OVIRONMENTAlArticlepubs.acs.org/estScience & lechnologyBiodegradationand Mineralization of Polystyreneby Plastic-EatingMealworms:Part 1.Chemical andPhysical CharacterizationandIsotopicTestsYu Yang, Jun Yang,*,t Wei-Min Wu, Jiao Zhao, Yiling Song," Longcheng Gao, Ruifu Yang,and Lei JiangfKey Laboratory of Bio-Inspired Smart Interfacial Science and Technology of Ministry of Education, School of Chemistry andEnvironment, and "school of Biological Science and Medical Engineering, Beihang University, Beijing 100191, People's Republic ofChina+Department of Civil and Environmental Engineering, William & Cloy Codiga Resource Recovery Research Center, CenterforSustainableDevelopment&Global Competitiveness,StanfordUniversity,Stanford,California94305-4020,United States‘Shenzhen Key Laboratory of Bioenergy, BGI-Shenzhen, Shenzhen, Guangdong 518083, People's Republic of ChinaSupporting InformationABSTRACT:Polystyrene (PS) is generally considered to beStyrofoam-chewingmeHworndurable and resistant to biodegradation.Mealworms (the10larvae of Tenebrio molitor Linnaeus) from different sourceschewandeatStyrofoam,a:commonPsproduct.TheoStyrofoam was efficiently degradedin thelarvalgutwithinaretention time of less than 24 h.Fed with Styrofoam as the6sole diet,the larvae lived as well asthose fed with a normal diet.suodo(bran) over a period of 1 month.The analysis of fecula egestedINIfrom Styrofoam-feeding larvae, using gel permeation chroma-tography (GPC), solid-state 13C cross-polarization/magicasangle spinningnuclear magnetic resonance(CP/MASNMR)Suptospectroscopy,and thermogravimetric Fourier transform infra-red (TG-FTIR) spectroscopy, substantiated that cleavage/depolymerization of long-chain Ps molecules and theformation of depolymerized metabolites occurred in the larval gut. Within a 16 day test period, 47.7% of the ingested司les/aosoesind//schyaosHStyrofoam carbon was converted into CO2 and the residue (ca. 49.2%) was egested as fecula with a limited fraction incorporatedinto biomass (ca. 0.5%). Tests with α 13c- or β 13C-labeled PS confrmed that the 13c-labeled PS was mineralized to 13CO, andincorporated into lipids.The discovery of the rapid biodegradation of Ps in the larval gut reveals a newfatefor plastic waste inthe environment.++fPrevious investigations have used tc-labeled PS tracers addedINTRODUCTIONto a variety of mixed microbial consortia from soil, sewageThe current global consumption of petroleum-based syntheticsludge, decaying garbage, or manure.8-10 The recovery ofplastic is approximately 299 Mt/year. Polystyrene (PS),14cO, ranged from 0.01% to less than 3% over periods of1-4molecular formula[-CH(CH,)CH2-], commonly knownas Styrofoam,accounted for approximately7.1% (21Mt/year)months, which does not yet constitute convincing results of theof the total plastic consumption in 20i3.Although PS isbiodegradation of PS because PS may contain a small fractionconsidered a durable plastic, PS products are often designed forof impurities, such as styrene.8-10 Although a few strains ofashortservicetimeand one-timeuseasaresultofthelowcostpure bacteria isolated from soils were capable of colonizing PSof this material.The sharp contrastbetween the remarkablesurfaces, the isolates have not proven that these bacteria weredurability of PS and the short service time of PS products haseffective in the biodegradation of PS, changing neither theled to the increasing accumulation of PSwaste in ourphysical nor chemical properties of its long-chain molecules.environment. Most of the collected PS waste is disposedFurther, no traces ofmetabolic activity werefound.2along with municipal solid waste in landills.2 Even moreproblematic is that a great amount of PS debris is also dispersedas “white pollutants"in the environment, becoming a globalReceived:May31,2015environmental concern.Revised:September 5,2015To date, it has generally been thought that PS is not subjectAccepted:September 21,2015Published:September 21,2015to biodegradation by microorganisms and soil invertebrates.12080AcsPublications 2015 American Chemical SocietyDOt: 10.1021/acsEmviron. Sci. Technol. 2015, 49, 1208012086
Biodegradation and Mineralization of Polystyrene by Plastic-Eating Mealworms: Part 1. Chemical and Physical Characterization and Isotopic Tests Yu Yang,† Jun Yang,*,† Wei-Min Wu,‡ Jiao Zhao,§ Yiling Song,∥ Longcheng Gao,† Ruifu Yang,§ and Lei Jiang*,† † Key Laboratory of Bio-Inspired Smart Interfacial Science and Technology of Ministry of Education, School of Chemistry and Environment, and ∥ School of Biological Science and Medical Engineering, Beihang University, Beijing 100191, People’s Republic of 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, People’s Republic of China *S Supporting Information ABSTRACT: Polystyrene (PS) is generally considered to be durable and resistant to biodegradation. Mealworms (the larvae of Tenebrio molitor Linnaeus) from different sources chew and eat Styrofoam, a common PS product. The Styrofoam was efficiently degraded in the larval gut within a retention time of less than 24 h. Fed with Styrofoam as the sole diet, the larvae lived as well as those fed with a normal diet (bran) over a period of 1 month. The analysis of fecula egested from Styrofoam-feeding larvae, using gel permeation chromatography (GPC), solid-state 13C cross-polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR) spectroscopy, and thermogravimetric Fourier transform infrared (TG−FTIR) spectroscopy, substantiated that cleavage/ depolymerization of long-chain PS molecules and the formation of depolymerized metabolites occurred in the larval gut. Within a 16 day test period, 47.7% of the ingested Styrofoam carbon was converted into CO2 and the residue (ca. 49.2%) was egested as fecula with a limited fraction incorporated into biomass (ca. 0.5%). Tests with α 13C- or β 13C-labeled PS confirmed that the 13C-labeled PS was mineralized to 13CO2 and incorporated into lipids. The discovery of the rapid biodegradation of PS in the larval gut reveals a new fate for plastic waste in the environment. ■ INTRODUCTION The current global consumption of petroleum-based synthetic plastic is approximately 299 Mt/year.1 Polystyrene (PS), molecular formula [−CH(C6H5)CH2−]n, commonly known as Styrofoam, accounted for approximately 7.1% (21 Mt/year) of the total plastic consumption in 2013.1 Although PS is considered a durable plastic, PS products are often designed for a short service time and one-time use as a result of the low cost of this material. The sharp contrast between the remarkable durability of PS and the short service time of PS products has led to the increasing accumulation of PS waste in our environment. Most of the collected PS waste is disposed along with municipal solid waste in landfills.2 Even more problematic is that a great amount of PS debris is also dispersed as “white pollutants” in the environment, becoming a global environmental concern.2−5 To date, it has generally been thought that PS is not subject to biodegradation by microorganisms and soil invertebrates.6−8 Previous investigations have used 14C-labeled PS tracers added to a variety of mixed microbial consortia from soil, sewage sludge, decaying garbage, or manure.8−10 The recovery of 14CO2 ranged from 0.01% to less than 3% over periods of 1−4 months, which does not yet constitute convincing results of the biodegradation of PS because PS may contain a small fraction of impurities, such as styrene.8−10 Although a few strains of pure bacteria isolated from soils were capable of colonizing PS surfaces, the isolates have not proven that these bacteria were effective in the biodegradation of PS, changing neither the physical nor chemical properties of its long-chain molecules. Further, no traces of metabolic activity were found.11,12 Received: May 31, 2015 Revised: September 5, 2015 Accepted: September 21, 2015 Published: September 21, 2015 Article pubs.acs.org/est © 2015 American Chemical Society 12080 DOI: 10.1021/acs.est.5b02661 Environ. Sci. Technol. 2015, 49, 12080−12086 Downloaded via BEIJING NORMAL UNIV on October 8, 2019 at 16:05:54 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles
ArticleEnvironmentalScience&TechnologySeveral soil invertebrates, including earthworms, isopods,bymealworm consumption was measured periodically.A test ofmillipedes, slugs, and snails, have also been tested to determinethe survival of mealworms reared in the laboratory solely on awhether they were able to degrade PS. These soil invertebratesStyrofoam diet in comparison to those reared on thewere fed with C-labeled PS tracers in their normal diets. Noconventional diet of bran was conducted, as described below.respired 14CO, was recovered during a 2 week test period.Mealworms (500)were reared with 5.8g of Styrofoam blocksSome mandibulate insects, as reported previously,areable toas a sole diet in a climate chamber (RQH-250, Shanghai,chew and eat plastic packages, including polyvinyl chlorideChina)under controlled conditions[25±1°C,80±2%(PVC), polyethylene (PE), and polypropylene (PP) packaginghumidity, and 16:8 (light/dark)photoperiod. Duringfilms.However,until recently,little was known aboutincubation, dead mealworms were removed immediately afterwhether the ingested plastic could be biodegraded in the gut oftheir death.The survival curves of mealworm groups fed onthe plastic-eating insect.Styrofoam were compared to those of the groups fed on branRecently,we reported that waxworms (the larvae of theusing a t test. Triplicate incubators were prepared for each test.Indian mealmothor Plodia interpunctella)werecapable ofCollection and Characterization of the Fecula.Thechewing and eating PE films, and two bacterial strains capablemealworms were fed with Styrofoam blocks as their sole dietof degrading PE were isolated from the gut of the worms, ie.,for 30 days. Subsequently, the mealworms were transferred to aEnterobacterasburiaeYTIand Bacllus spYPI16.17During theclean box to collect the fecula every12 h and to avoid carryoversame research period, we found that mealworms, the larvae ofof uningested Styrofoam morsels mixing with the accumulatedthe mealworm beetle or Tenebrio molitor Linnaeus (a species offecula.The collected fecula were immediately stored in liquiddarkling beetle), which are much larger in size than waxwormsnitrogen for further analysis.(typicallyapproximately 25 versus 12 mm in length), can eatFresh fecula of Styrofoam-feeding mealworms (ca. 1.0 g)Styrofoam as their sole diet. Mealworms are pests and have fourwere extracted with 150 mL of tetrahydrofuran (THF) as thelife stages: egg, larva, pupa, and adult. They are also a profitablesolvent in a Soxhlet extractor at 90 °C for 12 h. Then, theanimal food availableinmanyinsectmarketsandpet stores.extractedsolutionwasconcentratedtoSmL,ThemolecularThey can easily be reared on fresh oats, wheat bran, or grainweights and molecular weight distributions of the Styrofoamwith potato, cabbage, carrots, or apple. Here, we reportand the degraded products in the fecula were determined usingevidence that biodegradation and mineralization of PS doesGPC with a 50 μL injection each time. THF was used as anoccur in the gut of the mealworms based on the changes ineluent at a flowrate of 1.0mL/min at40°C.chemical and physical properties of egested residues (fecula)Solid-state 13C cross-polarization/magic angle spinningafter passage through the gut system compared to the originalnuclear magnetic resonance (CP/MAS NMR) analysis wasStyrofoam diet, with the conversion of ingested PS into COzcarried out at 100 MHz on a spectrometer (AVANCE III 400,and biomass. Our results confrmed PS biodegradation in theBruker, Billerica, MA) at ambient temperature.The operationallarval gut and indicated the presence of a promising petroleum-parameters were 1.5 ms contact time, 4 s recycle delay, 0.013 sbased plastic-degrading process in the environment.acquisition time, 4 μs 90° pulse, and 5 kHz MAS spin.The thermal characterization was performed using aMATERIALSANDMETHODSthermogravimetric (TG) analyzer (TGA-209F1, NETZSCH,Test Materials.The Styrofoam feedstock tested forSelb, Germany) interfaced with Fourier transform infrared(FTIR, Nicolet Magna IR-8700, Thermo Scientific, Waltham,biodegradation was obtained from SINOPEC Beijing YanshanMA) spectroscopy.Samples of the fecula and Styrofoam (ca.5Company, Beijing, China. The chemical composition of themg)were analyzed at a heating rate of 20 oC/min fromStyrofoam was identified as containing PS >98%with theambient temperature to 600 °C under high-purity nitrogennumber-average molecular weight(M.)of 40430 and weight-(99.999%)ataflowrateof 10mL/min.average molecular weight (M)of 124200 (Table S1 of theTestoftheCarbon Mass Balance.Carbonbalancefor theSupporting Information).No catalysts and additiveswereStyrofoam ingested by the worms was estimated using batchadded, as per the manufacturing standard in China (QB/T4009-2010).trails with incubators equipped with a pre-CO2 removal andBoth a 13C- and β 13C-labeled PS samples were purchasedsequential CO, trapping system (Figure S2 of the SupportingInformation).The worms were fed with Styrofoam as a solefrom Sigma-Aldrich, St. Louis, MO. Their material numbers arediet in 12 glass jars (500 mL in volume) in an incubator604445-SPEC and 604453-SPEC, respectively.The molecularcontaining 40 worms each. The incubators were sealed withweights of the two chemicals were characterized by gelrubber stoppers. Compressed air passed through two CO2permeation chromatography (GPC, Alliance V2000, Waters,trappers with 2 M NaOH solution (250 mL) in series toMilford,MA)and werefound to be51920 (M.)and133700remove CO, from the air, which was then moisturized before(Mw)for α13C-labeled PSand51690(M.)and 159000 (M.)for β 13C-labeled PS.entering the incubator.The off-airpassed through another twoMealworms were purchased from Daxing Insect BreedingCO, trappers in series to collect CO, produced from theincubator.Prior to the test, the weights of Styrofoam andPlant, Beijing, China, Insect Breeding Plant, Qinhuangdao,mealworms added were determined.CO2produced from eachHebei, China, and the Bug Company, Ham Lake, MN, for theincubator was collected in NaOH solutions and precipitatedinvestigation of Styrofoam-eating behavior (Figure Sl of theSupporting Information). The mealworms (growth age atwith BaCl, to BaCO, which was measured after being dried toapproximately 3-4 instars) from Daxing Insects Breeding Planta constant weight.The measured dryweight of BaCO,waswere used for all tests.used for the calculation of trapped CO. The incubation timeStyrofoam-Feeding Tests.The mealworms purchasedwas 4, 8, 12, and 16 days, respectively.At the end of eachfrom various sources reared on bran were placed in aincubation time, three incubators as a group were sacrificed.polypropylene plastic container with Styrofoam blocks.TheThemass changes in Styrofoam, weight ofworm biomass, CO2mass loss of the Styrofoam block as a function of time causedproduced, and fecula egested were determined. A lifeless12081DOt10.1021/acs.est5b02661Environ.Sci Technol 2015, 49, 1208012086
Several soil invertebrates, including earthworms, isopods, millipedes, slugs, and snails, have also been tested to determine whether they were able to degrade PS. These soil invertebrates were fed with 14C-labeled PS tracers in their normal diets.10 No respired 14CO2 was recovered during a 2 week test period. Some mandibulate insects, as reported previously, are able to chew and eat plastic packages, including polyvinyl chloride (PVC), polyethylene (PE), and polypropylene (PP) packaging films.13−15 However, until recently, little was known about whether the ingested plastic could be biodegraded in the gut of the plastic-eating insect. Recently, we reported that waxworms (the larvae of the Indian mealmoth or Plodia interpunctella) were capable of chewing and eating PE films, and two bacterial strains capable of degrading PE were isolated from the gut of the worms, i.e., Enterobacter asburiae YT1 and Bacillus sp. YP1.16,17 During the same research period, we found that mealworms, the larvae of the mealworm beetle or Tenebrio molitor Linnaeus (a species of darkling beetle), which are much larger in size than waxworms (typically approximately 25 versus 12 mm in length), can eat Styrofoam as their sole diet. Mealworms are pests and have four life stages: egg, larva, pupa, and adult. They are also a profitable animal food available in many insect markets and pet stores. They can easily be reared on fresh oats, wheat bran, or grain with potato, cabbage, carrots, or apple. Here, we report evidence that biodegradation and mineralization of PS does occur in the gut of the mealworms based on the changes in chemical and physical properties of egested residues (fecula) after passage through the gut system compared to the original Styrofoam diet, with the conversion of ingested PS into CO2 and biomass. Our results confirmed PS biodegradation in the larval gut and indicated the presence of a promising petroleumbased plastic-degrading process in the environment. ■ MATERIALS AND METHODS Test Materials. The Styrofoam feedstock tested for biodegradation was obtained from SINOPEC Beijing Yanshan Company, Beijing, China. The chemical composition of the Styrofoam was identified as containing PS > 98% with the number-average molecular weight (Mn) of 40 430 and weightaverage molecular weight (Mw) of 124 200 (Table S1 of the Supporting Information). No catalysts and additives were added, as per the manufacturing standard in China (QB/T 4009-2010). Both α 13C- and β 13C-labeled PS samples were purchased from Sigma-Aldrich, St. Louis, MO. Their material numbers are 604445-SPEC and 604453-SPEC, respectively. The molecular weights of the two chemicals were characterized by gel permeation chromatography (GPC, Alliance V2000, Waters, Milford, MA) and were found to be 51 920 (Mn) and 133 700 (Mw) for α 13C-labeled PS and 51 690 (Mn) and 159 000 (Mw) for β 13C-labeled PS. Mealworms were purchased from Daxing Insect Breeding Plant, Beijing, China, Insect Breeding Plant, Qinhuangdao, Hebei, China, and the Bug Company, Ham Lake, MN, for the investigation of Styrofoam-eating behavior (Figure S1 of the Supporting Information). The mealworms (growth age at approximately 3−4 instars) from Daxing Insects Breeding Plant were used for all tests. Styrofoam-Feeding Tests. The mealworms purchased from various sources reared on bran were placed in a polypropylene plastic container with Styrofoam blocks. The mass loss of the Styrofoam block as a function of time caused by mealworm consumption was measured periodically. A test of the survival of mealworms reared in the laboratory solely on a Styrofoam diet in comparison to those reared on the conventional diet of bran was conducted, as described below. Mealworms (500) were reared with 5.8 g of Styrofoam blocks as a sole diet in a climate chamber (RQH-250, Shanghai, China) under controlled conditions [25 ± 1 °C, 80 ± 2% humidity, and 16:8 (light/dark) photoperiod]. During incubation, dead mealworms were removed immediately after their death. The survival curves of mealworm groups fed on Styrofoam were compared to those of the groups fed on bran using a t test. Triplicate incubators were prepared for each test. Collection and Characterization of the Fecula. The mealworms were fed with Styrofoam blocks as their sole diet for 30 days. Subsequently, the mealworms were transferred to a clean box to collect the fecula every 12 h and to avoid carryover of uningested Styrofoam morsels mixing with the accumulated fecula. The collected fecula were immediately stored in liquid nitrogen for further analysis. Fresh fecula of Styrofoam-feeding mealworms (ca. 1.0 g) were extracted with 150 mL of tetrahydrofuran (THF) as the solvent in a Soxhlet extractor at 90 °C for 12 h. Then, the extracted solution was concentrated to 5 mL. The molecular weights and molecular weight distributions of the Styrofoam and the degraded products in the fecula were determined using GPC with a 50 μL injection each time. THF was used as an eluent at a flow rate of 1.0 mL/min at 40 °C. Solid-state 13C cross-polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR) analysis was carried out at 100 MHz on a spectrometer (AVANCE III 400, Bruker, Billerica, MA) at ambient temperature. The operational parameters were 1.5 ms contact time, 4 s recycle delay, 0.013 s acquisition time, 4 μs 90° pulse, and 5 kHz MAS spin. The thermal characterization was performed using a thermogravimetric (TG) analyzer (TGA-209F1, NETZSCH, Selb, Germany) interfaced with Fourier transform infrared (FTIR, Nicolet Magna IR-8700, Thermo Scientific, Waltham, MA) spectroscopy. Samples of the fecula and Styrofoam (ca. 5 mg) were analyzed at a heating rate of 20 °C/min from ambient temperature to 600 °C under high-purity nitrogen (99.999%) at a flow rate of 10 mL/min. Test of the Carbon Mass Balance. Carbon balance for the Styrofoam ingested by the worms was estimated using batch trails with incubators equipped with a pre-CO2 removal and sequential CO2 trapping system (Figure S2 of the Supporting Information). The worms were fed with Styrofoam as a sole diet in 12 glass jars (500 mL in volume) in an incubator containing 40 worms each. The incubators were sealed with rubber stoppers. Compressed air passed through two CO2 trappers with 2 M NaOH solution (250 mL) in series to remove CO2 from the air, which was then moisturized before entering the incubator. The off-air passed through another two CO2 trappers in series to collect CO2 produced from the incubator. Prior to the test, the weights of Styrofoam and mealworms added were determined. CO2 produced from each incubator was collected in NaOH solutions and precipitated with BaCl2 to BaCO3, which was measured after being dried to a constant weight. The measured dry weight of BaCO3 was used for the calculation of trapped CO2. The incubation time was 4, 8, 12, and 16 days, respectively. At the end of each incubation time, three incubators as a group were sacrificed. The mass changes in Styrofoam, weight of worm biomass, CO2 produced, and fecula egested were determined. A lifeless Environmental Science & Technology Article DOI: 10.1021/acs.est.5b02661 Environ. Sci. Technol. 2015, 49, 12080−12086 12081
Environmental Science&TechnologyArticlecontrol was also used to ensure that no CO, was generated(a)(Figure S2 of the Supporting Information).The carbon contentof the dried worm biomass and fecula was determined using an2elemental analyzer (Vario EL, Elementar AnalysensystemeGmbH, Hanau, Germany). The conversion of ingestedStyrofoam to COz and mealworm biomass was estimatedusing the procedures described in detail in Figure S2 of theSupporting Information.cm3c-Carbon IsotopeTracer Experiments.α13c-labeledor β 13C-labeled PS powder (20 mg)was mixed with branpowder (10 mg)and then wrapped in 50mL of 3% agar jellytofeed the mealworms(Figure S3 of the Supporting Informa-tion).The jelly food contained 0.4 mg of PS/mL and 0.2 mg of100(b)bran/mL. The glass jars (S00 mL in volume) were also used asOStyrofoam loss1.5OSR ofStyrofoam-feeding larvaeincubators with 40 mealworms each. The living control group-SRofbran-feedinglarvaeof triplicate incubators was fed only with unlabeled bran(%)ee1.295wrapped in agar jelly.13CO, in off-air from the incubator sealed:with a rubber stopper was trapped in the two-stage CO2trappers with 1MNaOH (250 mL)and precipitated with90BaCl, to BaCO3, as described above.The isotopic composition0.6(atom %)of carbon was analyzed using isotope ratiomassspectrometry (Finnigan MAT 253, Thermo Electron, Waltham,0.3MA).OThe incubation with 13C-labeled PS lasted 16 days. At the0.051015202530end of the incubation, mealworms fed both with and withoutTime (days)13C-labeled PS were harvested separately. The mealworms werefirst blown and then were washed and killed by submerging inFigure 1. Styrofoam-eating behavior of mealworms (T. molitor). (a)ethanol This step was to avoid contamination of non-Larvae of T.molitor chew and eat the Styrofoam block. (b) Styrofoammetabolized or partially metabolized 13c-labeled products onmass loss caused bya group of mealworms eating and the SR ofthe exterior of the mealworms. The washed mealworms thenStyrofoam-fed and conventional diet (bran)-fed mealworm popula-were lyophilized to produce dried bodies. After lyophilization,tions over 30 days [mean ± standard deviation (SD); n = 3 groups forthe whole gut tissue (which might contain fecula) was easilyeach condition; S00 mealworms for each group]. Survival curves areillustrated by the proportional shift in surviving mealworms over time.removed from the lyophilized body, which was then used forNo significant difference (t test; p = 0.944 > 0.05) in the survivallipid extraction. All lipids were extracted from the bodies usingcurves between the Styrofoam- and the bran-feeding mealworms waschloroform in a Soxhlet extractor for 6-8 h. The lipid-observed.chloroform solution was then evaporated under N2,and 100mg dried samples were resuspended with 4mL of MeOH/NaOH(0.5mol/L)at100Cfor5min.Aftercoolingtoroomtemperature, 5 mL of the mixture of MeOH/ethyl ether-boronnumber and growth stage of themealworms, and the batch oftrifluoride [(MEBT), 1:3, v/v] was added to the flask andmealwormspurchased.Forexample,agroupof S00mealwormsmethylated at 100 C for 2min.After cooling to room(n = 3 groups) from Beijing caused a total mass loss oftemperature, 8 mL of saturated NaCl aqueous solution wasStyrofoam accounting for 31.0± 1.7% of the initial mass (5.8added.Finally, 2 mL of n-hexane was added to extract theg) within 30 days (Figure 1b).methylated derivatives. Then, the extracted derivatized fattyA test for the determination of the survival rate (SR) over a 1acids (FAs) were separated by gas chromatography (GC) tomonth period using the same batch of mealworms from Beijingproduce individual FAs, which were then analyzed byshowed that the difference between the SR of Styrofoam-combustion-isotope ratio mass spectrometry (GC-C-irMS,feeding mealworms (s00 mealworms as a group; n =3 groups)Thermo Electron, Waltham, MA).and the SR of conventional diet (or bran)-feeding mealwormswas not significant (500 mealworms as a group; n =3groups; tRESULTSANDDISCUSSIONtest; p = 0.944 > 0.05) (Figure 1b). These Styrofoam-feedingMealworm Styrofoam-Eating Behavior.Feeding trialsmealworms survived for I month more until they stoppedwith Styrofoam were performed with mealworms from Beijingeating to become pupae, which then emerged as adult beetlesand Qinhuangdao, China, and Ham Lake, MN. The Styrofoamwithin 2 weeks. These observations imply that Styrofoamsamples used were not pretreated in any way and contained nofeeding did not pose a negative impact on the survivaladditives (Table Si of the Supporting Information).Thecapabilities of the mealworms.Changes in the Chemical Structure and Compositionmealworms from all sources ate Styrofoam as soon as it was fed(Figure la). The eating activity of the mealworms (20-25 cmmof Ingested Styrofoam. According to our observation, thein length) appeared high and created hollows in the Styrofoammealworms began to egest fecula 12-24h after ingestion ofblocks (Figure la).The same observations were repeated moreStyrofoam (inset of Figure 2a), suggesting a short retentionthan 3 times, regardless of the three different sites where thetime (<24 h) for the Styrofoam held in the gut. Fresh feculamealworms were purchased (Figure S1 of the Supportingwere collected and analyzed to determine whether changes inInformation). Their eating activity resulted in a decrease in thechemical structure and composition of the ingested Styrofoammass of Styrofoam, which depended upon the test period, thehad occurred after passage through the gut.12082DO:10.1021/acs.est.5b02661Environ, Sd. Technol 2015, 49, 1208012086
control was also used to ensure that no CO2 was generated (Figure S2 of the Supporting Information). The carbon content of the dried worm biomass and fecula was determined using an elemental analyzer (Vario EL, Elementar Analysensysteme GmbH, Hanau, Germany). The conversion of ingested Styrofoam to CO2 and mealworm biomass was estimated using the procedures described in detail in Figure S2 of the Supporting Information. 13C-Carbon Isotope Tracer Experiments. α 13C-labeled or β 13C-labeled PS powder (20 mg) was mixed with bran powder (10 mg) and then wrapped in 50 mL of 3% agar jelly to feed the mealworms (Figure S3 of the Supporting Information). The jelly food contained 0.4 mg of PS/mL and 0.2 mg of bran/mL. The glass jars (500 mL in volume) were also used as incubators with 40 mealworms each. The living control group of triplicate incubators was fed only with unlabeled bran wrapped in agar jelly. 13CO2 in off-air from the incubator sealed with a rubber stopper was trapped in the two-stage CO2 trappers with 1 M NaOH (250 mL) and precipitated with BaCl2 to BaCO3, as described above. The isotopic composition (atom %) of carbon was analyzed using isotope ratio mass spectrometry (Finnigan MAT 253, Thermo Electron, Waltham, MA). The incubation with 13C-labeled PS lasted 16 days. At the end of the incubation, mealworms fed both with and without 13C-labeled PS were harvested separately. The mealworms were first blown and then were washed and killed by submerging in ethanol. This step was to avoid contamination of nonmetabolized or partially metabolized 13C-labeled products on the exterior of the mealworms. The washed mealworms then were lyophilized to produce dried bodies. After lyophilization, the whole gut tissue (which might contain fecula) was easily removed from the lyophilized body, which was then used for lipid extraction. All lipids were extracted from the bodies using chloroform in a Soxhlet extractor for 6−8 h. The lipid− chloroform solution was then evaporated under N2, and 100 mg dried samples were resuspended with 4 mL of MeOH/ NaOH (0.5 mol/L) at 100 °C for 5 min. After cooling to room temperature, 5 mL of the mixture of MeOH/ethyl ether−boron trifluoride [(MEBT), 1:3, v/v] was added to the flask and methylated at 100 °C for 2 min. After cooling to room temperature, 8 mL of saturated NaCl aqueous solution was added. Finally, 2 mL of n-hexane was added to extract the methylated derivatives. Then, the extracted derivatized fatty acids (FAs) were separated by gas chromatography (GC) to produce individual FAs, which were then analyzed by combustion−isotope ratio mass spectrometry (GC−C−irMS, Thermo Electron, Waltham, MA).18 ■ RESULTS AND DISCUSSION Mealworm Styrofoam-Eating Behavior. Feeding trials with Styrofoam were performed with mealworms from Beijing and Qinhuangdao, China, and Ham Lake, MN. The Styrofoam samples used were not pretreated in any way and contained no additives (Table S1 of the Supporting Information). The mealworms from all sources ate Styrofoam as soon as it was fed (Figure 1a). The eating activity of the mealworms (20−25 cmm in length) appeared high and created hollows in the Styrofoam blocks (Figure 1a). The same observations were repeated more than 3 times, regardless of the three different sites where the mealworms were purchased (Figure S1 of the Supporting Information). Their eating activity resulted in a decrease in the mass of Styrofoam, which depended upon the test period, the number and growth stage of the mealworms, and the batch of mealworms purchased. For example, a group of 500 mealworms (n = 3 groups) from Beijing caused a total mass loss of Styrofoam accounting for 31.0 ± 1.7% of the initial mass (5.8 g) within 30 days (Figure 1b). A test for the determination of the survival rate (SR) over a 1 month period using the same batch of mealworms from Beijing showed that the difference between the SR of Styrofoamfeeding mealworms (500 mealworms as a group; n = 3 groups) and the SR of conventional diet (or bran)-feeding mealworms was not significant (500 mealworms as a group; n = 3 groups; t test; p = 0.944 > 0.05) (Figure 1b). These Styrofoam-feeding mealworms survived for 1 month more until they stopped eating to become pupae, which then emerged as adult beetles within 2 weeks. These observations imply that Styrofoam feeding did not pose a negative impact on the survival capabilities of the mealworms. Changes in the Chemical Structure and Composition of Ingested Styrofoam. According to our observation, the mealworms began to egest fecula 12−24 h after ingestion of Styrofoam (inset of Figure 2a), suggesting a short retention time (<24 h) for the Styrofoam held in the gut. Fresh fecula were collected and analyzed to determine whether changes in chemical structure and composition of the ingested Styrofoam had occurred after passage through the gut. Figure 1. Styrofoam-eating behavior of mealworms (T. molitor). (a) Larvae of T. molitor chew and eat the Styrofoam block. (b) Styrofoam mass loss caused by a group of mealworms eating and the SR of Styrofoam-fed and conventional diet (bran)-fed mealworm populations over 30 days [mean ± standard deviation (SD); n = 3 groups for each condition; 500 mealworms for each group]. Survival curves are illustrated by the proportional shift in surviving mealworms over time. No significant difference (t test; p = 0.944 > 0.05) in the survival curves between the Styrofoam- and the bran-feeding mealworms was observed. Environmental Science & Technology Article DOI: 10.1021/acs.est.5b02661 Environ. Sci. Technol. 2015, 49, 12080−12086 12082
Environmental Science&TechnologyArticle(a) (d)(Figure 2c). The, newly appearing alkyl- and methyl-C-6090resonance signals(s 10-40)could be assigned to aliphaticGhydrocarbons.21 The newly emerging resonance signals at FeculaPS175,104,99, 84, 75,73, 61, 55, and 23 were attributed to chitinolipapFeculaPS30from the insect cuticle.21The new aromatic C (8140,154, andFecula233327431160) resonance signals could be ascribed to phenyl derivatives,0.03.51002003004005006005.04.05.5as reported by Gilardi et al.22 The phenyl derivatives arelogMwTemperature (C)possible proxies forthe fragments or smaller molecules(b) e0.010produced during depolymerization/oxidation of Ps.8Thermal analysis can be used to compare the changes inchemical composition of the solid substrate by analyzing thePSgaseous compounds produced during substrate pyrolysis underanoxic conditions. TG coupling with the FTIR spectroscopy(c)0.100method is based on the precise study of the weight lossFecul(thermal decomposition) of the sample during programmedtemperature and online analysis of the evolved gaseouscompounds produced during thermal decomposition.TG/differential thermogravimetric (DTG)profiles during the'50251thermal decompositionof thefecula and the control StyrofoamS (ppm)as a function of the temperature were shown in Figure 2d.For the control, 98.0% of weight loss occurred during onlyFigure 2.Changes in the chemical structure and composition ofone stage,which ranged from360 to 480°C,and themaximumStyrofoam afterpassage through the mealworm gut as fecula.(a)Molecular weight distribution shift of the fecula extract versus thedecomposition rate occurred at 421 °C.In contrast, the feculacontrol PS. The inset picture is the control (up; scale bar =1 cm)showed three weight loss stages, stage 1 of 15.8% at 175-275versus the fecula (down; scale bar = 1 mm). (b and c) 13C CP/MASoC, stage 2 of 23.4% at 275-360 °C, and stage 3 of 26.6% atNMR spectra of the control and the fecula. The new appearance of360-480 °C.Themaximum decomposition ratesduring thephenyl derivatives at the 150-160 ppm resonance regions in thethree stages occurred at 233, 327, and 431 °C, respectively.fecula was indicated with a gray column. (d) TG/DTG curves of theUnder the same heating program, the fecula decomposed incontrol PS and the fecula (TG curves are solid lines, and DTG curvesmore stages than the control, indicating that the feculaare dashed lines). (e and f) Three-dimensional infrared (IR) spectra ofcontained not only Ps but also other new componentsgaseous compounds produced in the TG equipment during thermalproduced during digestion in the mealworm gut.Duringdecomposition of the control and the feculastage 3, the weight loss of fecula was obviously less than theweight loss of the control, demonstrating the depletion of PSThe change in the long-chain structure of PS molecules wascontent in the fecula.investigated by analyzing the whole molecular weightGaseous compounds produced in the TG process weredistributions and average molecular weights of the degradedanalyzed using FTIR. The three-dimensional'(3D) FTIRproducts in the fecula and the control PS using GPC.Theprofiles (panels e and f of Figure 2), compiled over the entiredegraded productswereextracted from the collected fecula (ca.temperature range of thermal decomposition, show that the1.0 g)with THF.The wholemolecular weight distributionevolved gaseous compounds generated from the controlcurve of the fecula extract shows a shift toward iower molecularStyrofoam and the fecula give different IR absorption.weight compared to the molecular weight distribution of theFor the control, the obvious absorptions were generated incontrol PS (Figure 2a).M, and Mw for the fecula extract alsothe temperature range from 360 to 480 C (Figure2e).Adecrease comparedto the control PS (M32260 versusrepresentative FTIR spectrum at 421 oC shows that all40430; Mw,98330 versus 124200).These results suggest thatdepolymerization/cleavage of the long-chain structure of PSabsorbance peaks are attributable to styrene, which representsthe main decomposition product of Ps (Figure S4a of thetook place and lower molecular weight fragments were newlySupportingInformation).formed in the mealworm gut. The observation of the decreaseFor the fecula, the obvious absorptions were generated in thein M, and Mw is a major indication of depolymerization andtemperature range from 175 to 480 °C (Figure 2f).degradation of polymers,19 which has been reported duringRepresentative FTIR spectra at 233, 327, and 431 °C showbiodegradation of PE films by the two bacterial strains isolated16,17that the strongest absorbance peaks at 2000-2250 and 2268-from theguts of waxworms in our laboratory.The chemical compositions of Styrofoam and fecula2395cm-couldbeassignedtocarbonmonoxideandcarbondioxide (Panels b to d of Figure S4 of the Supporting(residues of the Styrofoam egested through the gut of themealworms)were characterized using solid-state13CCP/MASInformation), respectively, which often represent the decom-NMR and thermal analysis. Analysis of the 13C CP/MAS NMRposition products of newly produced components in the fecula.The absorbance peaks attributed to styrene, the mainis usually applied to identifydirectlythe native composition of022Asdecomposition product of PS, were very weak, substantiatingthe solid substrate without separation of components.shown in Figure 2b, only four resonance signals were detectedthe depletion of PS content in the fecula (panels b-d of Figurein the spectrum of the control PS.Two resonance signals at S4 of the Supporting Information).As indicated by the NMR spectra (panels b and c of Figure146 and 128 were assigned to non-protonated and protonatedaromatic carbons,and tworesonance signals at 41 and 462) and thermal analysis (panels d-f of Figure 2), both nativecorresponded to the methylene and methyl (aliphatic) carbons.compositions and chemical components ofthe evolvedgaseousIn the spectrum of the fecula (Figure 2c), some newcompounds produced during thermal decomposition wereresonance signals were detected in the spectrum of the feculadifferent between the control and the fecula, indicating that the12083DOt10.1021/acs.est.5b02661Environ, Sd. Technol 2015, 49, 1208012086
The change in the long-chain structure of PS molecules was investigated by analyzing the whole molecular weight distributions and average molecular weights of the degraded products in the fecula and the control PS using GPC. The degraded products were extracted from the collected fecula (ca. 1.0 g) with THF. The whole molecular weight distribution curve of the fecula extract shows a shift toward lower molecular weight compared to the molecular weight distribution of the control PS (Figure 2a). Mn and Mw for the fecula extract also decrease compared to the control PS (Mn, 32 260 versus 40 430; Mw, 98 330 versus 124 200). These results suggest that depolymerization/cleavage of the long-chain structure of PS took place and lower molecular weight fragments were newly formed in the mealworm gut. The observation of the decrease in Mn and Mw is a major indication of depolymerization and degradation of polymers,19 which has been reported during biodegradation of PE films by the two bacterial strains isolated from the guts of waxworms in our laboratory.16,17 The chemical compositions of Styrofoam and fecula (residues of the Styrofoam egested through the gut of the mealworms) were characterized using solid-state 13C CP/MAS NMR and thermal analysis. Analysis of the 13C CP/MAS NMR is usually applied to identify directly the native composition of the solid substrate without separation of components.20−22 As shown in Figure 2b, only four resonance signals were detected in the spectrum of the control PS. Two resonance signals at δ 146 and 128 were assigned to non-protonated and protonated aromatic carbons, and two resonance signals at δ 41 and 46 corresponded to the methylene and methyl (aliphatic) carbons. In the spectrum of the fecula (Figure 2c), some new resonance signals were detected in the spectrum of the fecula (Figure 2c). The newly appearing alkyl- and methyl-C resonance signals (δ 10−40) could be assigned to aliphatic hydrocarbons.21 The newly emerging resonance signals at δ 175, 104, 99, 84, 75, 73, 61, 55, and 23 were attributed to chitin from the insect cuticle.21 The new aromatic C (δ 140, 154, and 160) resonance signals could be ascribed to phenyl derivatives, as reported by Gilardi et al.22 The phenyl derivatives are possible proxies for the fragments or smaller molecules produced during depolymerization/oxidation of PS.8 Thermal analysis can be used to compare the changes in chemical composition of the solid substrate by analyzing the gaseous compounds produced during substrate pyrolysis under anoxic conditions. TG coupling with the FTIR spectroscopy method is based on the precise study of the weight loss (thermal decomposition) of the sample during programmed temperature and online analysis of the evolved gaseous compounds produced during thermal decomposition. TG/ differential thermogravimetric (DTG) profiles during the thermal decomposition of the fecula and the control Styrofoam as a function of the temperature were shown in Figure 2d. For the control, 98.0% of weight loss occurred during only one stage, which ranged from 360 to 480 °C, and the maximum decomposition rate occurred at 421 °C. In contrast, the fecula showed three weight loss stages, stage 1 of 15.8% at 175−275 °C, stage 2 of 23.4% at 275−360 °C, and stage 3 of 26.6% at 360−480 °C. The maximum decomposition rates during the three stages occurred at 233, 327, and 431 °C, respectively. Under the same heating program, the fecula decomposed in more stages than the control, indicating that the fecula contained not only PS but also other new components produced during digestion in the mealworm gut. During stage 3, the weight loss of fecula was obviously less than the weight loss of the control, demonstrating the depletion of PS content in the fecula. Gaseous compounds produced in the TG process were analyzed using FTIR. The three-dimensional (3D) FTIR profiles (panels e and f of Figure 2), compiled over the entire temperature range of thermal decomposition, show that the evolved gaseous compounds generated from the control Styrofoam and the fecula give different IR absorption. For the control, the obvious absorptions were generated in the temperature range from 360 to 480 °C (Figure 2e). A representative FTIR spectrum at 421 °C shows that all absorbance peaks are attributable to styrene, which represents the main decomposition product of PS (Figure S4a of the Supporting Information). For the fecula, the obvious absorptions were generated in the temperature range from 175 to 480 °C (Figure 2f). Representative FTIR spectra at 233, 327, and 431 °C show that the strongest absorbance peaks at 2000−2250 and 2268− 2395 cm−1 could be assigned to carbon monoxide and carbon dioxide (Panels b to d of Figure S4 of the Supporting Information), respectively, which often represent the decomposition products of newly produced components in the fecula. The absorbance peaks attributed to styrene, the main decomposition product of PS, were very weak, substantiating the depletion of PS content in the fecula (panels b−d of Figure S4 of the Supporting Information). As indicated by the NMR spectra (panels b and c of Figure 2) and thermal analysis (panels d−f of Figure 2), both native compositions and chemical components of the evolved gaseous compounds produced during thermal decomposition were different between the control and the fecula, indicating that the Figure 2. Changes in the chemical structure and composition of Styrofoam after passage through the mealworm gut as fecula. (a) Molecular weight distribution shift of the fecula extract versus the control PS. The inset picture is the control (up; scale bar = 1 cm) versus the fecula (down; scale bar = 1 mm). (b and c) 13C CP/MAS NMR spectra of the control and the fecula. The new appearance of phenyl derivatives at the δ 150−160 ppm resonance regions in the fecula was indicated with a gray column. (d) TG/DTG curves of the control PS and the fecula (TG curves are solid lines, and DTG curves are dashed lines). (e and f) Three-dimensional infrared (IR) spectra of gaseous compounds produced in the TG equipment during thermal decomposition of the control and the fecula. Environmental Science & Technology Article DOI: 10.1021/acs.est.5b02661 Environ. Sci. Technol. 2015, 49, 12080−12086 12083
AricleEnvironmental Science&TechnologyTable1.CarbonBalance Estimates of theIngested StyrofoamConverted into Biomass, COz, and Fecula in theBatchStyrofoam-Feeding Trials with Different Incubation Periodsaincubation time (days)iteminitial carbon (mg)final carbon (mg)A = final - initial (mg)percentage of ingested styrofoam recovered (%)4592.1 ± 19.090.8styrofoam501.3 ± 24.00.50.6biomass933.0 ± 22.0933.5 ± 16.018.820.7CO20.018.8 ± 0.4fecula0.066.8 ± 14.866.873.694.9total recovery8610.0±22.0500.0 ± 39.0110.0styrofoam0.60.5biomass896.0±16.0896.6 ± 32.0CO,0.039.235.639.2 ± 1.00.0fecula65.7 ± 15.765.759.795.8total recovery12styrofoam720.0±5.0563.0 ± 26.0157.01.0biomass794.0±24.0795.0 ± 23.00.6CO20.065.0 ±12.065.041.4fecula0.089.056.789.0 ± 16.098.7total recovery16826.0 ± 54.0217.0styrofoam609.0 ± 47.0biomass1.00.5815.0 ± 36.0817.0 ± 6.0CO20.0103.647.7103.6 ± 3.00.0106.7fecula106.7±10.049.2total recovery97.4n = 3 incubators for each incubation time, with 40 mealworms for each incubator. The carbon contents of Styrofoam, biomass, and fecula werecalculated using their dry weight and carbon contents measured by an elemental analyzer.degradation of ingested Styrofoam and production of degraded(a)125FBiomassCO2products took place in the guts of the mealworms.eoosasaFeculaMineralization of Ingested Styrofoam.The conversion100woyso%of thecarbons of Styrofoam toCOmealwormbiomass,and75fecularesidueswasassessed byaseriesof carbonmassbalancetests with different incubation periods of 4, 8, 12, and 16 days50with 40 mealworms in each incubator (Figure S2 of theSupporting Information).The results showed that total carbonrecovery efficiencies were greater than 95% (Table 1). The25carbon balance estimates showed that the carbon of theingested Styrofoam recovered as CO, was increased from20.7-0481216to 47.7% and the carbon of the ingested Styrofoam egested asIncubation time (days)fecula was decreased from73.6 to49.2%fromday4today1512(b)(Figure 3a and Table 1), suggesting that the activity for the8digestion of ingested Styrofoam increased progressivelyThe mineralization of PS to CO,was furtherverified through(0%)"00e19Adetermination of the production of 13CO, by the mealwormsfed either α 13C-or β i3C-labeled PS-containing diet (Figure S3oFof the Supporting Information). The mealworms werecontinuously fed a 3% solidified jelly containing each of two413C-labeled PS (0.4 mg/mL) and bran (0.2 mg/mL) over a 16-8fday period.For the control, mealworms were fed on bran.CO2released in the off-air was trapped in 1 M NaOH solution and-12recovered as BaCO, for analysis. The mean 13C value of CO21°c-PSp"c-PSBranreleased by the mealworms fed on bran was -8.2 %c, while themean 13C values of CO2 released by mealworms fed on a andFigure3. Conversion of PS into CO,.(a)Carbon proportion of theβ 13C-labeled PS diets were 3.3% and 3.9%e,respectivelyingested Styrofoam recovered as COz, mealworms biomass, and fecula(Figure 3b), indicating that, in comparison to the controlresidues based on the carbon balance estimates over differentmealworms fed with bran, significant 13C enrichment (p< 0.05)incubation times of 4,8, 12, and 16 days (mean value; n -3groupswas observed in the CO2 released from 13C-labeled PS-feedingfor each condition; 40 mealworms for each group).Detailedcalculations are shown in Figure S2 of the Supporting Information.mealworms at the end of the 16 day period, confirming that(b) 3C signatures of CO, produced by the mealworms fed with 13C-13C-labeled PS was partially mineralized in 13CO2.labeled PS (a or β 1Bc-PS) versus unlabeled bran over a 16 dayAssimilation of 13c-PS by Styrofoam-Feeding Meal-incubationperiod (mean±SD; n=3groups foreach condition; 40worms. Carbon mass balance estimates showed that themealwormsforeachgroup)carbon oftheingestedStyrofoamrecoveredas mealwormbiomass remained at only approximately 0.5% and the biomass12084DOt10.1021/acs.est.5b02661Environ, Sd. Technol 2015, 49, 1208012086
degradation of ingested Styrofoam and production of degraded products took place in the guts of the mealworms. Mineralization of Ingested Styrofoam. The conversion of the carbons of Styrofoam to CO2, mealworm biomass, and fecula residues was assessed by a series of carbon mass balance tests with different incubation periods of 4, 8, 12, and 16 days with 40 mealworms in each incubator (Figure S2 of the Supporting Information). The results showed that total carbon recovery efficiencies were greater than 95% (Table 1). The carbon balance estimates showed that the carbon of the ingested Styrofoam recovered as CO2 was increased from 20.7 to 47.7% and the carbon of the ingested Styrofoam egested as fecula was decreased from 73.6 to 49.2% from day 4 to day 15 (Figure 3a and Table 1), suggesting that the activity for the digestion of ingested Styrofoam increased progressively. The mineralization of PS to CO2 was further verified through determination of the production of 13CO2 by the mealworms fed either α 13C- or β 13C-labeled PS-containing diet (Figure S3 of the Supporting Information). The mealworms were continuously fed a 3% solidified jelly containing each of two 13C-labeled PS (0.4 mg/mL) and bran (0.2 mg/mL) over a 16 day period. For the control, mealworms were fed on bran. CO2 released in the off-air was trapped in 1 M NaOH solution and recovered as BaCO3 for analysis. The mean δ 13C value of CO2 released by the mealworms fed on bran was −8.2 ‰, while the mean δ 13C values of CO2 released by mealworms fed on α and β 13C-labeled PS diets were 3.3% and 3.9‰, respectively (Figure 3b), indicating that, in comparison to the control mealworms fed with bran, significant 13C enrichment (p < 0.05) was observed in the CO2 released from 13C-labeled PS-feeding mealworms at the end of the 16 day period, confirming that 13C-labeled PS was partially mineralized in 13CO2. Assimilation of 13C-PS by Styrofoam-Feeding Mealworms. Carbon mass balance estimates showed that the carbon of the ingested Styrofoam recovered as mealworm biomass remained at only approximately 0.5% and the biomass Table 1. Carbon Balance Estimates of the Ingested Styrofoam Converted into Biomass, CO2, and Fecula in the Batch Styrofoam-Feeding Trials with Different Incubation Periodsa incubation time (days) item initial carbon (mg) final carbon (mg) Δ = final − initial (mg) percentage of ingested styrofoam recovered (%) 4 styrofoam 592.1 ± 19.0 501.3 ± 24.0 −90.8 biomass 933.0 ± 22.0 933.5 ± 16.0 0.5 0.6 CO2 0.0 18.8 ± 0.4 18.8 20.7 fecula 0.0 66.8 ± 14.8 66.8 73.6 total recovery 94.9 8 styrofoam 610.0 ± 22.0 500.0 ± 39.0 −110.0 biomass 896.0 ± 16.0 896.6 ± 32.0 0.6 0.5 CO2 0.0 39.2 ± 1.0 39.2 35.6 fecula 0.0 65.7 ± 15.7 65.7 59.7 total recovery 95.8 12 styrofoam 720.0 ± 5.0 563.0 ± 26.0 −157.0 biomass 794.0 ± 24.0 795.0 ± 23.0 1.0 0.6 CO2 0.0 65.0 ± 12.0 65.0 41.4 fecula 0.0 89.0 ± 16.0 89.0 56.7 total recovery 98.7 16 styrofoam 826.0 ± 54.0 609.0 ± 47.0 −217.0 biomass 815.0 ± 36.0 817.0 ± 6.0 1.0 0.5 CO2 0.0 103.6 ± 3.0 103.6 47.7 fecula 0.0 106.7 ± 10.0 106.7 49.2 total recovery 97.4 a n = 3 incubators for each incubation time, with 40 mealworms for each incubator. The carbon contents of Styrofoam, biomass, and fecula were calculated using their dry weight and carbon contents measured by an elemental analyzer. Figure 3. Conversion of PS into CO2. (a) Carbon proportion of the ingested Styrofoam recovered as CO2, mealworms biomass, and fecula residues based on the carbon balance estimates over different incubation times of 4, 8, 12, and 16 days (mean value; n = 3 groups for each condition; 40 mealworms for each group). Detailed calculations are shown in Figure S2 of the Supporting Information. (b) 13C signatures of CO2 produced by the mealworms fed with 13Clabeled PS (α or β 13C-PS) versus unlabeled bran over a 16 day incubation period (mean ± SD; n = 3 groups for each condition; 40 mealworms for each group). Environmental Science & Technology Article DOI: 10.1021/acs.est.5b02661 Environ. Sci. Technol. 2015, 49, 12080−12086 12084