Synthesis and characterization of proton-conducting membranes based on bacterial cellulose and human nail keratin

2.1 Materials

The materials used in this study were human nails, acetone (Sigma-Aldrich), sodium hydroxide (Merck), dimethyl formamide (Merck), ammonium sulfate (Merck), glacial acetic acid (Merck), Acetobacter xylinum, granulated sugar, and coconut water. Samples of human nails were obtained from family members, beauty salons, and other students. Meanwhile, coconut water was obtained from the Market in Bengkulu City.

2.2 Human nail powder preparation and keratin isolation

The keratin isolation process was as in previous research reports (13,14). In the first step, 30 g of the fine nail powder sample was dissolved in 150 mL of 1 M sodium hydroxide at 70°C until it completely dissolved. Then, 6 mL of glacial acetic acid was added to neutralize the solution. The mixture was heated at 70°C in a water bath until a gel was formed. Then, the gel was heated on a hotplate stirrer at 70°C until dry and then it was mashed using a blender. The keratin isolated from human nails was ready to be used for the next stage.

2.3 Production of bacterial cellulose

The bacterial cellulose was prepared according to the method by Sawitri et al. (10). First, 3 L of coconut water was boiled. Then, 300 g of granulated sugar and 15 g of ammonium sulfate were added, and the mixture was transferred to a container (27 cm × 21 cm × 3.5 cm) and covered with paper. When the mixture was almost cold, 30 mL of glacial acetate was added. After cooling to room temperature, bacterial cellulose was inoculated with 80 mL of Acetobacter xylinum starter and fermented at room temperature for 10 days.

2.4 Bacterial cellulose purification

The bacterial cellulose gel obtained from fermentation was soaked in water for 2 h. Then, it was soaked in distilled water at 80–90°C for 2 h. After that, it was washed with 2% NaOH at 80–90°C for 2 h. Finally, it was soaked again in distilled water at 80–90°C for 2 h.

2.5 Composite membrane synthesis

The synthesis of composite membranes with bacterial cellulose was carried out by blending the material in a blender until it was mushy and then it was filtered. The human nail keratin powder was dissolved in 2 mL of dimethyl formamide in a beaker. The synthesis was carried out based on the mass variation between cellulose and human nail keratin of 4.5:0.5, 4.3:0.7, and 4:1, respectively. After that, it was stirred until homogeneous and printed in a 5 cm × 5 cm Petri dish. Then, it was heated on a hotplate at a heating temperature of 40°C.

2.6 Characterization

Functional groups and interactions between bacterial cellulose and human nail keratin on proton conductive membranes were characterized using a Bruker Alpha-P FTIR spectrometer (Wismar, Germany) in the total attenuated reflectance range of 4,000–400 cm⁻¹. The diffraction patterns of the materials were analyzed using an X-ray diffractometer (Rigaku D-MAX 2200, Japan) with Cu Kα radiation (λ = 1.5406 A) over a 2θ range between 0° and 80°. The conductivity of the membrane was measured using IM 3590 Chemical Impedance Analyzer HIOKI. The membrane’s conductivity was evaluated at 1 kHz, 0.05 V, and at temperatures between 25°C and 80°C. The degree of swelling was determined by cutting the membrane to a size of 1 cm × 1 cm and was heated in an oven at 60°C for 5 h. It was then weighed to obtain the dry mass (m _dry). Then, the membrane was immersed in distilled water for 24 h at room temperature. The membrane surface was dried using tissue paper and weighed (m _wet). The following formula was used to calculate the value of the degree of swelling:

3.1 Human nail powder

The human nail powder was prepared by hydrolysis with 150 mL of 1 M sodium hydroxide at 70°C. Figure 1 shows the results of human nails that were made into powder (Figure 1a) and the results of keratin isolation (Figure 1b). After the nail powder was added to the sodium hydroxide solution, the pH of the mixture changes to alkaline. Then, glacial acetic acid was added until the pH was neutral. In this process, a hydrolysis reaction occurs between the strong base NaOH and glacial acetic acid, which produces sodium acetate. Finally, the mixture was heated to form a gel which was then dried and crushed to yield the keratin hygroscopic powder.

Figure 1

(a) Human nail powder and the (b) results of keratin isolation.

3.2 Bacterial cellulose production

The production of bacterial cellulose is the result of a fermentation process with the Acetobacter xylinum bacterium, which converts sugar into cellulose. It is this cellulose fiber that can make nata appear white. This result is similar to the ones that were reported earlier (9,10). In producing cellulose, these bacteria need nutrients such as carbon from glucose and nitrogen from ammonium sulfate. Then, acetic acid was added at pH 4 followed by glacial acetic acid, and then inoculated with Acetobacter xylinum and incubated for 10 days. The cellulose formed is then purified through a washing process with distilled water in the temperature range of 80–90°C. Then, the cellulose was washed with 2% NaOH at 80–90°C. The use of this temperature range is important because, at this temperature, the NaOH solution can penetrate better between the cellulose fibers to hydrolyze the bacterial cells and remove impurities in the formed bacterial cellulose. Then, the cellulose gel was soaked again in distilled water for 2 h in the same temperature range to get a whiter gel.

3.3 Composite membrane synthesis

The synthesis of composite membranes aims to improve physicochemical properties compared to its constituent polymers as well as provide better conductivity. It can also be applied as membranes in fuel cells. Composite membranes were made with three variations of human nail cellulose/keratin composition based on the mass ratio, 4.5:0.5, 4.3:0.7, and 4:1. The composite membrane synthesis begins with the purification of bacterial cellulose which aims to increase the reactivity of bacterial cellulose during the reaction. Then, the keratin powder was dissolved first in dimethyl formamide. Then, crushed cellulose was added, the membrane was made by imprinting it on a 6 cm diameter Petri dish and the solvent was evaporated on a hot plate at 35°C. A temperature of 35°C was then used to obtain a flat surface membrane.

The membranes (4.5:0.5, 4.3:0.7, and 4:1) obtained were in the form of brownish-yellow sheets with the thicknesses of each composite membrane, respectively, being 0.33, 0.42, and 0.51 mm. The membranes shown in Figure 2b and c have a better appearance, which has a flatter and smoother surface than the composite membrane (Figure 2d).

Figure 2

(a) Bacterial cellulose/composite membrane ratios: (b) 4.5:0.5, (c) 4.3:0.7, and (d) 4:1.

3.4 Characterization of the composite membrane

As shown in Figure 3, from the cellulose spectrum obtained, it can be seen that the characteristic absorption peak of bacterial cellulose has an absorption band of the hydroxyl group, namely the O–H stretching, which occurs at a wavenumber of 3,341 cm⁻¹. The peak of the stretching vibration of C–H appears in the wavenumber range 2,898–2,553.14 cm^-1 and that of the C═C stretching group at 1,584.539 cm⁻¹. The peak of CH buckling appears at 1,330.02 cm⁻¹, that of the bending C–C group at 1,157.61 cm⁻¹, and that of the stretching C–O–C vibrations at 1,050.88 cm⁻¹. The spectrum is similar to that previously reported by Sawitri et al. (10). While the spectrum for human nail keratin in Figure 3 shows a peak at 3,062.347 cm⁻¹ which is of the C–H stretching vibration absorption band. At a wavelength of 1,633.797 cm⁻, a peak of the stretching vibration of C═O of the amide I group is observed, and at 1,543.489 cm⁻¹, the peak of the bending vibration of N–H amide II is observed, which is a characteristic of keratin. At a wavelength of 1,239.717 cm⁻¹, the peak of the N–H absorption band of bending of amide III appears, and at a wavelength of 1,124.776 cm⁻¹, there is the absorption band of the stretching vibration of C–O–C. When compared with the previous studies of the same FTIR results, it can be concluded that the isolation results obtained were most likely of keratin (13,14).

Figure 3

FTIR spectra of bacterial cellulose, human nail keratin, composite membrane: 4.5:0.5, 4.3:0.7, and 4:1.

The functional group analysis of bacterial cellulose and human nail keratin composite membranes shows the effect of adding human nail keratin to the composite membrane. From the spectra of the functional group analysis, it is evident that the composite membrane contains constituent polymers, namely cellulose–keratin. However, there are some differences in wavenumbers when compared to the constituent materials. If there is a shift in the wavenumber, this occurs due to interactions between the polymers that make up the composite membrane. Thus, this interaction can also affect the characteristics of the composite membrane. The human nail keratin spectrum in the area around 3,341 cm⁻¹ shows that the band was widened. This is due to the influence of water on the hygroscopic nature of keratin. Previous research reports have shown the interaction of two materials linked by the presence of hydrogen bonding. The wavenumber between 3,000 and 2,000 cm⁻¹ showed a broadening of the band that can be related to hydrogen bonding network formation. The peaks near 1,100 and 979 cm⁻¹ are attributed to characteristic absorptions formation from the composite membrane (15) as shown in Figure 4. This report has examined the relationship between conductivity and temperature based on the following equation:

Figure 4

Bacterial cellulose–keratin interactions.

Further details are shown at the end of the discussion. Many studies related to polymer–polymer interactions have been reported, such as the performance and stability of mixed matrix membranes by thermodynamic compatibility review (16,17,18).

Figure 5 shows the results of the X-ray diffractogram pattern of bacterial cellulose, human nail keratin, and composite membrane: 4.5:0.5, 4.3:0.7, and 4:1. The results of X-ray analysis showed that the amorphous polymer produced broad peaks in bacterial cellulose. The purity was semicrystalline, which showed absorption peaks in the 2θ angle range of 10–30°. As previously reported (19), it is known that the intensity of cellulose shows a semi-crystalline structure. The semi-crystalline structure is due to the high intermolecular forces caused by the hydrogen bonding of adjacent hydroxyl groups.

Figure 5

X-ray diffractogram of bacterial cellulose, human nail keratin, composite membrane: 4.5:0.5; 4.3:0.7; and 4:1.

Human nail keratin diffractogram from XRD analysis shows that one of the highest peaks at an angle of 2θ was at 20.24°. Keratin isolated from human nails showed that there were no differences in the peaks of each area of the structure. Keratin from human nails has a wide peak with a low intensity of crystalline phase so it has a semicrystalline structure type. The predominance of the amorphous phase causes it to have more free space allowing an increase in ion movements, in agreement with the previously reported pattern of the keratin diffractogram (20).

Figure 5 shows that the X-ray diffractogram pattern of the composite membrane, 4.5:0.5, 4.3:0.7, and 4:1, indicates a semicrystalline phase. Materials with a strong structure due to high crystallinity certainly have high-pressure resistance, compared to materials with irregular structures, and provide a lot of space around them. The addition of amorphous properties to materials that have high crystallinity will convert compounds that were originally in the form of crystals to a semicrystalline phase.

The peak of the diffractogram pattern with variations in the 4.5:0.5 composite membrane showed angles of 16.97° and 22.77° with intensities of 446.82 and 679.15. In the diffractogram of the 4.5:0.5 composite membrane, the peaks are sharper than the diffractograms composite membrane 4.7:0.3 and 4:1. The diffractogram composite membrane 4.3:0.7 shows the maximum intensity at an angle of 23.08°, which is 361.71 and forms a semicrystalline phase. The results of the diffractogram 4:1 +composite membrane show the maximum intensity at 2θ with an angle of 22.97°, which is 399.41. Among the three variations of composite membrane 4.5:0.5, 4.3:0.7, and 4:1, the diffractogram composite membrane 4.5:0.5 has the sharpest peak compared to the other two composite membranes, which is due to the addition of bacterial cellulose and is more than the composite membrane 4.3:0.7 and 4:1. In the diffractogram composite membrane 4:1, the resulting peak is quite wide, which is the amorphous phase of the human nail keratin mass. It is higher compared to the diffractograms composite membrane of 4.5:0.5 and 4.3:0.7.

The degree of swelling is important because it can affect the flow of protons in the membrane. Figure 6 shows the degree of swelling of bacterial cellulose and composite membranes 4.5:0.5, 4.3:0.7, and 4:1. The degree of swelling of bacterial cellulose and composite membranes 4.5:0.5, 4.3:0.7, and 4:1 produced, respectively, 23.07%, 26.31%, 22.46%, and 32.50%. The average degree of swelling of the composite membrane obtained is close to the swelling degree of Nafion by 30%. This phenomenon indicates that the more keratin added, the higher the degree of swelling in each membrane. The increase in the degree of swelling is influenced by keratin, which has cellulose as the property of high water absorption capacity. Keratin is also hygroscopic or has the ability to absorb water molecules in the air. These results are almost similar to previous research reports (14).

Figure 6

Degree of swelling of bacterial cellulose, Nafion, composite membrane: 4.5:0.5, 4.3:0.7, and 4:1.

From Figure 7, it can be seen that the bacterial cellulose membranes have a proton conductivity of 2.213 × 10⁻⁵S·cm⁻¹ at 25°C. When compared to the results of cellulose synthesis in previous studies, the proton conductivity produced from cellulose is 1.7 × 10⁻⁵S·cm⁻¹ (6). Then, the proton conductivity results from this study are slightly larger so that this bacterial cellulose membrane can be applied as the proton conducting membrane. In a previous study, the proton conductivity of the Nafion membrane was 0.1 S·cm⁻¹ at a temperature below 100°C (21). The highest proton conductivity of the composite membrane was in the variation of the mass of 4:1, namely at a temperature of 25°C with proton conductivity of 4.572 × 10⁻⁵ S·cm⁻¹. In the mass ratio of the composite membrane of 4.3:0.7, the highest conductivity was 3.006 × 10⁻⁵ S·cm⁻¹ at 25°C. In the mass ratio of the composite membrane of 4.5:0.5, the highest proton conductivity was 2.213 × 10⁻⁵ S·cm⁻¹ at 25°C. This shows that the proton conductivity was influenced by the amount of bacterial cellulose and human nail keratin. Keratin could increase the proton conductivity due to the large number of amine groups in keratin so that protons would more easily move from one group to another. Proton conductivity on interactions between macromolecules containing hydrogen bonds has been reported in previous studies (22,23). The cellulose and keratin in the composite membrane are also the factors affecting the proton conductivity because the higher the degree of swelling, the higher the proton conductivity. This is due to the large number of water molecules in the membrane that can be a proton transfer medium. Based on the results of calculating the activation energy E _a of each mass ratio using σ = σ 0 exp − E a / RT , it can be seen that there is a tendency for an increase in the activation energy with increasing human nail keratin added to the composite membrane. This result is in line with the maximum proton conductivity that occurs when the mass ratio of bacterial cellulose/human nail keratin is 4:1. The effect of adding human nail keratin to bacterial cellulose on the activation energy is shown in Figure 8.

Figure 7

Proton conductivity of bacterial cellulose, composite membrane: 4.5:0.5, 4.3:0.7, and 4:1.

Figure 8

The effect of adding human nail keratin to bacterial cellulose on the activation energy.