AMIDATION AS A TOOL FOR MODULATING BACTERIAL CELLULOSE NANOFIBERS HYDROPHOBICITY

Autores

Gamboa Suárez, M.A. (UNIVERSIDAD INDUSTRIAL DE SANTANDER) ; Combariza, M.Y. (UNIVERSIDAD INDUSTRIAL DE SANTANDER) ; Blanco Tirado, C. (UNIVERSIDAD INDUSTRIAL DE SANTANDER)

Resumo

Bacterial cellulose (BC) is a biodegradable polymer that can be synthesized from agro-industrial waste. However, its hydrophilic nature limits its application in areas such as food packaging, where moisture control is essential. In this contribution, we test the effect of BC surface modification on the production of a hydrophobic material. The formation of amides, from amines coupled to carboxylic units of oxidized cellulose BC-TEMPO (BC-TOCN), allows modulating its hydrophilic properties. Characterization by IR, TGA, SEM and DLS suggests an effective modification of its surface. Contact angle (CA) and surface free energy (SFE) analysis indicate changes in hydrophobicity. Amidation increases the contact angle from 29° for BC-TOCN to 80° with BC-TOCN-AMD C-12 and 147° with BC-TOCN-AMD C-18.

Palavras chaves

Bacterial cellulose ; amidated cellulose ; hydrophobization

Introdução

Plastic waste's pollution of surface waters is rising at the planetary level. Synthetic plastics are recalcitrant to microbial degradation and generate CO2 emissions causing negative impacts on ecosystems (COSTELLO, et al., 2009; SHEN, et al., 2020). Biodegradable polymers (PBs) emerge as a solution to pollution by synthetic plastic waste. The production of PBs by biotechnological pathways using inexpensive carbon sources, especially by-products of agro-industrial processes, is a very active research area. Colombia is a country with an agricultural vocation with abundant residual biomass. Cacao beans production generates by-products such as cocoa mucilage exudate (CME), a liquid with high nutrient content, valuable as a carbon source for culture media in biotechnological processes. We previously demonstrated the use of CME for bacterial cellulose (BC) production. Structurally similar to plant cellulose, BC is a linear homopolymer formed by condensation of glucose molecules. BC is produced, by static or dynamic fermentations, by a group of microorganisms, among which the Gluconacetobacter xilynus stands out (SAAVEDRA SANABRIA, et al., 2021). BC exhibits good mechanical properties, high crystallinity, non-toxicity, biocompatibility, and biodegradability. BC finds uses in many areas, such as cosmetics, pharmaceuticals, biomaterials, and medicine (WANG, TAVAKOLI, & TANG, 2019). However, BC's hydrophilic nature restricts its use in specific applications such as food packaging, particularly for products susceptible to moisture damage. Thus, BC hydrophobization can modulate its hydrophobic character and improve its properties as an additive in biodegradable flim packaging materials (BALASUBRAMANIAM, PATEL, & NAYAK, 2020). Currently, several techniques are documented to improve BC´s moisture barrier capacity, such as esterification with organic acids (PENG, CHANG, KUMAR, MOON, & YOUNGBLOOD, 2016), amination (SIRVIO, VISANKO, LAITINEN, AMMALA, & LIIMATAINEN, 2016) and surface amidation (TAUBNER, COPÍKOVÁ, HAVELKA , & SYNYTSYA, 2013). We previously reported a surface amidation strategy to modify commercial microcrystalline cellulose, and Fique fibers cellulose using TBTU uronium salt [O-(1H benzotriazol-1-yl)-N, N,N',N'-tetramethyluronium tetrafluoroborate] as coupling agent, TEMPO-oxidized nanocellulose (BC-TOCN), and a primary amine (GÓMEZ, COMBARIZA, & BLANCO TIRADO, 2017). The modified material exhibits increased contact angle values, up to five times higher than the CA for the unmodified BC. This material displays favorable properties such as biodegradability and hydrophobicity, making it a potential additive for food packaging. On the other hand, BC is produced using CME, a residual by-product from the cocoa bean processing, thus avoiding the negative environmental impacts associated with discarding effluents with a high organic load into soils and surface water.

Material e métodos

• Bacterial cellulose The BC was biosynthesized by Gluconacetobacter xylinus in reactors with an operating volume of 3 L containing a supplemented medium and CME as a carbon source. BC is produced extracellularly; the film is washed and sterilized once the process finishes. • Bacterial cellulose TEMPO oxidation TEMPO ( 0.016 g) and NaBr (0.1 g) were added to a 1% (w/v) BC aqueous suspension. NaClO was added to the mixture dropwise, maintaining a molar ratio of 1:6 cellulose: NaClO. The reaction flask was placed in an ultrasonic bath for four hours while keeping the pH at 10.5 by NaOH addition. pH stabilization indicates reaction completion; at this point, ethanol is added to stop the catalytic reaction cycle. The reaction mixture was centrifuged, and the solid was washed with water and HCl (0.1 M) until neutral pH. Finally, an ultrasonic probe was used to obtain a uniform dispersion of BC-TOCN. • BC-TOCN amidation TBTU dissolved in dimethylformamide (DMF) was added to a BC-TOCN solution in a 2:1 TBTU:BC-TOCN molar ratio. The reaction mixture was stirred at room temperature for 30 minutes, keeping the pH basic. A primary amine solution (dodecylamine or octadecylamine) in DMF (molar ratio 4:1 amine group:COOH units in BC-TOCN), was added under constant stirring at room temperature for 2 hours. The solution was then filtered and washed with methanol, HCl, and water to eliminate unreacted amines. Finally, frozen and lyophilized materials were labeled BC-TOCN AMD C-12 and BC-TOCN AMD C-18. • Characterization The degree of oxidation and the colloidal stability of the BC-TOCN were evaluated by means of conductometric titration curves and ζ potential measurements, respectively. Thermal stability was also tested by thermogravimetry (TGA). The morphological changes and dimensions of the modified cellulose surface were observed by scanning electron microscopy (SEM). Likewise, its structure was studied by means of infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). Additionally, the hydrophobicity of the materials was evaluated with contact angle measurements.

Resultado e discussão

Bacterial cellulose TEMPO oxidized nanofibers (BC-TONC) exhibit a degree of oxidation (OD) of 0.26 and 1.53 mmol of COOH/g of cellulose. These characteristics indicate a reactive surface loaded with carboxylic groups, suitable for amide bonds formation. Figure 1 shows the IR spectrum of all the synthesized materials, together with a table showing the most characteristic bands. For BC, the broad band at 3341 cm-1 corresponds to stretching vibrations of the O-H bond that is present in the cellulose structure. The small signal seen at 2882 cm-1 is characteristic of stretching of the C-H bond (OH, et al., 2005). Additionally, the band at 1630 cm-1 is due to the bending of the H-O-H bond of water, given BC-TOCN´s highly hydrophilic nature. The band at 1316 cm-1 corresponds to the (C6) CH2 stretching. Additionally, at 1159 and 1107 cm-1 signs of stretching and symmetric and asymmetric elongations of the C-O-C were observed. The signals at 1056 and 1031 cm-1 are characteristic of skeletal vibrations involving C-O stretching of the pyranose ring (KONDO & SAWATARI, 1996; DUBEY, et al., 2017). On the other hand, for the BC-TOCN, the band at 1601 cm-1 corresponding to the COO-stretching is observed, which confirms the presence of carboxylate moieties in BC-TOCN. Additionally, in this band a shoulder is observed at 1632 cm-1, which is due to the presence of aldehyde and carbonyl groups, which end up overlapping (CHITBANYONG, PISUTPICHED, KHANTAYANUWONG, THEERAGOOL, & PUANGSIN, 2020). Additionally, in BC-TOCN-AMD-C12 and C18, as a product of the amidation of the material, a high increase in the signals attributed to the stretching of the methyl (–CH3) and methylene (–CH2) groups is observed at 2954 y 2850 cm-1 of the aliphatic chains of the amines. And significantly, the signals that confirm the presence of the amine group correspond to the overlapping broad band of amide I and amide II at 1643 y 1569 cm-1 respectively (LASSEUGUETTE, 2008; GÓMEZ, COMBARIZA, & BLANCO TIRADO, 2017). Likewise, Figure 1 shows the SEM images of these materials. In the BC (Figure 1.a) the morphology of the bacterias that produce bacterial cellulose, the gluconacetobacter xylinus, can be observed, even adhered to it since its production occurs extracellularly (SAAVEDRA SANABRIA, et al., 2021). In addition, when comparing the change that occurred after oxidation via TEMPO and mechanical defibrillation (Figure 1.b), the separated BC-TOCN can be observed forming networks, with diameters at the nanometric scale. Researchers report similar morphologies and sizes, such as those reported by OVALLE SERRANO, GÓMEZ, BLANCO TIRADO, & COMBARIZA (2018) who observed diameters around 100 nm, and those reported by LIU, et al., (2016) with diameters between 200 nm and 1.5 μm. Additionally, for BC-TOCN-AMD-C12 and C18 (Figure 1.c.d) the cellulose fibers are observed, some wider than others and adhered to them, which could correspond to the coupling of the grafted amines in the form of sheets. However, it can be inferred that its morphology does not change drastically as reported in similar studies GÓMEZ, COMBARIZA, & BLANCO TIRADO (2017). On the other hand, regarding thermal degradation processes, for BC the point of maximum decomposition of its structure occurs at 342 °C with a mass loss of 58.99% and for BC-TOCN it occurs at 303 °C , that is, there is a decrease in thermal stability (FUKUZUMI, SAITO, OKITA, & ISOGAI, 2010) with a mass loss of 19.71%. This is explained by the presence of carboxylic units that favor the decomposition of BC, facilitating the transitions from solid phase to gas through decarboxylation reactions. Also for the BC-TOCN, a shoulder can be seen at a temperature of 232 °C with a weight loss of 14.44%, which corresponds to the decomposition of the carboxylic units on the BC-TOCN surface. Additionally, in BC-TOCN-AMD-C12 and C18 thermal decomposition events were observed near 200 °C, possibly attributed to the volatilization of the hydrocarbon chains of the primary amines (CALDERÓN VERGARA, OVALLE SERRANO, BLANCO TIRADO, & COMBARIZA, 2019). In addition, the thermal degradation temperature for the amidated derivatives at 318 °C for AMD C-12 BC-TOCN and 317 °C for AMD C-18 BC-TOCN, is higher than for BC-TOCN. This indicates that the coupling of the primary amines leads to an apparent improvement in the thermal resistance of BC-TOCN. Additionally, the colloidal stability of BC-TOCN was analyzed by means of the Z potential. Thus, a value of -60.8 mV was obtained, inferring a good colloidal stability probably in response to the amount of negatively charged carboxyl groups COO- on the surface of the cellulose structure, which causes electrostatic repulsions that prevent the suspension from agglomerating (MAHENDRAN & RANJITHA KUMARI, 2016). On the other hand, the contact angle (CA) tests were performed with a double injector using the double sessile drop measurement method, depositing drops of water and diiodomethane, as shown in figure 2. For the BC-TOCN films, a contact angle to water of 29°, evidencing its highly hydrophilic nature corresponding to the presence of polar groups (–OH and –COOH) on the surface. As for the materials modified with primary amines, an increase from 29° to 80° was observed with BC-TOCN-AMD-C-12 and 147° with BC-TOCN-AMD-C-18, corresponding to the presence of the alkyl chains of dodecylamine and octadecylamine nonpolar nature, which coupled to the BC-TOCN surface. Comparing the results obtained, it is observed that the increase in hydrophobicity obtained with octadecylamine is higher than in other studies. However, with dodecylamine this increase was not as significant, but higher than that reported by GÓMEZ, COMBARIZA, & BLANCO TIRADO (2017). In addition, with the experimental values of the contact angles of water and diiodomethane with the films, the surface free energy (SFE) was obtained γ_s and the polar components γ_s^p and dispersive γ_s^d (Figure 2). Indeed, it is observed that BC-TOCN obtained values of SFE and its components very similar to those obtained by the investigation of CALDERÓN VERGARA, OVALLE SERRANO, BLANCO TIRADO, & COMBARIZA (2019) with BC-TOCN from fique. Additionally, they are within the ranges reported by authors such as KHOSHKAVA & KAMAL (2013) in nanocrystalline cellulose with SFE values of 66 mJ/m2 characteristic of materials with a hydrophilic nature. Likewise, as the higher value of the polar component on the dispersive that indicates hydrogen bond, inductive and acid-base interactions. Unlike the materials modified with amines, with which it was possible to demonstrate that amidation decreases the SFE of the materials, especially the one coupled with octadecylamine whose value was 7,5 mJ/m2, very similar to that reported by CALDERÓN VERGARA, OVALLE SERRANO, BLANCO TIRADO, & COMBARIZA (2019). Additionally, the polar component decreases drastically, being lower than the dispersive component, which indicates Van der Waals type interactions, characteristic of hydrophobic materials.

Conclusões

Contact angle measurement indicated the coupling of the primary amines with the procedures used. This is supported by the appearance of new signals in the IR spectrum attributed to the formation of amide bonds. Likewise, a slight increase in the decomposition temperature of the amidated materials with respect to TOCN was observed. On the other hand, morphological and dimensional changes of the BC surface and its modifications were observed, inferring that the oxidation via TEMPO combined with ultrasound effectively leads to the separation of the nanofibers. In addition, the apparent coupling of the amines to TOCN is observed. Likewise, as a result of CA and SFE, the hydrophobization of the material was corroborated, reaching contact angles with octadecylamine of up to 147° and SFE values of 7.5 mJ/m2, which are consistent with its chemical structure. Which indicates that the resulting materials have good potential to be used as additives in biopackaging given their biocompatibility, degradability, along with hydrophobicity and improvement in their properties.

Agradecimentos

We thank the Guatiguará Technology Park at Universidad Industrial de Santander for infrastructural support. To Minciencias and the Universidad Industrial de Santander for financing the postgraduate project of Maria Andrea Gamboa.

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