128 Chiang Mai J. Sci. 2012; 39(1)

Chiang Mai J. Sci. 2012; 39(1) : 128-132http://it.science.cmu.ac.th/ejournal/Short Communication

Poly(lactide) Degradation By Pseudonocardia alniAS4.1531T

Maytiya Konkit [a], Amnat Jarerat [b], Chartchai Khanongnuch [c,d],Saisamorn Lumyong [a,d] and Wasu Pathom-aree*[a,d][a] Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.[b] School of Interdisciplinary Studies, Mahidol University at Kanchanaburi, Saiyok, Kanchanaburi,

71150, Thailand.[c] Division of Biotechnology, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50200, Thailand.[d] Materials Science Research Center, Faculty of Science, Chiang Mai University, Chiang Mai 50200,

Thailand.*Author for correspondence; e-mail: wasu.p@cmu.ac.th

Received: 8 November 2011Accepted: 4 January 2012

ABSTRACTTwenty actinobacterial strains belong to the genus Pseudonocardia were

screened for their ability to degrade poly(lactide) plastic. Pseudonocardia alni AS4.1531T

was the only strain that decreased PLA film weight by more than 70% in eight days.This strain could degrade 35.8 mg out of 50 mg PLA films in liquid culture containing0.1% (w/v) gelatin. In addition, Pseudonocardia alni AS4.1531T assimilated the majordegradation product, lactic acid.

Keywords: Poly(lactide), Degradation, Pseudonocardia alni AS4.1531T.

1. INTRODUCTION Plastic wastes are currently a serious

environmental problem of concern.Bioplastics have been considered as a solutionto this problem. Several kinds such aspoly( -caprolactone)(PCL), poly(tetra-methylene succinate)(PTMS), poly(β-hydroxybutyrate)(PHB) and poly(lactide)(PLA)are now commercially available withpotential applications as food containers,foamed sheets and textiles [1,2]. PLA,an aliphatic polyester and biocompatiblethermoplastic, is currently the most promisingbiodegradable material since it can beproduced from renewable resource such as

starch, corn, cassava, through fermentationprocess [3]. PLA is being used in packagingmaterial, films, fiber and non-woven fabrics[4].

Many soil microorganisms were ableto degrade PLA plastics such as fungiTritirachium album [5], Fusarium moniliformeand Penicillium roqueforti [6] or bacteria suchas Bacillus smithi [7], Bacillus brevis [8], andPaenibacillus amylolyticus [2]. Members ofactinobacteria are also known to degradeplastics and polyester [9]. Amycolatopsisstrain HT-32 was the first isolatedPLA-degrading microorganism [10].

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PLA-degrading actinobacteria were re-ported to belong phylogenetically to thePseudonocardiaceae family and relatedgenera, including Amycolatopsis, Saccha-rothrix, Lentzea, Streptoalloteichus andKibdelosporangium [11]. Pseudonocardia isthe type genus of the family Pseudono-cardiaceae which accommodates 25 genera[12]. However, no information is availablefor PLA degradation by Pseudonocardia.

In this study, the degradation of PLAfilm in liquid culture by selected actino-bacteria in the genus Pseudonocardia wasinvestigated.

2. MATERIALS AND METHOD2.1 Materials

Poly(lactide)(PLA),4042D (Mw=74,000)was purchased from NatureWorks® LLC(U.S.). A PLA film was prepared by casting100 mg of PLA in 10 ml chloroform. Theresultant transparent film was dried undervacuum for 2 days at room temperature.

2.2 Microorganisms and Culture MediaSeventeen reference strains (Pseudono-

cardia acaciae TISTR1862T, P. alaniniphilaAS4.1536T, P. alni AS4.1531T, P. antarcticaDSM44749T, P. assaccharolytica DSM44247T,P. autotrophica AS4.1297T, P. benzinivoransDSM44703T, P. chloroethanivoran SL-1T,P. compacta AS4.1534T P. halophobicaAS4.1288T, P. hydrocarbonoxydans IMSNU22140T, P. petroleophila IMSNU 22072T, P.saturnea IMSNU22072T, P. thermophilaAS4.13039T, P. yunnannensis AS4.1542T, P.xinjiangensis AS4.1538T and P. zijingensisAS4.1545T) and 3 newly isolates fromrhizosphere soil samples (isolates 503-45,RM423 and RM452) putatively identifiedas Pseudonocardia were grown on basalmedium containing (Per liter): 1 g yeastextract, 4 g (NH4)2SO4, 2 g K2HPO4, 0.5 gMgSO4 × 7H2O, 2 g KH2PO4 (pH7.0) [13].

2.3 Degradation of PLA filmsDegradation of PLA was carried out in

duplicates using 250 ml Erlenmeyer flaskscontaining 50 ml liquid basal medium with0.1% (w/v) gelatin and 50 mg PLA films.The seed cultures were grown for 8 daysin liquid basal medium containing 0.1%(w/v) gelatin. The cell pellets were inoculatedat 10% (v/v) into the medium. Two controlexperiments were carried out: one was a filmcontrol, which cells were not inoculated;the other was a culture control in which thefilm was not added. Flasks were incubatedat 30°C with shaking at 180 rpm for 8 days.

2.4 Analytical MethodsThe culture broth was taken 10 ml every

2 days for measurement of pH and lacticacid. Lactic acid was measured by titrationmethod with NaOH according to AOAC(1998): Method 936.16 [14]. For dry cellweight, the culture broth was filtered throughWhatman No.1 filter paper and dried toconstant weight at 105oC.

The residual films were efficientlyrecovered from culture broth and washedto remove the bacterial cells then driedat room temperature for 2 days and weighed.

For the microscopic observation of thedegraded PLA films, the film samples(fromDay 4) were directly retrieved from thecultures, washed with distilled water toremove attached cells, and dried for 2 days.The films were coated with gold using aJEOL, JFC-1200 fine coater and observedusing a JEOL scanning electron microscope(SEM), Model JSM-5410LV, operating at15 kV.

3. RESULTS AND DISCUSSION3.1 PLA Film Degradation by Pseudono-cardia alni AS4.1531T

Among the 20 strains of Pseudonocardiatested only Pseudonocardia alni AS4.1531T could

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degrade PLA when grow in liquid basalmedium containing 0.1% (w/v) gelatin. Thisobservation suggested that PLA degradingability was not widely distributed in thisgenus unlike their taxonomically relatedtaxa Amycolatopsis. Several Amycolatopsisstrains were reported as PLA degrader [1, 10,11, 15, 16, 17]. However, PLA-degradingmicroorganisms were reported to be notwidely distributed in natural environment[15, 18]. Similar effect of gelatin on theinduction of PLA degrading activity was alsoobserved in other actinomycetes, Saccharothrixwaywayandensis [3] and Kibdelosporangium aridum[19]. PLA degrading ability was also reportedto increase by other protein eg. silk fibroin[1].

To study the degradation of PLA andassimilation of PLA by Pseudonocardia alniAS4.1531T, the time course of PLA filmdegradation was further investigated byculturing this strain in liquid basal mediumcontaining 50 mg PLA film and 0.1% (w/v) gelatin. After 4 days of cultivation,degradation was evident as the film samplewas disintegrated with an increase in cell

growth (Figure 1). P. alni AS4.1531T

degraded 35.8 mg out of 50 mg film (71.5%)within 8 days. PLA degradation by P. alniAS4.1531T was faster than previouslyreported Amycolatopsis strains. Forinstance, Amycolatopsis strain HT32degraded 60% of PLA film within 14 days[10] or Amycolatopsis strain No. 3118degraded 50% of PLA film in 8 days [16].A significant cell growth as indicated byhigh dry cell weight (50.5 mg) suggestedthat P. alni AS4.1531T could assimilate thedegradation products (Figure 1). Most ofthe PLA-degrading actinomycetes werefound to assimilate the degradationproducts [1, 2]. The degradation productscould be used by these strains for theirgrowth and eventually metabolized to CO2

and H2O [15]. The pH increased during4 days of cultivation, generally due to theammonium ions formation by microbialmetabolisms of gelatin. Thereafter, theamount of lactic acid increased as a resultof PLA film degradation and accumulatedin the culture broth leading to a decreasedpH value (Figure 1).

Figure 1. PLA degradation by Pseudonocardia alni AS4.1531T in liquid culture containing 0.1%(w/v) gelatin.

Film

wei

ght,

dry

cel

l

wei

ght,

Lac

tic

acid

(m

g)

pH

Culture time (d)

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3.2 SEM observation of PLA filmsSEM was used to observe the changes

of the remaining PLA film after 4 daysof cultivation with the strain. As show inFigure 2B, all over the surface of filmsample became rough and many irregular

holes were observed within the early periodof degradation. In contrast, the surfaceof film remained smooth in the controlwithout inoculation of the tested strain(Figure 2A).

Figure 2. Scanning electron micrographs of PLA films (magnification x 1,000); (A) filmcontrol without inoculation and (B) after cultivation with Pseudonocardia alni AS4.1531T for 4days at 30°C (bar = 10 μm).

A. B.

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4. CONCLUSIONOnly 1 of the 17 Pseudonocardia reference

strains was found to degrade PLA.Pseudonocardia alni AS4.1531T coulddegrade 71.5 % of PLA within 8 days inliquid basal medium containing 0.1% (w/v) gelatin. The information obtained fromthis study provides further evidence thatmembers of the family Pseudonocardiaceaeare good source for PLA degraders. PLAdegradation by Pseudonocardia alniAS4.1531T under extreme condition suchas high salt concentration is underinvestigation in our laboratory.

ACKNOWLEDGEMENTSWe wish to thank the National Research

University Project under Thailand’s Officeof the Higher Education Commission forfinancial support. Maytiya is also gratefulfor support from the Graduate School,Chiang Mai University.

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