Cellulose Source Tailors the Physical and Structural Properties of Double-Functionalized Aerogels

ABSTRACT Cellulose aerogels are solid materials with high porosity, low density, and large surface area, and show excellent absorption of mechanical stress. These characteristics are appropriate for the development of new high-value products. However, over the years, it has been increasingly difficult to compare different studies about this material due to the different approaches used. In the present work, three different raw materials, bacterial cellulose from synthetic medium (BC), bacterial cellulose from cashew juice permeate (BCP), and eucalyptus nanocellulose (EC) were double-functionalized with TEMPO oxidation followed by silanization and freeze-dried, resulting in three different types of cellulose aerogels. These aerogels were evaluated for their performance in the absorption of organic oils/solvents and mechanical properties. The raw materials lead to aerogels with different characteristics. BC aerogel has the best mechanical properties and absorption capacity after 7 cycles; BCP aerogel presented the highest values for oil absorption and EC aerogel for organic solvent absorption.


Introduction
Aerogels are solid materials with a large surface area and low density. The term aerogel can be used to define a material with such characteristics in which the fluid in the gel pores is replaced with air. If the replacement is performed with freeze-drying, the material can be named cryogel (Beh et al. 2020;Boukind et al. 2021;Garcia-Bordejé, Benito, and Maser 2021;Guan et al. 2020;Takeshita et al. 2021;Wang et al. 2020;Wu et al. 2020). Modifications in the aerogel structure affect the application of the material. Therefore, it is possible to produce an aerogel suitable for particle filtering, selective absorption for oil and solvent spills, and wastewater treatment, based on the type of used functionalization (Abouzeid et al. 2019;Heath and Thielemans 2010;Hosseini et al. 2019Hosseini et al. , 2020. The HS medium is a standard for research related to bacterial cellulose, but it represents about 30% of the overall cost and 65% of the costs with fermentation (Rivas et al. 2004). Cheaper raw materials like agricultural residues or byproducts may reduce the bottom-up nanocellulose production cost (Rathinamoorthy et al. 2021). One of these materials is the cashew juice permeate, a by-product of the extraction process of the yellow dye from the cashew apple juice. This permeated juice is an extract rich in sugars and nitrogen (BARROS 2016), which can replace the HS growth medium.
The effect of the type of cellulose (top-down or bottom-up) and the origin of the cellulose (from a synthetic or a natural medium), even after chemical functionalization, may impact the final properties of the material. In this research, three different types of nanocellulose were double functionalized, with a TEMPO-oxidation followed by silanization: BC of synthetic medium (BC), BC of cashew juice permeate (BCP), and eucalyptus cellulose (EC). To the best of our knowledge, no research has been published on the effect of cellulose sources on the properties of aerogels. Furthermore, no report examines the structure and mechanical properties of aerogels obtained from cashew juice permeate.

Raw materials and reagents
Bacterial cellulose obtained in synthetic medium HS (Schramm and Hestrin 1954) (BC) and those obtained in the cashew juice permeate medium (BCP) were produced by the strain Komagataeibacter xylinus ATCC 53,582 under static conditions in the Laboratory of Microbiology of Embrapa Tropical Agroindustry (Pereira et al. 2020). The nano fibrillated eucalyptus cellulose (EC), at a concentration of 3.0% (w/w), was supplied by Suzano Pulp and Paper (Microfibrillated cellulose; source: Eucalyptus bleached kraft pulp; Limeira, SP, Brazil, product code 13.465-970).

TEMPO-mediated oxidation
The BC, BCP, and EC were oxidized using the method proposed by Saito et al. (Saito et al. 2007) with adaptations (Pereira et al. 2020). After the complete dissolution of NaBr and TEMPO, 1 g of cellulose (dry basis) was added. After homogenization, NaOCl (5.0 mmol) was added dropwise, under stirring (1000 rpm) at 25 ± 1°C. The pH was adjusted to 10.0-10.5 with NaOH 1.0 mol. L −1 and the conditions were maintained for 150 min. Ethanol 95% v/v (60 mL) was added to stop the reaction, and the characteristic yellow color of the reactional medium changed to white. Each oxidized cellulose was washed with 200 mL of distilled H 2 O, centrifuged twice (4°C, 24,456 x g, 15 min; Hitachi Koki Himac CR22GIII, Japan), and the formed hydrogel stored in a refrigerator (4 °C).

Nanofibrillation
The BC (1.76 dry weight), BCP (1.76 dry weight), and EC (1.89 dry weight) were resuspended in 500 mL of distilled H 2 O at 1.0% (w/w) concentration and subjected to nanofibrillation in a high rotation blender (Vitamix, Vita-Prep 3 model) at maximum rotation (24,000 rpm, 38,707 x g) for 30 min. Finally, the three suspensions were stored in a refrigerator (4 °C) for further characterization and processing.

Production of cellulose aerogels
The BC, BCP, and EC suspensions were used to prepare the aerogels. The suspensions (30 mL) were deposited into 50 mL Falcon tubes, flash-frozen in liquid nitrogen, and stored in Ultrafreezer (SANYO, MDF-U33 V, Moriguchi, Japan) at −80°C. The samples were freeze-dried (K105 Liotop) at a pressure of 1000-1200 μHg, and temperature from −100 to −30°C for 48 h. All the aerogels were prepared in duplicate.

Determination of carboxylate content by conductometry
A titration method based on Besbes, Alila, and Boufi (2011), and Saito and Isogai (2004) (Besbes, Alila, and Boufi 2011;Saito and Isogai 2004) was performed to determine the -COO-content in the oxidized cellulose. An amount of 183.3 mL of distilled H 2 O and 16.67 mL of 0.01 mol. L −1 NaCl was added to 1.0 g of dry cellulose and the system was homogenized. Then, a solution of 0.1 mol·L −1 HCl was added to adjust the pH to 2.5-3.0. The titration was done by the addition of 0.04 mol·L −1 NaOH. The carboxylate content in mol·g −1 was determined by Equation (1): where C NaOH is the NaOH concentration used in the titration, m is the cellulose mass, V 0 is the volume of NaOH required for the HCl neutralization (strong acid), and V 1 , is for the -COOH neutralization (weak acid).

Fourier transform infrared absorption spectroscopy (FTIR)
The FTIR spectra were performed on a Shimadzu FTIR-8300 spectrophotometer, with 30 scans, in the range of 4000-400 cm −1 . The samples were oven-dried at 50°C and mixed with KBr at a ratio of 3.0% (w/w). Graphics were generated in OriginPro9.1 and modified in freeware Gimp 2.6 (version 2.10.14, https://www.gimp.org/).

Porosity and density
The aerogels obtained from each sample were sectioned into a cylinder shape to calculate the apparent density. The height, diameter, and weight of the cylinders were measured in duplicate. The bulk density (ρ S ) was calculated by the ratio between the sample mass and its volume obtained by its size. The porosity was calculated using Equation (2) ): where ρ S is the density of a similar solid material, in this case, cellulose for non-silanized samples, and ρ SM is a mean between the density of the cellulose and the polymethylsiloxane for silanized samples. The ρ SM density was calculated with the Equations (3) and (4)  : where w CELLULOSE is the mass fraction of cellulose in the sample and w POLI(MTMS) is the mass fraction of polymethylsiloxane. The ρ CELLULOSE was fixed at 1500 kg m −3 and ρ POLI(MTMS) at 1900 kg.m −3 , according to the literature . w POLI(MTMS) was estimated with the Equation (5): where w S and w NFC are the dry mass of the sample and the portion of cellulose nanofibrils in the sample, respectively. All measurements were performed in duplicate and the HSD Tukey's test was made at 5% level of significance.

Scanning electron microscopy (SEM)
Fragments of the aerogels were mounted on stubs, covered with a thin layer of gold in an Emitech K550 metallic cover apparatus, analyzed in an SEM Inspect-50 and Vega 3 Tescan, and visualized under a 15 kV voltage acceleration at different magnifications (1.000x and 20.000x). The diameter of 15 clearly visible fibers was measured using the ImageJ image analysis software (version 1.52a, Wayne Hasband, National Institute of Health, USA). The freeware Gimp 2.6 (version 2.10.14, https://www. gimp.org/) was used to make the figure.

Contact angle
The contact angle values were measured according to the ASTM D- 5725-99 (2003). Droplets of deionized water were deposited on the surface of the previously conditioned (25°C, under vacuum, 24 h) aerogels with a syringe coupled to the contact angle measurement system. The images were captured by a Nikon camera shortly after the formation of the drop. The angle obtained on the surface of the aerogel was calculated by the Angle Calculator program. The freeware Gimp 2.6 (version 2.10.14, https://www.gimp.org/) was used to make the figure. All measurements were performed at 25 ± 1°C, with three replicates and the HSD Tukey's test was made at 5% level of significance.

Mechanical compression tests
The compression tests were performed in a universal EMIC DL30000 mechanical test machine with a 100 kN load cell. The aerogel samples were cut in 12 mm height and 25 mm diameter pellets and arranged in a pair of fixed platters with a diameter of 110 mm. The compression speed was 1 mm·min −1 . The mechanical properties of the aerogels were evaluated using compression tests up to 50% deformation, based on the initial thickness of the sample. The shape recovery capacity was also evaluated, based on the initial and final thickness ). The tests were repeated twice and the HSD Tukey's test was made at 5% level of significance.

Absorption capacity
A volume of 20 mL of different liquids (ethanol, acetone, toluene, chloroform, mineral oil, and engine oil) was used to immerse the aerogels in a 50 mL beaker at room temperature (25 ± 1 °C). After the immersion for at least 1 min, the aerogel was removed, the excess or non-adsorbed liquid was removed with filter paper, and the sample was weighed. A total of three replications per sample were obtained. The tested aerogels had an average weight of 20 mg. The absorption capacity (C AB ) was calculated by the Equation (6): where m 0, and m S are respectively the mass of dry aerogel and after saturation. This analysis was done in triplicate ) and the HSD Tukey's test was made at 5% level of significance. A cycle absorption assay was performed using ethanol (Pereira et al. 2020). The same procedure of absorption was used, followed by drying the saturated aerogel in an oven at 60°C for 1 h. After new weighting, the second cycle of absorption/drying begins. The aerogels were submitted to seven absorption/drying cycles. Graphics were generated in OriginPro9.1 and modified in freeware Gimp 2.6 (version 2.10.14, https://www.gimp.org/).

TEMPO-oxidized nanofibrillated cellulose
Both BC and BCP purified membranes were deconstructed with a Vitamix blender, resulting in pastes. The BC (Figure 1) was lighter and thicker than the BCP (Figure 1), resembling the viscosity and color of the EC, a commercial product received as a nanofibrillated purified cellulose paste ( Figure 1). All materials presented oxidation yields around 80% after the TEMPO treatment and oxidation degrees of 0.25. The carboxylate group content ranged from 1.5 COO-•g −1 cellulose for BCP and EC to 1.6 COO 1-•g −1 cellulose for BC (Pereira et al. 2020), in agreement with the range of reported values (1.2-1.7 COO 1-•g −1 cellulose) (Isogai, Saito, and Fukuzumi 2011). These results indicate that the different cellulose sources presented low influence for the carboxylate group content after TEMPO oxidation.
About 500 g of oxidized BC and BCP suspensions, at a concentration of 1% (w/w), were fibrillated with a Vitamix blender at maximum rotation for 30 min. EC was resuspended in the same conditions. The obtained materials were translucent and slightly white hydrogels (Figure 1d,e,f). After the silanization step, the suspensions of oxidized nanocellulose did not change in appearance but became less viscous. The flowchart (Figure 2) represents all the process steps.

Obtaining the bacterial cellulose aerogel
After lyophilization, BC, BCP, and EC aerogel monoliths were obtained (Figure 3). The BCP is very similar to the BC aerogel, with no cracks, not very soft, and low brittleness. EC was obtained with cracks, but the non-collapsed region is as brittle as the BC aerogel. This is indicative of fractures during the freezing process.

Scanning electron microscopy (SEM)
The BCP aerogel has the lamellar configuration of the BC aerogel [33], but the lamellae are smaller (Figure 3e). At the edges of the structures, it is possible to see that they are made up of fibers organized in webs (Figure 3h), which were previously identified in BC aerogels (Zhang et al. , 2015. EC aerogel is similar to BC aerogel, consisting of lamellae and fibers, but of smaller lamellae (Figure 3f). There are spherical clusters in (Figure 3i), resulting from the condensation of the polymethylsiloxane (Zhang et al. 2015). This behavior was also reported by Sai et al. (2014) (Sai et al. 2014), who functionalized cellulose with Na 2 SiO 3 and MTMS. In the work reported by Zhang et al. (2015) (Zhang et al. 2015), the emergence of polymethylsiloxane condensates is linked to the spatial concentration of reagents, but in this work, the raw material seems to have a higher influence. This behavior was not seen in BC or BCP aerogel samples.
Aerogels produced from both types of bacterial cellulose substrate showed a lamellar configuration (Figure 3d-g). It seemed that the lamellae from the BCP aerogel were smaller than that of BC. It is noticeable at the edge of the lamellae that they presented fibers organized in webs, which were previously identified by Zhang et al. (2014Zhang et al. ( , 2015. Aerogels made of EC appears to be more compact, as if the microfibrils have irreversibly collapsed, as in the hornification process. There are spherical clusters on the cellulose, resulting from the condensation of the polymethylsiloxane (Sai et al. 2014;Zhang et al. 2015). The EC was produced by a plant cellulose synthase. Differences in the enzymatic machinery between eukaryotes and prokaryotes result in types of cellulose with different structures, impacting, for example, the nanofibril width, crystallinity degree, and cellulose allomorph content (Hussain et al. 2019;Klemm et al. 2011;Sacui et al. 2014). The bacterial cellulose is assembled without hemicellulose or lignin and the water incorporated within the net of bacterial cellulose stabilizes the 3D structure of fibrils and pores (Klemm et al. 2011). The pores formed in the EC were created during the freeze-drying. The pores in the BC hydrogel may have a more uniform distribution than the EC hydrogel, resulting in the micromorphology differences, without collapsing.
The comparison between the three types of aerogels indicated that although the three materials have the same chemical composition, the cellulose fibrils organization is different. The different visibility of the open web of submicron nanofibers for the aerogels (diameter of 140-156 nm for BC fibers, 162-237 nm for BCP fibers, and 272-337 nm for EC fibers) is indicative that the size distribution for the pores may vary as a function of the original biomass, impacting the absorption efficiency for the aerogels. In general, the aerogel samples of BCP and EC have smaller structures, which may result in more porous aerogels.
Published articles indicate that the native structure of bacterial cellulose may have different characteristics based on the cultivation conditions, such as crystallinity (Hussain et al. 2019;Jozala et al. 2015). Cellulose production is a complex process, from the intracellular synthesis of polyglucose building blocks to the secretion of ß-1,4-glucan chains (Ross et al. 1991). Many genes related to enzymatic synthesis and regulation are involved in cellulose metabolism Jang et al. 2019;Römling and Galperin 2015). K. xylinus is a versatile bacterial cellulose producer, which can be grown using more than two dozens of different sole carbon sources in the growth medium. Changes in the available nutrients change the genetic expression, impacting metabolic pathways from the tricarboxylic acid cycle to the bacterial cellulose synthase (Jang et al. 2019).
We hypothesize that macro and micronutrients in the BCP, not present in the synthetic medium HS, altered the genetic expression related to cellulose synthesis. These alterations may have changed the enzymatic efficiency, or the number of expressed enzymes, or even the structure of the enzymes. These differences do not impact major properties of bacterial cellulose from both media but may alter the microstructure of the fibril/pore networks, as detected on the micromorphology of different aerogels. These findings should be further investigated with a functional genomic analysis of the bacteria cultivated in different growth media to better understand the genetic impact on the final product.

Fourier transform infrared absorption spectroscopy (FTIR)
The FTIR analyses were performed to confirm the carboxylation as observed in the conductometric titration and the silanization of the materials. The Figure 4 shows bands at 1726 and 1623 cm −1 assigned to the presence of -COOH and -COO-, respectively. The bands at 780, 911, and 1275 cm −1 show the presence of chemical bonds with the silane (Chen et al. 2019;Jiang and Lo Hsieh 2016;Xiao et al. 2015;Zhang et al. 2015), confirming the successful double-functionalization of the different types of cellulose (Figure 4). The peak transmittance at 780 cm −1 may represent the presence of Si -O -Si bonds, which would result in polymethylsiloxane chains, or Si -C bonds, from methyl groups. The peak at 911 cm −1 denotes Si -OH bonds or Si -C bonds. The peak at 1275 cm −1 is attributed to the presence of CH 3 groups from polymethylsiloxane.
The presence of these five bands in the materials BC, BCP, and EC, in comparison with the untreated cellulose (Pereira et al. 2020), indicates that the double chemical functionalization was successfully achieved. Nevertheless, just the presence of silicon and carboxylic groups in the samples will not guarantee that the three different materials have the same mechanical and absorptive properties, and other analyses were performed.

Porosity, density, and mechanical tests of aerogels
The BCP and EC aerogels are very similar to the BC aerogel (Pereira et al. 2020), as shown in Table 1, demonstrating that the method can be applied successfully in different cellulose sources. The aerogels obtained have similar density and deformation. Aerogels also suffered a greater shrinkage but within the limit of 30% (Aegerter, Leventis, and Koebel 2011). The Table 1 values for apparent density, and porosity were very close to those reported in similar works: 11 kg·m −3 and 99.3% ), 6.18-16.39 kg·m −3 and 98.98-99.58% (Chen et al. 2019), 3.1-7.9 kg·m −3 and 99.48-99.81% (Laitinen et al. 2017), 11 kg·m −3 and 99.4% (Sai et al. 2014). It is interesting to highlight that the porosity within treatments is so uniform that even a difference of 0.1% is statistically significant (Table 1), indicating that this parameter has low variability. This is an indication that the different types of aerogels are not fully identical. When other mechanical properties are evaluated, the differences become more evident.
Compression tests were performed up to 50% deformation to evaluate the mechanical properties of the aerogels. The capacity to recover shape was also measured. The values for the maximum stress in the deformation of 50% and the modulus of elasticity were similar for the BCP and EC aerogels, and both values are smaller than those observed for BC. Samples of the aerogel made of plant cellulose and BC from the alternative source have less mechanical resistance but they can still recover about 85% of their initial shape. The tension values in this work (Table 1) were in the range of those reported in similar works: 4 kPa ) and 10 kPa (Nguyen et al. 2014). The deformation recovery is close to the value observed by Zhang et al. (2014)  ) (86%) and higher than that reported by Nguyen et al. (2014) The parameters with no statistical difference may be related to the chemical composition of the aerogels. The different types of cellulose were successfully modified by the proposed chemical double- functionalization (Section 3.1). Nevertheless, differences in the microstructure of aerogels (Figure 3) impact the mechanical properties, reinforcing the impact that the raw material has on the final product.

Contact angle
The BCP aerogel (126.8° ± 1.7°) is more hydrophobic than the BC aerogel (120.0° ± 2.5°) (Pereira et al. 2020) and the EC aerogel (129.6° ± 0.8°) is the most hydrophobic ( Figure 5). According to Wenzel's Law for wettability of a material, the rougher the surface, the greater the hydrophobicity (Sai et al. 2014). This can be understood in terms of surface area. The BCP and EC aerogels have a larger area, as they have much smaller structures than the large, flat lamellas of BC aerogel. Furthermore, the silane deposition on the fibers (Figure 3i) for the EC aerogel increases the roughness of the surface, having a higher impact on the hydrophobicity. The methods/processes used to obtain the aerogel can be applied for different cellulose sources, but the raw material can influence the properties of the doublefunctionalized aerogel. The observed values of contact angle ( Figure 5) were similar to those reported by other authors: 117.6°-124.6° (Sai et al. 2014), 136° , and 139° (Wan et al. 2015).

Absorption tests
BCP, EC, and BC aerogels (Pereira et al. 2020) have different absorption performances (Figure 6a). The EC aerogel absorbs a greater amount of organic solvents but does not show the same behavior for oils (acetone = 58 ± 3%, ethanol = 59 ± 4%, mineral oil = 41 ± 6%, motor oil = 45 ± 2%, toluene = 55 ± 7%, chloroform = 82 ± 1%). The BCP aerogel absorbs the same amount of acetone and ethanol, a lower Note: Values are the mean of two measurements ± standard deviation and means followed by a common superscript letter in the rows are not significantly different by the HSD Tukey's test at 5% level of significance. Figure 5. Average values of the contact angle between water and aerogel samples and test images. Means followed by a common letter are not significantly different by the HSD Tukey's test at 5% level of significance. BC: bacterial cellulose from the synthetic medium HS, BCP: bacterial cellulose from cashew juice permeate, EC: eucalyptus nanocellulose.
amount of toluene and chloroform, and a higher amount of oil (acetone = 42 ± 3%, ethanol = 44 ± 2%, mineral oil = 65 ± 4%, motor oil = 67 ± 3%, toluene = 45 ± 3%, chloroform = 70 ± 3%) than the BC (acetone = 40 ± 1%, ethanol = 45 ± 2%, mineral oil = 36 ± 3%, motor oil = 52 ± 6%, toluene = 35 ± 2%, chloroform = 77 ± 1%). The BCP and EC aerogels absorb almost the same amount of chloroform, demonstrating that for this liquid the double functionalization works regardless of the raw cellulosic material. The order of toluene absorption follows the order of hydrophobicity, EC> BCP> BC. EC aerogel is hydrophobic, but it is not as oleophilic as BCP aerogel, which absorbs a smaller amount of organic solvents. Although cellulose is amphiphilic, and double functionalization is applied in the same way for all of the raw materials, the cellulose source plays an important role in the oleophilicity of the aerogel. The three types of aerogels demonstrated different profiles of absorption behavior and there may be a different optimum application for each one. For example, to clean up motor or mineral oils, BCP is the best. For toluene, the EC aerogel is more appropriate. The absorption capacity values ( Figure 6A) were close to those reported in similar works: 12 and 22 times (ethanol and chloroform) , 40 to 60 times (ethanol, acetone, and motor oil) , 90-150 times (ethanol, acetone, toluene, chloroform, and motor oil) (Laitinen et al. 2017) and 49-102 times (ethanol, acetone, toluene, chloroform, motor oil, and mineral oil) . This is another evidence indicating that the choice of the raw material impacts the physical properties of the aerogel and may affect the final product.
The capacity for several absorption-drying cycles of the aerogel was evaluated ( Figure 6B). In comparison with the BC aerogel (Pereira et al. 2020), the BCP and EC aerogels have their capacity reduced to 40 and 55% already in the second cycle, respectively. The absorption for EC has the first decay to 55% and it continues to decline until 45%. BCP has a greater decay than EC, declining the absorption to 40%. The great shrinkage in drying is an indication that the microscopic structures collapse easily, but the macroscopic structures are maintained throughout the cycles, preserving around 45% of the initial absorption capacity.

Conclusion
Double-functionalized aerogels were successfully obtained using the oxidation-silanization approach to three different cellulosic raw materials: eucalyptus nanocellulose (EC), bacterial cellulose from HS synthetic medium (BC), and cashew juice permeate (BCP). EC and BCP have similar chemical Figure 6. a) Absorption capacity of BC aerogels for different organic oils/solvents (means followed by the same lowercase letter for each type of solvent are not significantly different by the HSD Tukey's test at 5% level of significance), and b) Cycles of ethanol absorption/drying of aerogels. BC: bacterial cellulose from synthetic medium, BCP: bacterial cellulose from cashew juice permeate, EC: eucalyptus nanocellulose.
composition, resistance to deformation, and density to the BC aerogel. The EC and BCP aerogels have lower mechanical resistance to stress than BC but higher absorption capacities for acetone and methanol (EC), and oils (BCP).
Considering the properties analyzed, different types of aerogels may have different potential uses. For example, BC aerogels can be applied in situations that demand robustness, BCP aerogel for oil spills, and EC aerogels for recovering organic solvents. These results are indicative that the cellulose origin impacts the mechanical and absorption properties of the obtained aerogels. On the one hand, the properties of the final product may demand a careful control of the raw material when the aerogel is produced, but on the other hand, the possibility to generate different products with the same chemical functionalization may expand the technological application for different natural materials.