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A top-down approach to produce protein-functionalized and highly thermally stable cellulose fibrils Franck Quero, Genesis Opazo, Yadong Zhao, Aymeric FeschotteParazon, Jeimy Fernández, Abraham Mackenna, and Marcos Flores Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00831 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 15, 2018

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A top-down approach to produce proteinfunctionalized and highly thermally stable cellulose fibrils Franck Quero1*, Genesis Opazo1, Yadong Zhao2*, Aymeric Feschotte-Parazon1, Jeimy Fernandez1, Abraham Mackenna1 and Marcos Flores3 1. Laboratorio de Nanocelulosa y Biomateriales, Departamento de Ingeniería Química, Biotecnología y Materiales, Facultad de Ciencias Físicas y Matemáticas, Universidad de Chile, Beauchef 851, Santiago, Chile. 2. Department of Fibre and Polymer Technology, KTH, Royal Institute of Technology, Teknikringen 56-58, 100 44 Stockholm, Sweden. 3. Laboratorio de Superficies y Nanomateriales, Departamento de Física, Facultad de Ciencias Físicas y Matemáticas, Universidad de Chile, Beauchef 850, Santiago, Chile. (*)

Corresponding Authors. F. Quero, Email: [email protected] and Y. Zhao E-mail:

[email protected].

KEYWORDS: Cellulose fibrils; top-down approach; proteins; tunicate; Pyura Chilensis; thermal stability.

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ABSTRACT

Protein-functionalized cellulose fibrils, having various amounts of covalently bonded proteins at their surface, were successfully extracted from the tunic of Pyura Chilensis tunicates using successive alkaline extractions. Pure cellulose fibrils were also obtained by further bleaching and were used as reference material. Extraction yields of protein-functionalized cellulose fibrils were within the range of 62-76% by weight based on the dry initial tunic powder. Fourier-transform infrared and Raman spectroscopy confirmed the preservation of residual protein at the surface of cellulose fibrils, which was then quantified by X-ray photoelectron spectroscopy. The proteinfunctionalized cellulose fibrils were found to have relatively high crystallinity and their cellulose I crystalline structure was preserved upon applying alkaline treatments. The extracted cellulosic materials were found to be constituted of fibrils having a ribbon-like morphology with widths ranging from ~30 nm up to ~400 nm. These protein-functionalized cellulose fibrils were found to have outstanding thermal stability with one of them having onset and peak degradation temperatures of ~350 °C and 374 °C, respectively. These values were found to be 24 and 41 °C higher than for bleached cellulose.

INTRODUCTION Materials obtained from renewable resources are currently attracting much attention owing to their sustainability among other characteristics they intrinsically possess.1,2 Cellulose belongs to this class of materials since it is a natural material that is obtained from renewable resources.3 It is commonly extracted from higher plant sources, such as wood,4 but other sources of cellulose


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including agricultural wastes,5 algae,4,6,7 bacteria,8 and sea animals called tunicate9,10 have also been reported. Cellulose obtained from tunicate sources has, to date, mainly been studied in the form of cellulose nanocrystals.11-17 Studies that focus on cellulose fibrils obtained from tunicates are, however, scarce with only a few works recently reported.10,18-20 For most of cellulose sources (including tunicate), cellulose fibrils are extracted from hierarchical structures that contain these fibrils, which are embedded in a natural polymer matrix material.20 Depending on the source and extraction conditions, the obtained cellulose fibrils show typically a width within the nano-micro scale. The width of these fibrils can be further tuned by adjusting processing methods.21 In order to be applied for a wider range of applications, the functionalization of cellulose fibrils has been proposed as a way to improve their properties.3,22 Proteins are biological macromolecules that have been utilized to functionalize the surface of cellulose and provide new surface characteristics.23-25 The methods that have been proposed have to date, considered only bottom-up approaches. These methods consist in using a highly pure source of cellulose and then immobilized proteins at the surface of cellulose. This can be done by chemical covalent attachment, biochemical affinity or physical adosption.25,26 Highly stable attachment of protein to the surface of cellulose fibrils can be expected by forming a covalent bond between both biomaterials. The formation of covalent bonds is, however, not always straightforward and requires tedious efforts involving first, the chemical modification of cellulose and secondly the covalent attachment of proteins to the modified cellulose.26,27 Another drawback of this approach is that a high number of toxic chemicals are necessary to modify the cellulose and then covalently link proteins to cellulose fibrils. Consequently, the search for approaches that use less steps involving toxic chemicals is necessary.27


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The covalent attachment of proteins at the surface of cellulose fibrils can potentially provide bioactive properties25, but also new chemical functionalities that do not possess cellulose owing to the presence of amine (-NH2) and carboxylic acid (-COOH) functional groups within the molecular structure of proteins. Another strategy, as opposed to bottom-up approaches, is to extract cellulose fibrils from sources that naturally contain proteins. In the present study, this method is referred to as a top-down approach. Recently, protein-functionalized cellulose fibrils have been successfully isolated from green macroalgae that initially contained a relatively low protein content of ~5%.6 These fibrils were subsequently used as building blocks to fabricate high performance cellulose nanopapers and unusually high thermal stability were reported.6 This work, however, is the only example of top-down approach that has been reported to date to obtain protein-functionalized cellulose fibrils. Tunicate is a source of cellulose fibrils that is naturally associated to high amounts of proteins. In these sea animals, cellulose fibrils can be found in their outer shell referred to as tunic. Tunics have much more abundant protein (~15-70% depending on the tunicate species) than macroalgae.10,18,28,29 As a result, the use of tunics could potentially allow obtaining cellulose fibrils with a wider range of protein content at their surface. Another interesting characteristics of tunics is that their proteins have been found to be naturally covalently bonded to cellulose fibrils through serine amino acids.30 This could provide high level of protein immobilization at the surface of cellulose fibrils. Also, the amino acid composition of proteins extracted from macroalgaes31 and microalgae32 is different from those of tunicates,18 indicating the presence of different protein types in these two aquatic organisms. This means that cellulose fibrils having residual proteins extracted from tunicates could potentially provide different bioactive properties to the ones obtained from macroalgae.


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These protein-functionalized cellulose fibrils could present attractive features due to their numerous potential applications including energy-related applications33-35 and could serve as building block to design high performance and renewable materials including cellulose nanopapers36,37, aerogels38 and composite materials.19,39 As a result, this study aims at extracting, for the first time, protein-functionalized cellulose fibrils using a top-down approach from a tunicate cellulose source referred to as Pyura Chilensis. This tunicate can be found exclusively along the southern Peruvian coasts and all along the Chilean coasts. Its current economical exploitation by the southern local seafood industry generates high amount of biological wastes (tunics). A way to manage this biomass could be through high value revalorization.

MATERIALS AND METHODS Materials and Reagents All chemicals, including sodium hydroxide and sodium hypochlorite, were of analytical grade and purchased from either Merck (Germany) and Sigma Aldrich (USA). Paper filters discs having a grammage of 84 g/m2 and retention of 12-15 µm were purchased from MunktellAhlstrom (Sweden) and were used whenever vacuum filtration was needed.

Chemical Isolation of Cellulosic Materials from the Initial Tunic Powder


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First of all, Pyura Chilensis tunicates were washed with fresh water to remove part of fooling organisms including algae and sand grains that were present at their surface. These were then opened using a sharp knife and the internal organs were removed and separated from the tunics. The tunics were subsequently cut into pieces of ~1 cm2 using a pair of sharp scissors. The pieces were then subjected to three successive alkaline pre-treatments at ambient temperature using an aqueous KOH solution at 5% w/v, to wash away the remaining fouling organisms and sand grains present on the tunics’ surface. In between each pre-treatment, the tunics were washed with distilled water. After that, the tunics were frozen at -18 °C for 48 h and lyophilized using a freeze dryer (Christ Alpha 1-2 LDplus, Germany) for 48 h at a temperature of ~-55 °C. The freeze-dried tunics were then immersed into liquid nitrogen for 10 min and subjected to a knife-milling process for 15 min at a speed of 5000 rpm (Retsch Grindomix GM200, Germany) to convert the dried pieces of tunics into a powder. The powder was then sieved and the fraction having a particle size < 200 µm was selected to carry out the alkaline extraction process. As shown in Figure 1, this initial tunic powder is referred to as PI.


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Figure 1. (a) Scheme showing the successive steps involved in the alkaline extractions of protein-functionalized cellulose fibrils (H1, H2, H3) and bleached cellulose (B) from the initial tunic powder (PI). (b) Digital photos showing the appearances of the initial tunic powder (PI) and the isolated cellulosic materials (H1, H2, H3, B). The black line is a scale and represents a dimension of 1 cm.


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Figure 1a reports a schematic representation of the three-step alkaline extraction process that was applied to PI. These were performed using an aqueous solution of KOH at 5% w/v at 85 °C for 24 h. The extraction was performed in a temperature-controlled bath with a reflux column to keep constant volume and concentration of the KOH solution. In-between each extraction step, some materials were removed for characterization. The materials obtained after the first, second and third extraction step are respectively referred to as H1, H2 and H3, as shown in Figure 1a. In-between each alkaline extraction, the materials were carefully vacuum-filtered using a pump (Rocker 400, Taiwan) and washed using subsequently distilled water, acetone and again distilled water. The materials were finally freeze-dried for 48 h at a temperature of ~-55 °C. The bleaching step was performed by adding H3 materials to a 100 mL aqueous solution containing sodium hypochlorite (>4% chlorine, ~670 µL) and acetic acid (~330 µL) for 24 h at 80 °C. The material obtained after the bleaching step is referred to as B (bleached cellulose), which was used as reference material for comparison with PI, H1, H2 and H3 materials. Finally, B was freezedried for 48 h at a temperature of ~-55 °C and stored in a desiccator until further use.

Gravimetric Yield Estimation The yield after each extraction step was obtained by using a gravimetric method. After the extraction of each cellulosic material, they were washed with distilled water, acetone and again water, freeze-dried for 48 h at a temperature of ~-55 °C and finally weighed using a precision scale (Shimadzu AUX120, Japan). The weight corresponding to the freeze-dried PI was taken as the initial weight and the extraction yield η for each extracted material was calculated using the equation


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= × 100

(1)

where wf is the weight of the extracted sample (H1, H2, H3 or B) and wi is the initial weight corresponding to dry PI. Care was taken to avoid as best as possible material loss during washing, filtration and freeze-drying. Experiments were performed in duplicate.

Characterization Chemical Composition Analysis and Molar Mass Distribution of Bleached Cellulose Ionic chromatography (IC) was used to determine the carbohydrate composition of B after complete hydrolysis using 72% sulphuric acid at room temperature and following autoclave in 3% H2SO4 at 125 °C for 1 h (TAPPI Test Method T 249). The calibration was performed on commercial glucose, galactose, mannose, xylose and arabinose. The ash content of the samples was gravimetrically measured after heating at 600 °C for 24 h. Shimadzu’s TNM-1 Total Nitrogen Module (Shimadzu, Japan) was utilized to determine the samples’ total nitrogen (TN) content after the complete hydrolysis with 6 M HCl at 110 °C for 24 h. 1–100 ppm KNO3 standard solutions were used for system calibration. The crude protein content was calculated from the nitrogen content by multiplying the conversion factor of 6.25.18 The molar mass distribution including weight average molecular weight (Mw), number average molecular weight (Mn) and polydispersity index (PDI) of B was determined by size exclusion chromatography (SEC) equipped with a Rheodyne injector (Shimadzu, Japan), a DGU-20A3 degasser (Shimadzu, Japan), a LC-20AD liquid chromatography (Shimadzu, Japan) and a RID10A refractive index detector (Shimadzu, Japan). ~30 mg of B was first mixed with water and stirred at 4 °C for 1 h for activation. Then the mixture was subjected to filtration to remove


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excess water. This was followed by a solvent exchange with methanol for three times and then with N,N-dimethylacetamide (DMAc) for another three times. Afterwards, 8% lithium chloride (LiCl)/DMAc was introduced to dissolve the samples to a concentration of 0.8% by stirring at 4 °C for 5 days. The completely dissolved samples were then diluted with DMAc to a final concentration of 0.5% before SEC analysis. Three 20 µm Mixed-A columns (7.5 × 300 mm, Polymer Laboratories, USA) and a guard column (7.5 × 50 mm, Polymer Laboratories, USA) were equipped to achieve molecular mass analysis under the conditions of 100 µL sample injection, 80 °C and a flow rate of 0.5 ml min-1 of 0.5% LiCl/DMAc. The pullulan standards with nominal masses from 320 to 800 kDa (Fluka/Riedel-de Haën, Seelze, Germany) were used for SEC system calibration. Data acquisition and evaluation was performed by using the LC Solution software installed in the SEC system (Shimadzu, Japan).

Molecular Structure Analysis by Fourier-Transform Infrared Spectroscopy Attenuated total reflection FTIR spectra were obtained using a FTIR spectrometer (Spectrum Two, Perkin Elmer, USA) equipped with the Perkin-Elmer Spectrum version 10.4.2 software. A spectral resolution of 4 cm-1 over a wavenumber range of respectively 450-4000 cm-1 was used for spectra acquisition as well as 32 scans. All spectra were baseline corrected and normalized. Experiments were performed in duplicate.

Molecular Structure Analysis by Raman Spectroscopy The molecular structure of all materials was characterized by using a Raman spectrometer (XploRATM, Horiba, France) equipped with a near infrared (785 nm) laser. A diffraction grating with groove density of 600 g mm-1 was selected and an optical microscope (Olympus BX41)


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with a ×50 long-working distance objective (PL Fluotar, NA = 0.55) was used to focus the laser at the sample surface. Prior to experiments, the Raman spectrometer was calibrated using silicon having a high intensity characteristic Raman band located at a Raman shift position of ~520 cm1

. A laser power of ~9 mW was used to obtain the spectra, which was found not to induce sample

burning. Raman spectra were acquired using an exposure time of 10 s with 3 accumulations. Baseline correction and normalization was also applied to each of the collected Raman spectrum using the LabSpec 6 version 6.3 software. Experiments were performed in duplicate.

Protein Quantification by X-ray Photoelectron Spectroscopy X-ray photoelectron spectroscopy (XPS) was used to assess the superficial elemental composition (including nitrogen from which the protein content is derived) and to quantify the protein content available at the surface of PI and the isolated cellulosic materials (H1, H2, H3 and B). First of all, the samples were deposited onto steal discs. Full range low resolution XPS spectra were obtained using a hemispheric analyzer (Physical Electronics 1257 System, USA) equipped with an Al K anode emitting an X-ray Al Kα radiation (1486.6 eV) at a constant power of 400 W and using an emission angle of 90°. Measurements were carried out under ultra-high vacuum at a pressure within the range of 10-6 Pa. The spectra were monitored using a binding energy range from 1200 down to 0 eV and energy step of 1.0 eV. High-resolution XPS spectra were subsequently acquired in the energy range of the corresponding detected specie and energy step of 1.0 eV. Detailed binding energy range and scan numbers used to obtain high-resolution XPS spectra for chemical elements are reported in Table S2. Prior to experiments, the binding energy was calibrated using the adventitious C1s (C-C) carbon signal having a binding energy of


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285.0 eV. Spectra were fitted using a mixed Gaussian-Lorentzian function for element quantification and bonding determination. The crude protein content Pc present at the surface of the cellulose fibrils expressed as percentage (%) was derived from the nitrogen content values and calculated using the equation:

(%) = (%) × K

(2)

where N is the amount nitrogen in percentage (%) and K is a conversion factor (6.25)40 that has been previously used for tunicates to convert the amount of detected nitrogen to a protein content.10

Crystalline Structure Measurement by Powder X-ray Diffraction All the powder materials were analysed using a powder X-ray diffractometer (D8 Advance, Bruker, UK). The X-ray generator was equipped with a copper tube operating at 40 kV and 30 mA and producing a CuKα radiation having a wavelength of 0.156 nm. The experimental settings were an incident angle 2θ from 5º to 40º at an angle step of 0.02º per 3 s. The rotational speed of the sample holder was set to 60 rpm. The obtained patterns were subtracted from the holder spectra and the baseline was corrected over the measurement scanning angles using the software Origin 8 SR0 V8.0724 (BT24, USA). Experiments were performed in duplicate. The crystallinity index (χc) of the cellulosic materials (PI, H1, H2, H3 and B) was determined by using the Integration Method41 and the equation:


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=

× 100

(3)

where Ac and Aa are the areas under the X-ray diffraction pattern corresponding to the contribution of crystalline and amorphous regions, respectively. The crystallite size was determined using Scherrer’s equation42:

=

(4)

!

where L002 is the crystallite size of the 002 reflection, K = 0.91 and β002 is the full width at half maximum of the 002 reflection.

Fibril Morphology and Width Size Distribution Analysis by Scanning Electron Microscopy A scanning electron microscopy (JSM JT-300, Jeol, Japan) was used to observe the surface morphology of PI and the isolated cellulosic materials (H1, H2, H3 and B). An acceleration voltage of 15 kV was used to acquire all the images. Prior to imaging, the powder samples (< 200 µm in size) were coated with gold using a sputtering coater. They were subsequently fixed onto metal stubs using carbon tabs. The gold layer that was deposited onto the samples’ surface was ~5 nm. The fibrils’ width was quantified from those images by generating width size distributions. Fibrils’ width was estimated by using an image processing software Image J version 1.45 (National Institutes of Health, USA). 200 measurements were performed on each


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image so as to estimate the fibril width size distribution of each material. The results are reported as frequency in percentage (%) as a function of fibril width size (nm).

Thermal Stability Measurement by Thermogravimetric Analysis Thermogravimetric analysis (TGA Q50, TA Instruments, USA) was carried out to study the thermal stability of PI, H1, H1, H3 and B materials. Sample mass of 5-10 mg were heated in a platinum crucible from room temperature up to 600 °C at a heating rate of 5 °C min-1 under a nitrogen atmosphere with a flow rate of 40 mL min-1. Onset and peak degradation temperatures were determined from the first derivative of the weight as a function of temperature using the software Universal Analysis 2000 (TA Instruments, USA). All measurements were performed in triplicate and average values were reported with their associated standard deviations used as error values.

RESULTS AND DISCUSSION Chemical Composition Analysis and Gravimetric Yield Estimation In tunicates, the cellulose is naturally embedded in a protein matrix and presents celluloseprotein fibrils cemented by non-cellulose glycans and lipids. It is almost exclusively present in the tunic fraction. In order to extract protein-functionalized cellulose fibrils from dry tunics, a top-down isolation approach based on successive alkaline extraction was applied. Figure 1b reports digital photos of PI and the cellulosic materials obtained upon performing the alkaline


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extractions (H1, H2 and H3) and bleaching (B). From left to right, a gradual color change from orangish red to white can be seen upon applying the chemical extractions. The orangish red color of PI could originate from the presence carotenoid pigments. For instance, it has been reported that a type of carotenoid is present within the tunic structure of orange tunicate Ecteinascidia Turbinata.43 After the following alkaline treatments, the colors of the cellulosic materials H1, H2 and H3 became lighter suggesting that some pigments were gradually removed by the successive alkaline extractions. The final bleaching step resulted in a white cellulose powder indicating the complete removal of pigment components. This material was used as a reference material. Table 1 reports yield values that were calculated by recording weight loss after performing each chemical treatment. B had a yield of 58%, which is in agreement with a previous work reporting that tunics from ascidians contain high levels of cellulose.10 The weight loss associated to the first alkaline treatment (PI to H1) might be mainly the result of the removal of sand grains or inorganic minerals but also due to the removal of protein, lipids and other non-cellulosic components. The further weight loss associated to the second and third alkaline treatments and following bleaching should mainly originate from the protein removal. This is evidenced by the chemical composition analysis of B as shown in Table 2, which suggests that B corresponds to highly pure cellulose (relative glucose content of 98.1%) with a negligible amount of residual protein (0.1%). By following the mass losses that occurred in-between each steps of the chemical isolation, the composition of the tunics was estimated to be ~24% of sand grains, ~18% of proteins, 58% of cellulose and a low amount of pigments, lipids and traces of other non-cellulose components.


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Table 1. Yield values (η) calculated for isolated cellulosic material (H1, H2, H3 and B) and the initial tunic powder (PI).

Material PI H1 H2 H3 B

η (%) 76 70 62 58

Table 2. Chemical composition of bleached cellulose (B). Content (%) Carbohydrate 88.0 Glucose* 98.1 Arabinose* 0.1 Galactose* 0.8 Mannose* 0.6 Xylose* 0.4 Protein 0.1 Ash 4.9 Recovery 93.0 *relative to total carbohydrate as 100%.

Molecular Structure Characterization The molecular structure of all the materials was analyzed by FTIR and Raman spectroscopy. As seen from FTIR spectrum of PI (Figure 2a), a band located at a wavenumber of ~874 cm-1 was observed and assigned to the vibrational motions of CH3 and OH moieties present in cellulose44 and carotenoids45 (Table S1). Another band located at a wavenumber position of ~1425 cm-1 originated from the vibrational motions of CH and CH3 moieties that are present within the molecular structure of carotenoids.46 The intensity of this band was found to significantly decrease upon the chemical isolation procedure indicating the gradual elimination


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of this pigment. This agrees well with the observed color change during chemical isolation, from a strong orangish red to a lighter one and finally to a white color as reported in Figure 1b. Four other bands observed at wavenumber positions of ~1029, 1053, 1110, and 1160 cm-1 can be observed and have been previously associated to the vibrational motions of C-O-C, C-OH, and C-O moieties that belong to cellulose.44,47 The band located at a wavenumber position of ~1648 cm-1 is attributed to the amide I moieties, which confirmed the presence of protein for the PI, H1, H2 and H3 materials.48 Upon applying the alkaline and the bleaching treatments, the intensities of the typical bands for cellulose increased while those for protein and carotenoids decreased. This suggests the gradual and controlled removal of these non-cellulose components from PI. These observations were further confirmed by Raman spectra as shown in Figure 2b. The intensity of the typical Raman bands for cellulose located at Raman shift positions of ~999, 1089, 1114, 1286, 1331 and 1372 cm-1, corresponding to the vibrational motions of C-O-C and C-O49, increased, indicating the gradual removal of non-cellulosic components including proteins and pigments. The intensities of the bands located at Raman shifts of ~1147, 1503 cm-1 and 1227 cm-1 originating from the vibrational motions of heavy atoms in carotenoids50-52 and vibrational motions of C-N and N-H moieties (amide III) in proteins53, decreased upon the chemical isolation process, further suggesting the removal of non-cellulose components. This also suggests that amine functional groups (-NH2) are present at the surface of the cellulose fibrils, originating from the presence of proteins. As a consequence, both FTIR and Raman analysis confirmed the presence of proteins at the surface of PI, H1, H2 and H3 materials. These analyses also confirmed that highly pure cellulose (B, reference material) was obtained after applying the bleaching treatment. The molecular weight distribution of B, was analyzed by SEC and numberaverage-molecular weight (Mn) and weight-average-molecular weight (Mw) were found to be


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~20 kDa and a of ~350 kDa, respectively. This corresponds to a polydispersity index (Mw/Mn) of 17.6 (Figure S1). This value is much higher than polydispersity values, ranging from 6.4-10.6, reported previously in the literature for other tunicate species.10

Figure 2. Typical (a) Fourier-transform infrared and (b) Raman spectra obtained for the initial tunicate powder (PI), and the isolated cellulosic materials (H1, H2, H3 and B). Dotted lines indicate FTIR and Raman band positions of interest.

Protein Surface Quantification The presence of protein available at the surface of PI, H1, H2, H3 and B materials was quantified by X-ray photoelectron spectroscopy. Figure S2 reports low-resolution survey XPS spectra for these materials, indicating (among other elements) the presence of a peak corresponding to the binding energy of the N1s chemical energy state. This peak was used to quantify the amount of nitrogen present at the surface of PI, H1, H2, H3 and B materials and to derive the protein content. Table 3 reports, among other elements, the nitrogen and protein


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contents obtained for PI, H1 and H2. The percentage of nitrogen atoms for PI was 2.9%, corresponding to a protein content of 18%. For the H1 material (first alkaline treatment), the nitrogen content decreased down to 0.6%, which corresponds to a protein content of 3.8%. For the H2 material (second alkaline treatment), the nitrogen content decreased down to 0.3%, which corresponds to a protein content of 1.9%. For the H3 and B materials, nitrogen was not sufficiently well detected to allow accurate quantification and so the protein content could not be derived. XPS confirmed the gradual removal of the proteins during the alkaline treatments as suggested by FTIR and Raman spectroscopy. Table 4 and 5 report binding energies associated to the C1s and N1s chemical energy state, respectively, for PI, H1, H2, H3 and B materials. Table 4 and Figure S3 indicate the presence of C-C or C-H, C-O, O=C-O or C-O-C chemical bonds for all the materials, which are typical for cellulose.24,54-56 On the other hand, Table 5 and Figure S4 confirm the presence of C-N and N-H chemical bonds indicating the presence of significant amounts of protein covalently bonded to the surface of PI, H1 and H2 materials.24,54,55 As previously mentioned, the presence of nitrogen in H3 and B materials was not sufficiently well detected and it was not possible to confirm whether proteins were covalently bonded to their surface. The presence of O=C-O and, C-N and N-H moieties suggests that carboxylic acid (-COOH) and amine (-NH2) functional groups, respectively, are present at the surface of the cellulose fibrils (PI, H1, H2), which is due to the presence of proteins. The identification and quantification for the presence of elements such as Na, Ca, Si and Mg may be mainly related to the presence of residual sand grains, whose presence has been reported before when using microalgae as a cellulose source.5 Elements such as S and Fe have been reported before to be present within the tunic of other tunicate species.18 The presence of K may


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originate from the successive KOH alkaline treatments, which were applied to the PI material to isolate protein-functionalized cellulose fibrils.

Table 3. Elemental atomic concentration obtained from high resolution X-ray photoelectron spectra for the initial tunic powder (PI) and the isolated cellulosic materials (H1, H2, H3 and B).

Material

Elemental atomic concentration (%)

Protein content (%)

C1s N1s O1s Na1s Mg2s Si2p S2p K2p Ca2p Fe2p PI H1 H2 H3 B

42.0 44.1 42.8 35.9 51.3

2.9 0.6 0.3 -

46.7 48.1 42.6 53.0 45.3

0.6 0.2 0.2 0.1 -

1.5 1.7 1.2 0.2 -

2.2 2.5 5.5 6.1 2.0

2.1 0.02 0.6 0.0 2.9 0.0 -

1.6 2.1 4.3 4.7 0.3

0.4 0.1 0.2 -

18.1 3.8 1.9 -

Table 4. Binding energy (BE) associated with C1s chemical energy states for the initial tunic powder (PI) and the isolated cellulosic materials (H1, H2, H3 and B). Material PI H1 H2 H3

BE (eV) C-C; C-H 284.9 284.8 284.8 284.9

BE (eV) C-O 286.6 286.3 286.6 286.5

BE (eV) O=C-O; O-C-O 288.7 288.5 288.5 288.5


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B

285.1

286.8

288.6

Table 5. Binding energy (BE) associated with N1s chemical energy states for the initial tunic powder (PI) and the isolated cellulosic materials (H1, H2, H3 and B). ND stands for not detected.

Material PI H1 H2 H3 B

BE (eV) N-H 397.8 398.9 397.9 ND ND

BE (eV) C-N 399.7 400.1 400.3 ND ND

Crystalline Structure Analysis Figure 3a reports powder X-ray diffraction patterns for PI, H1, H2, H3 and B. Two low intensity diffraction peaks located at diffraction angles between 26 and 27° can be observed for the PI material. These peaks must originate from the residual sand grains and indicated the possible presence of aragonite within its structure.57 After the first alkaline treatment, these peaks


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disappeared indicating the removal of these sand grains, which agrees well with the chemical composition analysis obtained by XPS. For all materials, four peaks at diffraction angles positions of ~14.8, 16.7, 22.9 and 34.5° were observed. These reflections correspond to the diffraction planes of (11"0), (110), (200) and (040), typical for cellulose I.10,44 No significant peak shift was observed for these diffraction patterns upon the alkaline treatments and bleaching, suggesting that the applied chemical extraction method did not influence significantly the crystalline I structure of the cellulose initially present in the tunics. The intensities of these diffraction peaks, however, increased upon applying more extraction steps, suggesting the gradual removal of proteins from the tunics. This observation is supported by an increase in crystallinity index as reported in Figure 3b. Detailed crystallinity index values are reported in Table S3. Upon applying the alkaline and bleaching steps, the crystallinity index increased from 58% for PI to 74% for B, indicating that cellulose was gradually isolated from proteins that were mostly present within the tunics. In addition, an increase in the crystallite size further indicated the removal of non-cellulose compounds including proteins as reported in Table S3. It is important to mention that the typical diffraction peak of collagen usually located at a diffraction angle position of ~8° was not observed. This could suggest that the protein present at the surface of PI, H1 and H2 materials is present in a denaturalized form.


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Figure 3. (a) Typical powder X-ray diffraction patterns and (b) crystallinity index obtained for the initial tunicate powder (PI) and the extracted cellulosic materials (H1, H2, H3 and B).

Morphological Observation and Width Size Distribution Analysis Figure 4 reports SEM images obtained from the surface of PI, H1, H2, H3 and B materials. In Figure 4a, corresponding to the PI material, one can observe the presence of fibrils having a ribbon-like morphology. It is very likely that these fibrils are actually a composite material containing mainly cellulose that is surrounded by a protein matrix material. This was confirmed by FTIR and Raman spectroscopy as well as XPS. In this image, one can also observe the presence of sand grains as shown by the white arrows. This is consistent with X-ray diffraction patterns where small diffraction peaks corresponding to sand grains were observed. Although the cellulosic materials obtained from alkaline treatments and bleaching showed similar ribbon-like structures (Figure 4b-4e), the typical fibrillar structure of cellulose became more obvious, possibly due to the gradual removal of the protein matrix material. This was confirmed by quantifying the fibril width distribution of PI, H1, H2, H3 and B materials as reported in Figure 5. For PI, H1 and H2, the fibril width distribution was wide with values ranging from ~30 nm up


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to 400 nm (Figure 5a-5c). After gradual protein elimination, the fibril width size distribution became significantly narrower for H3 and B materials (Figure 5d and 5e). The frequency of fibrils having a width size below 100 nm increased significantly from PI, H1 and H2 to H3 and B materials as reported in Figure 5. This means that, upon applying the alkaline and bleaching steps, the width size of the cellulose fibril is significantly reduced, which can be explained by the gradual removal of the protein matrix material that was original present in the PI material. This is also supported by the gravimetric yield determination, FTIR and Raman spectroscopy, XPS, and powder XRD.


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Figure 4. Scanning electron micrographs showing the morphology of (a) the initial tunic powder and (b) (c) (d) and (e) correspond, respectively, to the various extracted cellulosic materials (H1, H2, H3 and B). The white arrows in (a) indicate the presence of inorganic minerals.


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Figure 5. Fibril width size distribution estimated from scanning electron images obtained for (a) the initial tunic powder and (b) (c) (d) and (e) corresponding, respectively, to the various extracted cellulosic materials (H1, H2, H3 and B).


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Thermal Stability Analysis Figure 6a reports typical TGA traces for PI, H1, H2, H3 and B materials. For PI, the weight loss started at a temperature of ~170 °C may originate from the evaporation of residual moisture and to the degradation of low-molecular components, such as lipids, non-cellulose glycans and possibly low-molecular weight proteins as reported before in the literature.6 The major weight loss occurred at a temperature of ~300 °C due to the thermal degradation of cellulose. At a temperature of ~360 °C, the residual weight started getting stable and then reached a value of ~36%, which then further stabilized up to a temperature of 600 °C as reported in Figure 6a and Table S4. This relatively high residual weight could originate from the presence of residual sand as well as chars resulting from the thermal degradation. Figure 6a also reports TGA curves for H1, H2 and H3 materials, which showed similar degradation behavior with a first weight loss occurring at ~170 °C and a main weight loss occurring at ~370 °C. The residual weights for these materials were ~17% at a temperature of 600 °C and significantly lower than that for PI. B showed a main weight loss at ~310 °C, relatively close to PI but significantly lower than H1, H2 and H3 materials. This may be due to possible chemical degradation that might have occurred during bleaching. The residual weight for B was ~7% at 600 °C, originating from char formation due to cellulose thermal degradation. The thermal stability of the isolated materials was further analyzed based on parameters including the temperature at weight loss of 10% as well as the onset and peak degradation temperatures (T10%,Tonset and Tpeak). These values were derived from DTGA curves reported in Figure 6b. As shown in Table S4, H3 had the highest thermal stability with values of ~321, 350 and 374 °C for T10%, Tonset and Tpeak, respectively. These values


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(obtained at a heating rate of 5 °C min-1) are among the highest ever reported for cellulose fibrils20,58 and are 24 and 41 °C higher than B obtained in the present study (our reference material). H1 and H2 materials also displayed high thermal stability, significantly higher than for the bleached cellulose fibrils but significantly lower than the H3 material. The values reported in the present study for H3 are significantly higher than Tonset and Tpeak values of ~325 °C and ~360 °C reported earlier in the literature for enzymatically treated tunicate cellulose fibrils obtained (obtained at a heating rate of 10 °C min-1).10 Another study focusing on the extraction of tunicate cellulose from various species reported values within the range of 207-269 °C for Tonset and 354369 °C for Tpeak also using a heating rate of 10 °C min-1.9 These values are significantly lower than the values reported in the present study for H3. In a very recent study, the presence of residual proteins was associated to improvement in thermal stability for cellulose fibrils obtained from a macroalgae source.6 A Tpeak value of 349 °C was reported for nanopapers made of cellulose fibrils having a residual amounts of proteins of ~7% at their surface.6 In this work, the possible contribution of residual chlorophyll pigments on the thermal stability in these cellulose nanopapers was, however, not discussed. Another study focused on studying the thermal stability of acetylated bacterial cellulose nanofibers59 which were chemically modified by acetylation using a bottom-up approach. These chemically modified cellulose nanofibers derived maximum values of ~350 and 370 °C for T10% and Tpeak, respectively. The improved thermal stability obtained for the H3 material in the present study, compared to PI, H1, H2 and B, may arise from the very low amount of protein present at the cellulose fibril surface (compared to PI, H1 and H2) and due to the presence of residual carotenoid pigments (not identified in B by FTIR and Raman spectroscopy). It is known that carotenoid pigments possess in their molecular structure


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highly thermally stable rings and their presence could help in improving thermal stability. No direct evidence of this, however, is reported in the present study.

Figure 6. Typical (a) thermogravimetric analysis (TGA) and (b) DTGA traces obtained for the initial tunic powder (PI) and the various extracted cellulosic materials (H1, H2, H3 and B).

CONCLUSIONS


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As opposed to the environmentally unfriendly, costly and time consuming bottom-up approaches traditionally used to chemically modify the surface of cellulose fibrils with proteins, a top-down approach was used to obtain protein-functionalized cellulose fibrils. These fibrils were extracted from the tunic of Pyura Chilensis, a biomass waste from the Chilean seafood industry. These cellulose fibrils were found to have controlled amounts of covalently bonded protein at their surface, ranging from 2 up to 18% for PI, H1 and H2 materials. The extraction yields for these fibrils were high and ranged from 62 to 76% by weight. FTIR and Raman spectroscopy confirmed the presence of protein at the surface of the cellulose fibrils, which was then quantified by X-ray photoelectron spectroscopy. These techniques also suggested the presence of carboxylic acid and amine functional groups at the surface of the cellulose fibrils (PI, H1 and H2). Powder X-ray diffraction confirmed that the protein-functionalized fibrils had a relatively high crystallinity index and that their cellulose I crystalline structure was preserved upon applying successive chemical treatments. Scanning electron microscopy revealed that the extracted cellulosic materials were constituted of fibrils having a ribbon-like morphology. Their width-size distributions ranged from ~30 to 400 nm, and were found to get significantly narrower upon applying more chemical treatments, suggesting the gradual removal of proteins. The thermal stability of the protein-functionalized cellulose fibrils was significantly improved upon gradual removal of proteins (B being the exception). The H3 material showed outstanding thermal properties with onset and peak degradation temperature values of ~350 and 374 °C, respectively, which is significantly higher than the bleached cellulose fibrils (reference material).

SUPPORTING INFORMATION


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Detailed information on FTIR and Raman band assignment, XPS spectra acquisition, crystalline structure and termal properties data, molecular mass distribution and XPS spectra can be found in the supporting information.

AUTHOR INFORMATION (*)

Corresponding Authors. F. Quero, Email: [email protected] and Y. Zhao E-mail:

[email protected]. Notes. The authors declare no competing financial interest. Author Contributions. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT F.Q. acknowledges financial support from CONICYT/FONDECYT (No. 11160139) and from the Program U-INICIA VID 2016, GRANT UI11/16; University of Chile.

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functionalized and highly thermally stable cellulose fibrils - American

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