Characterization of Bacterial Cellulose fabricated with Vietnamese ingredients for potential textile applications

doi:10.21203/rs.3.rs-4200917/v1

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Characterization of Bacterial Cellulose fabricated with Vietnamese ingredients for potential textile applications

https://doi.org/10.21203/rs.3.rs-4200917/v1

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This research explores the fabrication and characterization of bacterial cellulose (BC), with a distinct emphasis on leveraging indigenous Vietnamese bio-mass sources. A diverse sample library of over 150 BC samples was gathered, with six samples selected for objective evaluation based on the standard test methods. These samples were subjected to characterization techniques including Scanning Electron Microscopy (SEM), Bursting Strength, Thickness, Fourier Transformation Infrared (FTIR) and pH level to explore potential applications in textiles. Moreover, the growth medium or SCOBY (Symbiotic Culture of Bacteria and Yeast) mother, from which the BC was cultivated, was analyzed to identify the constituent bacterial and yeast strains. The notable aspects of this study were: (a) the use of local Vietnamese ingredients (i.e., sugar and teas) as nutrient sources for BC cultivation, and (b) exploring the impact of local crafted solutions for drying food-based products akin to rice wraps, on the properties of BC. The study’s outcomes established a deeper comprehension of the morphological, mechanical, and chemical attributes of BC, as well as the microbial dynamics within the SCOBY mother. This exploration not only augments the existing knowledge on BC's potential in material design but also paves the way for further research on the influence of local ingredients on biomaterial production, thereby contributing to the burgeoning field of sustainable material design innovation within a localized context.

Sustainable material design

Bacterial cellulose

Vietnamese ingredients

Characterization

Textile application

Background of biomaterials and bacterial cellulose (BC).

Biobased materials represent a critical frontier in the field of material science, offering the promise of sustainable, biodegradable, and functional materials (Gorgieva & Trček, 2019). They are derived from biological sources and can be potential candidates to replace their petroleum-based counterparts in the field of material science. The newly developed biobased materials can completely replace the existing conventional materials from petroleum resources. Some biobased products are already being commercialized by global brands and others are paving their way through research and innovation. The surging interest in biobased materials stems from the global urgency to transition towards more sustainable, renewable, and ecofriendly material solutions. A significant innovation is the Bacterial cellulose (BC), derived from commonly used bio-ingredients such as tea, coffee, and sugar.

BC is a biobased material, with unique set of properties such as mechanical strength, permeability, porosity, and viscoelasticity (Klemm et al., 2001). BC is produced by certain strains of bacteria, notably the Acetobacter xylinum, which synthesizes cellulose chains and excretes them to form a three-dimensional (3D), nano-fibrillar network. The resultant BC boasts of exceptional purity compared to plant-derived cellulose, as it is devoid of lignin and hemicelluloses. Moreover, BC is characterized by high mechanical properties, excellent water-holding capacity, and a remarkable degree of crystallinity, making it a highly versatile material for a range of applications including fashion and textiles (Gama et al., 2016). For example, in the medical domain, BC has been utilized for wound dressing, tissue engineering, and drug delivery systems due to its biocompatibility and nontoxicity (Gama et al., 2016). In the realm of material design, BC offers a sustainable alternative for fabricating textiles, packaging materials, and acoustical devices among others (Gorgieva & Trček, 2019).

The current research aims at exploring the potential of BC in material design, with a focus on employing indigenous Vietnamese feedstock sources for its cultivation. This approach not only presents a pathway to producing BC with localized resources but also opens avenues for understanding the interplay between different cultivation conditions and the resultant BC properties. Moreover, the endeavor to incorporate local resources and traditional drying techniques accentuates the cultural integration and the potential for fostering sustainable material production practices within local communities (Lee, 2014 Klemm, D., Schumann, D., Udhardt, U., & Marsch, S. (2001). The study also focuses on exploring the potential applications of BC in fashion and textiles, creating a unique library of BC samples.

Objectives and scope of the current research

Several earlier research publications on BC have focused on the fabrication of biobased materials using commercially available ingredients for nitrogen and carbon sources (which includes tea, coffee, sugar, and vegetables). However, the number of studies using the local ingredients available in tropical countries is limited. Hence, the major aim of this research is to fabricate BC utilizing indigenous food sources available in tropical countries such as Vietnam in addition to using the traditional methods of drying similar products such as rice wrap. The BC samples developed through this research were characterized for their properties and investigated for potential applications in fashion and textiles, fostering sustainable material innovation. In tandem with the material characterization, a microbial analysis of the growing medium or SCOBY mother was conducted to identify the constituent bacteria and yeast strains, offering insight into the microbial dynamics integral to the BC production process.

In addition, the exploration of this research extends to traditional drying techniques and surface treatments in Vietnam. Documenting and assessing local crafted drying techniques highlight the efficacy of these indigenous methods in forming and drying BC, foregrounding the potential of traditional knowledge in modern material design applications (Lee, 2014). The recording and analysis of the data throughout the experimentation phase formed a critical part of the research. The data analysis endeavors to develop insights into the interplay between different cultivation techniques, conditions, and the resultant BC properties, offering a robust understanding of the parameters influencing material characteristics of BC.

More broadly, the research sits within the overarching discourse of sustainable and localized material production, aiming to contribute to the burgeoning field of biobased materials and sustainable design practices. By orchestrating the cultivation and characterization of BC with indigenous resources and knowledge, this study presents a model of material innovation that is ecologically viable, culturally resonant, and holds the promise of promoting sustainable design practices within and beyond the Vietnamese context (Wei et al., 2022; Bayer & Lamed, 1992).

BC production and applications.

Recently, researchers are focusing on fabricating new biobased materials produced from plants, in which cellulose is a major component. The demand for plant-based cellulose is rapidly increasing leading to deforestation of natural forests and intensification of farming, with its potential environmental impacts. Although the major source of natural cellulose is plant matter, several species of microorganisms (such as Acetobacter, Achromobacter, and Rhodobacter bacteria) can produce BC. The demand for BC in fashion and textile applications is growing due to its sustainability benefits. BC is a renewable, biocompatible, nontoxic, and biodegradable material, which leads to its growing applications in several areas such as healthcare, food, fashion, and textiles. Further, the scope of BC to be applied in various fashion and textile applications as a sustainable material has led to increased research on BC.

BC is prepared from a culture medium of carbon and nitrogen sources and the SCOBY. Numerous studies have sought to optimize the production process by altering parameters such as the culture medium (tea and sugar sources), type of bacterial species, and ambient conditions (temperature, growth period and pH levels). One such BC is Kombucha, which is widely used as a drink in several parts of the world including developing and developed countries. Kombucha is prepared using a range of nitrogen sources (such as black tea, white tea, and coffee), carbon sources (different types of sugar) in addition to different types of SCOBY. Kombucha has been a major research area in the material science domain for the last two decades.

A sustainable technology for the synthesis and modification of BC derived from kombucha was presented by Kamiński et al. (2020). They fabricated stable hydrogel bacterial cellulose (HGBC) with desirable physicochemical and mechanical properties for textile applications. Pedroso-Roussado (2023) explored the potential of BC as a textile material, shedding light on the initiatives by Suzanne Lee from BioCouture. This discourse extended to the development of BC fibers and yarns, broadening its applicability in the textile sector. The unique properties of BC, and its resemblance to natural leather, make it a potential candidate for several fashion and textile applications.

Sousa et al. (2021) investigated the purification of BC membranes from kombucha for diverse applications, including textiles. Their work highlighted the potential use of BC membranes in crafting sustainable leather-like fabrics and biodegradable packaging, thus contributing to the narrative of sustainable textile materials. Wood et al. (2022) accentuated the reproducibility of BC nanofibers for developing novel textiles, emphasizing the superior properties of BC over conventional materials like cotton, especially in terms of enhanced purity, crystallinity, tensile strength, and water retention. These attributes render BC a suitable candidate for various textile applications.

Existing methods for characterizing BC

The exploration of BC for textile applications necessitates a thorough evaluation of its properties and characteristics, employing both traditional and standard testing methods. Literature has increasingly acknowledged the distinct nature of BC and other bio-based materials, which may not always align completely with standard testing methods employed for traditional textiles. Hence, some modified test methods can be employed where the standard methods can’t be used.

Noteworthy contributions in the field have been made by researchers exploring the diverse aspects of BC testing and applications. Emam (2019) investigated organic antimicrobial reagents in cellulosic textile finishing, highlighting the importance of achieving a balance between comfort, easy care, health, and durability in antimicrobial textiles. This work provided a foundation for understanding the interplay of antimicrobial agents with cellulosic materials like BC, aiding in the development of health-centric textile products. Provin et al. (2021) tackled the challenge of wettability associated with BC in textile applications. Their comprehensive review gathers various studies and methods aimed at minimizing BC's hydrophilicity, thereby expanding its applicability in textiles. This discourse is crucial for addressing one of the inherent challenges in BC textile applications, paving the way for research focused on enhancing BC's compatibility with textile processes. Nayak et al. (Nayak, 2024) has recently summarized different sources and characterizations for fashion and textile applications. A range of standard test methods for evaluating chemical, mechanical, surface, thermal and biological properties has been discussed in this paper.

Feiguerias et al (2021) reviewed various technologies used to produce cellulose-based textiles, including BC. Their discussion on surface modification techniques unveils the potential of tailoring BC’s surface properties to meet specific applications. Moreover, the exploration of sustainable cellulose sources and life cycle assessment (LCA) of cellulose fiber production methods adds a sustainability dimension to the production and application of BC. The work by Kamiński et al. (2020) showcases a novel and ecofriendly technology for the synthesis and modification of BC for textile applications. By elucidating the manufacturing process of hydrogel bacterial cellulose (HGBC) from a yeast/bacteria culture, they highlighted a pathway for developing BC with desired mechanical and physicochemical properties suitable for textile applications. Costa et al. (2021) ventured into evaluating BC films as an alternative textile surface for developing clothing prototypes. Their exploration encompassed the production and characterization of BC films using different bacterial strains and assessing their compatibility with traditional sewing techniques. This work extends the discourse on BC’s potential as a textile material, especially in the realm of clothing design and fabrication.

Interweaving with these significant contributions, established methods like Scanning Electron Microscopy (SEM) for morphological analysis; tensile strength and bursting strength for mechanical properties; chemical modification techniques and several other techniques continue to play crucial roles in analyzing BC. However, the distinct nature of BC, especially its microbial composition and growth dynamics, calls for a blend of traditional textile testing and innovative biological testing approaches for fashion and textile applications.

This research acknowledges the limitation in existing textile testing methods in relation to BC. This stems from the fact that unlike traditional textile manufacturing processes, where the ‘farming’ of fibre is part of a distinct primary production phase (with its own agricultural testing processes), while fibre processing into yarns and textile manufacturing are further and separate stages of manufacturing (with their own specific testing regimes). While growing BC, the textile material can be produced directly by the organism itself, merging separate parts of a supply chain into a continuous and integrated process, requiring a more integrated, multidisciplinary testing regime.

Ghalachyan et al (2023) have identified and evaluated the sensory nature of new materials like BC, which is essential to their adoption in various products. While some traditional textile evaluation methods are employed, this study involved the analysis of bacteria and yeast strains integral to BC production, as well as exploring the influence of parameters such as growing duration, washing, and drying methods on the properties of BC. These dimensions, although not standard in textile testing, are pivotal for a holistic understanding of BC and its applicability. The diversification in testing throughout the textile production process is also reflected in the evaluation of different aspects of BC cultivation, like the pH levels of the growth medium, which can significantly impact the properties of the final material. Additionally, the assessment of BC’s compatibility with traditional sewing techniques by Costa et al. (2021) underscores the necessity of bridging traditional textile practices with the novel attributes of BC.

The discourse on BC testing and analysis hints at a broader need for the textile industry to evolve its testing paradigms to accommodate the unique attributes of biobased materials. Developing new methods or standards for testing and analyzing BC may not only enhance the understanding of this biomaterial but also pave the way for its standardized and broader application in the rapidly growing biobased textile industry. The existing methods for testing and analyzing BC, coupled with the innovative approaches seen in the recent research form a robust framework for comprehending BC’s properties and potential applications. The amalgamation of traditional testing methods with novel analytical approaches reflects the evolving narrative of BC testing, resonating with the broader transition towards biobased materials in textile applications. This study contributes to the emerging interdisciplinary field of biobased materials, identifying and bridging the divisions between scientific and aesthetic modes of evaluation, and between industrial and biological production processes.

Sample Library Creation

The creation of a rich and diverse sample library was the critical starting point in this research, laying the foundation for extensive recording, testing and analysis. Utilizing locally sourced ingredients from Vietnam, over 150 samples were meticulously cultivated, documented, and prepared for further investigation.

Ingredients and additives

The primary ingredients used to fabricate BC included various types of carbon and nitrogen biomass sources that were indigenous and easily available in Vietnam. Nitrogen sources (i.e., teas such as oolong tea, black tea, and green tea), and carbon sources (i.e., sugars such as white, brown, and sugar cane) were used in experiments to fabricate BC. A unique aspect of the research is an extension beyond these more traditional ingredients by utilizing local fruits like banana, mango, watermelon, and dragon fruit, either replacing or complementing the nitrogen and carbon sources. Additionally, the use of coconut water and apple juice in growing and influence was explored. The local ingredients used for fabricating BC are listed in Table 1.

Table 1

List of ingredients used for fabricating BC
Sample code	Carbon source (Sugar)	Nitrogen source (Tea)	Starter SCOBY	Growing duration (days)	pH after harvesting	pH at the beginning	Detergent Soaking	Fresh water soaking
S1	Brown sugar (300 g)	Black tea (30 g)	Mother 1	15	2.9	4.3	4 days	1 days
S2	Sugar cane + white sugar	Black tea	1	17	2.6	3	5 days	3 days
S3	White sugar	Black tea	1	7	2.8	3.2	5 days	3 days
S4	Red dragon fruit	Black tea	1	6	3.6	4	0 (wash with water only)
S5	White sugar	Cape jasmine seed (80 g)	1	18	3.3	4.2	0 (wash with water only)
S6	White sugar	Blue peas	1	14	3.6	4.4	0 (wash with water only)

Cultivation:

The starter liquids were derived from three distinct SCOBY mothers, each fed with different ingredient combinations, namely green tea with white sugar, black tea with brown sugar, and dragon fruit with no tea and no added sugar. The suspension proportions were standardized to 3 liters of water, 30 grams of tea, 300 grams of sugar, and 300 ml of starter liquid. Figure 1 shows the steps used for fabricating BC. The culture medium was prepared using the standardized proportions of ingredients. At first the nitrogen and carbon sources were mixed with boiling water. The mixture was then cooled to room temperature (25°C). Then, the starter culture from the mothers was added to the mixture and allowed to inoculate for the desired number of days as mentioned in Table 1.

The cultivation process involved the preparation of g a kombucha tea starter culture, comprising a blend of teas and sucrose, inoculated with a bacterial and yeast culture. The mixture could incubate at room temperature over 15 days, with periodic analyses to assess its chemical and microbiological properties. Bacterial cellulose samples grow on the surface of the culture medium due to fermentation as a floating layer, and the process is done in a static condition or static route.

Washing and drying

Once the growing period was reached, the BC samples were taken off the top of the culture medium and washed in running tap water to remove the residual deposits from its surface. They were then soaked in water for between 1 to 3 days followed by final washing and then drying directly in sunlight using bamboo-frames. Once dried, the BC samples were treated with coconut oil or wax to prevent rapid absorption and desorption of moisture that can lead to quick deterioration of the samples. Figure 2 shows the samples after washing (a), during drying (b) and dried samples ready for investigation (c).

A different protocol was employed for samples incorporating fruit. This involved mashing the fruit, mixing it with water, and heating it, which facilitated fermentation. This was followed by brewing tea, mixing the fruit pulp with the brewed tea, and adding the starter liquid for fermentation to initiate. This concoction was then allowed to ferment at room temperature for 10–15 days before harvesting the BC.

Growth Monitoring:

The growth of BC was monitored meticulously, recording parameters like growth duration, thickness of BC, pH levels at the start and end of cultivation, and visual observations of the BC's appearance. The growing duration ranged between 4 and 44 days, with an average of 6 to 18 days, influenced by factors including temperature, type of ingredients, and light exposure.

Drying and Coating:

Drying methods devised in this research was inspired by the local Vietnamese practices in drying rice wraps, utilizing bamboo racks directly under sunlight. Post drying, the BC samples were coated with a mixture of coconut oil and beeswax, allowing them to rest for two days for complete soaking before documentation, testing and evaluation.

Documentation:

Each sample was assigned a unique identifiable code, with an index card attached following the drying and coating process. This provided a detailed record of all the relevant parameters and observations. Some of the Kombucha samples are shown in Fig. 3.

Characterization

Once the BC samples were fabricated, they were subjected to a range of characterization techniques to evaluate their properties. It is essential to evaluate a range of properties to understand the behavior of a material for specific applications. In this research the surface morphology was analyzed by scanning electron microscopy (SEM); sample thickness was measured by a fabric thickness gauge; testing of pH was done using a pH meter to understand the acidity or alkalinity of the samples; Fourier Transform Infrared (FTIR) spectroscopy was used for understanding the chemical structure; mechanical properties were measure by bursting strength; and bacterial species were identified by using a mass spectrometer.

Scanning electron microscopy (SEM)

Field Emission Scanning Electron Microscopy (FESEM) was performed on the BC samples to investigate surface morphology using the Philips XL30 FESEM. SEM images were captured with a spot size of 4.0 µm and an accelerating voltage of 20 kV. The BC samples were placed on a stub and sputter coated by irradiating with gold using a high-resolution ion beam sputtering system. Coating thickness of 100 angstroms (Å) was achieved by applying a current of 50 mA for 20 s.

Thickness Testing

The thickness of BC samples was determined using a digital fabric thickness tester as per the ISO 5084:1996 Standard (Determination of thickness of textiles and textile products). Multiple measurements were taken across 10 different points on each sample to account for any thickness variations. The thickness values of BC samples are essential as it influences the drape, feel, and aesthetic appeal when utilized in textile applications.

pH level testing

The pH level was monitored in both the growing liquid and the final BC samples using a calibrated pH meter. The pH data, taken at the beginning and end of the cultivation period is crucial as pH level can mediate microbial activity during cultivation, affect the physical properties and comfort characteristics of the final material, especially in skin contact applications. Generally, a pH value of 7 (neutral pH) is appropriate for several textile applications. Higher acidic or alkalinity pH levels in the samples will lead to skin irritations.

Fourier Transform Infrared (FTIR) Spectroscopy

FTIR spectroscopy was used to determine the chemical composition of BC samples. A spectrophotometer (PerkinElmer Spectrum-400) was used to collect data on the absorbance of the samples to determine the functional groups, which was then analyzed to identify the chemical structure. The chemical structure of BC is crucial as it influences the chemical properties and reactivity of BC to chemicals. A complete understanding of chemical properties is essential to determine the potential application areas. A total of 16 scans per sample was used with the wave number range of 4000–650 cm^–1 used for scanning. The absorbance of the samples varies according to the functional groups, which is indicated in the spectra as a function of wave number.

Mechanical properties (Bursting strength)

The BC samples were subjected to a range of mechanical forces during their use. Hence, it was essential to measure the mechanical behavior to understand the nature of deformation and point of failure of the BC samples. Tensile strength is not an appropriate method for BC as it does not have a specific warp and weft direction like woven fabrics. Hence, the mechanical properties of the BC samples were evaluated by testing the bursting strength in a hydraulic bursting strength tester.

Bursting strength was measured by stressing the BC specimens in all directions at the same time. The bursting strength was evaluated using the SDL Auto-burst, digital bursting strength tester as per ISO13938-1 Standard (Bursting properties of fabrics Part 1: Hydraulic method for determination of bursting strength and bursting distension). The BC samples were subjected to multi-directional stress over a diaphragm that was inflated by a fluid at the rate of 100 cm³/min. The final pressure at which the specimens rupture is expressed as the bursting strength.

Identification of bacterial species

The bacterial species were identified using the Bruker Daltonik MALDI test at the Center for Bioscience and Biotechnology, Ho Chi Minh City, Vietnam. The mass spectrometer was used for identifying the bacterium species, which is more efficient than the traditional assays or sequencing method. The spectrometer used Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) technology to identify the bacteria accurately from a DNA analysis. For the bacterial count, the standard agar plate method was used to count the number of colony forming units per milliliter (CFUs/mL).

Statistical analysis

Single factor analysis of variance (ANOVA) statistical analyses was performed on the thickness and bursting strength results using Microsoft Excel 2016 (p ≤ 0.05). ANOVA is an essential test to understand whether there were statistically significant differences among the mean values of the test results. The difference between the test results was significant when the F_value was larger than F_critical. The F_value is the ratio of two mean square values, whereas the F_critical of the test results must exceed to reject the null hypothesis. A higher F_value results in greater variation among the group averages.

Observations during growth

The static formation route was used to fabricate BC in this study. The BC structure was developed by the intertwining of the cellulosic fibrillar structures forming uneven surfaces. It was observed that the growth of BC was quite slow at the beginning despite the high concentration of ingredients due to a lesser number of bacteria. As the time elapses, the bacterial quantity increases, and the growth process is faster. After some time, the concentration of ingredients starts to decrease, which leads to slower growth of BC and eventually to a stagnant phase. The BC samples grow as a floating membrane at the surface, which separates the air-liquid interface.

Surface morphology (SEM results)

The surface morphology of BC samples is shown in Fig. 4. The morphological structures are different due to the use of different ingredients. The presence of distinct particulate matter (of size 5 micron and 6 micron) was observed in the sample images of S4 and S5, respectively. This particulate matter can be attributed to the use of sugar (such as dragon fruit) and tea type (such as Cape Jasmine fruit) used for BC preparation. In samples S1, S2, S3 and S6, the particulate matters were not distinctly visible and were smaller in size as the nitrogen and carbon sources were well mixed. The distinct cellulosic structure was not visible in the SEM images due to insufficient purification process used during the washing of the BC to remove fermentation residues (Costa et al., 2021).

Thickness values

The thickness of textile materials is important in determining the types of applications as it influences draping behavior. The thickness results of dried BC specimens are shown in Fig. 5. It can be observed that the thickness of the BC sample fabricated with black tea, white sugar, and sugar cane (i.e., S2) is the highest. Similarly, the thickness of the samples produced with blue peas and white sugar (i.e., S6) is the lowest. The highest and lowest values of thickness values were 1.26 and 0.21 mm, respectively. The thickness order from highest to lowest was S2 > S3 > S5 > S1 > S4 > S6. The thickness result for the textile specimen (SS), that was used as a reference for comparison of bursting strength for textile applications, was collected from the research by Uyanik (Uyanik, 2020). The thickness result for sample ‘SS’ is indicative whether the BC samples can meet the desired results for textile applications.

The reason for the greater thickness of S2 can be attributed to the richer carbon source, due to the mixture of white sugar and sugar cane juice in addition to a rich nitrogen source. The richer carbon and nitrogen sources led to a sample with higher thickness. On the other hand, sample S6 showed the lowest thickness which might be due to the poor nitrogen source derived only from the blue peas. As there was no nitrogen source used in this experiment, the amount of nitrogen was insufficient for the growth of BC leading to the lowest thickness. Further it can be observed that the black tea samples produced higher thickness than that of the other sources. The difference between the thickness were statistically significant across the six BC samples (F = 4.21, at p < 0.05).

In general, several studies have established that the higher the number of days to grow BC, the higher is the thickness when keeping other parameters constant (Eslahi et al., 2020). However, factors such as the type of carbon and nitrogen sources greatly impact the thickness results. Sample S6 with blue peas as a nitrogen source showed the minimum thickness due to weaker source for nitrogen despite higher growing time.

pH results

The growth of BC samples also depends on the pH level of the culture medium. A lower pH (i.e., acidic medium) is favored for the microorganisms to grow. It was reported that a pH range of 4.0 to 5.0 produces the best result for the growth of BC (Verschuren et al., 2000). Klemm et al. (2001) had earlier reported that a pH of 3.5 was the optimum value for the growth of BC. Further, the pH value is important in relation to the application of BC in fashion and textiles. A neutral pH (pH value of 7) is needed for many applications that come in direct contact with the skin. The pH values of all the samples before the growth started and after the samples were harvested were measured, with the results shown in Fig. 6.

The results show that the pH of the samples before the fabrication of BC lie between 3.0-4.4. The pH values had decreased producing a more acidic medium when the samples were harvested, ranging from 2.6 to 3.6. Several publications have reported that the pH values decreased below 4.0 after the formation of BC (Lin et al., 2013). The formation of different types of organic acids such as ethanoic acid and gluconic acid has also been reported by several researchers. These acids are responsible for the lowering of the pH values. However, for fashion and textile applications, the low pH values of the harvested BC are not suitable. The samples would need to be treated with an alkaline solution such as calcium carbonate (CaCO₃) to raise the pH to a neutral value of 7 (Wang et al., 2019).

FTIR spectroscopy results

FTIR spectroscopy was used to understand the chemical composition of BC fabric. The transmittance peak analyses of the spectra were performed to check the appearance, disappearance and shifting of peaks to find the functional groups. The FTIR spectrographs of the BC samples are shown in Fig. 7. The major component of BC sample is cellulose, hence, one of the functional groups of cellulose is hydroxyl group (-O-H stretching), which was observed in the wave number region from 3240–3340 cm^− 1. The other functional group found in the spectra of cellulosic fiber is -C-H vibrations, observed in the wave number region of 2850–2920 cm^− 1.

The cellulosic peak for -C-O vibrations was observed in the region of 980–1060 cm^− 1. The presence of carboxylic acids was identified from the -C = O vibrations present at 1630 and 1730 cm^− 1. The -C-H vibrations of carboxylic acid were also identified from the peak vibrational peak present in the region 2850–2920 cm^− 1. Some of the spectrographs show lower intensity peaks, which might be due to the presence of some fruit residues in the BC structure. The characteristic peaks of all the six BC samples are identical, indicating the cellulosic structure across the BC specimens.

Table 2

FTIR results showing the functional groups present in BC samples (Berthomieu and Hienerwadel, 2009)
Wave number	Vibrations	Functional group
3240–3340	Stretching	-O-H (broad peak from alcohol or water)
2850–2920	Vibrations	-C-H (medium to strong peak from cellulose and carboxylic acid)
1730	Vibrations	-C = O (weak to strong peak from carboxylic acid)
1630	Vibrations	-C = O (weak peak from carboxylic acid)
980–1060	Vibrations	-C-O (from cellulose)

Mechanical properties (Bursting strength)

The mechanical properties of BC can be better evaluated by the bursting strength as the samples can break through multi-directional force rather than a unidirectional tensile force. The bursting strength is a measure of durability of the fabric or similar textile materials. The bursting strength results of BC samples are shown in Fig. 8. It can be observed that the bursting strength of sample S4 is the lowest (234.2 kPa) and the bursting strength of the sample S2 is the highest (671.3 kPa). The bursting strength of the samples are in the decreasing order of S2 > S3 > S5 > S6 > S1 > S4.

The bursting strength of a fabric (as shown by SS in the graph) used for clothing application was used to compare the values of BC samples. It can be observed that the BC samples showed lower bursting strength values compared to the SS fabric (100% cotton fabric), except the sample, S2. The thickness values (Fig. 5) for S2, S3 and S5 BC samples are higher than the reference cotton fabric (SS). However, the bursting strength of S3 and S5 are lower than the SS fabric. This shows that there is not a direct relationship between thickness and bursting strength. The difference among the bursting strength values were statistically significant among all the six BC samples (F = 6.7, at p < 0.05).

The textile fabric SS has higher bursting strength than the BC samples (except S2) due to intermeshing of yarns in the knitted fabric structure in the form of loops. The bursting strength of S2 is higher than SS, which might be due to the significantly higher thickness of the sample. The lower bursting strength of BC samples can be attributed to the uneven surface with thick and thin places. The presence of a thin place or a weak spot on the surface will lead to premature failure leading to lower bursting strength. It can be concluded that BC samples can be grown to the required thickness for achieving bursting strength results that are equivalent to the real textile fabrics made from 100% cotton.

Microbial Analysis Results

The results obtained from Bruker Daltonik MALDI tests of BC samples are shown in Table 3. It can be observed that Acetobacter Indonesiensis, a gram-negative bacterium, is found in all the three samples tested for bacterial analysis. The bacteria species Acetobacter Indonesiensis has also been used in several other research for growing BC (Jie et al., 2023, Tran et al., 2021, Yetiman and Kesmen, 2015). The bacteria of the genus Acetobacter are rod-shaped and elongated and belong to the group of acetic acid bacteria. This genus is the most widely used commercial bacteria and provides maximum growth of BC during the fermentation process. The bacteria of genus Acetobacter are purple, non-photosynthetic bacteria that can convert various sugar sources such as glucose, fructose, glycerol, and other organic substances into BC.

The other bacterial group found in the samples included Bacillus subtilis, Saccharomyces cerevisiae, Agrobacterium rubi, and Staphylococcus hominis. Bacillus subtilis is a rod-shaped and gram-positive bacteria, which was found in some BC (Savitskaya et al., 2019); Saccharomyces cerevisiae is a single-celled yeast, widely used in baking, brewing and wine making; Agrobacterium rubi is a mesophilic plant pathogen; and Staphylococcus hominis is a gram positive bacteria with round-shape. Hence, various types of bacteria and yeast were present in the samples, which originated from the parent SCOBY. The total bacterial count in colony forming unit per milliliter (CFU/ml) has been shown in Table 4. It can be observed that the number of CFUs in sample S6 was the highest and S4 was the lowest, with values of 2.16*10⁷ CFUs and 3.65*10⁶ CFUs, respectively. From the results it can be concluded that the thickness of the samples does not depend on the bacterial count, rather the type of carbon and nitrogen sources. Despite the highest CFU values for S6, the thickness was not the highest due to weaker carbon and nitrogen sources available for bacterial growth.

Table 3: Results obtained for bacterial type from Bruker Daltonik MALDI tests

Sample code	Test Type	Result	Gram positive or gram negative	MALDI Scores*
S1	Bruker Daltonik MALDI	Acetobacter indonesiensis	Gram negative	1.78
		Acetobacter indonesiensis	Gram negative	2.48
		Bacillus subtilis	Gram positive	1.82
		Saccharomyces cerevisiae	Yeast	2.20
S4	Bruker Daltonik MALDI	Acetobacter indonesiensis	Gram negative	2.02
		Agrobacterium rubi	Yeast	1.87
		Acetobacter indonesiensis	Gram negative	2.35
		Staphylococcus hominis	Gram positive	2.08
S6	Bruker Daltonik MALDI	Acetobacter indonesiensis	Gram negative	1.81
		Acetobacter indonesiensis	Gram negative	2.03
		Bacillus subtilis	Gram positive	2.12
		Acetobacter indonesiensis	Gram negative	1.18
*MALDI scores 2.00-3.00 means high-confidence identification of the bacterial species (symbol: +++ & level: green); a score of 1.70-1.99 means low-confidence identification (symbol: +, level: yellow); and scores between 0.00-1.69 are considered indicative results (symbol: -, level: red)

Meaning of Score Values
Range	Interpretation	Symbols	Color
2.00 - 3.00	High-confidence identification	(+++)	green
1.70 - 1.99	Low-confidence identification	(+)	yellow
0.00 - 1.69	No Organism identification possible	(-)	red

Table 4

Results showing bacterial count from Bruker Daltonik MALDI tests
Parameter	Sample code	Results (CFU/ml)	Average (CFU/ml)
Total bacteria	S1	1.38*10⁷	5.51*10⁶
Total bacteria	S1	2.73*10⁶
Total bacteria	S1	1.28*10²
Total bacteria	S4	8.90*10⁶	3.65*10⁶
Total bacteria	S4	2.06*10⁶
Total bacteria	S4	9.60*10¹
Total bacteria	S6	5.60*10⁷	2.16*10⁷
Total bacteria	S6	8.90*10⁶
Total bacteria	S6	3.22*10²

Significant findings

This paper discussed the findings from fabricating and characterizing BC samples. Various local ingredients available in Vietnam were used as Carbon and Nitrogen sources to grow BC fabrics. Further, the drying methods used in this research followed the traditional process of drying directly under the sunlight, which is used to dry food products such as paper rice in Vietnam. The unique drying process can produce patterned effects in the BC samples in addition to adequate drying. The BC samples were tested for their suitability aspects for textile applications. The morphological structures were different due to variations in the sources of sugar and nitrogen. The presence of distinct particulate matter in the SEM images was ascribed to the type of feedstock used to produce BC. The black tea produced the highest thickness of BC samples. Some of the BC samples showed higher thickness compared to commercially available fabric samples from 100% cotton.

The pH of BC samples was found to be acidic before, during and after fabrication. Hence, they would need to be treated with commonly available bases to bring the pH close to 7 for textile applications. FTIR spectroscopy showed various functional groups relating to the cellulosic structure of BC. Functional groups such as - O-H (broad peak from alcohol or water), -C-H (medium to strong peak from cellulose and carboxylic acid), -C = O (weak to strong peak from carboxylic acid), -C = O (weak peak from carboxylic acid) and -C-O (from cellulose) confirmed the cellulosic structure of BC.

The mechanical properties of BC samples were investigated by testing the bursting strength, which is multidirectional compared to the unidirectional tensile strength. It was found that the bursting strength of five of the BC samples were lower than the 100% commercial cotton fabric. The higher bursting strength of one sample can be attributed to the significantly higher thickness. The BC samples were tested by Bruker Daltonik MALDI test for identifying the type of bacteria present in the scoby. It was found that Acetobacter indonesiensis is the main bacteria present in the SCOBY. Other bacterial species included Bacillus subtilis, Saccharomyces cerevisiae, Agrobacterium rubi and Staphylococcus hominis.

Limitations and future directions

This study investigated the potential of some local ingredient indigenously available in Vietnam in producing bacterial cellulose. There are a wide range of local nitrogen and sugar sources, which could not be included in this research due to the project scope and time constraints. Future research can investigate these resources to fabricate BC. Another limitation was associated with the range of testing, for example, the BC samples were not characterized for thermal and crystalline properties to understand the applicability of textile products at high temperature and establishing the wash care methods. Hence, future studies should focus on these additional characterization techniques. Finally, given the focus of this initial research on investigating local feedstock sources, growing conditions and the testing of the resulting BC textile samples, it was beyond the scope of this project to address garment fabrication issues and strategies that could be used in fashion manufacturing using BC. Future research will investigate pre-shaping garment panels and ways of constructing and joining final garments.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. All co-authors agree with the contents of the manuscript and there is no financial interest.

Ethical Approval

(Applicable for both human and/ or animal studies. Ethical committees, Internal Review Boards and guidelines followed must be named. When applicable, additional headings with statements on consent to participate and consent to publish are also required)

Funding

This research was supported by a RMIT Vietnam’s Tier 1 Research Grant (IRG 2022–1).

Author Contribution

Donna Cleveland: Experimentation & result analysisRajkishore Nayak: Testing & Result analysis Frances Joseph: Technical input, reading and improvemens.

Acknowledgement

Not applicable

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Characterization of Bacterial Cellulose fabricated with Vietnamese ingredients for potential textile applications

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Abstract

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Introduction

Background of biomaterials and bacterial cellulose (BC).

Objectives and scope of the current research

Literature Review

BC production and applications.

Existing methods for characterizing BC

Materials and Methods

Growth Monitoring:

Drying and Coating:

Documentation:

Thickness Testing

Statistical analysis

Results and discussion

Conclusions

Declarations

Funding

Author Contribution

Acknowledgement

References

Additional Declarations

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