Biological Oxidative Mechanisms for Degradation ... - ACS Publications

Research Article pubs.acs.org/journal/ascecg

Biological Oxidative Mechanisms for Degradation of Poly(lactic acid) Blended with Thermoplastic Starch Carlos A. Rodrigues,† Aryane Tofanello,‡ Iseli L. Nantes,*,‡ and Derval S. Rosa*,† †

Centro de Engenharia, Modelagem e Ciências Sociais Aplicadas (CECS), Universidade Federal do ABC (UFABC), Av. dos Estados, 5001, Bairro Bangu, Santo André, São Paulo 09210-580, Brazil ‡ Centro de Ciências Naturais e Humanas (CCNH), Universidade Federal do ABC (UFABC), Av. dos Estados, 5001, Bairro Bangu, Santo André, São Paulo 09210-170, Brazil S Supporting Information *

ABSTRACT: In the present study, poly(lactic acid) (PLA) and their blends with 5%/wt and 10%/wt thermoplastic starch (TPS) were submitted to degradation in simulated soil. To investigate the mechanisms involved in the degradation, we also submitted the samples to degradation by tert-butyl hydroperoxide, myoglobin, and peroxide-activated myoglobin. The samples were analyzed by Fourier-transformed infrared spectrometry (FTIR), scanning electronic microscopy (SEM), contact angle analysis, and mass loss measurement. The FTIR results indicated a weak interaction between the two components (PLA and starch) in the blend’s amorphous structure. However, the corresponding SEM images showed that TPA increased ridges and roughness at the material surface associated with an increase of wettability evidenced by contact angle analysis. Consistently, TPS favored degradation of the material both in the simulated soil and pro-oxidant model systems. In the simulated soil, the occurrence of TPS hydrolysis provided glucose, a biological fuel, that contributed to the growth of the microorganisms. The similar degradation patterns observed in mimetic pro-oxidant biological systems and soil suggest that oxidative reactions catalyzed by heme proteins from biological sources as well as the presence of peroxides and transition metal traces in the original materials have a significant contribution to PLA and PLA/TPS degradation. KEYWORDS: Biodegradable polymers, Oxidant systems, Heme proteins, PLA, Simulated soil, TPS

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biopolymer.10,11 Thermoplastic starch (TPS) is an amorphous material or semicrystalline composite obtained by blending starch with synthetic polymers. The addition of TPS synthetic polymers results in degradable materials because some microbes utilize starch as a nutrient source and secrete enzymes that consume the polymer.12−14 There are many enzymes found in soil. These can be active in both biotic and abiotic environments. These enzymes are produced by native soil microorganisms as well as plant roots. Fungi are important producers of oxidative enzymes such as Mn-peroxidase and lignin peroxidase that can contribute to the degradation of synthetic and biopolymers.15−20 Soil enzymes are found in proliferating or latent cells, cell debris, clays, humic colloids, as well as the aqueous phase. The contribution of heme proteins and peroxides for PLA degradation modulated by the presence of TPS was the focus of the present study. The study of the oxidative mechanisms for PLA degradation associated with the effect of blended TPS combines the fundamental scientific

INTRODUCTION The disposal of a million tons of short-lived products, such as those destined for packaging, has been an important environmental problem.1 Therefore, scientists have been challenged to dedicate research focused on the development and evaluation of sustainable biobased polymers and composites for the substitution of petroleum-based polymers.2−4 Of these, poly(lactic acid) (PLA) has been widely studied due to its physicochemical features that are appropriated for the most common uses of these materials.2−4 PLA is a thermoplastic aliphatic polyester with two forms: (i) enantiomeric materials made from D- and L-lactic acid (PDLA and PLLA); these are semicrystalline polymers when enantiomerically pure; and (ii) amorphous materials that contain both the D and L forms of lactic acid (PDLLA).5 Therefore, the properties of PLA can be modulated by varying the proportion of the enantiomers in the material composition and by additives such as starch.6,7 Starch is a polysaccharide composed of the polysaccharides amylose and amylopectin and can be obtained from cereals and tuberous roots (Chart 1).8,9 Starch is a good replacement for petroleum-based plastics in many applications because it is an inexpensive and renewable © XXXX American Chemical Society

Received: July 7, 2015 Revised: September 28, 2015

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DOI: 10.1021/acssuschemeng.5b00639 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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10% TPS were processed in a single screw extruder with a diameter of 25 mm and an L/D = 25 with screens of 100 and 60 mesh on the headstock. Temperatures were 178 °C in zone 2, 183 °C in zone 2, and 174 °C in the headstock. These temperatures varied by ±5 °C. The extruded compositions were granulated and packed in bags to prevent moisture penetration. The granulated compositions were injected to form test specimens for type IV mechanical tests according to ASTM D-638 using a PIC-BOY model 22/10 injector provided by Peterson & Cia Ltd.a. (Sorocaba, SP) with an injection capacity of 22 g. The zone 1 (insertion) was 175 °C, and zone 2 was 180 °C. Fourier-Transform Infrared Spectroscopy (FTIR). FTIR spectra of the polymer plates were recorded with a PerkinElmer Spectrum RX1 FTIR spectrometer with a resolution of 4 cm−1. Contact Angle Measurement. The contact angle was determined using the sessile drop technique in an Attension Theta optical tensiometer. Angle measurements were performed every 6 s over 180 s of total time of analysis. Biodegradation in Simulated Soil. The soil was prepared by mixing sand, organic matter, and distilled water in equal parts by mass while keeping the carbon/nitrogen ratio between 10 and 40 (ASTM D 5988-96) along with microorganisms to promote biodegradation.21 The specimens (1.5 cm × 1.5 cm) were arranged in trays that were buried in simulated soil without incident light and kept at 25 °C with moisture controlled weekly during the year. The mass loss was measured at intervals of two months during the first six months and again at the end of the year. Oxidation of Polymers by tert-Butyl Hydroperoxide and/or Metmyoglobin. Plates of PLA and blends with TPS (5% and 10% mass) were immersed for 7 days in plastic vials containing 8 mL of 5 mM, pH 7.4 phosphate buffer in the absence and presence of 100 μM tert-butyl hydroperoxide, 10 μM metmyoglobin, or a mixture of equal concentrations of both. The enzymatic and nonenzymatic degradations were carried out at room temperature (∼25 °C). Identical concentrations of metmyoglobin and tert-butyl hydroperoxide were recharged every 2 days of incubation. Electronic absorption spectra of metmyoglobin were obtained before the incubation and after the recharge of the protein. Snapshots of

Chart 1. Structure of (a) Poly(lactic acid) and Starch: (b) Amylopectin and (c) Amylose

interest with the practical interest in developing new materials to ensure sustainability.

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MATERIALS AND METHODS Chemicals. Poly(lactic acid) (Ingeo 3801-X brand (lot 65389-01)) was obtained from Cargill Agricola S.A. (São Paulo, SP, Brazil). The modified starch, Penetrose 80 brand, in powdered form with 27% amylose and 73% amylopectin with a molar mass of 340 000 g mol−1 was supplied by Corn Products Brasil Ingredients Industriais Ltd.a. (Jundiai,́ SP, Brazil). Commercial glycerin was purchased from Cromoline Quimica Fina Ltd.a. (Diadema, SP, Brazil). Preparation of TPS and the PLA/TPS Composites. TPS was prepared in a mechanical helix mixer; 2000 g of modified starch (Penetrose 80) and 500 g of glycerin were stirred together for 8 min at 70 °C. The PLA composites with 5% and

Figure 1. Characterization of PLA and PLA blended with TPS. Top: from left to right, SEM images of the surface of PLA, PLA/TPS5%, and PLA/ TPS10%, respectively. Bottom: the corresponding FTIR spectra of PLA, PLA/TPS5%, and PLA/TPS10% (black, blue, and red lines, respectively) separated by five regions. B


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Figure 2. Superficial macroscopic changes in PLA (A), PLA/5%TPS (B), and PLA/10%TPS (C) surface associated with mass loss. In each panel, the percentage of mass relative to the initial mass was plotted overlapped on the respective snapshot of the material surface obtained at the indicated time of incubation.

have alkyl CH groups. However, some useful information about the size of the alkyl chain can be obtained from the intensity of these peaks in comparison to other peaks. The low-intensity peaks in this region for PLA and PLA/TPS are consistent with the absence of a long alkyl chain in PLA and TPS structures. For the PLA blended with 5% and 10% TPS, a broad and strong absorption peak near 3380 cm−1 appeared, which is usually assigned to the alcohol OH of the glucose residues. In this region, the overlapped contribution of δOH from water of starch hydration (3230 and 3114 cm−1) should not be discarded. The region from 1800 to 1700 cm−1 contained a band that peaked at 1752 cm−1 and was assigned to the strong CO stretching typical of PLA. For amorphous PLA, this band peaks at 1759 cm−1, and it is 10 cm−1 blue-shifted after thermal crystallization. The 1752 cm−1 peak found for this sample suggests the presence of a polymorphic material. This band was slightly less intense, and red-shifted in the presence of TPS, but without the appearance of additional bands. This result was consistent with a noncovalent modification of PLA in the blends. In the region from 1500 to 1300 cm−1, the FTIR spectra of PLA exhibited other typical bands of PLA, namely, δas(CH3) and δ(CH3) at 1453 and 1359 cm−1, respectively. These bands had decreased intensity in the presence of 5% and 10% TPS. A similar effect was observed for νas(COC) and νs(COC) at 1182 and 1037 cm−1, r(C−CH3) at 1017 cm−1, and (OCC) at 870 cm−1. The broadening of the band in the 960 cm−1 regions can be assigned to the overlapped contribution of the chair conformation of the glucopyranose units.25 Effect of TPS on PLA Wettability. The presence of TPS blended with PLA increased the wettability of the material as attested by changes in the contact angles (Figure 1S, provided in the Supporting Information). These results are similar to those obtained by Oromiehie26 for LDPE blended with TPS, and they are described in the Supporting Information.

the polymer plates were obtained before and after the incubation in different media as well as FTIR and MEV data. A Direct-Q 8UV MilliUNI water purification system was used to purify the water. Scanning Electron Microscopy (SEM). The images of the control and degraded polymer plates were obtained with a JSM6010LA (JEOL Ltd.a.) microscope to study the surface morphology of the samples before and after oxidative degradation. The samples were not polished or coated, and so low voltage (0.8 kV) was used.

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RESULTS AND DISCUSSION Spectral and Morphological Characterization of PLA and Blends. FTIR analysis of the polymers accompanied the five main regions described by Copinet et al., but the analysis was extended further.22 Figure 1 (top panels) shows, from left to right, the SEM images of the surface of pure PLA, PLA/5% TPS, and PLA/10% TPS. The bottom panels of Figure 1 show the corresponding FTIR spectra (black, blue, and red lines, respectively). The SEM image of pure PLA shows a homogeneous surface with low roughness. The presence of 5% starch in the material resulted in a surface with mainly parallel grooves along with some transversal ridges that were exacerbated in the material with 10% starch. This roughness can make PLA more biodegradable by facilitating binding and access of microorganisms in the material.23,24 The FTIR spectral analysis was separated into five regions: 950−800, 1300−950, 1500−1300, 1800−1700, and 3600−2600 cm−1. These regions were previously analyzed by Copinet et al.22 and encompass the principal chemical groups that were susceptible to modification and degradation. In the lower-energy Copinet region, the spectral region of 2998−2985 cm−1 corresponds to −CH− stretching of alkyl chains. This region usually does not provide significant information because almost all organic compounds C


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Figure 3. Changes in PLA and PLA/TPS after aging in the soil. (A) Bottom: FTIR spectra of PLA before and after degradation in soil. The black line is the spectrum obtained before the degradation step, and the blue and red lines are the spectra obtained for smooth and rough regions of the material surface observed after degradation. Top: representative SEM images (120× magnified) of the surface of PLA submitted to degradation in soil. (B) Bottom: FTIR spectra of PLA/5% TPS before and after degradation in soil. The black line is the spectrum obtained before the degradation step, and blue and red lines are the spectra obtained for smooth and rough regions of the material surface observed after degradation. Top: representative SEM images (170× and 200× magnified) of the surface of PLA/5%TPS submitted to degradation in soil. (C) Bottom: FTIR spectra of PLA/10% TPS before and after degradation in soil. The black line is the spectrum obtained before the degradation, and blue and red lines are the spectra obtained for smooth and rough regions of the material surface observed after degradation. Top: representative SEM images (75× and 120× magnified) of the surface of PLA/10% TPS submitted to degradation in soil.

Effect of TPS on the Biodegradation of PLA in Simulated Soil. Figure 2A−C shows the percentage of mass of PLA, PLA/5%TPS, and PLA/10%TPS relative to the respective initial values plotted as a function of the aging time. In this plot, each point is overlapped on the corresponding snapshots of PLA, PLA/5%TPS, and PLA/

10%TPS obtained at the indicated time of the incubation in the simulated soil.26 Before the incubation, the surfaces of PLA and PLA/5% TPS were smooth and homogeneously colored, while PLA/10% TPS had a smooth surface with some brown spots. The pure PLA did not feature significant macroscopic alterations on the D


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ACS Sustainable Chemistry & Engineering Scheme 1. Two Possible Pro-Oxidant Pathways of the Reaction of Myoglobin with Peroxidesa

a

Myoglobin promotes heterolytic cleavage of peroxides generating compound I that is converted into oxoferryl (steps a and b). The free radical of the heme protein could attack the polymer (step c) and generate myoglobin compound II. Compound II can be produced by homolytic cleavage of the peroxide (step a′). Myoglobin can also directly oxidize the polymer (step d).

the spectra of smooth and rough regions are presented as blue and red lines, respectively, and they present significant differences. When similar FTIR spectra were obtained for smooth and rough regions, a representative spectrum is presented as a dark green line. Figure 3A (lower panel) shows the changes in the FTIR spectra of PLA after 12 months under degradation in simulated soil; the upper panels show representative SEM images (120× magnified) and the lower panel the corresponding FTIR spectra. The black line is the spectrum obtained before the degradation step, and the blue and red lines correspond to smooth and rough regions of the material surface after degradation. On the basis of the visual changes, two regions were analyzed: a region without apparent modifications and a region with color and relief changes. In the altered regions, changes in the FTIR spectra were more evident and suggestive of PLA hydrolysis to produce lactic acid. The significant increase in the broad band peaking at 3400 cm−1 is assigned to the hydroxyl groups present in the lactic acid structure but not in PLA and corroborated by the bands peaking at 2995, 2944, 1383, and 1082 cm−1. The slight differences in energy relative to standard FTIR of lactic acid powder (2990, 2943, 1376, and 1046 cm−1) might be assigned to a different microenvironment as well as to the overlap with the spectral contribution of other compounds present in the sample. PLA hydrolysis is reinforced by the decreases in the bands at 914 and 870 cm−1 that are resulting from deformation vibrations of CCH coupled with CCO, OCO, and COC bending.32−36 PLA maintained in the soil also had a decrease in the band peaking at 1211 cm−1.

surface. However, both blends exhibited significant macroscopic surface changes after two months of aging in simulated soil. In PLA/5% TPS, the appearance of bubbles and color changes are evident. The PLA/10% TPS exhibited progressive roughness, bubbles, and large areas with brown color adjacent to bleached areas. The alterations of mass in the incubation span were consistent with macroscopic changes on the surface: this could be due to starch degradation.27 Increasing mass was observed during the first six months. The PLA exhibited a maximum mass increase four months after incubation while blends with TPS had a maximum mass gain after two months. After one year of incubation in the simulated soil, the blends with TPS exhibited a mass loss relative to the initial weight and proportional to the TPS content. Differently, the pure PLA remained with a slightly higher mass. The increase in mass in the first six months was proportional to the TPS content and could be related to wettability and colonization by microorganisms. For the TPS blends, the presence of amylopectin with branched hydroxylated chains favors hydration, water diffusion, and ultimately microorganism growth.28−31 The delay of the mass loss by degradation has been also related to the adaptation of microorganisms to the material after colonization. The higher mass content of PLA relative to baseline even after one year of aging in soil indicates that the material was hydrated and probably colonized by microorganisms. Determination of the effect of TPS on the degradation of PLA in the simulated soil was complemented by FTIR and SEM analysis. In the following figures, the FTIR spectra obtained before the degradation step of the material are presented as black lines. For the materials degraded in soil and oxidative model systems, E


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Figure 4. Changes in PLA incubated with pro-oxidant model systems. (A) Bottom: FTIR spectra of PLA before and after incubation with tert-butyl hydroperoxide. The black line is the spectrum obtained before incubation; the blue and red lines are the spectra obtained for smooth and rough regions of the material surface observed after degradation. Top: representative SEM images of the PLA surface after incubation with tert-butyl hydroperoxide. The central 50× magnified image covers a larger area for an overview of the degradation, and the lateral 200× and 220× magnified images show details about the changes in the material surface. (B) Bottom: FTIR spectra of PLA before and after incubation with myoglobin. The FTIR spectra of the two different patterns of PLA exhibited the same spectral features, and only a representative spectrum is shown. The black line is the spectrum obtained before incubation; the dark green line is the spectrum obtained for the material surface (dune regions) after degradation. Top: representative SEM images of the PLA surface after incubation with resting myoglobin. The central 50× magnified image covers a larger area for an overview of the degradation, and the lateral 200× and 220× magnified images show details about the changes in the material surface. (C) Bottom: FTIR spectra of PLA before and after incubation with peroxide-activated myoglobin. The FTIR spectra of the two different patterns of PLA exhibited the same spectral features, and only a representative spectrum is shown. The black line is the spectrum obtained before incubation; the dark green line is the spectrum obtained for the material surface after degradation. Top: representative SEM images of the PLA surface after incubation with peroxide-activated myoglobin. The central 50× magnified image covers a larger area for an overview of the degradation, and the lateral 220× magnified images show details about the changes in the material surface.

There was an increase in the bands peaking at 1264 and 1384 cm−1.36 The bands at 1300−1000 cm−1 were induced by ν(C− O−C), ν(CH3), ν(C−CH3), and their coupling vibrations. The SEM images of PLA obtained after degradation in soil, (Figure

3A, top panel) show surface erosion irregularly distributed on the surface. Consistent with the visible degradation, the FTIR of PLA with 5% TPS (Figure 3B) had a red shift and increase in the F


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ACS Sustainable Chemistry & Engineering 3300−3500 cm−1 band. After degradation, this band peaked at 3400 cm−1, which is consistent with the generation of lactic acid from PLA degradation. The PLA/5% TPS had a significant increase in the ν(CO) band at 1750 cm−1, which contrasts with what was observed for the degradation of pure PLA. This result is consistent with more efficient PLA degradation when blended with 5%TPS and also might reflect the overlapped contribution of amorphous PLA (peaking at around 1759 cm−1) and lactic acid (peaking at 1734 cm−1). The blend with 5% TPS aged in soil presented similar spectral changes to those observed for PLA (Figure 3B) in the 900−1300 and 1300− 1500 cm−1 regions. In this condition, we noted a significant increase in the 1264 and 1384 cm−1 bands as well as the bands peaking at 1014, 1085, 1183, and 1453 cm−1. Interestingly, the band peaking at 1211 cm−1 decreased and was accompanied by a significant increase in the intensity at 1207 cm−1 suggesting crystallization of the material.37,38 In the spectral region of 800−950 cm−1, the observed spectral changes after degradation were similar to that observed for PLA. The upper panel shows representative SEM images of the surface of PLA/5% TPS degraded in soil. The degradation due to soil components caused the appearance of pinholes, cracks, and some deep valleys. The FTIR spectra of PLA/10% TPS degraded in soil presented a striking difference in the region of 3200−3600 cm−1 when compared with the FTIR of PLA/5% TPS (Figure 3C, lower panel). For PLA/10% TPS, the loss of the band peaking at 3380 cm−1 was not accompanied by the appearance of a band peaking at 3400 cm−1. This event is probably because the higher TPS content favored the proliferation of microorganisms responsible for the degradation of PLA. SEM images (Figure 3C, upper panels) show that the increase in TPS percentage at 10% exacerbated the ridges and roughness that were present before degradation (Figure 1C). However, the incubation in soil promoted similar modifications on the surface as observed for PLA/5% TPS. The SEM images of PLA/10%TPS after degradation in soil show pinholes and hollows, and the fibrous patterns in the material not placed in soil disappeared due to erosion. Effect of TPS on the Degradation of PLA in Oxidant Systems. The degradation of PLA and blends in simulated soil is caused partly by enzymes secreted by living microorganisms and leaked from the dead ones. The biomolecules that can contribute to the degradation of PLA and blends include proteolytic enzymes,39 esterases,40 free radicals produced by the reaction of organic peroxides such as lipid-derived peroxides with transition metals, and heme proteins such as the cytochromes that are present in prokaryotic and eukaryotic cells. To investigate the mechanisms involved in the degradation of PLA and blends in the soil, and specifically whether free radical generation could be involved in this process, the materials were incubated with myoglobin activated by peroxide and with each reagent separately. Scheme 1 shows two possible pro-oxidant routes in the reaction of myoglobin with peroxides. Myoglobin predominantly promotes heterolytic cleavage of peroxides and generates myoglobin compound I (oxoferryl πcation), which is converted to oxoferryl and a tryptophancentered free radical (steps a and b). The free radical of the heme protein can attack the polymers (step c) and generate myoglobin compound II (oxoferryl form). Compound II can also be produced directly by the homolytic cleavage of peroxides (step a′). Regardless of the mechanism of generating

compound II, this high-valence form of myoglobin can also oxidize the polymers (step d). The homolytic cleavage of organic peroxides generates alkoxyl radicals that can also contribute significantly to the degradation of polymers. The samples were incubated for 7 days in solutions containing 100 μM tert-butyl hydroperoxide, 10 μM myoglobin, and myoglobin activated with tert-butyl hydroperoxide at the same concentrations. The 50× SEM image of PLA degraded by tert-butyl hydroperoxide revealed two principal patterns of attack: the presence of pinholes in a relatively smooth region and rough regions that are consistent with the efficiency of peroxides in promoting PLA degradation (Figure 4A, upper panels). Figure 4A, lower panel, shows changes in the corresponding FTIR spectra. On the basis of the visual changes in the PLA sample, two regions were analyzed: a region without apparent modifications and a region with darkening of the material. PLA treated with tert-butyl hydroperoxide did not present significant changes in the 2600−3600 cm−1 region, and there is no significant increase in the broad band peaking at 3400 cm−1. This result is consistent with the absence of PLA hydrolysis or secondary chemical reactions of lactic acid produced by PLA hydrolysis. A significant decrease was observed in the δ(CO) band in the rough region at 1750 cm−1 with complete disappearance of this spectral contribution in the smooth region. This result is consistent with PLA degradation to a compound in which the content of carbonyl-containing molecules is negligible or absent. A low-intensity band was also observed at 1640 cm−1 that was assigned to CC (both sp2). In contrast to what was observed for PLA degraded in soil, the polymer incubated in tert-butyl hydroperoxide showed a decrease in the band peaking at 1383 cm−1 without loss of the contribution of the band peaking at 1211 cm−1. The band peaking at 1186 cm−1 increased in the rough region and decreased in the smooth degraded region. Considering that the bands in this region are assigned to δ(C−O−C), δ(CH3), and δ(C−CH3) and their coupling vibration, the results were consistent with PLA degradation without contribution of lactic acid. This spectral feature is because the contribution of bands peaking at 2995, 2944, 1383, and 1082 cm−1 was not present after treatment with tert-butyl hydroperoxide. The FTIR analysis of the smooth region with pinholes suggested more extensive degradation of the material, and the vibrational spectrum in the region from 1100 to 1450 cm−1 exhibited only broad bands peaking around 1115, 1103, 1303, and 1430 cm−1. The 50× SEM image of PLA degraded by resting metmyoglobin (Figure 4B, upper panels) revealed a surface that resembles sand dunes and another that features several hills. The 200× image shows the region with hills leached while the area of dunes has deep grooves. However, both regions presented the same FTIR spectral features. Only the spectrum of the dune regions is shown (Figure 4B, dark green line in lower panel). Interestingly, spectral changes similar to that found in the material incubated in soil were observed when metmyoglobin was used as the degrading agent for PLA. Figure 4C, upper panel, shows the 50× image of PLA incubated with peroxide-activated myoglobin. This condition promoted a more homogeneous surface and fewer rough areas. The 220× magnification shows a smoother region with structures similar to volcano craters; the region has a very rough fibrous aspect. The activation of metmyoglobin by tertbutyl hydroperoxide led to complete degradation of PLA evidenced by the disappearance of the 1750 cm−1 band. G


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Figure 5. Changes in PLA/10% TPS incubated with pro-oxidant model systems. (A) Bottom: FTIR spectra of PLA/10% TPS before and after incubation with tert-butyl hydroperoxide. The black line is the spectrum obtained before incubation; blue and red lines are the spectra obtained for smooth and rough regions of the material surface observed after degradation. Top: representative SEM images of the PLA/10% TPS surface after incubation with tert-butyl hydroperoxide. The central 50× magnified image covers a larger area for an overview of the degradation, and the lateral 220× magnified images show details about the changes in the material surface. (B) Bottom: FTIR spectra of PLA/5% TPS before and after incubation with myoglobin. The FTIR spectra of the two different patterns of PLA/10% TPS exhibited the same spectral features and only a representative spectrum is shown. The black line is the spectrum obtained before incubation, and the dark green line is the spectrum of the material surface that was obtained after degradation. Top: representative SEM images of the surface of PLA/10% TPS surface after incubation with myoglobin. The central image 50× magnified covers a larger area for a general view of the degradation, and the lateral 220× magnified images show details about the changes in the material surface. (C) Bottom: FTIR spectra of PLA/5% TPS before and after incubation with peroxide-activated myoglobin. The black line is the spectrum obtained before incubation; blue and red lines are the spectra obtained for smooth and rough regions of the material surface after degradation. Top: representative SEM images of the surface of PLA/10% TPS surface after incubation with peroxideactivated myoglobin. The central 50× magnified image covers a larger area for an overview of the degradation, and the lateral 220× magnified image shows details about the changes in the material surface.

of PLA in the Copinet regions.22 In this condition, the highest peak with maximum at 2970 cm−1 is assigned to the vibration of the C−H axial strain.41

Significant spectral changes were also observed in other regions including the appearance of bands at 952, 1071, 1150, 1250, 1384, and 1470 cm−1. These bands replaced the typical bands H


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hydroperoxide were able to degrade the PLA and their blends. Therefore, the results show that the presence of peroxides and transition metals as additives to PLA products can significantly accelerate the degradation of these materials to make them environmentally sustainable. Another thing to point out is that the microorganisms normally secrete glycolytic and proteolytic enzymes but not heme proteins ,and thus, in the soil the contribution of heme proteins must come from the release of the internal contents of dead cells. Regarding the characteristics of the degradation process, the present study identified two principal patterns of degradation that could be identified by SEM and FTIR. The degradation process in which hydrolysis of PLA and TPS predominates led to the appearance of undulations, hollows, eruptions, and pinholes on the surface of the materials analyzed by SEM. In the corresponding FTIR spectra, the main change was the significant increase in the broad band at 3400 cm−1. The increases in the bands at 2995, 2944, 1383, and 1082 cm−1 are consistent with the formation of lactic acid as are the decreases at 914, 870, and 1211 cm−1 and the increases at 1264 and 1384 cm−1. This pattern was observed for the materials aged for one year in simulated soil and for the materials incubated with tertbutyl hydroperoxide and peroxide-activated myoglobin. The other pattern observed when the material was incubated in myoglobin solution was characterized by the predominance of a leached surface of the materials and complete changes in the FTIR spectra. Under these conditions, the bands at 3300−3500 cm−1 disappeared accompanied by significant increases in the bands at 953, 1073, 1150, 1252, 1384, 1453, and 2970 cm−1 (vibration of C−H axial strain). Taken together the results presented here demonstrated that additives such as peroxides and transition metals as well as the blend with TPS contribute significantly to the oxidative degradation of PLA. In addition, similar results obtained for the materials incubated in soil and in model systems suggest that the heme proteins leaked from dead microorganisms could significantly contribute to the oxidative degradation of the PLA and more favorably for PLA blended with TPS.

PLA blended with 5% and 10% TPS was also submitted to degradation by tert-butyl hydroperoxide, metmyoglobin, and peroxide-activated myoglobin. The presence of TPS at both percentages favored the degradation of PLA. The results obtained for PLA/5% TPS are shown in the Supporting Information, and the results obtained for PLA/10% TPS are described herein. The SEM images of PLA/10% TPS after treatment with tert-butyl hydroperoxide (Figure 5A, upper panels) show a degradation pattern with large holes with basrelief borders suggesting the formation of bubbles that were broken during incubation and are not homogeneously distributed. The corresponding FTIR spectra (Figure 5A, lower panel) are consistent with different degrees of PLA hydrolysis. In this condition, there is both a red shift and broadening of the 3300−3500 cm−1. This band is probably a composite resulting from an overlap of the lactic acid band at 3400 cm−1 with a 3315 cm−1 band that is assigned to the OH stretching of the glucose ring. The presence of glucose indicates that TPS was also hydrolyzed.42,43 In Figure 5B, upper panels, the SEM images of PLA/10% TPS after treatment with myoglobin show a leached surface with some sparse pinholes and eruptions and the presence of a large fissure crossing the surface. The corresponding FTIR (Figure 5B, lower panel) spectrum obtained after myoglobin treatment is very similar to the spectrum of PLA/5% TPS obtained after treatment with myoglobin and peroxide-activated myoglobin. Under this condition, the bands at 3300−3500 cm−1 (OH groups of TPS) as well as the 1750 cm−1 δ(CO) band of PLA and lactic acid disappeared. However, the other bands in the Copinet regions show a significant increase.23 In this condition, there was also a significant increase at 953, 1073, 1150, 1252, 1384, 1453, and 2970 cm−1 (vibration of C−H axial strain). The 50× and 200× images of PLA/10% TPS incubated with peroxide-activated myoglobin (Figure 5C, upper panels) resemble those obtained for the same material incubated with tert-butyl hydroperoxide. The images show undulations, eruptions, and hollows. Under these conditions, the FTIR spectra are consistent with PLA and TPS hydrolysis (Figure 5C, lower panel). There is a blue shift in the 3300−3500 cm−1 region: this peaked at 3315 cm−1. The presence of OH stretching from the glucose ring was only observed in this condition.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.5b00639. Contact angles and degradation of PLA/5% TPS by oxidant model systems (PDF)

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CONCLUSIONS The present study contributes to two aspects for the development of biodegradable materials. Regarding the development of blended materials, it was observed that the presence of the thermoplastic starch increased the wettability that, in turn, favored degradation in the simulated soil as well as in the pro-oxidant model systems. Evidence for the more efficient degradation of the blended material included mass loss, macroscopic surface changes, SEM images, and FTIR. The higher the TPS content, the greater the wettability and loss of mass of the materials incubated in simulated soil for one year. In the simulated soil, the presence of TPS contributed to the penetration and growth of the microorganisms because TPS offers glucose as a biological fuel. On the other hand, the similar results of degradation obtained in the soil and the model systems suggested that, in the former, heme proteins and peroxides could have a significant contribution. Another interesting fact is that lonely myoglobin and tert-butyl

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone +55-11-3356-7494. *E-mail: [email protected]. Phone +55-11-4996-8280. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to FAPESP (Proc. 2012/13445-8 and 2012/ 07456-7), CNPq, and CAPES for the grants. A.T. is a fellow of I


Research Article


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CAPES. We also thank Daniel Octaviano for preparing the samples and Hamilton Viana for initial discussions.

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ABBREVIATIONS FTIR, Fourier-transformed infrared; PDLA, poly D-(lactic) acid; PLA, poly(lactic)acid; PLLA, poly L-(lactic) acid; SEM, scanning electronic microscopy; TPS, thermoplastic starch

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