EOVIRONMENTAIArticlepubs.acs.org/estScience & lechnologuEvidenceofPolyethyleneBiodegradationbyBacterialStrainsfromtheGuts ofPlastic-EatingWaxworms*tllYuYang,g,ill Wei-Min Wu, Jiao Zhao,and Lei JiangJun YangtKey Laboratory of Bio-Inspired Smart Interfacial Science and Technology of Ministry of Education, School of Chemistry andEnvironment, Beihang University,Beijing 100191, P.RChina+Department of Civil and Environmental Engineering,William & Cloy Codiga Resource Recovery Research Center,CenterforSustainableDevelopment&GlobalCompetitiveness, Stanford University,Stanford,California94305-4020,United States$Shenzhen Key Laboratory of Bioenergy, BGI-Shenzhen, Shenzhen, Guangdong S18083, P. R. ChinaSupporting InformationABSTRACT:Polyethylene (PE)has been considered nonbiode-PEfilm-eatingwaxwormsgradablefor decades. Although the biodegradation of PE by bacterial8cultures has been occasionally described, valid evidence of PE1Ebiodegradation has remained limited in the literature. We found that：801ewaxworms, or Indian mealmoths (the larvae of Plodia interpunctella),were capable of chewing and eating PE films. Two bacterial strains0capableof degradingPE wereisolated from this worm's gut,Enterobacter asburiae YT1 and Bacillus sp. YP1. Over a 28-dayincubation period of the two strains on PE films, viable biofilmsLformed, and the PE films'hydrophobicity decreased. ObviousSdamage, including pits and cavities (0.3-0.4 μm in depth), wasoooobserved on the surfaces of the PE flms using scanning electron.5cmAINOading gutbacteriamicroscopy (SEM)and atomicforcemicroscopy(AFM).Theformation of carbonyl groups was verified using X-ray photoelectronNspectroscopy (XPS) and microattenuated total reflectance/Fourier transform infrared (micro-ATR/FTIR) imaging microscopeEaSuspension cultures of YT1 and YP1(10°cells/mL)were able to degrade approximately 6.1±0.3% and 10.7±0.2%of thePEfilms (100 mg),respectively, over a 60-day incubation period. The molecular weights of the residual PE films werelower, and therelease of 12 water-soluble daughter products was also detected. The results demonstrated the presence of PE-degrading bacteriain the guts of waxworms and provided promising evidencefor the biodegradation of PE in the environment.esaosoesi/schao8eooINTRODUCTIONoxidation and then through the biological activity of micro-organisms.15i6When PE samples are pretreated withUVlightPolyethylene (PE), the most common petroleum-based plastic,orthermo-oxidation,depolymerization ofthePEchainsoccursis expressed as “[CH,-CH,]." and is widely used in everydayand results in the formation of low molecular weight products,life,with an annual global production of approximately 140includingalkanes,alkenes,ketones,aldehydes,variousalcohols,million tons.12 However, the obvious contrast between theand fatty acids, which are then further degraded by selectremarkable durability and the short service time of PE products15-1s The pretreatments either decrease themicroorganisms.15leads to the accumulation of PE waste in the environment,hydrophobicity of the polymer, making it more compatible withwhich has generated international concern.microorganisms, or introducegroups such as C-Oor -OH,Several features of PE make it resistant tobiodegradationwhich are more prone to microbial degradation.19 In contrast,Among these features are a) PE's highly stable C-C and C-Halmost no biodegradation of PE through the biological activitiescovalent bonds; b) its high molecular weight, which makes itof select microorganisms can be observed without pretreat-too large to penetrate the cell walls of microbes; c) its lack of15 The poor biodegradation of virgin PE has beenments.readily oxidizable and/or hydrolyzable groups; and d) its highlyattributed to the lack of specific plastic-degrading micro-hydrophobic nature.7Since the early 1970s, tests on theorganisms with long carbon chain oxidization and depolyme-biodegradation of virgin PE (unpretreated and without anyrization capabilities. Certain researchers have assumed that theadditives)have been performed under natural environmentaltime for microbial evolution, which is required for plasticconditions, including soils, seawater, sludge, and compost,which harbor a multitude of diverse microbial communities.These studies have concluded that the biodegradation of virginReceived:August18,2014PE is extremely limited in selected mixed microbialRevised:October 31, 2014'It is believed that the environmentalcommunities.Accepted:November10,2014degradation of PE can be achieved by photo- or thermo-Published:November10,201413776ACS Publications2014 American Chemical Societydx.doLorg/10.1021/es504038a1Envron, Sdt Technot 2014, 48, 1377613784
Evidence of Polyethylene Biodegradation by Bacterial Strains from the Guts of Plastic-Eating Waxworms Jun Yang,*,†,∥ Yu Yang,†,∥ Wei-Min Wu,‡ Jiao Zhao,§ and Lei Jiang† † Key Laboratory of Bio-Inspired Smart Interfacial Science and Technology of Ministry of Education, School of Chemistry and Environment, Beihang University, Beijing 100191, P. R. China ‡ Department of Civil and Environmental Engineering, William & Cloy Codiga Resource Recovery Research Center, Center for Sustainable Development & Global Competitiveness, Stanford University, Stanford, California 94305-4020, United States § Shenzhen Key Laboratory of Bioenergy, BGI-Shenzhen, Shenzhen, Guangdong 518083, P. R. China *S Supporting Information ABSTRACT: Polyethylene (PE) has been considered nonbiodegradable for decades. Although the biodegradation of PE by bacterial cultures has been occasionally described, valid evidence of PE biodegradation has remained limited in the literature. We found that waxworms, or Indian mealmoths (the larvae of Plodia interpunctella), were capable of chewing and eating PE films. Two bacterial strains capable of degrading PE were isolated from this worm’s gut, Enterobacter asburiae YT1 and Bacillus sp. YP1. Over a 28-day incubation period of the two strains on PE films, viable biofilms formed, and the PE films’ hydrophobicity decreased. Obvious damage, including pits and cavities (0.3−0.4 μm in depth), was observed on the surfaces of the PE films using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The formation of carbonyl groups was verified using X-ray photoelectron spectroscopy (XPS) and microattenuated total reflectance/Fourier transform infrared (micro-ATR/FTIR) imaging microscope. Suspension cultures of YT1 and YP1 (108 cells/mL) were able to degrade approximately 6.1 ± 0.3% and 10.7 ± 0.2% of the PE films (100 mg), respectively, over a 60-day incubation period. The molecular weights of the residual PE films were lower, and the release of 12 water-soluble daughter products was also detected. The results demonstrated the presence of PE-degrading bacteria in the guts of waxworms and provided promising evidence for the biodegradation of PE in the environment. ■ INTRODUCTION Polyethylene (PE), the most common petroleum-based plastic, is expressed as “[CH2−CH2]n” and is widely used in everyday life, with an annual global production of approximately 140 million tons.1,2 However, the obvious contrast between the remarkable durability and the short service time of PE products leads to the accumulation of PE waste in the environment, which has generated international concern.3−6 Several features of PE make it resistant to biodegradation. Among these features are a) PE’s highly stable C−C and C−H covalent bonds; b) its high molecular weight, which makes it too large to penetrate the cell walls of microbes; c) its lack of readily oxidizable and/or hydrolyzable groups; and d) its highly hydrophobic nature.7 Since the early 1970s, tests on the biodegradation of virgin PE (unpretreated and without any additives) have been performed under natural environmental conditions, including soils, seawater, sludge, and compost, which harbor a multitude of diverse microbial communities.8−14 These studies have concluded that the biodegradation of virgin PE is extremely limited in selected mixed microbial communities.2−7 It is believed that the environmental degradation of PE can be achieved by photo- or thermooxidation and then through the biological activity of microorganisms.15,16 When PE samples are pretreated with UV light or thermo-oxidation, depolymerization of the PE chains occurs and results in the formation of low molecular weight products, including alkanes, alkenes, ketones, aldehydes, various alcohols, and fatty acids, which are then further degraded by select microorganisms.15−18 The pretreatments either decrease the hydrophobicity of the polymer, making it more compatible with microorganisms, or introduce groups such as CO or −OH, which are more prone to microbial degradation.19 In contrast, almost no biodegradation of PE through the biological activities of select microorganisms can be observed without pretreatments.15 The poor biodegradation of virgin PE has been attributed to the lack of specific plastic-degrading microorganisms with long carbon chain oxidization and depolymerization capabilities. Certain researchers have assumed that the time for microbial evolution, which is required for plastic Received: August 18, 2014 Revised: October 31, 2014 Accepted: November 10, 2014 Published: November 10, 2014 Article pubs.acs.org/est © 2014 American Chemical Society 13776 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784 Downloaded via BEIJING NORMAL UNIV on October 9, 2019 at 08:31:02 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles
Environmental Science&TechnologyArticledegradation, might be much longer than the history of PEmolecular weight was analyzed using high-temperature gel20 To date, researchers are still attempting toapplications.2permeation chromatography (GPC, PL220, Agilent, USA), anddiscover PE-degrading microorganisms through tests invoivingthe average molecular weight (M) and number-averageplastic-waste-contaminated soils, landflls, compost, and marinemolecular weight (M.) were found to be 88,200 and 27,700,life and have isolated several bacterial strains (SI Tablerespectively.S1).20-39 Some of these strains have shown a moderate abilityThePEfilmwascut into50mmXS0mm squaresheetsforto use virgin PE as a carbon source based on theincubation inanagarmediumplateandsmall3mmX3mmcharacterization of biofilm formation on PE films, weight losspieces for incubation in a liquid medium.The PE materialsof PE materials, surface deterioration, and changes in thewere weighed, disinfected in 75% ethanol, and then air-dried inmechanical and thermal properties of PE (SI Table S1).Fora laminar-flow clean bench prior to use.example, it has been reported that a Pseudomonas strain causesThe liquid carbon-free basal medium (LCFBM) preparedweight losses of up to 20% in the tested PE within 120 days.with deionized water contained (per 1,000 mL) 0.7 g ofAdditionally,a Canadian Web sitehas reported the isolation ofKH,PO0.7gofK,HPO0.7gofMgSO,7H,O,1.0goftwo novel bacteria that rapidly degrade 22% or more of theNH,NO,0.005g of NaC, 0.002g of FeSO4-7H,O, 0.002g oftested PE weight over a six week period39 However, theseZnSO,7H,O,and 0.001 g of MnSO,H,O, according to thepromising reports of PE degradation based on weight loss haveASTM standard for determining the resistance of plastics tonot been confirmed due to a failure to provide additionalbacteria (G22-76, 1996).1The carbon-free basal agar mediumsupporting evidence, such as changes in surface hydrophobicity,(CFBAM)wasprepared byadding15gof agar to1,000mLoftheformation ofoxidized carbonyl groups, the scission of longLCFBM.These media were sterilized by autoclaving at 121 °Cchain molecules, or the release of low molecular weightfor 20 min.products. No report on the deposits of the aforementionedEnrichment and Isolation of PE-Degrading Bacteria.bacteria in any culture collection center has been provided.Once the waxworms had chewed enough of the PE bags toMore studies are needed to confirm PE degradation bycause significant observable damage,approximately200 of themicroorganisms via sequential two-step reactions, which havelarvae were collected from the PE bags containing millet grainsbeen achieved by the synergistic actions of physicochemical(SI, Figure S1).To obtain the gut contents for the enrichmentoxidation and through the biological activity ofmicroorganisms,of PE-degrading bacteria, the surfaces of the larvae wereas proposed by Albertsson et alissterilized via immersion in 75% ethanol for 1 min and thenWe found that the grain pest larvae of Plodia interpunctellarinsed 2 times with sterile saline water (SW). Next, the guts of(Hubner), commonly known as waxworms or Indian meal-the larvae were drawn out and pooled into a S0 mL centrifugemoths, can damage PE packing films by chewing and eating thetube containing 40 mL of SW. After being shaken on a vortexfilm (SI, Figure S1). A similar observation stating that variousmixer for S min, the gut tissues were carefully removed with aspecies of stored-product insect pests like to chew andpipet. The remaining suspension, used as a microbial inoculum,penetrate PE packaging flms has been previously reported.40wastransferredintoa2S0mLErlenmeyerflaskthatcontainedHowever, the focus of the previous report was to test for and1gof thesmall PEpieces and 80mLof LCFBM.This flaskwasdiscover a package film material that was resistant to the insects'incubated on a rotary shaker (120 rpm) at 30 °C. After 60 days,destructive tendencies. We were inspired by the insect used inthe residual PE pieces were removed, and the cultures werethat test and began to wonder whether a microbial symbiontspread across plates with three different agar media containingcapable of digesting PE was present in the waxworm gut andcomplex organic carbon substrates: potato dextrose agarwhether the gut bacteria could act as a source for the isolation(PDA), bean sprout agar (BSA), and beef extract-peptoneof PE-degrading microorganisms.agar (BPA). After an incubation period of 24 h, the coloniesIn this study,with PE film as a sole carbon source, wethat formed were transferred to plates with fresh agar medium,isolated twoPE-degradingbacterial strainsfromtheenrichmentwhere they were kept until pure colonies of isolates (based on aof waxworm gut contents and identified that thesebacteria weremicrobial purity test) could be obtained.These isolates havecapable of degrading PE within a limited incubation periodbeen deposited at the China General Microbiological Culturebased on the characterization of biofilm formation, changes inCollection Center (CGMCCC) in Beijing, China.the PE's physical properties (tensile strength and surfacePreliminary Screening of Isolates for PE-Degradingtopography), chemical structure (hydrophobicity and appear-Ability.The bacterial isolates were grown in liquid nutrientance of carbonyl groups), molecular weight (accompanied bybroth (NB) medium for 12 h. The cells that grew werethe formation of daughter products), and weight loss. Ourcollected via centrifugation (10,000 rpm) and rinsed with SWresults confirmed PE biodegradation by the two gut bacteriato remove the residual medium. This procedure was repeatedisolatedfromwaxwormsandindicatedthatthebacteriafromthree times. Next, the collected cells were resuspended in SWthese plastic-chewing insect larvae were a promising source ofto obtain a cell suspension of approximately 10°cells per mL.plastic-degrading microorganisms.Themelted sterileCFBAM(15mL)was poured into a Petridish (90 mm diameter) and cooled to ambient temperature.MATERIALSANDMETHODSThe cell suspension (0.5 mL)was spread homogeneouslyPE Film and Medium. Linear low-density PE (LDPE) flmacross the surface of a CFBAM plate,which was subsequently(DFDA-9020,22.5 μm thickness)was purchased fromcovered with a PE sheet (50 mm X 50 mm).In the sterileSINOPEC Beijing Yanshan Company in China. No catalystscontrols, PE sheets were added without inoculation of the cellor additives were added to this PE product, according to thesuspension.CFBAM plates inoculated with the cell suspensionmanufacturer's standard(Q/SH3180014).The chemicalwithout added PE film were used as a control to check whethercomposition was characterized by a thermogravimetric analyzertheisolates could grow on agar alone. All plates were then(TGA, 209F1, NETZSCH, Germany) and Fourier transformsealed with Parafilm and incubated at 30 °C and 85% r.h.for 28infrared spectroscopy (FTIR, iN10MX, Nicolet, USA). Thedays. Three plates were prepared for each isolated strain. The13777dx.doi.org/10.1021/es504038alEmviron.Sc.Technol. 2014,48,1377613784
degradation, might be much longer than the history of PE applications.20 To date, researchers are still attempting to discover PE-degrading microorganisms through tests involving plastic-waste-contaminated soils, landfills, compost, and marine life and have isolated several bacterial strains (SI Table S1).20−39 Some of these strains have shown a moderate ability to use virgin PE as a carbon source based on the characterization of biofilm formation on PE films, weight loss of PE materials, surface deterioration, and changes in the mechanical and thermal properties of PE (SI Table S1). For example, it has been reported that a Pseudomonas strain causes weight losses of up to 20% in the tested PE within 120 days.32 Additionally, a Canadian Web site has reported the isolation of two novel bacteria that rapidly degrade 22% or more of the tested PE weight over a six week period.39 However, these promising reports of PE degradation based on weight loss have not been confirmed due to a failure to provide additional supporting evidence, such as changes in surface hydrophobicity, the formation of oxidized carbonyl groups, the scission of longchain molecules, or the release of low molecular weight products. No report on the deposits of the aforementioned bacteria in any culture collection center has been provided. More studies are needed to confirm PE degradation by microorganisms via sequential two-step reactions, which have been achieved by the synergistic actions of physicochemical oxidation and through the biological activity of microorganisms, as proposed by Albertsson et al.15 We found that the grain pest larvae of Plodia interpunctella (Hü bner), commonly known as waxworms or Indian mealmoths, can damage PE packing films by chewing and eating the film (SI, Figure S1). A similar observation stating that various species of stored-product insect pests like to chew and penetrate PE packaging films has been previously reported.40 However, the focus of the previous report was to test for and discover a package film material that was resistant to the insects’ destructive tendencies. We were inspired by the insect used in that test and began to wonder whether a microbial symbiont capable of digesting PE was present in the waxworm gut and whether the gut bacteria could act as a source for the isolation of PE-degrading microorganisms. In this study, with PE film as a sole carbon source, we isolated two PE-degrading bacterial strains from the enrichment of waxworm gut contents and identified that these bacteria were capable of degrading PE within a limited incubation period based on the characterization of biofilm formation, changes in the PE’s physical properties (tensile strength and surface topography), chemical structure (hydrophobicity and appearance of carbonyl groups), molecular weight (accompanied by the formation of daughter products), and weight loss. Our results confirmed PE biodegradation by the two gut bacteria isolated from waxworms and indicated that the bacteria from these plastic-chewing insect larvae were a promising source of plastic-degrading microorganisms. ■ MATERIALS AND METHODS PE Film and Medium. Linear low-density PE (LDPE) film (DFDA-9020, 22.5 μm thickness) was purchased from SINOPEC Beijing Yanshan Company in China. No catalysts or additives were added to this PE product, according to the manufacturer’s standard (Q/SH3180 014). The chemical composition was characterized by a thermogravimetric analyzer (TGA, 209F1, NETZSCH, Germany) and Fourier transform infrared spectroscopy (FTIR, iN10MX, Nicolet, USA). The molecular weight was analyzed using high-temperature gel permeation chromatography (GPC, PL220, Agilent, USA), and the average molecular weight (Mw) and number-average molecular weight (Mn) were found to be 88,200 and 27,700, respectively. The PE film was cut into 50 mm × 50 mm square sheets for incubation in an agar medium plate and small 3 mm × 3 mm pieces for incubation in a liquid medium. The PE materials were weighed, disinfected in 75% ethanol, and then air-dried in a laminar-flow clean bench prior to use. The liquid carbon-free basal medium (LCFBM) prepared with deionized water contained (per 1,000 mL) 0.7 g of KH2PO4, 0.7 g of K2HPO4, 0.7 g of MgSO4·7H2O, 1.0 g of NH4NO3, 0.005 g of NaCl, 0.002 g of FeSO4·7H2O, 0.002 g of ZnSO4·7H2O, and 0.001 g of MnSO4·H2O, according to the ASTM standard for determining the resistance of plastics to bacteria (G22-76, 1996).41 The carbon-free basal agar medium (CFBAM) was prepared by adding 15 g of agar to 1,000 mL of LCFBM. These media were sterilized by autoclaving at 121 °C for 20 min. Enrichment and Isolation of PE-Degrading Bacteria. Once the waxworms had chewed enough of the PE bags to cause significant observable damage, approximately 200 of the larvae were collected from the PE bags containing millet grains (SI, Figure S1). To obtain the gut contents for the enrichment of PE-degrading bacteria, the surfaces of the larvae were sterilized via immersion in 75% ethanol for 1 min and then rinsed 2 times with sterile saline water (SW). Next, the guts of the larvae were drawn out and pooled into a 50 mL centrifuge tube containing 40 mL of SW. After being shaken on a vortex mixer for 5 min, the gut tissues were carefully removed with a pipet. The remaining suspension, used as a microbial inoculum, was transferred into a 250 mL Erlenmeyer flask that contained 1 g of the small PE pieces and 80 mL of LCFBM. This flask was incubated on a rotary shaker (120 rpm) at 30 °C. After 60 days, the residual PE pieces were removed, and the cultures were spread across plates with three different agar media containing complex organic carbon substrates: potato dextrose agar (PDA), bean sprout agar (BSA), and beef extract-peptone agar (BPA). After an incubation period of 24 h, the colonies that formed were transferred to plates with fresh agar medium, where they were kept until pure colonies of isolates (based on a microbial purity test) could be obtained. These isolates have been deposited at the China General Microbiological Culture Collection Center (CGMCCC) in Beijing, China. Preliminary Screening of Isolates for PE-Degrading Ability. The bacterial isolates were grown in liquid nutrient broth (NB) medium for 12 h. The cells that grew were collected via centrifugation (10,000 rpm) and rinsed with SW to remove the residual medium. This procedure was repeated three times. Next, the collected cells were resuspended in SW to obtain a cell suspension of approximately 108 cells per mL. The melted sterile CFBAM (15 mL) was poured into a Petri dish (90 mm diameter) and cooled to ambient temperature. The cell suspension (0.5 mL) was spread homogeneously across the surface of a CFBAM plate, which was subsequently covered with a PE sheet (50 mm × 50 mm). In the sterile controls, PE sheets were added without inoculation of the cell suspension. CFBAM plates inoculated with the cell suspension without added PE film were used as a control to check whether the isolates could grow on agar alone. All plates were then sealed with Parafilm and incubated at 30 °C and 85% r.h. for 28 days. Three plates were prepared for each isolated strain. The Environmental Science & Technology Article 13777 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784
Environmental Science&TechnologyArticleevidence of PE biodegradation was preliminarily determined byATR/FTIR microscope was 400 × 400 μm, and the scanningcharacterizing the changes in the tensile strength of thewas performed at a step size of 10 μm under a contact pressureincubated PE sheets on a universal test machine (AGS-X-IKN,of 3 MPa.Shimadzu, Japan), according to the standard method.2Biodegradation Assays. The PE biodegradation wasTo examine changes in the PE flm's hydrophobicity over 28characterized by the timing of its weight loss, molecular weightdays, the sole carbon source of the isolated strains was a PEshift, and the formation of soluble daughter products in thesample in the LCFBM. Afterward, the biofilm on the PE sheetLCFBM.PEpieces(100mg)and bacterial suspensions (10was completely removed by mixing it with 2% w/v aqueousmL) were added to a 150 mL Erlenmeyer flask with 40 mL ofsodium dodecyl sulfate (SDS) for 4 h and then rinsing it withLCFBM.The final bacterial concentration was approximatelydeionized water, according to the washing procedure reported10° cells per mL, and a flask without inoculation served as aby Sivan A et al.21,22 Following the treatment, no cells weresterile control. Three samples were prepared for eachobserved on the surface of the PE film.The change in the PEincubation period of7,14, 21,28, and 60 days, and the flaskssurface's hydrophobicitywas determined by measuring thewere incubated on a rotary shaker (120 rpm) at 30 °C. Forwater contact angle (WCA) using a contact angle measuringeach incubation period, the residual PE pieces were collected,washed using the procedure reported by Sivan et al,21,22 anddevice (OCA40, Dataphysics, Germany)Characterization of Biofilm Formation and Celldried at ambient temperature for the measurement of residualViability.The isolates with positive results obtained duringweight.The washed PE residues collected at the end of the test werepreliminary screen tests were further tested for their ability toform biofilms on PE film as a sole carbon source andfor the cellrandomly sampled for molecular weight distribution analysisviability in the biofilm. A cell suspension (10° cells per mL)wasusing the HT-GPC (PL220, Agilent, USA).23 The liquid culturespread across the CFBAM plate, which was subsequentlywas centrifuged at 10,000 rpm for 15 min, and the supernatantcovered with thePEfilm sheet(50 mm×50mm),as describedwas filtered through a 0.22-μm membrane filter. The solubleabove.Threesamplesweretested at incubationperiods of O, 3,daughter products in the fltrate were analyzed using electro-7,14, 21, and 28 days at 30 °C and 85% r.h.For the incubationspray ionization-mass spectrometry (ESI-MS, LCQ, Finnigan,of zero days, samples were collected after 3 h of inoculation.USA) in the positive ion mode.The plates without inoculation served as a sterile control.Sequencing and Phylogenetic Analysis.The genomicAfter a biofilm formed on the PE surface, the cell number ofDNA needed for 16S rDNA amplification of the isolated strainsbacteria that had grown on the PE film surface was countedwas extracted from cells grown in the late log phase usingausing the following procedure.The PE film sheet containing aconventionalproteinaseKtreatmentandphenol-chloroformbiofilm was transferred into a 50 mL centrifuge tube with 40extraction.Amplification of the S end of the gene wasmL of SW, and the tube was shaken on a vortex mixer for 5performed with the universal primers 8-F (5'-AGAGTTTGA-min. The PE film was transferred into another centrifuge tubeTYMTGGCTCAG-3")and 1942-R (S'-GGTTACCTTGT-with 40 mL of SW,and thistubewas alsoshaken on thevortexTACGACTT-3'). The obtained sequences were aligned withmixer for S min.The suspensions of the two tubes were thenorganisms present on the GenBank database using the Basicpooled in a centrifuge tube, which was centrifuged at 10,000Local Alignment Search Tool (BLAST) created by the Nationalrpm for 15 min. The supernatant was discarded, and the pelletCenter for Biotechnology Information, USAwas resuspended in5 mL of SW.Thecell number of thesuspension was counted using the series dilution method ofRESULTSANDDISCUSSION.platecounting.ThebiofilmonthePEfilmsheetwasexaminedPreliminary Screen of PE-Degrading Isolates. Eightusing a scanning electron microscopy(SEM,Quanta FEG250,bacterial strains were isolated during the enrichment of the gutFEI, USA),21 and the viability of the bacterial cells wascontents of the PE-eating waxworms when using PE as a solecharacterized in situ using a fluorescence microscope (BXS1,carbon source (SI, Figure S1). Screenings for PE-degradingOlympus, Japan) after staining with the LIVE/DEAD BacLightmicroorganisms among these isolates were subsequentlyBacterial Viability Kit.22Microscale Analysis of Surface Topography andperformed in terms of the colonization on the PE film sheetsin the CFBAM plates, the changes in tensile strength of the PEChemistry. After a 28-day incubation period, the biofilm onsheets, the floating behavior of the PE pieces in the LCFBM,the PE sheet was completely removed using a washingand the WCA of the PE surfaces.procedure with 2% w/y aqueous sodium dodecyl sulfate(SDS) solution for 4h.2i22 The PE sheets in the sterile controlThe Gram-positive strain YP1,identified by 16S rDNAsequencing as Bacillus sp-, grew as relatively thick, opaquewere also treated using the same procedure. Consequently,thecolonies visible to the naked eye on the PE film sheet. Thesurface topography of all PE sheets was characterized by SEMobservation(QuantaFEG250,FEI,USA)and withan atomicGram-negative strain YTl,identified as Enferobacter asburiae,force microscope (AFM,Dimension Icon, Veeco, USA).21,26grew as visible, thin,translucent colonies (Figure la and TableThe surface chemical components were investigated using X1).Theothersix isolated bacterial strainsdid not developvisible colonies on the PE film sheets. In addition, no visiblerayphotoelectron spectroscopy(XPS,Thermo Scientific, USA)colonization occurred on the two control plates: a)the CFBAMand a microattenuated total reflectance/Fourier transformwith a PE flm sheet but without inoculation or b) the CFBAMinfrared imaging,microscope (Nicolet iN1o MX, ThermoScientific, USA).43 During the XPS spectra analysis, scanningwithout a PE sheet but with inoculation.was carried out over a broad-band energy range (0-1,200 ev)After a 28-day incubation period, all of the incubated PE filmat an electron takeoff angleof 90°from the sample areas lesssheetswere collectedtotesttheirtensile strength.The samplesspread with the culture of Bacillus sp. YP1 or E. asburiae YT1than 1 mm in diameter. The overlapping peaks in the C lsregion were resolved into their individual components using aexhibitedtensilestrengthdecreasesofoverS0%comparedtopeak-ftting program (XPSPEAK v3.0). The view field of thethat of the sterile controls, while the samples with the other six13778dx.doi.org/10.1021/es504038alEmviron.Sc.Technol. 2014,48,1377613784
evidence of PE biodegradation was preliminarily determined by characterizing the changes in the tensile strength of the incubated PE sheets on a universal test machine (AGS-X-1KN, Shimadzu, Japan), according to the standard method.42 To examine changes in the PE film’s hydrophobicity over 28 days, the sole carbon source of the isolated strains was a PE sample in the LCFBM. Afterward, the biofilm on the PE sheet was completely removed by mixing it with 2% w/v aqueous sodium dodecyl sulfate (SDS) for 4 h and then rinsing it with deionized water, according to the washing procedure reported by Sivan A et al.21,22 Following the treatment, no cells were observed on the surface of the PE film. The change in the PE surface’s hydrophobicity was determined by measuring the water contact angle (WCA) using a contact angle measuring device (OCA40, Dataphysics, Germany). Characterization of Biofilm Formation and Cell Viability. The isolates with positive results obtained during preliminary screen tests were further tested for their ability to form biofilms on PE film as a sole carbon source and for the cell viability in the biofilm. A cell suspension (108 cells per mL) was spread across the CFBAM plate, which was subsequently covered with the PE film sheet (50 mm × 50 mm), as described above. Three samples were tested at incubation periods of 0, 3, 7, 14, 21, and 28 days at 30 °C and 85% r.h. For the incubation of zero days, samples were collected after 3 h of inoculation. The plates without inoculation served as a sterile control. After a biofilm formed on the PE surface, the cell number of bacteria that had grown on the PE film surface was counted using the following procedure. The PE film sheet containing a biofilm was transferred into a 50 mL centrifuge tube with 40 mL of SW, and the tube was shaken on a vortex mixer for 5 min. The PE film was transferred into another centrifuge tube with 40 mL of SW, and this tube was also shaken on the vortex mixer for 5 min. The suspensions of the two tubes were then pooled in a centrifuge tube, which was centrifuged at 10,000 rpm for 15 min. The supernatant was discarded, and the pellet was resuspended in 5 mL of SW. The cell number of the suspension was counted using the series dilution method of plate counting. The biofilm on the PE film sheet was examined using a scanning electron microscopy (SEM, Quanta FEG250, FEI, USA),21 and the viability of the bacterial cells was characterized in situ using a fluorescence microscope (BX51, Olympus, Japan) after staining with the LIVE/DEAD BacLight Bacterial Viability Kit.22 Microscale Analysis of Surface Topography and Chemistry. After a 28-day incubation period, the biofilm on the PE sheet was completely removed using a washing procedure with 2% w/v aqueous sodium dodecyl sulfate (SDS) solution for 4 h.21,22 The PE sheets in the sterile control were also treated using the same procedure. Consequently, the surface topography of all PE sheets was characterized by SEM observation (Quanta FEG250, FEI, USA) and with an atomic force microscope (AFM, Dimension Icon, Veeco, USA).21,26 The surface chemical components were investigated using Xray photoelectron spectroscopy (XPS, Thermo Scientific, USA) and a microattenuated total reflectance/Fourier transform infrared imaging microscope (Nicolet iN10 MX, Thermo Scientific, USA).43 During the XPS spectra analysis, scanning was carried out over a broad-band energy range (0−1,200 eV) at an electron takeoff angle of 90° from the sample areas less than 1 mm in diameter. The overlapping peaks in the C 1s region were resolved into their individual components using a peak-fitting program (XPSPEAK v3.0). The view field of the ATR/FTIR microscope was 400 × 400 μm, and the scanning was performed at a step size of 10 μm under a contact pressure of 3 MPa. Biodegradation Assays. The PE biodegradation was characterized by the timing of its weight loss, molecular weight shift, and the formation of soluble daughter products in the LCFBM. PE pieces (100 mg) and bacterial suspensions (10 mL) were added to a 150 mL Erlenmeyer flask with 40 mL of LCFBM. The final bacterial concentration was approximately 108 cells per mL, and a flask without inoculation served as a sterile control. Three samples were prepared for each incubation period of 7, 14, 21, 28, and 60 days, and the flasks were incubated on a rotary shaker (120 rpm) at 30 °C. For each incubation period, the residual PE pieces were collected, washed using the procedure reported by Sivan et al.,21,22 and dried at ambient temperature for the measurement of residual weight. The washed PE residues collected at the end of the test were randomly sampled for molecular weight distribution analysis using the HT-GPC (PL220, Agilent, USA).23 The liquid culture was centrifuged at 10,000 rpm for 15 min, and the supernatant was filtered through a 0.22-μm membrane filter. The soluble daughter products in the filtrate were analyzed using electrospray ionization-mass spectrometry (ESI-MS, LCQ, Finnigan, USA) in the positive ion mode.44 Sequencing and Phylogenetic Analysis. The genomic DNA needed for 16S rDNA amplification of the isolated strains was extracted from cells grown in the late log phase using a conventional proteinase K treatment and phenol-chloroform extraction. Amplification of the 5′ end of the gene was performed with the universal primers 8-F (5′-AGAGTTTGATYMTGGCTCAG-3′) and 1942-R (5′-GGTTACCTTGTTACGACTT-3′). The obtained sequences were aligned with organisms present on the GenBank database using the Basic Local Alignment Search Tool (BLAST) created by the National Center for Biotechnology Information, USA. ■ RESULTS AND DISCUSSION Preliminary Screen of PE-Degrading Isolates. Eight bacterial strains were isolated during the enrichment of the gut contents of the PE-eating waxworms when using PE as a sole carbon source (SI, Figure S1). Screenings for PE-degrading microorganisms among these isolates were subsequently performed in terms of the colonization on the PE film sheets in the CFBAM plates, the changes in tensile strength of the PE sheets, the floating behavior of the PE pieces in the LCFBM, and the WCA of the PE surfaces. The Gram-positive strain YP1, identified by 16S rDNA sequencing as Bacillus sp., grew as relatively thick, opaque colonies visible to the naked eye on the PE film sheet. The Gram-negative strain YT1, identified as Enterobacter asburiae, grew as visible, thin, translucent colonies (Figure 1a and Table 1). The other six isolated bacterial strains did not develop visible colonies on the PE film sheets. In addition, no visible colonization occurred on the two control plates: a) the CFBAM with a PE film sheet but without inoculation or b) the CFBAM without a PE sheet but with inoculation. After a 28-day incubation period, all of the incubated PE film sheets were collected to test their tensile strength. The samples spread with the culture of Bacillus sp. YP1 or E. asburiae YT1 exhibited tensile strength decreases of over 50% compared to that of the sterile controls, while the samples with the other six Environmental Science & Technology Article 13778 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784
EnvironmentalScience&TechnologyArticlestudy. Sequences of the two strains were deposited in GenBank(a)bControYP1YTunder references KJ466896 to KJ466897.Biofilm Formation Process on PE Film.The biofilmformation on PE film determines its potential biodegradationbecauseabiofilmenablesthemicroorganismstoefficientlyutilize the nonsoluble substrate.2122The biofilm formationprocesses by the two select strains on the PE film in this studywere characterized by cell number counting,morphotypes, andthe biofilm's viability.The biofilm development on the PE film sheet in a CFBAMplate was monitored by counting the number of cells colonizingon the PE film sheet via series dilution during the 28-dayincubation.The results showed that both strains almostimmediately adhered to the PE film sheets and built detectablebiofilms within the first 3 h of incubation (Figures 2a-c).The(C)97.2±1.6969.3±3.8%67.1±1.6cell density of the initial biofilm of strain YT1 was 3.8X 107CFU/cm2.Thecelldensityof strainYP1was1.4×106CFU/cm’, which was approximately 1 order of magnitude lower thanthat of strain YT1.The biofilm densities of strains YT1 andYP1increased from day 3 to day 21, reaching mature biofilms of16.0and9.4×107CFU/cm2,respectively,onday28(FiguresFigure 1. Assay of the strains YT1 and YP1 isolated for their PE-2b and c).degrading activity. (a)Formation of colonies on the PE film sheets inAt the end of the 28-day incubation, the biofilm morphotypeCFBAM plates. No colony was present on the sterile control. (b) ThePE pieces became suspended in the inoculated liquid medium, but thewasobserved using SEM.Thecells of strain YT1wererodscontrol remained unchanged.()Decreased water contactanglesmeasuring 0.2 × 0.8 μm.This strain developed a denser biofilm(WCA) of PE samples after inoculation.but produced less extracellular polymeric substance(EPS)thanthe cells of strain YP1 (Figures 2e-f). The cells of strain YP1were mainly short irregular rods measuring 0.5 × 3 μm, andisolated bacterial strains exhibited no reduction in tensilefewer spore-like cells were observed in its biofilm (Figure 2f).strength (SI,Figure S2)The viability of the biofilms'bacterial cells grown on the PEAt the beginning of incubation, the PE pieces floated in thefilm was estimated in situ using a fluorescent microscope afterupper portion of the liquid medium because its density (0.915-staining with the LIVE/DEAD BacLight Bacterial Viability0.940g/cm)waslower than thatof water (1g/cm*) (FigureKit.21 Under fluorescence (Figure 2g-i), the live cells (green)Ib).After a 28-day incubation period with strains YT1 anddominated in the biofilms of the two strains, and only a limitedYP1, the PE pieces did not float and became suspended in thenumberofdeadcells(red)wasobserved.ThepredominanceofLCFBM, whereas the PE pieces in the sterile controlslive cells in the biofilm indicated that these cells receivedcontinued to float (Figure 1b).The PE film pieces incubatedenoughgrowth substrate,21226 which likely camefrom thewith the other six strains also continued to float in the liquidmetabolism of the PE material and its daughter products.medium (data not shown).This result was likely because theChanges inPhysicochemical SurfaceProperties.Aftercells in strains YT1 and YPI were able to adhesively grow ona 28-day incubation period, the changes in the physicochemicalthe PE species, and this cell adhesion increased the weight ofsurface properties of the incubated PE film sheets on thethePEspecies,resultingintheformationofasuspensionCFBAM plates were observed after the biofilm was completelyAn analysis of the water contact angles of the PE piecesremoved from the PE samples. The change in surfaceshowed that the WCAs of the samples inoculated with strainstopography was examined using SEM and AFM, and theYT1andYP1were69.3±3.8and67.1±1.6°(n=5)surface deterioration was estimated based on the formation ofrespectively,which were much lower than the control WCAofpits and cavities observed on the surfaces of the PE samples97.2 ± 1.6° (n = 5) (Figure lc). The other six isolated strainswith bacterial inoculation (Figures 3c-f). The surfaces of thedid not significantly change the WCA of the PE pieces (datacontrols remained smooth, without any defects (Figures 3a-b).not shown). The results indicated that the inoculation ofThe cavities on the surfaces of the PE sheets had maximumbacterial strainsYT1andYPldecreasedthehvdrophobicityofthe tested PE materials. As the PE materials became lessdepths of approximately 0.3 and 0.4 μm after inoculation withhydrophobic, they became less resistant to subsequentstrains YT1 and YP1, respectively (Figures 3d and f). This typedegradation by the bacterial cells.26of deep cavity has not been reported by other researchersBased on these results, we selected the E.asburiae strain YT1working on the characterization of PE-degrading microorganisms.2631The results obtained from the microscaleand Bacillus sp. strain YP1 as potential PE-degraders for furtherTable 1.Isolation and Identification of PE-Degrading Bacterial Strainsstrainacolorisolation mediumhighest-homology organismmaximum identity, %Gramsize,μmYTI99yellow0.2 × 0.8BPAEnterobacter asburiae-+YP199PDABaillus sp.yellow0.5 × 3aThe two strains have been preserved at the China General Microbiological Culture Collection Center (CGMCCC) in Bejing, China as patentedcultures. °BPA = beef extract-peptone agar; PDA = potato dextrose agar.13779dx.doi.org/10.1021/es504038alEmviron.Sc.Technol. 2014,48,1377613784
isolated bacterial strains exhibited no reduction in tensile strength (SI, Figure S2) At the beginning of incubation, the PE pieces floated in the upper portion of the liquid medium because its density (0.915− 0.940 g/cm3 ) was lower than that of water (1 g/cm3 ) (Figure 1b). After a 28-day incubation period with strains YT1 and YP1, the PE pieces did not float and became suspended in the LCFBM, whereas the PE pieces in the sterile controls continued to float (Figure 1b). The PE film pieces incubated with the other six strains also continued to float in the liquid medium (data not shown). This result was likely because the cells in strains YT1 and YP1 were able to adhesively grow on the PE species, and this cell adhesion increased the weight of the PE species, resulting in the formation of a suspension. An analysis of the water contact angles of the PE pieces showed that the WCAs of the samples inoculated with strains YT1 and YP1 were 69.3 ± 3.8° and 67.1 ± 1.6° (n = 5), respectively, which were much lower than the control WCA of 97.2 ± 1.6° (n = 5) (Figure 1c). The other six isolated strains did not significantly change the WCA of the PE pieces (data not shown). The results indicated that the inoculation of bacterial strains YT1 and YP1 decreased the hydrophobicity of the tested PE materials. As the PE materials became less hydrophobic, they became less resistant to subsequent degradation by the bacterial cells.26 Based on these results, we selected the E. asburiae strain YT1 and Bacillus sp. strain YP1 as potential PE-degraders for further study. Sequences of the two strains were deposited in GenBank under references KJ466896 to KJ466897. Biofilm Formation Process on PE Film. The biofilm formation on PE film determines its potential biodegradation because a biofilm enables the microorganisms to efficiently utilize the nonsoluble substrate.21,22 The biofilm formation processes by the two select strains on the PE film in this study were characterized by cell number counting, morphotypes, and the biofilm’s viability. The biofilm development on the PE film sheet in a CFBAM plate was monitored by counting the number of cells colonizing on the PE film sheet via series dilution during the 28-day incubation. The results showed that both strains almost immediately adhered to the PE film sheets and built detectable biofilms within the first 3 h of incubation (Figures 2a-c). The cell density of the initial biofilm of strain YT1 was 3.8 × 107 CFU/cm2 . The cell density of strain YP1 was 1.4 × 106 CFU/ cm2 , which was approximately 1 order of magnitude lower than that of strain YT1. The biofilm densities of strains YT1 and YP1 increased from day 3 to day 21, reaching mature biofilms of 16.0 and 9.4 × 107 CFU/cm2 , respectively, on day 28 (Figures 2b and c). At the end of the 28-day incubation, the biofilm morphotype was observed using SEM. The cells of strain YT1 were rods measuring 0.2 × 0.8 μm. This strain developed a denser biofilm but produced less extracellular polymeric substance (EPS) than the cells of strain YP1 (Figures 2e-f). The cells of strain YP1 were mainly short irregular rods measuring 0.5 × 3 μm, and fewer spore-like cells were observed in its biofilm (Figure 2f). The viability of the biofilms’ bacterial cells grown on the PE film was estimated in situ using a fluorescent microscope after staining with the LIVE/DEAD BacLight Bacterial Viability Kit.21 Under fluorescence (Figure 2g-i), the live cells (green) dominated in the biofilms of the two strains, and only a limited number of dead cells (red) was observed. The predominance of live cells in the biofilm indicated that these cells received enough growth substrate,21,22,26 which likely came from the metabolism of the PE material and its daughter products. Changes in Physicochemical Surface Properties. After a 28-day incubation period, the changes in the physicochemical surface properties of the incubated PE film sheets on the CFBAM plates were observed after the biofilm was completely removed from the PE samples. The change in surface topography was examined using SEM and AFM, and the surface deterioration was estimated based on the formation of pits and cavities observed on the surfaces of the PE samples with bacterial inoculation (Figures 3c-f). The surfaces of the controls remained smooth, without any defects (Figures 3a-b). The cavities on the surfaces of the PE sheets had maximum depths of approximately 0.3 and 0.4 μm after inoculation with strains YT1 and YP1, respectively (Figures 3d and f). This type of deep cavity has not been reported by other researchers working on the characterization of PE-degrading microorganisms.26,31 The results obtained from the microscale Figure 1. Assay of the strains YT1 and YP1 isolated for their PEdegrading activity. (a) Formation of colonies on the PE film sheets in CFBAM plates. No colony was present on the sterile control. (b) The PE pieces became suspended in the inoculated liquid medium, but the control remained unchanged. (c) Decreased water contact angles (WCA) of PE samples after inoculation. Table 1. Isolation and Identification of PE-Degrading Bacterial Strains straina highest-homology organism maximum identity, % Gram color size, μm isolation mediumb YT1 Enterobacter asburiae 99 − yellow 0.2 × 0.8 BPA YP1 Bacillus sp. 99 + yellow 0.5 × 3 PDA a The two strains have been preserved at the China General Microbiological Culture Collection Center (CGMCCC) in Beijing, China as patented cultures. b BPA = beef extract-peptone agar; PDA = potato dextrose agar. Environmental Science & Technology Article 13779 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784
Environmental Science&TechnologyAricle(b)(c)YT1YP1(a)Control202020soanoo10106550n00610152030251715202510152025Time (days)Time (days)Time (days)(d)22μm2μm(g)30μmFigure 2. Biofilm formation and viability. (a-c) Changes in bacterial cell number on the PE film sheets. (d-f) Morphotypes of the cells in the maturebiofilm on the PE sheet. (g-i) Fluorescent microscopic images of biofilms, which show cell viability after the 28-day incubation. Live cells are greenand dead cells are red. The PE sheets were covered in CFBAM.(Figures 4c-e) were constructed based on the transmittancesurface topography indicated that the two strains did causedamage to the PE's physical integrity.of carbonyl bands (1715 cm-'), with pixels ranging in colorXPS and micro-ATR/FTIR imaging were used to analyze thefromblue to red (transmittance:100-96%).Compared to thechanges to the surface chemical components and functioncontrols, the surfaces of the inoculated samples showedgroups.Figure 4a shows the XPS spectra of C Is in theincreased amounts of color ranging from light blue to redinoculated PE sheet surface versus the sterile control. Theindicating heterogeneous oxidation by the bacterial strains.peaking-fitting result of C 1s for the control showed only oneA previous study on PE degradation after pretreatment haspeak at 284.8 eV, which was attributed toa -C-C-group indemonstrated that the appearance of carbonyl groups is anthe long chain skeleton of PE (Figure 4a). In the incubatedessential sign of PE biodegradation.19Wasserbauer et al.havesamples, in addition to the peak at 284.8 eV, another peakindicated that PE foils, which were exposed to bacterialappeared at 288.2 eV and was assigned to the carbonyl bands(Bacillus brevis andPseudomonas putida)andliver homogenates,(-C-O-), which is generally considered suggestive of PEshowed oxidation structures and signs of carbonyl-like groupsoxidation(Figure4a).177Accordingly,the O/C ratios andin FTIR spectra*$ However, these researchers have notrelative abundance of -C-O- group peaks in the incubatedreported follow-up work on PE degradation. To the best ofsamples are remarkably higher than those of the control (Tableourknowledge,noother researchers havereportedthe2).appearanceof carbonyl groups in theXPSandFTIR spectraThe ATR/FTIR spectra of the inoculated samples surfacesof PE foils or films inoculated with microorganisms.In thiswere different from that of the control (Figure 4b).All samplesstudy,we observed an increasing number of carbonyl grouphad a common peak at 1450 cm-, which was attributed to thepeaks in the samples inoculated with strains YT1 and YP1 usingC-H bending vibration in the PE's long chain skeletonXPS spectra and micro-ATR/FTIR image analysis, indicatingHowever, the inoculated samples had a peak at 1715 cm-1thattheisolatedstrainswerecapableof attackingoroxidizingwhich was assigned to the carbonyl bands (-C-O-). Thisobservation was consistent with the XPs spectra resultsPE structures to produce carbonyl groups without anydescribedabove (Figure4a). Micro-ATR/FTIRimagespretreatment, such as photo- or thermo-oxidation.13780dx.dol.org/10.1021/es504038alEmviron.Sc.Technol. 2014,48, 1377613784
surface topography indicated that the two strains did cause damage to the PE’s physical integrity. XPS and micro-ATR/FTIR imaging were used to analyze the changes to the surface chemical components and function groups. Figure 4a shows the XPS spectra of C 1s in the inoculated PE sheet surface versus the sterile control. The peaking-fitting result of C 1s for the control showed only one peak at 284.8 eV, which was attributed to a −C−C− group in the long chain skeleton of PE (Figure 4a). In the incubated samples, in addition to the peak at 284.8 eV, another peak appeared at 288.2 eV and was assigned to the carbonyl bands (−CO−), which is generally considered suggestive of PE oxidation (Figure 4a).17 Accordingly, the O/C ratios and relative abundance of −CO− group peaks in the incubated samples are remarkably higher than those of the control (Table 2). The ATR/FTIR spectra of the inoculated samples’ surfaces were different from that of the control (Figure 4b). All samples had a common peak at 1450 cm−1 , which was attributed to the C−H bending vibration in the PE’s long chain skeleton. However, the inoculated samples had a peak at 1715 cm−1 , which was assigned to the carbonyl bands (−CO−). This observation was consistent with the XPS spectra results described above (Figure 4a). Micro-ATR/FTIR images (Figures 4c-e) were constructed based on the transmittance of carbonyl bands (1715 cm−1 ), with pixels ranging in color from blue to red (transmittance: 100−96%). Compared to the controls, the surfaces of the inoculated samples showed increased amounts of color ranging from light blue to red, indicating heterogeneous oxidation by the bacterial strains. A previous study on PE degradation after pretreatment has demonstrated that the appearance of carbonyl groups is an essential sign of PE biodegradation.19 Wasserbauer et al. have indicated that PE foils, which were exposed to bacterial (Bacillus brevis and Pseudomonas putida) and liver homogenates, showed oxidation structures and signs of carbonyl-like groups in FTIR spectra.45 However, these researchers have not reported follow-up work on PE degradation. To the best of our knowledge, no other researchers have reported the appearance of carbonyl groups in the XPS and FTIR spectra of PE foils or films inoculated with microorganisms. In this study, we observed an increasing number of carbonyl group peaks in the samples inoculated with strains YT1 and YP1 using XPS spectra and micro-ATR/FTIR image analysis, indicating that the isolated strains were capable of attacking or oxidizing PE structures to produce carbonyl groups without any pretreatment, such as photo- or thermo-oxidation. Figure 2. Biofilm formation and viability. (a-c) Changes in bacterial cell number on the PE film sheets. (d-f) Morphotypes of the cells in the mature biofilm on the PE sheet. (g-i) Fluorescent microscopic images of biofilms, which show cell viability after the 28-day incubation. Live cells are green and dead cells are red. The PE sheets were covered in CFBAM. Environmental Science & Technology Article 13780 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784
Environmental Science&TechnologyArticle(b)(a)200nma0nm4.0 μm4.0um￥3.03.02.02.01.01.02μmX(d)IC200nmOnA4.8 μm4.8um363.62.42.41.21.2(f)200 nmOnnIOA404.0um3.03.02.02.01.01.0Figure 3. SEM and AFM observations of the physical surface topography of the sterile control versus the PE sheets incubated with strains YT1 andYP1 after 28 days.Weight Loss and Molecular Weight Decrease of PEresults in a net loss in the samples of only up to 5%, based on26,28 In thisSamples.PE's degradation efficiency can be directly measuredour estimate of their 60-day removal efficiencyby the weight loss of samples, while the material'sstudy,theincubation withYTl resulted in anet weightloss ofdepolymerization can be documented by the shift of its6.1±0.3%.Todate, this is the first reported Enterobacter strainmolecular weight and the release of lower molecular weightwith the ability to degrade PE in the literature. The growthcompounds.yields of YP1 and YT1 on PE as a sole carbon source wereThe weight loss over time of the PE samples inoculated withestimated to be 0.82 ± 0.05 and 0.66 ± 0.04 g cells per g PE,the two strains is presented in Figure Sa. During a 60-dayrespectivelyThe molecular weight and molecular weight distributionincubationperiod inLCFBM,theweightloss of the PE(MWD)of thePE samples after a 60-day incubation werespecimens with inoculation increased consistently,whereas thatof the controls did not (Figure Sa). On day 60, the incubationdetermined using HT-GPC. The MWDs of the PE sampleswith strain YP1 resulted in a net loss in the PE sample of 10.7±incubatedwith strainsYT1 andYP1showed a clearnegative0.2%, which was higher than the 7.5% weight loss reported overtrend compared to the controls (Figure Sb). The moleculara 56-day period for Rhodococcus rubber C208, a previouslyweights (M./M.) of the incubated PE samples containingstudied PE-degrading strain.21-23 Other researchers havestrainsYT1andYP1were82,500/24,700and78,200/23,900,reported that incubation with four PE-degrading Bacillus strainsrespectively, which represents a ~6-13% reduction from that13781dx.dol.org/10.1021/es504038alEmviron.Sc.Technol. 2014,48, 1377613784
Weight Loss and Molecular Weight Decrease of PE Samples. PE’s degradation efficiency can be directly measured by the weight loss of samples, while the material’s depolymerization can be documented by the shift of its molecular weight and the release of lower molecular weight compounds. The weight loss over time of the PE samples inoculated with the two strains is presented in Figure 5a. During a 60-day incubation period in LCFBM, the weight loss of the PE specimens with inoculation increased consistently, whereas that of the controls did not (Figure 5a). On day 60, the incubation with strain YP1 resulted in a net loss in the PE sample of 10.7 ± 0.2%, which was higher than the 7.5% weight loss reported over a 56-day period for Rhodococcus rubber C208, a previously studied PE-degrading strain.21−23 Other researchers have reported that incubation with four PE-degrading Bacillus strains results in a net loss in the samples of only up to 5%, based on our estimate of their 60-day removal efficiency.26,28 In this study, the incubation with YT1 resulted in a net weight loss of 6.1 ± 0.3%. To date, this is the first reported Enterobacter strain with the ability to degrade PE in the literature. The growth yields of YP1 and YT1 on PE as a sole carbon source were estimated to be 0.82 ± 0.05 and 0.66 ± 0.04 g cells per g PE, respectively. The molecular weight and molecular weight distribution (MWD) of the PE samples after a 60-day incubation were determined using HT-GPC. The MWDs of the PE samples incubated with strains YT1 and YP1 showed a clear negative trend compared to the controls (Figure 5b). The molecular weights (Mw/Mn) of the incubated PE samples containing strains YT1 and YP1 were 82,500/24,700 and 78,200/23,900, respectively, which represents a ∼6−13% reduction from that Figure 3. SEM and AFM observations of the physical surface topography of the sterile control versus the PE sheets incubated with strains YT1 and YP1 after 28 days. Environmental Science & Technology Article 13781 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784
AricleEnvironmental Science&Technology(a)(b)-C-C-Control1450(s) sunoC=O-YT11715-C=OYP1(T1YP1Control120090029028828628428228018001500Wavenumbers (cm*1)Binding Energy (eV)(c)(d)YT1YP1Control(e)营营oan ameoaeomy onESousodT0Position (um)Position(um)osition(um96%97%98%99%100%TransmittanceFigure 4. Surface chemical analysis of the sterile control and PE samples after the 28-day incubation with strains YP1 and YT1. Samples withbacterial strains had -C-=O-bonds, but the control did not. (a) XPS spectra of theC 1s of PE samples. (b)ATR-FTIR spectra of PE samples. (c-e) Distribution of carbonyl bands (1715 cm-l) imaged with a micro-FTIR microscope with pixels of 1 point per 10 μm.The changes in colorindicate the heterogeneous oxidation of PE samples by the bacterial strains.Table2.O/CAtomicRatiosandRelativeClsPeakAreasofother 12compounds were more abundant in the PE samples-C-C- and -C-O- Groupsinoculated with strains YT1 and YP1 (SI, Table S2). Amongthese12 compounds, four with an m/z range between 400 andrelative area (%)500 were ten times more abundant in all incubated samplesmaterialO/C-C-C--C=O-than in the control (SI, Table S2).These potential daughtercontrol0.021000products occurred in the media containing an inoculation but0.10973treated by YT1were absent in the control. Although the bacterial cells could4treated by YP10.1196secrete a diverse mixture of secondary metabolic products inthe medium, causing peaks, the ESI-MS results stll provideadditional information on the occurrence of PE's heterotrophicof the control (Figure Sb internal figure).The decrease inmicrobial metabolism.molecular weight suggested that depolymerization/cleavage ofIn summary, the weight loss and molecular weight decreasethe PE's long chain structure occurred, and lower molecularof the PE samples and the appearance of daughter productsweight fragments were formed in the presence of these bacterialindicate that the bacterial strains isolated from waxworm gutsstrainswere capable of degrading PE.The formation of water-soluble daughter products wasImplications.To date, photo- and thermal-based pretreat-characterized by ESI-MS.+ The control exhibited a simplements have been considered a critical step in thespectrumwithonlytwopeaksatm/z110.1and130.1.Inthebiodegradation of PE, and the studies of PE-degradingsamples inoculated with the two strains, 12 new peaksmicroorganisms have focused on plastic-contaminated environ-appeared, covering an m/z range from 100 to 600 (Figurements, such as soils, compost, landfills, and marine environ-5c).Most ofthese compounds containedelements ofC,H, andments.2 This study is the first to report the presence of PE-O in their assumed formulas (SI, Table S2). Fold-changedegrading bacteria in theguts of waxworms, which eat PE films.analysis was used to identify these14 compounds withPE degradation was confirmed not only by thebacterial growthdifferential abundances in each of the incubated samples versusthe products released from the controls (SI, Table S2).Inon PEfilms,whichacted as solecarbon sourceandthePEaddition to the two compounds at m/z 110.1 and 130.1, thesubstrate's weight loss, but also by identification of key13782dx.dol.org/10.1021/es504038alEmviron.Sc.Technol. 2014, 48, 1377613784
of the control (Figure 5b internal figure). The decrease in molecular weight suggested that depolymerization/cleavage of the PE’s long chain structure occurred, and lower molecular weight fragments were formed in the presence of these bacterial strains. The formation of water-soluble daughter products was characterized by ESI-MS.44 The control exhibited a simple spectrum with only two peaks at m/z 110.1 and 130.1. In the samples inoculated with the two strains, 12 new peaks appeared, covering an m/z range from 100 to 600 (Figure 5c). Most of these compounds contained elements of C, H, and O in their assumed formulas (SI, Table S2). Fold-change analysis was used to identify these 14 compounds with differential abundances in each of the incubated samples versus the products released from the controls (SI, Table S2). In addition to the two compounds at m/z 110.1 and 130.1, the other 12 compounds were more abundant in the PE samples inoculated with strains YT1 and YP1 (SI, Table S2). Among these 12 compounds, four with an m/z range between 400 and 500 were ten times more abundant in all incubated samples than in the control (SI, Table S2). These potential daughter products occurred in the media containing an inoculation but were absent in the control. Although the bacterial cells could secrete a diverse mixture of secondary metabolic products in the medium, causing peaks, the ESI-MS results still provide additional information on the occurrence of PE’s heterotrophic microbial metabolism. In summary, the weight loss and molecular weight decrease of the PE samples and the appearance of daughter products indicate that the bacterial strains isolated from waxworm guts were capable of degrading PE. Implications. To date, photo- and thermal-based pretreatments have been considered a critical step in the biodegradation of PE, and the studies of PE-degrading microorganisms have focused on plastic-contaminated environments, such as soils, compost, landfills, and marine environments.2 This study is the first to report the presence of PEdegrading bacteria in the guts of waxworms, which eat PE films. PE degradation was confirmed not only by the bacterial growth on PE films, which acted as sole carbon source and the PE substrate’s weight loss, but also by identification of key Figure 4. Surface chemical analysis of the sterile control and PE samples after the 28-day incubation with strains YP1 and YT1. Samples with bacterial strains had −CO− bonds, but the control did not. (a) XPS spectra of the C 1s of PE samples. (b) ATR-FTIR spectra of PE samples. (ce) Distribution of carbonyl bands (1715 cm−1 ) imaged with a micro-FTIR microscope with pixels of 1 point per 10 μm. The changes in color indicate the heterogeneous oxidation of PE samples by the bacterial strains. Table 2. O/C Atomic Ratios and Relative C 1s Peak Areas of −C−C− and −CO− Groups relative area (%) material O/C −C−C− −CO− control 0.02 100 0 treated by YT1 0.10 97 3 treated by YP1 0.11 96 4 Environmental Science & Technology Article 13782 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784
AricleEnvironmental Science&Technology(a) (c) 2.0Control1.521.04680.50.0eontre+YT12.0-YP1YT11.5o102030405060M1.0Time (days)0 0.8453.2(b)Control174.1 202.8.41592140.08--YT1213.3274.498.6.YP10.60.0,012.0+460.7BRYP11.50.21.04150.5274.4318.0213.30.05.55.04.54.03.5200400600logMwm/zFigure 5. Characterization of PE biodegradation by strains YT1 and YPI in LCFBM. (a) Changes in the dry weight of the PE specimens over the 60-day period (mean value ± SD, n = 3). (b) Molecular weight distribution (MWD) and molecular weight (weight-averaged molecular weight (Mw)/number-averaged molecular weight (Mn) shift (internal figure) of the PE residues versus the controls after 60 days. (c) ESI-MS analysis showingdegraded oligomeric intermediates in the PE residues after 60 days of incubation versus the controls.reactions similar to those in photo- and thermal-basedAUTHORINFORMATIONpretreatments,suchaschangesinsurfacetopography,decreasesCorresponding Authorin hydrophobicity, the formation of carbonyl groups, decrease*Phone/Fax: 86-10-8233-8552.E-mail: yangjun@buaaedu.cn.in molecular weight, and release of soluble daughter products.Corresponding author address:School of Chemistry andThe soluble daughter products were not identified in this study,Environment, Beihang University (BUAA),37Xueyuan Road,and future study will be needed to identify the daughter and/orBeijing 100191, P. R China.intermediate products of PE degradation and the associatedAuthor Contributionsmetabolic pathways. Further research should also focus on theI'j.Y. and Y.Y. contributed equally to this work.ecophysiological relationship between the PE-degrading bac-Notesteria and waxworms, which may be mutually beneficial, similarThe authors declare no competing financial interest.to the microbial symbionts in cow rumens.The discovery of thepresence of PE-degradingmicroorganisms in waxworm guts hasACKNOWLEDGMENTSprovided a promising direction for further study of theThis research was supported by the National Natural Sciencemicrobial degradation of both PE and other plastics, alongFoundation of China (51373006, 20477002),the State Basicwith new insights into the life cycle of synthetic plastics inResearch Program of China (2014CB931800),and thenature. In addition, the isolation and characterization of moreShenzhenKeyLaboratory of Bioenergy(grantPE-degrading microorganisms from this source and a betterCXB201005240001A).TheauthorsthankMs.AndriaWuforunderstanding of the enzymatic system involved in PEher help with manuscript preparation.degradation could be helpful in the development ofremediation approaches for plastic wastes, which couldREFERENCESeliminate plastic pollution concerns.(1) Yang, J, Song, Y. L., Qin, X. Y. Biodegradation of polyethylene.Environ. Sci. (in Chinese)2007, 28 (5), 1165-1168.(2) Restrepo-Florez, J. M; Bassi, A., Thompson, M. R. MicrobialASSOCIATEDCONTENT第degradation and deterioration of polyethylene-A review. Int.SupportingInformationBiodeterior. Biodegrad. 2014, 88, 83-90.Summary of the updated reported virgin PE-degrading(3) Roy, P. K; Hakkarainen, M, Varma, I K; Albertsson, A. C.Degradable polyethylene: fantasy or reality.Environ. Sci. Technol. 2011,microorganisms (Table S1). Fold-change analysis of the45 (10), 4217-4227.compounds released from the biodegradation of PE (Table(4) Zettler, E. R; Mincer, T. J; Amaral-Zettler, L. A. Life in theS2). Photograph of waxworms eating PE film (Figure S1)."Plastisphere":microbial communities on plastic marine debris.Changes in tensile strength of the PE film samples incubatedEnviron.Sci.Technol. 2013, 47 (13),7137-7146.with eight bacterial strains (Figure S2). This material is($) Ammala, A; Bateman, S., Dean, K., Petinakis, E, Sangwan, P.;available free of charge via the Internet at http://pubs.acs.org.Wong, S.;Yuan, Q;Yu, L.; Patrick, C.,Leong, KH. An overview of13783dx.dol.org/10.1021/es504038alEmviron.Sc.Technol. 2014,48, 1377613784
reactions similar to those in photo- and thermal-based pretreatments, such as changes in surface topography, decreases in hydrophobicity, the formation of carbonyl groups, decrease in molecular weight, and release of soluble daughter products. The soluble daughter products were not identified in this study, and future study will be needed to identify the daughter and/or intermediate products of PE degradation and the associated metabolic pathways. Further research should also focus on the ecophysiological relationship between the PE-degrading bacteria and waxworms, which may be mutually beneficial, similar to the microbial symbionts in cow rumens. The discovery of the presence of PE-degrading microorganisms in waxworm guts has provided a promising direction for further study of the microbial degradation of both PE and other plastics, along with new insights into the life cycle of synthetic plastics in nature. In addition, the isolation and characterization of more PE-degrading microorganisms from this source and a better understanding of the enzymatic system involved in PE degradation could be helpful in the development of remediation approaches for plastic wastes, which could eliminate plastic pollution concerns. ■ ASSOCIATED CONTENT *S Supporting Information Summary of the updated reported virgin PE-degrading microorganisms (Table S1). Fold-change analysis of the compounds released from the biodegradation of PE (Table S2). Photograph of waxworms eating PE film (Figure S1). Changes in tensile strength of the PE film samples incubated with eight bacterial strains (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org. ■ AUTHOR INFORMATION Corresponding Author *Phone/Fax: 86-10-8233-8552. E-mail: yangjun@buaa.edu.cn. Corresponding author address: School of Chemistry and Environment, Beihang University (BUAA), 37 Xueyuan Road, Beijing 100191, P. R. China. Author Contributions ∥ J.Y. and Y.Y. contributed equally to this work. Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (51373006, 20477002), the State Basic Research Program of China (2014CB931800), and the Shenzhen Key Laboratory of Bioenergy (grant CXB201005240001A). The authors thank Ms. Andria Wu for her help with manuscript preparation. ■ REFERENCES (1) Yang, J.; Song, Y. L.; Qin, X. Y. Biodegradation of polyethylene. Environ. Sci. (in Chinese) 2007, 28 (5), 1165−1168. (2) Restrepo-Florez, J. M.; Bassi, A.; Thompson, M. R. Microbial ́ degradation and deterioration of polyethylene-A review. Int. Biodeterior. Biodegrad. 2014, 88, 83−90. (3) Roy, P. K.; Hakkarainen, M.; Varma, I. K.; Albertsson, A. C. Degradable polyethylene: fantasy or reality. Environ. Sci. Technol. 2011, 45 (10), 4217−4227. (4) Zettler, E. R.; Mincer, T. J.; Amaral-Zettler, L. A. Life in the “Plastisphere”: microbial communities on plastic marine debris. Environ. Sci. Technol. 2013, 47 (13), 7137−7146. (5) Ammala, A.; Bateman, S.; Dean, K.; Petinakis, E.; Sangwan, P.; Wong, S.; Yuan, Q.; Yu, L.; Patrick, C.; Leong, K. H. An overview of Figure 5. Characterization of PE biodegradation by strains YT1 and YP1 in LCFBM. (a) Changes in the dry weight of the PE specimens over the 60- day period (mean value ± SD, n = 3). (b) Molecular weight distribution (MWD) and molecular weight (weight-averaged molecular weight (Mw)/ number-averaged molecular weight (Mn)) shift (internal figure) of the PE residues versus the controls after 60 days. (c) ESI-MS analysis showing degraded oligomeric intermediates in the PE residues after 60 days of incubation versus the controls. Environmental Science & Technology Article 13783 dx.doi.org/10.1021/es504038a | Environ. Sci. Technol. 2014, 48, 13776−13784
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