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Soil Science and Plant Nutrition (2007) 53, 568–574 doi: 10.1111/j.1747-0765.2007.00169.x ORIGINAL ARTICLE Blackwell Publishing Ltd Fungal communities on biodegradable plastics ORIGINAL ARTICLE 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 Abstract 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) http://www.plasticseurope.org). 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: email@example.com 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. MATERIALS AND METHODS 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 (http://www.ncbi.nlm.nih.gov). 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 microfungi.truman.edu/). 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 fung5r. 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. REFERENCES Benedict CV, Cameron JA, Huang SJ 1983: Polycaprolactone degradation by mixed and pure cultures of bacteria and a yeast. J. Appl. Polym. Sci., 28, 335–342. Hoshino A, Isono Y 2002: Degradation of aliphatic polymer films by commercially available lipases with special refer- ence to rapid and complete degradation of poly(L-lactide) film by lipase derived from Alcaligenes sp. Biodegradation., 13, 141–147. Hoshino A, Sawada H, Yokota M, Tsuji M, Fukuda K, Kimura M 2001: Influence of weather conditions and soil properties on degradation of biodegradable plastics in soil. Soil Sci. Plant Nutr., 47, 35– 43. Nakayama K, Saito T, Fukui T, Shirakura Y, Tomita K 1985: Purification and properties of extracellular poly Figure 6 DGGE patterns of PBSA degrading fungal communities (3-hydroxybutyrate) depolymerases from Pseudomonas in the Anjo and university soils. As the reference, DGGE lemoignei. Biochim. Biophys. Acta., 827, 63–72. mobilities of PBSA-W and PBSA-Y were also shown. Nishide H, Toyata K, Kimura M 1999: Effects of soil temper- ature and anaerobiosis on degradation of biodegradable plastics in soil and their degrading microorganisms. Soil Sci. Plant Nutr., 45, 963 –972. Perriere G, Guoy M 1996: WWW-Query: An on-line retrieval In contrast, no discernible degradation of PCL and PBS system for biological sequence banks. Biochimie, 78, films could be attributed to the property of the univer- 364–369. sity soil: lower fungal activities because of lower total C Smit E, Leeflang P, Glandorf B, Elsas JD, Wernars K 1999: Analysis of fungal diversity in the wheat rhizosphere by content in the university soil and pH than in Anjo soil. sequencing of cloned PCR-amplified genes encoding 18S PCR-DGGE analysis of fungal communities RNA and temperature gradient gel electrophoresis. Appl. Environ. Microbiol., 65, 2614 – 2621. developed on PBSA films in the university soil Tanio T, Fukui T, Shirakura Y, Saito T, Tomita K, Kaiho T, The PCR-DGGE patterns of fungal communities devel- Masamune S 1982: An extracellular poly(3-hydroxybutyrate) oped on PBSA films in the university farm soil consisted depolymerase from Alcaligenes faecalis. Eur. J. Biochem., of 12 bands, and they were constantly present except 119, 152–161. for four bands (d, e, f, l) on four sampling dates (Fig. 6). Tokiwa Y, Suzuki T 1977: Hydrolysis of polyesters by lipases. Six DGGE bands (a, b, g, h, i, j) were located at the Nature, 270, 76–78. same positions to the DGGE bands from the Anjo Tokiwa Y, Suzuki Y, Takeda K 1986: Hydrolysis of polyesters by Rhizopus arrhizus lipase. Agric. Biol. Chem., 50, paddy soil. In addition, band j was also broad, indi- 1323–1325. cating an assemblage of several fungal members as Tomita K, Kuroki Y, Hayashi N, Komukai Y 2000: Isolation observed in the Anjo soil. Therefore, phylogenetically of a thermophile degrading poly(butylene succinate- close fungal members are considered to inhabit and pre- co-butylene adipate). J. Biosci. Bioeng., 90, 350– 352. sumably take roles in the degradation of PCL, PBS and Torres A, Li SM, Roussos S, Vert M 1996: Screening of micro- PBSA, irrespective of soil type. organisms for biodegradation of poly(lactic acid) and lactic acid-containing polymers. Appl. Environ. Microbiol., 62, 2393–2397. 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 JOURNAl, OF MATERIALS SCIENCE 14 (1979) 9 LETTERS Letters Coarse shear bands and fracture in polystyrene 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 . 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 Stre55 | . . . . . . . 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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