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Home , Beeswax, Galleria mellonella, Ideonella sakaiensis

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A Correspondence B C

Polyethylene B bio-degradation by 1.2 of the 13% -2 Galleria 1.0 mellonella mg cm

Paolo Bombelli1, ~100 wax worms 0.2 92 mg mass loss 1, Christopher J. Howe *, ~12 hours exposure 0.0 and Federica Bertocchini2,3,* Untreated Treated

D E

Plastics are synthetic polymers derived 1.0 from fossil oil and largely resistant to Untreated Untreated 0.8 . (PE) and Treated T(%) Treated polypropylene (PP) represent ~92% of 0.6 -C-H total plastic production. PE is largely Abs 0.4 utilized in packaging, representing -C=O ~40% of total demand for plastic 0.2 products (www.plasticseurope.org) 0.0 with over a trillion plastic bags used 4000 3500 3000 2500 1800 1500 1200 900 every year [1]. Plastic production has cm-1 cm-1 increased exponentially in the past F G 50 years (Figure S1A in Supplemental Information, published with this article Untreated Treated online). In the 27 EU countries plus Z[nm] Z[nm] 500.0 Norway and Switzerland up to 38% 0.0 of plastic is discarded in landfi lls, 0.0 -6.0 8.0 -10.0 with the rest utilized for recycling -5.0 7.0 -9.0 (26%) and energy recovery (36%) via -4.0 6.0 -8.0 9.0 -3.0 5.0 -7.0 8.0 combustion (www.plasticseurope. Y[m] 7.0 X[m] -2.0 4.0 X[m] -6.0 6.0 Y[m] org), carrying a heavy environmental 3.0 -5.0 -1.0 5.0 2.0 impact. Therefore, new solutions 0.0 -4.0 4.0 for plastic degradation are urgently needed. We report the fast bio- Figure 1. Polyethylene degradation by . degradation of PE by larvae of the wax (A) Plastic bag after exposure to ~100 wax worms for 12 hours. (B) Magnifi cation of the area moth Galleria mellonella, producing indicated in A. (C) Gravimetric analysis of homogenate-treated versus untreated polyethylene . (PE), showing a reduction (13%) of mass per unit of area in the former. (D,E) FTIR analysis of the homogenate-treated and control PE fi lms. (F,G) Atomic Force Microscopy on homogenate- PE comprises a linear backbone treated (G) and untreated (F) PE fi lm (representative examples of 3 topographic maps each). of carbon atoms (Figure S1B), which is resistant to degradation. Although (FTIR) analysis of treated samples of another plastic, poly(ethylene PE is believed not to be susceptible revealed formation of an absorbance terephthalate) (PET) by a microbial to bio-degradation, a few attempts peak around 3,300 cm-1, a signature consortium including a newly isolated have been made, as PE is the most for ethylene glycol, confi rming PE bacterium, sakaiensis, was common packaging plastic. Slow degradation. More recently, Yang described recently [5]. Although PET is (weeks/months) PE biodegradation et al. reported bacterial degradation a resistant material, one might expect has been observed, given appropriate of PE over several weeks [4]. its biodegradation to be easier than conditions. For example, modest However, no production of ethylene PE, as PET has a polyester backbone degradation of PE was observed after glycol from the biodegradation was and can be hydrolysed. We report nitric acid treatment and incubation described. The authors reported that here the fast biodegradation of PE for 3 months in a liquid culture of the PE biodegradation depended on the by the wax worm, the fungus Penicillium simplicissimum activity of microorganisms present of the wax moth Galleria mellonella of [2]. Slow PE degradation was in the gut of the larvae of the Indian the snout moth () family of also recorded after 4 to 7 months mealmoth Plodia interpunctella (two . exposure to the bacterium Nocardia bacterial strains, sp. YP1 and When a PE fi lm was left in direct asteroides [3]. In both cases, fourier asburiae YT1). Faster contact with wax worms, holes started transform infrared spectroscopy biodegradation (~0.13 mg cm-2 day-1) to appear after 40 minutes, with an

R292 Current Biology 27, R283–R293, April 24, 2017 © 2017 Elsevier Ltd. Current Biology Magazine

estimated 2.2±1.2 holes per worm wax worm extract three new peaks Nevertheless, given the fast rate of per hour (Table S1A). Figure 1A,B appeared at the lower end of the biodegradation reported here, these shows the result of leaving ~100 wax m/z region (110.0, 122.9 and 170.0). fi ndings have potential for signifi cant worms in contact with a commercial The chemical identity of these lighter biotechnological applications. PE shopping bag for ~12 hours, which fractions was not confi rmed but their caused a mass loss of 92 mg. To presence supports the hypothesis SUPPLEMENTAL INFORMATION exclude the possibility that mechanical of PE degradation by the wax worm action of the masticatory system was homogenate. Supplemental Information contains solely responsible for the observed To analyse further the effect of experimental procedures, one fi gure and PE breakdown, worm homogenate wax worm homogenate on the PE one table and can be found with this was smeared on and left in contact surface, Atomic Force Microscopy article online at http://dx.doi.org/10.1016/j. with PE fi lms. Gravimetric analysis (AFM) was performed (Figure 1F,G). cub.2017.02.060. of the treated samples confi rmed After treatment with homogenate, ACKNOWLEDGMENTS a signifi cant mass loss of 13% PE we observed an obvious change in over 14 hours of treatment (one-way the topography of the PE surface F.B. is a Ramon y Cajal Fellow at the ANOVA, p = 0.029) compared to the (Figure 1G), corresponding to a IBBTEC, in Santander, Spain. We thank Dr. untreated samples (Figure S1C and signifi cant (one-way ANOVA = 0.005) Simone Ruggeri and Dr. Jenny Zhang for Table S1B,C). This corresponds to greater than 140% increase in surface technical help. This work was funded by the an average degradation rate of 0.23 roughness (Figure S1H and Table Leverhulme . P.B. and F.B. are -2 -1 mg cm h , which is markedly higher S1D). These results indicate that the co-founders of the company Baky.ltd. The than the rate of PET biodegradation physical contact of the wax worm authors declare no competing interest. by a microbial consortium recently homogenate with the PE surface reported [5]. modifi ed the integrity of the polymer REFERENCES To test if the PE polymer was surface. chemically degraded by contact with What allows the wax worm to 1. The Economist (2016). Single-use refuse. http://www.economist.com/news/ the worm homogenate, we carried out degrade a chemical bond not generally international/21670516-over-trillion-plastic- FTIR analysis. When the FTIR probe susceptible to bio-degradation? The bags-are-used-every-year-does-charging- was pointed on untreated samples, answer may lie in the ecology of them-help-single-use-refuse. 2. Yamada-Onodera, K., Mukumoto, H., the spectroscopic results confi rmed the wax worm itself. They feed on Katsuyaya, Y., Saiganji, A., and Tani, Y. (2001). the identity of the PE fi lm, with peaks beeswax, and their natural niche is Degradation of polyethylene by a fungus. -1 Penicillium simplicissimum YK. Polym. Degrad. at 2,921 and 2,852 cm being the the ; the moth lays its Stab. 72, 323–327. classical signatures of PE (Figure 1D, eggs inside the , where the 3. Bonhomme, S., Cuer, A., Delort, A.-M., black line). However, when the probe worms grow to their stage, Lemaire, J., Sancelme, M., and Scott, C. (2003). Environmental biodegradation of was pointed on sample smeared with eating beeswax [7]. Beeswax is polyethylene. Polym. Degrad. Stab. 81, worm homogenate, an additional composed of a highly diverse mixture 441–452. -1 4. Yang, J., Yang, Y., Wu, W-M., Zhao, J., and peak at ~3,350 cm was seen of lipid compounds, including alkanes, Jiang, L. (2014). Evidence of polyethylene (Figure 1D, red line). This FTIR peak alkenes, fatty acids and [8]. biodegradation by bacterial Strains from the corresponds to the one previously The most frequent guts of plastic-eating . Environ. Sci. Tech. 48, 13776–13784. described as the ethylene glycol bond is the CH2–CH2, as in PE 5. Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, signature (also compare Figure 1E (Figure S1B). Although the molecular I., Yamaji, H., Maeda, Y., Toyohara, K., Miyamoto, K., Kimura, Y., and Oda, K. (2016). with Figure 4B in [4]) [3,6]. In addition, details of wax biodegradation require A bacterium that degrades and assimilates a peak at 1,700 cm-1 appeared in the further investigation, it seems likely poly(ethylene terephthalate). Science 351, treated sample, which is the classical that the C–C single bond of these 1196–1199. 6. Zuchowska, D., Hlavata, D., Steller, R., signature of the carbonyl bond (Figure aliphatic compounds is one of the Adamiah, W., and Meissner, W. (1999). Physical 1E, red line). The ethylene glycol targets of digestion. The appearance structure of polyolefin-starch after ageing. Polym. Degrad. Stab. 64, 339–346. signature was also seen when the of holes when PE fi lms are left in 7. Dickman, R. (1933). Studies on the waxmoth probe was pointed close to holes in direct contact with wax worms, and Galleria mellonella, with particular reference PE caused by intact worms, but not the FTIR analysis of degraded PE, to the digestion of wax by the larvae. J. Cell. Comp. Physiol. 3, 223–246. when the probe was pointed at a indicate chemical breakdown of 8. Maia, M., and Nunes, F.M. (2013). distance (Figure S1C–E). the PE, including breakage of C–C Authentication of beeswax (Apis mellifera) by high-temperature gas chromatography The formation of products after bonds. It is not clear whether the and chemometric analysis. Food Chem. 136, treatment with wax worm extract hydrocarbon-digesting activity of G. 961–968. was also characterised by high mellonella derives from the organism performance liquid chromatography itself, or from enzymatic activities 1Department of Biochemistry, University coupled with mass spectrometry of its intestinal fl ora [7], as with PE of Cambridge, Downing Site, Tennis (HPLC–MS), covering a mass/ digestion by Plodia interpunctella [4]. Court Road, Cambridge, UK. 2Instituto de charge (m/z) range from 100 to 600 Further investigation is also required to Biomedicina y Biotecnologia de Cantabria- (Figure S1F,G). Figure S1G shows determine if related species have the CSIC-Universidad de Cantabria-SODERCAN, Av.da A. Einstein, Santander, Spain. the spectra for untreated PE (top, capacity for PE degradation, and to 3Lead contact. black) and the treated PE (bottom, analyse its molecular basis including *E-mail: [email protected] (C.J.H.); red). In the samples treated with the the detailed nature of the products. [email protected] (F.B.)

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