope (BX50, Olympus, Tokyo, Japan)              The PCR-DGGE analysis was carried out on PCL, PBS
at the appropriate magnification. Identification on              and PBSA films periodically recovered from the incu-
morphology was roughly carried out according to the              bated Anjo paddy soil. In general, the number of DGGE
database of the Microfungi Research website (http://             bands was a few (5 bands each) in comparison with                                         that of the bulk soil and their position on the DGGE
                                                                 gel was stable during the incubation period (Fig. 3).
                                                                 These findings were different from an observation for
RESULTS AND DISCUSSION                                           rice straw (a natural substance having the surface),
                                                                 where many restriction fragment length polymorphism
Degradation of plastic films in Anjo paddy soil                  (RFLP) bands of fungal origins were observed during its
Degradation was estimated from the weight loss of                decomposition (Tun et al. 2002). This was attributed to
plastic films (Fig. 1). The PBSA underwent the fastest           the homogeneity of the constituents of the plastic films
degradation among the four plastic films, and it was             throughout the incubation period, which was in con-
degraded by 60% during the 20-day incubation at                  trast to the change in constituents of rice straw along
25°C. The PCL and PBS were intermediate, and they                with its decomposition. Among seven bands detected in
were degraded by 56% and 46% during the 50-day and               the gel, three bands (a, g, j) were common, irrespective
34-day incubation, respectively. Many fungal hyphae              of the kind of plastics and the incubation period, which
and hollows along hyphae were observed on the surface            indicates that a few phylogenetically similar kinds of
of the PCL, PBS and PBSA films using SEM (Fig. 2).               fungi contribute to the degradation of every plastic film.
However, no circular holes or pits indicating bacterial             Four bands (g, h, i, j) with strong intensity were
decomposition were observed on the films, indicating             successfully sequenced and DNA sequences of bands g,
the monopolization of fungi in plastic degradation.              h and i were the same irrespective of the kind of plastics

© 2007 Japanese Society of Soil Science and Plant Nutrition
572 M. Kamiya et al.

Figure 2 SEM observation of degraded PCL, PBS and PBSA in Anjo soil. a) PCL after 20 days (×500), b) PCL after 50 days (×500),
c) PBS after 10 days (×400), d) PBS after 20 days (×1000), e) PBSA after 10 days (×500), and f) PBSA after 20 days (×500).

                                                                 appearance, irrespective of the kind of plastic films and
                                                                 the origin of isolation (plastic film or milli-Q water used
                                                                 for cleaning the film). Five colonies of white and yellow
                                                                 colors, respectively, were randomly isolated from each
                                                                 film and milli-Q water (60 colonies in total), and were
                                                                 subjected to DGGE analysis after PCR amplification
                                                                 with the primer set of EF4 and fung5r with GC-clamp.
                                                                 All PCR-products moved to the same position as the
                                                                 position of band j (data not shown). Therefore, one of
                                                                 each colony was chosen from the white and yellow
                                                                 colonies developed on the medium with PCL and PBSA
                                                                 films and designated as PCL-W, PCL-Y, PBSA-W and
Figure 3 DGGE patterns of fungal communities on PCL, PBS,        PBSA-Y, respectively.
and PBSA films placed in Anjo paddy soil. DGGE pattern              The abilities of PCL, PBS and PBSA degradation by
of the Anjo soil was shown as the reference. As the pattern      PCL-W, PCL-Y, PBSA-W and PBSA-Y were examined
was taken separately, direct comparison of DGGE patterns
between plastics samples and the soil was not possible. The      in sterilized Anjo soil. PCL-W and PCL-Y degraded
arrow symbol indicates the position of band j.                   every film within 70 days, while PBSA-W and PBSA-Y
                                                                 degraded PBS and PBSA, but did not degrade PCL
                                                                 within 40 days. The hollows on those degrading films
                                                                 by PCL-W, PCL-Y, PBSA-W and PBSA-Y were linear
and the incubation period (Fig. 4). In contrast, the closest     and similar to those observed on respective films in the
relative of band j was affiliated to Cyphellophora sp.           paddy field soil (data not shown). As PCL-W, PCL-Y,
for PCL and to Exophiala sp. for PBS and PBSA. Inter-            PBSA-W and PBSA-Y were phylogenetically close to
esting was the very high similarity of the DNA sequence          each other, it was not clear whether the inability of
(more than 94%) among the four bands, and all of them            PBSA-W and PBSA-Y to degrade PCL resulted from
belonged to the order of Chaetothyriales or Ascomycota           the property of the responsible enzymes released from
incertaesedis in Ascomycota.                                     those isolates or the experimental soil conditions.
                                                                    All of these strains were closely related to each other
Isolation and identification of fungal degraders                 in DNA sequence (the similarity was more than 96%)
of plastic films                                                 and belonged to Mitosporic Ascomycota (PCL-W,
All the fungi that appeared on Rose Bengal agar plates           PBSA-W and PBSA-Y) or Eurotiales (PCL-Y) as shown
formed either white or yellow colonies with similar              in Fig. 4. The closest relative of PCL-W, PBSA-W and

                                                                   © 2007 Japanese Society of Soil Science and Plant Nutrition
                                                                      Fungal communities on biodegradable plastics   573

Figure 4 Phylogenetic relationships of
18S rRNA gene sequences retrieved by
DGGE and fungal isolates with EF4 and

Figure 5 Degradation of PBSA films in
the university soil. a) Degradation of
PBSA films with incubation time, b) after
7 days (×1500), c) after 18 days (×1500),
and d) after 50 days (×1500).

PBSA-Y was Eladia saccula AB031391. While, the                Degradation of plastic films in the university
closest relative was Penicillim verruculosum for PCL-Y.       farm soil
Eladia saccula was formerly named Penicillium saccu-
lum. Thus, the four isolates belonged to other orders         The PBSA films were degraded by 50% during the
from DGGE band j. When band j in Fig. 3 was closely           50-day incubation, but no degradation was observed
examined again, it was broad for every lane and con-          for PCL and PBS over this period in the university
sidered to be an assemblage of phylogenetically remote        soil (Fig. 5a). The SEM observations of degraded PBSA
fungal members with very similar mobility. Thus, fungal       films also detected fungal growth on the films with
decomposers representing the band j were not isolated.        hollows by degradation on the film along the fungal
As Penicillim spp. produces many spores in their late         mycelia (Fig. 5). Hoshino et al. (2001) found that the
stages, they might be isolated on Rose Bengal medium          degradation of PHB/V, PCL, PBS, PBSA and PLA sheets
preferentially to the strains of DGGE band j sequences.       was generally faster in soils with larger total N content.

© 2007 Japanese Society of Soil Science and Plant Nutrition
574 M. Kamiya et al.

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ACKNOWLEDGMENTS                                                Tun CC, Ikenaga M, Asakawa S, Kimura M 2002: Com-
The authors thank Daicel Chemical Industries, Showa                 munity structure of bacteria and fungi responsible for rice
High Polymer Company, Showa High Polymer Com-                       straw decomposition in a paddy field estimated by PCR-RFLP
                                                                    analysis. Soil Sci. Plant Nutr., 48, 805–813.
pany and Unitika for kindly preparing the plastic film
                                                               Watanabe T, Asakawa S, Nakamura A, Nagaoka K,
specimens. This study was conducted with financial
                                                                    Kimura M 2004: DGGE method for analyzing 16S rDNA
support from the Biodegradable Plastics Society of                  of methanogenic archaeal community in paddy field soil.
Japan. We are also grateful to Professor Kazumi                     FEMS Microbiol. Lett., 232, 153 –163.
Hattori of the Graduate School of Bioagricultural              Zhou J, Bruns MA, Tiedje JM 1996: DNA recovery from soils
Sciences, Nagoya University, for his help with the SEM              of diverse composition. Appl. Environ. Microbiol., 62,
observations.                                                       316 – 322.

                                                                 © 2007 Japanese Society of Soil Science and Plant Nutrition

Coarse shear bands and fracture in

Unlike the inorganic glasses, most glassy polymers
can undergo appreciable plastic deformation at
room temperature and moderate strain rates be-
fore fracture occurs. In most cases this deformation
develops inhomogeneously, i.e. only local regions
in the material are plastically stretched. Two
deformation modes are possible, depending upon
the conditions of stress and the ambient. These
two modes are shear yielding' and normal stress
yielding (crazing) [1 ].
   The process of craze formation, which only
occurs under tensile-like loading, and the influence
of crazes on crack propagation and fracture in
polymers have been clarified to a large extent in
recent years [2, 3]. There are very. few studies                           Figure 2 Discrete displacement of scratches on the speci-
which consider in detail the very closely related                          men surface by coarse shear bands viewed in the scanning
                                                                           electron microscope.
phenomenon, the formation of shear bands. It is
well known that when crazing is suppressed,                                Fine slip bands arranged in a broad diffuse shear
amorphous polymers such as polystyrene deform                              zone are found in low speed deformation and/or
by localized shear with the appearance of intense                          at higher temperatures, The authors mentioned
shear bands [4].                                                           that brittle fracture occurred in the coarse bands
   More recently Wu and Li [5, 6] have reported                            after they had extended across the specimen
that two slip processes during the compression                             (Fig. l), while the diffuse shear zone caused
of bulk atactic polystyrene are characteristic.                            ductile fracture behavionr after large strains.
Individual, coarse shear bands appear in high                                 The purpose of this work is to investigate
speed deformation. They are also observed when                             the brittle shear fracture process in more detail,
deformation is carried out at low temperatures.                            mainly by scanning electron microscopic (SEM)
 Compressive    d

                                               .   .   .   .   .   .   .

                                                                                                Figure l Schematic representation
                                                                                                of the stress-strain curve during
                                                                                                compression era notched specimen.
                                                                                                The stages of deformation inside
                                                                                                the specimen are indicated: (1)
                                                                                                Shear band initiation; (2) Maximum
                                                                                                at a band length of about ~ of the
                                                                                                way across the specimen; (3) Mini-
                                                                                                mum when one band packet has
                                                                                                crossed the specimen ; (4) Sliding of
                                                                                                the specimen pieces to each other
                                                                             Strain     r_      combined with final fracture.
480                                                                          9 1 9 79 Chapman and Hall Ltd. Printed in Great Britain.

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