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Biodegradation of poly(l -lactide) (PLA) exposed to UV irradiation by a mesophilic bacterium
International Biodeterioration & Biodegradation 85 (2013) 289e293
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Biodegradation of poly(L-lactide) (PLA) exposed to UV irradiation by a mesophilic bacterium Hyun Jeong Jeon, Mal Nam Kim* Department of Life Science, Sangmyung University, Seoul 110-743, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 July 2013 Received in revised form 23 August 2013 Accepted 24 August 2013 Available online 17 September 2013
Degradation of PLA was examined with a mesophilic bacterium, which was identified as Stenotrophomonas maltophilia LB 2-3. The PLA degradation activity of S. maltophilia LB 2-3 was accessed using the modified Sturm test. PLA lost molecular weight and tensile properties quickly when exposed to UV irradiation. The biodegradability of PLA was enhanced as UV irradiation was increased to 8 h and then decreased with a further increase in UV irradiation. This contrasted with the general expectation that longer UV exposure would lower the molecular weight of PLA and, thus, increase biodegradability. The same behavior was also observed when PLA degradation was carried out in compost. A brittle white solid which may be poorly assimilated by microorganisms was formed during UV irradiation and was thought to be at least, in part, responsible for the biodegradability behavior. The GPC chromatogram of PLA powder suspended in mineral medium with S. maltophilia LB 2-3 was higher in intensity than that without the strain in the low molecular weight region, but at the same time, the reverse was true in the high molecular weight zone, revealing that the cleavage of PLA molecules was accelerated by enzymes secreted by S. maltophilia LB 2-3. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: PLA Biodegradation Stenotrophomonas maltophilia LB 2-3 UV exposure
1. Introduction Poly(L-lactide) (PLA) is one of the most economically competitive and environmentally benign polymers. Its production does not increase greenhouse gases as much as other fossil resource-based plastics, and it is classified as a renewable material, so PLA is currently receiving considerable attention not only for single-use applications such as packaging but also for more durable applications such as car interior parts, textile fibers, flooring materials, and wallpaper. Therefore, the use of PLA is expected to increase greatly in the future (Sangwan and Wu, 2008). Therefore, its degradation should be studied comprehensively to cope with potential widespread use of this polymer, which may lead to environment contamination due to discharge of a huge amount of waste. PLA is a biodegradable polymer. However, the complete disappearance of PLA in a natural environment may take several years (Kimura, 1999), because only a limited number of microorganisms are capable of degrading PLA. Suyama et al. (1998) found that 39 bacterial strains in the classes Firmicutes and Proteobacteria isolated from soil degrade aliphatic polyesters such as PHB, PCL, and PBS, but none are capable of degrading PLA (Tokiwa and Calabia, 2006). * Corresponding author. Tel.: þ82 2 2287 5150; fax: þ82 2 287 0070. E-mail address: [email protected] (M.N. Kim). 0964-8305/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2013.08.013
The first reported PLA degrader is Amycolatopsis sp. isolated from soil by Pranamuda et al. (1997). As summarized by Tokiwa and Calabia (2006), PLA degraders isolated so far are Amycolatopsis sp., Cryptococcus sp., Lentzea waywayandensis, Kibdelosporangium aridum, Tritirachium album, Bacillus brevis, Bacillus sinithii, Bacillus stearothermophilus, Geobacillus thermocatenulatus and Paenibacillus amylolyticus. Recently Bacillus licheniformis and Pseudomonas sp. were isolated for PLA degradation by Van et al. (2012) and Wang et al. (2011) from soil and activated sludge, respectively. In the present study, a new mesophilic PLA degrader was isolated and the biological degradation of PLA was examined with the isolated strain by minimizing concomitant abiotic degradation such as hydrolysis. Biodegradation of PLA exposed to UV irradiation as well as intact PLA was examined in mineral medium and in compost sterilized prior to inoculation with the isolated strain. 2. Materials and methods 2.1. Materials A 0.3 mm thick PLA sheet, which was made of NatureWorksÒ PLA Polymer 2003D, was donated by Green Chemical Co. (Seoul,
290
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Korea). Because biological degradation of PLA may be affected by a releasing agent such as silicone, which is usually sprayed on commercial PLA sheets, neat PLA powder with about 50 mm in diameter was prepared by grinding NatureWorksÒ PLA Polymer 4032D pellets. 2.2. Isolation of the PLA degrading strain Compost top-dressed for pear trees was harvested from a farm field in Siheung City, Korea, as a microbial source to isolate the PLA degrading strains. The compost sample (10 g) was suspended in 100 ml sterilized distilled water for 10 min and then settled for 30 min. The supernatant (0.5 ml) was inoculated into 20 ml of the enrichment medium with 0.1 g of PLA powder followed by incubation at 37 C, 120 rpm for 7 days in a rotary shaker. The enrichment medium was prepared according to the method proposed by Hadad et al. (2005) with slight modifications. The composition of the enrichment medium in g L1 was as follows: K2HPO4, 2.34; KH2PO4, 1.33; MgSO4$7H2O, 0.2; (NH4)2SO4, 1; NaCl, 0.5; yeast extract, 0.06; and 1 ml of trace element solution per l L of distilled water (pH 7.0). The trace element solution contained 11.9 mg of CoCl2, 11.8 mg of NiCl2, 6.3 mg of CrCl2, 15.7 mg of CuSO4, 0.97 g of FeCl3, 0.78 g of CaCl2, and 10.0 mg of MnCl2 per l L of distilled deionized water. The subcultures were made five times by inoculating 0.5 ml of the original culture into fresh medium with PLA as a carbon source. After subculturing, the enrichment culture broth was spread on agar plates containing 0.1% PLA and then cultured at 37 C for 7 days. Strains forming clear zones on the agar plate were selected as PLA degrading strains. Agar plates containing 0.1% PLA were made by dissolving 1 g PLA in 40 ml dichloromethane (Samchun, Seoul, Korea). The solution was added to 1 L of enrichment medium with 1 ml of trace element solution and then dispersed using an ultra-sonicator. The emulsified medium was mixed with 15 g of agar (Samchun). 2.3. Identification of PLA degrading strains The isolated strain was identified through 16S rRNA amplification using the universal primers 518F (50-CCAGCAGCCGCGG TAATACG-30) and 800R (50-TACCAGGGTATCTAATCC-30). Sequence analyses were carried out at Macrogen (Seoul, Korea) using the amplified PCR products. The sequences were aligned with Ez Taxon together with those of genera possessing sequences similar to those of the isolated strain using the CLUSTAL W program (Thompson et al., 1994). The phylogenetic tree was inferred with the neighbor-joining method using MEGA version 5.0 (Kumar et al., 2004). 2.4. Sturm test Biodegradation of PLA by the isolated strain at 37 C was monitored by the modified Sturm test using the facility assembled according to ASTM D 5209-91(1991). 2.5. Biodegradability test in compost The biodegradation tests in compost were carried out according to KS M 3100-1:2002; MOD ISO 14855:1999 at 37 C. The isolated strain was incubated in nutrient broth at 37 C for 24 h. Compost made from animal fodder was used as the medium for the tests. The culture broth (1 ml) was inoculated into compost previously sterilized at 121 C for 30 min in an autoclave followed by drying at 100 C for 24 h. During the biodegradation tests, the air flow rate was controlled at 40 ml min1.
2.6. Measurements Average molecular weight and molecular weight distribution were measured by using a size exclusion chromatography (Waters 2414, Milford, MA, USA) equipped with a refractive index, ultraviolet detectors, and four linear columns packed with m-Styragel. The flow rate of THF was 1.0 ml min1, and the columns were calibrated with polystyrene standards. Tensile properties were measured using a universal testing machine (Hounsfield Co. H 10KS-0061, Horsham, PA, USA). Specimens were prepared according to ASTM D638 type 5. Cross-head speed was 10 mm min1. The results of at least five measurements were averaged. Fourier transformed infrared spectra were recorded on a PerkineElmer Spectrum 2000 spectrometer (Waltham, MA, USA) over a wave number range of 4000e400 cm1. UV intensity was measured by the UV-MO3A UV light measuring device (ORC Manufacturing, Tokyo, Japan) at 254 nm and with a UV power meter C8026 (Hamamatsu Photonics K.K, Tokyo, Japan) at 185 nm. 1H nuclear magnetic resonance spectra were obtained using Varian Inova 400 NMR (Santa Clara, CA, USA). 3. Results and discussion 3.1. Isolation of the PLA-degrading mesophilic bacterium A mesophilic bacterium capable of PLA degradation was isolated from a compost sample harvested from a pear farm field in Siheung City, Korea. The isolated strain was a Gram negative rod-shaped bacterium. The biochemical and physiological characteristics of the isolated strain were similar to the API 20NE test results reported by Urszula et al. (2009) and Johnson et al. (2003) who isolated Stenotrophomonas maltophilia from activated sludge and animals to treat different aromatic substrates and to examine strain pathogenicity, respectively. The 16S rRNA gene sequence analyses revealed that the isolated strain possessed 99.59% similarity to Stenotrophomonas maltophilia ATCC 13637. The phylogenetic tree was drawn based on the 16S rRNA coding gene sequence analysis and the nearest relatives. From the results, it was reasonable to identify the present isolated strain as Stenotrophomonas maltophilia LB 2-3. S. maltophilia is a bacterium frequently detected in moist habitats such as plants, animals and food, and is an opportunistic pathogen causing respiratory disease in humans (Looney et al., 2009; Brooke, 2012). It is powerful for bio-treating wastewater and soil contaminated by aromatic compounds (Urszula et al., 2009). Only a limited number of microbial PLA-degraders have been isolated so far, as summarized in Table 1. Most of the isolated strains are thermophilic. Because PLA is degraded in the natural environment mostly by mesophilic bacteria, they are more useful than thermophilic strains for bioremediation of waste PLA. In this
Table 1 Microorganisms capable of PLA degradation as reported in the literature. Strains
Substrate
Bacillus licheniformis Pseudomonas sp. Geobacillus thermocatenulatus Paenibacillus amylolyticus
PLA powder Soil sample Van et al., 2012 PLA film Activated sludge Wang et al., 2011 PLA film Soil sample Tomita et al., 2004 DL-PLA
Bacillus stearothermophilus D-PLA film Bacillus smithii, L-PLA Bacillus brevis L-PLA film
Source
Soil sample Soil sample. Compost Soil Sample
References
Akutsu-Shigeno et al., 2003 Tomita et al., 2003 Sakai et al., 2001 Tomita et al., 1999
H.J. Jeon, M.N. Kim / International Biodeterioration & Biodegradation 85 (2013) 289e293 Table 2 Variations in the molecular weight of PLAa powder suspended in mineral medium at 37 C with and without inoculation with Stenotrophomonas maltophilia LB 2-3.
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Table 4 Variations in the physical properties of PLAa as a function of ultraviolet irradiation time.
Sample
Mn
Mw
Sample
Mn
Mw
PLA-0 day PLA-10 days PLA-20 days PLA-30 days PLA-40 days PLA-10 days-LB PLA-20 days-LB PLA-30 days-LB PLA-40 days-LB
86,500 87,000 84,700 83,200 80,500 85,400 84,000 81,100 73,900
198,200 195,900 188,900 185,000 184,100 193,700 187,900 182,700 173,200
Contact angle ( )
Tensile strength (kgf cm2)
Strain at break (%)
PLA-0 h PLA-4 h PLA-8 h PLA-12 h PLA-16 h PLA-21 h PLA-24 h
59,400 54,000 45,400 33,700 29,500 22,400 18,700
188,700 122,700 97,800 74,600 66,300 52,800 41,400
84.5 78.5 77.4 76.2 75.5 74.3 73.9
631 617 538 115 e e e
25.1 12.8 8.4 4.2 e e e
a
2-3 2-3 2-3 2-3
PLA: ‘NatureWorksÒ PLA Polymer 402D’.
a PLA sheets with 0.3 mm thickness were made of ‘NatureWorksÒPLA Polymer 2003D’.
regard, the mesophilic bacterium, S. maltophilia, may be used successfully for this purpose. 3.2. Biological degradation of PLA Conflicting results have been reported by different researchers concerning the mechanism of PLA degradation. Some studies have claimed that PLA is degraded entirely by abiotic processes, whereas others have argued that microorganisms or enzymes play a vital role in PLA degradation (Suyama et al., 1998). It is generally agreed that abiotic hydrolysis of PLA proceeds first until the molecular weight of PLA decreases sufficiently to be assimilated by microorganisms (Suyama et al., 1998; Tokiwa and Calabia, 2006). Table 2 compares the variations in the molecular weight of PLA powder suspended in a mineral medium at 37 C with and without S. maltophilia LB 2-3. The molecular weight of PLA decreased monotonously with increasing incubation time in the absence of S. maltophilia LB 2-3, but decreased faster in the presence of the strain (Table 2). The GPC profiles of neat PLA, PLA-40 days, and PLA-40 days-LB 2-3 were all monodisperse. Notably, the GPC chromatogram of PLA40 days-LB 2-3 (PLA powder suspended in mineral medium for 40 days in the presence of the PLA degrader) compared to that of PLA40 days (PLA powder suspended in the mineral medium for 40 days in the absence of the PLA degrader) was higher in intensity at a longer elution time (lower molecular weight region). But at the same time, the reverse was true at a shorter elution time, clearly revealing that the PLA molecules were cleaved not only by abiotic hydrolysis but also that cleavage was accelerated by enzymes secreted from S. maltophilia LB 2-3.
strongly on the wavelength of irradiated light, we fabricated a UV lamp emitting light with similar intensity at 245 nm to that of sunlight. The UV chamber was equipped with four UV lamps emitting 6.41 103 mW cm2 and 3.22 mW cm2 of intensity at 185 nm and 245 nm, respectively. The intensity of sunlight at 245 nm was 0.19e2.55 mW cm2 in October 2012, in Seoul, Korea, depending on the time of measurement and cloudiness but that at 185 nm was zero indicating that the light at 185 nm was completely absorbed by the atmosphere. We attempted to block the light at 185 nm from our UV source by shielding the specimens with polypropylene (PP) films because PP film was reported by Ojeda et al. (2009) to effectively block UV-C but allow UV-A and UV-B to pass through. However, even after shielding with 10 layers of PP 50 mm film, the transmitted light at 185 nm was as intense as one fourth of 6.41 103 mW cm2, revealing that complete blockage of UV at 185 nm by PP film was infeasible. When UV intensity was 6.41 103 mW cm2 and 3.22 mW cm2 at 185 nm and 245 nm, respectively, the molecular weight decreased from 188,700 to 41,400, and the contact angle decreased from 84.5 to 73.9 as the PLA sheet was UV irradiated for 24 h (Table 4), indicating that PLA became more hydrophilic as a result of UV exposure. The tensile properties became sharply worse, and thereby became impossible to measure as soon as UV irradiation time exceeded 12 h. Fig. 1 shows the GPC chromatograms of the PLA sheets before and after UV exposure. The peak at short elution times decreased in intensity quickly and moved to a longer elution time indicating that the long PLA chains were fragmented by UV irradiation. 3.4. Biodegradation of PLA before and after UV exposure
3.3. UV degradation of PLA PLA sheets were exposed to UV irradiation at room temperature. UV-photo-degradation of plastics has mostly been carried out with UV wavelengths >300 nm to imitate photo-degradation by sunlight. However, as shown in Table 3, the intensity of sunlight was still strong at 245 nm. Because photochemical reactions depend
Degradation of PLA was examined in the modified Sturm test apparatus set up according to ASTM D 5209-91 by measuring the net amount of CO2 evolved from mineral medium loaded with PLA. A 500 ml aliquot of S. maltophilia LB 2-3 cells was inoculated into 100 ml of stock solution. Fig. 2 shows the modified Sturm test results for the PLA exposed to UV irradiation for different times. It was
Table 3 Ultraviolet intensity in mW cm2 of sunlight at 185 nm and at 245 nm measured in Seoul, Korea. Time
Date Oct. 10, 2012
9 am 12 pm 3 pm
Oct. 11, 2012
Oct. 12, 2012
185 nm
245 nm
185 nm
245 nm
185 nm
245 nm
0 0 0
0.190 0.005 0.655 0.064 2.107 0.086
0 0 0
2.503 0.034 2.536 0.011 2.540 0.002
0 0 0
2.448 0.007 2.517 0.005 2.510 0.003
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Fig. 1. GPC profiles of PLA exposed to ultraviolet irradiation for different times.
generally expected that the longer the exposure, the lower the molecular weight of PLA and, thus, the higher the biodegradability. However, the apparent biodegradability of PLA reached a maximum at 8 h of UV exposure and then decreased with further increase in UV exposure (Fig. 2). We replicated the experiments three times and obtained the same conclusion. Here, the apparent biodegradability was defined as follows: Apparent biodegradability ¼ (A B)D1where A is the accumulated amount of CO2 evolved from the suspension loaded with PLA, and B is the accumulated amount of CO2 evolved from the suspension without PLA. D, 1.83 g of CO2/g of PLA, is the amount of CO2 evolved when all the carbons in the PLA samples were assumed to be mineralized into CO2 (Jeon and Kim, 2013). Apparent biodegradability is used here, because all the PLA carbons may not be consumed entirely as CO2 but part of them may be locked up in the biomass or in other metabolites (Gu et al., 1993a,b). Fig. 3 shows the PLA biodegradability measured in the compost at 37 C under controlled conditions according to KS M3100-1,
Fig. 3. Biodegradability of PLA measured in compost under controlled conditions according to KS M3100-1, 2002; MOD ISO 14855, 1999.
2002; MOD ISO 14855 (1999). PLA biodegradability was higher in Fig. 3 than that in Fig. 2, indicating more favorable conditions for biodegradation in the compost than in mineral medium. The evolution of CO2 from PLA buried in the sterilized compost was negligible, but PLA was transformed into CO2 at a considerable rate even at 37 C when the compost was inoculated with S. maltophilia LB 2-3. Gu et al. (1992) also observed that weight loss of PLA occurred much faster in a compost as compared to when the compost was sterilized or poisoned by KCN. Hence it can be concluded that the presence of S. maltophilia LB 2-3 accelerated PLA degradation. The results in Fig. 3 demonstrate again that the apparent biodegradability of PLA was the highest at 8 h of UV exposure. We do not have a clear answer for this biodegradation behavior of PLA. One plausible explanation is that a Norrish type reaction as shown in Fig. 4 may transform PLA into a structure more recalcitrant to biodegradation. However, as demonstrated in Fig. 5, the peaks at w3.6 ppm, which should appear if the Norrish type reaction occurred, did not appear in the 1H nuclear magnetic resonance spectrum of PLA exposed to UV irradiation for 24 h. UV irradiation may transform PLA into a brittle white solid which may be poorly assimilated by microorganisms. Because apparent biodegradability is determined based on the assumption that all carbon in the test specimen is metabolized to CO2 by microorganisms, the formation of the poorly assimilable solid should contribute to the biodegradability behavior of the PLA exposed to UV for longer than 8 h. Acknowledgments This study was supported by a 2012 Research Grant from Sangmyung University.
Fig. 2. Modified Sturm test results for PLAs exposed to ultraviolet irradiation for different times.
Fig. 4. Plausible Norrish type reaction of PLA during ultraviolet irradiation.
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293
Fig. 5. 1H nuclear magnetic resonance spectra of PLA exposed to ultraviolet irradiation for 0 and 24 h.
References Akutsu-Shigeno, Y., Teeraphatpornchai, T., Teamitisong, K., Nomura, N., Uchiyama, H., Nakahara, T., Nakajima-Kambe, T., 2003. Cloning and sequencing of a poly(dl-Lactic Acid) depolymerase gene from Paenibacillus amylolyticus strain TB-13 and its functional expression in Escherichia coli. Appl. Environ. Microbiol. 69, 2498e2504. ASTM D 5209e91, 1991. Annual Book of ASTM Standards, vol. 08.03. American Society for Testing and Materials, Philadelphia, U.S.A. Brooke, J.S., 2012. Stenotrophomonas maltophilia: an emerging global opportunistic pathogen. Clin. Microbiol. Rev. 25, 2e41. Gu, J.D., Gada, M., Kharas, G., Eberiel, D., McCarthy, S.P., Gross, R.A., 1992. Degradability of cellulose acetate and polylactide in simulated composting bioreactors. Polym. Mat. Sci. Eng., Proc. ACS Div. Polym. Mat. Sci. Eng. 67, 351e352. Gu, J.D., Coulter, S., Eberiel, D., McCarthy, S.P., Gross, R.A., 1993a. A respirometric method to measure mineralization of polymeric materials in a matured compost environment. J. Environ. Polym. Degrad. 1, 293e299. Gu, J.D., Eberiel, D., McCarthy, S.P., Gross, R.A., 1993b. Cellulose acetate biodegradability upon exposure to simulated aerobic composting and anaerobic bioreactor environments. J. Environ. Polym. Degrad. 1, 143e153. Hadad, D., Geresh, S., Sivan, A., 2005. Biodegradation of polyethylene by the thermophilic bacterium Brevibacillus borstelensis. J. Appl. Microbiol. 98, 1093e1100. Jeon, H.J., Kim, M.N., 2013. Isolation of a thermophilic bacterium capable of lowmolecular-weight polyethylene degradation. Biodegradation 24, 89e98. Johnson, E.H., Al-Busaidy, R., Hameed, M.S., 2003. An outbreak of Lymphadenitis associated with Stenotrophomonas (Xanthomonas) maltophilia in Omani Goats. J. Vet. Med. 50, 102e104. Kimura, T., 1999. High speed degradation of biodegradable plastics by composting of biological wastes. Biosci. Ind. 57, 35e36. Kumar, S., Tamura, K., Nei, M., 2004. Mega 3: integrated software for molecular evolutionary genetic analysis and sequence alignment. Brief. Bioinform. 5, 150e163. Looney, W.J., Narita, M., Mühlemann, K., 2009. Stenotrophomonas maltophilia: an emerging opportunist human pathogen. Lancet Infect. Dis. 9, 312e323. KS M3100-1:2002; MOD ISO 14855, 1999. Determination of the Ultimate Aerobic Biodegradability and Disintegration of Plastic Materials under Controlled Composting Conditions Part 1: Analysis of Evolved Carbon Dioxide by Titration Method. Korean Agency for Technology and Standards, Seoul, South Korea.
Ojeda, T.F.M., Dalmolin, E., Forte, M.M.C., Jacques, R.J.S., Bento, F.M., Camargo, F.A.O., 2009. Abiotic and biotic degradation of oxo-biodegradable polyethylenes. Polym. Degrad. Stabil. 94, 965e970. Pranamuda, H., Tokiwa, Y., Tanaka, H., 1997. Polylactide degradation by an Amycolatopsis sp. Appl. Environ. Microbiol. 63, 1637e1640. Sakai, K., Kawano, H., Iwami, A., Nakamura, M., Moriguchi, M., 2001. Isolation of a thermophilic poly-L-lactide degrading bacterium from compost and its enzymatic characterization. J. Biosci. Bioeng. 92, 298e300. Sangwan, P., Wu, D.Y., 2008. New insights into polylactide biodegradation from molecular ecological techniques. Macromol. Biosci. 8, 304e315. Suyama, T., Tokiwa, Y., Ouichanpagdee, P., Kanagawa, T., Kamagata, Y., 1998. Phylogenetic affiliation of soil bacteria that degrade aliphatic polyesters available commercially as biodegradable plastics. Appl. Environ. Microbiol. 64, 5008e5011. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids. Res. 22, 4673e4680. Tokiwa, Y., Calabia, B.P., 2006. Biodegradability and biodegradation of poly(lactide). Appl. Microbiol. Biotechnol. 72, 244e251. Tomita, K., Kuroki, Y., Nagai, K., 1999. Isolation of thermophiles degrading poly(Llactic acid). J. Biosci. Bioeng. 87, 752e755. Tomita, K., Tsuji, H., Nakajima, T., Kikuchi, Y., Ikarashi, K., Ikeda, N., 2003. Degradation of poly(D-lactic acid) by a thermophile. Polym. Degrad. Stabil. 81, 167e171. Tomita, K., Nakajima, T., Kikuchi, Y., Miwa, N., 2004. Degradation of poly(L-lactic acid) by a newly isolated thermophile. Polym. Degrad. Stabil. 84, 433e438. Urszula, G., Izabela, G., Danuta, W.S., Sylwia, A.E., 2009. Isolation and characterization of a novel strain of stenotrophomonas maltophilia possessing various dioxygenases for monocyclic hydrocarbon degradation. Braz. J. Microbiol. 40, 285e291. Van, L.H., Yen, P.B., Huy, N.Q., 2012. Polylactic acid biodegradability study of a thermophilic bacterium isolated in Vietnam. J. Eng. Technol. Educ., 104e110. The 2012 International Conference on Green Technology and Sustainable Development (GTSD2012). Wang, Z., Wang, Y., Guo, Z., Li, F., Chen, S., 2011. Purification and characterization of poly(L-lactic acid) depolymerase from Pseudomonas sp. strain DS04-T. Polym. Eng. Sci. 51, 454e459.
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Biodegradation of poly(L-lactide) (PLA) exposed to UV irradiation by a mesophilic bacterium Hyun Jeong Jeon, Mal Nam Kim* Department of Life Science, Sangmyung University, Seoul 110-743, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 July 2013 Received in revised form 23 August 2013 Accepted 24 August 2013 Available online 17 September 2013
Degradation of PLA was examined with a mesophilic bacterium, which was identified as Stenotrophomonas maltophilia LB 2-3. The PLA degradation activity of S. maltophilia LB 2-3 was accessed using the modified Sturm test. PLA lost molecular weight and tensile properties quickly when exposed to UV irradiation. The biodegradability of PLA was enhanced as UV irradiation was increased to 8 h and then decreased with a further increase in UV irradiation. This contrasted with the general expectation that longer UV exposure would lower the molecular weight of PLA and, thus, increase biodegradability. The same behavior was also observed when PLA degradation was carried out in compost. A brittle white solid which may be poorly assimilated by microorganisms was formed during UV irradiation and was thought to be at least, in part, responsible for the biodegradability behavior. The GPC chromatogram of PLA powder suspended in mineral medium with S. maltophilia LB 2-3 was higher in intensity than that without the strain in the low molecular weight region, but at the same time, the reverse was true in the high molecular weight zone, revealing that the cleavage of PLA molecules was accelerated by enzymes secreted by S. maltophilia LB 2-3. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: PLA Biodegradation Stenotrophomonas maltophilia LB 2-3 UV exposure
1. Introduction Poly(L-lactide) (PLA) is one of the most economically competitive and environmentally benign polymers. Its production does not increase greenhouse gases as much as other fossil resource-based plastics, and it is classified as a renewable material, so PLA is currently receiving considerable attention not only for single-use applications such as packaging but also for more durable applications such as car interior parts, textile fibers, flooring materials, and wallpaper. Therefore, the use of PLA is expected to increase greatly in the future (Sangwan and Wu, 2008). Therefore, its degradation should be studied comprehensively to cope with potential widespread use of this polymer, which may lead to environment contamination due to discharge of a huge amount of waste. PLA is a biodegradable polymer. However, the complete disappearance of PLA in a natural environment may take several years (Kimura, 1999), because only a limited number of microorganisms are capable of degrading PLA. Suyama et al. (1998) found that 39 bacterial strains in the classes Firmicutes and Proteobacteria isolated from soil degrade aliphatic polyesters such as PHB, PCL, and PBS, but none are capable of degrading PLA (Tokiwa and Calabia, 2006). * Corresponding author. Tel.: þ82 2 2287 5150; fax: þ82 2 287 0070. E-mail address: [email protected] (M.N. Kim). 0964-8305/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2013.08.013
The first reported PLA degrader is Amycolatopsis sp. isolated from soil by Pranamuda et al. (1997). As summarized by Tokiwa and Calabia (2006), PLA degraders isolated so far are Amycolatopsis sp., Cryptococcus sp., Lentzea waywayandensis, Kibdelosporangium aridum, Tritirachium album, Bacillus brevis, Bacillus sinithii, Bacillus stearothermophilus, Geobacillus thermocatenulatus and Paenibacillus amylolyticus. Recently Bacillus licheniformis and Pseudomonas sp. were isolated for PLA degradation by Van et al. (2012) and Wang et al. (2011) from soil and activated sludge, respectively. In the present study, a new mesophilic PLA degrader was isolated and the biological degradation of PLA was examined with the isolated strain by minimizing concomitant abiotic degradation such as hydrolysis. Biodegradation of PLA exposed to UV irradiation as well as intact PLA was examined in mineral medium and in compost sterilized prior to inoculation with the isolated strain. 2. Materials and methods 2.1. Materials A 0.3 mm thick PLA sheet, which was made of NatureWorksÒ PLA Polymer 2003D, was donated by Green Chemical Co. (Seoul,
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Korea). Because biological degradation of PLA may be affected by a releasing agent such as silicone, which is usually sprayed on commercial PLA sheets, neat PLA powder with about 50 mm in diameter was prepared by grinding NatureWorksÒ PLA Polymer 4032D pellets. 2.2. Isolation of the PLA degrading strain Compost top-dressed for pear trees was harvested from a farm field in Siheung City, Korea, as a microbial source to isolate the PLA degrading strains. The compost sample (10 g) was suspended in 100 ml sterilized distilled water for 10 min and then settled for 30 min. The supernatant (0.5 ml) was inoculated into 20 ml of the enrichment medium with 0.1 g of PLA powder followed by incubation at 37 C, 120 rpm for 7 days in a rotary shaker. The enrichment medium was prepared according to the method proposed by Hadad et al. (2005) with slight modifications. The composition of the enrichment medium in g L1 was as follows: K2HPO4, 2.34; KH2PO4, 1.33; MgSO4$7H2O, 0.2; (NH4)2SO4, 1; NaCl, 0.5; yeast extract, 0.06; and 1 ml of trace element solution per l L of distilled water (pH 7.0). The trace element solution contained 11.9 mg of CoCl2, 11.8 mg of NiCl2, 6.3 mg of CrCl2, 15.7 mg of CuSO4, 0.97 g of FeCl3, 0.78 g of CaCl2, and 10.0 mg of MnCl2 per l L of distilled deionized water. The subcultures were made five times by inoculating 0.5 ml of the original culture into fresh medium with PLA as a carbon source. After subculturing, the enrichment culture broth was spread on agar plates containing 0.1% PLA and then cultured at 37 C for 7 days. Strains forming clear zones on the agar plate were selected as PLA degrading strains. Agar plates containing 0.1% PLA were made by dissolving 1 g PLA in 40 ml dichloromethane (Samchun, Seoul, Korea). The solution was added to 1 L of enrichment medium with 1 ml of trace element solution and then dispersed using an ultra-sonicator. The emulsified medium was mixed with 15 g of agar (Samchun). 2.3. Identification of PLA degrading strains The isolated strain was identified through 16S rRNA amplification using the universal primers 518F (50-CCAGCAGCCGCGG TAATACG-30) and 800R (50-TACCAGGGTATCTAATCC-30). Sequence analyses were carried out at Macrogen (Seoul, Korea) using the amplified PCR products. The sequences were aligned with Ez Taxon together with those of genera possessing sequences similar to those of the isolated strain using the CLUSTAL W program (Thompson et al., 1994). The phylogenetic tree was inferred with the neighbor-joining method using MEGA version 5.0 (Kumar et al., 2004). 2.4. Sturm test Biodegradation of PLA by the isolated strain at 37 C was monitored by the modified Sturm test using the facility assembled according to ASTM D 5209-91(1991). 2.5. Biodegradability test in compost The biodegradation tests in compost were carried out according to KS M 3100-1:2002; MOD ISO 14855:1999 at 37 C. The isolated strain was incubated in nutrient broth at 37 C for 24 h. Compost made from animal fodder was used as the medium for the tests. The culture broth (1 ml) was inoculated into compost previously sterilized at 121 C for 30 min in an autoclave followed by drying at 100 C for 24 h. During the biodegradation tests, the air flow rate was controlled at 40 ml min1.
2.6. Measurements Average molecular weight and molecular weight distribution were measured by using a size exclusion chromatography (Waters 2414, Milford, MA, USA) equipped with a refractive index, ultraviolet detectors, and four linear columns packed with m-Styragel. The flow rate of THF was 1.0 ml min1, and the columns were calibrated with polystyrene standards. Tensile properties were measured using a universal testing machine (Hounsfield Co. H 10KS-0061, Horsham, PA, USA). Specimens were prepared according to ASTM D638 type 5. Cross-head speed was 10 mm min1. The results of at least five measurements were averaged. Fourier transformed infrared spectra were recorded on a PerkineElmer Spectrum 2000 spectrometer (Waltham, MA, USA) over a wave number range of 4000e400 cm1. UV intensity was measured by the UV-MO3A UV light measuring device (ORC Manufacturing, Tokyo, Japan) at 254 nm and with a UV power meter C8026 (Hamamatsu Photonics K.K, Tokyo, Japan) at 185 nm. 1H nuclear magnetic resonance spectra were obtained using Varian Inova 400 NMR (Santa Clara, CA, USA). 3. Results and discussion 3.1. Isolation of the PLA-degrading mesophilic bacterium A mesophilic bacterium capable of PLA degradation was isolated from a compost sample harvested from a pear farm field in Siheung City, Korea. The isolated strain was a Gram negative rod-shaped bacterium. The biochemical and physiological characteristics of the isolated strain were similar to the API 20NE test results reported by Urszula et al. (2009) and Johnson et al. (2003) who isolated Stenotrophomonas maltophilia from activated sludge and animals to treat different aromatic substrates and to examine strain pathogenicity, respectively. The 16S rRNA gene sequence analyses revealed that the isolated strain possessed 99.59% similarity to Stenotrophomonas maltophilia ATCC 13637. The phylogenetic tree was drawn based on the 16S rRNA coding gene sequence analysis and the nearest relatives. From the results, it was reasonable to identify the present isolated strain as Stenotrophomonas maltophilia LB 2-3. S. maltophilia is a bacterium frequently detected in moist habitats such as plants, animals and food, and is an opportunistic pathogen causing respiratory disease in humans (Looney et al., 2009; Brooke, 2012). It is powerful for bio-treating wastewater and soil contaminated by aromatic compounds (Urszula et al., 2009). Only a limited number of microbial PLA-degraders have been isolated so far, as summarized in Table 1. Most of the isolated strains are thermophilic. Because PLA is degraded in the natural environment mostly by mesophilic bacteria, they are more useful than thermophilic strains for bioremediation of waste PLA. In this
Table 1 Microorganisms capable of PLA degradation as reported in the literature. Strains
Substrate
Bacillus licheniformis Pseudomonas sp. Geobacillus thermocatenulatus Paenibacillus amylolyticus
PLA powder Soil sample Van et al., 2012 PLA film Activated sludge Wang et al., 2011 PLA film Soil sample Tomita et al., 2004 DL-PLA
Bacillus stearothermophilus D-PLA film Bacillus smithii, L-PLA Bacillus brevis L-PLA film
Source
Soil sample Soil sample. Compost Soil Sample
References
Akutsu-Shigeno et al., 2003 Tomita et al., 2003 Sakai et al., 2001 Tomita et al., 1999
H.J. Jeon, M.N. Kim / International Biodeterioration & Biodegradation 85 (2013) 289e293 Table 2 Variations in the molecular weight of PLAa powder suspended in mineral medium at 37 C with and without inoculation with Stenotrophomonas maltophilia LB 2-3.
291
Table 4 Variations in the physical properties of PLAa as a function of ultraviolet irradiation time.
Sample
Mn
Mw
Sample
Mn
Mw
PLA-0 day PLA-10 days PLA-20 days PLA-30 days PLA-40 days PLA-10 days-LB PLA-20 days-LB PLA-30 days-LB PLA-40 days-LB
86,500 87,000 84,700 83,200 80,500 85,400 84,000 81,100 73,900
198,200 195,900 188,900 185,000 184,100 193,700 187,900 182,700 173,200
Contact angle ( )
Tensile strength (kgf cm2)
Strain at break (%)
PLA-0 h PLA-4 h PLA-8 h PLA-12 h PLA-16 h PLA-21 h PLA-24 h
59,400 54,000 45,400 33,700 29,500 22,400 18,700
188,700 122,700 97,800 74,600 66,300 52,800 41,400
84.5 78.5 77.4 76.2 75.5 74.3 73.9
631 617 538 115 e e e
25.1 12.8 8.4 4.2 e e e
a
2-3 2-3 2-3 2-3
PLA: ‘NatureWorksÒ PLA Polymer 402D’.
a PLA sheets with 0.3 mm thickness were made of ‘NatureWorksÒPLA Polymer 2003D’.
regard, the mesophilic bacterium, S. maltophilia, may be used successfully for this purpose. 3.2. Biological degradation of PLA Conflicting results have been reported by different researchers concerning the mechanism of PLA degradation. Some studies have claimed that PLA is degraded entirely by abiotic processes, whereas others have argued that microorganisms or enzymes play a vital role in PLA degradation (Suyama et al., 1998). It is generally agreed that abiotic hydrolysis of PLA proceeds first until the molecular weight of PLA decreases sufficiently to be assimilated by microorganisms (Suyama et al., 1998; Tokiwa and Calabia, 2006). Table 2 compares the variations in the molecular weight of PLA powder suspended in a mineral medium at 37 C with and without S. maltophilia LB 2-3. The molecular weight of PLA decreased monotonously with increasing incubation time in the absence of S. maltophilia LB 2-3, but decreased faster in the presence of the strain (Table 2). The GPC profiles of neat PLA, PLA-40 days, and PLA-40 days-LB 2-3 were all monodisperse. Notably, the GPC chromatogram of PLA40 days-LB 2-3 (PLA powder suspended in mineral medium for 40 days in the presence of the PLA degrader) compared to that of PLA40 days (PLA powder suspended in the mineral medium for 40 days in the absence of the PLA degrader) was higher in intensity at a longer elution time (lower molecular weight region). But at the same time, the reverse was true at a shorter elution time, clearly revealing that the PLA molecules were cleaved not only by abiotic hydrolysis but also that cleavage was accelerated by enzymes secreted from S. maltophilia LB 2-3.
strongly on the wavelength of irradiated light, we fabricated a UV lamp emitting light with similar intensity at 245 nm to that of sunlight. The UV chamber was equipped with four UV lamps emitting 6.41 103 mW cm2 and 3.22 mW cm2 of intensity at 185 nm and 245 nm, respectively. The intensity of sunlight at 245 nm was 0.19e2.55 mW cm2 in October 2012, in Seoul, Korea, depending on the time of measurement and cloudiness but that at 185 nm was zero indicating that the light at 185 nm was completely absorbed by the atmosphere. We attempted to block the light at 185 nm from our UV source by shielding the specimens with polypropylene (PP) films because PP film was reported by Ojeda et al. (2009) to effectively block UV-C but allow UV-A and UV-B to pass through. However, even after shielding with 10 layers of PP 50 mm film, the transmitted light at 185 nm was as intense as one fourth of 6.41 103 mW cm2, revealing that complete blockage of UV at 185 nm by PP film was infeasible. When UV intensity was 6.41 103 mW cm2 and 3.22 mW cm2 at 185 nm and 245 nm, respectively, the molecular weight decreased from 188,700 to 41,400, and the contact angle decreased from 84.5 to 73.9 as the PLA sheet was UV irradiated for 24 h (Table 4), indicating that PLA became more hydrophilic as a result of UV exposure. The tensile properties became sharply worse, and thereby became impossible to measure as soon as UV irradiation time exceeded 12 h. Fig. 1 shows the GPC chromatograms of the PLA sheets before and after UV exposure. The peak at short elution times decreased in intensity quickly and moved to a longer elution time indicating that the long PLA chains were fragmented by UV irradiation. 3.4. Biodegradation of PLA before and after UV exposure
3.3. UV degradation of PLA PLA sheets were exposed to UV irradiation at room temperature. UV-photo-degradation of plastics has mostly been carried out with UV wavelengths >300 nm to imitate photo-degradation by sunlight. However, as shown in Table 3, the intensity of sunlight was still strong at 245 nm. Because photochemical reactions depend
Degradation of PLA was examined in the modified Sturm test apparatus set up according to ASTM D 5209-91 by measuring the net amount of CO2 evolved from mineral medium loaded with PLA. A 500 ml aliquot of S. maltophilia LB 2-3 cells was inoculated into 100 ml of stock solution. Fig. 2 shows the modified Sturm test results for the PLA exposed to UV irradiation for different times. It was
Table 3 Ultraviolet intensity in mW cm2 of sunlight at 185 nm and at 245 nm measured in Seoul, Korea. Time
Date Oct. 10, 2012
9 am 12 pm 3 pm
Oct. 11, 2012
Oct. 12, 2012
185 nm
245 nm
185 nm
245 nm
185 nm
245 nm
0 0 0
0.190 0.005 0.655 0.064 2.107 0.086
0 0 0
2.503 0.034 2.536 0.011 2.540 0.002
0 0 0
2.448 0.007 2.517 0.005 2.510 0.003
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Fig. 1. GPC profiles of PLA exposed to ultraviolet irradiation for different times.
generally expected that the longer the exposure, the lower the molecular weight of PLA and, thus, the higher the biodegradability. However, the apparent biodegradability of PLA reached a maximum at 8 h of UV exposure and then decreased with further increase in UV exposure (Fig. 2). We replicated the experiments three times and obtained the same conclusion. Here, the apparent biodegradability was defined as follows: Apparent biodegradability ¼ (A B)D1where A is the accumulated amount of CO2 evolved from the suspension loaded with PLA, and B is the accumulated amount of CO2 evolved from the suspension without PLA. D, 1.83 g of CO2/g of PLA, is the amount of CO2 evolved when all the carbons in the PLA samples were assumed to be mineralized into CO2 (Jeon and Kim, 2013). Apparent biodegradability is used here, because all the PLA carbons may not be consumed entirely as CO2 but part of them may be locked up in the biomass or in other metabolites (Gu et al., 1993a,b). Fig. 3 shows the PLA biodegradability measured in the compost at 37 C under controlled conditions according to KS M3100-1,
Fig. 3. Biodegradability of PLA measured in compost under controlled conditions according to KS M3100-1, 2002; MOD ISO 14855, 1999.
2002; MOD ISO 14855 (1999). PLA biodegradability was higher in Fig. 3 than that in Fig. 2, indicating more favorable conditions for biodegradation in the compost than in mineral medium. The evolution of CO2 from PLA buried in the sterilized compost was negligible, but PLA was transformed into CO2 at a considerable rate even at 37 C when the compost was inoculated with S. maltophilia LB 2-3. Gu et al. (1992) also observed that weight loss of PLA occurred much faster in a compost as compared to when the compost was sterilized or poisoned by KCN. Hence it can be concluded that the presence of S. maltophilia LB 2-3 accelerated PLA degradation. The results in Fig. 3 demonstrate again that the apparent biodegradability of PLA was the highest at 8 h of UV exposure. We do not have a clear answer for this biodegradation behavior of PLA. One plausible explanation is that a Norrish type reaction as shown in Fig. 4 may transform PLA into a structure more recalcitrant to biodegradation. However, as demonstrated in Fig. 5, the peaks at w3.6 ppm, which should appear if the Norrish type reaction occurred, did not appear in the 1H nuclear magnetic resonance spectrum of PLA exposed to UV irradiation for 24 h. UV irradiation may transform PLA into a brittle white solid which may be poorly assimilated by microorganisms. Because apparent biodegradability is determined based on the assumption that all carbon in the test specimen is metabolized to CO2 by microorganisms, the formation of the poorly assimilable solid should contribute to the biodegradability behavior of the PLA exposed to UV for longer than 8 h. Acknowledgments This study was supported by a 2012 Research Grant from Sangmyung University.
Fig. 2. Modified Sturm test results for PLAs exposed to ultraviolet irradiation for different times.
Fig. 4. Plausible Norrish type reaction of PLA during ultraviolet irradiation.
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293
Fig. 5. 1H nuclear magnetic resonance spectra of PLA exposed to ultraviolet irradiation for 0 and 24 h.
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