thermoplastic starch blends

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Original Article

Fungal degradation of reprocessed PP/PBAT/thermoplastic starch blends Thainá Araújo de Oliveira a , Renata Barbosa b , Avilnete B.S. Mesquita c , Josie H.L. Ferreira c , Laura Hecker de Carvalho d , Tatianny Soares Alves b,∗ a

Graduate Program in Materials Science and Engineering, Technology Center, Federal University of Piauí, Teresina, PI, 64049-550, Brazil Course of Materials Engineering and Graduate Program in Materials Science and Engineering, Technology Center, Federal University of Piauí, Teresina, PI, 64049-550, Brazil c Department of Parasitology and Microbiology, Health Sciences Center, Federal University of Piauí, Teresina, PI, 64049-550, Brazil d Federal University of Campina Grande – Center Science and Technology – Graduate Program in Materials Science and Engineering, UAEMa, Campina Grande, PB, Brazil b

a r t i c l e

i n f o

a b s t r a c t

Article history:

It is well known that the inadequate disposal of polymeric materials causes significant

Received 10 May 2019

environmental problems. Recycling and the use of biodegradable polymers are among the

Accepted 20 December 2019

methods used to minimize these problems. However, the low mechanical performance of the

Available online xxx

majority of biodegradable polymers hinders the direct substitution of synthetic polymers,

Keywords:

Blending synthetic and biodegradable polymers is a way to develop recyclable biodegradable

such as polyethylene (PE), polypropylene (PP) and polystyrene (PS), by biodegradable ones. Fungi degradation

products with a good set of mechanical properties. Therefore, in this work we investigate the

Mechanical recycling

influence of extrusion cycles on the degradation of a polypropylene/poly (butylene adipate-

Hydrophilicity

co-terephthalate)/thermoplastic starch blend before and after inoculation with Aspergillus sp. and Penicillium sp. fungi degradaded for 30 days. The samples were exposed to the fungi and their mass loss, chemical structure, hydrophilicity, and morphology were observed as a function of inoculation time and processing cycles. Our results indicate that thermomechanical degradation favors the deposition of fungi in the samples and enables changes in film morphology and hydrophilicity. © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.

Introduction

The number of studies aiming to control the “lifetime” of synthetic polymers after being reprocessed and/or discarded in the environment is increasing as these polymers tend to resist thermo-mechanical and biotic degradations. Therefore,

∗

investigations on the effects of recycling as well as on the manufacture of blends and composites with biodegradable matrices by academic and industrial communities, has been growing steadily [1–4]. Classic plastics waste disposal causes significant environmental problems and still needs to be studied extensively. Blending synthetic polymers with matrices containing hydrolysable groups enhances biodegradation, as degradation starts at the biodegradable component, compromises the structural integrity of the mixture and increases its sur-

Corresponding author. E-mail: [email protected] (T.S. Alves). https://doi.org/10.1016/j.jmrt.2019.12.065 2238-7854/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Oliveira TA, et al. Fungal degradation of reprocessed PP/PBAT/thermoplastic starch blends. J Mater Res Technol. 2020. https://doi.org/10.1016/j.jmrt.2019.12.065

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face area, thus enabling enzyme attack [5]. This behavior was reported by Passos et al. [6], when studying the biodegradation of low-density polyethylene (LDPE)/poly (hydroxybutyrate-cohydroxyvalerate) (PHBV) blends, which proved an increase in the biodegradation of the blend when compared to neat LDPE. However, caution should be exercised when using this approach, as degradation of these materials may not be complete and can generate microplastics. Biodegradation may occur in diverse environments and in the presence of a wide range of microorganisms. It depends on several factors which include the polymer’s molecular weight, chain flexibility, crystallinity, the presence of hydrolysable groups, the types of microorganisms present, temperature and exposure medium [1,2,5,7–10]. Several investigations on the effects of environmental conditions and the most efficient microorganisms to cause polymer biodegradation are reported in the literature. da Costa Reis et al. [11] used the Sturm Test to evaluate biodegradation of bionanocomposites of poly(3-hydroxybutyrate-co3-hydroxyvalerate)/vermiculite for a period of 28 days; Hermanová et al. [12] reported the biodegradability of a series of copolyesters of poly(ethylene terephthalate-co-lactate) by inoculating them in digested mud; Nowak et al. [13] exposed LDPE films to Aspergillus niger, Aspergillus terreus, Aureobasidium pullulans, Paecilomyces varioti, Penicillium funiculosum, Penicillium ochrochloron, Scopulariopsis brevicaulis, Trichoderma viride fungi and of a mixture of these fungi to evaluate the microbial degradation of these samples. Among the fungi used in biodegradation tests, Aspergillus sp. and Penicillium sp. are two of the most economically important, as they are responsible for food and environment deterioration and production of mycotoxins. These are filamentous fungi which are able to form biofilms, i.e., they can organize themselves in a communal structure and, by secreting extracellular enzymes, they can stick to inert surfaces [14,15]. Canché-Escamilla et al. [16] studied the biodegradation of thermoplastic starch and grafted thermoplastic starch, inoculated with Aspergillus niger. The authors state that the poly(acrylate) moiety in methyl butyl-co-poly(methacrylate) limits the access of the fungus to starch and reduces the system´s biodegradation rate. Chandra and Rustgi [17] inoculated a blend of linear low-density polyethylene (LLDPE) and starch modified with a consortium of fungi (Aspergillus niger, Penicillium funiculosum, Chaetomium globosum, Gliocladium virens, and Pullularia pullulans) for 28 days, and observed that biodegradation rate increased with starch content. The degradation mechanisms of the major commercial thermoplastics, when mechanically recycled, are well defined in the literature [18–20]. However, the behavior of these polymers when mixed with biodegradable matrices and reprocessed for several cycles is not properly understood. Peres et al. [21] investigated the effect of extrusion reprocessing on the structure, mechanical, rheological and thermal properties of LDPE/thermoplastic starch and stated that an increase in extrusion cycles reduces the size of the starch phase in the blend and causes discrete changes in the mechanical and rheological properties of the blend. The authors also state that the transition temperatures of the blend are not affected by increasing extrusion cycles, which suggests that recycling

does not significantly change these two polymers and that the substitution of LDPE-based products by LDPE/thermoplastic starch blends is viable. Oliveira et al. [22] studied the effect of reprocessing cycles on a PP/PBAT/thermoplastic starch blend and observed that, due to the low degradation temperatures of the PBAT/thermoplastic starch system, this phase degrades before the PP matrix and that recycling resulted in improved elastic modulus and yield strength and decreased impact strength of the blend. This work investigates alternatives to the challenges found in the disposal of polymers such as polypropylene (PP) and polyethylene (PE). Since PP is one of the most recycled polymers; this paper proposes to study the relationship between thermomechanical degradation, which occurs during recycling, and possible biodegradation when PP is mixed with a biodegradable matrix. Until now, only Sikorska, et al. [23] have evaluated the influence of multiple reprocessing cycles on the biodegradation of poly (L-lactic acid) in industrial composting conditions. Thus, the aim of the present work is to expand the findings of a previous work of ours [22] and evaluate the influence of consecutive extrusion cycles in the biodegradation of films of PP/PBAT/thermoplastic starch blends promoted by the fungi Aspergillus sp. and Penicillium sp. after 30 days inoculation. The biodegradation essay was carried out according to ASTM G21-13 standard and the properties of the inoculated films were evaluated by means of mass loss (ML), Fourier transform infrared (FTIR), contact angle, optical microscopy (OM) and scanning electron microscopy (SEM). Results indicate that thermo-mechanical degradation favors the deposition of fungi on the samples leading to changes in film morphology and hydrophilicity.

2.

Experimental

2.1.

Materials

The polymer used was virgin polypropylene provided by Braskem/SA under the commercial name RP-347. It is a random copolymer of propene and ethene with a melt index of 1 g min−1 measured at 230 ◦ C and under a 2.16 kg weight according to the ASTM D1238 standard. Its density, determined according to the ASTM D792A standard, is 0.902 g cm−3 . Ecobras® RD 704, a PBAT/thermoplastic starch blend containing 48% PBAT and 52% w/w thermoplastic starch (TPS) provided by BASF was used to make the PP/PBAT/TPS blend. The thermoplastic starch, manufactured by Corn Products, is composed of at least two different polysaccharides, linear amylose and branched amylopectin, and low molecular weight plasticizers.

2.2.

Methods

2.2.1.

Reprocessing

For the simulation of the recycling processes, both virgin PP and 85/15% PP/PBAT/thermoplastic starch blend were extruded seven times (seven extrusion cycles) in a lab model AX Plastic single-screw extruder (model Lab-16) with a temperature profile of 175, 180 and 190 ◦ C at a screw speed of

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60 rpm. Samples were collected after cycles 1 and 7, followed by specimen molding, biodegradation, and characterization.

40× magnification in a Leica Microsystems MD500 equipment, with ICC 50E capture camera.

2.2.2.

2.3.4.

Compression molding

Samples of neat PP and of the blend extruded once were dried for 7 h at 70 ◦ C, while those of neat PP and of the blend, subjected to seven extrusion cycles, were dried for 24 h at the same temperature. The films for biodegradation were compression molded at 160–165 ◦ C under 0.5 T for 90 s. The films were pressed between two 50 mm2 aluminum sheets. All samples have the same average thickness.

2.2.3. Biodegradation assay: inoculation of samples with the microorganisms Biodegradation was performed according to ASTM G21-13, which evaluates the resistance of synthetic polymers to fungal attack. The glassware used to prepare the culture medium and spore suspension was autoclaved at 121 ◦ C for 15 min, the same conditions used to autoclave the solidified culture medium of the Agar salt. The spore suspension containing a fungi consortium of Aspergillus sp. and Penicillium sp. was pulverized over the film’s surface, which was then placed in 150 mm diameter Petri dishes. The samples thus inoculated were incubated for 30 days in an oven operating at 29 ◦ C and 85% average humidity. After the incubation period, the films were removed from the Petri dishes, washed with distilled water and dried in an oven for 24 h at 60 ◦ C before being characterized.

2.3.

Characterizations

2.3.1.

FTIR

An IRAffinity-1 Fourier transform infrared spectrometer (FTIR), operating in attenuated total reflectance (ATR) mode and 4000–400 cm−1 wavelength range, was used to record the spectra of the samples before and after thermo-mechanical degradation. The spectra were recorded after 64 scans. The carbonyl index (%) was used to o compare the extent of degradation of neat PP and of PP/PBAT/thermoplastic blends. The carbonyl index is given by the ratio of band intensity from the carbonyl group at 1723 cm−1 to that of the band at 2721 cm−1 , i.e., the group that was not changed during degradation [24].

2.3.2.

Contact angle

The Sessile Drop method was used to perform contact angle measurements. In this procedure, a 10 ␮L drop of distilled water is placed on the film surface attached on a flat base and monitored over time. The real time procedure is recorded with a Logitech camera, HD 1080p and with Media Player Classic software on a computer. Contact angle was estimated by image analysis obtained with a version 4.5 Surftens software. Contact angle readings were taken at 60 s intervals after the deposition of the drop onto the film surface. Each contact angle value reported corresponds to an average of three images.

2.3.3.

Optical microscopy

Optical microscopy was used to analyze the effect of the fungal attack on the films’ surface. The images were taken with

SEM

SEM morphological analysis was performed on films inoculated for 30 days with the Aspergillus sp. and Penicillium sp. fungi. A FEI Quanta FEG 250 scanning electron microscope was used at an operating voltage of 20.00 kV. The samples were metallized with gold, and the images captured at magnifications of 100×, 500×, 1000× and 10,000×.

2.3.5.

Mass loss

Gravimetric analysis was used for monitoring the mass loss of neat PP and of PP/PBAT/thermoplastic starch blend after reprocessing and of exposure to spore suspension. The samples were weighed before and after the 30-day incubation period. The values of mass loss were calculated by means of Eq. (1). % Mass Loss = Initial mass−final mass⁄Initial mass x100

3.

Results and discussion

3.1.

FTIR

(1)

The infrared spectra of neat PP and of the PP/PBAT/thermoplastic starch blend after 1 and 7 processing cycles, before (black) and after 30 days (traced red line) inoculation with Aspergillus sp. and Penicillium sp. fungi are shown in Fig. 1. Characteristic bands of PP, i.e., bands at 2920 cm−1 , 1457 cm−1 and 1373 cm−1 corresponding, respectively, to the sp3 carbon stretching vibration of the CH group, to the angle deformation vibration of CH2 and the symmetric deformation of the CH3 group present in the polypropylene chain [22] are shown in Fig. 1(a) and (b). No new bands, nor the disappearance or displacement of PP characteristic bands were observed in the neat PP spectra as a function of reprocessing cycles (1 and 7). After inoculation with the fungi consortium, however, a slight decrease is seen in the intensity of the bands after 30 days inoculation. This decrease is more prominent in the seventh cycle and likely occurs due to the formation of biofilm in the samples’ surface. Sheik et al. [10] demonstrated that the abiotic degradation of polyolefins increases the quantity of terminal CH3 groups of these macromolecules, allowing microorganisms colonize these synthetic polymers. Previous results, reported by our research group by Oliveira et al. [22], indicate that most significant changes in mechanical properties, morphology and thermal stability are observed at the most extreme processing conditions used. Thus, in this work, the blends chosen for degradation were those processed under mild and extreme conditions i.e., after 1 and 7 cycles. Fig. 1(c)–(d) exhibits the spectra of the PP/PBAT/thermoplastic starch blend, where characteristic bands of each material were observed. According to Oliveira et al. [22], the PBAT-related vibrations may be observed in the regions 1723 cm−1 , corresponding to the stretching of the carbonyl ester group, at 1271 cm−1 attributed to vibration stretch of the C O bond directly bonded to the carbonyl

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Fig. 1 – FTIR spectrum at the 4000–400 cm−1 interval for the extrusion cycles (a) 1 and (b) 7 of neat PP, and (c) 1 and (d) 7 of the PP/PBAT/thermoplastic starch blend, before (black) and after 30 days (red) of inoculation.

and at 725 cm−1 the deformation outside the aromatic ring plane appears. The thermoplastic starch-related vibrations are found at 3400 and 1100 cm−1 corresponding to the O H stretching and the stretch vibration of the hydroxyl-bonded carbon, respectively. A comparison of the spectra of the blend after one and seven extrusion cycles incubated with fungi for 30 days, show an inversion in the intensity of O H stretching vibration (3400 cm−1 ), bands while that attributed to the stretching the ester group carbonyl (1723 cm−1 ) remains unaltered in the first cycle, and slightly decreases in the seventh cycle. The effects of fungal degradation were also observed by changes in carbonyl index (CI). Attention was focused on the region of the carbonyl group at 1723 cm−1 , which is susceptible to degradation. Carbonyl indexes (CI) of, respectively, 2.24 and 2.13 were obtained for the 1 cycle PP/PBAT/thermoplastic starch blend before and after fungal attack, corresponding to a discrete oxidation and low fungal attack. In contrast, the reduction in CI was more significant for 7 cycle PP/PBAT/thermoplastic starch blend before and after fungal attack, with CI values of 2.65 and 1.83, respectively. The decrease in the CI observed suggests that the microorganisms preferentially attacked more oxidized molecules. During continuous contact with biotic factors, polymers decompose, resulting in a reduced number of carbonyl bonds and hence reducing the carbonyl index [25]. In general, it is accepted that, in the presence of microorganisms, the concentration of these surface functional groups will decrease, which is commonly reported as a decrease in the carbonyl index [26]. Harding et al. [15] stated that filamentous fungi go through six stages for the formation of biofilms: (a) the deposition of spores onto the substrate’s surface; (b) secretion of adhesive

substances of spores and active germination; (c) formation of biofilms, which is the growth and initial colonization stage of fungi – in this stage, there is the ramification of hyphae in the samples’ surface layers; (d) the formation of a more compact hyphae and micelles network; (e) adhesion amid hyphae as a reproduction occurs, characterized by an enlargement in the aerial growth of the colony, fundamental for the reproduction and dispersion of fungi; (f) the release of spores so the formation cycle restarts. Sheik et al. [10] while explaining the mechanism of polymeric biodegradation, pointed out that the first stage of the attack of microorganisms is the release of extracellular enzymes for oxidation or hydrolysis of the polymers. This creates functional groups capable of improving the hydrophilicity of the polymer and consequently increases the adhesion of the fungi to the polymeric matrix. The reaction produced by the enzymes released by the microorganism on the sample surface causes polymer chain scission. The decrease in molecular weight facilitates the consumption of polymers by microorganisms. The proposed mechanism described is depicted in Fig. 2. According to Sheik et al. [10], the increase in intensity of the band at 3400 cm−1 , in the first cycle after the 30-day fungal inoculation, may be an indicative of an increase in vibrations associated with functional groups that could be associated to proteins/enzymes. If this is true, it suggests that the fungi present in these samples would be in the first stage of biodegradation. However, it is also a region of the FTIR spectrum in which humidity or oxidation of the sample could result in increased intensity. The seven times reprocessed samples, however, have already suffered thermo-mechanical degradation, as reported by Oliveira et al. [22]. Therefore, the microorganisms present in these samples are already in the

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Fig. 2 – Proposed polymer biodegradation mechanism. Source: Adapted Sheik, Chandrashekar, Swaroop and Somashekarappa [10].

polymer assimilation stage, changing the macromolecules by ␤-oxidation of the chains of the carbonyls present in the PBAT/TPS system and consequently decreasing the intensity of the band at 1723 cm−1 [6,10]. The infrared spectra of the degraded materials indicated that, in neat PP, the fungi adhered to the sample’s surface and that, in the once processed blend, the fungi released the extracellular enzymes necessary to the break of macromolecules easing assimilation by the microorganisms. The fungi present in the blend reprocessed seven times could likely assimilate the polymer chains.

3.2.

Contact angle

Fig. 3 shows the average values of contact angle for the reprocessed films of (a) neat PP and (b) PP-PBAT/TPS blend, before and after 30 days of inoculation with the Aspergillus sp. and Penicillium sp. fungi. The average value of the contact angle for the films of neat PP extruded once was 52.07 ± 0.73◦ , while for that extruded 7 times a value 7.18% lower was obtained. The result found for the neat PP differs from those reported in the literature. Butnaru et al. [27] tested the degradation of polypropylene-based biocomposites by the Bjerkandera adusta fungus and found values of 93◦ for the contact angle of neat PP with no exposure to the fungus. Long and Chen [28] studied the surface characteristics of polypropylene and found the

initial contact angle of PP was approximately 84◦ . According to Suresh et al. [29], surface rugosity is one of the factors influencing contact angle values; the author explains that an increase in rugosity leads to higher contact angle. Thus, the discordance between the values found in this work and those reported in the literature may be an effect of different irregularities on the sample’s surface. Oliveira et al. [22] suggest that the thermo-mechanical degradation imposed by reprocessing cycles induces ␤scission of polypropylene chains, generating free-radical carbons. The 7.18% reduction in contact angle for the seventimes reprocessed neat PP is in accordance with these statements, as the presence of free radicals in these samples improves the interaction between the film’s surface and the distilled water drop [29]. After 30 days exposure to the fungi, the contact angle of one time extruded neat PP decreased by 11.46% when compared to that of a non-exposed single extruded PP sample. Neat PP subjected to seven reprocessing cycles and exposed to the fungi displayed a 24.68% reduction in contact angle compared to a seven times extruded PP sample not exposed to the fungi. Thermo-mechanical degradation causes chain scission and increases the availability of the CH3 chain terminal groups susceptible to enzymatic oxidation. The fungi present in the samples can secrete the extracellular enzymes and form water channels, so the maturation stage of biofilms occurs. This is justified as there is a larger increase of hydrophilic-

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et al. [32] when studying biodegradation of the poly (vinyl alcohol)/poly (vinyl chloride) (PVA/PVC) and poly (vinyl alcohol)/poly (caprolactone) (PVA/PCL) blends in soils. Therefore, contact angle measurements indicate that increasing extrusion cycles render the samples more hydrophilic, as the thermo-mechanical degradation breaks the macromolecular chains and create polar groups that can interact with water. Furthermore, it is noteworthy that when fungi consume polymer chains, they cause an increase in the samples’ surface rugosity and, consequently, decrease water contact angle over the film surface. The choice of the number of processing cycles (1 and 7) to perform biodegradation by fungal inoculation was based on the results reported by Oliveira et al. [22]. Increased number of processing cycles altered blend morphology, rendered the starch phase more homogeneous and partially coalescing in the 7th cycle.

3.3.

Fig. 3 – Average values of contact angle for the samples of (a) neat PP and for (b) PP-PBAT/TPS blend after 1 and 7 extrusion cycles, before and after 30 days of inoculation with the fungi Aspergillus sp. and Penicillium sp.

ity of the samples extruded seven times and exposed to fungi [15,30]. Addition of PBAT/thermoplastic starch caused a reduction of 3.70% of the contact angle with respect to the one-cycled extruded neat PP. This reduction is associated with the insertion of hydroxyl groups present in the thermoplastic starch and carbonyl groups present in the PBAT ester group, which tend to increase sample polarity. For the seven times reprocessed blend, the value of the contact angle was 44.40 ± 1.20◦ . This 11.40% reduction with regard the first cycle is probably associated with improvement of the blend’s interfacial interaction. The miscibility increase of the PBAT/thermoplastic starch system with PP makes the polar functional groups more available to interact with the drop of distilled water deposited on the film surface [22,31]. The contact angle reduction of the fungi exposed blends were 27.40 and 36.37% for the first and seventh extrusion cycles, respectively. These results indicate that there was an increase in rugosity of the samples due to the action of microorganisms. Similar results were found by Campos

Optical microscopy

Fig. 4 shows optical micrographs of films of neat PP reprocessed 1–7 times after 10 and 30 days of fungal inoculation. No morphological changes were observed for the neat PP films after biotreatment. However, micrographs showed evidence of adhesion of fungal spores (black dots) on the samples’ surface. These results corroborate contact angle and infrared spectra data. Nowak et al. [13] and Passos et al. [6] showed the adhesion of spores to the surface of neat LDPE films by scanning electron microscopy; Longo et al. [33] used optical microscopy to detect the adherence of microorganisms on the surface of PP and bi-oriented PP films buried two meters underground in a landfill. These results reinforce the capacity of microorganisms of adhering to the surfaces of polyolefins, and this is the first step in the biodegradation process. Optical micrographs of PP/PBAT/thermoplastic starch films reprocessed 1 and 7 times after 10 and 30 days of inoculation with the fungi are shown in Fig. 5. In addition, a sample of PP/PBAT/thermoplastic starch films before fungal attack was added to the Fig. 5a. Fig. 5a shows a homogeneous surface even after exposure to 7 consecutive reprocessing cycles. Fungal attack caused the appearance of fissures (black smears) on the films’ surface within 10 days of incubation, both in the first (Fig. 5b) and the seventh extrusion cycles (Fig. 5d). The microorganisms keep increasing fissure size as the incubation time increases (Fig. 5c–e). Additionally, for the same period, fungal attack is more aggressive in the seven-times reprocessed blend than in the blend processed only once. Passos et al. [6] observed significant changes in the morphology of PE/PHBV blends caused by the action of microbial enzymes incubated for 30-days. Optical micrograph results corroborate the values found for contact angle; demonstrating that thermo-mechanical degradation, which occurred during extrusion, facilitates microbial attack and consequently increases sample roughness.

3.4.

Scanning electron microscopy

Fig. 6(a–b) shows the electron micrographs of blends processed 1 and 7 times without the fungal attack. Fig. 6a indicates

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Fig. 4 – Optical microscopy of the neat PP after 1(a–b) and 7(c–d) extrusion cycles after 10 and 30 days of inoculation with the fungi, respectively. Magnification: 40×.

a regular surface with no cracks or cavities present for the blend undergoing single processing (extruded once). Increased reprocessing (7 times extruded, Fig. 6b), renders film surface more irregular, probably due to polymer thermo-mechanical degradation, which facilitated fungal deposition in the samples. Fig. 7(a–d) corresponds to the images of the blend extruded a single time and inoculated with the fungi consortium for a 30-day period, with magnifications of 100×, 500×, 1000× and 10,000×. The third stage of degradation mechanism involves the formation of micro-colonies, from the multiplication, the apical enlargement and the ramification of hyphae. Fig. 7(b) shows the growth and ramification of hyphae. In this stage of degradation, the microorganisms cause physical changes in the film such as voids (white circles). These results indicate that the fungi consortium applied over the sample surface can use the PBAT/thermoplastic starch as a carbon source. Similar results were found by Khan et al. [9] when studying the biodegradation of polyurethane by Aspergillus tubingensis for 20 days. Fig. 7(c) and (d) indicated that despite the presence of microorganisms causing physical changes on the film’s surface, the adhesion of fungi is still poor, probably due to the film’s low hydrophilicity as, the lower the hydrophilic characteristic of the surface, the poorer the adhesion and interaction between the substrate and the microbial cells. Fig. 8(a–d) shows micrographs of the seven times reprocessed PP/PBAT/thermoplastic starch blend after a 30 days

exposure to the fungi Aspergillus sp. and Penicillium sp., with magnifications of 100×, 500×, 1000× and 10,000×. A comparison of the amount of spores and voids between Figs. 8(a) and (b) and 7(a) and (b), indicates that fungal growth is more expressive for the seven times reprocessed blend than for the blend processed only once. In these micrographs, the presence of spores (white dots) scattered all over the sample’s surface is visualized as well as voids (white circles) indicating that the fungi could penetrate deeper in the substrates as well as the formation of networks of hyphae and micelles (black arrows) in the film’s surface. These results suggest that for these samples, the fungi could complete the six formation stages of the biofilm and that the blend’s thermo-mechanical degradation accelerates biodegradation, as confirmed by FTIR data. The heterotrophic nutrition mode, the secretion of extracellular enzymes, the absorption of nutrients through the cell wall and the apical growth are characteristics that demand the fungi to keep close contact with the substrate. In Fig. 8(c) and (d), adhesions are shown amid hyphae-spores, spore–spore and spore–substrate. These interactions are a consequence of the formation of biofilms, and thus the microorganisms inoculated in the samples with seven consecutive extrusion cycles reached deeper layers of the films. That is because in the seven-times reprocessed samples there is a decrease of the PBAT/thermoplastic starch phase making the break and assimilation of these molecules easier and assuring the colonization and survival of the fungi consortium [34].

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Fig. 5 – Optical microscopy of the neat PP/PBAT/thermoplastic starch blend (a) before fungal attack, after 1(b–c) and 7(d–e) extrusion cycles after 10 and 30 days of inoculation with the fungi, respectively. Magnification: 40×.

Fig. 6 – Scanning electron microscopy of blends processed 1 (a) and 7 (b) times without the fungal attack. Magnifications: 1000×.

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Fig. 7 – Scanning electron microscopy of the single time extruded PP/PBAT/thermoplastic starch after 30 days of inoculation with the fungi Aspergillus sp. and Penicillium sp. Magnifications: (a) 100×, (b) 500×, (c) 1000× and (d) 10,000×.

Fig. 8 – Scanning electron microscopy of the PP/PBAT/thermoplastic starch blend with 7 extrusion cycles, after 30 days of inoculation with the fungi Aspergillus sp. and Penicillium sp. Magnifications: (a) 100×, (b) 500×, (c) 1000× and (d) 10,000×.

3.5.

Mass loss

Table 1 shows the results of mass loss of the neat PP samples and of the blend processed 1 and 7 times, after the incubation period with the fungi consortium of Aspergillus sp. and Penicillium sp.

A discrete mass gain is observed with the inoculation of the PP samples. This gain may be attributed to the initial stages of the formation of biofilms, which involves adhesion of spores on the substrates, followed by an active adhesion to the surface, in which there is secretion of adhesive substances by the germinated spores and hyphae [15].

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Table 1 – Mass loss of the neat PP and the PP/PBAT/thermoplastic starch blend after 30 days of inoculation with the fungi Aspergillus sp. and Penicillium sp. Sample

Mass loss (%)

Standard deviation

Neat PP 1 cycle Neat PP 7 cycles PP/PBAT-thermoplastic starch 1 cycle PP/PBAT-thermoplastic starch 7 cycles

−0.262 −0.620 1.04 2.32

0.015 0.053 0.080 0.160

Once the suspension of spores is sprayed over the films’ surface, the mass increase of the neat polymer indicates that the fungi secrete the adhesive substances from the spores. Since there was a larger mass gain for the samples reprocessed 7 times, a suggestion is made that there was a larger adhesion of the spores on these samples. Optical microscopy images confirm these results. Passos et al. [6] investigated the biodegradation of PHBV, LDPE films and the LDPE/PHBV blend after a 28-day inoculation period with the fungus Paecilomyces variotii and observed fungal adhesion to the surface of polyolefin films without causing significant damages to them. Karlsson and Stromberg [35] showed that after 98 days of incubation of neat and recycled polypropylene with a mixture of algae and fungi, there was formation of biofilm on the samples’ surface and the growth extension of the biofilm was larger on the recycled matrix. The authors suggest that the additives and the recycled polymer degradation products are accessible to microorganisms. Similar results were observed in this work. In the samples containing 15% of PBAT/thermoplastic starch, a mass reduction was observed during degradation, indicating that the microorganisms used the system as a carbon source. Similar results were found by Canché-Escamilla et al. [16] who evaluated the degradation of thermoplastic starch samples incubated with the fungus Aspergillus niger for 45 days. In the work presented here, the degradation rate of the blend for the period studied was low. Only 1.04% of mass was lost for the samples processed once and 2.32% mass loss for the samples reprocessed 7 times. Nikazar et al. [36] and Chandra and Rustgi [17] investigated the influence of starch content on the degradation of LDPE/corn starch blend and concluded that increased degradation rate was observed for the starch richer samples, which was attributed to the larger amounts of natural polymer facilitating fungal access to the carbon source. Additionally, an increase in the number of extrusion cycles doubled the percentage of mass loss of the blend. This behavior is likely due to the decrease of PBAT/thermoplastic starch domains caused by the thermo-mechanical degradation suffered by the material during reprocessing [22]. Thus, mass loss analysis shows that the action of microorganisms is affected both by the presence of PBAT/thermoplastic starch and the thermo-mechanical degradation imposed by the reprocessing. The low degradation index displayed by the blends, even after 7 reprocessing cycles, can be attributed to the low rate of enzymatic activity of the fungi in the first 4 weeks. According

to Arutchelvi et al. [37] the addition of natural polymers such as starch promotes blend degradation, but the rate of degradation and microbial activity are strongly dependent on the type of synthetic polymer. Thus, it is necessary to employ more suitable characterization techniques to confirm the biodegradation of the blends investigated. Microscopy results indicate that there was a slight fungal attack on the seven times reprocessed mixture after 30 days of incubation. Contact angle results corroborate this observation, since the fungal attack causes the appearance of cracks in the samples promoting an increase in roughness of the material. These results may justify the decrease in intensity of the band at 1723 cm−1 , corresponding to the carbonyl ester group, as these are the main functional groups degraded during polymer consumption by microorganisms. Despite these observations, it is important to note that this is an initial study, that it is necessary to assess the longterm impacts of these fungi on these blends, and whether this is the best approach to improve the solid waste problem caused by polymers such as PP.

4.

Conclusions

The influence of multiple extrusions on the biodegradation of inoculated PP films was slight and taken as an indication that the microorganisms used here cannot significantly consume this polymer. PBAT/TPS addition to PP did not lead to significant changes in mass loss, but these results associated with FTIR spectra, microscopy images and contact angle analysis suggest a slight degradation, which was attributed to eased fungal attack in these blends. Infrared analyses suggest that the blends are in different stages of the degradation by fungal attack compared to neat PP. Microscopic analysis (OM and SEM), show differences in the growth extension and the fungal attack and show that these are more aggressive in the samples extruded seven times.

Conflict of interest All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version. This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue. The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript The following authors have affiliations with organizations with direct or indirect financial interest in the subject matter discussed in the manuscript:

Funding This work was supported by the CNPq [process numbers 446655/2014-7; 306312/2015-8]; CAPES; and the FAPEPI.

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Acknowledgement [13]

The authors acknowledge the support from UFPI. [14]

Appendix A. Supplementary data [15]

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j. jmrt.2019.12.065.

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