128 Chiang Mai J. Sci. 2012; 39(1)
Chiang Mai J. Sci. 2012; 39(1) : 128-132 http://it.science.cmu.ac.th/ejournal/ Short Communication
Poly(lactide) Degradation By Pseudonocardia alni AS4.1531T 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: [email protected]
Received: 8 November 2011 Accepted: 4 January 2012
ABSTRACT Twenty 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 containing 0.1% (w/v) gelatin. In addition, Pseudonocardia alni AS4.1531T assimilated the major degradation product, lactic acid.
Keywords: Poly(lactide), Degradation, Pseudonocardia alni AS4.1531T.
1. INTRODUCTION Plastic wastes are currently a serious starch, corn, cassava, through fermentation environmental problem of concern. process [3]. PLA is being used in packaging Bioplastics have been considered as a solution material, films, fiber and non-woven fabrics to this problem. Several kinds such as [4]. poly( -caprolactone)(PCL), poly(tetra- Many soil microorganisms were able methylene succinate)(PTMS), poly(β-hydro to degrade PLA plastics such as fungi xybutyrate)(PHB) and poly(lactide)(PLA) Tritirachium album [5], Fusarium moniliforme are now commercially available with and Penicillium roqueforti [6] or bacteria such potential applications as food containers, as Bacillus smithi [7], Bacillus brevis [8], and foamed sheets and textiles [1,2]. PLA, Paenibacillus amylolyticus [2]. Members of an aliphatic polyester and biocompatible actinobacteria are also known to degrade thermoplastic, is currently the most promising plastics and polyester [9]. Amycolatopsis biodegradable material since it can be strain HT-32 was the first isolated produced from renewable resource such as PLA-degrading microorganism [10].
128-132Wasu.pmd 128 10/2/2555, 9:06 Chiang Mai J. Sci. 2012; 39(1) 129
PLA-degrading actinobacteria were re- 2.3 Degradation of PLA films ported to belong phylogenetically to the Degradation of PLA was carried out in Pseudonocardiaceae family and related duplicates using 250 ml Erlenmeyer flasks genera, including Amycolatopsis, Saccha- containing 50 ml liquid basal medium with rothrix, Lentzea, Streptoalloteichus and 0.1% (w/v) gelatin and 50 mg PLA films. Kibdelosporangium [11]. Pseudonocardia is The seed cultures were grown for 8 days the type genus of the family Pseudono- in liquid basal medium containing 0.1% cardiaceae which accommodates 25 genera (w/v) gelatin. The cell pellets were inoculated [12]. However, no information is available at 10% (v/v) into the medium. Two control for PLA degradation by Pseudonocardia. experiments were carried out: one was a film In this study, the degradation of PLA control, which cells were not inoculated; film in liquid culture by selected actino- the other was a culture control in which the bacteria in the genus Pseudonocardia was film was not added. Flasks were incubated investigated. at 30°C with shaking at 180 rpm for 8 days.
2. MATERIALS AND METHOD 2.4 Analytical Methods 2.1 Materials The culture broth was taken 10 ml every Poly(lactide)(PLA),4042D (Mw=74,000) 2 days for measurement of pH and lactic was purchased from NatureWorks® LLC acid. Lactic acid was measured by titration (U.S.). A PLA film was prepared by casting method with NaOH according to AOAC 100 mg of PLA in 10 ml chloroform. The (1998): Method 936.16 [14]. For dry cell resultant transparent film was dried under weight, the culture broth was filtered through vacuum for 2 days at room temperature. Whatman No.1 filter paper and dried to constant weight at 105oC. 2.2 Microorganisms and Culture Media The residual films were efficiently Seventeen reference strains (Pseudono- recovered from culture broth and washed cardia acaciae TISTR1862T, P. alaniniphila to remove the bacterial cells then dried AS4.1536T, P. alni AS4.1531T, P. antarctica at room temperature for 2 days and weighed. DSM44749T, P. assaccharolytica DSM44247T, For the microscopic observation of the P. autotrophica AS4.1297T, P. benzinivorans degraded PLA films, the film samples(from DSM44703T, P. chloroethanivoran SL-1T, Day 4) were directly retrieved from the P. compacta AS4.1534T P. halophobica cultures, washed with distilled water to AS4.1288T, P. hydrocarbonoxydans IMSNU remove attached cells, and dried for 2 days. 22140T, P. petroleophila IMSNU 22072T, P. The films were coated with gold using a saturnea IMSNU22072T, P. thermophila JEOL, JFC-1200 fine coater and observed AS4.13039T, P. yunnannensis AS4.1542T, P. using a JEOL scanning electron microscope xinjiangensis AS4.1538T and P. zijingensis (SEM), Model JSM-5410LV, operating at AS4.1545T) and 3 newly isolates from 15 kV. rhizosphere soil samples (isolates 503-45, RM423 and RM452) putatively identified 3. RESULTS AND DISCUSSION as Pseudonocardia were grown on basal 3.1 PLA Film Degradation by Pseudono- medium containing (Per liter): 1 g yeast cardia alni AS4.1531T
extract, 4 g (NH4)2SO4, 2 g K2HPO4, 0.5 g Among the 20 strains of Pseudonocardia T MgSO4 × 7H2O, 2 g KH2PO4 (pH7.0) [13]. tested only Pseudonocardia alni AS4.1531 could
128-132Wasu.pmd 129 10/2/2555, 9:06 130 Chiang Mai J. Sci. 2012; 39(1)
degrade PLA when grow in liquid basal growth (Figure 1). P. alni AS4.1531T medium containing 0.1% (w/v) gelatin. This degraded 35.8 mg out of 50 mg film (71.5%) observation suggested that PLA degrading within 8 days. PLA degradation by P. alni ability was not widely distributed in this AS4.1531T was faster than previously genus unlike their taxonomically related reported Amycolatopsis strains. For taxa Amycolatopsis. Several Amycolatopsis instance, Amycolatopsis strain HT32 strains were reported as PLA degrader [1, 10, degraded 60% of PLA film within 14 days 11, 15, 16, 17]. However, PLA-degrading [10] or Amycolatopsis strain No. 3118 microorganisms were reported to be not degraded 50% of PLA film in 8 days [16]. widely distributed in natural environment A significant cell growth as indicated by [15, 18]. Similar effect of gelatin on the high dry cell weight (50.5 mg) suggested induction of PLA degrading activity was also that P. alni AS4.1531T could assimilate the observed in other actinomycetes, Saccharothrix degradation products (Figure 1). Most of waywayandensis [3] and Kibdelosporangium aridum the PLA-degrading actinomycetes were [19]. PLA degrading ability was also reported found to assimilate the degradation to increase by other protein eg. silk fibroin products [1, 2]. The degradation products [1]. could be used by these strains for their
To study the degradation of PLA and growth and eventually metabolized to CO2
assimilation of PLA by Pseudonocardia alni and H2O [15]. The pH increased during AS4.1531T, the time course of PLA film 4 days of cultivation, generally due to the degradation was further investigated by ammonium ions formation by microbial culturing this strain in liquid basal medium metabolisms of gelatin. Thereafter, the containing 50 mg PLA film and 0.1% (w/ amount of lactic acid increased as a result v) gelatin. After 4 days of cultivation, of PLA film degradation and accumulated degradation was evident as the film sample in the culture broth leading to a decreased was disintegrated with an increase in cell pH value (Figure 1). pH Film weight, dry cell weight, Lactic acid (mg)
Culture time (d)
Figure 1. PLA degradation by Pseudonocardia alni AS4.1531T in liquid culture containing 0.1% (w/v) gelatin.
128-132Wasu.pmd 130 10/2/2555, 9:06 Chiang Mai J. Sci. 2012; 39(1) 131
3.2 SEM observation of PLA films holes were observed within the early period SEM was used to observe the changes of degradation. In contrast, the surface of the remaining PLA film after 4 days of film remained smooth in the control of cultivation with the strain. As show in without inoculation of the tested strain Figure 2B, all over the surface of film (Figure 2A). sample became rough and many irregular
A. B.
Figure 2. Scanning electron micrographs of PLA films (magnification x 1,000); (A) film control without inoculation and (B) after cultivation with Pseudonocardia alni AS4.1531T for 4 days at 30°C (bar = 10 μm).
4. CONCLUSION REFERENCES Only 1 of the 17 Pseudonocardia reference strains was found to degrade PLA. [1] Pranamuda H. and Tokiwa Y., Pseudonocardia alni AS4.1531T could Degradation of poly(L-lactide) by strains belonging to genus Amycola- degrade 71.5 % of PLA within 8 days in topsis, Biotechnol. Letts., 1999; 21: 901- liquid basal medium containing 0.1% (w/ 905. v) gelatin. The information obtained from this study provides further evidence that [2] Teeraphatpornchai T., Nakajima A. T., Shigeno A.Y., Nakayama M., members of the family Pseudonocardiaceae Nomura N., Nakahara T. and are good source for PLA degraders. PLA Uchiyama H., Isolation and characteri- degradation by Pseudonocardia alni zation of a bacterium that degrades T AS4.1531 under extreme condition such various polyester-based biodegradable as high salt concentration is under plastics, Biotechnol. Letts., 2003; 25: investigation in our laboratory. 23-28. [3] Jarerat A. and Tokiwa Y., Poly (L- ACKNOWLEDGEMENTS lactide) degradation by Saccharothrix We wish to thank the National Research waywayandensis, Biotechnol. Letts., University Project under Thailand’s Office 2003; 25: 401-404. of the Higher Education Commission for [4] Lunt J., Large-scale production, financial support. Maytiya is also grateful properties and commercial application for support from the Graduate School, of polylactic acid polymer, Polym. Chiang Mai University. Degrad. Stabil., 1997; 59: 145-152.
128-132Wasu.pmd 131 10/2/2555, 9:06 132 Chiang Mai J. Sci. 2012; 39(1)
[5] William D. F., Enzymatic hydrolysis [13] Kim N.M., Kim G.W., Weon Y.H. and of polylactic acid, Eng. Med., 1981; 10: Lee H.S., Poly (L-lactide)-degrading 5-7. activity of newly isolated bacterium, J. Appl. Polymer Sci., 2008; 109: [6] Torres A., Li S. M., Roussos S. and 234-239. Vert M., Screening of microorganism for biodegradation of poly(lactic acid) [14] AOAC. 1998. Official Method of and lactic acid-containing polymer, Analysis. 16th ed.-Rev. 4. Association Appl. Environ. Microbiol., 1996; 62: of Official Analytical Chemists 2393-2397. International, Maryland. [7] Sakai K., Kawano H., Nakamura M. [15] Tokiwa Y. and Calabia B.P., Biodegrad- and Moriguchi M., Isolation of a ability and biodegradation of poly thermophilic poly-L-lacide degrading (lactide), Appl. Microbiol. Biotechnol., bacterium from compost and its 2006; 72: 244-251. enzymatic characterization, J. Biosci. [16] Ikura Y. and Kudo T., Isolation of a Bioeng., 2001; 92: 298-300. microorganism capable of degrading [8] Tomita K., Kuroki Y. and Nakai K., poly-(L-lactide), J. Gen. Appl. Microbiol. Isolation of thermophiles degrading 1999; 45: 247-251. poly(L-lactic acid), J. Biosci. Bioeng., [17] Chomchoei, A., Pathom-aree, W., 1999; 87: 752-755. Yokota, T., Kanongnuch C. and S. [9] Tokiwa Y. and Pranamuda H., Lumyong., Amycolatopsis thailandensis Microbial degradation of aliphatic sp. nov., a poly(L-lactic acid)-degrading polymer, In: Doi Y, Steinbuchel A, actinomycete, isolated from soil. eds, Biopolymer., 2001; 3: 85-103. Int. J. Syst. Evol. Microbiol. 2011; 61: 839-843. [10] Pranamuda H., Tokiwa Y. and Tanaka H., Polylactide degrading by an [18] Suyama T., Tokiwa Y., Ouichanpagdee Amycolatopsis sp, Appl. Environ. P., Kanagawa T. and Kamagata Y., Microbiol., 1997; 63: 1637-1640. Phylogenetic affiliation of soil bacteria that degrade aliphatic polyesters [11] Jarerat A., Pranamuda H. and Tokiwa available commercially as biodegrad- Y., Poly(L-lactide) degrading activity able plastics, Appl. Environ. Microbiol., in various actinomycetes, Macromol. 1998; 64: 5008-5011. Biosci., 2002; 2: 420-428. [19] Jarerat A., Tokiwa Y. and Tanaka [12] Labeda D.P., Goodfellow M., Chun H., Poly(L-lactide) degradation by J., Zhi Y.X. and Li W.J., Reassessment Kibdelosporangium aridum, Biotechnol. of the systematic of the suborder Letts., 2003; 25: 2035-2038. Pseudonocardineae : transfer of the genera within the family Actinosyn- nemataceae Labeda and Kroppenstedt 2000 emend. Zhi et al. 2009 into an emended family Pseudonorcardiaceae Embley et al. 1989 emend. Zhi et al. 2009, Int. J. Syst. Evol. Microbiol. 2011; 61:1259-1264.
128-132Wasu.pmd 132 10/2/2555, 9:06