3.1 Proof of oxidation
FTIR confirmed chemical modification by periodate oxidation of the BC membrane. Figure 1 shows the spectra of the samples, with emphasis on the vibration bands 3400 cm−1 (O-H symmetrical stretching), 2895 cm−1 (C-H, symmetrical stretching), 1420 and 1370 cm−1 (C-OH and C-H2 coupled to OH, angular deformation), and 1162 cm−1 (asymmetrical stretching the β-glycosidic bond C-O-C) characteristic of the chemical structure of cellulose (Chang & Chen, 2016, Tabarsa et al., 2017). The 830 cm− 1 band is present only in BC and has been associated with the vibration of glycosidic bonds (Atykyan, Revin & Shutova, 2020). Sugiyama, Persson & Chanzy (1991) reported that signals close to 750 cm−1 and 3240 cm−1 indicate the existence of type Iα crystalline cellulose, as observed by XRD.
The vibration pattern of periodate oxidized cellulose has been well documented since 1960 (Spedding, 1960). Oxidized cellulose is characterized by symmetrical elongation at 1730 cm− 1, which corresponds to the carbonyl vibration (C=O) of the aliphatic aldehydes that were substituted into C2 and C3 in the cellulose structure (Fan et al., 2001). The presence of this band confirms the chemical modification of the BC membrane. The vibrational band at 880 cm− 1 is generally attributed to the formation of hemiacetals (C=O(-O)C*(-OC-)H) between newly formed aldehyde groups and neighboring hydroxyl groups (Kim et al., 2000; Li et al., 2011). This band is absent in the BC spectrum and evidences the oxidation process in cellulose. The loss of vibrational band intensity at 3400 cm− 1 for OxBCs is related to converting secondary alcohol groups into aldehyde groups.
The relative atomic concentrations and the electronic state of carbons on the sample surface were evaluated by XPS. This technique allows obtaining the relative surface concentrations of carbon and oxygen atoms (the O/C ratio) on the surface (analysis depth up to 10 nm), giving essential information about the efficiency of chemical modification in the membrane. Table 1 presents the values of O/C ratios for BC and OxBCs.
Table 1 - Oxygen/carbon ratio, level of oxidation of carbons, degree of oxidation (OD), thermal degradation temperature (Tonset), mass loss (%) and crystallinity index (CrI) for membranes of BC and OxBCs.
Sample
|
XPS
|
Spectrophotometric
|
TGA
|
XRD
|
O/C ratio
|
(C3 + C4)/C2
|
OD* (%)
|
Tonset (ºC)
|
Loss mass (%)
|
CrI (%)
|
BC
|
0.57
|
0.39
|
n.d.
|
315
|
58
|
86
|
OxBC-MW-0.75%
|
0.66
|
0.54
|
25.8 ± 0.03a
|
283
|
59
|
86
|
OxBC-MW-1%
|
0.59
|
0.80
|
73.6 ± 3.01b
|
273
|
63
|
87
|
OxBC-WB-0.75%
|
0.73
|
0.44
|
19.2 ± 0.02c
|
276
|
57
|
74
|
OxBC-WB-1%
|
n.d.
|
n.d.
|
36.0 ± 0.33d
|
274
|
59
|
69
|
n.d. – not determined
* Values are expressed as mean ± standard deviation (n = 3) and different superscript letters (a, b, c and d) show the statistical difference (α = 0.05) by Tukey test.
The surface O/C ratio for purified BC was lower than the theoretical value for pure cellulose (O/C = 0.83, as Topalovic et al. (2007) described. This difference between the theoretical and experimental O/C ratio is associated with contaminants on the BC membrane surface, such as other polymers, hydrocarbons or protein residues resulting from the fermentation process (Matuana et al., 2001). The BC membrane had an atomic composition of: 60.2% carbon, 34.4% oxygen, 4.9% nitrogen, and 0.5% sodium (residual traces of purification). The presence of N on the membrane surface is associated with amino acids, constituents of the protein structure of bacteria still present in the membrane structure after its purification. It has been reported that O/C values are influenced by process efficiency and purification methods (Fras et al., 2005; Li et al., 2009; Topalovic et al., 2007).
The surface O/C ratio of OxBC membranes increased compared to BC. This is due to the removal of protein contaminants by the periodate oxidation reaction. BC oxidation occurs by modifying only the bonds and valence states of the atoms and maintains the same number of carbon and oxygen atoms as the non-oxidized cellulose structure. OxBC-MW-0.75% (60.1 % of carbon and 39.9% of oxygen), OxBC-MW-1% (62.5% of carbon and 37.5% of oxygen), and OxBC-WB-0.75% (56.1% of carbon and 43.9% of oxygen) showed only carbon and oxygen in their atomic composition, confirming the absence of protein residues and other contaminants on their surfaces.
Vasconcelos et al., (2020) reported a decrease in the O/C ratio of periodate oxidized OxBC membrane (O/C = 0.40). This decrease is related to a hydrolysis process due to the acidic reaction medium (pH = 1), which results in cellulosic material degradation. Li et al. (2009) reported a decrease in the O/C ratio in BC membranes that were degraded in physiological buffers immersed for a long time, suggesting that such behavior is directly associated with the physical degradation of the material.
The oxidation conditions performed by Vasconcelos et al. (2020) employed wet BC membranes, reaction time of 6 hours, temperature of 55 ºC and periodate concentration of 1% (w/v). All the OxBCs obtained in our study had a higher surface O/C ratio than the OxBC obtained by Vasconcelos et al. (2020), indicating that the conditions studied (NaOI4 concentration and heating method) promoted a lower degradation capacity to the material, although it used a higher temperature (90 ºC) and a shorter reaction time (30 minutes). OxBC-MW-0.75% and OxBC-WB-0.75% membranes exhibited the highest surface O/C ratio values compared to the other samples. Therefore, the oxidation of wet BC membranes using a lower concentration of NaIO4 reduces the degradation process, preserving the physical structure of the cellulose membrane.
To better understand the surface composition of membranes, high-resolution C1s spectra were recorded, as shown in Figure 2. The deconvolution of the spectra exhibited four C1s contributions with different intensities, indicating a change in the valence band of the carbon occupied by the oxygen after the periodate oxidation reaction. The first contribution (C1) at 284.8 eV represents the C-H and C-C- bonds (Miller et al., 2002). Therefore, C1 was used as a binding energy reference for the deconvolution of the spectra. The second (C2) at 286.4 eV and the third (C3) at 287.8 eV contributions represent the C-O and O-C-O/C=O bonds, respectively. The fourth (C4) at 289.9 eV contribution represents the most oxidized carbon, O-C=O.
The XPS spectrum for purified BC showed three types of contributions: C1 (33.64%), C2 (47.79%) and C3 (18.57%) on its surface structure. These contributions in BC are attributed to the bonds resulting from the formation of the D-glucopyranose ring, in the alcohol groups, and in the acetal groups (glycosidic bond), respectively (Matuana et al. 2001; Coseri et al. 2013; Barbosa et al. 2019). C2 is the contribution with the highest relative intensity observed in the structure due to the presence of alcohol groups that prevail in the D-glucopyranose ring. Several BC studies report the presence of these binding energy values in high-resolution C1s core level spectra (Shao et al. 2017; Wan et al. 2019; Sun et al. 2019).
The XPS spectra for the OxBC samples showed four types of contributions: C1, C2, C3 and C4 and the percentage of these contributions can be seen in Figure 2b, 2c and 2d. For all samples, C2 is the major peak. OxBC-WB-0.75% and OxBC-MW-0.75% show an increase in the contribution of C2 when compared to BC. On the other hand, OxBC-MW-1% presented a reduction in the percentage of C2 compared to BC due to the oxidation of the aldehyde groups to carboxylic acid, contributing to an increase in the percentage of C4. A considerable increase in the percentage of C3 in all oxidized samples is also seen, attributed to the formation of aldehyde (H-C=O) in the structure of OxBCs. Therefore, the increase in the percentage C3 demonstrates the oxidation efficiency on the OxBC membrane surface. C4 is present in the spectra of OxBCs, but not present in BC and indicates a secondary oxidation of aldehyde groups to carboxyl groups (O-C=O). The presence of carboxylic acid in OxBCs may result from the high temperature used in the oxidation reaction, as Klemm et al. (1998) reported.
Carbons with a higher binding energy (such as O-C-O/C=O and O-C=O bonds) are also in a higher oxidation state. The change in oxidation state was illustrated by the ratio (C3 + C4) / C2 (Luz et al., 2020), as shown in Table 1. The progressive increase in these values corresponds to a greater oxidation level in the wet BC membranes, confirming the difference in the oxidation profile promoted by studied reaction parameters.
3.2 Oxidation degree
The OD values of the OxBC samples are shown in Table 1. The use of microwave irradiation as a heating method promoted higher degrees of oxidation when compared to the water bath (commonly used heating method). OxBC-MW-0.75% has an aldehyde content 35% higher than OxBC-WB-0.75%, while the aldehyde content of OxBC-MW-1% is double the value obtained for OxBC-WB-1%. The periodate concentration associated with the heating method also promoted a significant difference in the OD values, with the samples oxidized to 1% (w/v) of NaIO4 the ones with the highest degree of oxidation.
The water bath (conventional heating) is a combination of conduction and convection. These heat transfer methods depend on the existence of a temperature gradient. The microwave (dielectric heating) uses radiation from electromagnetic waves, which induce the rotation of polar molecules, transforming kinetic energy into heat. This heating method is direct, speedy and independent of temperature gradients (Nüchter et al., 2004). These particular aspects of the heat transfer process promoted by microwave use allow a chemical reaction to achieve the desired yield in shorter time intervals than conventional heating methods. Successful reduction of overall reaction times has been previously reported for other cellulose modification methods (Lin et al., 2017; Satgé et al., 2002).
Vasconcelos et al. (2020) carried out periodate oxidation at 1% (w/v) of wet BC membranes using 6 hours of reaction at 55ºC and obtained a lower degree of oxidation (OD = 50%) when compared to OxBC-MW-1%. Thus, the concentration of 1% (w/v) of NaIO4 and microwave use at 90 ºC for 30 minutes was adequate to oxidize wet BC membranes, promoting a considerable reduction in reaction time and energy expenditure.
Oxidation of the BC wet membrane tends to occur heterogeneously. During cellulose biosynthesis by bacteria, membranes are organized into layers, forming a porosity gradient along their thickness (Vasconcelos et al., 2020). The membrane's outer and most porous layers have a higher surface/volume ratio and are more exposed to the reaction medium, suffering more oxidation than the inner and less porous layers. Thus, the wet membrane tends to form a more functionalized surface ("wrap") with distinct properties concerning the interior and center of the membrane.
3.3 Physical and morphological characteristics
XRD analyses were performed to assess the crystallinity of the material after chemical modification, which is an essential factor influencing the membrane's mechanical properties. The X-ray diffractograms for BC and OxBC samples are shown in Figure 3. The XRD patterns showed three major 2θ peaks at 14.4º, 16.7º and 22.6º, which correspond to crystallographic planes with Miller indices (100) (010) and (110). These peaks were similar for all samples and characterized the type Iα cellulose polymorphism with a triclinic unit structure (Hult et al., 2003). Polymorph Iα is more common in BC while Iβ, which has similar peak positions, is mostly common in pant cellulose (Horii et al. 1987; Sugiyama et al. 1991; Nishiyama et al. 2003). For the crystal structure of cellulose Iα given by Nishiyama et al. (2003) and , the peaks (100) and (010) they have approximately the same intensity, whereas in the diffractograms obtained from the samples the reflection (100) is considerably stronger. This difference in peak intensities (100) and (010) can be attributed to a preferential degree of orientation of the unit cells, which couple/structure favorably in a worsening direction to form energetically effective crystal structures. Similar cases can be seen in other studies on cellulose, such as
The intensity of the peaks in the XRD patterns empirically describes the organization of crystalline blocks along the structure. As already reported in the literature, cellulose has ordered regions interspersed with disordered (amorphous) regions, so that chemical changes occur preferentially in disordered regions, as it has less stability (Calvini et al., 2006; Vasconcelos et al., 2017). For the crystal structure of cellulose Iα given by Nishiyama et al. (2003) and French (2014), the peaks (100) and (010) they have approximately the same intensity, whereas in the diffractograms obtained from the samples the reflection (100) is considerably stronger. This difference in peak intensities (100) and (010) can be attributed to a preferential degree of orientation of the unit cells, which couples/structures favorably in a prioritized direction to form crystalline packings. Similar cases can be seen in other studies on cellulose, such as Vasconcelos et al. (2017) for BC/nanowhiskers and Vasconcelos et al. (2020) for BC and OxBC.
The insertion or replacement of clusters in the cellulose promotes a decrease in the relative intensities of these peaks, which reveals an alteration in the geometry or symmetry of the crystal, altering its preferential orientation of the unit cells and its crystalline packing (Vasconcelos et al., 2019). The peak intensities (110) of BC and OxBCs did not change significantly, inducing that periodate oxidation of the wet membrane of BC did not change the crystal structure of the material. However, comparing the peaks (100) and (010) between BC and OxBC, it is possible to observe a difference in their intensities, indicating that the oxidation affected the preferential orientation of the unit cells, with the processes being at 1% (w/v) periodate the most remarkable.
The crystallinity index allows the assessment of the change in crystal structure numerically. The CrI values obtained for BC and OxBCs are shown in Table 1. The samples oxidized by a water bath had a reduction in CrI values, changing the orientation of their atoms and the integrity of the crystal structure (Castro et al. 2011). Periodate oxidation promotes the cleavage of the C – C bond between vicinal diols of D-glucopyranose molecules, forming an open structure with two aldehyde groups. Due to this ring-opening, the crystal packaging structure is altered, affecting its crystal structure (Kim et al. 2000). The more substitutions that occur in the crystal structure (the higher the OD value) the smaller the CrI value will be, as can be seen by comparing BC-BW-0.75% and BC-BW-1%. On the other hand, the microwave oxidized samples presented CrI values close to BC, indicating that the chemical substitutions occurred mainly in the available hydroxyl groups located on the surface of the crystalline blocks, without altering their packing.
The CrI result obtained for BC was similar to that reported by Vasconcelos et al. (2020). However, the oxidation conditions (55°C for 6 hours) by the authors provided OxBCs with a lower crystallinity index (CrI = 62%) compared to the samples OxBC-MW-0.75% (CrI = 86%) and OxBC-MW-1% (CrI = 87%).
Thermogravimetry provides information about chemical interactions in the molecule, thermal stability of cellulose and inorganic content (purity) (Liu & Yu, 2006). Figure 4 shows the TG curves and their respective DTG for purified BC and OxBCs.
The thermogravimetric curves of the samples showed three mass loss events, as seen through the DTG curves. The presence of these three thermal mass loss events for oxidized BC (DAC) has been reported in the literature (Kim & Kuga, 2001). The first event occurs between 50 and 150 ºC and is due to the evaporation of wastewater from the material, given the hydrophilic characteristic of cellulose. This event is more pronounced for OxBCs and indicates that oxidized samples had a higher residual water content in their structure, due to the presence of hydrated aldehydes (Siller et al., 2015). This grouping promotes a more hydrophilic characteristic to oxidized cellulose than the hydroxyl groups present in non-oxidized cellulose.
The second event occurs between 150 and 350 ºC, where the most remarkable mass loss is observed, and corresponds to the depolymerization of the carbon chain (including breakage in intermolecular and intramolecular interactions) and decomposition of the short chains and monomeric units of the cellulosic material. The initial degradation temperatures (Tonset) of BC and OxBCs were observed in this event, shown in Table 1. The Tonset observed for the OxBC samples was lower (about 32 ºC) than the purified BC, suggesting that the oxidation process decreased the material's thermal stability due to changes in the crystal structure, as seen in the XRD data. These structural changes may occur due to cleavage of D-glucopyranose rings, breakage of hydrogen bonds between cellulose chains and breakage of β1,4-glycosidic bonds (Agustin et al., 2016). Vasconcelos et al. (2020) reported a 54 ºC decrease in the thermal stability of purified BC after periodate oxidation. The literature reported that the greater the degree of substitution in cellulose, the lower the Tonset values. However, although microwave irradiation generated higher oxidation levels in wet BC membranes, it did not promote reductions in Tonset values. A similar result was published by Siller et al. (2015) with the carboxymethylation of cellulose using microwaves. In general, all samples showed thermal stability that allows the performance of physical processes involving temperatures above 200 ºC, such as extrusion (production of plastics) and sterilization by autoclave (necessary for biological applications).
The third and last event occurs between 350 – 500 ºC and can be attributed to the oxidation and degradation of the products generated in the second event, in which they are carbonized, producing gases and low molecular weight residues (inorganic products). BC and OxBCs had a residual mass of less than 1% ash, indicating the high purity of membranes after purification and oxidation processes.
Scanning electron microscopy (SEM) allows for assessing the morphology of the membranes surface, which can be seen in Figure 5. BCs oxidized in 1% (w/v) periodate showed the highest OD values. Therefore, the samples OxBC-BW-1% and OxBC-MW-1% were selected to verify if the heating methods used in the oxidation promoted morphological changes in the membranes.
Figure 5a corresponds to purified BC in which it presents a uniform and intertwined nanofibrils, forming a three-dimensional and porous network, as reported in the literature (Shao et al., 2017; Vasconcelos et al., 2020). The random orientation of the fibrils responsible for the structure of the network is due to the sporadic movement of bacteria in the fermentation medium (Wang et al., 2018). BC nanofibrils ranged from 17 to 110 nm, presenting an average diameter of 48 ± 15 nm. This nanometric structure of nanofibrils is due to the diameter of the pores of the bacterial perplasmic membrane that varies from 1.5 – 3.5 nm (Römling & Galperin, 2015). These fibrils are randomly excreted and aggregate to form nanofiber strands with a width of 20 to 100 nanometers (Wang et al., 2019).
Figures 5b and 5c correspond to OxBC membranes oxidized to 1% (w/v) of NaOI4 obtained by water bath and microwave, respectively. The oxidized samples kept the three-dimensional network of fibrils; however, it is possible to observe an aggregation and shortening of the fibrils. These factors are responsible for the "shrinkage" of the wet BC membrane after periodate oxidation.
The fibrils of OxBC-BW-1% ranged from 30 – 123 nm, with a mean diameter of 68 ± 22 nm and the fibrils of OxBC-MW-1% ranged from 37 – 197 nm, with a mean diameter of 86 ± 22 nm. This increase in the mean diameter concerning BC is related to the aggregation process observed in SEM micrographs. The higher the OD value (more oxidized) the greater will be the aggregation of fibrils and, consequently, the greater will be its average diameter, as observed in the samples.
The shortening of fibrils results from the hydrolysis process that breaks the glycosidic bonds that form the cellulose chain. This process promotes the loss of mass in the wet membrane (as reported by Vasconcelos et al. (2020) and is accentuated by the association of two factors: 1) the oxidation reaction occurs in a highly acidic medium and 2) the use of a temperature above 40 °C (Margutti et al., 2001). The shortening of nanofibrils was more expressive in OxBC-WB-1%. This suggests that oxidation by a water bath (convection transfer) at 90 ºC is more severe and favors the membrane hydrolysis process. This process causes changes in the porosity of never-dried membranes, one of BC’s more important properties in various applications.
Microwave-assisted periodate oxidation allows reaching high degrees of oxidation, using a shorter reaction time than the reaction conditions usually employed, such as Vasconcelos et al. (2020). In addition, this approach promotes the obtainment of a wet OxBC membrane, with a more preserved nanofibrillar structure, which leads to mechanical and thermal properties close to those of non-oxidized BC.