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Abstract

In this study, bacterial cellulose was produced through the fermentation of a mixture of black tea, ginger, and sugar, and used to create bio-textile films on sugarcane-based media. Characterization included ribosomal RNA gene sequencing, FTIR spectroscopy, XRD, and SEM was used to examine surface morphology. The bio-textile films showed increasing UV resistance beyond 10 days of cultivation (T.UVA%; 0.13 ± 0.02, T.UVB%; 0.22 ± 0.01, UPF; 629 ± 2.12) and antimicrobial resistance was assessed by quantifying Colony-Forming Units (CFU), resulting in a 100% reduction in growth for both Escherichia coli and Staphylococcus aureus. Subsequently, after 15 days of cultivation, antimicrobial activity was evaluated using the disc agar diffusion method, yielding noteworthy outcomes. E. coli displayed a 25 mm zone of inhibition, S. aureus exhibited a 31 mm zone of inhibition, Candida albicans showed a 35 mm zone of inhibition, and Aspergillus niger presented a 22 mm zone of inhibition.

Keywords

Bacterial cellulose bio-textile films biosynthesized black tea and ginger sugar cane media

Introduction

To mitigate the environmental impact of the textile and leather industries, designers are increasingly exploring textiles created from renewable raw materials. We are well aware of the ethical and environmental challenges associated with rearing animals for leather and fur, tanning leather using hazardous methods, exploiting silkworms, and relying on petroleum-based materials to produce synthetic fibres, which are widely used in fabric and artificial leather production.¹ Consequently, the textile and garment industries rank among the world’s most polluting sectors. Therefore, bio-fabrics present an excellent alternative to non-eco-friendly textiles and leather materials.

Bio-fabrics, especially those produced through biological processes, represent the future of sustainable textiles on a global scale. These materials are cultivated by microorganisms such as bacteria, fungi, yeast, and others. Through bioengineering techniques, we can engineer these microorganisms to synthesize biopolymers that can be extruded into filaments using a spindle or molded into materials that can be retrieved without generating waste. During the biosynthesis process, bacteria or a consortium of microorganisms produce a film, known as a pellicle or membrane, consisting of a random nanofibrillar network of cellulose chains (as depicted in Figure 1).² This network is interspersed with regions of water, which account for 90%–98% of the material’s total volume. The structure, thickness, color, and texture of the final materials can be controlled through the microorganisms’ DNA. These microorganisms are nourished with sugar and transformed into biological fiber factories.

Figure 1.

Random nanofibrillary network of bacterial cellulose chains.

Bacterial cellulose (BC) is the most abundant biopolymer on the planet. BC is synthesized by bacteria belonging to the genus Acetobacter, with “Gluconobacter xylinus” being the most efficient producer.³ BC consists of a network of self-assembled nanofibers, leading to a BC structure with higher crystallinity, tensile strength, and water retention capacity compared to plant cellulose.⁴

Due to its unique properties, BC is emerging as a promising biopolymer for various applications. For instance, owing to its biocompatibility,⁵ BC finds numerous applications in the biomedical field, including the development of artificial blood vessels for microsurgery,⁶ scaffolds for cartilage tissue engineering,⁷ and wound dressings.⁸ It is also utilized in the manufacture of acoustic diaphragms for audio speakers and headphones,⁹ electronic paper,¹⁰ and optically transparent nanocomposites.^11,12 Purified and dried BC is transformed into membranes for use in separation processes such as ultrafiltration, gas permeation, and vapor permeation, as well as in paper production.^13,14 Food-related applications of BC encompass additives, emulsifiers, dietary fibers, edible preservatives, and barriers against bacterial growth.^15,16

Despite the growing interest in BC, there is a limited number of publications addressing modifications in culture conditions, including strain selection, growth temperature, and carbon source. Existing studies predominantly focus on chemical modifications like acetylation,^17,18 nitration, amination with diethylene-triamine,¹⁹ and phosphorylation.²⁰ In this work, we present the production and investigation of bio-textile films derived from BC biosynthesis using black tea and ginger, cultivated on sugar cane media. The primary objective was the manufacturing of bio-textile films with the ability to resist microbes and ultraviolet radiation. To be considered a sustainable alternative for specific textiles and leather, especially in medical and technical applications, it should be both environmentally friendly and cost-effective. The cultivation process for obtaining biotextile films ranged from 4 to 10 days, depending on the desired thickness. It’s worth noting that the harvest time in this work is relatively shorter compared to most publications, where harvest times of 10–14 days are commonly reported.^21,22

Materials and methods

Description of the biotextile films production process

The bio-textiles production is divided into four stages.

- Biosynthesis for bacterial celluloses. Bacterial cellulose was biosynthesised by fermenting black tea and ginger and creating BC wet membranes (95% humidity, three-dimensional network of nano- and microfibrils) for this study.

- Cultivation conditions on sugar cane media. The BC previously described has been cultured on sugar cane media. Sterilised by boiling at 110°C for 5 min, then cooling to a room temperature range of 25 to 29°C.

- Production of bio-textile films. Four bio-textile films were biosynthesized according to the culture/fermentation time on sugar cane media Sterilized.

- Washing and drying. The films were washed with distilled water and dried in atmospheric air.

Biosynthesis for bacteria celluloses

To biosynthesize bacterial nanocellulose (BC), we combined black tea from Lipton and 100% organic Indian ginger rhizome powder from “Imtenan- Egypt” in the first phase. This recipe involves using 1 L of distilled water, 150 g of sucrose, 10 mL of apple cider vinegar, 5 g of black tea, and 200 mL of ginger drink. These components were carefully blended before being placed in an airtight bottle, as shown in Figure 2(a). During the first phase, incubation and fermentation occurred over a 3-month period under room conditions (with temperatures ranging from 25 to 29°C and relative humidity between 45 and 50%). This process was conducted in a dark and elevated location. In the second phase, the biosynthesis of BC wet membranes required the following ingredients: 1/2 L of hot water, 4 g of black tea, 1/2 L of room temperature water, 150 g of sugar, and 1/2 L of the pre-prepared material. These ingredients were placed in a glass jar and covered securely with a breathable cloth, as illustrated in Figure 2(b). During the second phase, incubation and fermentation continued for 30 days at temperatures ranging from 25 to 29°C, with relative humidity levels maintained between 45 and 50%. This process also took place in a dark and elevated location. The BC wet membrane was formed, resulting in gel-like materials, as depicted in Figure 2(c).

Figure 2.

(a) Brewing ginger and black tea together; (b) Biosynthesis for BC; (c) BC wet membranes created by floating gel-like material.

Cultivation conditions on sugar cane media

On sugar cane media grown in Upper Egypt, the BC was cultured. The seller was “Al-Qubaisi Company of Ramses Street, Ghamra, Cairo, Egypt.” All the protocols for the use of plant material were compliant with relevant institutional, national, and international guidelines and legislation. In static culture conditions, it was Incubation for 7 days at room temperature in a dark and high place. in a temperature range of 25 to 29°C and a relative humidity range of 45 to 50%. BC film was created by floating gel-like material. The fermentation process is depicted in Figure 3(a). The findings after 5 days are shown in Figure 3(b), and the cellulose bacteria after 7 days are shown in Figure 3(c).

Figure 3.

(a) Process of fermentation on sugar cane medium, (b) after 5 days of fermentation on sugar cane medium, (c) BC wet membrane, created by floating gel-like material after 7 days of cultivation on the medium of sugar cane.

Production of bio-textile films

We made four bio-textile films with varying culture durations (4, 6, 8, and 10 days) by incubating 4 L of sugar cane juice with 50 g of bacterium cellulose generated in a 30 × 50 cm container, as shown in Figure 4(a). Then it is covered well with a breathable cloth and fermented in static cultivation conditions in a In a high place that looks like a basement. As illustrated in Figure 4(b) biotextile films were generated by floating a gel-like substance.

Figure 4.

(a) Incubating 4 L of sugar cane juice with 50 g of BC generated; (b) Bio-textile films were generated following incubation by floating a gel-like substance.

Washing and drying

The films were rinsed multiple times with distilled water and squeezed to minimise its water content, as shown in Figure 5(a) and (b). The washed film was dried for 2 days on a horizontal surface in the sun and atmospheric air, as illustrated in Figure 5(c), Figure 6 shows Scheme of bio-textile film production in sugar cane media.

Figure 5.

(a) Rinsed films multiple times with distilled water, (b) squeeze to minimise its water content, (c) dry on a horizontal surface in the sun and atmospheric air for 2 days.

Figure 6.

Scheme of bio-textile films stages production in sugar cane media.

Natural dyeing

Bacterial cellulose can be dyed not only after it is produced (ex situ), but also while it is being produced by adding dye during cultivation (in situ). Both dyeing methods produced smooth surfaces and kept their inherent nanostructures during dye penetration, but the in situ method produced a smoother surface and more even colour than the ex-situ method.²³ Based on this, the bio-textile film was dyed by adding dye during cultivation (in situ)14 mL of food colouring was added to the carbon source (sugarcane juice). using blue food dye from Surya International, Ahmedabad, India. Figure 7(a) shows the dyed bio-textile film during washing, Figure 7(b) shows the dyed bio-textile film after drying.

Figure 7.

(a) The dyed bio-textile film is rinsed multiple times with distilled water, (b) the dyed bio-textile film after drying.

Figure 8 depicts bio-textile films created at various cultivation times. Figure 8(a) after 4 days of cultivation, Figure 8(b) after 6 days of cultivation, Figure 8(c) after 8 days of cultivation, and Figure 8(d) after 10 days of cultivation.

Figure 8.

(a) Bio-textile film after 4 days of culture time; (b) bio-textile film after 6 days of culture time; (c) bio-textile film after 8 days of culture time; (d) bio-textile film after 10 days of culture time.

Characterization

Isolation and characterization of microbes the cultivation on sugar cane media

Nutrient Agar (NA) and Potato Dextrose Agar (PDA) obtained from Sigma-Aldrich were utilized. Both NA and PDA were prepared following the manufacturer’s recommended procedures. For Hestrin-Schramm (HS) agar, a mixture was created by combining 20 g per liter of glucose, 5 g per liter of peptone, 5 g per liter of yeast extract, 2.7 g per liter of Na₂HPO₄, and 1.15 g per liter of citric acid. To prepare plates for enumerating microorganisms in BC fermentations, a solution containing 0.5 mg per milliliter of cycloheximide in ethanol was added to the NA and HS agar while they were in their liquid state and had cooled down. For yeast enumeration in BC fermentations, plates were prepared differently. A solution containing 25 mg per milliliter of chloramphenicol in ethanol was added to the cooled, molten PDA agar.

Isolation of dominant microbes (bacteria and yeast) culture adapted in sugar cane media

A fermentation broth was subjected to culture on three different agar types: PDA, NA, and HS. To begin, a 10 mL sample of the fermentation broth was serially diluted in sterile phosphate-buffered saline (PBS). The bacteria were isolated on NA media with the addition of cycloheximide to hinder yeast growth. This mixture was then incubated at room temperature for a duration of 24–48 h. In contrast, yeast isolation took place on PDA agar, which was supplemented with chloramphenicol to prevent bacterial growth. The yeast samples were incubated at room temperature for a period of 72 h. Additionally, microbial samples were grown on HS agar media at room temperature for 72 h. The dominant isolates from each petri dish were chosen based on their colony morphology and the results of Gram staining.

Characterization and identification of bacteria and yeast isolates using sequence analysis of ribosomal RNA genes

Bacterial isolates were identified using a PCR method along with gene sequencing of the 16S rRNA gene, employing universal primers (27F/1492R) with the following sequence:

(5′-AGAGTTTGATCCTGGCTCAG-3′ and 5′-GGTTACCTTGTTACGACTT-3′).^24,25 In parallel, yeast isolates underwent identification through a PCR method, focusing on the D1/D2 region of the 26S rRNA gene, using NL-1 and NL-4 primers with the following sequence:

(5′-GCATATCAATAAGCGGAGGAAAAG-3′ and 5′-GGTCCGTGTTTCAAGACGG-3′).²⁶ The resulting PCR products were then subjected to electrophoresis on a 1% agarose gel with Tris-borate-EDTA. Subsequently, the agarose gel was treated with ethidium bromide and visualized under UV light. Following this, the pure PCR results obtained from the amplification of the bacterial 16S rRNA gene and yeast 26S rRNA gene were sent for sequencing.

Scanning electron microscopy (SEM)

SEM analysis was performed using scanning electron microscope. was carried out using a high-resolution scanning electron microscopy (SEM) EDAX AMETEK Quanta FEG 250 with field emission gun attached with an accelerating voltage 30 kV, FEI company - Netherlands in the central lab, National Research Centre, Egypt. the samples cut from the dried bio-textile film alized were coated with gold film before analysis.

X-ray diffraction (XRD)

The X-ray diffraction (XRD) was used to study the crystallinity and microstructure of the sample bio-textile film. The XRD pattern was captured over a 2-h period with an angle range of 0–40°. The Cu/Ka radiation source employed had a wavelength of 0.154 nm and was created at an acceleration voltage of 40 kV and a filament emission of 40 mA.

Fourier transform infrared spectroscopy (FTIR)

Fourier transform infrared spectroscopy (FTIR) Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was performed using a Thermo Scientific Spectrometer equipped with a Smart Orbit ATR accessory. The reflectance element was a dia-mond crystal. Spectra were collected in the 4000–650 cm⁻¹ range with 16 scans and at 16 resolution.

Contact angle test

Contact angle analysis was performed in the Desert Research Centre of the Egyptian Ministry of Agriculture and Land Reclamation using the Dataphysics model tension metre and the OCA-15EC. The sample was subjected to one drop of deionized water (3 μL) at room temperature. The evaluations were conducted at 0, 1, 3, 4, and 5 s.

Percentage of water retention (PWR)

All four wet bio-textile film were weighed and dried in atmospheric air in order to completely remove the water and maintain a constant weight. Then the PWR was obtained using equation (1).²⁷

PWR (%) = (\frac{M e a n o f t h e w e t w e i g h t s - M e a n o f t h e d r y w e i g h t s}{M e a n o f t h e w e t w e i g h t s}) x 100 .

(1)

Yield and thickness

To calculate the yield of the biosynthesis process, we used equation (2)

Y i e l d (g / l) = \frac{M}{C}

(2)

where M is the dry/wet weight of BC (g), and C is the weight of the carbon source (L) used in the production medium (sugar cane juice).

The thickness test was carried out according to DIN EN ISO 5084 by using a digital thickness gauge with a roller insert measuring tool type (0.01 mm–12.5 mm).

Ultraviolet resistance analysis (UVA)

The ultraviolet resistance properties of bio-textile films were determined using the UV/Visible Spectrophotometer JASCO V-750 (from 280 to 400 nm) in accordance with the Australian/New Zealand standard method AATCC Test Method 183:2010.

Antimicrobial activity studies

Antimicrobial activity was determined in two ways.

1- Determination of antibacterial activity by measuring colony forming units (CFU)

2- antimicrobial activity the disc agar diffusion method (Zone of inhibition)

Where the antimicrobial activities of microbes were investigated Staphylococcus aureus ATCC 6538-P (G+ve) and Escherichia coli ATCC 25933 (G−ve), Candida albicans ATCC 10231 (yeast), and Aspergillus niger NRRL-A326 (fungus).

Staphylococcus aureus (bacteria G+ve)

A peptidoglycan layer and teichoic acids compose the cell wall of gramme-positive bacteria.²⁸ Peptidoglycans, or mureins, are composed of linear glycans that are cross-linked by short peptides. Glycans are disaccharides comprised of N-acetylglucosamine and N-acetylmuramic acids linked β (1,4) glycosidic linkages.^28–30 Teichoic acids are anionic glycopolymers that give gramme-positive cells a negative charge [80]. Two types of teichoic acids exist: those that covalently attach to the N-acetylmuramic acid of peptidoglycans and those that anchor to cytoplasmic membranes.²⁸

Escherichia coli (bacteria G-ve)

A Gramme-negative bacteria’s cell wall consists of a thin layer of 2–7-nm-thick peptidoglycan and an outer membrane, with lipoproteins binding these two together.^31,28 The outer membrane, on the other hand, is composed of an asymmetrical lipid bilayer with an inner side comparable to a cytoplasmic membrane but an outside side distinct due to the presence of lipopolysaccharides.^32,31 There are proteins in this outer membrane that tend to be organised into trimers called “porins,” and these porins contain a hydrophilic channel that allows low-molecular-weight molecules from the external environment to enter the bacterial cell.^32,31

Candida albicans (yeast)

C. albicans is a common pathogen that causes mucosal infections. It also causes life-threatening bloodstream infections that spread to internal organs in certain types of immunocompromised patients. It is a polymorphic fungus that can grow in yeast, hyphal, or pseudohyphal forms. The hyphal form enters the bloodstream by penetrating epithelia and endothelia, causing tissue injury and gaining access to the bloodstream.³³

Aspergillus niger (fungus)

Niger is a widespread contaminant of food, soil, and the indoor environment due to its propensity to thrive on a wide range of substances. Despite the fact that its spores are widespread, the fungus has been considered a less likely source of human disease than other Aspergillus species.³⁴ A. niger typically infects tissues that have already been rendered vulnerable by bacterial infections, physical damage, or cerumen deposition in the external auditory canal. A. niger, like other Aspergillus species, is a causal agent of otomycosis, a subacute or chronic fungal infection of the ear, throat, or nose. Immune deficient individuals, like most filamentous fungal species, are prone to A. niger infections, which cause invasive pulmonary aspergillosis.^35,34 characterised by a chronic productive cough with blood coughing.

1- Determination of antibacterial activity by measuring colony forming units (CFU)

The antimicrobial activities of bio-textile films against S. aureus and E. coli were investigated using the colony forming unit technique (CFU). Bacterial stocks, totaling (100 µl) in volume, contained an estimated CFU count of around 10⁸. were inoculated into a 20-ml freshly prepared liquid nutrient broth containing 5 g/L peptone and 3 g/L beef extract at pH 6.8 in 100 mL Erlenmeyer flasks and incubated for 24 h. Bio-textile films (250 mg) were added to the inoculated flasks (with 20 µl of inoculums), leaving the control inoculated flasks without samples) The reduction (R%) in growth was measured after 24 h of incubation at 37°C by detecting the reduction in absorbance of the inoculated flasks compared to the untreated controls. Equation (3) was used to calculate the growth reduction.

R (%) = \frac{A - B}{A} \times 100

(3)

where A represents the number of colonies in the culture control and B represents the number of colonies in the bio-textile films sample.³⁶

2- Determination of antimicrobial activity the disc agar diffusion method (zone of inhibition)

The disc agar diffusion method was used to investigate the antimicrobial activities of the four representative test microbes used: S. aureus ATCC 6538-P (G +ve) and E. coli ATCC 25933 (G−ve), C. albicans ATCC 10231 (yeast), and A. niger NRRL-A326 (fungus). Bacteria and yeast were regularly introduced to NA plates through heavy inoculation, with each plate receiving 0.1 mL of cells per milliliter in the range of 10⁵–10⁶ cells/ml. PDA plates seeded with 0.1 mL (10⁶ cells/mL) of fungal inoculum were used to evaluate the antifungal activities. The inoculated plates were covered with bio-textile film discs (15 mm in diameter). After that, the plates were kept at 4°C for 2–4 h to allow for maximum diffusion. The plates were then incubated in an upright position for 24 h at 37°C for bacteria and 48 h at 30°C for organism growth. The test agent’s antimicrobial activity was determined by measuring the diameter of the zone of inhibition in millimetres (mm). The experiment was repeated several times, and the mean of the readings was recorded.

Statistical analysis

We conducted statistical analysis using Microsoft Excel 365 from Microsoft, USA. The data were analyzed using trend lines, their equations and values of the correlation coefficient (R²) determination coefficient values, and one-way analysis of variance (ONE WAY-ANOVA).

Results and discussion

BC material formation occurred by enveloping native cellulose polyglucosan chains with water molecules from the growth medium. Subsequent drying of the BC material led to the establishment of hydrogen bonds among cellulose hydroxyl groups, transforming the gel material into a film.²⁴ In the static cultivation process, a BC film formed on the surface of the sugar cane medium. Within 3 days of fermentation initiation, the BC film exhibited an increase in both weight and thickness, gradually covering the entire liquid surface. Consequently, harvesting was conducted at intervals of 4, 6, 8, and 10 days to investigate the influence of culture time on various film properties, such as antimicrobial resistance, ultraviolet resistance, water retention percentage, yield, and thickness. Additionally, the bio-textile films underwent thorough characterization using diverse analytical techniques, including the sequencing of ribosomal RNA genes, FTIR spectroscopy, XRD, and SEM.

Bacterial cellulose-producing isolates screening

Bacteria that had been isolated and preserved from a previously adapted culture were selected for screening based on their distinctive colony characteristics. Before the screening process, six different colony types underwent microscopic examination, revealing that all of them exhibited uniform visual features, appearing as rod-shaped and pink when subjected to a Gram staining procedure Figure 9. These characteristics indicated that the bacteria belonged to the acetic acid bacteria group,³⁷ which is a common producer of BC. Following a 14-day period of static incubation, the most productive bacterium was chosen based on its notably thick appearance, determined through visual assessment. This selected isolate became the sole bacterial inoculum used for subsequent experiments.

Figure 9.

Light microscope at 1000× magnification of gramme-stained bacteria.

Sequence analysis of ribosomal RNA genes

The PCR product was subjected to DNA sequencing, and the resulting sequencing data were processed using FinchTV software to select high-quality peaks for both the forward and reverse primers. Subsequently, the forward primer was aligned with the reverse-complement of the reverse primer to verify the accuracy of both sequences. This alignment was performed using CLC Viewer software, resulting in the generation of a “Consensus” sequence, the consensus sequence was then screened using nucleotide BLAST to determine the microbial identity. The choice of microbial identity from the BLAST results was made based on the journal status of the accession link, whether it was published or submitted. Any unpublished BLAST results were disregarded. A total of three bacteria and one yeast were successfully sequenced and identified, as outlined in Table 1. All three bacteria belong to closely related genera, while the single isolated yeast falls within the Brettanomyces genus.

Table 1.

Names of bacteria and yeast isolated.

No	Forward primer	Reverse primer	Identity	Accession number
1	27F	1492R	Komagataeibacter sp. DS1MA.62A	LN884048.1
2	27F	1492R	Komagataeibacter xylinus	MH511551.1
3	27F	1492R	Komagataeibacter saccharivorans	KY287776.1
4	NL1	NL4	Brettanomyces bruxellensis	MH930867.1

Primers, Identities, and the Accession.

Scanning electron microscopy (SEM) for bio-textile film

Bacterial cellulose’s unique properties are determined by the ultrastructure and size of its fibrils. The surface microstructure of BC pellicles is revealed by SEM images Figure 10(a) to be layered nets of varying densities made of randomly oriented cellulose microfibrils. This is typical BC morphology, as described by Yamanaka and Sugiyama (2000).³⁸ BC nanofibrils can be seen on the surface in a multilayered structure, as shown in Figure 10(b), confirming the structure of a well-organized three-dimensional network. The crystallization of cellulose in a single direction confirms that the molecules are linear glucose linked by a β (1 → 4) glycosidic connection. When glycosidic chains join, microfibrils are formed that are oriented by intramolecular hydrogen bonds.³⁹ Because of the presence of binding agents (mainly sucrose from the growth medium), the structure of the BC sample was bulky, making determining cellulose fibers challenging. However, using SEM, it was possible to determine the cellulose fibers and their diameter. As shown in Figure 10(b) the diameter of microfibrils in the BC specimens varied between 20 and 95 nm.

Figure 10.

(a) SEM image of the bio-textile film; (b) SEM of the diameter of cellulose fibres.

X-ray diffraction (XRD) analysis

Here are four crystallographic polymorphisms in cellulose (I, II, III, and IV). Cellulose I is the crystalline cellulose produced naturally by a wide range of species, including trees, plants, tunicates, algae, and bacteria. Cellulose I has a thermodynamically metastable structure and can be changed to cellulose II or III. All of the cellulose strands are arranged in a highly organised parallel pattern.⁴⁰ Regeneration (solubilization and recrystallization) and mercerization (alkaline solution) can be used to convert cellulose I to cellulose II. The structure of cellulose II is monoclinic. Cellulose III is created by combining liquid ammonia and thermal treatments with Cellulose I and II. Cellulose IV is created by combining cellulose III.^41,42 Diffraction patterns obtained for the bio-textile film obtained are shown in Figure 11 three distinguished peaks at 2h (2θ) = 14.35, 22.4, and 35.2, corresponding to the (101), (200), and (040) crystallographic plane. These peaks are typical reflections of cellulose I.^43,44 Cellulose I is the crystal structure with the highest axial elastic modulus.⁴⁵ The peaks also appear narrow and pointed, which shows that they have a high degree of crystallinity.⁴⁶ Other diffraction peaks appear at 2h = 12.5, and 19.9, corresponding to the (110), (200), and (040) crystallographic planes, typical for type-II structure.⁴⁷ When compared to cellulose I particles, cellulose II particles lead to improved dispersibility in water, increased compatibility with materials such as proteins and lipids, and are beneficial as an excipient in tablet manufacturing.⁴⁸ Furthermore, cellulose II particles with a lower crystalline structure can improve ductility in rigid polymeric matrices.⁴⁹

Figure 11.

X-ray diffractograms (XRD) of bio-textile film.

Fourier transform infrared spectroscopy (FTIR) analysis

Table 2, shows the assignment of absorption bands for the FTIR spectrum of bio-textile film obtained from the biosynthesis process. Figure 12 the spectrum of the FTIR spectra of The broad peak at 3242.7 cm⁻¹ mainly corresponds to O–H stretching of the CH2–OH structure on cellulose.⁵⁰ The peaks at 767.8 and 1006.3 cm⁻¹ were due to the C–H vibration and bending; the peaks at 1148 cm⁻¹ correspond to the glycosidic bonds (C–O–C), the peak at 1617.6 cm⁻¹ is due to C–C stretching.^51–54 The bands at 1744.39 and 1423.84 cm⁻¹ could be associated with ester group (C = O) vibration of triglycerides, and stretching –C = O inorganic carbonate, respectively.⁵⁵ The band at 2855.14 cm⁻¹ is assigned to the CH and CH2 stretching aliphatic groups.^56,57 The peak at 2922.23 cm⁻¹ CH2 str., asym. saturated fatty acids,⁴⁸ The peak is at 1207.65 cm⁻¹ PO2 asymmetric (phosphate I) collagen.⁵⁸ The band at 849.83 cm⁻¹ which could be left-handed helix DNA (Z form) indicated a transient localized conformational change.^59,51

Table 2.

List of band assignments for FTIR spectra.

Wavenumber cm⁻¹	Functional group	References
767.83159	Out-of-plane bending vibrations	⁴¹
849.83303	Left-handed helix DNA (Z form)	⁴¹
1006.38122	Ring stretching vibrations mixed strongly with CH in-plane bending	⁴²
1148.02005	C–O–C polysaccharide	⁴³
1207.65746	PO₂ asymmetric (phosphate I) collagen	⁴⁵
1423.84305	Stretching –C = O inorganic carbonate	⁴⁶
1617.66462	C = C unsaturated compounds	⁴⁴
1744.39411	Ester group (C = O) vibration of triglycerides	⁴⁷
2855.14078	CH and CH2 stretching aliphatic group	⁴⁶
2922.23286	CH2 str., asym. Saturated fatty acids	⁴⁸
3242.78392	O–H stretching (water)	⁴²

Figure 12.

FTIR spectra of bio-textile film.

Contact angle test

Table 3 presents contact angle data, comparing to those from previous literature. Figure 13 illustrates that the contact angles measured were below 90°, indicating complete wettability of the sample. From after first second of analysis, when it was no longer possible to verify the contact angle with the equipment. In this case, the drop of water added to the sample was completely absorbed by it, resulting in thermodynamic equilibrium at the interface of materials. This balance is probably due to the interactions between the solid and liquid particles.⁶⁰ The contact angle values found for the bio-textile film were from zero 40.75°, as shown in Figure 13(a). After 1 s of analysis, were 21.65° as shown in Figure 13(b), after 3 s of analysis, were 15.01° as shown in Figure 13(c), after 4 s of analysis, were 14.3° as shown in Figure 13(d), and after 5 s of analysis, were 0° as shown in Figure 13(e). It was evident that the membrane produced using the method presented in this paper showed the best results in terms of wettability when compared to the standard BC.

Table 3.

Time and contact angle for bio-textile film.

Bio-textile film		Reference
Time (s)	Angle (°)	Reference
0	40.75
1	21.65
3	15.01	This work
4	14.3
5	-
0	32.55
5	30.45	⁶¹
30	26.70

Figure 13.

Analysis of contact angle for bio-textile film: (a) zero time; (b) 1 s; (c) 3 s; (d) 4 s; (e) 5 s.

Effect of culture time on percentage of water retention (PWR)

In this study, mass values for the production of bio-textile films were determined using equation (1). Based on the experimental findings presented in Table 4, it was established that the optimal culture period for producing bio-textile films was observed in Sample Number 4, which required a duration of 10 days. During this period, it yielded 3250.31 g of cellulose (in wet weight) and 147.79 g of cellulose (in dry weight). Furthermore, when comparing our results with data from previous literature, our findings consistently demonstrate a high-water activity in the bio-textile film. This aligns perfectly with the observations from contact angle tests, as outlined in Table 4, indicating that the bio-textile film exhibits exceptional hydrophilicity. This characteristic is of paramount importance for potential applications in the medical field, such as its use as a material for hydration face masks and other related purposes. Figure 14, depicting the relationship between culture time and the Percentage of Water Retention, clearly illustrates that as the culture duration is extended, there is a proportional increase in the percentage of water retention in the bio-textile films. This outcome is consistent with the findings reported in Bibliography.^62,63 Furthermore, the calculation reveals a correlation coefficient (R²) with a value of 0.993. This R² value confirms a robust positive correlation between the extension of the culture period and the increase in water retention percentage within biotextile films.

Table 4.

Water retention percentage of bio-textile film obtained.

Culture time (day)	Wet weight (g)	Dry weight (g)	PWR (%)	Reference
4	233.26	20.84	91.06
6	488.1	36.89	92.44	This work
8	990.81	55.29	94.41
10	3250.31	147.79	95.45
10	240.83	9.67	96.01	⁶²
10	276.43	13.2	95.22
10	11.31	0.62	94.52
10	165.24	6.98	95.77
10	125.32	4.67	96.27
10	73.88	3.56	95.18
10	93.31	3.36	96.39
10	43.35	2.79	93.56
10	122.99	5.42	95.60
10	49.47	1.52	93.71
10	34.22	1.52	95.55
14	179.25	2.72	98.48	⁶³

Figure 14.

Effect of culture time on the water retention percentage of bio-textile films.

Effect of culture time on yield and thickness of bio-textile films

Based on the experimental outcomes presented in Table 5, it is evident that the yield and thickness of bio-textile film samples experienced growth as the culture time increased. The thickness data were subjected to statistical analysis using an ANOVA test. For each bio-textile film, thickness measurements were taken at 10 different positions and then averaged to assess whether there were any significant differences in the thickness of the samples. The ANOVA test results, as detailed in Table 6, revealed a significant distinction among the samples with extended culture time (p = 1.5293E-20). As indicated by Figures 15 and 16 illustrate the correlation coefficient, denoted as (R²). The R² value affirms a positive correlation between thickness and yield, both of which rise with extended culture duration. This is linked to the bacteria having more time to produce cellulose, as a longer culture period results in increased cellulose production.⁶⁴ The yield and thickness of bio-textile film samples increased in the following order: sample 1 (4 days), sample 2 (6 days), sample 3 (8 days), and sample 4 (10 days). The thickness of sample 2 was increased by approximately 36% and increased yield by approximately 77%; the thickness of sample three was increased by approximately 56% more than that of sample 2 and increased yield by approximately 50%; and the thickness of sample four was increased by approximately 98% more than that of sample three and increased yield by approximately 167%. The results show that after the eighth day of culture on sugar cane medium, the thickness and yield significantly increased. According to Bibliography,⁶⁵ the yield obtained in this investigation was eight times greater than the results reported by The quoted authors investigated BC production in various culture media containing fruit juice and discovered that culture media with orange juice yielded a greater cellulose yield (6 g/L) after 4 days of cultivation. The authors According to the literature,⁶⁶ the yield obtained in this investigation was approximately 18 times greater than the authors’ reported values. After 7 days of cultivation, BC production in HS broth supplemented with thin stillage (TS) increased to a concentration of 10.38 g/L with a 100% TS supplement. According to Bibliography,⁶⁷ used molasses and maize steep liquor to reduce culture media costs and achieved the greatest BC yield of 2.21 mg/mL, which corresponds to the dry weight of cellulose per millilitre of culture media. In our study, the best result was 650 g/L after 10 days of culture, which was approximately 29 times higher than that achieved in the bibliography.⁶⁸ Low-cost carbon sources such as glycerol from biodiesel production and grape bagasse, a wine byproduct, were investigated. Maximum BC production was attained after 14 days of cultivation by using roughly 10 mg/mL glycerol and 8 mg/mL grape bagasse. Moreover, Table 7 shows the comparison of different samples from previous literature.

Table 5.

Effect of culture time on yield and thickness.

Culture time (days)	Thickness of dried (mm)	Bacterial cellulose yield of wet (g/l)	Bacterial cellulose yield of dried (g/l)
4	0.25	46.65	5.21
6	0.34	97.62	9.22
8	0.53	198.16	13.82
10	1.05	650.06	36.94

Table 6.

ANOVA test results.

Source of variation	SS	Df	MS	F	p-value	F crit
Between groups	3.145282	3	1.048427	127.9989	1.5293E-20	2.838745
Within groups	0.327636	40	0.008191
Total	3.472918	43

Figure 15.

Effect of culture time on thickness.

Figure 16.

Effect of culture time on yield.

Table 7.

Effect of culture time, and media type on yield from previous literature.

Carbon and nitrogen sources	Microorganism	Dry weight yield (g L⁻¹)	Time (days)	References
Fruit residues	Gluconacetobacter hansenii, (ATCC 53582)	6.98	10	⁶⁹
Corn steep liquor	Gluconacetobacter hansenii UCP1619	2.69	6	⁷⁰
Distillery effluent	Gluconacetobacter oboediens MTCC 5610	8.50	8	⁷¹
Cheese whey	Komagataeibacter medellinensis NBRC 3288	2.37	10	⁷²
Rotten banana juice	Komagataeibacter medellinensis NBRC 3288	4.81	10	⁷²
Potato peel wastes	Gluconacetobacter xylinus ATCC 10,245	4.70	6	⁷³
Tomato juice	Acetobacter pasteurianus MTCC 25117	7.80	7	⁷⁴
Tobacco waste extract	Acetobacter xylinum ATCC 23767	5.20	16	⁷⁵
Lipid fermentation wastewater	Gluconacetobacter xylinus CH001	0.66	5	⁷⁶
Sugarcane molasses	Gluconacetobacter intermedius SNT-1	12.60	6	⁷⁰
Corn steep liquor	Gluconacetobacter hansenii UCP1619	9.63	10	²³
Rotten mango juice	Komagataeibacter medellinensis NBRC 3288	1.95	10	⁷⁷
Pullulan fermentation wastewater	Gluconacetobacter xylinus BC-11	1.18	10	⁷⁸
Durian shell hydrolysate	Gluconacetobacter xylinus CH001	2.67	10	⁷⁹
Cashew tree residues	Komagataeibacter rhaeticus	6.00	7	⁸⁰
Mature black spear grass (Heter- opogon contortus) hydrolysate	Gluconacetobacter xylinus isolated from rot- 10 banana juice	4.88	15	⁸¹

Effect of culture time on ultraviolet resistance analysis

Various UPF ratings and grades are compiled in Table 8. According to the National Research Center, Egypt.

Table 8.

UPF rating and protection grades.

Protection grade	Rating	UPF range	UV- rays transmission
Good	No value	15–24	6.7–4.2
Very good	30	25–39	4.1–2.6
Excellent	40	40–50 (>50)	<2.5

The results in Table 9 describe the UV-resistance characteristics of different bio-textile films. Depending on the percentage value, ultraviolet resistance characteristics will vary for T (UVA) and T (UVB) and the UPF value. A lower percentage value of T (UVA) and T (UVB) in a material indicates that it has good ultraviolet resistance properties, whereas a higher value of the ultraviolet protection factor (UPF) indicates that the material has been enhanced towards excellent UV resistance characteristics. Table 9 shows the comparison values for our targets. UV resistance improves with increased culture time. This phenomenon can occur during the production process, where protective envelopes are created around the microbial cells. These envelopes serve as a shield, safeguarding the cells from both ultraviolet radiation and drying processes.^82,83 As well, the results showed that cell growth increased with increasing culture time, which led to increased BC production. Generally, the production of many microfibrils often leads to a compact structure and increased thickness, which leads to increased ultraviolet resistance. Figure 17 shows the value of the correlation coefficient (R²)This R² value confirms a demonstrates a negative correlation between culture time and T (UVA) and T (UVB). As the culture time lengthens, the bio-textile films exhibit a reduced percentage value for both T (UVA) and T (UVB). while In Figure 18, R² value confirms a demonstrates a it is evident that there exists a positive correlation between culture time and the UPF. As the culture time increases, the bio-textile films demonstrate higher UPF values. To summarize, bio-textile films cultivated on sugar cane media exhibit exceptional ultraviolet resistance properties, making them suitable for applications that demand materials with UV protection.

Table 9.

UV-resistance characteristics of bio-textile films.

Sample number	culture time (day)	Transmission (%)		UPF
Sample number	culture time (day)	UVA	UVB	UPF
1	4	0.53	0.67	225
2	6	0.42	0.56	320
3	8	0.21	0.39	478
4	10	0.13	0.22	629

Figure 17.

Effect of culture time on Transmission of (UVA, UVB).

Figure 18.

Effect of culture time on ultraviolet protection factor.

Effect of culture time on antibacterial activity by measuring colony-forming units (CFU)

Tables 10 and 11 illustrate the influence of culture time on antibacterial activity, assessed through CFU. Furthermore, the statistical analysis presented in Figure 19 displays the correlation coefficient (R²). The R² value validates a positive correlation between the culture time and the reduction (R%) in the number of bacterial colonies. When comparing the four samples, it becomes evident that the rate of reduction (R%) in bacterial growth increases as the culture time lengthens, particularly in the case of E. coli bacteria.

Table 10.

Effect of culture time on antibacterial activity by measuring colony forming units (CFU).

		Colony forming unit (CFU)
Sample number	Culture time (day)	Staphylococcus aureus		Escherichia coli
Sample number	Culture time (day)	No. of colonies	Reduction (R%)	No. of colonies	Reduction (R%)
Control	-	163	-	404	-
1	4	42	74.23	316	21.78
2	6	0	100	7	98.27
3	8	0	100	4	99.09
4	10	0	100	0	100

Table 11.

Photograph images of the Determination of antibacterial activity by measuring colony forming unit (CFU).

Figure 19.

Effect of culture time on antibacterial activity by (CFU).

Effect of culture time on antimicrobial activity by the disc agar diffusion method (zone of inhibition)

The data in Table 12 clearly show that increasing the culture time improves its antimicrobial properties; where it showed assay by zone of inhibition, the samples 3 and 4 (8 days and 10 days, respectively) from bio-textile films exhibited an obvious inhibition zone against S. aureus, E. coli bacteria, and the yeast C. albicans. It was observed that the sample 2 (6 days) from bio-textile films had very low inhibition zones compared to the samples three and four in terms of both inhibition zones. In addition, it showed no inhibition zone for the four samples with A. niger. Table 13 displays photographs depicting the impact of culture time on antimicrobial activity, measured by the disc agar diffusion method (zone of inhibition). In conclusion, bio-textile films grown on sugar cane media have good antimicrobial properties and should be good antimicrobial candidates for many everyday applications, especially with increased culture time. According to Bibliography,⁸⁴ Cellulosic materials such as BC do not show any type of bactericidal property. In order to acquire the bactericidal characteristic from BC, many scientists have worked on BC, that work encompassing several types of metals and metal oxide nanoparticles, among which Cu, CuO and ZnO. In our study, the findings indicated that bio-textile films cultivated using sugar cane media demonstrated significant resistance to antimicrobial activity without requiring any additional treatments.

Table 12.

Effect of culture time on the antimicrobial activity against different test microbes.

		Zone of inhibition (ϕmm)
Sample number	Culture time (day)	Staphylococcus aureus	Escherichia coli	Candida albicans	Aspergillus niger
1	4	0	0	0	0
2	6	15	13	16	0
3	8	21	16	19	0
4	10	25	22	24	0

Table 13.

Photograph images of the inhibition zone of against different test microbes.

To explore the influence of culture time on antimicrobial resistance, we measured the zone of inhibition after a 15-day culture period to assess its impact. Table 14 provides clear evidence that extending the culture time enhances the antimicrobial properties. In Figure 20, we observe the inhibition zone of the bio-textile film after 15 days of culture, revealing a substantial increase in the clear zone and the presence of an inhibition zone for the A. niger fungus. This inhibition zone was absent in bio-textile films cultured for up to 10 days, Additionally, the statistical analysis portrayed in Figures 21 and 22 elucidates the correlation coefficient (R²). It highlights a positive correlation between the culture time and the presence of an inhibition zone. These results emphasize the robust antimicrobial properties of bio-textile films cultivated on sugar cane media, suggesting their viability for diverse applications, especially when the culture time is extended.

Table 14.

Antimicrobial activity against different test microbes after 15 days of culture.

Culture time (day)	Zone of inhibition (ϕmm)
Culture time (day)	Staphylococcus aureus	Escherichia coli	Candida albicans	Aspergillus niger
15	31	25	35	22

Figure 20.

Photograph images of the inhibition zone of against different test microbes after 15 days of culture.

Figure 21.

Effect of culture time on antifungus activity by the (zone of inhibition).

Figure 22.

Effect of culture time on antibacterial activity by the (zone of inhibition).

Conclusions

Bacterial cellulose biosynthesis, isolated from black tea and ginger, is an exciting area of research that has the potential to revolutionise the way we think about and use cellulose. According to the results reported, this bacterial strain is capable of producing quality cellulose at high yields. Up to (650 g/L wet weight, 36 g/L dry weight) after 10 days of culture time with sugar cane as the culture medium. They also contributed to the economy of the bioprocess, as high values of productivity were obtained without the use of more expensive raw materials such as yeast extract. X-ray patterns and FTIR spectra confirmed the presence of cellulose I and II crystalline polymorphisms. The contact angle test was conducted to confirm if the bio-textile films created are extremely hydrophilic or hydrophobic. The results showed that the films are exceptionally hydrophilic. The study proved that the effect of culture time on BC production is significant. As the culture time increases, the amount of cellulose produced increases. This is because the bacteria have more time to produce cellulose, and the longer the culture time, the more cellulose. As a result for cultivation increase period, several positive changes were observed in the bio-textile film. After 10 days of cultivation, there was an increase in film thickness, a rise in the percentage of water retained by the bio-textile film, and an enhancement in its antimicrobial properties. This was evident in the antibacterial activity measured by CFU, where for E. coli and S. aureus, the reduction in growth (R%) was 100%. Subsequently, after 15 days of cultivation, the antimicrobial activity was assessed using the disc agar diffusion method, which revealed notable results with E. coli displaying a 25 mm zone of inhibition, S. aureus with a 31 mm zone of inhibition, C. albicans exhibiting a 35 mm zone of inhibition, and A. niger displaying a 22 mm zone of inhibition. and the possession of excellent UV resistance characteristics Up to (T.UVA%; 0.13 ± 0.02, T.UVB%; 0.22 ± 0.01, UPF; 629 ± 2.12). The experiment also demonstrated the feasibility of naturally dyeing the bio-textile films produced. Making it ideal for use in a wide range of applications. Bio-textile films produced in this study hold significant promise for utilisation in various domains, including:

- Healthcare and Medical Applications: Bio-textile films can have applications in healthcare and medicine, such as wound dressings, tissue engineering, and drug delivery systems. These films can potentially improve patient care and medical treatments.

- UV Protection: Bio-textile films with enhanced UV resistance can provide better protection against harmful UV radiation, benefiting individuals who spend time outdoors and reducing the risk of skin damage and skin cancers.

- Antimicrobial Properties: Bio-textile films with antimicrobial properties can have applications in hygiene and healthcare, reducing the risk of infections and improving overall health outcomes.

- Sustainable Agriculture: Bio-textile films can also have applications in agriculture, such as in crop protection and soil improvement. They can help reduce the use of synthetic chemicals and enhance agricultural sustainability.

- Economic Opportunities: The production and commercialization of bio-textile films can create economic opportunities, including job creation and revenue generation, particularly in regions where raw materials like BC, tea, or ginger are readily available.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Hanaa Abouzaid Khalil Abouzaid

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Production and investigation of bio-textile films produced from bacterial cellulose biosynthesis from black tea and ginger,and cultivation on sugar cane media

Abstract

Keywords

Introduction

Materials and methods

Description of the biotextile films production process

Biosynthesis for bacteria celluloses

Cultivation conditions on sugar cane media

Production of bio-textile films

Washing and drying

Natural dyeing

Characterization

Isolation and characterization of microbes the cultivation on sugar cane media

Isolation of dominant microbes (bacteria and yeast) culture adapted in sugar cane media

Characterization and identification of bacteria and yeast isolates using sequence analysis of ribosomal RNA genes

Scanning electron microscopy (SEM)

X-ray diffraction (XRD)

Fourier transform infrared spectroscopy (FTIR)

Contact angle test

Percentage of water retention (PWR)

Yield and thickness

Ultraviolet resistance analysis (UVA)

Antimicrobial activity studies

Staphylococcus aureus (bacteria G+ve)

Escherichia coli (bacteria G-ve)

Candida albicans (yeast)

Aspergillus niger (fungus)

1- Determination of antibacterial activity by measuring colony forming units (CFU)

2- Determination of antimicrobial activity the disc agar diffusion method (zone of inhibition)

Statistical analysis

Results and discussion

Bacterial cellulose-producing isolates screening

Sequence analysis of ribosomal RNA genes

Scanning electron microscopy (SEM) for bio-textile film

X-ray diffraction (XRD) analysis

Fourier transform infrared spectroscopy (FTIR) analysis

Contact angle test

Effect of culture time on percentage of water retention (PWR)

Effect of culture time on yield and thickness of bio-textile films

Effect of culture time on ultraviolet resistance analysis

Effect of culture time on antibacterial activity by measuring colony-forming units (CFU)

Effect of culture time on antimicrobial activity by the disc agar diffusion method (zone of inhibition)

Conclusions

Footnotes

Declaration of conflicting interests

Funding

ORCID iD

References