# Novel and Highly Efficient Antibacterial PLA Composites Prepared with Liquidambar Orientalis Oil and Ag@g-C3N4 Nanocomposite

Zehra Durmus, Roberto Köferstein, Arzu Özgen, Titus Lindenberg, A. Wouter

Maijenburg, Ali Durmus

► To cite this version:

Zehra Durmus, Roberto Köferstein, Arzu Özgen, Titus Lindenberg, A. Wouter Maijenburg, et al. Novel and Highly Efficient Antibacterial PLA Composites Prepared with Liquidambar Orientalis Oil and Ag@g-C3N4 Nanocomposite. Journal of Polymers and the Environment, 2025, 33 (5), pp.2249-2266. 10.1007/s10924-025-03520-9. hal-05041901

HAL Id: hal-05041901

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Submitted on 30 Apr 2025

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# Novel and Highly Efficient Antibacterial PLA Composites Prepared with *Liquidambar orientalis* Oil and Ag@g-C₃N₄ Nanocomposite

Zehra Durmus<fnref n="1" />,<fnref n="2" /> · Roberto Köferstein<fnref n="3" /> · Arzu Özgen<fnref n="4" /> · Titus Lindenberg<fnref n="5" /> · A. Wouter Maijenburg<fnref n="3" />,<fnref n="5" /> · Ali Durmus<fnref n="6" />

Accepted: 28 January 2025 / Published online: 18 February 2025
© The Author(s) 2025

## Abstract

Flexible PLA-based antibacterial composite films were prepared using a natural oil, *Liquidambar orientalis*, as bio-based plasticizer, and 2D graphitic carbon nitride (g-C₃N₄) decorated with Ag nanoparticles with a particle size of 10–30 nm as antibacterial agent (Ag@g-C₃N₄). This structurally designed antibacterial nanocomposite was synthesized with the preparation of g-C₃N₄ by high-temperature annealing followed by the reduction of silver salt onto g-C₃N₄. The Ag@g-C₃N₄ nanocomposite exhibited a surface area value of 18 g/m<fnref n="2" />. PLA/Ag@g-C₃N₄ composite films were prepared with solution casting method by introducing 30 phr of *L. orientalis* oil and various amounts (1, 2 and 4 phr) of Ag@g-C₃N₄. It was found that 30 phr of *L. orientalis* oil successfully plasticized the PLA and reduced its glass transition temperature from 60 °C to 43 °C and its melting temperature more than 10 °C by reducing the strong interactions and hydrogen bonds between PLA chains. *L. orientalis* oil also acted as a dispersion agent for the Ag@g-C₃N₄ nanocomposite particles and significantly improved their antibacterial activity. Antibacterial tests performed using Gram-positive bacteria (*Staphylococcus aureus* ATCC 25923) and Gram-negative bacteria (*Escherichia coli* ATCC 25922 and *Acinetobacter baumannii* ATCC BAA 747) indicated that introducing a small amount of 2D Ag@g-C₃N₄ nanocomposite particles into PLA yielded superior antibacterial activity.

**Keywords** Polylactic acid · Antibacterial Composite · Graphitic Carbon Nitride (g-C₃N₄) · Silver (Ag) Nanoparticle · *Liquidambar orientalis*

## Introduction

Polylactic acid (PLA) is a thermoplastic polyester and the most extensively used biodegradable plastic in various applications such as textile and medical technologies, automotive plastics, electronics, and mainly in food packaging, due to its superior structural and physical properties [1]. The most prominent advantages of PLA are its biodegradability, good transparency, mechanical durability and easy processing like other commodity thermoplastics. These features make PLA and PLA-based compounds promising and green alternatives to commercially used packaging polymers; polyethylene and polypropylene. However, it is also a well-known fact that the relatively high glass transition temperature (*T*<sub>g</sub>) of PLA (about 60 °C) makes this polymer very brittle in very wide range of temperature and limits its application potential for the areas required flexibility. Therefore, a considerable research effort has emerged to reduce the *T*<sub>g</sub> of PLA and expand its application areas by compounding with different plasticizers, polymers, and additives [2–12]. Majority of

<footnote>Ali Durmus
durmus@iuc.edu.tr</footnote>

<footnote n="1">¹ Faculty of Engineering and Natural Sciences, Department of Biomedical Engineering, Biruni University, Istanbul 34015, Türkiye</footnote>

<footnote n="2">² Biruni University Research Center (BAMER), Biruni University, Istanbul 34015, Türkiye</footnote>

<footnote n="3">³ Inorganic Chemistry, Institute of Chemistry, Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany</footnote>

<footnote n="4">⁴ Vocational School of Health, Istanbul Gelisim University, Istanbul 34310, Türkiye</footnote>

<footnote n="5">⁵ Center for Innovation Competence (ZIK) SiLi-nano®, Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany</footnote>

<footnote n="6">⁶ Faculty of Engineering, Department of Chemical Engineering, Istanbul University-Cerrahpasa, 34320 Avcılar/Istanbul, Türkiye</footnote>
commercially available plasticizers used in polymer industry, are synthetic chemicals obtained from petrochemicals and thus crude-oil feed stocks. Regarding the recent trends in chemistry and materials science such as sustainability, environmental friendly materials and processes, and reducing carbon foot-print etc., nontoxic and green plasticizers and additives obtained from biodegradable and renewable resources have been attracted a great scientific and technical attention. Mele et al. used cardanol oil as a bio-based plasticizer in PLA obtained from a renewable resource, cashew agro-industry [13]. Carpintero et al. showed that egg yolk oil acted as a plasticizer for PLA and increased its flexibility and also provided the PLA films antioxidant and light barrier properties [14]. Hasanoglu et al. recently reported that *L. orientalis* oil as a nontoxic, environmentally friendly, and green additive can be successfully used as a natural, renewable, and sustainable plasticizer to produce flexible PLA parts [15]. They also informed about the planting of Anatolian sweetgum trees, harvesting of *L. orientalis* gum and oil, and biochemical and physical features of oil.

Green plasticizers can contribute to produce completely biodegradable PLA compounds. But, functional additives must be introduced into polymer compounds to manufacture parts for particular applications. Antibacterial plastics have been attracted great industrial demands in several applications such as producing synthetic fibers, textiles, and fabrics, food packaging materials, medical parts. Silver nanoparticles and their derivatives are the most extensively and favorably used antibacterial agents in thermoplastics as well as paints, surface coatings, and fibers [16–18]. Several studies have been reported about the preparation, characterization, and specifically food packaging applications of antibacterial PLA-based compounds and composites [19, 20]. Yalcinkaya et al. synthesized Ag grafted cellulose nanocrys-tals (Ag-g-CNC) and loaded this hybrid filler into PLA [21]. They compared the antibacterial activities of PLA nanocomposites containing 1% wt. of CNC (PLA/1CNC), 0.5% wt. of Ag nanoparticles (PLA/0.5Ag) and hybrid system containing CNC and Ag in the same amount (PLA/1CNC/0.5Ag). They found that the Ag-g-CNC positively affected the thermal degradation and cold crystallization of PLA and the hybrid filler loaded nanocomposite (PLA/1CNC/0.5Ag) showed the best antibacterial performance. Velásquez et al. reviewed the processing techniques used to develop active PLA packaging films included various active agents (AAs), such as essential oils and polyphenols as antibacterial and antioxidant natural compounds and compared their feasibility, advantages, limitations, and relevant processing aspects [22].

In this study, antibacterial and flexible PLA films were prepared using 30 phr of *L. orientalis* oil as natural plasticizer and various amounts (1, 2, and 4 phr) of

Ag nanoparticle decorated g-C₃N₄ as antibacterial agent. Structural, physical, and thermal properties of Ag@g-C₃N₄ nanocomposite and PLA/Ag@g-C₃N₄ composite films were studied, in detail.

# Experimental

## Materials

Melamine (Alfa-Aesar), N,N-Dimethylformamide (Merck), AgNO₃ (Carl Roth), NaBH₄ (Chemsolute), and Polyvinylpyrrolidone (Carl Roth) were analytical reagent grades and used as received without further purification. Poly (lactic acid) (PLA) was a commercial grade polymer, Luminy® L130, with a melt flow index (MFI) of 10 g/10 min (190 °C, 2.16 kg), density of 1.24 g/cm³, and L-isomer of 99%, kindly supplied by Amco Polymers, LLC. The *L. orientalis* oil was kindly provided from a local herbalist and essential oil producer in Muğla Province, Turkey. Chemical composition and some other properties of *L. orientalis* oil used in this study was previously reported, in detail [15].

# Synthesis and Preparation Studies

## Synthesis of Graphitic Carbon Nitride (g-C3N4)

Graphitic carbon nitride (g-C₃N₄) was synthesized according to the previously reported route [23]. In the synthesis, a proper amount of melamine was dispersed into a diluted H₂SO₄ solution. The suspension was placed into a Teflon-lined stainless-steel autoclave and heated up to 180 °C and kept at this temperature for 12 h. Then, the acid-treated melamine precursor was cooled to room temperature and washed repeatedly with distilled water and ethanol. After drying at 80 °C for 12 h, it was heated up to 550 °C in a muffle furnace (Carbolite P310) with a heating rate of 2 °C/min and kept at this temperature for 4 h. Afterwards, the resulting pale yellow g-C₃N₄ powder was obtained.

## Synthesis of Ag Decorated g-C3N4 Nanocomposite (Ag@g-C3N4)

Silver (Ag) nanoparticle decorated graphitic carbon nitride (g-C₃N₄) was synthesized following a previously reported procedure [24, 25]. 0.2 g of g-C₃N₄ was added into a flask containing 20 mL of an aqueous solution containing 0.04 mmol AgNO₃, which was then vigorously stirred on a magnetic stirrer at room temperature for 24 h. Then, a fresh aqueous solution containing sodium borohydride (NaBH₄) was added dropwise into the mixture at 0 °C under vigorous stirring (molar concentration ratio of AgNO₃:NaBH₄ = 1:50). A
proper amount of poly(vinyl pyrrolidone) (PVP), as steric capping agent, was used as a surface stabilizer for reducing the aggregation. The resulting material, g-C₃N₄ flakes decorated with Ag nanoparticles, was obtained by centrifugation and washed with distilled water and dried in vacuum at 100 °C for 12 h.

## Preparation of Polylactic Acid (PLA)/Ag@g-C3N4 Composites

PLA films plasticized with *L. orientalis* oil were prepared with solution mixing method. Sample notation and compositions are defined in Table 1. Before the sample preparation, PLA granules were dried in an oven for 24 h at 80 °C.

In the solution mixing method, PLA was completely dissolved in chloroform (polymer: solvent ratio of 1:10, w/v) then proper amount of *L. orientalis* oil (denoted as L in the sample names) was introduced into PLA solution and rigorously mixed at ambient temperature for 3 h. In another beaker, Ag@g-C₃N₄ powder (denoted as Ag in the sample names) was completely dispersed into a chloroform using ultrasonic homogenizer (Bandelin Sonopuls HD2200) equipped with a horn and KE76 probe with titanium flat tip, for 5 min. Then the PLA solution were gradually poured into the Ag@g-C₃N₄ suspension in 4 parts within one hour and under a vigorous stirring on a magnetic stirrer (300 rpm) at room temperature. A short and intense ultrasonication (2 min.) was also applied to the final mixture to ensure uniform dispersion of particles then the mixture was immediately poured in petri dishes and kept at room temperature for 3 days for evaporating solvent slowly. PLA films were dried in a vacuum oven at 60 °C for 24 h for completely evaporating remaining solvent. A blank PLA film (L0) was also prepared as control sample.

## Analysis and Testing Studies

### Structural Characterization of Ag@g-C3N4 Nanocomposite

Chemical structures of samples were characterized with Fourier-Transform Infrared Spectroscopy (FTIR) (Tensor 27, Bruker Co.) equipped with an ATR (Attenuated Total Reflectance) device.

<table><caption>**Table 1** Sample notations and compositions</caption><thead><tr><th>Samples</th><th>PLA</th><th>L. orientalis oil (L)</th><th>Ag@g-C<sub>3</sub>N<sub>4</sub> (Ag)</th></tr></thead><tbody><tr><td>L0</td><td>100</td><td>-</td><td>-</td></tr><tr><td>L30</td><td></td><td>30</td><td>-</td></tr><tr><td>L30-Ag1</td><td></td><td>30</td><td>1</td></tr><tr><td>L30-Ag2</td><td></td><td>30</td><td>2</td></tr><tr><td>L30-Ag4</td><td></td><td>30</td><td>4</td></tr><tr><td>L0-Ag4</td><td></td><td>-</td><td>4</td></tr></tbody></table>

The chemical composition of the samples was determined by an X-ray diffractometer (XRD, Bruker D8 Advance, Cu-Kα, λ=0.15406 nm, 40 kV, 40 mA) in the 2θ range of 5°–70° with a constant shutter speed, a time step of 0.5 s and a step size of ca. 0.01°. The powders were measured on a silicon (Si) sample holder to reduce the background. The software DiffracPlus EVA. Bruker was used for data evaluation and background correction.

The specific surface area was determined using nitrogen physisorption and five-point Brunauer-Emmett-Teller (BET) method (Nova 1000, Quantachrome Corporation).

Thermal properties of samples were analyzed in a thermogravimetric analyzer (TGA, Netzsch Jupiter STA 449F5) with a heating rate of 10 °C/min under air atmosphere with a flow rate of 50 mL/min.

### Morphological and Microstructural Characterization of PLA/Ag@g-C3N4 Composites

Infrared spectra of PLA and composite samples were recorded with a Fourier-transform infrared spectrophotometer (FT-IR, Perkin Elmer Spectrum II) equipped with an ATR (Attenuated Total Reflection) unit in the wavenumber range of 4000–400 cm⁻¹ at room temperature to characterize possible reactions and physical and/or chemical interactions between the components.

The morphological features and microstructural properties of the powder and film samples were investigated by a scanning electron microscope (SEM, Carl Zeiss Merlin) and a field emission scanning electron microscope (FE-SEM, FEI Quanta FEG 450) operated at 10 kV, respectively. Cryo-fractured cross-sections of the films were prepared by using liquid nitrogen, after which they were directly prepared by golf sputtering and imaged in the electron microscope.

Contact angle measurements were performed with a KSV Attension®, Theta Lite Goniometer device using the pendant drop method (5 µL of water) taken at least five points on the film sample.

### Melting and Crystallization Behaviors of PLA/Ag@g-C3N4 Composites

Thermal behaviors of samples were investigated using a differential scanning calorimeter (SII Nanotechnology, Hitachi ExStar 6200) equipped with an electrical cooling device (Thermo-Scientific intracooler: EK90C/SII). Calibration of the instrument was conducted using indium (In), tin (Sn), and zinc (Zn) metals. Samples weighing 7–8 mg in an aluminum pan were heated from 0 °C to 220 °C with a heating rate of 10 °C/min and equilibrated at this temperature for 3 min to eliminate thermal history. The samples were then cooled to 0 °C with a cooling rate of 30 °C/min and kept at
this temperature for 3 min. Finally, they were heated up to 220 °C with a heating rate of 10 °C/min. DSC runs were performed under nitrogen atmosphere with a flow rate of 50 mL/min, to avoid thermo-oxidative degradation. Degree of crystallinity (Xc, %) values of samples were calculated using the following equation;

$$ \%X_c = \frac{\Delta H_m}{\Delta H_m^0(1 - \alpha)} \times 100 \quad (1) $$

where ΔHm is the melting enthalpy of samples (J/g) recorded during second heating, ΔHm° is the melting enthalpy value of 100% crystalline form of PLA (93.6 J/g) [26], and α is the sum of the weight fractions of plasticizer and additive (L. orientalis oil and Ag@g-C3N4).

## Dynamic Mechanical Analysis of PLA/Ag@g-C3N4 Composites

Solid state viscoelastic properties of samples were analyzed with a test protocol performed in a dynamic mechanical analyzer (DMA, SII Nanotechnology, Hitachi, ExStar DMS 6100). The temperature sweep test was performed in uniaxial tension mode with a heating rate of 3 °C/min and frequency of 1.0 Hz in the temperature range of 20–160 °C. Rectangular film specimens with dimensions of 20 × 5 × 0.2 mm were used.

## Antibacterial Tests

### Bacterial strains.

In the antibacterial activity assays, we used Gram-positive bacteria (Staphylococcus aureus ATCC 25923) and Gram-negative bacteria (Escherichia coli ATCC 25922 and Acinetobacter baumannii ATCC BAA 747), which were stored in our laboratory.

### Disc Diffusion Method.

The disc diffusion method was used to determine the antibacterial activities of composite films. The samples were tested on three types of bacteria, including S. aureus ATCC 25,923, E. coli ATCC 25,922, and A. baumannii ATCC BAA 747. PLA film and L30 (PLA film containing LO essential oil) were used as negative controls, while VA: vancomycin (30 µg/ml), AMC: amoxicillin-clavulanate (20/10 µg/ml), and AMC: amoxicillin-clavulanate (20/10 µg/ml) were used as positive controls for each respective bacteria type. First, a 10⁸ CFU/mL dilution was prepared by growing microorganisms in Luria-Bertani liquid media (Merck) for 16 h. The concentration was then adjusted to correspond to a 0.5 McFarland concentration, and this dilution was used as the inoculum. Next, 60 µl of the bacterial dilution was added to petri dishes containing Luria-Bertani

(Merck) agar and spread using an L-loop stick. The films were prepared as square pieces with 6 mm edges, sterilized under UV irradiation (both sides) for 20 min, and placed appropriately in petri dishes. The petri dishes were then incubated at 37 °C for 24 h, and the diameters of inhibition zones were measured in millimeters. The experiments were repeated thrice to ensure accuracy [27, 28].

## Results and Discussion

### Structural Characterizations of Ag@g-C3N4 Nanocomposite

Figure 1(a) and 1(b) show the FT-IR spectra of pristine g-C3N4, Ag@g-C3N4, and Ag@g-C3N4 after heating at 340 °C as TGA residue in different wavenumber range. All samples exhibit the characteristic structure of g-C3N4. Each sample shows an absorption peak around 810 cm⁻¹, indicating the presence of a tri-s-triazine-based structure. The peaks observed in the 1200–1700 cm⁻¹ range correspond to the typical stretching vibrations of C-N heterocycles. Additionally, peaks near 3400 cm⁻¹ are attributed to N-H and O-H stretching vibrations, which imply amino groups and adsorbed water. A comparison between and the TGA residue at 340 °C (intermediate) shows the disappearance of bands at 1570, 1534, 1453, and 1308 cm⁻¹. This result indicates that the heating yields change in the Ag@g-C3N4 structure. The TGA residue after full decomposition at 900 °C only shows reflections of silver and the broad reflection at 28° disappears, which supports the assumption that the peak originated from g-C3N4.

Figure 1(c) compares the XRD patterns of pristine g-C3N4, Ag@g-C3N4, intermediate residue of Ag@g-C3N4 (black-brown powder after heating up to 340 °C in TGA), and final residue of Ag@g-C3N4 (white shining powder after heating up to 900 °C in TGA). Pristine g-C3N4 exhibits two broad crystallinity peaks indexed (100) at 13° and (002) at 28° with PDF #01–087–1523 in the DiffracPlus Eva software library. These broad peaks could be attributed to the fact that the strong acid treatment partially destroys the graphitic structure and produces some fragments in the g-C3N4 sample [23]. It is seen that the XRD pattern of intermediate residue is identical to the pattern of Ag@g-C3N4 and shows strong reflections of elemental silver. The very broad and weak peak of intermediate, observed at 28°, indicates low crystalline g-C3N4. This peak is completely disappeared after heating the Ag@g-C3N4 up to 900 °C because all the organic content (carbon nitride structure) is decomposed. It was also obtained that peak (100) at 13° seemed in the pristine g-C3N4 and appeared in none of Ag@g-C3N4, intermediate, and final residues. This result could be attributed
<figure><img src="image_2.png" /><figcaption>**Fig. 1** (a) Full scale FTIR spectra of g-C₃N₄, Ag@g-C₃N₄, and Ag@g-C₃N₄ after heating up to 340 °C, (b) focusing their spectra in the wave-number range of 1700–700 cm⁻¹ and (c) XRD patterns of g-C₃N₄, Ag@g-C₃N₄, and Ag@g-C₃N₄ after heating up to 340 °C and 900 °C</figcaption></figure>

to the same issue that the low crystalline g-C₃N₄ shows a strong preferred orientation in the Ag@g-C₃N₄ structure thus the (100) peak is not appeared. Some researchers also concluded that the Ag introducing into the g-C₃N₄ matrix

may disturb the g-C₃N₄ structure by altering the in-plane structural repeating network [29–31].

Morphologies of g-C₃N₄ and Ag@g-C₃N₄ nanocomposite are given in Fig. 2. SEM images of g-C₃N₄ clearly implies that it is formed with layered structure. These curved layers, showing a convex and spherical form in many parts and having a few tens nanometer in thickness are stacked to form macro pores with larger diameter than a few hundreds. Surface of layers are quite smooth while the cracked edges of stacks includes some roughness. On the other hand, SEM images of Ag@g-C₃N₄ nanocomposite is highly different from the pristine g-C₃N₄. As especially seen in the SEM image below on the right, Ag nanoparticles (as shiny white points) and agglomerates are distributed well onto the surface of g-C₃N₄. It can be assumed that the size of Ag nanoparticles varied in a wide range, approximately in 10–30 nm.

Based on the BET measurements, surface area values of g-C₃N₄ and Ag@g-C₃N₄ were determined to be 25 and 18 m²/g, respectively. It was found that the decoration of g-C₃N₄ surfaces with silver nanoparticles yielded only to a small reduction in its specific surface area probably due to the blocking of mesopores with largely agglomerated Ag nanoparticles.

Figure 3 represents the TGA, DTG, and DSC thermograms of pristine and Ag decorated g-C₃N₄. Figure 3(a) shows that the g-C₃N₄ is stable in air up to 580 °C. Further heating leads to a decomposition process with a strong weight loss of 80%. The decomposition process is completed at approximately 730 °C. The 20% residue is probably caused by oxygen capturing from air onto the protonated surfaces, as pointed out in our recent paper [23]. In general, the thermo-oxidative decomposition of g-C₃N₄ roughly remains a 10% residue [32]. In this study, the Ag@g-C₃N₄ nanocomposite shows a different decomposition behavior. The first weight loss step about 7.1% appeared in the temperature range of 200–300 °C probably corresponds to decomposition of organic compound (PVP) used as steric capping agent. Intermediate Ag@g-C₃N₄ is stable up to 410 °C then the second two-step decomposition begins above 410 °C and proceeds to 540 °C. This second decomposition corresponds to collapsing the structure and formation of volatile carbon and nitrogen species. It was found that the main decomposition process of Ag@g-C₃N₄ begun approximately 120 °C earlier than the g-C₃N₄. It might be originated from the catalytic effect of Ag nanoparticles for the decomposition organic structure because it is a well-known fact that the active metal sites onto a support initiate and accelerate the decomposition behaviors of organic molecules [33]. Total weight loss of nanocomposite was found to be 33.5%. XRD analysis of the residue of Ag@g-C₃N₄ nanocomposite as given in Fig. 1(c) only showed characteristic peaks of Ag;
<figure><img src="image_3.png" /><figcaption>**Fig. 2** SEM images of g-C₃N₄ (a and b) and the Ag@g-C₃N₄ nanocomposite (c and d)</figcaption></figure>

(111) at 38.1°, (200) at 44.3°, and (220) at 64.5, indexed as the cubic phase of Ag (JCPDS No. 04-0783), consistently with the literature [34]. DSC thermograms of g-C₃N₄ and Ag@g-C₃N₄ given in Fig. 3(c) display similar peak shapes to their DTG curves and clearly exhibit oxidative decomposition exotherms. It is noticeable that the exotherm of g-C₃N₄ spread over a very wide temperature range, starting at 100 to end up at 750 °C. It should be noted that DSC signal is associated with the change in heat capacity of specimens with temperature. Thus, such an extremely wide range of exotherm is related to the heat capacity change of g-C₃N₄ and the main decomposition exotherm is seen as a sharp and small peak above 700 °C. The DSC thermogram of Ag@g-C₃N₄ implies three distinctive peaks at 300, 440 and 500 °C which correspond to the ends of thermo-oxidative decompositions of PVP, first step of g-C₃N₄ and second step of g-C₃N₄ as mentioned above.

Structural and Morphological Properties of PLA/
Ag@g-C3N4 Nanocomposites

Figure 4(a-c) illustrate the photographs of solution casted PLA/Ag@g-C₃N₄ nanocomposite films. It is seen that the neat PLA and PLA plasticized with the *L. orientalis* oil yielded much transparent films compared to the Ag@g-C₃N₄ loaded ones. PLA is more transparent than the plasticized counterpart. It was possibly due to the effect of *L. orientalis* on the dynamics of PLA chains by enhancing the chain mobility thus degree of crystallinity during solvent evaporation and solidification of polymer solution.

One can also notice that the addition of 30 phr *L. orientalis* oil slightly turned the film color into yellowish in addition to the higher opacity. Increase in the loading amount of Ag@g-C₃N₄ resulted in the dark brown color thus the logo image cannot be clearly seen under any of these samples.

Figure 4(d) shows the flexibilities of plasticized PLA with *L. orientalis* oil in the absence and presence of 4 phr of Ag@g-C₃N₄ additive. It was found that the introducing of
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3** (a) TGA, (b) DTG, and (c) DSC thermograms of g-C₃N₄ and Ag@g-C₃N₄</figcaption></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

a solid functional additive did not inversely affect the flexibility of sample.

Figure 5(a), 5(b), and 5(c) show the FTIR spectra of PLA, *L. orientalis* oil and composite samples, respectively.

FTIR spectra of PLA film shows the characteristic bonds of polymer chain as denoted on the spectrum. The natural plasticizer, *L. orientalis* oil exhibited many characteristic

<caption>**Fig. 4** Photographs of solution casted film samples</caption>
<figure><img src="image_12.png" /><figcaption>**Fig. 5** FTIR spectra of (a) PLA, (b) *L. orientalis* oil, and (c) composite samples</figcaption></figure>

peaks below the wavenumber of 1700 cm⁻¹. Peaks in the range of 1000–650 cm⁻¹ and 1650–1450 cm⁻¹ correspond to vibrations of C=C and C-H bonds of aromatic units, respectively, as previously reported [35, 36]. It was reported that the main components (wt%) of LO oil were styrene (70.4%), α-pinene (19.4%), β-pinene (4.3%), and β-caryophyllene (0.2%) [37]. Therefore, the distinctive and characteristic

peak of *L. orientalis* oil could be considered as the C=C bond of styrene seen at 1635 cm⁻¹. Figure 5(c) compares the FTIR spectra of composites to the plactized PLA specimen (L30). The peaks appeared at 1715 and 1635 cm⁻¹ in FTIR spectra of composites corresponded to ketonic species in the *L. orientalis* oil and C=C bonds of styrene, as the main components of *L. orientalis* oil, respectively. The absence of other new peaks in the spectra of the composite samples pointed out that there was no chemical interaction between the components, PLA, natural plasticizer, and functional nanocomposite. Any characteristic peak (C-Cl strecthing at 773 cm⁻¹ and C-Cl bending at 670 cm⁻¹) belong to the solvent, chloroform or trichlorometan (CHCl₃), used in film preparation did not detected in the FTIR spectra of PLA and composites. This result indicated that the solvent was completely removed from the samples.

Figure 6 compares the SEM images of cryo-fractured cross-sections of L0(PLA), L30-Ag4, and L0-Ag4 samples taken with the magnification of x5000. It appears that many fracturing points have formed on the surface of L0 but the cross-section of L30-Ag4 possesses a smooth fracturing surface although it includes 4 phr of solid additive. It is also interesting that solid particles are not clearly observed in the SEM image of L30-Ag4 at this magnification. On the other hand, agglomerated particles are clearly observed on the fractured surface of L0-Ag4 which indicates poor dispersion of functional additive. Surface topography of samples implies that the addition of *L. orientalis* oil improves the dispersion of Ag@g-C₃N₄ particles into PLA matrix. Based on the SEM images, it can be concluded that the *L. orientalis* oil acted as a dispersion agent or compatibilizer for the Ag@g-C₃N₄ particles. Therefore, this natural oil might be a dual-function material into PLA-based composites as bio-based plasticizer and dispersion agent.

Figure 7 represents the SEM images of L0, L30 and samples prepared with different amounts of Ag@g-C₃N₄ additive, taken with the magnification of x10.000, except the L30-Ag2 (x5000). This figure also reports the elemental composition of L30-Ag2 sample based on the semi-quantitative EDS analysis on a particular region of sample seen as gray domain at the center of image. Higher magnification SEM images of composites specify the well dispersion of Ag@g-C₃N₄ due to the contributing effect of *L. orientalis* oil to particle dispersion as mentioned before.

It was found that the weight fraction of Ag was 0.82 in the L30-Ag2 sample regarding the EDS analysis. TGA analysis indicated that the g-C₃N₄ ratio of Ag@g-C₃N₄ was about 33.5 wt%. The remaining 66.5 wt% should be silver oxide (Ag₂O) due to easy formation of silver oxide by reacting of elemental silver (Ag) with oxygen above the 200 °C under oxidative atmosphere. Atomic composition of Ag₂O can be transformed into weight ratio; as 93 wt% of silver
<figure><img src="image_13.png" /><figcaption>**Fig. 6** SEM images of PLA (L0), L30-Ag4, and L0-Ag4</figcaption></figure>

and 7 wt% of oxygen. Considering the weight ratio of Ag₂O and TGA result, it can be determined that the 93 wt% of remaining amount (66.5 wt% as Ag₂O) should be Ag (about 62 wt%). The weight% of Ag@g-C₃N₄ in the L30-Ag2 sample must be about 1.5 wt% regarding the 2 part of additive in the total 132 part of sample (100+30+2) and 62 wt% of 1.5 wt% corresponds to 0.93 wt% of Ag. EDS analysis indicated the 0.82 wt% of Ag in this sample. The compositional deviation about 12% can be accepted within the reliable limits for such a semi-quantitative approach and this result confirms the compositional consistency of samples.

Figure 8 shows the representative images of contact angle measurements and the average contact angle values of samples are also listed in Table 2. Contact angle measurements were performed at least five different points on a specimen and the average values of left and right angles were reported with the standard deviations.

The contact angle value of neat PLA film was about 75 that is very consistent with the previously reported values [17, 38, 39]. Addition of 30 phr *L. orientalis* significantly increased the contact angle value of PLA. This increase clearly refers to a substantial improvement in hydrophilicity of PLA as the natural oil is composed of various aromatic and cyclic organic compounds as reported before [15]. It could be assumed that the improvement in hydrophilicity of PLA may reduce its hydrolytic decomposition or biodegradation rate and enhance the physical durability of PLA parts plasticized with *L. orientalis* oil. Introducing of functional additive, Ag@g-C₃N₄, into plasticized specimen decreased the contact angle value of L30. But, the contact angle values of all composites were still quite higher than that of PLA because the amount of *L. orientalis* oil was much higher than the amount of Ag@g-C₃N₄. It was also found that the addition of 4 phr Ag@g-C₃N₄ without natural oil did not change the contact angle value of PLA.

## Thermal Properties of PLA/Ag@g-C3N4 Composites

Figure 9(a-c) exhibit the DSC thermograms of samples recorded at first heating, cooling, and second heating cycles. The DSC parameters of samples calculated from corresponding thermograms are also listed in Table 3. In Fig. 9(a), it is seen that all samples exhibit single melting endotherms. During the first heating, none of the samples showed glass transition and cold crystallization peak clearly because all the samples were crystallized during solvent evaporation and solidification process. It is a well-known fact that the first heating run in DSC analysis of polymers is applied for deleting the thermal history of specimens thus excessive and speculative arguments should not be constructed on the first heating thermograms. But, it should be noted that the most interesting and obvious finding about the first heating thermograms is the fact that the melting endotherms of plasticized specimens significantly shift to lower temperatures, about 15 °C compared to the PLA and L30. This decrease can be attributed to plasticization effect of *L. orientalis* oil on PLA, well. Figure 9(b) indicates that none of the samples can crystallize during the relatively fast cooling rate (30 °C/min.). This result implied that all samples formed highly amorphous structure during the cooling step. The Tg values of unplasticized samples, L0 and L0-Ag4, were found to be 52.5 and 52.8 °C, respectively. But, the Tg values of plasticized samples varied in the range of 30–37 °C. This result
<figure><img src="image_14.png" /><figcaption>Fig. 7 SEM images of PLA (L0), L30, composites, and EDS results of L30-Ag2</figcaption></figure>

also marks the plasticization effect of *L. orientalis* oil as previously reported [15]. The most quantitative results about the effect of compositional variations on the thermal properties of semi-crystalline thermoplastic based systems can be extracted from the second heating thermograms because the thermal history of polymer chains and processing related microstructural features have been deleted. In Fig. 9(c), it is seen that all samples exhibit glass transition temperature, cold crystallization exotherm and melting endotherm. A

smaller, low temperature shoulder can also be noticed in the thermograms of L30-Ag2 and L30-Ag4 samples just before the main melting peak. During the second heating, $T_g$ values of unplasticized specimens, P0 (PLA) and L0-Ag4, were found to be 60.2 and 60.5 °C, respectively. Introducing 30 phr of *L. orientalis* oil into PLA reduced its $T_g$ value about 17 °C. Addition of Ag@g-C$_3$N$_4$ into the plasticized PLA resulted in further reduce in glass transition temperature.
<figure><img src="image_15.png" /><figcaption>**Fig. 8** Representative images for the contact angle measurements of samples</figcaption></figure>

<table><caption>**Table 2** Contact angle values of film samples</caption><thead><tr><th>Samples</th><th>Left</th><th>Right</th></tr></thead><tbody><tr><td>L0</td><td>75.3±1.30</td><td>74.7±0.73</td></tr><tr><td>L30</td><td>92.9±2.06</td><td>91.3±2.69</td></tr><tr><td>L30-Ag1</td><td>89.8±0.57</td><td>88.9±0.89</td></tr><tr><td>L30-Ag2</td><td>85.6±0.22</td><td>83.8±0.44</td></tr><tr><td>L30-Ag4</td><td>84.9±0.33</td><td>84.1±0.56</td></tr><tr><td>L0-Ag4</td><td>74.3±1.57</td><td>74.7±1.61</td></tr></tbody></table>

As seen in Fig. 9(c), PLA exhibited a cold crystallization behavior in the temperature range of 100–135 °C. Similar cold crystallization behavior was also observed for the L0-Ag4. Plasticization of PLA with the *L. orientalis* oil significantly decelerated the cold crystallization of PLA based on the following findings; (i) apparently broadening the cold crystallization exotherm, (ii) shifting the peak maximum temperature (T<sub>cc-peak</sub>) to higher value, and (iii) decreasing the intensity of crystallization exotherm and enthalpy value (ΔH<sub>cc</sub>). In the specific case of *L. orientalis* oil, Hasanoglu et al. explained this effect by the excellent distribution of aromatic and cyclic compounds of *L. orientalis* oil into the PLA phase and restriction effect of these organic molecules for

ordering of PLA chains [15]. It was found that introducing Ag@g-C<sub>3</sub>N<sub>4</sub> into the plasticized PLA with 30 phr of *L. orientalis* oil dramatically accelerated the cold crystallization behavior of PLA. Ag@g-C<sub>3</sub>N<sub>4</sub> addition led to decrease the crystallization peak onset and peak maximum temperature and increased the crystallization enthalpy values compared to L30. This result can be attributed to the characteristic nucleation effect of 2D solid materials on the phase transition of polymer chains due to the formation of physical network with solid particles and resulting restriction effect for the mobile PLA chains then easy folding into ordered structure. Although the crystallization mechanism of semi-crystalline polymers as the phase transition from unordered (or amorphous) state to ordered state is a well-known phenomenon, dynamics of cold crystallization is quite different from the most commonly discussed melt crystallization issue under non-isothermal conditions because chain folding proceeds with the increase in temperature during cold crystallization. In this transition, the effect of temperature and effect of solid additives on the chain mobility work oppositely. Therefore, it has been accepted that the cold crystallization is a more
<figure><img src="image_16.png" /><figcaption>**Fig. 9** (a) DSC first heating, (b) cooling, and (c) second heating thermograms of samples</figcaption></figure>

complex phenomenon than the melt crystallization. In this study, both the converse effects of plasticizer (*L. orientalis* oil) and nucleating agent (Ag@g-C₃N₄) on such a complex transition must be taken into account.

Consequently, it has been inferred that these functional additives do not induce the melt crystallization of PLA under fast cooling conditions. This result refers that any

<table><caption>**Table 3** DSC parameters of samples</caption><thead><tr><th rowspan="2">Samples</th><th colspan="2">1st heating</th><th colspan="2">cooling</th><th colspan="2">2nd heating</th><th rowspan="2">T<sub>cc-peak</sub> (°C)</th><th rowspan="2">ΔH<sub>cc</sub> (J/g)</th><th rowspan="2">T<sub>m</sub> (°C)</th><th rowspan="2">ΔH<sub>m</sub> (J/g)</th><th rowspan="2">X<sub>c</sub> (%)</th></tr><tr><th>T<sub>m</sub> (°C)</th><th>ΔH<sub>m</sub> (J/g)</th><th>T<sub>g</sub> (°C)</th><th>ΔH<sub>g</sub> (J/g)</th><th>T<sub>cc-onset</sub> (°C)</th><th>T<sub>cc-peak</sub> (°C)</th></tr></thead><tbody><tr><td>L0</td><td>174.2</td><td>38.0</td><td>52.5</td><td>60.2</td><td>101.6</td><td>116.1</td><td>41.2</td><td>174.5</td><td>41.5</td><td>44.3</td></tr><tr><td>L30</td><td>163.0</td><td>30.6</td><td>36.7</td><td>43.0</td><td>99.2</td><td>125.6</td><td>10.0</td><td>162.4</td><td>13</td><td>18.1</td></tr><tr><td>L30-Ag1</td><td>159.0</td><td>33.5</td><td>35.3</td><td>39.6</td><td>92.1</td><td>104.6</td><td>29.6</td><td>162.7</td><td>33.2</td><td>46.5</td></tr><tr><td>L30-Ag2</td><td>160.8</td><td>32.4</td><td>35.0</td><td>39.4</td><td>90.7</td><td>99.6</td><td>27.0</td><td>162.8</td><td>32.0</td><td>45.1</td></tr><tr><td>L30-Ag4</td><td>161.3</td><td>30.2</td><td>30.3</td><td>33.3</td><td>83.7</td><td>90.8</td><td>25.6</td><td>159.8</td><td>28.5</td><td>40.8</td></tr><tr><td>L0-Ag4</td><td>175.0</td><td>33.5</td><td>52.8</td><td>60.5</td><td>100.0</td><td>112.0</td><td>32.0</td><td>175.0</td><td>34.6</td><td>38.4</td></tr></tbody></table>

<figure><img src="image_17.png" /></figure>
of these compositions can be solidified in amorphous state with the melt state processing operations such as film blowing or injection molding. But, the composite samples may crystallize when they are exposed to relatively high temperatures about 80 °C. This phenomenon brings about the physical deformations such as shrinking and deflection on parts and changes their physical properties. From the realistic point of view, it can be concluded that a dominantly evident cold crystallization behavior might narrow down the application area and service conditions of PLA-based parts. As concluded for the melting endotherms recorded during the first heating run, it was obtained that the melting temperatures of plasticized samples (L30, L30-Ag1, L30-Ag2, and L30-Ag4) were lower than unplasticized ones (L0 and L0-Ag4) during second heating. Low melting temperature of semi-crystalline polymers can be related to two phenomena; (i) smaller crystal size formed with the thinner lamellae and lower degree of crystallinity values (Xc, %) and/or (ii) lower interactions between chains in folded and ordered domains. Comparing the Xc values of composite samples, calculated with the Eq. 1 and listed in Table 3, it is seen that their degree of crystallinity values are not lower than the L0 and L0-Ag4. This result implies that lower melting temperatures of such samples are obviously originated from the latter case. *L. orientalis* oil can effectively plasticize PLA by reducing the strong interactions and hydrogen bonds between PLA chains and lead to transform easier from ordered solid to melt state than the unplasticized counterparts. This relationship may allow us to expect another role from *L. orientalis* oil as processing aid in melt compounding operations in addition to aforementioned ones; plasticizer and dispersion agent.

## Dynamic Mechanical Analysis of PLA/Ag@g-C3N4 Composites

Figure 10(a) shows the storage modulus (E') curves of samples as a function of temperature. As the DMA analyses were performed with the specimens taken from the solution casted samples seen in Fig. 4, DMA thermograms directly reflected the solid state viscoelastic and mechanical properties of samples.

Cold crystallization phenomenon was not observed in the samples because they were already crystallized during solvent evaporation and solidification step. Their Xc% values, calculated with the enthalpy values of samples recorded at first heating and listed in Table 3, were found to be in the range of 40.6–46.9. All the compositional variations yielded a relatively small difference in Xc%. This result pointed out that variation in the viscoelastic and mechanical properties of samples was not related to their different degree of crystallinity values. As these samples do not show cold

crystallization behavior, E' values of samples can be compared at rubbery plateau. Figure 10(b) illustrates the comparison of E' values at 100 °C as a function of compositional variation. It is seen that the addition of 30 phr plasticizer decreases the E' of PLA while the addition of 4 phr Ag@g-C3N4 without plasticizer increased the modulus. In the plasticized samples, increasing amount of Ag@g-C3N4 readily improves the modulus, as expected.

## Antibacterial Properties of PLA/Ag@g-C3N4 Composites

Antibacterial activity results of samples on *A. baumannii* ATCC BAA 747, *E. coli* ATCC 25,922, and *S. aureus* ATCC 25,923 are given in Table 4. Additionally, inhibition zones are also shown in Fig. 11.

The antibacterial activity of samples was evaluated using the disk diffusion method. The study revealed that PLA and L30 films did not show any antibacterial activity. However, the unplasticized sample including 4 phr of antibacterial agent (L0-Ag4) exhibited significant antibacterial activity against all types of bacteria tested. The L0-Ag4 film created growth inhibition zones of 11, 11, and 13 mm in diameter on *A. baumannii*, *E. coli*, and *S. aureus* bacteria, respectively. It is worth noting that silver is a well-known antibacterial agent and is widely used in various fields, such as food packaging, medical materials, devices, and cosmetics [31, 40, 41].

It was interestingly found that the L30-Ag1, L30-Ag2, and L30-Ag4 samples showed the same or slightly higher antibacterial activity than L0-Ag4 for *A. baumannii* and *S. aureus*. A similar trend was found for *E. coli*, except the L30-Ag2. Similar or higher antibacterial activity of composites even though lower amount of Ag was probably due to the dispersing agent effect of *L. orientalis* oil as mentioned before based on the SEM images. It can be concluded that the natural plasticizer contributed to antibacterial agent for dispersing well into PLA structure and yielded higher antibacterial activity. L30-Ag1 film resulted in an inhibition zone of 14, 12, and 12 mm against *S. aureus*, *E. coli*, and *A. baumannii* bacteria, respectively. The inhibition zone of L30-Ag2 film against *S. aureus*, *A. baumannii* and *E. coli* bacteria was found to be 13, 11 and 7 mm, respectively. The inhibition zone of the L30-Ag4 film against *S. aureus*, *E. coli* and *A. baumannii* bacteria was found to be 14, 13, and 11 mm respectively.

According to the results, only *L. orientalis* added and both plasticizer and antibacterial agent loaded samples are more effective on G (+) bacteria than G (-) bacteria. This situation is consistent with the literature [42–45]. G (-) bacteria have lipopolysaccharide (LPS) layers in their cell walls.
<figure><img src="image_18.png" /><figcaption>**Fig. 10** (a) Storage modulus (E') curves of samples as a function of temperature and (b) E' values of samples at a particular temperature in rubbery region</figcaption></figure>
<table><caption>**Table 4** Antibacterial activity of samples against pathogenic microorganisms</caption><thead><tr><th rowspan="2">Samples</th><th colspan="3">Inhibition Zone (mm)</th></tr><tr><th><i>A. baumannii</i></th><th><i>E. coli</i></th><th><i>S. aureus</i></th></tr></thead><tbody><tr><td>L0 (PLA)</td><td>0</td><td>0</td><td>0</td></tr><tr><td>L30</td><td>0</td><td>0</td><td>0</td></tr><tr><td>L30-Ag1</td><td>12</td><td>12</td><td>14</td></tr><tr><td>L30-Ag2</td><td>11</td><td>7</td><td>13</td></tr><tr><td>L30-Ag4</td><td>11</td><td>13</td><td>14</td></tr><tr><td>L0-Ag4</td><td>11</td><td>11</td><td>13</td></tr><tr><td>AMC<sup>a</sup></td><td>19</td><td>-</td><td>-</td></tr><tr><td>AMC<sup>a</sup></td><td>-</td><td>35</td><td>-</td></tr><tr><td>VA<sup>b</sup></td><td>-</td><td>-</td><td>17</td></tr></tbody></table>

plasticized the PLA and significantly reduced its glass transition temperature. *L. orientalis* oil also acted as an agent to improve the dispersion of nanocomposite particles into the PLA matrix and enhanced the antibacterial activity of the Ag@g-C₃N₄ nanocomposite. It can be concluded that a PLA-based nanocomposite, possessing a composition of PLA/L. orientalis oil/Ag@g-C₃N₄ (100/30/2), could be successfully used in various engineering applications that requires antibacterial and biodegradable plastic parts such as food packaging films, medical materials like tissue scaffolds, textile and automotive plastics etc.

This lipopolysaccharide and protein part are more resistant to EOs [46].

Nowadays, a significant increase has been observed in foodborne diseases. This condition is often associated with raw food consumption. It is known that the bacteria used in this study cause foodborne infections. A. baumannii is associated with multidrug resistance (MDR) and has been reported to be detected in animal foods and vegetables [47, 48]. It has been concluded that our findings on the antibacterial activity against A. baumannii make a significant contribution to the existing literature. It was also observed that the L0 (PLA) and L30 films did not exhibit any antibacterial activity on S. aureus, A. baumannii, and E. coli bacteria. However, the literature suggests that L. orientalis extracts have antibacterial activity [49–53]. Another crucial point of the current study is the fact that the introducing the combinations of L. orientalis oil and 2D designed antibacterial agent (Ag@g-C3N4) into PLA showed better antibacterial activity than the only antibacterial agent loaded film (L0-Ag4). This indicates that the synergy between silver and L. orientalis extract enhances the antibacterial activity.

## Conclusions

This study reports the structure-property relationships of flexible antibacterial PLA-based films formulated by using *L. orientalis* oil as a bio-based and sustainable plasticizer harvested from an endemic Anatolian sweetgum tree. Additionally, composite films containing PLA and 2D g-C₃N₄ sheets decorated with Ag nanoparticles were prepared. It has been shown that Ag nanoparticles with an average diameter of 10–30 nm were synthesized by in-situ chemical reduction onto the 2D nano sheets. Then a functional additive, Ag@g-C₃N₄ nanocomposite, was successfully obtained and used as antibacterial agent into a thermoplastic matrix. Furthermore, it has been shown that *L. orientalis* oil

<footnote><sup>a</sup> AMC: amoxicillin-clavulanate (20/10 µg/ml)</footnote>

<footnote><sup>b</sup> VA: vancomycin (30 µg/ml)</footnote>
<table><caption>**Fig. 11** Photographs of inhibition zones of samples and antibiotics as the control samples against three different pathogenic microorganisms</caption><thead><tr><th>Samples</th><th>A. baumannii</th><th>E. coli</th><th>S. aureus</th></tr></thead><tbody><tr><td>L0 (PLA)</td><td></td><td></td><td></td></tr><tr><td>L30</td><td></td><td></td><td></td></tr><tr><td>L30-Ag1</td><td></td><td></td><td></td></tr><tr><td>L30-Ag2</td><td></td><td></td><td></td></tr><tr><td>L30-Ag4</td><td></td><td></td><td></td></tr><tr><td>L0-Ag4</td><td></td><td></td><td></td></tr></tbody></table>

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**Acknowledgements** Financial supports received from the MLU Halle-Wittenberg and from the BMBF (Grant number FKZ:03Z22HN11) are greatly acknowledged.

**Author Contributions** Author 1 tooks part in “Conceptualization”, “Data Curation”, “Investigation”, “Methodology in material synthesis work” and “Writing – original draft” Authors 2, 3, and 4 made “Investigation in characterization/analysis studies”, “Formal analysis”, and “Writing – original draft” Author 5 was responsible for “Funding acquisition”, “Project administration” and “Writing – review &

editingAuthor 6 (corresponding author) made “reviewing and editing original draft”, “Supervision”, and “Conceptualization”.

**Funding** Open access funding provided by the Scientific and Technological Research Council of Türkiye (TÜBİTAK).

**Data Availability** No datasets were generated or analysed during the current study.

<figure><img src="image_37.png" /></figure>
## Declarations

**Conflict of interest** There are no conflicts to declare.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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