Trends Sci. 2026; 23(2): 11128

Bacterial cellulose (BC) is a nanostructured exopolysaccharide synthesized by specific acetic acid bacteria, particularly those from the Komagataeibacter genus. Compared to plant-derived cellulose, BC exhibits superior characteristics, including high crystallinity, tensile strength, purity, water-holding capacity, and biocompatibility. These features make it a valuable biomaterial across various industries, including food, biomedical, packaging, and cosmetic [1-4].

Kombucha fermentation has recently gained interest as an alternative biotechnological platform for BC production. This fermentation process involves a symbiotic culture of bacteria and yeast (SCOBY) cultivated in a tea-based medium under static aerobic conditions. The yeasts hydrolyze sucrose into glucose and fructose, which are then utilized by acetic acid bacteria for cellulose biosynthesis. The microbial synergy within the SCOBY plays a critical role in cellulose production, particularly when supported by suitable carbon sources and nutrient-rich tea infusions [5-7].

Among available substrates, tea infusions derived from Camellia sinensis —such as green, black, and oolong teas—have been widely used due to their content of polyphenols, amino acids, and minerals that promote microbial growth and activity [8]. In our previous work, Thai red tea ( Camellia sinensis ) was identified as a highly effective and economical fermentation medium for BC production, offering both strong microbial support and cost advantages over other tea types.

While the fermentation matrix contributes essential nutrients, the type and composition of the carbon source remain primary determinants of BC yield and quality. Sugars not only serve as energy sources but also act as direct precursors in the cellulose biosynthetic pathway via the UDP-glucose route [9,10]. Although many studies have investigated the impact of individual sugars—such as glucose, fructose, dextrose, glycerol, or sucrose—on BC production, the use of sugar combinations remain relatively unexplored, particularly in kombucha systems. Considering that sucrose is naturally hydrolyzed during kombucha fermentation, combining it with other sugars may influence microbial interactions, carbon utilization, and ultimately, cellulose biosynthesis [11-13].

Building on this perspective, the present study investigates the effects of various sucrose-based sugar combinations—specifically with glucose, fructose, dextrose, and glycerol—on the production and characteristics of BC in Thai red tea kombucha fermentation. By systematically evaluating these combinations, this work aims to provide new insights into optimizing carbon source formulations for enhanced BC yield and quality. The findings are expected to support future optimization studies and contribute to the development of low-cost, scalable BC production systems using tea-based fermentation platforms.

Materials and methods

The materials used in this study included a commercial kombucha starter (SCOBY) from Neo Cold Brew Shop (Thailand), Thai red tea-vanilla flavor ( ChaTraMue brand), and commercial white sugar (sucrose). Additional chemicals were NaOH (Q RëC™), D-glucose (UniVAR, Merck), D-fructose (Carlo Erba), sucrose (ACI Labscan), dextrose monohydrate (KC, Bangkok Chemical), and glycerol (Merck). Reverse osmosis (RO) and deionized (DI) water were used as water sources. The equipment included a cheesecloth, coffee filter, glass jar, funnel, autoclave (Biobase), laboratory glassware, biosafety cabinet (Cryste, Puricube Neo), analytical balance (AE Nimbus NBL 84E), incubator, pH meter (Oakton pH 700), refractometer, oven dryer (XUE058, France-Etuves), FT-IR (Bruker VERTEX 70), XRD (Bruker D8 Advance), SEM (Zeiss AURIGA, Germany), nanoindenter (NanoTest Vantage, Micro Materials Limited, UK), and HPLC (Hitachi Chromaster).

Bacterial cellulose production

The fermentation medium was prepared by dissolving 20 g of sucrose in 180 mL of Thai red tea extract (10 g/L), which was obtained by steeping 2 g of tea in 180 mL of hot deionized water (90 °C, 15 min), followed by filtration and mixing with sucrose. The volume was adjusted to 180 mL, stirred, autoclaved (121 °C, 15 min), and cooled to 30 - 35 °C.

For starter regeneration, 20 mL of commercial kombucha was added to the prepared medium and incubated statically at 30 °C for 14 days. For BC production, 20 mL of the regenerated starter was inoculated into fresh media containing different carbon sources and incubated statically at 30 °C for 15 days.

To investigate the effect of carbon source combinations on BC production, five types of fermentation media were used. The control treatment (RTC-C) contained 10 g/100 mL sucrose. The other treatments consisted of a 1:1 mixture of sucrose (5 g/100 mL) with either fructose (RTC-SF), dextrose (RTC-SD), glycerol (RTC-SGly), or glucose (RTC-SGlu), keeping the total carbon source concentration at 10 g/100 mL in all cases.

Following fermentation, BC pellicles were harvested, boiled for 30 min, treated with 2% NaOH at 90 °C for 120 min, rinsed with deionized water to neutral pH, drained, weighed (wet weight), and oven-dried at 40 °C to constant weight using a method modified from [14]. All treatments were performed in triplicate to ensure reproducibility.

Culture medium characterization

Before and after fermentation, the culture medium was analyzed for Brix, pH, and sugar content, including sucrose, glucose, fructose, and glycerol. Brix was measured using a refractometer, and pH was determined using a pH meter. Sugar analysis was performed using an HPLC system equipped with an Aminex HPX-42A column and a refractive index detector (RI). Filtered deionized water (0.22 μm) served as the mobile phase, operated at a flow rate of 0.6 mL/min for 22 min with the column maintained at 45 °C. Samples were diluted (1:10), filtered through a 0.22 μm membrane, and quantified against a standard curve (0 - 15 g/L). All measurements were carried out in triplicate to ensure reproducibility and accuracy after system stabilization.

Bacterial cellulose characterization

Visual appearance of BC

The visual appearance of BC samples was assessed before and after purification with NaOH to evaluate any changes in color. All observations were conducted under uniform lighting conditions through direct visual inspection. Each sample was compared to the control (RTC-C) to identify noticeable differences in coloration.

Yield and water holding capacity analysis

Following purification, the BC samples were drained for approximately 10 min before being weighed to determine the wet weight (wet yield). For dry weight (dry yield) measurement, the samples were oven-dried at 40 °C until a constant weight was reached, following the method described by Aswini et al. [14]. The water holding capacity (WHC) was calculated based on the amount of water lost during drying and expressed as the ratio of water weight to dry BC weight (g/g), as described by Schrecker and Gostomski [15]. All measurements were performed in triplicate.

Morphology and fiber size analysis

The surface morphology of BC was analyzed via scanning electron microscopy (SEM). Dried BC sheets were cut into ~5×5 mm pieces, mounted on SEM stubs, and gold-coated. Fiber diameters were measured from SEM images using ImageJ software, based on 50 data points per sample [16]. Statistical analysis was performed using one-way ANOVA followed by an LSD test ( p < 0.05) to compare differences between groups.

FT-IR spectroscopy analysis

The functional groups present in the samples were analyzed using Fourier-transform infrared (FT-IR) spectroscopy. Spectra were collected in transmittance mode across the range of 400 to 4,000 cm⁻¹, with a resolution of 4 cm⁻¹. Each spectrum was obtained by averaging 64 scans for both the background and the sample. All measurements were conducted at ambient (room) temperature.

X-Ray diffraction analysis

X-ray diffraction (XRD) analysis was performed using a Bruker D8 Advance diffractometer to investigate the crystalline characteristics of the samples, including diffraction peaks (2θ) and crystallinity index (CI). The instrument operated at 40 kV and 40 mA (1,600 W), scanning across a 2θ range of 10° to 60°. Data were collected in 2,446 steps, with each step sized at 0.0204° and a scan duration of 0.4 s, totaling approximately 1,035.2 seconds per sample. The CI was determined using Equation 1, while crystallite size was calculated via the Scherrer equation Eq. (2). Further analysis of crystallinity and crystallite dimensions was carried out using OriginLab and Microsoft Excel.

where CI is the crystallinity index, d is the crystallite size, K is the Scherrer constant (0.89), λ is the X-ray wavelength (1.5406 Å), β is the full width at half maximum (FWHM) in radians, and θ is the Bragg angle [17] .

Thermogravimetric analysis

A thermogravimetric analyzer (Mettler-Toledo, Model TGA/DSC1) was used to assess the dynamic weight loss and thermal degradation behavior of BC samples. The samples were heated in an alumina pan at a temperature of 10 °C per min from 28 °C to 600 °C while flowing at a rate of 30 mL/min in a nitrogen (N₂) environment. OriginLab software was used to process TGA/DTG data, and Microsoft Excel was used to create the associated graphs.

Nano-indentation

The mechanical characteristics of BC films were examined using a nanoindenter. Circular samples approximately 0.8 cm in diameter were prepared and securely attached to the holder with white adhesive latex. Indentation testing involved applying a maximum load of 50 mN with a loading and unloading speed of 0.40 mN/s. To ensure reliability, 6 indentations were conducted on each sample, and the average values were recorded. Young’s modulus ( E ) was determined using the following formula Eq. (3):

where ν represents the Poisson’s ratio of the BC sample (0.3), E _{r ​} is the reduced modulus, ν _i ​ denotes the Poisson’s ratio of the indenter (0.07), and E _i ​ is the Young’s modulus of the indenter (1141 GPa) [18,19].

Statistical analysis

All experiments were conducted with an appropriate number of replications depending on the parameter measured. Most measurements were performed in triplicate, fiber diameter analysis involved 50 measurements per sample, and nanoindentation tests were conducted with 6 replications. Data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using Microsoft Excel. One-way analysis of variance (ANOVA) was used to assess differences among treatments, followed by the Least Significant Difference (LSD) test for post-hoc comparisons. Statistical significance was determined at a 95% confidence level ( p < 0.05).

Results and discussion

Characteristics of culture medium

The change of pH and total soluble solid (TSS)

This study investigated pH and TSS changes during fermentation ( Table 1 ). The initial medium pH (4.57 - 4.84) dropped slightly to 3.61 - 3.66 after inoculation, then declined significantly to 2.17 - 2.66 over 15 days. Among carbon sources, RTC-SGLU yielded the lowest final pH, while RTC-SGly had the highest. The initial pH drop is attributed to the starter culture’s acidic nature, with further reduction driven by organic acid production (e.g., acetic, gluconic, lactic, and citric acids) [14,20]. Similar trends were reported in kombucha fermentation, with pH decreasing from 3.04 to 2.63 after 10 days [21]. Other studies noted final pH ranges of 3.36 - 3.82 [22] and 2.72 - 2.79 [23], consistent with typical acidification during fermentation.

This study monitored changes in pH and total soluble solids (TSS) during fermentation ( Table 1 ). The initial pH of the medium (ranging from 4.57 to 4.84) decreased slightly to 3.61 - 3.66 after inoculation, then dropped significantly to 2.17 - 2.66 by day 15. Among the carbon source treatments, RTC-SGlu resulted in the lowest final pH, while RTC-SGly had the highest. The initial pH reduction is likely due to the acidity of the starter culture, while the continued decline is attributed to the production of organic acids such as acetic, gluconic, lactic, and citric acids [14,20]. Similar acidification patterns have been reported in previous kombucha studies, with pH decreasing from 3.04 to 2.63 after 10 days [21], and final pH values ranging from 3.36 to 3.82 [22] and 2.72 to 2.79 [23], aligning with the typical pH decline observed during fermentation.

Table 1 Changes in pH and Brix degree during kombucha RTC fermentation with different additives.

Different lowercase superscript letters within the same column indicate significant differences among the tea samples (LSD test, p < 0.05).

For the TSS observation, initial values ranged from 9.70 ± 0.00 to 10.70 ± 0.00° Brix before inoculation. After adding the inoculum, the TSS remained relatively stable, ranging from 9.70 ± 0.00 to 10.60 ± 0.00° Brix. However, by the end of the fermentation process, TSS values decreased significantly, reaching a range of 8.57 ± 0.05 to 8.60 ± 0.00° Brix.

This reduction reflects microbial sugar consumption, where microorganisms metabolize sugars for growth, energy, and the production of metabolites such as organic acids, ethanol, and BC [13]. Carbon sources like glucose, fructose, and glycerol are converted into intermediates that fuel BC biosynthesis, a process driven by microbial activity [24]. Similar trends have been reported in other fermentations, such as snake fruit kombucha (TSS decline from 13.30 - 14.08 to 12.43° - 12.97° Brix [25]) and cascara kombucha (10.97° to 9.97° Brix over 6 days [24]). These findings highlight the consistent role of microbial activity in reducing sugar content during fermentation [26] .

The change of sugar composition

HPLC analysis revealed significant changes in sugar profiles ( Table 2 ). Initial concentrations ranged from 4.41% -9.05% sucrose, 0.82% - 5.16% glucose, 0% - 4.92% fructose, and 5.40% glycerol (RTC-SGly). Glucose and fructose in samples without added glucose/dextrose resulted from sucrose hydrolysis during autoclaving and kombucha culture activity. Previous studies confirm similar findings, with autoclaving a 3% sucrose solution yielding 0.7% - 0.9% glucose [27] and full sucrose hydrolysis at pH 2 [28].

Sucrose serves as the primary carbon source, enzymatically hydrolyzed to glucose and fructose by yeast invertase [29,30]. Microorganisms exhibit preferential uptake of monosaccharides, with glucose being rapidly metabolized through glycolysis [29]. Under anaerobic conditions, yeasts ferment hexoses to ethanol, which acetic acid bacteria subsequently oxidize to acetic acid [31]. This metabolic hierarchy explains the differential sugar utilization rates: Glucose (57.0% ± 0.5%) and dextrose (51.9% ± 0.3%) were most rapidly consumed, followed by fructose (41.1% ± 0.4%), while sucrose (22.1% ± 0.2%) and glycerol (19.6% ± 0.1%) showed significantly lower reductions ( p < 0.05, ANOVA). These results align with Neffe-Skocińska et al. [21] , who reported 92.6% sucrose depletion (9.97% - 0.74%), minimal glucose change (0.09% - 0.10%), and 12.4-fold fructose accumulation (0.07% - 0.87%) in kombucha fermentation.

Table 2 The change of sugar composition during Thai red tea kombucha fermentation with different types of carbon sources.

Different lowercase letters in columns indicate significant differences among tea samples ( p < 0.05), while uppercase letters in rows indicate significant differences between pre- and post-fermentation ( p < 0.05, LSD test). ND = not detected.

Characteristics of bacterial cellulose

The appearance of bacterial cellulose

The appearance of BC produced from Thai red tea kombucha fermentation with different uses of carbon source combinations is presented in Figure 1. Unpurified BC from RTC-SD, RTC-SGlu, and RTC-SF appears darker red-orange compared to RTC-C and RTC-SGly ( Figures 1(a) and 1(b) ). This color likely results from natural tea pigments and artificial colorants in Thai red tea, which contains black tea, artificial flavor, and FD&C Yellow No. 6 (INS 110). Black tea catechins oxidize into theaflavins (reddish-orange) and thearubigins (brown) [32,33], while FD&C Yellow No. 6 enhances the orange-red hue [32-34]. After purification with 2% w/v sodium hydroxide, the BC turns white ( Figure 1(c) ) and retains a consistent white, opaque color after oven drying at 40 °C ( Figure 1(d) ), matching findings in most BC studies. Sodium hydroxide effectively removes tannins, polyphenols, residual bacteria, yeast cells, proteins, nucleic acids, and other organic compounds from the kombucha pellicle [35,36].

Figure 1 BC appearance at different stages of Thai red tea kombucha fermentation with various carbon source combinations: (a) during fermentation, (b) before purification, (c) after purification with sodium hydroxide, and (d) after oven drying.

BC productivity and water holding capacity

Figure 2 demonstrates the wet yield, dry yield, and WHC of BC in Thai red tea fermentation with different carbon source combinations. Among the treatments, RTC-SGlu showed the highest wet and dry BC yields, significantly exceeding RTC-C, RTC-SF, and RTC-SGly ( p  < 0.05), but not differing from RTC-SD ( p  > 0.05). This result likely reflects the efficient microbial metabolism of glucose, a direct precursor in cellulose biosynthesis. Glucose, dextrose, and fructose, as simple sugars, are metabolized more efficiently than disaccharides like sucrose, accelerating the formation of UDP-glucose [29]. Although glucose and dextrose are chemically identical, both were used in this study to reflect their different commercial uses and to explore any practical variation in BC production. In RTC-SD and RTC-SGlu, sucrose was likely hydrolyzed by invertase into glucose and fructose, with glucose preferentially utilized [31]. As shown in Table 2 , higher carbon consumption in these treatments likely contributed to increased BC production.

Figure 2 Wet yield (g/L), dry yield (g/L), and water holding capacity (WHC) (g water/g cellulose) of BC produced from RTC kombucha using different combinations of carbon sources. Different lowercase letters indicate significant differences among treatments ( p < 0.05, LSD test).

RTC-SF showed a moderate but non-significant yield increase compared to the control. While fructose supports BC formation, its efficiency is generally lower than glucose [29]. The control (RTC-C) produced lower yields, suggesting that monosaccharide addition improves sugar utilization and BC synthesis. RTC-SGly had the lowest yields, indicating glycerol was least effective under the tested conditions, possibly due to slower conversion into cellulose precursors or inhibitory effects. Although previous studies reported high yields with glycerol [37-42], variations in strains, pH, or media likely account for the difference. These findings align with [8], who reported the highest BC yields with glucose and dextrose (301.81 g/L and 11.19 g/L; 300.74 g/L and 12.12 g/L, respectively), followed by fructose and sucrose. Similarly, [13] reported that glucose resulted in higher BC yields compared to other carbon sources such as raffinose and glycerol. K. rhaeticus produced a maximum BC yield (~8.7 g/L) at 3% glycerol, but the yield declined at concentrations above 4% [40]. Likewise, Adnan et al. [43] observed that BC production peaked at 2% glycerol, with a subsequent decrease at concentrations of 3% and higher [43].

WHC showed no significant differences among treatments, indicating the carbon source primarily affects yield, not water-holding ability. WHC depends more on nanofiber structure, porosity, and surface area, which remained consistent across conditions. Values (99.07 - 112.44 g/g) were consistent with literature: 98.5 g/g from distillery wastewater [44], ~114 g/g from black tea kombucha [45], and 90 - 200 g/g ranges reported elsewhere [8,46-48]. In summary, while the carbon source influenced BC yield, it did not affect WHC, which remained a stable property across conditions.

Morphology analysis

Figure 3 shows SEM images of BC produced with different carbon sources. All samples were captured at 10,000× and 30,000× magnification, while RTC-C and RTC-SGlu were also imaged at 50,000× for a closer look at their fiber structures. The BC samples display a consistent fiber pattern, aligning with previous studies [49-51].

The SEM images reveal how different carbon sources affect BC structure. Overall, glucose (RTC-SGlu) and dextrose (RTC-SD) produce denser, more organized networks, while glycerol (RTC-SGly) results in a looser structure. The thicker BC layers in RTC-SD and RTC-SGlu before drying likely contribute to their denser fibrils, aligning with their higher wet yields. In contrast, RTC-SGly’s thinner network reflects its lower wet yield. Some impurities, especially in RTC-C, are visible in the SEM images, likely residues from alkali purification.

Figure 3 SEM image of BC (a) RTC-control; (b) RTC-SF; (c) RTC-SD; (d) RTC-SG; and (e) RTC-SGly; (1) magnification of 10,000 × ; (2) magnification of 30,000 × ; (3) magnification 50,000 × .

The SEM analysis focused on the polydispersity of BC fiber diameters, as illustrated in Figure 4 . The control sample (RTC-C) exhibited the smallest average fiber diameter (29.70 nm), whereas the RTC-SD sample (sucrose and dextrose) showed the largest (40.39 nm). The RTC-SF sample (sucrose and fructose) also significantly increased the fiber diameter to 38.56 nm. Intermediate values were observed for RTC-Gly (35.86 nm) and RTC-Glu (34.34 nm). Based on LSD analysis ( p  < 0.05), RTC-Gly and RTC-Glu did not differ significantly from each other, but both exhibited significantly larger fiber diameters than the control. These results suggest that the type of sugar used as a carbon source can influence the nanofibrillar structure of BC. The broader polydispersity observed in Figure 4 for sugar-treated samples further supports the impact of the carbon source on BC fiber morphology.

Figure 4 Graph of poly distribution size of BC samples diameter from RTC kombucha fermentation with various types of carbon source combinations.

These findings align with previous studies. BC fibers produced by K. hansenii and a strain from grape juice ranged from 10 to 60 nm [49], while K. saccharivorans in HS medium with palm date supplementation had diameters between 10 and 90 nm [52]. Similarly, BC nanofibers from HS medium ranged from 18 to 69 nm (avg. 36 nm), and those from waste fig medium ranged from 23 to 90 nm (avg. 44 nm) [53]. Low-cost media, such as date syrup and cheese whey, synthesized BC with an average diameter of 45 - 55 nm [54]. Overall, the BC fiber diameters from Thai red tea kombucha align with those from various strains and media, indicating similar morphological characteristics.

Fourier transform infrared spectroscopy

Figure 5 presents the FTIR spectra of BC samples derived from RTC kombucha fermentation using various carbon sources. All samples display similar absorption bands, although their intensities differ. The spectra confirm characteristic cellulose absorption bands, with variations in band intensity consistent with previous studies [55-57].

Figure 5 FTIR spectra of BCs from RTC kombucha fermentation with various of carbon sources combinations.

The spectra are divided into the feature region (4,000 - 1,330 cm⁻¹) and the fingerprint region (1,330 - 500 cm⁻¹) [58]. In the feature region, absorption bands at ~3,340 cm⁻¹ and 3,234 cm⁻¹ correspond to O–H stretching vibrations, with intensity differences indicating variations in hydrogen bonding. The band at 2,895 cm⁻¹, attributed to C–H stretching, shows slightly reduced absorption in RTC-SD, suggesting altered C–H interactions. A faint band at ~2,340 cm⁻¹ may indicate the presence of C≡C or C≡N functional groups, potentially originating from polyphenols, fermentation byproducts, or microbial activity in kombucha [59].

In the 1,350 - 2,000 cm⁻¹ range, bands at 1,645 cm⁻¹ (C=O stretching), 1,427 cm⁻¹ (CH₂ symmetric bending), and 1,364 cm⁻¹ (C–H symmetric bending) further characterize the BC structure [60,61]. The fingerprint region features bands at 1,315 cm⁻¹ (CH₂ wagging or C–OH deformation), 1,247 cm⁻¹ (unidentified, but similar to BC produced from glucose and other sugars), 1,159 cm⁻¹ (C–O–C antisymmetric bridge stretching), 1,106 cm⁻¹ (ring asymmetric stretching), 1,053 and 1,028 cm⁻¹ (C–O–C and C–O–H stretching), and 898 - 894 cm⁻¹ (C–O–C stretching in β-1,4-glycosidic linkages), indicating an amorphous cellulose structure [58,62].

X-Ray diffraction analysis

The XRD patterns of BC samples from Thai red tea fermentation with different carbon sources are shown in Figure 6 , with crystallinity index (CI) and crystallite size detailed in Table 3 . All samples exhibit three distinct peaks at 2θ ≈ 14.74°, 17.03° and 22.90°, corresponding to the 100, 110, and 200 planes of cellulose type I [63,64]. The strongest peak near 23° confirms the crystalline structure of cellulose type I [64,65], confirming its crystalline nature [66]. While band positions are consistent, variations in relative intensities and crystallinity indicate differences in cellulose chain orientation and structural properties [64].

Figure 6 XRD spectra of BC from Thai red tea kombucha with different carbon source combinations.

The crystallinity index (CI) and crystallite size of BC samples were determined from XRD data using the Segal method ( Table 3 ). CI values ranged from 84.74% (RTC-SGly) to 89.54% (RTC-C), with RTC-SD and RTC-SF showing relatively similar values. All mixed carbon source samples had lower CI than sucrose-only RTC-C. BC crystallinity depends on multiple factors, including carbon/nitrogen sources, additives, bacterial strain, fermentation conditions, and post-processing [67,68]. These results agree with previous studies: K. medellinensis produced BC with 83% - 90% CI using different sugars [69], K. hansenii achieved >80% CI with glucose/glycerol [13], and Komagataeibacter sp. W1 showed 74% - 89% CI across various carbon sources [55]. These findings confirm that carbon source selection significantly impacts BCʼs crystallinity and structural properties.

Table 3 Crystallinity index and average crystallite size of dried BC from Thai red tea kombucha fermentation with different additives.

Samples	Crystallinity Index (%)	Average Crystallite Size (nm)
RTC-C	89.54	3.194
RTC-SF	88.35	3.112
RTC-SD	88.17	3.181
RTC-SGlu	86.00	3.327
RTC-SGly	84.74	3.069

Analysis was performed once; no replication was conducted for this measurement.

Building on the crystallinity findings, the crystallite size analysis revealed similar dependencies on carbon sources and fermentation conditions. Our BC samples showed crystallite sizes ranging from 3.07 to 3.33 nm ( Table 3 ), consistent with the 3.06 nm crystals reported for G. xylinus [70]. The observed variations align well with literature values: K. xylinus produced larger crystallites (4.7 - 6.8 nm) using simple sugars [71], while Lactobacillus plantarum in green tea medium formed 5.36 - 5.98 nm crystallites over extended fermentation [72]. Other studies report comparable ranges, from black tea kombucha BC (3.29 - 4.80 nm) [73] to larger crystallites (4.76 - 8.36 nm) under different conditions [17,74,75]. Overall, these findings suggest that while crystallite size remains within a narrow range, it is subtly influenced by the type of carbon source and the resulting microbial activity during fermentation.

Thermogravimetric analysis (TGA/DTG)

Figure 7 shows the thermogravimetric (TG) ( Figure 7(a) ) and derivative thermogravimetric (DTG) ( Figure 7(b) ) curves of BC samples (RTC-C, RTC-SF, RTC-SD, RTC-Glu, RTC-Gly), with decomposition data summarized in Table 4 . These results illustrate the thermal stability and degradation behavior of BC, which are essential for applications requiring chemical resistance and strong fiber–matrix interactions [76]

Figure 7 TGA (a) and DTG (b) thermographs of BC samples from Thai red tea fermentation with different combinations of carbon sources.

Table 4 The details of data decomposition during the TGA process of BC samples from kombucha fermentation with different types of tea.

Samples	First stage weight loss (%)	Second stage weight loss (%)	Residue (%)	DTG peak range ( o C)	DTG T Max ( o C)
RTC-C	6.03	71.01	23.01	256 - 384	353.67
RTC-SD	1.34	70.10	28.52	260 - 391	349.50
RTC-SF	3.60	73.43	22.94	267 - 390	353.33
RTC-SGlu	5.14	73.90	20.92	268 - 389	356.83
RTC-SGly	4.15	71.92	23.90	258 - 388	359.50

Analysis was performed once; no replication was conducted for this measurement.

The first-stage weight loss, ranging from 1.34% to 6.03%, corresponds to moisture loss and the volatilization of low-molecular-weight components [77,78]. RTC-SD exhibited the lowest weight loss (1.42%), suggesting reduced moisture retention, a result consistent with previous studies reporting first-stage losses of 1% - 2% [1] and 5% - 9% [77]. The second-stage weight loss, associated with cellulose decomposition, ranged from 70.10% in RTC-SD to 73.90% in RTC-SGlu. Residual weights at 600 °C varied from 20.92% (RTC-SGlu) to 28.52% (RTC-SD), indicating differences in the formation of thermally stable by-products. This stage causes the biggest weight loss, mainly due to the breakdown of β-glucan chains and the oxidation of cellulose into carbon residues [77,80].

These findings are consistent with previous studies. For example, BC from Acetobacter xylinum AGR 60 exhibited a two-stage thermal decomposition: An initial weight loss of 6%, followed by a second-stage loss of 74%, leaving 20% residue at 700 °C. The main decomposition occurred between 300 °C and 360 °C [81]. Similarly, BC from Gluconacetobacter xylinus AGR 60 showed a 6.2% first-stage loss, a 64% second-stage loss, and 22.8% residue at 600 °C, with a DTG_max of 339.6 °C [82]. BC synthesized from different carbon sources—glucose, fructose, sucrose, and glycerol—also displayed a comparable decomposition pattern, with residual mass ranging from 15.2% to 23.1% at 600 °C [83]. In contrast, BC produced from green tea kombucha undergoes three decomposition stages at 152, 267 and 359 °C, resulting in a total weight loss of 74.42% and a residue of 25.58%, which differs from the two-stage decomposition observed in the present study [84].

DTG analysis showed a peak temperature range of 256 - 391 °C, with maximum degradation temperatures (DTG Max) between 349.50 °C and 359.50 °C. RTC-SGly exhibited the highest DTG Max (359.50 °C), suggesting slightly enhanced thermal stability. Comparisons with previous studies indicate some variation in DTG Max values, such as 328.36 °C for G. xylinus BC [74], 354.5 - 355.4 °C for G. hansenii BC in HS medium [85], and 327 - 368 °C for K. medellinensis BC from various waste sources [86]. Additionally, BC treated with freeze-drying and hot-press drying methods yielded DTG Max values of 343.6 °C and 313.4 °C, respectively, with DTG peak ranges of 186 - 363 °C and 199 - 347 °C [77]. BC produced through kombucha fermentation of green tea showed a DTG Max of 366 °C [84]. Overall, while RTC-SGly demonstrated the highest DTG Max in this study, the narrow range of differences suggests that carbon source selection has a relatively minor impact on the thermal properties of BC.

Nano-indentation analysis

Nano-indentation was used to evaluate the mechanical properties of BC samples at the nanoscale. Samples RTC-SD and RTC-SGlu, selected for their high yields, were compared with the control (RTC-C) ( Table 5 ). No significant differences were found in maximum depth, plastic indentation depth, hardness, reduced modulus, or Young’s modulus ( p  > 0.05), indicating that the carbon source—sucrose alone or combined with dextrose or glucose—had minimal impact. While carbon sources can influence BC properties [87,88], this effect was not statistically significant here.

Table 5 Mechanical properties of BC from kombucha fermentation of RTC-SD, RTC-SGlu, and RTC-C analyzed using a nano-indenter.

MD: maximum depth, Pl: plastic, ML: maximum load, H: hardness, RM: reduced modulus, ERP: elastic recovery parameters, CC: contact compliance, PW: plastic work, EW: elastic work, and YM: Young’s Modulus. Based on the statistical analysis, there were no significant differences among the samples across all parameters ( p > 0.05).

The mechanical properties of bacterial cellulose (BC) are affected by various factors, including the type of microorganism, how the BC is grown, how it is purified, and how it is dried [87-91]. Using different concentrations of NaOH during purification can damage or preserve the fiber structure [92,93]. The drying method also changes the porosity, density, and fiber arrangement of the final product [90,94]. Crystallinity is another important factor. A higher CI usually leads to greater stiffness and Young’s modulus but lower flexibility [57,95]. In this study, all treatments had similar CI values, which match their similar Young’s modulus values.

The measured Young’s modulus values fall within ranges reported in the literature: 1.10 - 5.56 GPa for Komagataeibacter strains [91], up to 9.14 GPa for A. xylinum AGR60 in coconut water [81], and 8.0 ± 1.9 GPa in kombucha-derived BC [96]. In conclusion, while carbon sources can affect mechanical behavior, crystallinity and other processing factors appeared to play a more dominant role in this study.

This study reveals that substituting part of the sucrose with either glucose or dextrose significantly improves BC production in terms of both wet and dry yield. The sucrose-only control (RTC-C) produced a lower yield compared to the RTC-SGlu and RTC-SD combinations, indicating enhanced fermentation efficiency with the addition of these monosaccharides. Although glucose and dextrose yielded similar results, the lower cost of dextrose makes it a more practical and economical option for large-scale production. In contrast, sucrose alone was less effective, suggesting limited suitability as a sole carbon source under these conditions.

From a value perspective, the increase in BC yield translates to more efficient fiber production, which is critical for applications requiring high quantities of cellulose material. While the addition of glucose or dextrose increases the raw material cost compared to using sucrose alone, the improvement in yield—particularly when using the more affordable dextrose—can offset this cost in industrial settings. Glucose, though slightly more expensive, remains valuable for controlled research environments due to its predictable behavior during fermentation. Overall, these findings support the use of sucrose–dextrose or glucose combinations as the cost-effective and high-performing formulation for optimizing BC yield and scalability. Future optimization and cost-benefit analysis will further clarify the best carbon source strategies for commercial BC production.

Conclusions

This study demonstrated that carbon source combinations significantly influenced BC production from Thai red tea kombucha, with wet yields ranging from 74.53 g/L (RTC-SGly) to 259.54 g/L (RTC-SGlu) and dry yields from 0.66 g/L to 2.59 g/L. Water-holding capacity ranged from 99.07 to 112.44 g/g, and fiber diameters from 29.70 ± 5.17 nm (control) to 40.39 ± 8.65 nm (RTC-SD), while all samples exhibited comparable functional groups and crystallinity
(84.74% - 89.54%). Thermal and mechanical properties showed no significant differences among treatments. These findings highlight that combining sucrose with glucose or dextrose can substantially boost BC yield without compromising its structural and functional integrity.

Acknowledgements

We gratefully acknowledge the financial support for this research provided by Suranaree University of Technology (SUT), Thailand Science Research and Innovation (TSRI), the National Science, Research and Innovation Fund (NSRF) under contract no. 195650, and the National Research and Innovation Agency, Indonesia (BRIN).

Declaration of generative AI in scientific writing

This manuscript was edited for language and readability using generative AI tools under human supervision. The authors take full responsibility for its content.

CRediT author statement

Wawan Agustina : conceptualization, methodology, writing—original draft, and visualization. Apichat Boontawan : writing - review & editing, supervision, and funding acquisition.

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