Top-down fabrication of biodegradable multilayer tunicate cellulose films with controlled mechanical properties

Owing to non-degradable waste plastics causing serious environmental pollution, the development of natural polymeric materials has become an important topic. Herein, multilayer tunicate cellulose films (MTCFs) were fabricated by a top-down approach, where the alkali treated mantles of tunicate were layer-by-layer hot-pressed. Strong hydrogen bonds that formed between tunicate cellulose layers resulted in good interfacial compatibility of multilayer films. The as-prepared MTCFs exhibited improved thermal stabilities, excellent mechanical properties, and good degradability in natural soil. This work provided a top-down approach for the preparation of biodegradable multilayer films as packaging materials.


Introduction
Owing to their low cost and high performance, traditional plastics derived from petroleum such as polyethylene (PE), polypropylene (PP), polyvinylchloride (PVC), and polyethylene terephthalate (PET) have been increasingly used as packaging materials (Geyer et al. 2017;Siracusa et al. 2008). However, the tremendous consumption of plastics has led to the production of a large amount of plastic waste, which has triggered global environmental problems ). In the marine environment, the accumulation of plastic debris has become a major global issue which seriously threatened ecological environment, food safety, and human health (Barboza et al. 2018). For sustainable development, it is urgent to seek biodegradable materials to replace petroleum-based plastics (Mohanty et al. 2018). Therefore, natural polymers have been widely concerned for the construction of environmentally friendly materials, due to their abundant, low cost, safety, and biodegradability (Klemm et al. 2005).
Cellulose is the quintessential sustainable resource and the most abundant natural polymer on the earth, and has various applications from energy to multifunctional materials in the society (Cheng et al. 2016;Peng et al. 2020). Transparent films composed of cellulose nanofibers that formed in aqueous solution exhibit high transparency, mechanical flexibility, high strength, excellent thermal stability, non-toxicity, outstanding printability, and biodegradability (Ye et al. 2019). This bottom-up technique for the construction of cellulose-based materials usually requires a complicated preparation process. Recently, a top-down strategy has been employed to create functional, widely used, materials that are cellulosebased in building materials, flexible electronics, packaging, and electronics storage devices (Gao et al. 2016;Hu et al. 2018;Jia et al. 2017;Jiang et al. 2018;Jung et al. 2015;Koppolu et al. 2019;Li et al. 2016Li et al. , 2018Li et al. , 2019Wang et al. 2017Wang et al. , 2018Zhu et al. 2016aZhu et al. , 2016bZhu et al. , 2017Zhu et al. , 2018. For example, isotropic paper obtained from anisotropic wood through a top-down approach with high transmittance and high haze is an excellent substrate for disposable electronic and optical devices . To develop functional materials, scientists have filled the delignified wood with polymers, such as polyvinylpyrrolidone and epoxy resin, so these composite materials have good light management effects and can improve the efficiency of solar cells and serve as energy efficient building materials (Li et al. 2016;Zhu et al. 2016b). However, the fabrication of woodderived cellulose materials may require delignification or other processing steps, because the tight association of cellulose, hemicellulose, and lignin can make it difficult to selectively remove lignin from the wood via simple chemical treatment (Keplinger et al. 2020). Unlike wood, tunicate is a kind of sea animal with unique integumentary tissue (tunic) that is composed of crystalline cellulose embedded with protein, lipids, sulfated glycan and mucopolysaccharides (Quero et al. 2018;Zhao and Li 2014). After removal of protein and other components, tunicate cellulose exhibits larger molecular weight, higher crystallinity (I b ), better thermal stability, and higher modulus (143 GPa) in comparison with plant cellulose (Cheng et al. 2017(Cheng et al. , 2018Zhan et al. 2018a, b;Zhan et al. 2018a, b). To the best of our knowledge, tunicate cellulose films with good tensile strength and toughness prepared from purified tunics are scarcely reported.
Herein, we developed a top-down approach to fabricate transparent tunicate cellulose-based films. Firstly, tunicate cellulose films were obtained by alkaline treatment of the tunics from Ciona intestinalis, according to the reported method . Secondly, multilayer tunicate cellulose films (MTCFs) were prepared by layer-by-layer hot pressing of above tunicate cellulose films. This top-down strategy is a simple, efficient, and environmentally friendly pathway to prepare high performance cellulose films. The structure and morphology of multilayer tunicate cellulose films (MTCFs) were characterized by using Fourier transfer infrared spectroscopy (FTIR) and scanning electron micrograph (SEM) measurements. The transparency and mechanical properties of samples were investigated by UV-Vis spectrophotometer and universal testing machine. Moreover, the biodegradation of multilayer films was also studied by soil burial method.

Materials
Tunicate (Ciona intestinalis) was purchased from Weihai Evergreen Marine science and technology Co. Ltd (Shandong, China) and used as raw material. The microcrystalline cellulose (DP: 15-375, particle size: 50 lm) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd (Shanghai, China) and used as a comparative sample. All other reagents were analytical grade purchased from Shanghai Chemical Agents Co. Ltd (Shanghai, China) and used without further purification.

Fabrication of multilayer films (MTCFs)
The tunics were obtained by removing the internal organs from tunicates (Ciona intestinalis) with a scalpel. The wet tunics were rinsed three times with deionized water and immersed in NaOH solution (5% w/v) at 80°C for 3 h . After treatment, proteins, lipids and sediment were removed and the films remained intact without dispersing into individual fiber. Finally, tunicate cellulose films were obtained by rinsing the films three times with deionized water.
Multilayer tunicate cellulose films (MTCFs) were prepared by layer-by-layer hot pressing of above tunicate cellulose films. A tunicate cellulose film was put between two Teflon films at 100°C under a pressure of about 5 MPa for 1 h to obtain single layer tunicate cellulose film (MTCF-1). Then MTCF-1 was wetted and superimposed with another tunicate cellulose film and repeated the above operation to obtain MTCF-2. By analogy, a series of samples, including MTCF-4, MTCF-8, MTCF-10, and MTCF-12, were fabricated, respectively.

Characterization
Fourier transfer infrared spectroscopy (FTIR) was carried out using a NICOLET 5700 FTIR spectrometer (Thermo Fisher Scientific, USA), the samples were ground to powders and placed in a 40°C vacuum oven for 12 h and tested via a KBr method. Scanning electron micrograph (SEM) measurements were taken with the field emission scanning electron microscopy (FESEM, Zeiss, Germany) at an accelerating voltage of 8 kV. The mechanical properties of multilayer tunicate cellulose films (MTCFs) were carried out using a universal material testing machine with a 1000 N load cell (Instron 5967, USA) at room temperature with a tensile speed of 5 mm min -1 . The optical transmittance of films was measured using Evolution 201 UV-Vis spectrometer (Thermo Fisher Scientific, USA). Thermal gravimetric analysis was conducted using a Q500 (TA instrument, USA) under air at a heating rate of 10°C/min from 80 to 600°C. Wide-angle X-ray diffraction (WXRD) measurement was carried out on an XRD diffractometer (XPertPro, Panalytical, Netherlands). The crystallinity index (CrI) of the samples is caculated from the equation as follows: where I 200 is the intensity of the (200) peak and I am is the minimum intensity between the (110) and the (200) peak (French and Santiago Cintrón 2013;French 2014French , 2020Ling et al. 2019).

Biodegradation test
Multilayer films (5 9 4 cm 2 ) were buried about 20 cm beneath in natural soil. The average temperature and moisture of the soil were about 25°C and 35%, respectively. The degraded samples were carefully taken out from soil each week, rinsed 3 times with deionized water, and dried at 25°C for 2 days under vacuum before photograph. The weight loss of samples degraded in soil at one-week intervals was measured for 12 weeks. SEM measurements of samples were conducted after degradation for 3 weeks.

Results and discussion
Preparation and structure of MTCFs Tunicate is well known as the only animal source of cellulose in the world and tunicate cellulose is superior to plant cellulose in terms of molecular weight and crystallinity . Therefore, tunicate cellulose nanocrystals with high aspect ratio and elastic modulus could be used as reinforcements to effectively improve the mechanical performance of polymeric materials ). Here, multilayer tunicate cellulose films were fabricated by a top-down approach, as shown in Fig. 1. First, tunic was separated from Ciona intestijnalis, and tunicate cellulose films were obtained by alkaline treatment to remove proteins, lipids, and sediment. Then, multilayer tunicate cellulose films were obtained by hotpressing of tunicate cellulose films. To confirm the effectiveness of alkaline treatment, the FTIR spectra of microcrystalline cellulose, tunic before and after alkaline treatments are compared in Fig. S1. The characteristic peaks at 1240 cm -1 and 1540 cm -1 appeared in the spectrum of tunic which could be assigned to the C-O stretching of lipids and N-H stretching from amide groups of proteins, respectively. For tunicate cellulose film, only the typical peaks of cellulose could be observed, such as the absorption peaks at 3420 cm -1 and 2900 cm -1 corresponding to -OH and C-H stretching, respectively; the peaks at 1161 cm -1 and 1112 cm -1 were attributed to the C-O anti-symmetric bridge stretching and the C-OH skeletal vibration, respectively; the peaks at 1057 cm -1 and 1032 cm -1 correspond with the C-O-C pyranose ring skeletal vibration, and the small peak at 895 cm -1 originated from the glycosidic -CH deformation with a ring vibration and -OH bending, which was characteristic of b-glycosidic linkages between glucoses in cellulose (Nakashima et al. 2008).
These results indicated that the lipids and proteins had been completely removed from tunic and tunicate cellulose film was successfully fabricated. After removal of protein and lipid, only tunicate cellulose was left in the form of nanofibers (Fig. S2).
The interaction between the film layers in the films was characterized by FTIR spectra. As shown in Fig. S3, we use the intensity of absorption peak at 2900 cm -1 corresponding to C-H stretching to normalize all the curves, large absorption bands at 3420 cm -1 were observed in the spectra of all samples, which could be attributed to -OH stretching. With the increase of layer number, these characteristic peaks become broader, revealing that strong hydrogen bonds formed between the layers during hot pressing process. However, the intensity of characteristic peaks decreased with increasing layers. This result indicated that the hydrogen bonds in the layer of sample weakened during the hot-pressing process. The WXRD patterns and crystallinity index of tunic and tunicate cellulose film are shown in Fig. S4. Four main diffraction peaks located at 14.7°, 16.5°, 22.8°and 26.7°could be observed in the WXRD patterns of tunic (Fig. S4a), reflecting the crystalline peaks of cellulose and the residual sand grain (Quero et al. 2018). For tunicate cellulose film, only three typical crystalline peaks at 14.7°, 16.5°and 22.8°were observed, corresponding to the (110), (110), and (200) crystal planes of cellulose I, respectively (Zhao and Li 2014). Moreover, upon applying the alkaline treatment, the crystallinity index increased from 55.6% for tunic to 85.0% for tunicate cellulose film, indicating that the lipids and proteins have been completely removed from tunic (Fig. S4b). These results were consistent with results of FTIR, revealing that only tunicate cellulose was retained after alkaline treatment.
Moreover, the morphologies of the cross section of multilayer tunicate films were investigated, as shown in Fig. 2. MTCF-1 exhibited uniform and compact morphology because slow evaporation of water during hot pressing caused the reassembly of tunicate cellulose nanofibers and the formation of dense structure (Fig. 2a). As the number of layers increased, no obvious boundary could be observed in the cross section of MTCF samples, indicating their good interfacial compatibility (Fig. 2b-d). However, MTCF-12 had the layered structure in its crosssection, demonstrating that the reassembly of hydrogen bonds was limited by evaporating too much water during hot pressing (Fig. 2e). The thickness of samples linearly raised with the increase of the number of layers from 4.65 to 62.3 lm (Fig. 2f).

Mechanical, optical, thermal and degradable property of MTCFs
The mechanical properties of materials are significantly dependent on its morphology. The tensile strength and toughness of multilayer tunicate cellulose films are shown in Fig. 3. The tensile strength and elongation at break of MTCF-1 were 41.8 MPa and 1.31%, respectively, indicating its high stiffness and rigidity. Interestingly, both tensile strength and elongation at break of samples increased as the numbers of layers in the films. For MTCF-8, the tensile strength reached 140.3 MPa that was 3.4 times of MTCF-1, while the fracture strain was as high as 20.8% that was 15.9 times of MTCF-1 (Fig. 3a). The toughness of MTCF-8 was 13.2 MJ m -3 , which was the highest value of all samples. The improved mechanical properties of films would benefit and widen their application in packaging field ). However, the mechanical property of MTCF-12 was sharply decreased (Fig. 3b), which could be attributed to the defects in its layered structure. This results revealed that the layers with excellent interfacial compatibility could share the mechanical load, resulting in higher tensile strength. On the other hand, the sliding between the layers in the films led to the rearrangement of hydrogen bonds and the improvement of their break at elongation, leading to their higher toughness (Fig. S5a). From the SEM images for fracture surface of MTCFs ( Fig. S5b-g), it can be seen that these samples with good interfacial compatibility (from MTCF-1 to MTCF-8) showed flat fracture with tunicate cellulose nanofibers that had been pulled out, whereas MTCF-12 exhibited apparent defects in its fracture surface.
For packaging application, the optical property of film is also an important parameter. Figure 4a displays the optical transmittance of MTCF samples from 400 to 800 nm. MTCF-1 had the highest transmittance, but the transparency of films decreased as the increase of the number of layers. The transmittance of MTCF samples descended from 83.6 to 62.8% at wavelength of 550 nm, when the number of layers increased from 1 to 12 (Fig. 4b). Light reflection and scattering occurred in the interfaces of films, resulting in the more layers had in the films the lower transparency was. We also compared the thickness and transparency of MTCFs with wood-based materials (Table S1). The transparency of MTCFs was slightly lower than that of transparent wood with similar thicknesses. In future work, it may be a feasible method to add suitable polymers to increase its transparency (Jacucci et al. 2020).
Furthermore, the thermal stabilities of MTCFs were also studied and compared. Figure 4c shows the thermogravimetric analysis (TGA) curves of MTCFs. Two steps of active weight loss of all samples could be observed with the elevating temperature. The main decomposition of all films into small molecules and condensable volatile macromolecules occurred from 200 to 350°C. The second step at 350-500°C could be assigned to the further decomposition of residues. After 500°C, all samples were completely decomposed in air and reached constant weights. To investigate the temperature of maximum decomposition rates, DTG curves of samples are shown in Fig. 4d. For the first decomposition step, the T d1 values of all samples were 283, 295, 298, 300, and 301°C for MTCF-1, MTCF-2, MTCF-4, MTCF-6, MTCF-8, respectively. These results demonstrated that the hydrogen bonding interaction between tunicate cellulose layers slightly enhanced the thermal stability of samples (Song et al. 2015). The T d1 of MTCF-12 decreased to 298°C could be attributed to the presence of defects in the film. In the second decomposition step, T d2 values of samples were 419, 429, 430, 438 and 442°C, for MTCF-1, MTCF-2, MTCF-4, MTCF-6, MTCF-8, respectively, suggesting similar trend to the first stage. Due to its poor interfacial compatibility, MTCF-12 had a T d2 of 437°C. These results indicated that strong hydrogen bonds between the tunicate cellulose layers improved their interfacial compatibility, resulting in good thermal stabilities of samples.
In the perspective of environmental protection, biodegradation of films is key factor for their application as packaging materials. Figure 5a shows the photographs of various MTCFs after biodegradation in natural soil for different times. With the extension of degradation time, the size of the sample decreased gradually and finally disappeared completely. The biodegradation rate of samples depended on the number of tunicate cellulose layers in the films. For example, MTCF-1 completely degraded within 5 weeks but MTCF-12 needed 12 weeks (Fig. 5b). The weeks for complete degradation of various MTCF samples are shown in Fig. 5c. The complete degradation time are 5, 5, 7, 9, 11 and 12 weeks, for MTCF-1, MTCF-2, MTCF-4, MTCF-8, MTCF-10, and MTCF-12, respectively. When the samples had same area, it took longer time to degrade the thicker sample. To understand the degradation process, the SEM images of samples after being buried in the soil for 3 weeks are shown in Fig. 5d. The porous morphology of samples indicated that microorganisms in the soil obtained nutrients by penetrating the surface of MTCFs and directly decompose tunicate cellulose. These results indicated that all samples were Fig. 5 Biodegradation of MTCFs: a digital pictures, b weight loss vs. degradation time, c time for complete degradation and d SEM images for the surface of MTCF samples from MTCF-1 to MTCF-12 after degrading for 3 weeks biodegradable, which fulfilled the environmental concern as potential packaging materials.

Conclusion
We have successfully fabricated transparent and biodegradable multilayer films by hot pressing of tunicate cellulose film layers. This top-down approach efficiently promoted the formation of hydrogen bonds between two layers that was composed of tunicate cellulose nanofibers. The strong hydrogen bonding interactions offered good interfacial compatibility of materials, resulting in their improved thermal stabilities and excellent mechanical properties. Moreover, these resultant multilayer films could be completely degraded by microorganism in natural soil, and the biodegradation rate was dependence of the thickness of films. This work provided a top-down approach to manufacture degradable multilayer films from Ciona intestijnalis, which could be used as potential packaging materials instead of traditional plastics.

Supporting Information
FTIR spectra of pure cellulose, tunicate cellulose film and tunic; SEM images of the tunicate cellulose film; FTIR spectra of MTCF-1, MTCF-2, MTCF-4, MTCF-6, MTCF-8 and MTCF-12; XRD patterns and crystallinity index of tunic and tunicate cellulose film; Mechanism diagram of toughness enhancement and SEM images for fracture surface of films.