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Biodegradation of polylactide-based composites with an addition of a compatibilizing agent in different environments
International Biodeterioration & Biodegradation 147 (2020) 104840
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Biodegradation of polylactide-based composites with an addition of a compatibilizing agent in different environments
T
Ewa Olewnik-Kruszkowskaa,∗, Aleksandra Burkowska-Butb, Iwona Taracha, Maciej Walczakb, Ewelina Jakubowskaa a
Nicolaus Copernicus University in Toruń, Faculty of Chemistry, Chair of Physical Chemistry and Physicochemistry of Polymers. Gagarin 7, 87-100, Torun, Poland Faculty Biology and Environment Protection, Department of Environmental Microbiology and Biotechnology, Nicolaus Copernicus University in Toruń, Lwowska 1, Toruń, Poland
b
A R T I C LE I N FO
A B S T R A C T
Keywords: Polylactide Biodegradation Compatibilizer Nanocomposites Soil Compost Lake water
Presented work describes the influence of a compatibilizing agent on the degradation process of polylactidebased composites in different environments. Biodegradation of composites, containing polymer matrix in form of polylactide (PLA), nanofillers montmorillonite and Nanofil2 and poly(ε-caprolactone) (PCL) acting as a compatibilizing agent, was carried out for 28 days in lake water, soil and compost. OXI TOP Control analysis as well as the test for the activity of hydrolytic enzymes were performed and revealed that the presence of PCL significantly increases activity of hydrolytic enzymes and oxygen consumption during biodegradation of the studied materials in the above mentioned environments. The influence the PCL has got on the decomposition process was studied during the analysis of thermal properties of the degraded films as well as by means of a scanning electron microscopy, atomic force and epifluorescence microscopy. In summary, the compatibilizing agent accelerates the degradation of polylactide-based composites in all of the examined scenarios. Results of studies constitute a valuable source of knowledge on effective biodegradation process of polylactide-based composites containing a compatibilizing agent as well as the influence of poly(ε-caprolactone) on the decomposition progress of the studied materials.
1. Introduction Polylactide (PLA) is one of the most widely known biodegradable polymers which has currently got various applications such as food packaging, grocery bags and sacks as well as bioresorbable implants and surgical threads, braces, clips, surgical masks, dressings, compresses and medical staff clothing. For this reason PLA, being a very promising material, is modified by introducing different types of additives in the form of fillers, nanofillers, flame retardants and plasticizers which can improve thermal stability, affect stress resistance and brittleness (Chávez-Montes et al., 2016; Olewnik-Kruszkowska et al., 2016; Chow et al., 2018). Compatibilizing agents are added during the processing of PLA which also contains additives. It is well known that a compatibilizing agent allows the blending of components which are incompatible - in this case - polylactide and different types of additives. The compatibilizing agent is introduced in aim to lower surface tension and improve interfacial adhesion between the additive and the polymer matrix. Various attempts have been made in aim to combine a compatibilizer, polylactide and a nanofiller. One of the most popular
∗
compatibilizing agent used for polylactide is poly(ε-caprolactone) (Zenkiewicz and Richert, 2008; Olewnik and Richert, 2015). The presence of additives, nanofillers, plasticizers and stabilizing agents in the polymer matrix significantly influences the degradation process (Gu et al., 2002; Roy et al., 2012; Fukushima et al., 2013; Richert et al., 2013; Stepczyńska and Rytlewski, 2018). Other factors which affect decomposition of biodegradable polymers are molecular mass, degree of crystallinity of the materials, surface roughness, thickness and porosity. Materials based on biodegradable polymers are often exposed to external conditions such as temperature, moisture, UV-radiation as well as microorganisms. Combination of the above mentioned factors is present in different environments for example: water, compost and soil (Gu, 2003). Monitoring of decomposition of polymer materials in selected environments allows to establish their biodegradability and determine the influence of the compatibilizer on the changes occurring in physicochemical properties of the materials (Gu et al., 1994). Biodegradation of polylactide and polylactide-based composites was described by a number of researchers. The diagram of biochemical
Corresponding author. E-mail address: [email protected] (E. Olewnik-Kruszkowska).
https://doi.org/10.1016/j.ibiod.2019.104840 Received 26 March 2019; Received in revised form 12 November 2019; Accepted 13 November 2019 0964-8305/ © 2019 Elsevier Ltd. All rights reserved.
International Biodeterioration & Biodegradation 147 (2020) 104840
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Table 1 Physicochemical properties of the studied environments. Environment Water Soil Compost
pH 8.4 ± 0.09 7.1 ± 0.15 7.6 ± 0.11
TOC
TN −3
a
12,7 ± 0.97 mg dm 21.4 ± 1.8 g kg DM−1 259.8 ± 28.3 g kg DM−1
1.3 ± 0.11 mg dm−3 2.03 ± 0.3 g kg DM−1 25.32 ± 3,7 g kg DM−1
TOC – total organic carbon, TN – total nitrogen, DM – dry matter. a - mean ± SD (n = 5).
Nanofil2 were described as follows: LS and LN respectively. Poly(ε-caprolactone) CAPA 6506 (Solvay Caprolactones), UK as the plasticizing agent was introduced (5%wt) into the studied composites – symbol K. Samples containing 5% of PCL were named LNK and LSK. Formation of investigated materials has been presented in previous work (Olewnik and Richert, 2014).
processes in PLA biodegradation was presented in the work of Qi et al. (2017). According to Qi et al. (2017) as well as Mueller (2006) biochemical processes of polylactide degradation consist of chemical hydrolysis and biodegradation in natural environment which is ultimate mineralization. In the first stage, as a result of chemical hydrolysis, the ester bonds of PLA are fragmented into carboxylic acid and alcohol. In the second stage a microbial degradation is observed involving mineralization to end-products such as CO2, H2O and biomass. Most of the papers indicate that abiotic hydrolysis determines the rate of PLA biodegradation, moreover it constitutes the main degradation mechanism. In the work of Walczak et al. (2015) a biofilm was allowed to form on the surface of PLA during its biodegradation in soil, compost and lake water, while the degradation of polylactide-based composites in water was analyzed by M. A. Paul and K. Fukushima (Paul et al., 2005; Fukushima et al., 2013). Obtained results indicated that nanofillers increase the rate of hydrolysis and diffusion of water into the polymer matrix. However described above materials have not contained compatibilizing agent. It is known that the introduction of a nanofiller can increase the polylactide biodegradation rate. It is caused by the presence of hydroxyl groups which belong to the silicate layers of the nanofiller. The modification of the nanofiller can also influence the decomposition of materials (Fukushima et al., 2011, 2013; Roy et al., 2012). Another factor which can affect the kinetic of degradation process is a compatibilizing agent. It was previously described that a compatibilizer can influence the kinetic of degradation process induced by ozone as well as UVC radiation (Olewnik-Kruszkowska et al., 2015, 2017). However there are no publications describing the influence of a compatibilizing agent on the decomposition of polylactide-based materials in lake water, compost and soil. Undoubtedly, incorporated additives, compatibilizing agents as well as various biodegradation conditions affect decomposition of PLA-based composites. The novelty of the presented research is a comparison of the degradation of PLA films with and without the addition of a compatibilizing agent affected by different factors in various environments such as lake water, compost and soil. For this reason obtained results are important for being a source of valuable information on potential applications of composites as well as on its eventual disposal. In particular, the data is really useful because influence of a compatibilizing agent on the biodegradation of PLA-based composites in water, soil as well as in compost have not been the subject of scientific discussion. After the degradation in all of the above mentioned environments, changes in physicochemical properties were analyzed comprehensively, by means of microscopic and thermal methods. Moreover the activity of hydrolytic enzymes produced by microorganisms colonizing the surface of polylactide and the polylactide-based materials was studied.
2.2. Incubation of samples The research and control samples (1 cm2, 5 replicates for each material) were incubated in a glass containers filled with lake water from a eutrophic lake (Lake Chełmżyńskie), compost and soil. The most important parameters of the used environments have been depicted in Table 1. a). Biodegradation in lake water -The lake water (97 ml) and the PLA films (1 g of material cut into pieces with an area of approximately 1 cm2) were incubated in 500 ml glass bottles at 20 °C for 28 days in the dark. b). Biodegradation of modified PLA in compost and soil -The compost (100 g, 89 ml)/soil (100 g, 56 ml) and the PLA based films (1 g of material cut into pieces with an area of approximately 1 cm2) were placed in 1 dm3 glass containers in the dark. All samples were incubated at 20 °C for 28 days. 2.3. Methods of analysis 2.3.1. Oxygen uptake using the WTW OXI TOP method Biodegradation of modified PLA in different environments (lake water, compost, soil) was determined using a respirometric technique with a WTW OXI TOP—Control 110 set, which analyzed the microbial respiration activity (the oxygen uptake). In the case of biodegradation in lake water, the measurement of Biological Oxygen Demand (BOD) with OXI TOP—Control in lake water was carried out according to the operating instructions provided by the supplier. Lake water which did not contain polylactide based materials was used as a control sample (endogenous respiration). Microbial respiration activity was shown as mgO2/dm3 of lake water after 28 days. In the case of compost and soil the measured values were recorded by the OXI TOP —Control system in the “Pressure p” mode. The compost/soil which did not contain PLA films was used as control samples (endogenous respiration). All samples were analyzed in five replicates. Microbial respiration activity was shown as mgO2/kg of compost/soil after 28 days. 2.3.2. Viability of the biofilm determined by the LIVE/DEAD method Material samples (1 cm2) after incubation in lake water/compost/ soil were dyed with LIVE/DEAD ® BacLight ™ Bacterial Viability Kit containing SYTO 9 and propidium iodide. The samples were washed gently with sterile water to remove excess mineral and organic particles as well as microbes unbound, then the surface of the sample was covered with the aqueous solutions of dyes and left for 15 min at the temperature of 20 °C (in the dark). After mentioned time the excess dye was removed, and the studied materials were analyzed by means of Nikon H550S epifluorescence microscope (1000× magnification,
2. Materials and methods 2.1. Materials For the preparation of the PLA-based materials polylactide – symbol L, type 2002D (NatureWorks®, USA), with Mn of 156 kDa was applied. As nanofillers: Montmorillonite - symbol S (Acros Organics, Belgium) and Nanofil2 –symbol N (Southern Clay Products, USA) were applied. PLA-based composites containing 5% (w/w/) of montmorillonite and 2
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100 Oxygen uptake [mg O2 · dm-3]
470–490-nm excitation filter, 520-nm barrier filter). For each fragments of the materials 10 fields of view were evaluated. Color microphotographs were taken using the digital image processor (Olympus XC50). Viable cells were seen as a green, while non-viable cells were red. 2.3.3. Activity of hydrolytic enzymes in the biofilm The overall hydrolase activity was determined by measuring the fluorescein release from fluorescein diacetate (FDA). The FDA is a substrate for many hydrolytic enzymes, e.g. esterases, lipases and certain proteases. This method can be successfully used to determine the hydrolytic activity of microorganisms that form biofilms on the surface of various materials (Peeters et al., 2008). A method modified by Adam and Duncan (2001) was applied. After incubation in lake water/compost/soil, the samples were washed gently with sterile water to remove excess mineral and organic particles as well as microbes unbound to the surface of the sample. The samples were then placed in 10 ml of 0.85% NaCl solution. After the addition of 0.1 ml FDA, the samples were incubated for 1 h at temperature 30 °C (in the dark). The amount of released fluorescein was analyzed using a Hitachi F-2500 spectrophotometer.
(a) L LS LSK LN LNK
80 60 40 20 0
Oxygen uptake [mg O2 · kg-1]
250
2.3.4. Analysis of surface morphology by means of SEM and AFM techniques Changes in the geometric structure of polylactide and its composites were recorded using a scanning electron microscope LEO Electron Microscopy Ltd, UK, type 1430 VP. The surface analysis of all studied materials was performed in air, at room temperature by means of the NanoScope MultiMode microscope (Veeco Metrology, Inc., USA). The roughness parameters such as the root mean square (Rq) and arithmetical mean deviation of the assessed profile (Ra) were calculated for 5 μm × 5 μm area.
200 150
L LS LSK LN LNK
(b)
100 50
0
2.3.5. Analysis of thermal properties by means of thermogravimetric analysis and DSC method Thermal stability of materials with and without PCL was analyzed by means of Simultaneous TGA-DTA Thermal Analysis type SDT 2960 (TA Instruments, UK). All measurements were performed in a range from room temperature to 600 °C, under air flow. A heating rate of the analyses was 10 °C/min. Measurements of changes in thermal properties of studied materials were carried out using a differential scanning calorimeter -Polymer Laboratories type, Epson, GB. The studied samples were tested in temperatures ranging from 25 °C to 180 °C in nitrogen atmosphere.
Fig. 1. Oxygen uptake on the biodegradation of PLA and PLA based composites in: (a) lake water, (b) compost, (c) soil.
compatibilizing agent and unmodified montmorillonite (S), while the lowest oxygen uptake was recorded during the decomposition of unmodified PLA. According to Fukushima (Fukushima et al., 2009) the addition of a nanoclay, especially an unmodified nanofiller, increases PLA degradation rate. This can be assigned to the presence of hydroxyl groups belonging to the layers of silica. It should be emphasised that the influence of a compatibilizing agent on the biodegradation of PLA-based materials was not studied before. Introduction of the compatibilizer into PLA-nanofiller systems improves the dispersion of the nanofiller and for this reason it can facilitate the degradation process of studied composites (OlewnikKruszkowska et al., 2016). Moreover is should be mentioned that PCL introduced into PLA matrix can act as a plasticizer. For this reason PCL decreases the attraction between PLA chains and improves contact between the polymer matrix and the environmental factors, i.e. water, soil and compost. The other factor which significantly influences the decomposition of studied materials is easier biodegradation of PCL in comparison to PLA. A as result of PCL biodegradation, the disintegration of PLA-based composites with an addition of compatibilizer occurs much more rapidly. Taking into account obtained results it is clearly visible that samples containing PCL (LNK and LSK) are characterized by the highest values of oxygen consumption. Moreover, it should be
3. Results and discussion 3.1. Assessment of oxygen consumption and activity of the hydrolases during biodegradation In aim to establish the extent of oxygen consumption during degradation of the above mentioned materials in different environments the OXI TOP technique was applied. Oxygen consumption, known as biological oxygen demand test, is strongly connected with the respiratory activity of microorganisms. During the analyses, the air pressure is measured after trapping the produced carbon dioxide into a strong base solutions monitoring. This technique started to be popular because it allows to observe the transformation of organic matter in a particular environment (Gu and Gu, 2005; Walczak et al., 2015). Fig. 1 depicts the respiratory activity of microorganisms in soil, compost and lake water after degradation of PLA and PLA based composites without and with an addition of poly(ε-caprolactone) as the compatibilizing agent. Taking into account the composition of the studied materials it is clearly visible that the highest oxygen consumption among all of the studied environments was observed during the biodegradation of LSK material consisting of PLA, PCL – as 3
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Table 2 DSC parameters of PLA and its composites after degradation in soil (S), compost (C) and water (W).
Fig. 2. Biological oxygen demand (BOD) in different environments in presence PLA and its composites.
mentioned that according to the results presented in Fig. 1 all studied materials are characterised by the highest biological oxygen demand during degradation in soil. In this case the highest oxygen consumption was recorded in relation to the LSK sample (504.3 mgO2/kg), while the lowest was observed in the case pristine polylactide (98.4 mgO2/kg). Results obtained by means of OXI TOP method are almost entirely consistent with the activity of the hydrolases. It is known that bacteria present in compost, soil and lake water can form a biofilm on the surface of a polylactide and play an active role in its biodegradation (Walczak et al., 2015). For this reason in aim to establish the effect of additives in the form of a varying nanofiller (pure montmorillonite and Nanofil2) and a compatibilizing agent (poly(ε-caprolactone)) they were introduced to determine the hydrolytic activity of microorganisms that form biofilms on the surface of studied materials. Obtained results of the activity of hydrolytic enzymes in the biofilm on the surface of pure and modified PLA are shown in Fig. 2. It was established that the enzymatic degradation of aliphatic polyesters proceeds according to the mechanism proposed by Tokiwa et al. (Tokiwa and Calabia, 2006). The first stage is connected with the adsorption of the enzyme on the polymer surface while the second stage involves the hydrolysis of bond in ester groups. Among the factors influenced by the presence of hydrolase, the most important are: temperature, pH, presence of activators and inhibitors. In the case of some hydrolases its activity is connected with the presence of nitrogen in soil especially in form of NH4+. For this reason it can be observed that the highest values of activity of hydrolytic enzymes in soil were registered in the case of samples comprising a nanofiller modified by means of quaternary ammonium salt. However the same tendency was not observed in the case of degradation in compost and water. During the degradation process in compost as well as in water the highest values of hydrolases activity were observed in the case of LSK samples, with the LNK materials falling shortly behind. For this reason, it is reasonable to claim that the addition of a compatibilizer (K) positively influenced the activity of hydrolytic enzymes, which indicates a better biodegradability of materials enriched with this component. The lowest activity of hydrolytic enzymes of biofilm, which had formed on samples incubated in either of the environments, was recorded in the case of unmodified polylactide (L). Taking into account the type of used environment the highest values of hydrolases activation for all PLA-based composites were observed during degradation in soil, while in the case of neat PLA lowest values had been observed.
Sample
Tg [°C]
Tc [°C]
ΔHc [J/g]
Tm [°C]
ΔHm [J/g]
Xc [%]
T5 [°C]
T10 [°C]
PLA-S PLA-C PLA-W LN-S LN-C LN-W LNK-S LNK-C LNK-W LS-S
68.2 68.6 68.9 67.5 67.8 68.0 66.3 67.0 67.4 67.4
123.4 124.0 125.3 124.2 125.7 125.7 101.1 102.8 103.0 114.7
23.3 18.3 17.9 23.7 22.2 20.9 21.4 21 20.8 25.5
25.9 20.4 19.6 26 24.3 23.3 27.7 24.6 24.2 28.6
27.0 21.3 20.4 27.1 25.3 24.3 28.9 25.6 25.2 29.8
315.9 316.7 319.3 319.0 321.4 323.2 312.3 315.9 313.0 303.4
326.5 328.2 329.2 330.1 331.2 333.8 324.9 328.8 326.7 315.7
LS-C
67.9
116.0
24.1
27.5
28.6
305.2
317.9
LS-W
66.9
115.2
23.9
27
28.1
302.8
313.4
LSK-S
62.4
98.8
25.7
29.7
30.9
297.5
310.8
LSK-C
63.9
99.7
25.2
28.4
29.6
299.3
313.3
LSK-W
63.2
99.7
25.1
153.9 154.3 154.8 153.5 154.8 154.3 154.1 154.8 155.0 150.1/ 156.1 150.9/ 156.3 150.4/ 155.5 145.5/ 153.9 145.9/ 154.4 145.7/ 155.0
28.1
29.3
289.7
304.9
compost and soil, the changes in thermal properties of the investigated materials after 28 days of degradation have been analyzed. It is well know that during degradation, the thermal properties of biodegradable polymers significantly decrease. This phenomenon is related to scission of the polymer chains and the formation of PLA oligomers during decomposition. The obtained results indicate that an addition of a nanofiller (montmorillonite and Nanofil2) influences thermal properties of PLA during degradation (Table 2). However an addition of poly(ε-caprolactone) to LN and LS systems significantly decreases glass transition (Tg) and cold crystallization (Tc) temperatures of studied composites in comparison with materials without a compatibilizer. It has been noted that the most significant decrease of all mentioned parameters was observed in the case of materials with an addition of PCL degraded in lake water, soil as well as in compost. The lowest values of Tg -as well as Tc were observed in the case of LSK sample degraded in soil. This suggests that not only the addition of PCL influences the degradation process but also the conditions in which the degradation occurs significantly influence the decomposition of PLA. Moreover, obtained results indicate that the addition of a nanoadditive as well as a compatibilizer decreases the melting temperature of degraded composites in comparison with neat polylactide, with the lowest values of Tm similarly observed after degradation in soil. It should be mentioned that in the case of materials containing pure montmorillonite (S) after degradation process in all environments two endothermic peaks were observed. It is known that formation of bimodal peak is connected with the formation of different crystalline forms of polylactide, however it can be also related to the formation of fractions differing in molecular weight (Olewnik-Kruszkowska et al., 2017). Recently it was established that a lower molecular weight can form the α’ structure which is characterized by a lower temperature in comparison with the α form (Tábi et al., 2010). Irrespectively of the environment in which the degradation had taken place, the samples containing PCL were characterized by higher degree of crystallinity in reference to the polylactide-nanofiller systems. It is well known that the degradation in the amorphous phase of polymers takes place preferentially, moreover shorter chains of PLA are able to reorganize and form the crystalline phase (Calafel et al., 2010; Tábi et al., 2010; Gupta et al., 2012). The above discussed results indicate that an addition of a compatibilizing agent facilitates the degradation of an unordered structure of PLA and the formation of a crystal phase. However it can clearly be observed that the highest values of the degree of crystallinity (Xc) were observed
3.2. Changes in thermal properties During the decomposition of PLA and its composites in lake water, 4
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significantly after degradation processes. All films containing PCL were characterized by a more corrugated surface. Research of samples comprising a compatibilizing agent (LNK and LSK) revealed that the most significant changes in surface morphology after degradation had occurred in soil. The increase in surface roughness of the above mentioned samples was also observed after degradation in compost and water, however the changes were not as substantial as the ones observed after decomposition in soil. It is well known that during the degradation process polymer disintegration takes place which leads to a formation of mainly oligomers. Cracks and pores form on the surface of polylactide improve contact with the environment and in this way can influence the kinetic decomposition of a PLA. The increase in contact surface of a polymer with the environment leads to erosion of the studied materials and to the decomposition of the amorphous polymer phases (Rydz et al., 2013). In the case of materials containing PCL, acting as a compatibilizer, an improved dispersion of nanoadditives in the polymer matrix promotes contact between the materials and the environment. All these processes result in the increased efficiency of degradation after PCL is introduced into the polylactide-nanofiller systems. Summarizing, the PLA presents the most smooth surface while the LSK sample is characterized by the most rugged surface, regardless of the degradation environment. Taking into account the type of environmental conditions in which the degradation occurs the same tendency as revealed in the case of DSC technique can clearly be noticed. It means that the highest surface roughness parameters were observed in the case of samples undergoing degradation in soil.
in the case of materials degraded in soil, while the lowest values of Xc were obtained for materials degraded in water. Summarizing, based on the analysis of the above discussed results (Table 2) it is reasonable to assume that the introduction of poly(ε-caprolactone) accelerates the degradation of polylactide-based composites, however the most significant changes in discussed properties are observed after degradation carried out in soil. This phenomenon is caused by improved dispersion of the nanoadditives in the polylactide as well as the plasticizing effect of PCL (Olewnik and Richert, 2014; Olewnik-Kruszkowska et al., 2016). The same tendency was observed during thermogravimetric analysis. The results of the measurements of thermal stabilities of the PLA-based composites after degradation in different conditions are presented in Table 2. It was found that introduction of PCL significantly decreases the temperature of 5 and 10% mass loss of the analyzed materials in comparison with PLA-nanoadditive systems. The reduction in T5 and T10 values can be explained by better dispersion of the nanofiller in polymer matrix by means of a compatibilizing agent. Consequently the microorganisms and water can readily diffuse into the studied materials. As mentioned above, in the case of the DSC analysis the most significant changes in thermal parameters were noticed in the case of samples stored in soil. Taking into account the results obtained by means of TG technique it can also be observed that the lowest values of thermal stability were obtained in relation to LNK and LSK composites degraded in soil. Moreover it is interesting that samples containing unmodified montmorillonite (S) degrade significantly faster in comparison with a composite comprising a modified nanofiller (N). For this reason it is justified to assume that the modification of the nanofiller, and the resulting increased hydrophobicity of the nanoadditive, impedes the degradation process of polymer composites (Olewnik-Kruszkowska et al., 2016). Summarizing, the analysis of the thermal properties and stability indicates that during the degradation process values of the studied parameters decreased in the following order: soil < compost < water (Table 2).
3.4. Changes in surface morphology by means of SEM and LIVE/DEAD techniques It was indicated by other researchers that the degradation processes often influences the surface structure of studied materials (Gu et al., 2002; Gu, 2003). In relation to the changes in surface morphology and the effect different environmental conditions have got on degradation of PLA-based composites, the SEM analysis can be used to study these phenomena. Obtained results for PLA and PLA-nanofiller systems without (LN and LS) and with an addition of a compatibilizing agent (LNK and LSK) are shown in Fig. 4. Based on the obtained results it needs to be emphasis that in the case of all samples a biofilm had formed on their surface. The most noticeable surface changes have been observed in the case of materials degraded in soil. Among the samples degraded in soil the most radical modification of surface has occurred in the case of LS and LSK samples, where unmodified, pure MMT was introduced as nanofiller into the PLA matrix. Inspection of the surface of all samples stored in soil have not revealed, apart from the formation of biofilms, any other changes like cavities, cracks or pinholes. In the case of samples subjected to degradation in compost fragments of fungus as well as bacteria have been found on the surface of degraded materials. The least noticeable changes in the surface morphology have been observed for the samples stored in lake water. In addition in the case of neat PLA it can be noticed that sample is almost clear in comparison to the PLA-based composites. Moreover it should be mentioned that neat polylactide sample exhibits the smoothest and flattest surface not only after degradation in lake water but also after storage in other environments: compost and soil. The same tendency was observed by Stepczyńska et al. (Stepczyńska and Rytlewski, 2018) where PLA and PLA filled with flax-fibers were subjected to the enzymatic degradation process. In aim to establish whether living bacteria were present on the surface of the studied materials, the LIVE/DEAD technique was applied. Fig. 5 depicts biofilm formation incubated in the mentioned environments. Obtained results clearly indicate that in the case of LSK sample comprising polylactide, pure montmorillonite and PCL, acting as the as compatibilizing agent, viable cells colored by fluorescent to green are especially visible. This is strongly related to the distribution of
3.3. Assessment of surface morphology by means of AFM technique Atomic force microscopy was applied in aim to establish the influence a compatibilizing agent has got on surface changes of the polylactide-based composites during degradation process in different environments. It is well known that the AFM technique provides detailed information on the surface morphology. Moreover it should be mention that the morphology of the studied samples during biodegradation depends on their composition and conditions of the degradation process. Fig. 3 presents AFM photographs of the surface areas of the pure PLA and PLA-based composites without or with an addition of PCL as a compatibilizing agent after biodegradation had been carried out in soil, compost and lake water. The values of the different roughness parameters (Ra, Rq) for all samples stored in described media are summarized in Table 3. The AFM images show differences in the surface roughness parameters during degradation process as well as the dependence on the composition of materials and the environmental conditions in which the degradation process occurs. After degradation the surface morphology of PLA-nanofiller systems (LN and LS) was characterised by increased roughness in comparison to pure polylactide. It is reasonable to assume that this phenomenon is connected with the agglomeration of nanoparticles, especially in the case of an unmodified nanofiller. In the case of composites the increase in roughness parameters can be attributed to the macro-chains rearrangement in the polymer. The highest values of the Ra, and Rq for LN and LS samples were observed during the degradation in soil, where in the case of water and composites the described parameters are comparable. Moreover the Ra and Rq values of LN and LS samples after degradation in soil were at least three times higher compared to those measured after degradation in water and compost. After the addition of PCL into the PLA-nanofiller systems surface roughness of obtained composites increased 5
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Fig. 3. AFM images of studied materials after degradation in different environments (S-soil, C-compost, W-water).
nanoadditives in the polymer matrix, which allows penetration of the sample by bacteria. However it should be noted that bacteria are able to form a biofilm on the surface of all of the degraded materials in any of the three environments. Summarizing, the results discussed above, obtained by means of the LIVE/DEAD technique as well as the SEM analysis, have confirmed the presence of microorganisms on the surface of studied materials. As predicted the results obtained using scanning electron microscopy and LIVE/DEAD techniques correspond with the variations in surface morphology determined using the AFM method.
Table 3 Values of roughness parameters of polylactide after 28 days of degradation in different environments. Sample
L-S LN-S LNK-S LS-S LSK-S L-C LN-C LNK-C LS-C LSK-C L-W LN-W LNK-W LS-W LSK-W
Roughness parameters [nm] Ra
Rq
6.5 39.1 46.7 52.4 56.8 6.3 10.9 13.4 10.6 16.6 7.3 9.6 13.5 17.8 20.1
11.9 53.2 68.6 72.8 87.0 13.6 15.1 24.2 13.8 26.3 18.2 13.5 18.1 22.9 38.3
4. Conclusions Evaluation of the surface of the polylactide and polylactide-based composites with and without poly(ε-caprolactone) showed that a compatibilizing agent plays an important role during degradation process in different environments. It can be clearly noticed that an addition of a compatibilizing agent significantly increases oxygen consumption as well as the activity of hydrolytic enzymes. 6
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Fig. 4. SEM images of PLA and its composites after degradation in different environments (S-soil, C-compost, W-water).
the degradation process of materials containing PCL is more profound in comparison with the degradation occurring in the polylactide and PLA-nanofiller systems.
Simultaneously the changes in thermal properties as well as in thermal stability of materials consisting of polylactide, poly(ε-caprolactone) and nanofillers are characterized by lower values than composites without an addition of a compatibilizing agent. Moreover it should be mentioned that the results of the present study demonstrate that regardless of the environmental conditions, may it be soil, lake water or compost, 7
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Fig. 5. The biofilm formed on the materials incubated in different environments. Bacteria stained by using the LIVE/DEAD method (microscope Olympus BX50, magnification 1000 x, live bacteria – green, dead bacteria – red). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Biodegradation of polylactide-based composites with an addition of a compatibilizing agent in different environments
T
Ewa Olewnik-Kruszkowskaa,∗, Aleksandra Burkowska-Butb, Iwona Taracha, Maciej Walczakb, Ewelina Jakubowskaa a
Nicolaus Copernicus University in Toruń, Faculty of Chemistry, Chair of Physical Chemistry and Physicochemistry of Polymers. Gagarin 7, 87-100, Torun, Poland Faculty Biology and Environment Protection, Department of Environmental Microbiology and Biotechnology, Nicolaus Copernicus University in Toruń, Lwowska 1, Toruń, Poland
b
A R T I C LE I N FO
A B S T R A C T
Keywords: Polylactide Biodegradation Compatibilizer Nanocomposites Soil Compost Lake water
Presented work describes the influence of a compatibilizing agent on the degradation process of polylactidebased composites in different environments. Biodegradation of composites, containing polymer matrix in form of polylactide (PLA), nanofillers montmorillonite and Nanofil2 and poly(ε-caprolactone) (PCL) acting as a compatibilizing agent, was carried out for 28 days in lake water, soil and compost. OXI TOP Control analysis as well as the test for the activity of hydrolytic enzymes were performed and revealed that the presence of PCL significantly increases activity of hydrolytic enzymes and oxygen consumption during biodegradation of the studied materials in the above mentioned environments. The influence the PCL has got on the decomposition process was studied during the analysis of thermal properties of the degraded films as well as by means of a scanning electron microscopy, atomic force and epifluorescence microscopy. In summary, the compatibilizing agent accelerates the degradation of polylactide-based composites in all of the examined scenarios. Results of studies constitute a valuable source of knowledge on effective biodegradation process of polylactide-based composites containing a compatibilizing agent as well as the influence of poly(ε-caprolactone) on the decomposition progress of the studied materials.
1. Introduction Polylactide (PLA) is one of the most widely known biodegradable polymers which has currently got various applications such as food packaging, grocery bags and sacks as well as bioresorbable implants and surgical threads, braces, clips, surgical masks, dressings, compresses and medical staff clothing. For this reason PLA, being a very promising material, is modified by introducing different types of additives in the form of fillers, nanofillers, flame retardants and plasticizers which can improve thermal stability, affect stress resistance and brittleness (Chávez-Montes et al., 2016; Olewnik-Kruszkowska et al., 2016; Chow et al., 2018). Compatibilizing agents are added during the processing of PLA which also contains additives. It is well known that a compatibilizing agent allows the blending of components which are incompatible - in this case - polylactide and different types of additives. The compatibilizing agent is introduced in aim to lower surface tension and improve interfacial adhesion between the additive and the polymer matrix. Various attempts have been made in aim to combine a compatibilizer, polylactide and a nanofiller. One of the most popular
∗
compatibilizing agent used for polylactide is poly(ε-caprolactone) (Zenkiewicz and Richert, 2008; Olewnik and Richert, 2015). The presence of additives, nanofillers, plasticizers and stabilizing agents in the polymer matrix significantly influences the degradation process (Gu et al., 2002; Roy et al., 2012; Fukushima et al., 2013; Richert et al., 2013; Stepczyńska and Rytlewski, 2018). Other factors which affect decomposition of biodegradable polymers are molecular mass, degree of crystallinity of the materials, surface roughness, thickness and porosity. Materials based on biodegradable polymers are often exposed to external conditions such as temperature, moisture, UV-radiation as well as microorganisms. Combination of the above mentioned factors is present in different environments for example: water, compost and soil (Gu, 2003). Monitoring of decomposition of polymer materials in selected environments allows to establish their biodegradability and determine the influence of the compatibilizer on the changes occurring in physicochemical properties of the materials (Gu et al., 1994). Biodegradation of polylactide and polylactide-based composites was described by a number of researchers. The diagram of biochemical
Corresponding author. E-mail address: [email protected] (E. Olewnik-Kruszkowska).
https://doi.org/10.1016/j.ibiod.2019.104840 Received 26 March 2019; Received in revised form 12 November 2019; Accepted 13 November 2019 0964-8305/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Physicochemical properties of the studied environments. Environment Water Soil Compost
pH 8.4 ± 0.09 7.1 ± 0.15 7.6 ± 0.11
TOC
TN −3
a
12,7 ± 0.97 mg dm 21.4 ± 1.8 g kg DM−1 259.8 ± 28.3 g kg DM−1
1.3 ± 0.11 mg dm−3 2.03 ± 0.3 g kg DM−1 25.32 ± 3,7 g kg DM−1
TOC – total organic carbon, TN – total nitrogen, DM – dry matter. a - mean ± SD (n = 5).
Nanofil2 were described as follows: LS and LN respectively. Poly(ε-caprolactone) CAPA 6506 (Solvay Caprolactones), UK as the plasticizing agent was introduced (5%wt) into the studied composites – symbol K. Samples containing 5% of PCL were named LNK and LSK. Formation of investigated materials has been presented in previous work (Olewnik and Richert, 2014).
processes in PLA biodegradation was presented in the work of Qi et al. (2017). According to Qi et al. (2017) as well as Mueller (2006) biochemical processes of polylactide degradation consist of chemical hydrolysis and biodegradation in natural environment which is ultimate mineralization. In the first stage, as a result of chemical hydrolysis, the ester bonds of PLA are fragmented into carboxylic acid and alcohol. In the second stage a microbial degradation is observed involving mineralization to end-products such as CO2, H2O and biomass. Most of the papers indicate that abiotic hydrolysis determines the rate of PLA biodegradation, moreover it constitutes the main degradation mechanism. In the work of Walczak et al. (2015) a biofilm was allowed to form on the surface of PLA during its biodegradation in soil, compost and lake water, while the degradation of polylactide-based composites in water was analyzed by M. A. Paul and K. Fukushima (Paul et al., 2005; Fukushima et al., 2013). Obtained results indicated that nanofillers increase the rate of hydrolysis and diffusion of water into the polymer matrix. However described above materials have not contained compatibilizing agent. It is known that the introduction of a nanofiller can increase the polylactide biodegradation rate. It is caused by the presence of hydroxyl groups which belong to the silicate layers of the nanofiller. The modification of the nanofiller can also influence the decomposition of materials (Fukushima et al., 2011, 2013; Roy et al., 2012). Another factor which can affect the kinetic of degradation process is a compatibilizing agent. It was previously described that a compatibilizer can influence the kinetic of degradation process induced by ozone as well as UVC radiation (Olewnik-Kruszkowska et al., 2015, 2017). However there are no publications describing the influence of a compatibilizing agent on the decomposition of polylactide-based materials in lake water, compost and soil. Undoubtedly, incorporated additives, compatibilizing agents as well as various biodegradation conditions affect decomposition of PLA-based composites. The novelty of the presented research is a comparison of the degradation of PLA films with and without the addition of a compatibilizing agent affected by different factors in various environments such as lake water, compost and soil. For this reason obtained results are important for being a source of valuable information on potential applications of composites as well as on its eventual disposal. In particular, the data is really useful because influence of a compatibilizing agent on the biodegradation of PLA-based composites in water, soil as well as in compost have not been the subject of scientific discussion. After the degradation in all of the above mentioned environments, changes in physicochemical properties were analyzed comprehensively, by means of microscopic and thermal methods. Moreover the activity of hydrolytic enzymes produced by microorganisms colonizing the surface of polylactide and the polylactide-based materials was studied.
2.2. Incubation of samples The research and control samples (1 cm2, 5 replicates for each material) were incubated in a glass containers filled with lake water from a eutrophic lake (Lake Chełmżyńskie), compost and soil. The most important parameters of the used environments have been depicted in Table 1. a). Biodegradation in lake water -The lake water (97 ml) and the PLA films (1 g of material cut into pieces with an area of approximately 1 cm2) were incubated in 500 ml glass bottles at 20 °C for 28 days in the dark. b). Biodegradation of modified PLA in compost and soil -The compost (100 g, 89 ml)/soil (100 g, 56 ml) and the PLA based films (1 g of material cut into pieces with an area of approximately 1 cm2) were placed in 1 dm3 glass containers in the dark. All samples were incubated at 20 °C for 28 days. 2.3. Methods of analysis 2.3.1. Oxygen uptake using the WTW OXI TOP method Biodegradation of modified PLA in different environments (lake water, compost, soil) was determined using a respirometric technique with a WTW OXI TOP—Control 110 set, which analyzed the microbial respiration activity (the oxygen uptake). In the case of biodegradation in lake water, the measurement of Biological Oxygen Demand (BOD) with OXI TOP—Control in lake water was carried out according to the operating instructions provided by the supplier. Lake water which did not contain polylactide based materials was used as a control sample (endogenous respiration). Microbial respiration activity was shown as mgO2/dm3 of lake water after 28 days. In the case of compost and soil the measured values were recorded by the OXI TOP —Control system in the “Pressure p” mode. The compost/soil which did not contain PLA films was used as control samples (endogenous respiration). All samples were analyzed in five replicates. Microbial respiration activity was shown as mgO2/kg of compost/soil after 28 days. 2.3.2. Viability of the biofilm determined by the LIVE/DEAD method Material samples (1 cm2) after incubation in lake water/compost/ soil were dyed with LIVE/DEAD ® BacLight ™ Bacterial Viability Kit containing SYTO 9 and propidium iodide. The samples were washed gently with sterile water to remove excess mineral and organic particles as well as microbes unbound, then the surface of the sample was covered with the aqueous solutions of dyes and left for 15 min at the temperature of 20 °C (in the dark). After mentioned time the excess dye was removed, and the studied materials were analyzed by means of Nikon H550S epifluorescence microscope (1000× magnification,
2. Materials and methods 2.1. Materials For the preparation of the PLA-based materials polylactide – symbol L, type 2002D (NatureWorks®, USA), with Mn of 156 kDa was applied. As nanofillers: Montmorillonite - symbol S (Acros Organics, Belgium) and Nanofil2 –symbol N (Southern Clay Products, USA) were applied. PLA-based composites containing 5% (w/w/) of montmorillonite and 2
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100 Oxygen uptake [mg O2 · dm-3]
470–490-nm excitation filter, 520-nm barrier filter). For each fragments of the materials 10 fields of view were evaluated. Color microphotographs were taken using the digital image processor (Olympus XC50). Viable cells were seen as a green, while non-viable cells were red. 2.3.3. Activity of hydrolytic enzymes in the biofilm The overall hydrolase activity was determined by measuring the fluorescein release from fluorescein diacetate (FDA). The FDA is a substrate for many hydrolytic enzymes, e.g. esterases, lipases and certain proteases. This method can be successfully used to determine the hydrolytic activity of microorganisms that form biofilms on the surface of various materials (Peeters et al., 2008). A method modified by Adam and Duncan (2001) was applied. After incubation in lake water/compost/soil, the samples were washed gently with sterile water to remove excess mineral and organic particles as well as microbes unbound to the surface of the sample. The samples were then placed in 10 ml of 0.85% NaCl solution. After the addition of 0.1 ml FDA, the samples were incubated for 1 h at temperature 30 °C (in the dark). The amount of released fluorescein was analyzed using a Hitachi F-2500 spectrophotometer.
(a) L LS LSK LN LNK
80 60 40 20 0
Oxygen uptake [mg O2 · kg-1]
250
2.3.4. Analysis of surface morphology by means of SEM and AFM techniques Changes in the geometric structure of polylactide and its composites were recorded using a scanning electron microscope LEO Electron Microscopy Ltd, UK, type 1430 VP. The surface analysis of all studied materials was performed in air, at room temperature by means of the NanoScope MultiMode microscope (Veeco Metrology, Inc., USA). The roughness parameters such as the root mean square (Rq) and arithmetical mean deviation of the assessed profile (Ra) were calculated for 5 μm × 5 μm area.
200 150
L LS LSK LN LNK
(b)
100 50
0
2.3.5. Analysis of thermal properties by means of thermogravimetric analysis and DSC method Thermal stability of materials with and without PCL was analyzed by means of Simultaneous TGA-DTA Thermal Analysis type SDT 2960 (TA Instruments, UK). All measurements were performed in a range from room temperature to 600 °C, under air flow. A heating rate of the analyses was 10 °C/min. Measurements of changes in thermal properties of studied materials were carried out using a differential scanning calorimeter -Polymer Laboratories type, Epson, GB. The studied samples were tested in temperatures ranging from 25 °C to 180 °C in nitrogen atmosphere.
Fig. 1. Oxygen uptake on the biodegradation of PLA and PLA based composites in: (a) lake water, (b) compost, (c) soil.
compatibilizing agent and unmodified montmorillonite (S), while the lowest oxygen uptake was recorded during the decomposition of unmodified PLA. According to Fukushima (Fukushima et al., 2009) the addition of a nanoclay, especially an unmodified nanofiller, increases PLA degradation rate. This can be assigned to the presence of hydroxyl groups belonging to the layers of silica. It should be emphasised that the influence of a compatibilizing agent on the biodegradation of PLA-based materials was not studied before. Introduction of the compatibilizer into PLA-nanofiller systems improves the dispersion of the nanofiller and for this reason it can facilitate the degradation process of studied composites (OlewnikKruszkowska et al., 2016). Moreover is should be mentioned that PCL introduced into PLA matrix can act as a plasticizer. For this reason PCL decreases the attraction between PLA chains and improves contact between the polymer matrix and the environmental factors, i.e. water, soil and compost. The other factor which significantly influences the decomposition of studied materials is easier biodegradation of PCL in comparison to PLA. A as result of PCL biodegradation, the disintegration of PLA-based composites with an addition of compatibilizer occurs much more rapidly. Taking into account obtained results it is clearly visible that samples containing PCL (LNK and LSK) are characterized by the highest values of oxygen consumption. Moreover, it should be
3. Results and discussion 3.1. Assessment of oxygen consumption and activity of the hydrolases during biodegradation In aim to establish the extent of oxygen consumption during degradation of the above mentioned materials in different environments the OXI TOP technique was applied. Oxygen consumption, known as biological oxygen demand test, is strongly connected with the respiratory activity of microorganisms. During the analyses, the air pressure is measured after trapping the produced carbon dioxide into a strong base solutions monitoring. This technique started to be popular because it allows to observe the transformation of organic matter in a particular environment (Gu and Gu, 2005; Walczak et al., 2015). Fig. 1 depicts the respiratory activity of microorganisms in soil, compost and lake water after degradation of PLA and PLA based composites without and with an addition of poly(ε-caprolactone) as the compatibilizing agent. Taking into account the composition of the studied materials it is clearly visible that the highest oxygen consumption among all of the studied environments was observed during the biodegradation of LSK material consisting of PLA, PCL – as 3
International Biodeterioration & Biodegradation 147 (2020) 104840
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Table 2 DSC parameters of PLA and its composites after degradation in soil (S), compost (C) and water (W).
Fig. 2. Biological oxygen demand (BOD) in different environments in presence PLA and its composites.
mentioned that according to the results presented in Fig. 1 all studied materials are characterised by the highest biological oxygen demand during degradation in soil. In this case the highest oxygen consumption was recorded in relation to the LSK sample (504.3 mgO2/kg), while the lowest was observed in the case pristine polylactide (98.4 mgO2/kg). Results obtained by means of OXI TOP method are almost entirely consistent with the activity of the hydrolases. It is known that bacteria present in compost, soil and lake water can form a biofilm on the surface of a polylactide and play an active role in its biodegradation (Walczak et al., 2015). For this reason in aim to establish the effect of additives in the form of a varying nanofiller (pure montmorillonite and Nanofil2) and a compatibilizing agent (poly(ε-caprolactone)) they were introduced to determine the hydrolytic activity of microorganisms that form biofilms on the surface of studied materials. Obtained results of the activity of hydrolytic enzymes in the biofilm on the surface of pure and modified PLA are shown in Fig. 2. It was established that the enzymatic degradation of aliphatic polyesters proceeds according to the mechanism proposed by Tokiwa et al. (Tokiwa and Calabia, 2006). The first stage is connected with the adsorption of the enzyme on the polymer surface while the second stage involves the hydrolysis of bond in ester groups. Among the factors influenced by the presence of hydrolase, the most important are: temperature, pH, presence of activators and inhibitors. In the case of some hydrolases its activity is connected with the presence of nitrogen in soil especially in form of NH4+. For this reason it can be observed that the highest values of activity of hydrolytic enzymes in soil were registered in the case of samples comprising a nanofiller modified by means of quaternary ammonium salt. However the same tendency was not observed in the case of degradation in compost and water. During the degradation process in compost as well as in water the highest values of hydrolases activity were observed in the case of LSK samples, with the LNK materials falling shortly behind. For this reason, it is reasonable to claim that the addition of a compatibilizer (K) positively influenced the activity of hydrolytic enzymes, which indicates a better biodegradability of materials enriched with this component. The lowest activity of hydrolytic enzymes of biofilm, which had formed on samples incubated in either of the environments, was recorded in the case of unmodified polylactide (L). Taking into account the type of used environment the highest values of hydrolases activation for all PLA-based composites were observed during degradation in soil, while in the case of neat PLA lowest values had been observed.
Sample
Tg [°C]
Tc [°C]
ΔHc [J/g]
Tm [°C]
ΔHm [J/g]
Xc [%]
T5 [°C]
T10 [°C]
PLA-S PLA-C PLA-W LN-S LN-C LN-W LNK-S LNK-C LNK-W LS-S
68.2 68.6 68.9 67.5 67.8 68.0 66.3 67.0 67.4 67.4
123.4 124.0 125.3 124.2 125.7 125.7 101.1 102.8 103.0 114.7
23.3 18.3 17.9 23.7 22.2 20.9 21.4 21 20.8 25.5
25.9 20.4 19.6 26 24.3 23.3 27.7 24.6 24.2 28.6
27.0 21.3 20.4 27.1 25.3 24.3 28.9 25.6 25.2 29.8
315.9 316.7 319.3 319.0 321.4 323.2 312.3 315.9 313.0 303.4
326.5 328.2 329.2 330.1 331.2 333.8 324.9 328.8 326.7 315.7
LS-C
67.9
116.0
24.1
27.5
28.6
305.2
317.9
LS-W
66.9
115.2
23.9
27
28.1
302.8
313.4
LSK-S
62.4
98.8
25.7
29.7
30.9
297.5
310.8
LSK-C
63.9
99.7
25.2
28.4
29.6
299.3
313.3
LSK-W
63.2
99.7
25.1
153.9 154.3 154.8 153.5 154.8 154.3 154.1 154.8 155.0 150.1/ 156.1 150.9/ 156.3 150.4/ 155.5 145.5/ 153.9 145.9/ 154.4 145.7/ 155.0
28.1
29.3
289.7
304.9
compost and soil, the changes in thermal properties of the investigated materials after 28 days of degradation have been analyzed. It is well know that during degradation, the thermal properties of biodegradable polymers significantly decrease. This phenomenon is related to scission of the polymer chains and the formation of PLA oligomers during decomposition. The obtained results indicate that an addition of a nanofiller (montmorillonite and Nanofil2) influences thermal properties of PLA during degradation (Table 2). However an addition of poly(ε-caprolactone) to LN and LS systems significantly decreases glass transition (Tg) and cold crystallization (Tc) temperatures of studied composites in comparison with materials without a compatibilizer. It has been noted that the most significant decrease of all mentioned parameters was observed in the case of materials with an addition of PCL degraded in lake water, soil as well as in compost. The lowest values of Tg -as well as Tc were observed in the case of LSK sample degraded in soil. This suggests that not only the addition of PCL influences the degradation process but also the conditions in which the degradation occurs significantly influence the decomposition of PLA. Moreover, obtained results indicate that the addition of a nanoadditive as well as a compatibilizer decreases the melting temperature of degraded composites in comparison with neat polylactide, with the lowest values of Tm similarly observed after degradation in soil. It should be mentioned that in the case of materials containing pure montmorillonite (S) after degradation process in all environments two endothermic peaks were observed. It is known that formation of bimodal peak is connected with the formation of different crystalline forms of polylactide, however it can be also related to the formation of fractions differing in molecular weight (Olewnik-Kruszkowska et al., 2017). Recently it was established that a lower molecular weight can form the α’ structure which is characterized by a lower temperature in comparison with the α form (Tábi et al., 2010). Irrespectively of the environment in which the degradation had taken place, the samples containing PCL were characterized by higher degree of crystallinity in reference to the polylactide-nanofiller systems. It is well known that the degradation in the amorphous phase of polymers takes place preferentially, moreover shorter chains of PLA are able to reorganize and form the crystalline phase (Calafel et al., 2010; Tábi et al., 2010; Gupta et al., 2012). The above discussed results indicate that an addition of a compatibilizing agent facilitates the degradation of an unordered structure of PLA and the formation of a crystal phase. However it can clearly be observed that the highest values of the degree of crystallinity (Xc) were observed
3.2. Changes in thermal properties During the decomposition of PLA and its composites in lake water, 4
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significantly after degradation processes. All films containing PCL were characterized by a more corrugated surface. Research of samples comprising a compatibilizing agent (LNK and LSK) revealed that the most significant changes in surface morphology after degradation had occurred in soil. The increase in surface roughness of the above mentioned samples was also observed after degradation in compost and water, however the changes were not as substantial as the ones observed after decomposition in soil. It is well known that during the degradation process polymer disintegration takes place which leads to a formation of mainly oligomers. Cracks and pores form on the surface of polylactide improve contact with the environment and in this way can influence the kinetic decomposition of a PLA. The increase in contact surface of a polymer with the environment leads to erosion of the studied materials and to the decomposition of the amorphous polymer phases (Rydz et al., 2013). In the case of materials containing PCL, acting as a compatibilizer, an improved dispersion of nanoadditives in the polymer matrix promotes contact between the materials and the environment. All these processes result in the increased efficiency of degradation after PCL is introduced into the polylactide-nanofiller systems. Summarizing, the PLA presents the most smooth surface while the LSK sample is characterized by the most rugged surface, regardless of the degradation environment. Taking into account the type of environmental conditions in which the degradation occurs the same tendency as revealed in the case of DSC technique can clearly be noticed. It means that the highest surface roughness parameters were observed in the case of samples undergoing degradation in soil.
in the case of materials degraded in soil, while the lowest values of Xc were obtained for materials degraded in water. Summarizing, based on the analysis of the above discussed results (Table 2) it is reasonable to assume that the introduction of poly(ε-caprolactone) accelerates the degradation of polylactide-based composites, however the most significant changes in discussed properties are observed after degradation carried out in soil. This phenomenon is caused by improved dispersion of the nanoadditives in the polylactide as well as the plasticizing effect of PCL (Olewnik and Richert, 2014; Olewnik-Kruszkowska et al., 2016). The same tendency was observed during thermogravimetric analysis. The results of the measurements of thermal stabilities of the PLA-based composites after degradation in different conditions are presented in Table 2. It was found that introduction of PCL significantly decreases the temperature of 5 and 10% mass loss of the analyzed materials in comparison with PLA-nanoadditive systems. The reduction in T5 and T10 values can be explained by better dispersion of the nanofiller in polymer matrix by means of a compatibilizing agent. Consequently the microorganisms and water can readily diffuse into the studied materials. As mentioned above, in the case of the DSC analysis the most significant changes in thermal parameters were noticed in the case of samples stored in soil. Taking into account the results obtained by means of TG technique it can also be observed that the lowest values of thermal stability were obtained in relation to LNK and LSK composites degraded in soil. Moreover it is interesting that samples containing unmodified montmorillonite (S) degrade significantly faster in comparison with a composite comprising a modified nanofiller (N). For this reason it is justified to assume that the modification of the nanofiller, and the resulting increased hydrophobicity of the nanoadditive, impedes the degradation process of polymer composites (Olewnik-Kruszkowska et al., 2016). Summarizing, the analysis of the thermal properties and stability indicates that during the degradation process values of the studied parameters decreased in the following order: soil < compost < water (Table 2).
3.4. Changes in surface morphology by means of SEM and LIVE/DEAD techniques It was indicated by other researchers that the degradation processes often influences the surface structure of studied materials (Gu et al., 2002; Gu, 2003). In relation to the changes in surface morphology and the effect different environmental conditions have got on degradation of PLA-based composites, the SEM analysis can be used to study these phenomena. Obtained results for PLA and PLA-nanofiller systems without (LN and LS) and with an addition of a compatibilizing agent (LNK and LSK) are shown in Fig. 4. Based on the obtained results it needs to be emphasis that in the case of all samples a biofilm had formed on their surface. The most noticeable surface changes have been observed in the case of materials degraded in soil. Among the samples degraded in soil the most radical modification of surface has occurred in the case of LS and LSK samples, where unmodified, pure MMT was introduced as nanofiller into the PLA matrix. Inspection of the surface of all samples stored in soil have not revealed, apart from the formation of biofilms, any other changes like cavities, cracks or pinholes. In the case of samples subjected to degradation in compost fragments of fungus as well as bacteria have been found on the surface of degraded materials. The least noticeable changes in the surface morphology have been observed for the samples stored in lake water. In addition in the case of neat PLA it can be noticed that sample is almost clear in comparison to the PLA-based composites. Moreover it should be mentioned that neat polylactide sample exhibits the smoothest and flattest surface not only after degradation in lake water but also after storage in other environments: compost and soil. The same tendency was observed by Stepczyńska et al. (Stepczyńska and Rytlewski, 2018) where PLA and PLA filled with flax-fibers were subjected to the enzymatic degradation process. In aim to establish whether living bacteria were present on the surface of the studied materials, the LIVE/DEAD technique was applied. Fig. 5 depicts biofilm formation incubated in the mentioned environments. Obtained results clearly indicate that in the case of LSK sample comprising polylactide, pure montmorillonite and PCL, acting as the as compatibilizing agent, viable cells colored by fluorescent to green are especially visible. This is strongly related to the distribution of
3.3. Assessment of surface morphology by means of AFM technique Atomic force microscopy was applied in aim to establish the influence a compatibilizing agent has got on surface changes of the polylactide-based composites during degradation process in different environments. It is well known that the AFM technique provides detailed information on the surface morphology. Moreover it should be mention that the morphology of the studied samples during biodegradation depends on their composition and conditions of the degradation process. Fig. 3 presents AFM photographs of the surface areas of the pure PLA and PLA-based composites without or with an addition of PCL as a compatibilizing agent after biodegradation had been carried out in soil, compost and lake water. The values of the different roughness parameters (Ra, Rq) for all samples stored in described media are summarized in Table 3. The AFM images show differences in the surface roughness parameters during degradation process as well as the dependence on the composition of materials and the environmental conditions in which the degradation process occurs. After degradation the surface morphology of PLA-nanofiller systems (LN and LS) was characterised by increased roughness in comparison to pure polylactide. It is reasonable to assume that this phenomenon is connected with the agglomeration of nanoparticles, especially in the case of an unmodified nanofiller. In the case of composites the increase in roughness parameters can be attributed to the macro-chains rearrangement in the polymer. The highest values of the Ra, and Rq for LN and LS samples were observed during the degradation in soil, where in the case of water and composites the described parameters are comparable. Moreover the Ra and Rq values of LN and LS samples after degradation in soil were at least three times higher compared to those measured after degradation in water and compost. After the addition of PCL into the PLA-nanofiller systems surface roughness of obtained composites increased 5
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Fig. 3. AFM images of studied materials after degradation in different environments (S-soil, C-compost, W-water).
nanoadditives in the polymer matrix, which allows penetration of the sample by bacteria. However it should be noted that bacteria are able to form a biofilm on the surface of all of the degraded materials in any of the three environments. Summarizing, the results discussed above, obtained by means of the LIVE/DEAD technique as well as the SEM analysis, have confirmed the presence of microorganisms on the surface of studied materials. As predicted the results obtained using scanning electron microscopy and LIVE/DEAD techniques correspond with the variations in surface morphology determined using the AFM method.
Table 3 Values of roughness parameters of polylactide after 28 days of degradation in different environments. Sample
L-S LN-S LNK-S LS-S LSK-S L-C LN-C LNK-C LS-C LSK-C L-W LN-W LNK-W LS-W LSK-W
Roughness parameters [nm] Ra
Rq
6.5 39.1 46.7 52.4 56.8 6.3 10.9 13.4 10.6 16.6 7.3 9.6 13.5 17.8 20.1
11.9 53.2 68.6 72.8 87.0 13.6 15.1 24.2 13.8 26.3 18.2 13.5 18.1 22.9 38.3
4. Conclusions Evaluation of the surface of the polylactide and polylactide-based composites with and without poly(ε-caprolactone) showed that a compatibilizing agent plays an important role during degradation process in different environments. It can be clearly noticed that an addition of a compatibilizing agent significantly increases oxygen consumption as well as the activity of hydrolytic enzymes. 6
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Fig. 4. SEM images of PLA and its composites after degradation in different environments (S-soil, C-compost, W-water).
the degradation process of materials containing PCL is more profound in comparison with the degradation occurring in the polylactide and PLA-nanofiller systems.
Simultaneously the changes in thermal properties as well as in thermal stability of materials consisting of polylactide, poly(ε-caprolactone) and nanofillers are characterized by lower values than composites without an addition of a compatibilizing agent. Moreover it should be mentioned that the results of the present study demonstrate that regardless of the environmental conditions, may it be soil, lake water or compost, 7
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Fig. 5. The biofilm formed on the materials incubated in different environments. Bacteria stained by using the LIVE/DEAD method (microscope Olympus BX50, magnification 1000 x, live bacteria – green, dead bacteria – red). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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