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Effects of soil temperature and anaerobiosis on

degradation of biodegradable plastics in soil and theirdegrading microorganismsHiroyo Nishide

ab

, Koki Toyotaa

& Makoto Kimuraa

aComputer Room, National Institute for Basic Biology, Okazaki, 444-8585, Japan

bNagoya University, Laboratory of Soil Biology and Chemistry, Graduate School of

Bioagricultural Sciences, Nagoya, 464-8601, Japan

Published online: 04 Jan 2012.

To cite this article: Hiroyo Nishide , Koki Toyota & Makoto Kimura (1999): Effects of soil temperature and anaerobiosis on

degradation of biodegradable plastics in soil and their degrading microorganisms, Soil Science and Plant Nutrition, 45:4,

963-972

To link to this article: http://dx.doi.org/10.1080/00380768.1999.10414346

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Soil Sci. Plant Nutr., 45 (4), 963-972, 1999

Effects of Soil Temperature and Anaerobiosis onDegradation of Biodegradable Plastics in Soiland Their Degrading Microorganisms

Hiroyo Nishidel, Koki Toyota, and Makoto KimuraLaboratory of Soil Biology and Chemistry, Graduate School of Bioagricultural Sciences,Nagoya University, Nagoya, 464-8601 Japan

Received July 9, 1999; accepted in revised form October 7, 1999

The bioplastic PHB/HV (copolymer of 3-hydroxybutyrate an d 3-hydroxyvalerate) underwent a faster degradation at 30"C than at 52C in soil under aerobicconditions, while there was no remarkable difference between 30C an d 52C inth e degradation rate of PCL [ p o l y ( ~ - c a p r o l a c t o n e ) ] , PBSA (polybutylenesuccinate an d agipate), and PBS (polybutylene succinate). PH B showed thefastest degree of degradation among th e four plastics at 30C and PBSA th efastest at 52C. Degradation of al l the four plastics was no r observed both at30C and 52C under anaerobic conditions fo r 50 d. Microorganisms on th edegrading plastics appeared to be diverse at 30"C, including bacteria and fungi.However, among th e several to ca. 10 kinds of bacterial and fungal strainsisolated from th e degradation sites of each plastic at 30"C, only one or twofungal strains were able to degrade th e respective plastics in vitro. The degraders were identified as Mucor sp. (PHB), Paecilomyces sp. (PCL), Aspergil-lus sp. (PBSA), an d Cunninghamella sp. (PBSA). In contrast, only a singletype of fungus was observed at th e degradation sites of PCL and PBSA at 52C.The fungus isolated from PCL an d PBSA was identified as Thermomyces sp.This study demonstrated that soil temperature and anaerobiosis exerted significant effects on the degradation of the plastics, and that fungi were mainlyresponsible fo r th e degradation of th e plastics in soil.Key Words: biodegradable plastics, degrading microorganisms, fungi, soilconditions.

963

Synthetic plastics have been used for various purposes, and the production of plasticshas been one of the key industries. However, synthetic plastics with high performance andstability cause serious problems in waste management (Hrabak 1992; Lee 1995). Recently,biodegradable plastics have become a cause for public concern. A number of biodegradableplastics derived from natural products or polymerized from naturally occurring monomers(Brandl and Puchner 1992; Takiyama and Fujimaki 1994) are commercially available. PI-IB[poly(3-hydroxybutyrate)] is typical of the former group, and several plastics such aspoly(vinyl alcohol) (PV A), poly(methyl-glutamate) (PGA), polY(E-caprolactone) (peL),polybutylene succinate (PBS), polybutylene succinate and agipate copolymer (PBSA)belong to the latter group.1 Present address: Computer Room, National Institute for Basic Biology, Okazaki, 444-8585 Japan.


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964 H. NISHIDE, K. TOYOTA, and M. KIMURAI t was reported that a variety of microbial strains, including bacteria and fungi,

degraded polyhydroxyalkanoates (PHAs) (Brandl et al. 1995; Mergaert and Swings 1996).Most of the plastic-degrading microorganisms were screened for their potential to degradethem in vitro (Delafield et al. 1965; Fields and Finn 1974; Tanaka et al. 1976; Matavulj andMolitoris 1992; lendrossek et al. 1993; Od a et al. 1995; Pranamuda et al. 1995; Mergaert andSwings 1996), and in several studies it was shown that plastic-degrading microorganismswere isolated from the plastics that were degraded in the environment (Mergaert et al. 1992,1993). Much less attention has been paid to determine whether isolates from the degradedplastics actually degrade the respective plastics in situ.

Degradation of PHAs was mainly performed by bacteria (e.g., Matavuli and Molitoris1992; Mergaert and Swings 1996). I t was also found that fungi, rather than bacteria, weremainly responsible for the degradation of PHB in soil (Kimura et al. 1994; Od a et al. 1995).PHA-degrading microorganisms have been screened for their in vitro ability to degrade inmost of the studies. It is, therefore, necessary to isolate degrading microorganisms fromdegraded plastics in the environment in order to determine what kinds of microorganismsare mainly responsible for the in situ degradation. We successfully isolated several newplastic-degrading microorganisms from soil under different conditions. Here, we report theeffect of soil conditions on the degradation of biodegradable plastics (PHB/HV, PCL,PBSA, and PBS), and the microorganisms that degraded the plastics in soil.

MATERIALS AND METHODSSoil an d biodegradable plastics. The soil used (Hapludults; total C = 1.2%, total

N=O.ll%, pH(HzO)=6.2) was collected from the Nagoya University Farm, and passedthrough a 2-mm mesh screen. Farmyard manure (FYM) was also obtained from the NagoyaUniversity Farm, and passed through a 4-mm mesh screen. The soil was mixed with 2% (w /w) of FYM prior to use. The plastic film samples used were as follows: PHB/HV (poly-3-hydroxybutyrate, (10%) 3-hydroxyvalerate, Zeneca Co.), PCL (poly-caprolactone, DaicelChemical Industries), PBSA (poly-butylene succinate and agipate copolymer, ShowaHighpolymer Co.), and PBS (poly-butylene succinate, Showa Highpolymer Co.).

Effect of soil conditions on degradation of biodegradable plastics. Four kilograms of soil that was adjusted to 40% of maximum water holding capacity was placed in10 L of an air-tight plastic bag. Films (1 cmX 1 cm, 10 to 15 mg in weight) of the plasticsamples were buried in soil, and incubated at 30"C or 52"C for 30 to 50 d. Degradation ofthe plastics under anaerobic conditions was also examined as follows. A piece of film wasburied in 20 g of soil placed in a 50 mL flask with an air tight cap and then the air wasreplaced with nitrogen. Th e films were taken out periodically, washed gently in water toremove adhering soil particles, and the weight of the recovered films was determined. Fourpieces of films were used for each plastic on each sampling day.

Isolation and identification of degrading microorganisms. Microorganisms wereisolated from degradation sites (holes) with a sterile toothpick and purified on nutrient agar(NA: Eiken Chemical Co.). To ensure their ability to degrade plastics, isolated microorganisms were inoculated into a minimum medium (0.1% NH 4N0 3 , 0.1% KH zP0 4 , 0.05% MgS04 7HzO, 0.02% KCl) or autoclaved (12l"c for 15 min) soil with respective plas tic films as a solecarbon source and incubated either at 30"C or 52"C. Plastic degradation was estimated basedon microbial growth in the medium and holes formed on the films.

Identification of the fungal strains was performed by the Japan Food Research Lab.,


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Degradation of Biodegradable Plastics in Soil 965a) 30"C, aerobic conditions b) 52"C, aerobic condit ions

100 100

~ 80 ~ 80~ ~~ ~CI Ci&:: &::"2 "2ii i 60 pe l ii i 60E EG/ G/... ...... .....c .cCI CI PBSi 40 Gi 403: 3:

20 HH 20 tLSD (P < 0,05) PBSA LSD (P < 0,05)

0 00 5 10 15 20 25 30 0 5 10 15 20 25 30Incubation days Incubation days

Fig, 1. Degradation of the plastics PHB/HV, PCL, PBS, and PBS A in soil under aerobic conditions at30T (a) and S2C (b). Each symbol is the mean of four replications and bars indicate the least significancedifference (LSD: p


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966 H. NISHIDE, K. TOYOTA, and M. KIMURA

Fig. 3. Microscopic obser-vation (X 6.3 to 40) of theplastics buried in soil underaerobic conditions at 30'C. A:PHB/HV. B: peL. C: PBSA.


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Degradation of Biodegradable Plastics in Soil 967

Fig. 4. Microscopic obser-vation (x 6.3 to 40) of theplastics buried in soil underaerobic conditions at 52'C. A:PHB/HV.B: PCL. C: PBSA.


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Fig. 5. Degradation of theplastics by isolated micro-organisms under aerobic con-ditions at 30"C. A: PHB/HV,by Mucor sp. B: peL, byPaecilomyces sp. C: PBSA, byCunninghamella sp.


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Degradation of Biodegradable Plastics in Soil 969mainly based on morphology.

RESULTS AND DISCUSSIONEffect of soil conditions on degradation of PBB/BV, peL, PBS, an d PBSA

Degradation was estimated from the weight loss of the plastics. PHB underwent thefastest degradation among the four plastics at 30C under aerobic conditions and PBS theslowest: the estimated half-life (the day when the weight of the recovered films was reducedto half) was ca. 10 d for PHB and over 25 d for PBS (Fig. 1). At 52C, PBSA underwent thefastest degradation with a half-life of ca. 7 d, and PHB, PCL, and PBS showed a similardegradation rate under aerobic conditions. PHB/HV underwent a faster degradation at 30Cthan at 52T under aerobic conditions, while PCL, PBS, and PBS A showed a fasterdegradation at 52C (Fig. 1).

The plastics were not degraded under anaerobic conditions at both 30C and 52C [Fig.2; the weight of PC L after 50 d of incubation at 52C was larger than that at a-time, becauseof the adhesion of soil particles due to partial melting of PCL (the melting point of PC Lis 5YC)]. These results suggested that the degradation ofPHB, PCL, PBS, and PBSA at 52Twas not physical, bu t biological. Most of the reports on the biodegradation of PHB indifferent environments have been conducted under aerobic conditions. Only a few studieswere reported on the anaerobic degradation of PHB in an estuarine sediment (Janssen andHarfoot 1990) an d sludges (Budwill et al. 1992; Mergaert and Swings 1996). There has been,hitherto, no report on the anaerobic degradation of PHB in soil.Isolation of plastic-degrading microorganisms

Two types of degradation of the plastics were observed. In one case, various kinds offungi were observed under a microscope (X40) on/around the degradation sites, suggestingthat these fungi may be responsible for the degradation of the plastics. In the other case, nomycelium was observed around the degradation sites, suggesting that degradation was dueto bacteria, rather than fungi. Under aerobic conditions at 30C, different types of fungi,based on the color of spores, were observed on the plastics, irrespective of the kinds ofplastics, and they appeared to be the organisms that degraded the plastics (Fig. 3). Incontrast, fungal mycelia were hardly observed on the degradation sites of PHB at 52C (Fig.4A), indicating that bacteria may be the major microorganisms that degraded PHB at 5TC.In the cases of PC L and PBSA, only a single type of fungus was observed on the degraded

Table 1. Relative abundance of fungi on degraded plastics and identificaiton of theplastic-degrading microorganisms.

Plastics Temperature Microscopic observation Identified microorganisms as plastic degradersPHB/HV 30"C Fungi+ + + * Mucor sp.52"C Fungi+ Talaromyces sp., an actinomycete strainPCL 30"C Fungi + + + Paecilomyces sp.

5TC Fungi+ + + Thermomyces sp.PBSA 30"C Fungi+ + + Cunninghamella sp., Aspergillus sp.

5TC Fungi+ + + Thermomyces sp.PBS 30"C Fungi+ + + not isolated52"C Fungi+ + + no t isolated

*Relative amount of fungal mycelia on the films. +: present, + + +: abundant.


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Fig. 6. Degradation of theplastics by isolated micro-organisms under aerobic con-ditions at 52T. A: PHB/HV,by Talaromyces sp. B: peL,by Thermomyces sp. C:PBSA, by Thermomyces sp.


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Degradation of Biodegradable Plastics in Soil 971plastics at 52e with a marked proliferation around the degradation sites (Fig. 4B, e).

Several to ca. 10 kinds of bacterial and fungal strains were isolated from the degradationsites of each plastic at 30C. However, only several strains, exclusively fungi, were able todegrade the respective plastics in inoculation experiments. They showed a similar pattern ofdegradation to that observed in soil (Fig. 5), and were considered to be the microorganismsresponsible for the degradation of the plastics in soil. Mucor sp. and Paecilomyces sp. wereidentified as PHB/HV- and PeL-degrading microorganisms, respectively (Table 1). Asper-gillus sp. and Cunninghamella sp. were identified as PBS A-degrading microorganisms.Unlike the degradation at 30o e , a single type of fungus, based on the colony morphology,was isolated from the degradation sites of peL and PBSA at 52T, and the fungus alsoshowed a similar way of degradation to that observed in soil (Fig. 6B, e). Both fungi thatdegraded either peL or PBSA were identified as Thermomyces sp. Five bacterial and onefungal strains were isolated from the degradation sites of PHB/HV at 52T, but only oneactinomycete and one fungal strain were able to degrade PHB/HV at 52e in the inoculationexperiments. The fungus was identified as Talaromyces sp.

Although the degradation of PHAs was mainly performed by bacteria, fungal degradation was also reported (e.g., Matavulj and Molitoris 1992; Mergaert et al. 1993; Oda et al.1995; Mergaert and Swings 1996). Degradation of PHB/HV, peL, and PBSA in soil at 300 ewas remarkably suppressed by the addition of a fungicide Daconil (chlorothalonil,2,4,5,6-tetrachloroisophthalonitrile), while the addition of streptomycin did no t affectappreciably the degradation of the plastics (PHB/HV, Kimura et al. 1994; peL and PBSA,data not shown). These results suggest that fungi might be mainly responsible for thebiodegradation of the plastics in soil.

Reports on the microbial degradation of biodegradable plastics other than PH As arerelatively limited compared with those of PHAs. Paecilomyces sp. (Oda et al. 1995),Penicillium sp. (Tokiwa et al. 1976), and actinomycetes (Mergaert and Swings 1996) werereported to be PeL-degrading microorganisms. Paecilomyces sp., Acremonium sp., Aspergil-lus sp. (Nishioka et al. 1994), Paecilomyces sp., an d Sphingobacterium sp. (Imada et al. 1998)and actinomycetes (Mergaert and Swings 1996) were reported to be PBSA-degradingmicroorganisms. Talaromyces sp., Thermomyces sp., and Cunninghamella sp. were for thefirst time reported to be plastic-degrading fungi in this study.

In conclusion, this study suggested that fungi were mainly responsible for the degradation of the plastics PHB/HV, peL, PBS, and PBSA in soil, and that microorganismsresponsible for the degradation were different depending on the kinds of plastics and soilconditions.

Acknowledgments. This study was partly funded by the Biodegradable Plastics Society. We thank thechairman Mr. A. Hoshino for his encouragement in the study, and Zeneca Co., Daicel Chemical Industries,and Showa Highpolymer Co. for supplying the plastics.

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972 H. NISHIDE, K. TOYOTA, and M. KIMURAAppl. Environ. Microbiol., 58, 1398-1401

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