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Dyeing of bacterial cellulose films using plant-based natural dyes
Accepted Manuscript Dyeing of bacterial cellulose films using plant-based natural dyes
Andréa Fernanda de S. Costa, Júlia D.P. de Amorim, Fabíola Carolina G. Almeida, Ivo Diego de Lima, Silva Sérgio C. de Paiva, Maria Alice V. Rocha, Glória M. Vinhas, Leonie A. Sarubbo PII: DOI: Reference:
S0141-8130(18)35172-9 doi:10.1016/j.ijbiomac.2018.10.066 BIOMAC 10717
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
28 September 2018 11 October 2018 14 October 2018
Please cite this article as: Andréa Fernanda de S. Costa, Júlia D.P. de Amorim, Fabíola Carolina G. Almeida, Ivo Diego de Lima, Silva Sérgio C. de Paiva, Maria Alice V. Rocha, Glória M. Vinhas, Leonie A. Sarubbo , Dyeing of bacterial cellulose films using plantbased natural dyes. Biomac (2018), doi:10.1016/j.ijbiomac.2018.10.066
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ACCEPTED MANUSCRIPT Dyeing of bacterial cellulose films using plant-based natural dyes
Andréa Fernanda de S. Costaa,b, Júlia D. P. de Amorimb,f, Fabíola Carolina G. Almeidab,d, Ivo Diego de Limac, Silva Sérgio C. de Paivad, Maria Alice V. Rochae, Glória M. Vinhasf and Leonie A. Sarubbo b,d*
a
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Design and Communication Centre, Federal University of Pernambuco, Caruaru, Brazil;
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Advanced Institute of Technology and Innovation, Recife, Brazil
Center for Exact and Nature Sciences, Federal University of Pernambuco, Recife, Brazil
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e
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Centre for Sciences and Technology, Catholic University of Pernambuco, Recife, Brazil
Department of Domestic Sciences, Federal Rural University of Pernambuco, Brazil;
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Department of Chemical Engineering, Federal University of Pernambuco, Recife, Brazil
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Correspondence to: Leonie Asfora Sarubbo, Center for Sciences and Technology; Catholic University of
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E-mail: [email protected]
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Pernambuco; Recife; Brazil
ACCEPTED MANUSCRIPT Abstract The aim of this work was to test the use of plant-based natural dyes on bacterial cellulose (BC) to add aesthetic value to dyed pellicles while maintaining the mechanical properties. Natural pigments from Clitoria ternatea L. and Hibiscus rosa-sinensis were tested. The commercial ARAQCEL RL 500 was also used for comparison purposes. The behaviour of biocellulose regarding dye fixation, rehydration, tensile strength, and elasticity was
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evaluated in comparison to the dried biomaterial, showing that dyeing is a process that can be performed on hydrated BC. Dyeing the BC films through an innovative process maintained the crystallinity, thermal stability
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and mechanical strength of the BC and confirmed the compatibility of the membrane with the dyes tested, from
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the observed Scanning Electron Microscopy (SEM) morphology of nanofibers. Dyed biomaterial can be applied to various products, as confirmed by the results of the mechanical tests. As environmental awareness
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an attractive new material for the textile industry.
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and public concern regarding pollution increase, the combination of natural dyes and BC pellicles can produce
Keywords: Gluconacetobacter hansenii; bacterial cellulose; dyeing; Hibiscus rosa-sinensis; Clitoria ternatea
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1. Introduction
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The microfibrils of bacterial cellulose (BC) were first described on 1949 [1], and were found to be about 100 times smaller than those of vegetable cellulose (VC) [2]. The fibrils are made of three dimensional
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nanofribrils, which, when arranged, form of a highly porous hydrogel sheet with a large surface area [3]. Visually, the difference between VC and BC resides in the appearance and water content. VC has a fibrous appearance, whereas BC resembles a gel. Although the functional groups that characterize BC are the same as those in VC, the formation of the reticularly interconnected tridimensional pellicle is reported under static conditions, whereas irregular sphere-shaped cellulose particles are found under agitated conditions [4,5]. The formation of cellulose under static conditions is governed by the air supply on the surface of the medium and the yield depends on the concentration of the carbon source [6]. Acetic acid bacteria, especially those of the genus Gluconacetobacter [7-9], produce BC through oxidative fermentation because such bacteria can
ACCEPTED MANUSCRIPT assimilate various sugars, producing high yields of cellulose in a liquid medium [10,11]. The potential of BC goes beyond its existing applications, especially in big scale production using lowcost raw material, and includes special textile materials, such as functional materials and even packaging [12]. “Eco-driven” fashion or “sustainable clothing” is a global concern for producers and consumers. BC is a renewable raw material that can serve as the basis for “green clothing” [13].
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During the production of BC, the colour of the biomaterial varies depending on the substrate used in the culture medium. Formulations involving tea, molasses, beer, wine, and residual waste have been reported. The
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aim is to reduce the production cost and aggregate sensory, chemical, and physical properties to biocellulose
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for industrial applications [14–18].
Colours found in nature have a profound effect on humans and are crucial to the acceptance of textiles.
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[19] Colour aggregates both aesthetic and symbolic value. In pre-historic times, natural pigments were extracted from ores, insects, plants, and microorganisms and used as dyes for numerous materials. Such dyes
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have chromophore groups with different chemical structures that confer sufficient stability for the purposes of staining [20]. Synthetic dyes became popular in the middle of the 19th century and continue to dominate the
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market despite having properties that are harmful to humans, animals, and the environment [21,22].
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Natural pigments differ chemically from their synthetic counterparts and can be used to add colour to food, drugs, cosmetics, and textiles without compromising health [23]. Although natural pigments have the same
market.
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technological and economic advantages as synthetic pigments, they do not yet enjoy the same share of the
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Paint is a US$1.25 billion market for which synthetic dyes account for 40%, natural dyes account for 28% and caramel dyes account for 12%. The annual growth of synthetic dyes is 2 to 5%, while the annual growth of natural dyes is 5 to 10% [24]. The major environmental problem with synthetic dyes regards the large volume of water required for processing and the disposal of toxic effluents in bodies of water, which has a huge impact on the environment, causing harm to the reproduction of the fauna and flora and compromising the health of human populations [13]. Intense colour, shine, stability when exposed to light and washing, and the vast variety of shades are characteristics with aesthetic appeal. Natural pigments from ores, bugs, plants, and microbes are considered safe for the environment due to their non-toxic, non-carcinogenic, and biodegradable properties. Sources of
ACCEPTED MANUSCRIPT pigments include natural products, such as flavonoids and anthraquinones, which are produced in plants and animals. The use of natural pigments has tremendous advantages, such as abundance in the environment, simple extraction methods, biodegradability and the absence of toxicity [24–26]. Vegetal pigments have different molecular functional groups (tetrahydrides, tetraterpenes, quinones, Nheterocyclics and O-heterocyclics) and are classified as carotenoids, porphyrins, flavonoids, phenolics,
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indigoids, etc., which are found in the leaves, flowers, fruit, seeds, trunk and roots of plants [27]. In the present study, natural pigments were extracted from the flowers from Clitoria ternatea L. and Hibiscus rosa-sinensis.
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Clitoria ternatea L., commonly known as the blue pea or butterfly pea, is a member of the family Leguminosae
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(Fabaceae; tribe: Phaseoleae; subtribe: Clitoriinae). This plant is native to tropical equatorial Asia (Indonesia and Malaysia), but has been introduced in Africa, Australia and the Americas [28]. Chemical compounds
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isolated from C. ternatea include various triterpenoids, flavonol glycosides, anthocyanins and steroids [29]. Cyclic peptides known as cliotides have been isolated from the heat-stable fraction of C. ternatea extracts [30].
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Hibiscus rosa-sinensis, known colloquially as the Chinese hibiscus, is a tropical flowering plant from the Hibisceae tribe of the family Malvaceae native to tropical Asia (China). This plant is commonly found
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throughout the tropics and as a house plant throughout the world. Most ornamental varieties are hybrids. The
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present wide range of cultivars is considered to be a complex of inter-specific hybrids between eight or more different species originating from the eastern coast of Africa and islands in the Indian and Pacific Oceans. The flowers contain cyanidin diglucoside, flavonoids, thiamine, riboflavin, niacin, and ascorbic acid [29,31].
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In this work, the usability of flowers from Clitoria ternatea L. and Hibiscus rosa-sinensis as novel natural
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dyes was tested to add aesthetic value while maintaining the properties of BC pellicles.
2. Materials and Methods
2.1. Materials Glucose, peptone, yeast extract, citric acid monohydrate, agar, and sodium hydroxide were purchased from Merck Ltd., USA. Corn steep liquor (CSL) was acquired from a local company in the state of Pernambuco, Brazil. Dehydrated flowers from Hibiscus rosa-sinensis were obtained from a local market while the flowers from Clitoria ternatea L. were obtained directly form a local garden. Both flowers were used to stain BC
ACCEPTED MANUSCRIPT pellicles. The synthetic dye ARAQCEL RL 500 blue (ExtraCor Dyes and Chemicals, Brazil) was used for comparison.
2.2. Microorganism Gluconacetobacter hansenii UCP1619 was obtained from the culture collection of the Center for
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Environmental Sciences of the Catholic University of Pernambuco, Brazil, and used for the production of BC. The strain was maintained in the HS medium described on 1954 [32] and modified on 2010 [33], containing
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glucose agar slants with 2% glucose (w/v), 0.5% yeast extract (w/v), 0.5% peptone, 0.27% Na2HPO4 (w/v),
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1.5% citric acid (v/v), and 1.8% agar (w/v). The pH was adjusted to 5.0 using NaOH 1.0 M. The medium was stored in a refrigerator at 4 °C and sub-cultured every two months for inoculum development or stored at -80
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°C using 20% (v/v) glycerol instead of agar for long-term storage.
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2.3. Culture conditions
For the production of BC, the bacterium was cultivated in an alternative medium [14], in which the carbon
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source was reduced by 25% and synthetic nitrogen was completely replaced with corn steep liquor (CSL):
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1.5% glucose (w/v), 2.5% CSL (w/v), 0.27% Na2HPO4 (w/v), and 0.15% citric acid (v/v). The culture was prepared by transferring the G. hansenii cell suspension stored at -80 °C to the HS medium, followed by static cultivation at 30 °C for two days. The statically grown culture was then shaken vigorously
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to detach cells from the cellulose pellicle. The resulting cell suspension (3 mL) was inoculated in a semi-
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capped glass vessel (250 mL) containing 100 mL of the alternative medium and statically incubated at 30 °C for 10 days in triplicate experiments. After the fermentation process, the pellicles were collected, rinsed with tap water and purified in a 4% NaOH solution at 80 °C for 30 minutes to remove the microbial and excess substrate. After purification, the pellicles were washed in distilled water at neutral pH [14,34].
2.4. Extraction of pigments A kilogram of Clitoria ternatea L. flowers was placed in 200 mL of 80% cold ethanol (5°C) and stirred at 150 rpm for three hours. The solution was then filtered with No. 1 Whatman filter paper, resulting in a light blue solution, which was concentrated through lyophilization (Advantage Plus EL-85, SP SCIENTIFIC, USA).
ACCEPTED MANUSCRIPT The Clitoria ternatea L. dye solution was prepared by diluting 1 g of the light blue concentrated pigment released by the flower in 500 ml of deionized water. A solution with a burgundy colour was obtained from an infusion prepared using 50 g of dehydrated Hibiscus rosa-sinensis flowers in 500 mL of deionized water at 90 °C for 20 min. The solution was then filtered with No. 1 Whatman filter paper.
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The ARAQCEL RL 500 solution was prepared with 5 g of the dye diluted in 10% deionized water (w/v),
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resulting in an indigo blue solution.
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2.5. Dyeing procedure
The three pigment solutions prepared as described in section 2.4 were heated to 90 °C to speed up the
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reaction of the dye with the cellulose fiber and a 10% (w/v) solution of sodium chloride and sodium ferrocyanide (COMSAL, Rio Grande do Norte, Brazil) was added as the mordant. The batch process was used
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for the impregnation of the BC pellicles with the dyes. Round pellicles measuring 25 cm in diameter were submerged in the heated dye solutions for 30 minutes in 500 mL beakers. The experiments were conducted in
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triplicate.
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After dyeing, the films were rinsed twice in running water. To fix the colour, the hydrated films were submerged for 15 minutes in a solution containing 1% of ARAQFIX WE (ExtraCor Dyes and Chemicals, São Paulo, Brazil) fixer and 2% of the cationic SOFTEX MTZ softener (Texpal Química, São Paulo, Brazil), which
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is commonly used in conventional dyeing processes and dry cleaners for cotton cellulose fibers. The dyed BC
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films were rinsed on running water at pH 7.0 and stored in glass vials containing 250 mL of deionized water. The water was replaced every 24 hours for five days until the dye was no longer observed in the water. Samples were dried in an oven (SX1.2 Sterilifer, São Paulo, Brazil) at 60°C for 12 hours. After drying, the films were submerged in 200 mL of distilled water for two hours to simulate delicate hand washing and observe the release of the dye into the water. Some samples were kept hydrated in water for further analysis.
2.6. Determination of water activity The water contained in BC film can influence the fixing of dye to cellulose fibrils. Therefore, water activity was measured in the hydrated and oven-dried natural and dyed BC films using a water activity analyzer
ACCEPTED MANUSCRIPT with an internal sample temperature control (AQUALAB SERIES 4TE, São Paulo, Brazil). Water activity values between 0 and 0.20 indicate that the water is strongly bound, whereas values in the range of 0.70 to 1.00 indicate that most of the water is free and available for chemical reactions, enzymes, and the development of microorganisms. As water activity is entirely linked to the moisture of the biomaterial, this analysis enabled
2.7. Spectrophotometric evaluation of amount of dye retained in BC films
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the determination of its ability to adsorb and fix the dyes.
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The amount of dye retained in the dyed BC films was determined using UV-Vis light spectrophotometry.
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To determine the percentage of dye adsorbed to the BC fibres and nanofibrils, the three dyeing solutions (Clitoria ternatea L., Hibiscus rosa-sinensis and ARAQCEL RL 500) were analysed before submerging the
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films. After the dyeing process, the films were removed from the solutions and analysed again. The solution obtained after the pigment-fixing step pigments was also analysed. It was therefore possible to determine the
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percentage of dye retained and released in the different stages of the dyeing of the films. A digital spectrophotometer SP-22 (Biospectro, Paraná, Brazil) matched with 1 cm quartz cell was used for the
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absorbance measurement. Absorbance readings were made for each dye and the best wavelength (ʎ nm) was
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identified using the range from 375 to 700 nm. Dilutions were then performed with the dyeing solutions, the solutions generated after the dye fixation and the solutions obtained after the hand wash simulation to construct
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the calibration curves and equations for all dyes.
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2.8. Scanning Electron Microscopy (SEM) 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 (MIRA3 LM, Tescan, USA) operating at 10.0 kV at room temperature.
2.9. X-ray Diffractrometry (XRD)
ACCEPTED MANUSCRIPT X-ray diffraction patterns of all BC membranes were measured using a diffractometer (Bruker D8 Advance Davinci) with Cu Ka radiation. The crystallinity percentage was measured by equation (1) of Segal given below: CrI = [I002 – Iam] / I002 x 100
(1)
Where Crl expresses the relative degree of crystallinity, I002 is the maximum intensity (in arbitrary units) of the
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002 lattice diffraction, and Iam is the intensity of diffraction in the same units at 28 = 18°.
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2.10. Thermogravimetric Analysis
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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
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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
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measurements [35].
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2.11. Mechanical test
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Tensile strength (N), tensile strength at peak load (MPa), elongation at break (ε) (%) and Young’s modulus of elasticity (MPa) were determined for dyed BC films, according to Callister and William [36]. The BC sheets (thickness: 0.48 ± 0.0 and 8.7 ± 0.01 mm for dried and hydrated sheets, respectively) were cut into rectangular
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strips (80 x 25 mm). Tensile testing was performed at room temperature (28°C) at a speed of 1 mm/second and
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a static load of 0.5 N using a universal testing machine (EMIC DL – 500 Mf, Brazil) following the ASTM D882 Method for Tensile Properties of Thin Plastic Sheeting. The Bluehill LiteTM software program was used to calculate the stress-strain relationship and modulus of elasticity. A crosshead displacement sensor was used for the deformation measurements [14].
2.12. Statistical Analysis The analyses were performed in triplicate. Mean and standard deviation values were calculated and tested. Analysis of variance (ANOVA) was performed for all values and a p-value < 0.05 was considered indicative of statistical significance.
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3. Results and discussion
3.1. Production and dyeing of BC films After washing, purification and neutralization, the wet BC film produced with the alternative medium (Fig.
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1a) had a uniform, beige appearance and smooth texture and was very flexible. The yield of the wet and dried BC pellicles was 381.10 ± 0.81 and 9.63 ± 0.90 g, respectively. After five days submerged in water, the colour
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was off white (Fig. 1b) and uniformity in the off white tone was found throughout the pellicle after the drying
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process (Fig. 1c). The surface had a smooth, paper-like appearance and was neither flexible nor brittle. Dyes are highly detectable without the aid of lenses and, in some cases, are visible even at concentrations
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as low as 1 ppm [37]. When a fiber is dipped in a dye bath, the dye is adsorbed to the surface of the fiber and disseminates into the fiber. This phenomenon was observed in the hydrated BC films dyed with Clitoria
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ternatea L. (Fig. 2a), Hibiscus rosa-sinensis (Fig. 2b) and ARAQCEL RL 500 (Fig. 2 c). During the five days of submersion in water, the BC films dyed with the light blue solution (Fig. 3a)
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demonstrated a reduction in the colour intensity. After the subsequent drying and hand washing processes, a
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lack of uniformity of the light blue colour was found on the surface of the BC film. The dried film was fragile and brittle and its texture resembled paper (Figs. 3a’ and 3a’’). On the other hand, the pellicles dyed with the burgundy colour solution from Hibiscus rosa-sinensis flowers retained colouration after five days of
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immersion in water (Figure 3b). The oven drying process caused changes in colour intensity and uniformity as
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well as the texture of the biomaterial. The pellicle appeared as a soft, flexible film similar to processed animal leather (Fig. 3b’). Rehydration of the film during hand washing for two hours did not cause significant changes in coloration, although the film became more flexible and elastic (Fig. 3b"). The solution prepared with the synthetic ARAQCEL RL 500 dye was very concentrated and resulted in a BC film with an uniform indigo blue colour across the surface (Fig. 2c). This shade was retained after five days submerged in water (Fig. 3c). Drying caused a change in colour and made the film darker (Fig 3c’). The texture was soft. After hand washing for two hours at 28 °C, the film slightly lost colour intensity compared to the dry film but demonstrated greater flexibility and elasticity similar to processed animal leather (Fig. 3c’’).
ACCEPTED MANUSCRIPT 3.2. Characterization of dyed samples The penetration of dyes in individual fibers to a practical degree of durability is fundamental to the uniform application of colours [38]. The dyeing process occurs due to a chemical reaction between the material and dye. Permeability is an intrinsic property of materials that facilitates the diffusion of dye molecules in the fiber [39].
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Water activity in the BC films was analyzed to identify the amount of residual moisture in the BC nanofibers. This moisture is due to either capillary condensation or multilayer formation in spaces of cellulose
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fiber that allow occupation by water molecules. The calorimetric method described indicates the limit of
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hydroxyl interaction and provides evidence regarding the nature of the capillary spaces in which condensation occurs [39,40]. The authors state that, during the dyeing process, all adsorbed water below 4.3% for a cotton
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vegetal cellulose sample is connected to free hydroxyl groups of glucose units in the non-crystalline cellulose plant fiber. The X-ray of the BC films confirms the existence of crystalline regions in biocellulose, as described
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in a previous research [13].
Fibers that swell in contact with water have a hydrophilic nature and are able to accept water soluble dyes.
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For the three dyes used in the hydrated BC films, colour uniformity and fastness were found after fixing (Fig.
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3a, 3b and 3c). High residual moisture was detected in the wet BC films, with water activity of 0.985± 0.002, 0.982±0.001, 0.992±0.006 and 0.982±0.001 for the natural colour of the film and the light blue, burgundy and indigo blue dyed films, respectively. After drying, the moisture content was reduced by more than 40% in all
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three dyed BC films. This result indicates that, after dyeing and drying, the BC films exhibit residual moisture
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that does not interfere with usability. The hand washing simulation confirmed that the residual water activity of the dried cellulose biomaterial assisted in the release of dye that was not chemically bound to the BC fibres when the films were submerged in water. Since the swelling of fibers facilitates the migration of dye that has not been adsorbed, the colour intensity decreases (Figs. 3a’’, 3b’’ and 3c’’). The best wavelengths identified for the Clitoria ternatea L., Hibiscus rosa-sinensis and ARAQCEL RL 500 dyes were 375, 520 and 560 nm, respectively. Equations from calibration graphs (i.e., y = 89.915x + 0.2567 and R2 = 0.9651 for the Clitoria ternatea L dye solution, y = 91441X - 0.0008 and R2 = 0.9472 for the Hibiscus rosa-sinensis dye solution and y = 50993x +0.0048 and R2 = 0.9983 for the ARAQCEL RL 500 dye
ACCEPTED MANUSCRIPT solution) enabled the identification of the amount of dye retained in BC films in the various stages of the biocellulose dyeing process. After the dyeing step, the wet BC films absorbed 11.01±0.15, 31.09±0.31 and 48.05±0.20% of the Clitoria ternatea L., Hibiscus rosa-sinensis and ARAQCEL RL 500 dyes, respectively. These rates were increased to 17.13±0.05, 60.21±0.04 and 95.09±0.09%, respectively, after the fixing step. Submersion in distilled water for
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five days led to a 28.31±0.11% reduction in fixation for the Clitoria ternatea L. dye and a 12.08±% reduction for the Hibiscus rosa-sinensis dye. In contrast, the ARAQCEL RL 500 dye lost only 5.04±0.30% of the amount
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absorbed, demonstrating excellent fixation.
3.3. Scanning Electron Microscopy (SEM)
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The surface morphology of BC films produced in alternate medium was elucidated by SEM technique to highlight the influence and modification of the natural dyes extracted from Clitoria ternatea L. and Hibiscus
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rosa-sinensis and of the synthetic dye ARAQCEL RL 500 (Fig. 4). In contrast to the surface of natural BC (Fig. 4a), the sample of light blue dyed BC showed the presence of varying amounts of peculiar matter
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deposited on its surface (Fig. 4b). The burgundy color BC showed that the deposition of crystals became more
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apparent (Fig. 4c) whereas the indigo blue dyed BC presented morphological characteristics of BC fibers rather impregnated with the dye, showing that this dye can diffuse more in the fibers, reducing its accumulation on the surface (Fig. 4d). Davarpanah et al. [41] also showed the presence of peculiar matter firmly adhered to the
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surface of the silk surface after modification and influence of the chitosan on the degummed and acylated
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samples grafted with acylates.
3.4. X-ray Diffractrometry (XRD) The crystallinity index was calculated based on peak intensity data determined using the Segal et al. [42] method described by Cheng et al. [43]. The crystallinity index found in this study was 84.37±0.06% for the natural BC produced in the alternative medium (Fig. 5). Costa et al. [14] confirmed that the BC produced with the alternative medium shows high crystallinity. These results show that the composition of the medium did not affect the crystalline morphology of the polymer and the physical characteristics of the membrane were maintained.
ACCEPTED MANUSCRIPT The crystallinity indexes of the BC films produced in the same culture medium and dyed with Clitoria ternatea L. flowers (light blue), Hibiscus rosa-sinensis flowers (burgundy) and ARAQCEL RL 500 (indigo blue) were 82.23±0.21, 80.72±0.19 and 73.22±0.15%, respectively. These results show a tendency for the decrease in crystallinity of BC after dyeing using both naturals and synthetic dyes. The presence of the dyes weakens the intermolecular forces that maintains an organized structural arrangement proper of crystalline
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domains and favors a structural disarrangement, thus creating amorphous regions. This behavior may be associated with the breakdown of the hydrogen bonds between the chains of BC and the formation of
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difficult due to the incorporation of other compounds into BC [44].
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intermolecular interactions between BC and the dyes, making molecular orientation of the polymer chains
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3.5. Thermogravimetric Analysis
Fig. 6 shows the thermogravimetric degradation curves of the BC films in percentage of the mass of the
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original sample as a function of temperature. The natural film had a mass loss of approximately 42.11±0.21%, in the temperature range between 302 °C and 355 °C, while the dyed films had more than one stage relevant
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to mass loss. The burgundy BC film lost 55.23±0.13%, % mass between 74-330°C. The ligth blue BC showed
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a significant mass loss of 41.09±0.12%, in the bands of 292-350°C, while the blue indigo BC film lost 36.15±0.17%, mass between 95-354°C. This large initial mass loss can be attributed to the dehydration of physically adsorbed water molecules as well as chemically bound water molecules. The biomaterial
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degradation process begins to occur from the maximum temperature, which defines the thermal stability
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according to Costa et al. [14]. In these experiments, the T max were 333°C for the natural BC, 334°C for the ligth blue BC, 308°C for the burgundy BC and 333°C for the indigo blue colour BC. These T max are attributed to simultaneous events such as the removal of chemically bound water molecules and the decomposition of specific compounds of the dyes used in BC staining. The results of the thermogravimetric analysis confirmed that 96.09±0.11% of the natural biocellulose was degraded, against 59.23±0.09%% of the indigo blue BC film. This result is of extreme relevance for the studies that aim to apply dyed BC membranes as an industrial raw material. The BC film dyed with the dye extracted from Hibiscus rosa-sinensis (burgundy) presented the lowest thermal stability, with degradation beginning at lower temperatures than the other films. Probably, the major
ACCEPTED MANUSCRIPT components identified in this extract, such as carboxylic acids and others oxidants substances resulting from the extraction with ethanol, may have accelerated the thermal degradation of the polymer [45,46].
3.6. Tensile Strength Fig. 7 shows the results of the tensile assay used to characterize the mechanical properties of the dry and
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dyed pellicles. The dry natural pellicle had a mean tensile strength of 48.17 ± 15.38 MPa when the average maximum force was 115.59 ± 16.89 N at the breaking point. Considerable variations in the results were found
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when the dry samples in natural colour were compared to the dyed biomaterial submitted to the mechanical
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tests. The dyes may have altered the mechanical properties of the dyed BC pellicles by reducing the intermolecular force between the fibers, generating the disordered regions, as seen in the XRD analysis, which
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shows a decrease in crystallinity [27,38]. The dyed pellicles with Clitoria ternatea L and ARAQCEL RL 500 dyes exhibited a greater elongation at the breaking point than the natural film and the film dyed with
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ARAQCEL RL 500 dye demonstrated 92±0.07% minor stiffness in comparison to the natural film. During tensile testing, a decrease in strength occurs, beginning with a fracture followed by cracks, which
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are stabilized for a few seconds by microfibrils and nanofibrils until the onset of the rupture [40]. The increase
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in delamination or crack propagation occurs very quickly in dry pellicles due to the lack of elasticity. Young's modulus of elasticity (E) is an intrinsic property used as a mechanical parameter directly related to polymer stiffness. In the present study, the mean modulus of elasticity of the dyed pellicles was 199.08 ± 12.06, 145.94
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± 8.91 and 37.06 ± 8.01 MPa for the pellicles with Hibiscus rosa-sinensis, Clitoria ternatea L and ARAQCEL
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RL 500 dyes, respectively, while the natural BC pellicle showed an elasticity value of 458.38 ± 5.59 MPa. It is important to emphasize that the elasticity of the material is a characteristic very important for the moldings of pieces of fabric for the textile industry. The values also show that the natural dyes cause less change in the plasticity of the BC film compared to the synthetic dye. Depending on the use of the biomaterial, such as the manufacture of products that require less stiffness, natural dyeing can aggregate value to the product. On the other hand, an excessive load can break the dyed biomaterial more easily. The mechanical and physical properties of the dyed BC pellicles produced with the alternative medium indicate possible applications of this biomaterial in many industrial sectors, since colour is an aesthetic element
ACCEPTED MANUSCRIPT that adds symbolic value to products. Thus, the development of new products, such as hats and bags, with fashion value will be tested in the next step of this research.
4. Conclusions Although synthetic dyes are more widely used than natural dyes, the latter are more eco-friendly, less toxic,
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and less allergenic. The natural dyes used in this work can be successfully applied as pigments for BC films by controlling conditions and the supporting cast used in the dyeing and fixation process. The combination of
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natural and biodegradable compounds, such as the plant pigments and BC pellicles, can furnish new biotech
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products that meet the needs of the world market, which seeks safe, environmentally friendly options to currently used chemical compounds. Studies physicochemical characterization of the films obtained and the
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improvement of the dyeing methods will allow obtaining a commercial product for the textile industry as well
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as other industrial sectors, such as packaging and pharmaceutics.
Acknowledgements
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This study was funded by the following Brazilian fostering agencies: State of Pernambuco Assistance to
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Science and Technology Foundation (FACEPE), National Council for Scientific and Technological Development (CNPq), Coordination for the Advancement of Higher Education Personnel (CAPES) and the
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National Electrical Energy Agency (ANEEL).
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[40] L.R. Almeida, A.R. Martins, E.M. Fernandes, M.B. Oliveira, V.M. Correlo, I. Pashkuleva, A.P. Marques, A.S. Ribeiro, N.F. Durães, C.J. Silva, G. Bonifácio, R.A. Sousa, A.L. Oliveira, R.L. Reis, New biotextiles for tissue engineering: Development, characterization and in vitro cellular viability, Acta Biomaterialia 9
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[41] S. Davarpanah, N.M. Mahmoodi, M. Arami, et al. Modificação de superfície de fibra de seda a favor do meio ambiente: Enxerto e tingidura da quitosana, Ciência de Superfície Aplicada, 255 (2009) 4171-4176. [42] L. Segal, J.J. Creely, A.E. Martin, M.C. Conrad, An empirical method for estimating he degree of crystallinity of native cellulose using the X-ray diffractometer. Textile Research Journal. 29 (1959) 786794. https://doi.org/10.1177/004051755902901003 [43] K.C. Cheng, J.M. Catchmark, A. Demirci, Effect of different additives on bacterial cellulose production by Acetobacter xylinum and analysis of material property, Cellulose 16 (2009) 1033-1045.
ACCEPTED MANUSCRIPT [44] C. Gao, G.Y. Xiong, H.L. Luo, K.J. Ren, Y. Huang, Y.Z. Wan, Dynamic interaction between the growing Ca–P minerals and bacterial cellulose nanofibers during early biomineralization process, Cellulose 17 (2010) 365-373. [45] R.T. Chen, S.D. Fang, On the chemical constituents of Cotton rose Hibiscus (Hibiscus mutabilis), Chinese Traditional and Herbal Drug, 24 (1993) 227-229.
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[46] M.I.S. Melecchi, M.M. Martinez, F.C. Abad, P.P. Zini, I.N. Filho, E.B. Cramão, Chemical composition of Hibiscus tiliaceus L. flowers: A study of extraction methods, Journal Separation Science, 25 (2002)
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86-90. https://doi.org/10.1002/1615-9314(20020101)25:1/2<86::AID-JSSC86>3.0.CO;2-7
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Fig. 1. BC film produced by G. hansenii UCP1619 in alternative medium formulated with 1.5% glucose, 2.5% CSL, 0.27% Na2HPO4 and 0.15% citric acid for 10 days; (a) natural pellicle; (b) oven-dried pellicle and (c)
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pellicle after being submerged in water for five days.
Fig. 2. BC films dyed with Clitoria ternatea L. flowers (a), Hibiscus rosa-sinensis flowers (b) and ARAQCEL
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RL 500 (c).
Fig. 3. Dyed BC films with Clitoria ternatea L. (a), Hibiscus rosa-sinensis (b) and ARAQCEL RL 500 (c)
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after five days submerged in water. Images a', b' and c' are the results of oven dried pellicles. Images a'', b'' and
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c'' are the results after delicate hand washing with water.
Fig. 4. SEM images of the natural BC film (a) and of the BC films dyed with Clitoria ternatea L. flowers –
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light blue color (b), Hibiscus rosa-sinensis flowers – burgundy color (c) and ARAQCEL RL 500 – indigo blue
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color (d).
Fig. 5. X-ray diffraction diagrams of the natural BC film and of the BC films dyed with Clitoria ternatea L.
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blue color.
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flowers – light blue color, Hibiscus rosa-sinensis flowers – burgundy color and ARAQCEL RL 500 – indigo
Fig. 6. TG curves of the natural BC film and of the BC films dyed with Clitoria ternatea L. flowers – light blue color, Hibiscus rosa-sinensis flowers – burgundy color and ARAQCEL RL 500 – indigo blue color.
Fig. 7. Mechanical properties of BC pellicles. Tensile strength (MPa), elongation at the break point (% and mm) and Young’s modulus (MPa) were determined for natural and dyed BC pellicles.
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ACCEPTED MANUSCRIPT HIGHLIGHTS
1. Plant base natural dyes were tested in bacterial cellulose (BC) pellicles dying. 2. Clitoria ternatea L. and Hibiscus rosa-sinensis pigments were tested.
4. Dyeing can be performed on the hydrated BC pellicles.
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5. The dyed biomaterial can be applied to various artifacts.
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3. Dye fixation, tensile strength and elasticity was observed for BC pellicles.
Andréa Fernanda de S. Costa, Júlia D.P. de Amorim, Fabíola Carolina G. Almeida, Ivo Diego de Lima, Silva Sérgio C. de Paiva, Maria Alice V. Rocha, Glória M. Vinhas, Leonie A. Sarubbo PII: DOI: Reference:
S0141-8130(18)35172-9 doi:10.1016/j.ijbiomac.2018.10.066 BIOMAC 10717
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
28 September 2018 11 October 2018 14 October 2018
Please cite this article as: Andréa Fernanda de S. Costa, Júlia D.P. de Amorim, Fabíola Carolina G. Almeida, Ivo Diego de Lima, Silva Sérgio C. de Paiva, Maria Alice V. Rocha, Glória M. Vinhas, Leonie A. Sarubbo , Dyeing of bacterial cellulose films using plantbased natural dyes. Biomac (2018), doi:10.1016/j.ijbiomac.2018.10.066
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ACCEPTED MANUSCRIPT Dyeing of bacterial cellulose films using plant-based natural dyes
Andréa Fernanda de S. Costaa,b, Júlia D. P. de Amorimb,f, Fabíola Carolina G. Almeidab,d, Ivo Diego de Limac, Silva Sérgio C. de Paivad, Maria Alice V. Rochae, Glória M. Vinhasf and Leonie A. Sarubbo b,d*
a
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Design and Communication Centre, Federal University of Pernambuco, Caruaru, Brazil;
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Advanced Institute of Technology and Innovation, Recife, Brazil
Center for Exact and Nature Sciences, Federal University of Pernambuco, Recife, Brazil
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c
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e
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Centre for Sciences and Technology, Catholic University of Pernambuco, Recife, Brazil
Department of Domestic Sciences, Federal Rural University of Pernambuco, Brazil;
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Department of Chemical Engineering, Federal University of Pernambuco, Recife, Brazil
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Correspondence to: Leonie Asfora Sarubbo, Center for Sciences and Technology; Catholic University of
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E-mail: [email protected]
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Pernambuco; Recife; Brazil
ACCEPTED MANUSCRIPT Abstract The aim of this work was to test the use of plant-based natural dyes on bacterial cellulose (BC) to add aesthetic value to dyed pellicles while maintaining the mechanical properties. Natural pigments from Clitoria ternatea L. and Hibiscus rosa-sinensis were tested. The commercial ARAQCEL RL 500 was also used for comparison purposes. The behaviour of biocellulose regarding dye fixation, rehydration, tensile strength, and elasticity was
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evaluated in comparison to the dried biomaterial, showing that dyeing is a process that can be performed on hydrated BC. Dyeing the BC films through an innovative process maintained the crystallinity, thermal stability
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and mechanical strength of the BC and confirmed the compatibility of the membrane with the dyes tested, from
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the observed Scanning Electron Microscopy (SEM) morphology of nanofibers. Dyed biomaterial can be applied to various products, as confirmed by the results of the mechanical tests. As environmental awareness
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an attractive new material for the textile industry.
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and public concern regarding pollution increase, the combination of natural dyes and BC pellicles can produce
Keywords: Gluconacetobacter hansenii; bacterial cellulose; dyeing; Hibiscus rosa-sinensis; Clitoria ternatea
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1. Introduction
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The microfibrils of bacterial cellulose (BC) were first described on 1949 [1], and were found to be about 100 times smaller than those of vegetable cellulose (VC) [2]. The fibrils are made of three dimensional
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nanofribrils, which, when arranged, form of a highly porous hydrogel sheet with a large surface area [3]. Visually, the difference between VC and BC resides in the appearance and water content. VC has a fibrous appearance, whereas BC resembles a gel. Although the functional groups that characterize BC are the same as those in VC, the formation of the reticularly interconnected tridimensional pellicle is reported under static conditions, whereas irregular sphere-shaped cellulose particles are found under agitated conditions [4,5]. The formation of cellulose under static conditions is governed by the air supply on the surface of the medium and the yield depends on the concentration of the carbon source [6]. Acetic acid bacteria, especially those of the genus Gluconacetobacter [7-9], produce BC through oxidative fermentation because such bacteria can
ACCEPTED MANUSCRIPT assimilate various sugars, producing high yields of cellulose in a liquid medium [10,11]. The potential of BC goes beyond its existing applications, especially in big scale production using lowcost raw material, and includes special textile materials, such as functional materials and even packaging [12]. “Eco-driven” fashion or “sustainable clothing” is a global concern for producers and consumers. BC is a renewable raw material that can serve as the basis for “green clothing” [13].
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During the production of BC, the colour of the biomaterial varies depending on the substrate used in the culture medium. Formulations involving tea, molasses, beer, wine, and residual waste have been reported. The
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aim is to reduce the production cost and aggregate sensory, chemical, and physical properties to biocellulose
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for industrial applications [14–18].
Colours found in nature have a profound effect on humans and are crucial to the acceptance of textiles.
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[19] Colour aggregates both aesthetic and symbolic value. In pre-historic times, natural pigments were extracted from ores, insects, plants, and microorganisms and used as dyes for numerous materials. Such dyes
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have chromophore groups with different chemical structures that confer sufficient stability for the purposes of staining [20]. Synthetic dyes became popular in the middle of the 19th century and continue to dominate the
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market despite having properties that are harmful to humans, animals, and the environment [21,22].
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Natural pigments differ chemically from their synthetic counterparts and can be used to add colour to food, drugs, cosmetics, and textiles without compromising health [23]. Although natural pigments have the same
market.
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technological and economic advantages as synthetic pigments, they do not yet enjoy the same share of the
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Paint is a US$1.25 billion market for which synthetic dyes account for 40%, natural dyes account for 28% and caramel dyes account for 12%. The annual growth of synthetic dyes is 2 to 5%, while the annual growth of natural dyes is 5 to 10% [24]. The major environmental problem with synthetic dyes regards the large volume of water required for processing and the disposal of toxic effluents in bodies of water, which has a huge impact on the environment, causing harm to the reproduction of the fauna and flora and compromising the health of human populations [13]. Intense colour, shine, stability when exposed to light and washing, and the vast variety of shades are characteristics with aesthetic appeal. Natural pigments from ores, bugs, plants, and microbes are considered safe for the environment due to their non-toxic, non-carcinogenic, and biodegradable properties. Sources of
ACCEPTED MANUSCRIPT pigments include natural products, such as flavonoids and anthraquinones, which are produced in plants and animals. The use of natural pigments has tremendous advantages, such as abundance in the environment, simple extraction methods, biodegradability and the absence of toxicity [24–26]. Vegetal pigments have different molecular functional groups (tetrahydrides, tetraterpenes, quinones, Nheterocyclics and O-heterocyclics) and are classified as carotenoids, porphyrins, flavonoids, phenolics,
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indigoids, etc., which are found in the leaves, flowers, fruit, seeds, trunk and roots of plants [27]. In the present study, natural pigments were extracted from the flowers from Clitoria ternatea L. and Hibiscus rosa-sinensis.
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Clitoria ternatea L., commonly known as the blue pea or butterfly pea, is a member of the family Leguminosae
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(Fabaceae; tribe: Phaseoleae; subtribe: Clitoriinae). This plant is native to tropical equatorial Asia (Indonesia and Malaysia), but has been introduced in Africa, Australia and the Americas [28]. Chemical compounds
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isolated from C. ternatea include various triterpenoids, flavonol glycosides, anthocyanins and steroids [29]. Cyclic peptides known as cliotides have been isolated from the heat-stable fraction of C. ternatea extracts [30].
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Hibiscus rosa-sinensis, known colloquially as the Chinese hibiscus, is a tropical flowering plant from the Hibisceae tribe of the family Malvaceae native to tropical Asia (China). This plant is commonly found
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throughout the tropics and as a house plant throughout the world. Most ornamental varieties are hybrids. The
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present wide range of cultivars is considered to be a complex of inter-specific hybrids between eight or more different species originating from the eastern coast of Africa and islands in the Indian and Pacific Oceans. The flowers contain cyanidin diglucoside, flavonoids, thiamine, riboflavin, niacin, and ascorbic acid [29,31].
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In this work, the usability of flowers from Clitoria ternatea L. and Hibiscus rosa-sinensis as novel natural
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dyes was tested to add aesthetic value while maintaining the properties of BC pellicles.
2. Materials and Methods
2.1. Materials Glucose, peptone, yeast extract, citric acid monohydrate, agar, and sodium hydroxide were purchased from Merck Ltd., USA. Corn steep liquor (CSL) was acquired from a local company in the state of Pernambuco, Brazil. Dehydrated flowers from Hibiscus rosa-sinensis were obtained from a local market while the flowers from Clitoria ternatea L. were obtained directly form a local garden. Both flowers were used to stain BC
ACCEPTED MANUSCRIPT pellicles. The synthetic dye ARAQCEL RL 500 blue (ExtraCor Dyes and Chemicals, Brazil) was used for comparison.
2.2. Microorganism Gluconacetobacter hansenii UCP1619 was obtained from the culture collection of the Center for
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Environmental Sciences of the Catholic University of Pernambuco, Brazil, and used for the production of BC. The strain was maintained in the HS medium described on 1954 [32] and modified on 2010 [33], containing
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glucose agar slants with 2% glucose (w/v), 0.5% yeast extract (w/v), 0.5% peptone, 0.27% Na2HPO4 (w/v),
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1.5% citric acid (v/v), and 1.8% agar (w/v). The pH was adjusted to 5.0 using NaOH 1.0 M. The medium was stored in a refrigerator at 4 °C and sub-cultured every two months for inoculum development or stored at -80
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°C using 20% (v/v) glycerol instead of agar for long-term storage.
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2.3. Culture conditions
For the production of BC, the bacterium was cultivated in an alternative medium [14], in which the carbon
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source was reduced by 25% and synthetic nitrogen was completely replaced with corn steep liquor (CSL):
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1.5% glucose (w/v), 2.5% CSL (w/v), 0.27% Na2HPO4 (w/v), and 0.15% citric acid (v/v). The culture was prepared by transferring the G. hansenii cell suspension stored at -80 °C to the HS medium, followed by static cultivation at 30 °C for two days. The statically grown culture was then shaken vigorously
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to detach cells from the cellulose pellicle. The resulting cell suspension (3 mL) was inoculated in a semi-
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capped glass vessel (250 mL) containing 100 mL of the alternative medium and statically incubated at 30 °C for 10 days in triplicate experiments. After the fermentation process, the pellicles were collected, rinsed with tap water and purified in a 4% NaOH solution at 80 °C for 30 minutes to remove the microbial and excess substrate. After purification, the pellicles were washed in distilled water at neutral pH [14,34].
2.4. Extraction of pigments A kilogram of Clitoria ternatea L. flowers was placed in 200 mL of 80% cold ethanol (5°C) and stirred at 150 rpm for three hours. The solution was then filtered with No. 1 Whatman filter paper, resulting in a light blue solution, which was concentrated through lyophilization (Advantage Plus EL-85, SP SCIENTIFIC, USA).
ACCEPTED MANUSCRIPT The Clitoria ternatea L. dye solution was prepared by diluting 1 g of the light blue concentrated pigment released by the flower in 500 ml of deionized water. A solution with a burgundy colour was obtained from an infusion prepared using 50 g of dehydrated Hibiscus rosa-sinensis flowers in 500 mL of deionized water at 90 °C for 20 min. The solution was then filtered with No. 1 Whatman filter paper.
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The ARAQCEL RL 500 solution was prepared with 5 g of the dye diluted in 10% deionized water (w/v),
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resulting in an indigo blue solution.
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2.5. Dyeing procedure
The three pigment solutions prepared as described in section 2.4 were heated to 90 °C to speed up the
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reaction of the dye with the cellulose fiber and a 10% (w/v) solution of sodium chloride and sodium ferrocyanide (COMSAL, Rio Grande do Norte, Brazil) was added as the mordant. The batch process was used
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for the impregnation of the BC pellicles with the dyes. Round pellicles measuring 25 cm in diameter were submerged in the heated dye solutions for 30 minutes in 500 mL beakers. The experiments were conducted in
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triplicate.
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After dyeing, the films were rinsed twice in running water. To fix the colour, the hydrated films were submerged for 15 minutes in a solution containing 1% of ARAQFIX WE (ExtraCor Dyes and Chemicals, São Paulo, Brazil) fixer and 2% of the cationic SOFTEX MTZ softener (Texpal Química, São Paulo, Brazil), which
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is commonly used in conventional dyeing processes and dry cleaners for cotton cellulose fibers. The dyed BC
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films were rinsed on running water at pH 7.0 and stored in glass vials containing 250 mL of deionized water. The water was replaced every 24 hours for five days until the dye was no longer observed in the water. Samples were dried in an oven (SX1.2 Sterilifer, São Paulo, Brazil) at 60°C for 12 hours. After drying, the films were submerged in 200 mL of distilled water for two hours to simulate delicate hand washing and observe the release of the dye into the water. Some samples were kept hydrated in water for further analysis.
2.6. Determination of water activity The water contained in BC film can influence the fixing of dye to cellulose fibrils. Therefore, water activity was measured in the hydrated and oven-dried natural and dyed BC films using a water activity analyzer
ACCEPTED MANUSCRIPT with an internal sample temperature control (AQUALAB SERIES 4TE, São Paulo, Brazil). Water activity values between 0 and 0.20 indicate that the water is strongly bound, whereas values in the range of 0.70 to 1.00 indicate that most of the water is free and available for chemical reactions, enzymes, and the development of microorganisms. As water activity is entirely linked to the moisture of the biomaterial, this analysis enabled
2.7. Spectrophotometric evaluation of amount of dye retained in BC films
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the determination of its ability to adsorb and fix the dyes.
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The amount of dye retained in the dyed BC films was determined using UV-Vis light spectrophotometry.
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To determine the percentage of dye adsorbed to the BC fibres and nanofibrils, the three dyeing solutions (Clitoria ternatea L., Hibiscus rosa-sinensis and ARAQCEL RL 500) were analysed before submerging the
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films. After the dyeing process, the films were removed from the solutions and analysed again. The solution obtained after the pigment-fixing step pigments was also analysed. It was therefore possible to determine the
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percentage of dye retained and released in the different stages of the dyeing of the films. A digital spectrophotometer SP-22 (Biospectro, Paraná, Brazil) matched with 1 cm quartz cell was used for the
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absorbance measurement. Absorbance readings were made for each dye and the best wavelength (ʎ nm) was
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identified using the range from 375 to 700 nm. Dilutions were then performed with the dyeing solutions, the solutions generated after the dye fixation and the solutions obtained after the hand wash simulation to construct
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the calibration curves and equations for all dyes.
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2.8. Scanning Electron Microscopy (SEM) 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 (MIRA3 LM, Tescan, USA) operating at 10.0 kV at room temperature.
2.9. X-ray Diffractrometry (XRD)
ACCEPTED MANUSCRIPT X-ray diffraction patterns of all BC membranes were measured using a diffractometer (Bruker D8 Advance Davinci) with Cu Ka radiation. The crystallinity percentage was measured by equation (1) of Segal given below: CrI = [I002 – Iam] / I002 x 100
(1)
Where Crl expresses the relative degree of crystallinity, I002 is the maximum intensity (in arbitrary units) of the
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002 lattice diffraction, and Iam is the intensity of diffraction in the same units at 28 = 18°.
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2.10. Thermogravimetric Analysis
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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
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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
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measurements [35].
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2.11. Mechanical test
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Tensile strength (N), tensile strength at peak load (MPa), elongation at break (ε) (%) and Young’s modulus of elasticity (MPa) were determined for dyed BC films, according to Callister and William [36]. The BC sheets (thickness: 0.48 ± 0.0 and 8.7 ± 0.01 mm for dried and hydrated sheets, respectively) were cut into rectangular
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strips (80 x 25 mm). Tensile testing was performed at room temperature (28°C) at a speed of 1 mm/second and
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a static load of 0.5 N using a universal testing machine (EMIC DL – 500 Mf, Brazil) following the ASTM D882 Method for Tensile Properties of Thin Plastic Sheeting. The Bluehill LiteTM software program was used to calculate the stress-strain relationship and modulus of elasticity. A crosshead displacement sensor was used for the deformation measurements [14].
2.12. Statistical Analysis The analyses were performed in triplicate. Mean and standard deviation values were calculated and tested. Analysis of variance (ANOVA) was performed for all values and a p-value < 0.05 was considered indicative of statistical significance.
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3. Results and discussion
3.1. Production and dyeing of BC films After washing, purification and neutralization, the wet BC film produced with the alternative medium (Fig.
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1a) had a uniform, beige appearance and smooth texture and was very flexible. The yield of the wet and dried BC pellicles was 381.10 ± 0.81 and 9.63 ± 0.90 g, respectively. After five days submerged in water, the colour
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was off white (Fig. 1b) and uniformity in the off white tone was found throughout the pellicle after the drying
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process (Fig. 1c). The surface had a smooth, paper-like appearance and was neither flexible nor brittle. Dyes are highly detectable without the aid of lenses and, in some cases, are visible even at concentrations
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as low as 1 ppm [37]. When a fiber is dipped in a dye bath, the dye is adsorbed to the surface of the fiber and disseminates into the fiber. This phenomenon was observed in the hydrated BC films dyed with Clitoria
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ternatea L. (Fig. 2a), Hibiscus rosa-sinensis (Fig. 2b) and ARAQCEL RL 500 (Fig. 2 c). During the five days of submersion in water, the BC films dyed with the light blue solution (Fig. 3a)
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demonstrated a reduction in the colour intensity. After the subsequent drying and hand washing processes, a
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lack of uniformity of the light blue colour was found on the surface of the BC film. The dried film was fragile and brittle and its texture resembled paper (Figs. 3a’ and 3a’’). On the other hand, the pellicles dyed with the burgundy colour solution from Hibiscus rosa-sinensis flowers retained colouration after five days of
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immersion in water (Figure 3b). The oven drying process caused changes in colour intensity and uniformity as
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well as the texture of the biomaterial. The pellicle appeared as a soft, flexible film similar to processed animal leather (Fig. 3b’). Rehydration of the film during hand washing for two hours did not cause significant changes in coloration, although the film became more flexible and elastic (Fig. 3b"). The solution prepared with the synthetic ARAQCEL RL 500 dye was very concentrated and resulted in a BC film with an uniform indigo blue colour across the surface (Fig. 2c). This shade was retained after five days submerged in water (Fig. 3c). Drying caused a change in colour and made the film darker (Fig 3c’). The texture was soft. After hand washing for two hours at 28 °C, the film slightly lost colour intensity compared to the dry film but demonstrated greater flexibility and elasticity similar to processed animal leather (Fig. 3c’’).
ACCEPTED MANUSCRIPT 3.2. Characterization of dyed samples The penetration of dyes in individual fibers to a practical degree of durability is fundamental to the uniform application of colours [38]. The dyeing process occurs due to a chemical reaction between the material and dye. Permeability is an intrinsic property of materials that facilitates the diffusion of dye molecules in the fiber [39].
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Water activity in the BC films was analyzed to identify the amount of residual moisture in the BC nanofibers. This moisture is due to either capillary condensation or multilayer formation in spaces of cellulose
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fiber that allow occupation by water molecules. The calorimetric method described indicates the limit of
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hydroxyl interaction and provides evidence regarding the nature of the capillary spaces in which condensation occurs [39,40]. The authors state that, during the dyeing process, all adsorbed water below 4.3% for a cotton
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vegetal cellulose sample is connected to free hydroxyl groups of glucose units in the non-crystalline cellulose plant fiber. The X-ray of the BC films confirms the existence of crystalline regions in biocellulose, as described
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in a previous research [13].
Fibers that swell in contact with water have a hydrophilic nature and are able to accept water soluble dyes.
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For the three dyes used in the hydrated BC films, colour uniformity and fastness were found after fixing (Fig.
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3a, 3b and 3c). High residual moisture was detected in the wet BC films, with water activity of 0.985± 0.002, 0.982±0.001, 0.992±0.006 and 0.982±0.001 for the natural colour of the film and the light blue, burgundy and indigo blue dyed films, respectively. After drying, the moisture content was reduced by more than 40% in all
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three dyed BC films. This result indicates that, after dyeing and drying, the BC films exhibit residual moisture
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that does not interfere with usability. The hand washing simulation confirmed that the residual water activity of the dried cellulose biomaterial assisted in the release of dye that was not chemically bound to the BC fibres when the films were submerged in water. Since the swelling of fibers facilitates the migration of dye that has not been adsorbed, the colour intensity decreases (Figs. 3a’’, 3b’’ and 3c’’). The best wavelengths identified for the Clitoria ternatea L., Hibiscus rosa-sinensis and ARAQCEL RL 500 dyes were 375, 520 and 560 nm, respectively. Equations from calibration graphs (i.e., y = 89.915x + 0.2567 and R2 = 0.9651 for the Clitoria ternatea L dye solution, y = 91441X - 0.0008 and R2 = 0.9472 for the Hibiscus rosa-sinensis dye solution and y = 50993x +0.0048 and R2 = 0.9983 for the ARAQCEL RL 500 dye
ACCEPTED MANUSCRIPT solution) enabled the identification of the amount of dye retained in BC films in the various stages of the biocellulose dyeing process. After the dyeing step, the wet BC films absorbed 11.01±0.15, 31.09±0.31 and 48.05±0.20% of the Clitoria ternatea L., Hibiscus rosa-sinensis and ARAQCEL RL 500 dyes, respectively. These rates were increased to 17.13±0.05, 60.21±0.04 and 95.09±0.09%, respectively, after the fixing step. Submersion in distilled water for
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five days led to a 28.31±0.11% reduction in fixation for the Clitoria ternatea L. dye and a 12.08±% reduction for the Hibiscus rosa-sinensis dye. In contrast, the ARAQCEL RL 500 dye lost only 5.04±0.30% of the amount
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absorbed, demonstrating excellent fixation.
3.3. Scanning Electron Microscopy (SEM)
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The surface morphology of BC films produced in alternate medium was elucidated by SEM technique to highlight the influence and modification of the natural dyes extracted from Clitoria ternatea L. and Hibiscus
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rosa-sinensis and of the synthetic dye ARAQCEL RL 500 (Fig. 4). In contrast to the surface of natural BC (Fig. 4a), the sample of light blue dyed BC showed the presence of varying amounts of peculiar matter
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deposited on its surface (Fig. 4b). The burgundy color BC showed that the deposition of crystals became more
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apparent (Fig. 4c) whereas the indigo blue dyed BC presented morphological characteristics of BC fibers rather impregnated with the dye, showing that this dye can diffuse more in the fibers, reducing its accumulation on the surface (Fig. 4d). Davarpanah et al. [41] also showed the presence of peculiar matter firmly adhered to the
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surface of the silk surface after modification and influence of the chitosan on the degummed and acylated
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samples grafted with acylates.
3.4. X-ray Diffractrometry (XRD) The crystallinity index was calculated based on peak intensity data determined using the Segal et al. [42] method described by Cheng et al. [43]. The crystallinity index found in this study was 84.37±0.06% for the natural BC produced in the alternative medium (Fig. 5). Costa et al. [14] confirmed that the BC produced with the alternative medium shows high crystallinity. These results show that the composition of the medium did not affect the crystalline morphology of the polymer and the physical characteristics of the membrane were maintained.
ACCEPTED MANUSCRIPT The crystallinity indexes of the BC films produced in the same culture medium and dyed with Clitoria ternatea L. flowers (light blue), Hibiscus rosa-sinensis flowers (burgundy) and ARAQCEL RL 500 (indigo blue) were 82.23±0.21, 80.72±0.19 and 73.22±0.15%, respectively. These results show a tendency for the decrease in crystallinity of BC after dyeing using both naturals and synthetic dyes. The presence of the dyes weakens the intermolecular forces that maintains an organized structural arrangement proper of crystalline
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domains and favors a structural disarrangement, thus creating amorphous regions. This behavior may be associated with the breakdown of the hydrogen bonds between the chains of BC and the formation of
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difficult due to the incorporation of other compounds into BC [44].
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intermolecular interactions between BC and the dyes, making molecular orientation of the polymer chains
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3.5. Thermogravimetric Analysis
Fig. 6 shows the thermogravimetric degradation curves of the BC films in percentage of the mass of the
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original sample as a function of temperature. The natural film had a mass loss of approximately 42.11±0.21%, in the temperature range between 302 °C and 355 °C, while the dyed films had more than one stage relevant
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to mass loss. The burgundy BC film lost 55.23±0.13%, % mass between 74-330°C. The ligth blue BC showed
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a significant mass loss of 41.09±0.12%, in the bands of 292-350°C, while the blue indigo BC film lost 36.15±0.17%, mass between 95-354°C. This large initial mass loss can be attributed to the dehydration of physically adsorbed water molecules as well as chemically bound water molecules. The biomaterial
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degradation process begins to occur from the maximum temperature, which defines the thermal stability
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according to Costa et al. [14]. In these experiments, the T max were 333°C for the natural BC, 334°C for the ligth blue BC, 308°C for the burgundy BC and 333°C for the indigo blue colour BC. These T max are attributed to simultaneous events such as the removal of chemically bound water molecules and the decomposition of specific compounds of the dyes used in BC staining. The results of the thermogravimetric analysis confirmed that 96.09±0.11% of the natural biocellulose was degraded, against 59.23±0.09%% of the indigo blue BC film. This result is of extreme relevance for the studies that aim to apply dyed BC membranes as an industrial raw material. The BC film dyed with the dye extracted from Hibiscus rosa-sinensis (burgundy) presented the lowest thermal stability, with degradation beginning at lower temperatures than the other films. Probably, the major
ACCEPTED MANUSCRIPT components identified in this extract, such as carboxylic acids and others oxidants substances resulting from the extraction with ethanol, may have accelerated the thermal degradation of the polymer [45,46].
3.6. Tensile Strength Fig. 7 shows the results of the tensile assay used to characterize the mechanical properties of the dry and
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dyed pellicles. The dry natural pellicle had a mean tensile strength of 48.17 ± 15.38 MPa when the average maximum force was 115.59 ± 16.89 N at the breaking point. Considerable variations in the results were found
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when the dry samples in natural colour were compared to the dyed biomaterial submitted to the mechanical
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tests. The dyes may have altered the mechanical properties of the dyed BC pellicles by reducing the intermolecular force between the fibers, generating the disordered regions, as seen in the XRD analysis, which
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shows a decrease in crystallinity [27,38]. The dyed pellicles with Clitoria ternatea L and ARAQCEL RL 500 dyes exhibited a greater elongation at the breaking point than the natural film and the film dyed with
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ARAQCEL RL 500 dye demonstrated 92±0.07% minor stiffness in comparison to the natural film. During tensile testing, a decrease in strength occurs, beginning with a fracture followed by cracks, which
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are stabilized for a few seconds by microfibrils and nanofibrils until the onset of the rupture [40]. The increase
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in delamination or crack propagation occurs very quickly in dry pellicles due to the lack of elasticity. Young's modulus of elasticity (E) is an intrinsic property used as a mechanical parameter directly related to polymer stiffness. In the present study, the mean modulus of elasticity of the dyed pellicles was 199.08 ± 12.06, 145.94
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± 8.91 and 37.06 ± 8.01 MPa for the pellicles with Hibiscus rosa-sinensis, Clitoria ternatea L and ARAQCEL
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RL 500 dyes, respectively, while the natural BC pellicle showed an elasticity value of 458.38 ± 5.59 MPa. It is important to emphasize that the elasticity of the material is a characteristic very important for the moldings of pieces of fabric for the textile industry. The values also show that the natural dyes cause less change in the plasticity of the BC film compared to the synthetic dye. Depending on the use of the biomaterial, such as the manufacture of products that require less stiffness, natural dyeing can aggregate value to the product. On the other hand, an excessive load can break the dyed biomaterial more easily. The mechanical and physical properties of the dyed BC pellicles produced with the alternative medium indicate possible applications of this biomaterial in many industrial sectors, since colour is an aesthetic element
ACCEPTED MANUSCRIPT that adds symbolic value to products. Thus, the development of new products, such as hats and bags, with fashion value will be tested in the next step of this research.
4. Conclusions Although synthetic dyes are more widely used than natural dyes, the latter are more eco-friendly, less toxic,
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and less allergenic. The natural dyes used in this work can be successfully applied as pigments for BC films by controlling conditions and the supporting cast used in the dyeing and fixation process. The combination of
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natural and biodegradable compounds, such as the plant pigments and BC pellicles, can furnish new biotech
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products that meet the needs of the world market, which seeks safe, environmentally friendly options to currently used chemical compounds. Studies physicochemical characterization of the films obtained and the
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improvement of the dyeing methods will allow obtaining a commercial product for the textile industry as well
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as other industrial sectors, such as packaging and pharmaceutics.
Acknowledgements
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This study was funded by the following Brazilian fostering agencies: State of Pernambuco Assistance to
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Science and Technology Foundation (FACEPE), National Council for Scientific and Technological Development (CNPq), Coordination for the Advancement of Higher Education Personnel (CAPES) and the
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[40] L.R. Almeida, A.R. Martins, E.M. Fernandes, M.B. Oliveira, V.M. Correlo, I. Pashkuleva, A.P. Marques, A.S. Ribeiro, N.F. Durães, C.J. Silva, G. Bonifácio, R.A. Sousa, A.L. Oliveira, R.L. Reis, New biotextiles for tissue engineering: Development, characterization and in vitro cellular viability, Acta Biomaterialia 9
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[41] S. Davarpanah, N.M. Mahmoodi, M. Arami, et al. Modificação de superfície de fibra de seda a favor do meio ambiente: Enxerto e tingidura da quitosana, Ciência de Superfície Aplicada, 255 (2009) 4171-4176. [42] L. Segal, J.J. Creely, A.E. Martin, M.C. Conrad, An empirical method for estimating he degree of crystallinity of native cellulose using the X-ray diffractometer. Textile Research Journal. 29 (1959) 786794. https://doi.org/10.1177/004051755902901003 [43] K.C. Cheng, J.M. Catchmark, A. Demirci, Effect of different additives on bacterial cellulose production by Acetobacter xylinum and analysis of material property, Cellulose 16 (2009) 1033-1045.
ACCEPTED MANUSCRIPT [44] C. Gao, G.Y. Xiong, H.L. Luo, K.J. Ren, Y. Huang, Y.Z. Wan, Dynamic interaction between the growing Ca–P minerals and bacterial cellulose nanofibers during early biomineralization process, Cellulose 17 (2010) 365-373. [45] R.T. Chen, S.D. Fang, On the chemical constituents of Cotton rose Hibiscus (Hibiscus mutabilis), Chinese Traditional and Herbal Drug, 24 (1993) 227-229.
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[46] M.I.S. Melecchi, M.M. Martinez, F.C. Abad, P.P. Zini, I.N. Filho, E.B. Cramão, Chemical composition of Hibiscus tiliaceus L. flowers: A study of extraction methods, Journal Separation Science, 25 (2002)
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86-90. https://doi.org/10.1002/1615-9314(20020101)25:1/2<86::AID-JSSC86>3.0.CO;2-7
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Fig. 1. BC film produced by G. hansenii UCP1619 in alternative medium formulated with 1.5% glucose, 2.5% CSL, 0.27% Na2HPO4 and 0.15% citric acid for 10 days; (a) natural pellicle; (b) oven-dried pellicle and (c)
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pellicle after being submerged in water for five days.
Fig. 2. BC films dyed with Clitoria ternatea L. flowers (a), Hibiscus rosa-sinensis flowers (b) and ARAQCEL
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RL 500 (c).
Fig. 3. Dyed BC films with Clitoria ternatea L. (a), Hibiscus rosa-sinensis (b) and ARAQCEL RL 500 (c)
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after five days submerged in water. Images a', b' and c' are the results of oven dried pellicles. Images a'', b'' and
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Fig. 4. SEM images of the natural BC film (a) and of the BC films dyed with Clitoria ternatea L. flowers –
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light blue color (b), Hibiscus rosa-sinensis flowers – burgundy color (c) and ARAQCEL RL 500 – indigo blue
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color (d).
Fig. 5. X-ray diffraction diagrams of the natural BC film and of the BC films dyed with Clitoria ternatea L.
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blue color.
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flowers – light blue color, Hibiscus rosa-sinensis flowers – burgundy color and ARAQCEL RL 500 – indigo
Fig. 6. TG curves of the natural BC film and of the BC films dyed with Clitoria ternatea L. flowers – light blue color, Hibiscus rosa-sinensis flowers – burgundy color and ARAQCEL RL 500 – indigo blue color.
Fig. 7. Mechanical properties of BC pellicles. Tensile strength (MPa), elongation at the break point (% and mm) and Young’s modulus (MPa) were determined for natural and dyed BC pellicles.
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1. Plant base natural dyes were tested in bacterial cellulose (BC) pellicles dying. 2. Clitoria ternatea L. and Hibiscus rosa-sinensis pigments were tested.
4. Dyeing can be performed on the hydrated BC pellicles.
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5. The dyed biomaterial can be applied to various artifacts.
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3. Dye fixation, tensile strength and elasticity was observed for BC pellicles.