Glucose, peptone, yeast extract, citric acid monohydrate, agar, and sodium hydroxide were purchased from Merck Ltd., United States. Corn steep liquor (CSL), sugarcane molasses, and laundry effluent of jeans processing were procured from local companies in the state of Pernambuco, Brazil. The jeans laundry effluent was characterized by macroscopic (visual), physical–chemical (pH, conductivity, turbidity, and color by spectrophotometry at 455 nm), and microbiological (bacteria, filamentous fungi, and yeasts counts) analyses and presented favorable results to allow the possible obtaining of a colored BC pellicle.

The effluent sample had the following characteristics: pH in the range of 5.9–7.0, electrical conductivity of 208 μS/cm, COD of 608 mg O₂/L, and turbidity of 19.5 UNT. The effluent presented high values for the color parameter. The apparent color ranged from 1240 to 1360 mg Pt/L and true color from 229 to 235 mg Pt/L. The following maximum CFU/mL counts were obtained as follows: 3 × 10⁷ for bacteria, 2 × 10² for yeasts, and 4 × 10 for filamentous fungi. The effluent was sterilized by autoclaving at 121°C for 20 min prior to use.

For BC production, a strain of Gluconacetobacter hansenii UCP1619, obtained from the culture collection of Nucleus of Resource in Environmental Sciences, Catholic University of Pernambuco, Brazil, was used. The standard culture medium used in the experiments was the HS described by Hestrin and Schramm (1954) and modified by Hungund and Gupta (2010). The liquid medium contained 2.0% glucose (w/v), 0.5% yeast extract (w/v), 0.5% peptone, 0.27% Na₂HPO₄ (w/v), and 0.15% citric acid (v/v). To formulate desolid medium, 1.8% agar (w/v) was added. The pH was adjusted to 5.0 using NaOH 1.0 M. The medium was stored at 4°C in a refrigerator and sub-cultured every 2 months for inoculum development.

The inoculum culture was prepared by transferring the cellulose film containing the G. hansenii adhered cells stored at -80°C in HS agar to the liquid HS medium, followed by static cultivation at 30°C for 2 days. The statically grown culture was then shaken vigorously to homogenize the cell distribution in the inoculum. The resulting cell suspension (3 mL) was inoculated in a semi-capped glass vessel (250 mL) containing 100 mL of the different liquid media, as described below, and then statically incubated at 30°C for 10 days in triplicate experiments (Gomes et al., 2013; Wu et al., 2014). After cultivation, the BC pellicles were sent for cleaning, purification as well as the determination of thickness, hydrated mass, and dry mass, as it will be described in the Section “Harvesting and Weighing of BC.” The experiments were carried out under sterile conditions.

Different carbon (glucose, acetylated glucose, and molasses) and nitrogen (yeast extract, peptone, and CSL) sources were used in the HS medium for the comparison of the total yield (dry mass) of cellulose produced by the bacterial isolate. A jeans laundry effluent was also tested. Table 1 lists the compositions of the different media (pH adjusted to 5–6) used for cellulose production.

After selection of the alternative medium (modified HS medium) consisting of 20 g/L of glucose and 10 g/L of CSL (Medium N° 3 in Table 1), further experiments were conducted under the same cultivation conditions, varying the concentrations of glucose and CSL, as described in Table 2. After the static cultivation of the bacterium for 10 days, the BC pellicles produced in the surface of the cultivation medium were harvest with the aid of a metal clamp. The mass and the thickness of the BC pellicles were determined using a precision balance and a micrometer, respectively, to compare the quality of the films obtained with the different cultivation media. After selection of the best concentration of glucose and CSL, the BC membranes were purified and characterized in terms of chemical structure, crystallinity, and morphology using XRD and SEM.

After cultivation, the BC membranes were washed with water and soaked in 0.1 M NaOH at 80°C for 2 h to remove bacterial cells possibly attached to the BC pellicles. Then, the pellicles were washed with deionized water several times to warrant the complete remove the alkali, leaving the pellicle at neutral pH. The purified cellulose was dried at 60°C for 12 h until reaching a constant mass. Triplicate experiments were performed for each membrane and mean values were calculated (Wu et al., 2014).

For SEM, the dried BC membrane was mounted on a copper stub using double adhesive carbon conductive tape and coated with gold for 30 s (SC-701 Quick Coater, Japan). The SEM photographs were obtained using a scanning electron microscope (550 Superscan, Shimadzu, Japan) operating at 15 kV at room temperature.

X-ray diffraction (XRD) patterns of all BC membranes were measured using a diffractometer (Bruker D8 Advance Davinci) with Cu Kα radiation. The crystallinity percentage was measured by equation of Segal given below.

In this equation, where Crl expresses the relative degree of crystallinity, I₀₀₂ is the maximum intensity (in arbitrary units) of the 002 lattice diffraction, and I_am is the intensity of diffraction in the same units at 28 = 18°.

Thermogravimetric analysis (TGA) measurements were performed using a simultaneous thermal analyzer (STA 6000, PerkinElmer) on samples of about 8 mg. Each sample was scanned over a temperature range from room temperature to 800°C with a heating rate of 10°C/min under nitrogen with a flow rate 50 mL min^-1 to avoid oxidation. Six randomly selected ground (fine particles) samples were used for the TGA measurements (Kiziltas et al., 2015).

For the films produced in the alternative medium and in the HS medium, tensile strength (N), tensile strength at peak load (MPa), elongation at break (𝜀) (%), and Young’s modulus of elasticity (E) were determined based on Rethwisch and William (2016). The BC sheets (thickness of 0.48 ± 0.0 and 8.7 ± 0.01 mm for dried and hydrated sheets, respectively) were cut into rectangular strips (70 mm × 20 mm). Tensile testing was performed at room temperature at a speed of 1 mm/min and a static load of 0.5 N using a universal testing machine (EMIC DL – 500MF, Brazil) following the ASTM D882 Method for Tensile Properties of Thin Plastic Sheeting. The Bluehill Lite^TM software program was used to calculate the stress–strain relationship and modulus of elasticity. A crosshead displacement sensor was used for the deformation measurements.

In order to verify the existence of differences between the average responses of the treatments, analysis of variance (ANOVA) tests were performed. Before, however, the data that composed each midpoint were preliminarily compared with the help of box plot. The one-way ANOVA test was used, and it was concluded that there were significant differences between the means of the treatments at the significance level of p < 0.05.

Bacterial cellulose (BC) pellicles were produced through fermentation at 30°C for 10 days. The pH, temperature, and the ratio of substrate surface area to volume proved to be very important to optimal BC production, since the surface area of the culture medium is inversely proportional to the thickness of the wet pellicle. For the production of membranes with a surface area of 44.17 cm² and a mean thickness of 0.62 ± 0.08 mm, it was necessary to use a volume of culture medium equal to 100 mL and Schott vessels of 500 mL and diameter of 7.50 mm.

The replacement of synthetic compounds with industrial residues in the HS medium enabled the production of BC under static conditions in some of the media tested. The residues used in this study did not undergo treatment prior to use. However, the jeans laundry effluent was sterilized to avoid contamination of the medium. After 10 days of fermentation, the media containing acetylated glucose (Media 1 and 9) and laundry effluent (Media 5, 6, 7, and 8) did not produce BC. Regarding the acetylated glucose, it was observed that it had a low solubility in the medium and after autoclaving the degradation of this compound occurred.

The objective of using the jeans laundry effluent was to produce an already colored pellicle for industrial application, thus avoiding the dyeing step, which represents a high environmental impact. According to the State Agency of Environment and Water Resources of the State of Pernambuco, Brazil, the volume of wastewater generated in the laundries is close to 55985.25 m³/month (Lima et al., 2005). Another reason would be to give the effluent generated in the laundries an applicability, avoiding their disposal in the water bodies. The visual observation of the effluent allowed to detect textile fibers dispersed in the liquid. These fibers could be entangled together with the BC fibers produced in the medium, thus forming a biocomposite. Although the sterilized effluent has maintained its initial color and pH, the results obtained with the alternative media formulated in its presence were not satisfactory. Probably, the artificial dyes present inhibited the production of BC by the bacterium, as also reported in the literature (Bertazzoli and Pelegrini, 2002; Kunz et al., 2002; Pinheiro et al., 2004).

The hydrated BC pellicles were obtained in the presence of glucose, molasses, and CSL (Media 2, 3, and 4). Medium N° 3 – formulated with glucose as the carbon source and CSL as the nitrogen source to replace yeast extract and peptone – was selected for further study based on the yields in hydrated (367.63 ± 0.5) and dry (5.80 ± 0.5) mass shown in Table 3.

The availability of the carbon and nitrogen sources in the culture medium favors the production of BC, especially noting that the yields of BC were dependent on the carbon source. Thus, the media which presented toxic compounds as dyes, i.e., the media containing the laundry effluent, did not allow the production of BC, as there was an inhibition or destruction of the G. hansenii cells. Acetylation of glucose, on the other hand, caused physical and chemical modifications to glucose, making the availability of the substrate difficult to the microorganism. The substitution of 100% of glucose by sugarcane molasses, in turn, resulted in a minimal production of BC. The substitution of the synthetic nitrogen source by the CSL residue allowed a 25% reduction of the glucose favoring the production of films of excellent quality.

Further fermentations were conducted varying the concentrations of glucose and CSL, as shown in Table 4. The alternative modified HS medium denominated N° 14, containing 15 g/L glucose and 25 g/L CSL, achieved the highest yields in terms of dry (9.63 ± 0.9 g/L) and hydrated (381.10 ± 0.8 g/L) mass. The hydrated pellicle produced with this alternative medium corresponded to 73% of the production achieved with the standard HS medium. The result obtained in this medium is satisfactory especially considering the results described for other media formulated with industrial residues as it will be described below. Statistical analysis indicated significant differences (p < 0.05) in the yields of the pellicles showed in Tables 3 and 4.

The pellicles produced in the alternative medium exhibited similar thickness as well as visual and tactile texture to the control (HS medium) and demonstrated a yield 3.53% higher than that achieved previously (Table 3), with a 25% reduction in the carbon source and 100% replacement of the nitrogen source with CSL in comparison with the HS medium (Figures 1A–D). As shown in Figure 1A, a light brownish pellicle was obtained with the alternative culture medium. After the pellicles were treated in NaOH to remove the G. hansenii biomass, purified white pellicles were obtained (Figure 1C). Membrane thickness was estimated to be 0.2–0.5 mm. The color intensity of the pellicles produced in media containing CSL was slightly higher than that produced with the HS medium.

Figure 2 displays the micrographs of the surface of the BC produced with the alternative medium under the optimal conditions selected in this study and the standard HS medium, showing the dispersion of the fibers and interfacial adhesion. It is notable that the BC pellicles synthesized with the alternative medium were stable. The compact cellulose network structure consisted of a random assembly of fibrils. Both purified BC pellicles exhibited a reticulated structure consisting of ultrafine nanofibrils and no significant difference in size, as illustrated in the SEM images. Greater microfibril and nanofibril production was achieved when CSL was used in place of synthetic compounds.

The BC produced in the modified HS medium in the present study exhibited fine cellulose fibers, which formed a porous three-dimensional network structure. It is important to note that the application of heat for drying the films was instrumental in the concentration of the network structure. According to Santos et al. (2015), high temperatures shorten the distance between the layers of BC and assist in the evaluation of porosity. The visualization of the fiber network magnified 8,000 and 20,000× times demonstrated a smooth surface of the fibers produced with both media.

Figure 3 shows the thermogravimetric degradation curves of the dried pellicles in percentage of the mass of the original sample as a function of temperature. During the initial thermal treatment, the samples exhibited slight mass loss beginning with room temperature up to 200°C due to the loss of water. A second, significant mass loss occurred around 300°C, which may be attributed to the decomposition of the samples. This event also may be associated with the degradation of cellulose, including the depolymerization, dehydration, and decomposition of glucose units.

The BC produced in the alternative medium (Figure 3) did not decompose before 250°C, while the BC produced in the HS medium exhibited a 2.6% decomposition rate at 195°C. Above 700°C, mass loss rates were low and the charred ashes corresponded to approximately 37% for the BC produced with the HS medium and 42% for that produced with the alternative modified HS medium. The agro-industrial residue conferred more thermal stability to the BC pellicle produced in the alternative medium than that produced in the standard HS medium. The use of an industrial waste by-product in the production of BC can help minimize environmental impacts and reduce BC production costs, thereby aggregating economic value to the pellicle.

Maximum decomposition temperature is a criterion of thermal stability. Thermal degradation is affected by structural variables, such as molecular mass, crystallinity, and the arrangement of the fibers (Vazquez et al., 2013). Maximum decomposition occurred at 310°C for the BC produced with the alternative medium and 290°C for that produced with the standard HS medium. These results indicate that greater thermal stability of the BC produced in the alternative medium.

X-ray diffraction (XRD) was used to evaluate the crystalline structure as well as the change in crystallinity of the BC produced with the two media. As shown in Figure 4, the peaks at 2𝜃 angles of 14.422 and 22.996° for the BC produced with the alternative medium and at 2𝜃 angles of 14.422 and 22.747° for the BC produced with the standard HS medium corresponded to the (-1 1 0) and (2 0 0) crystal planes, respectively, demonstrating that the products obtained from both media were cellulose I.

The crystallinity index was calculated based on peak intensity data determined using the Segal method (Cheng et al., 2009). The crystallinity index for the BC produced with the alternative medium (84.9%) was practically the same of that obtained with the HS medium (86.1%). These results show that the composition of the medium did not affect the crystalline morphology of the polymers and the physical characteristics of the membranes were maintained. Moreover, crystallinity has an inversely proportional relationship with the porosity of the cellulose surface, as a lower throughput denotes greater pore size in the cellulose structure. The fact that the pores increase in size allows a greater amount of water molecules to penetrate and be absorbed by the membrane, demonstrating an increase in the degree of hydrophilicity.

Moreover, crystallinity has an inversely proportional relationship with the porosity of the cellulose surface, i.e., the larger the size of the crystals and the proximity between them, the smaller the number of pores and the lower the hydrophilicity of the polymer. The fact that the pores increase in size allows a greater amount of water molecules to penetrate and be absorbed by the membrane, demonstrating an increase in the degree of hydrophilicity. The images presented in Figures 2A,C show a higher concentration of fibers and nanofibers of BC in the pellicle produced in the alternative medium, justifying the difference in mass between the films, even though they have very close Crl values.

Another important aspect concerns the type of membrane purification. Heat treatment with NaOH facilitates the removal of certain metabolites and increases viscosity, promoting purification and the elimination of low molecular mass cellulose, which confers better characteristics to the biomaterial.

Figure 5 shows the results of the tensile assay used to characterize the mechanical properties of the hydrated and dry pellicles produced with the alternative medium and with the HS medium. The results were similar between the pellicles. The main difference was observed in the Young’s modulus of the dry BC pellicle produced in the HS medium, which was superior when compared to the pellicle obtained in the alternative medium. The hydrated pellicles obtained in the alternative medium had mean tensile strength of 2.29 ± 0.94 MPa when subjected to an average force of 25.67 N while the dry pellicle had mean tensile strength of 48.17 ± 15.38 MPa when the average maximum force was 115.59 N at the breaking point. Considerable variation in the results is found when hydrated and dry samples of the same polymer are submitted to mechanical tests. According to Almeida et al. (2013) and Gao et al. (2016), water acts as a plasticizer. The hydrated pellicle from the alternative medium had greater elongation at break, stretching 56.60% more than the dry pellicle.

The water and waste by-products in the culture medium acted as plasticizers and altered the mechanical performance of the biomaterial. The water in hydrated pellicles decreases the number of hydrogen bonds between molecules and reduces intermolecular forces, as demonstrated by the results of the mechanical tests, in which the hydrated pellicle exhibited greater elongation and lower tensile strength than the dry pellicle.

Considerable plastic deformation of the pellicle was observed before rupture in the form of an irreversible ductile fracture of the biopolymer. The propagation of cracks happened slowly and accompanied by necking during the tensile test.

Regarding deformation, the hydrated and dry pellicles did not return to the natural (non-deformed) state. The increase in delamination or crack propagation occurs in dry pellicles faster than hydrated pellicles due to the presence of water and residues from the culture medium, demonstrating that the hydrogel does not have elasticity even when subjected to low stress. Young’s modulus of elasticity (E) is an intrinsic property used as a mechanical parameter directly related to polymer stiffness (Almeida et al., 2013). The amount of water held between BC fibers is a determining factor of the greater deformation of hydrated pellicles than dry pellicles, since water acts as a plasticizer and its absence makes biomaterial brittle and hard. In the present study, the mean modulus of elasticity of the dry pellicle was 96.98% higher than that of the hydrated pellicle, which demonstrates the mechanical strength of the biomaterial.

Macroscopically, it was difficult to identify the point of rupture and the alignment behavior of the fibers of the BC pellicle obtained from the alternative medium when submitted to stress (Figure 6). The hydrated and dry pellicles initially broke transversely at a point, but remained whole with the increase in load during the tear process until the complete rupture of the film. This occurred because the pellicles had microfibers and nanofibers that were disorderly arranged as a non-woven fibrous network that contained a significant amount of water and residues from the culture medium.