APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/97/$04.0010

Apr. 1997, p. 1637–1640 Vol. 63, No. 4

Copyright q 1997, American Society for Microbiology

Polylactide Degradation by an Amycolatopsis sp.HARDANING PRANAMUDA,1 YUTAKA TOKIWA,2* AND HIDEO TANAKA1

Institute of Applied Biochemistry, University of Tsukuba,1 and National Institute ofBioscience and Human-Technology,2 Tsukuba, Ibaraki 305, Japan

Received 16 August 1996/Accepted 6 January 1997

By applying the plate count and clear-zone methods, it was confirmed that polylactide (PLA)-degradingmicroorganisms are sparsely distributed in soil environments. An Amycolatopsis isolate was successfullyisolated. Microbial degradation of PLA film was demonstrated; i.e., about 60% of the 100-mg film initiallyadded was degraded by the strain after 14 days of liquid culture.

Polylactide or poly(lactic acid) (PLA) was formerly knownas a hydrolyzable and unstable polyester with limited use as abiodegradable material in medical applications (3, 5). Polymerprocessing technology has advanced quite remarkably, how-ever, so PLA film and fiber with great stability and good phys-ical properties are now available. Hence, the applications ofPLA have expanded to include its use as a biodegradableplastic for solving the worldwide plastic waste problem. Unlikecommercially available biodegradable plastic materials synthe-sized from petrochemicals, such as poly(ε-caprolactone) (PCL)and poly(tetramethylene succinate) (PTMS), PLA is polymer-ized from lactic acid, which can be prepared effectively byfermentation with renewable resources such as starchy mate-rials and cellulose. Moreover, PLA has other good properties,such as a high melting point (1808C), a high degree of trans-parency, and ease of fabrication. Some commercial manufac-turers such as Shimadzu Co. Ltd. (Kyoto, Japan), MitsuiToatsu Co. (Tokyo, Japan), and Cargill Inc. (Minneapolis,Minn.) have already started producing this renewable biode-gradable polymer on an industrial scale.In spite of the great need for PLA as a biodegradable plastic,

until now studies on PLA degradation have focused only onenzymatic (1, 2, 10, 11) or hydrolytic (8, 9) degradation, and nostudy of microbial degradation has been reported. Since mi-croorganisms are involved in the biodegradation of plasticpolymers in the environment, however, the microbial degrad-ability of PLA must be confirmed before it is widely distrib-uted. In this report, we investigate the distributions of PLA-degrading microorganisms and compare their distributionswith those of microorganisms that degrade PCL and PTMS.The isolation of a microbial strain with a high level of PLA-degrading activity was carried out, and the degradation of PLAfilms by this microbial strain was demonstrated.Distribution of PLA-degrading microorganisms. We previ-

ously established plate count and clear-zone methods to eval-uate the distributions of polymer-degrading microorganisms indifferent soil environments (4, 6). By using agar plates contain-ing 0.1% (wt/vol) emulsified polyester (PLA, PCL, or PTMS),total and polyester-degrading microorganisms in soil sampleswere counted within a 30-day period. Forty-five soil samples(taken at about 5 cm below the soil surface) were collectedfrom various places in Tsukuba City, such as paddy fields, weedfields, roadsides, and dumping grounds, etc. From all the sam-ples, we confirmed that the total colonies formed on PLA,PCL, and PTMS plates were of roughly the same order ofmagnitude, i.e., approximately 106 to 108 CFU per gram of

sample. However, clear zones were observed in only 1 sampleon PLA plates versus 33 samples on PCL plates and 19 sampleson PTMS plates. In the sample for which clear-zone formationwas observed on all plates, namely, sample 32 taken from aweed field, the ratios of clear zones to the total colonies were0.04, 0.8, and 0.2% for PLA, PCL, and PTMS plates, respectively.Isolation and characterization of PLA-degrading microor-

ganisms. From the colonies forming clear zones on a PLA-emulsified agar plate, actinomycete strain HT-32 was success-fully isolated (Fig. 1). Strain HT-32 was then grown on yeastmalt agar for 3 days, and its morphology was observed with aphase-contrast Nikon Optiphot HFX-IIA microscope. Physio-logical tests, e.g., cell wall analysis, oxidase and catalase pro-duction, and nitrate reduction, were carried out by conven-tional methods. Biochemical properties were determined withthe Bio Chemical test (CTA Medium; Nissui, Tokyo, Japan).The taxonomic characteristics of strain HT-32 are summarizedin Table 1. On the basis of Bergey’s Manual of DeterminativeBacteriology (2a), strain HT-32 was assigned to the genusAmycolatopsis. Strain HT-32 has now been deposited in theNational Institute of Bioscience and Human-Technology-Agency of Industrial Science and Technology culture collec-tion and has been registered as FERM P-14921.It has been reported that proteinase K produced by a strain

of the mold Tritirachium album can degrade PLA (1, 10) andthat the hydrolysis of biocompatible materials from PLA isaccelerated by autocatalysis of lactic acid (9). By filling peni-cillin cups located on a PLA plate with either proteinase K or1% lactic acid, we confirmed that proteinase K formed a clearzone but that lactic acid did not. However, when the samespecies of T. album obtained from the American Type CultureCollection (ATCC 22563) was streaked on a PLA plate, wefound that no clear zone was formed around the colonies. Onthe other hand, as shown in Fig. 1, strain HT-32 formed obvi-ous clear zones on a PLA plate, suggesting that it secreted anunidentified PLA-degrading agent. By streaking the strain onother plates, we observed that clear zones were formed on PCLplates but not on PTMS plates.Degradation of PLA films by an Amycolatopsis sp. Liquid

cultures of an Amycolatopsis sp. were carried out with twokinds of PLA film with different L-lactic acid contents. PLAwith 100% L-lactic acid content was obtained from ShimadzuCo. Ltd., while PLA with 94% L-lactic acid content was ob-tained from Cargill Inc. The number-average molecularweights (Mn) were 1.88 3 105 and 1.22 3 104, respectively.PLA films were prepared by the heat-pressing method at 150kgf/cm2 and 2108C. The thicknesses of the Shimadzu andCargill films were 100 and 350 mm, respectively. Five-hundred-milliliter Erlenmeyer flasks containing 100 ml of the basal* Corresponding author.

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medium and 100 mg of PLA film were incubated on a rotaryshaker (180 rpm) at 308C (6). After the culture, a 5-ml samplewas taken from the culture broth for analysis of water-solubletotal organic carbon (TOC) and pH. TOC was measured witha TOC-5000 analyzer (Shimadzu Corp.). As shown in Table 2,in a film control (without cell inoculation), neither a significantincrease in TOC nor a pH drop was observed. However, in theculture inoculated with the Amycolatopsis sp., the TOC in-creased about sevenfold and the pH dropped. In the case ofthe cell control (with no film added), TOC decreased as theyeast extract in the medium was consumed by the cells. How-ever, the pH remained unchanged. The strain could degradeboth Shimadzu and Cargill films equally well. The surfacestructures of the PLA films after culture were observed undera JEOL scanning electron microscope, model JSM-T220, with15-kV acceleration. The films were directly retrieved from theculture broths and air dried overnight before observation. Fig-ure 2 shows the surfaces of PLA films after the cultures. Thefilm surfaces were smooth in the controls, for which there wasno inoculation of the Amycolatopsis sp. On the other hand, inthe inoculated cultures, the surfaces became rough and hemi-spherical holes were formed. It seems that there was a differ-ence between the Shimadzu and Cargill films in the diametersof the holes formed. This may be caused by the difference inthe high-order molecular structures of the films due to thedifferent contents of L-lactic acid. However, further investiga-tion is required to clarify this point.The time course of PLA film degradation by the Amycola-

topsis sp. was investigated. Liquid cultures with 100 mg ofShimadzu PLA film were grown. Residual PLA film was re-covered from the culture broth by extraction with 100 ml ofchloroform. The recovered polymer was dried at 808C in avacuum overnight. The cells were collected by centrifugation at4,500 3 g for 10 min and dried at 1058C for 16 h. Experimentswere done in triplicate, and the mean value for each set ofexperiments was used. The result is shown in Fig. 3. The weightof the film decreased with the disintegration of the film. TheTOC increased and the pH dropped. However, there was no

decrease in TOC afterward, and cell growth was very slow.After 14 days of cultivation, of the 100 mg of PLA film initiallyadded, about 40 mg remained dispersed as fragments in theculture broth, but only 4 mg of cells was formed. It seems thatthe degradation products were hardly utilized by the strain. Wealso confirmed that in liquid culture with lactic acid as a carbonsource, only a small amount of cell weight was produced (datanot shown). On the other hand, in a control not inoculatedwith the strain, neither a decrease in film weight, nor a pH

FIG. 1. Colonies of strain HT-32 on an agar plate with emulsified PLA. Theplate was incubated at 308C for 14 days.

TABLE 1. Biochemical characteristics of strain HT-32

Characteristic Result forHT-32

Colony morphology ...........................................................Round, cream,wrinkled,1 mm indiameter

Gram stain.......................................................................... 1Spores.................................................................................. 2Motility................................................................................ 2Aerial mycelium................................................................. 1Cell wall compositionmeso-DAPa ..................................................................... 1Mycolic acids .................................................................. 2

Catalase............................................................................... 1Oxidase................................................................................ 1Fermentative in glucose OFb ........................................... No changeNitrate reduction ............................................................... 1H2S production .................................................................. 2Utilization of carbohydratesGlucose ........................................................................... 1Maltose ........................................................................... 1Fructose .......................................................................... 1Raffinose ......................................................................... 1Rhamnose....................................................................... 2Sucrose ............................................................................ 1Xylose.............................................................................. 1Mannitol.......................................................................... 1Inositol ............................................................................ 1

Growth418C ................................................................................. 2NaCl (7%) ...................................................................... 2Phenol (0.1%) ................................................................ 2Rifampin ......................................................................... 1Neomycin ........................................................................ 1Arbutin............................................................................ 2

a DAP, diaminopimelic acid.b OF, oxidation-fermentation solution.

TABLE 2. TOC and pH of the culture broth ofstrain HT-32 with PLA filmsa

Film

Before culture After culture

pH TOC(ppm) pH TOC

(ppm)

Shimadzu filmFilm control (no inoculation) 7 43 7 60Culture of strain HT-32 7 42 6 303

Cargill filmFilm control (no inoculation) 7 43 7 68Culture of strain HT-32 7 42 7 308

Cell control (no film) 7 40 7 11

a Culture was carried out with 100 ml of the basal medium containing 0.01%(wt/vol) yeast extract and 100 mg of film at 180-rpm shaking and 308C for 20 days.

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drop, nor a TOC increase was observed, suggesting that thePLA film is so stable that almost no hydrolysis process oc-curred. It is known that polymer degradation at temperaturesbelow the glass transition temperature (Tg) occurs slowly be-cause of the inactivity of the polymer’s molecules. Since the Tgof PLA is approximately 53 to 598C, it has been demonstratedthat the Amycolatopsis sp. degrades PLA even at temperaturesbelow the Tg.The problem of nondegradable plastic waste is getting more

and more serious, so the development of biodegradable plasticpolymers has become essential. PLA is one of the polyesterspotentially applicable as biodegradable plastic. However, thestability of PLA in water and the scarcity of PLA-degradingmicroorganisms in soil environments may cause the degrada-tion of PLA in natural environments to be very slow. There-fore, it is necessary to develop a treatment process for plasticwaste from PLA in order to prevent a new waste problem. Ourisolate, an Amycolatopsis sp., is an effective strain for the treat-ment of PLA plastic waste and for the evaluation of degrad-ability of plastics containing PLA.

We thank Akio Tsuchii for helpful advice and technical assistance.

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FIG. 2. Surface structures of PLA films after degradation by the Amycolatopsis sp. Bar, 5 mm. (A) Surface structures without cell inoculation; (B) surface structureswith cell inoculation.

FIG. 3. Time course of PLA film degradation by the Amycolatopsis sp. Solidsymbols, culture with cell inoculation; open symbols, control without cell inoc-ulation. Bars show standard errors.

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