Low-cost bacterial cellulose production from agricultural waste for antibacterial applications

Materials

The bacterial strains Acetobacter xylinus NCIM 2526, Staphylococcus aureus NCIM 2492, and Escherichia coli NCIM 2092 were obtained from the National Collection of Industrial Microorganisms (NCIM), Pune, India. Analytical-grade media components, including peptone, yeast extract, disodium phosphate, hydrochloric acid, sulfuric acid, sodium hydroxide, D-sorbitol, agar, and chitosan (low molecular weight, with a viscosity of 10–150 mPa·s and a degree of acetylation of 90%), were sourced from Sisco Research Laboratories (SRL) Pvt. Ltd., India. Bamboo dust particles (an agricultural by-product) were procured from a local market in Kanpur, Uttar Pradesh, India.

Characterization of agro-residues

Energy-dispersive X-ray (EDX) spectroscopy was used to determine the carbon, oxygen, and nitrogen content of various agro-residues, including cotton stalk, paddy straw, soybean stalk, groundnut shell, turn stalk, and bamboo dust particles. Among the six agro-residues evaluated, bamboo dust exhibited the highest carbon, oxygen, and nitrogen content, with 49% carbon, 38% oxygen, and 0.5% nitrogen. The chemical composition of bamboo dust was further analyzed using TAPPI test methods, revealing a cellulose content of 64.6%, a moisture content of 7.6%, an ash content of 1.9%, a lignin content of 23.4%, and 2.5% of other extractives, including wax and pectin. ²⁶

Preparation of modified medium from extracted (glucose) reducing sugars

In this study, 60 g of bamboo dust was initially treated with 1% NaOH solution (1500 ml), autoclaved at 121°C for 30 min, thoroughly washed, and dried at 60°C, following the protocol adapted from Sharma et al. ²⁶ Subsequently, 20 g of alkali-treated bamboo dust was treated with 1% and 2% (v/v) sulfuric acid solutions (1500 ml each) according to the TAPPI T222 method. ²⁷ After acid treatment, the solutions were neutralized to pH 5 using NaOH pellets, filtered to separate the bamboo residue, and centrifuged at 12,000 rpm for 10 min to obtain a clear solution. The concentration of reducing sugars in these solutions was determined using the DNS (dinitrosalicylic acid) assay, ²⁸ as illustrated in Figure 1.

OPEN IN VIEWER

Figure 1.

Process flow of extraction of glucose from bamboo fiber waste.

Synthesis of bacterial cellulose (BC)

The Hestrin-Schramm (HS) medium was prepared by autoclaving a solution containing D-glucose (as the carbon source), 5 g of peptone and 5 g of yeast extract (as nitrogen sources), 2.7 g of disodium hydrogen phosphate, and 1.15 g of citric acid. ¹⁸ The modified medium was then prepared by combining the autoclaved Hestrin-Schramm medium with additional D-glucose extracted from bamboo waste material, as shown in Figure 2. For BC synthesis, 140 ml of the modified medium was inoculated with 3–5 mm colonies of Acetobacter xylinus NCIM 2526 in each of 7 flat-bottomed cylindrical flasks (250 ml). The flasks were incubated on a shaker at 110 rpm for 2 days at 30°C, followed by static incubation at the same temperature for 5 days to facilitate BC membrane formation. After incubation, the BC membranes from each flask were treated with 100 ml of 0.1 N NaOH solution at 60°C for 30 min to remove any residual medium. The treated membranes were then thoroughly washed with hot and cold double-distilled water and dried at 60°C for 4 h.

OPEN IN VIEWER

Figure 2.

Synthesis of pure BC and chitosan incorporated BC.

In situ synthesis of chitosan-incorporated BC

Transparent chitosan solutions (2%, 6%, and 10%) were prepared by dissolving chitosan in a 1% acetic acid solution and stirring vigorously at 80°C for 8 h. For the in situ synthesis of chitosan-incorporated BC, 126 ml of the modified medium was inoculated with 3–5 mm colonies of Acetobacter xylinus NCIM 2526 in each of 7 flat-bottomed cylindrical flasks (250 ml). The flasks were incubated on a shaker at 110 rpm for 2 days at 30°C. Following this, 14 ml of the prepared chitosan solutions (2%, 6%, and 10%) were added to the flasks, and dynamic incubation was continued at the same temperature and shaking conditions for an additional day to ensure proper distribution of chitosan within the BC-synthesizing suspension. After a 24-h period, the chitosan-containing bacterial cultures were further incubated under static conditions at 30°C for 4 days to produce chitosan-incorporated bacterial cellulose.

The initial concentrations of chitosan in the culture medium were 0.2%, 0.6%, and 1%. BC films were successfully formed in the culture media containing 0.2% and 0.6% chitosan, whereas the medium with 1% chitosan showed negligible or no BC yield, as depicted in Figure 2. The BC membranes obtained from the media with 0.2% and 0.6% chitosan content were treated with 100 ml of 0.1 N NaOH solution at 60°C for 30 min, washed thoroughly with double-distilled water, and dried at 60°C for 4 h, following the same protocol as described above.

Fourier transform infrared (FTIR) spectroscopy

FTIR spectroscopy was performed in transmission mode using a Bruker Alpha FTIR spectrometer to identify and validate the presence of functional groups in bacterial cellulose (BC) pellicles and films. This technique helps to determine the chemical structure and possible modifications in the functional groups after incorporating different substances into the BC matrix.

Scanning Electron Microscopy (SEM)

A JOELJCM-7000 SEM was used to examine the surface morphology of BC pellicles and membranes. This analysis provided detailed information on the microstructural characteristics, surface topology, and fiber arrangement, which are crucial for understanding the material’s mechanical properties and potential applications in biomedical fields.

X-ray diffraction (XRD)

X-ray diffraction analysis was conducted using a Panalytical X-ray diffractometer with CuKα radiation (λ = 1.5406 Å), operating at 40 mA and 45 kV. The diffraction patterns were recorded over a 2θ range of 10° to 40°. The crystallite size was calculated using the Scherrer equation, while the crystallinity index was determined from the ratio of the crystalline peak area to the total reflection area. This analysis aimed to evaluate the crystalline nature of the BC samples, which is critical for determining the strength, rigidity, and thermal stability of the material.

Water holding ability

To assess the water-holding capacity of the BC membranes, the samples were first conditioned at 25°C for 24 h. Approximately 0.5 g of each type of membrane was then placed in a sample pan and heated at 105°C using a digital moisture meter (Model No. 101). The percentage of water-holding ability was calculated by comparing the weight of the moist BC to that of the oven-dried BC. This property is essential for applications requiring moisture retention, such as wound dressings and biomedical implants.

Mechanical analysis

Tensile strength measurements were conducted using an Instron tensile testing instrument to determine the mechanical properties of the BC samples. Samples were cut to dimensions of 10 × 50 mm and subjected to a 5 kN load cell at a traverse speed of 2 mm/min. The mechanical strength of BC is a key factor in its applicability for various structural and biomedical applications, ensuring that the material can withstand mechanical stresses in practical use.

Statistical analysis

Each experimental treatment was performed in triplicate, with all experiments repeated at least twice to ensure accuracy and reproducibility. The statistical significance of the data was analyzed using one-way analysis of variance (ANOVA). Results were considered statistically significant at a p-value of less than 0.05, unless stated otherwise.

Antibacterial activity analysis

The antibacterial efficacy of chitosan-incorporated BC against Staphylococcus aureus (NCIM 2492) and Escherichia coli (NCIM 2092) was evaluated using the agar disk diffusion method. BC membranes were placed on agar plates inoculated with the bacteria, and the diameter of the inhibition zone (in mm) surrounding the BC membrane pellicles was measured. This analysis was conducted to determine the potential of chitosan-incorporated BC as an antibacterial agent, which is particularly relevant for its application in wound care and medical devices.

Yield of reducing sugars

A sustainable approach was employed to extract reducing sugars from bamboo dust particles using a mild alkali (initial treatment) followed by an acid hydrolysis process, in accordance with the TAPPI T222 procedure. The glucose yields from bamboo dust particles were estimated to be 52.3% with a 1% sulfuric acid solution and 75% with a 2% sulfuric acid solution. The 2% sulfuric acid solution was found to be optimal for obtaining the highest glucose yield from agro-residues after 5 h of hydrolysis, as reported by Wood et al. ²⁹ However, studies by Qi et al. ³⁰ and Shi et al. ³¹ reported glucose yields of 65%–70% from agricultural waste using a more costly enzymatic hydrolysis process. The sustainable approach employed in this study reduces the cost of glucose extraction while enhancing the glucose yield, aligning with the goals of sustainable and economical bioprocessing.

Analysis of BC and chitosan-treated BC production yield

Statistical analysis indicates a significant difference in bacterial cellulose (BC) production yields (g/l) between media with and without chitosan incorporation. The average production yields were 5.8 ± 0.21 g/l for pure BC, 5.2 ± 0.21 g/l for BC containing 0.2% chitosan, and 4.6 ± 0.21 g/l for BC treated with 0.6% chitosan. A probability (p) value of less than 0.05 suggests that the mean BC production yields differ significantly with chitosan incorporation. The addition of low molecular weight chitosan slightly inhibits the growth of Acetobacter xylinus, reducing the production yield of chitosan-incorporated BC, as reported by Phisalaphong and Jatupaiboon. ³²

This study demonstrates a notable advancement in using bamboo dust, an agricultural waste material, as a novel and efficient substrate for BC production. Compared to earlier studies (Table 1) utilizing various organic waste substrates,^32,33 such as sugarcane molasses, apple pomace, and potato peel waste, this research achieved the highest BC yield of 5.8 ± 0.21 g/l under optimized conditions with Acetobacter xylinus, as indicated by the bold entries in Table 1. This high yield was achieved by initially employing a dynamic incubation phase for 2 days, followed by a 5-day static incubation at 30°C and pH 5. The superior yield obtained in this study highlights the potential of bamboo dust as a cost-effective and sustainable feedstock for BC synthesis and emphasizes the importance of optimizing incubation conditions to improve BC production efficiency. These findings support the use of bamboo dust in industrial-scale BC production, promoting sustainable practices and waste valorization.

OPEN IN VIEWER

Table 1.

Utilization of different waste material as additional carbon sources from previous studies.

Sr. No.	Waste material	Bacterium used	pH	Incubation temperature	Incubation condition and time	Yield (g/l)	Ref.
1	Baggases waste	Gluconacetobacter xylinus CH001	6.7	28 °C	Statical for 20 days.	1.09	Qi et al. 30
2	Apple pomace and sugarcan	Gluconacetobacter medellinensis	4	28 °C	Statical for 14 days.	2.5	Urbina et al. 37
3	Sugercane molasses	Komagataeibacter rhaeticus	6	30 °C	Statical for 14 h.	3.46–4.01	Machado et al. 38
4	Rotten banana and rotten mango juice	Komagataeibacter medellinensis	3.5	30 °C	Static for 3 days.	4.81	Molina-Ramírez et al. 39
5	Potato peel waste	Gluconacetobacter xylinum ATCC 10,245	9	35 °C	Static for 6 days.	4.70	Abdelraof et al. 40
6	Tobacco waste	Acetobacter xylinum ATCC 23,767	6.5	30 °C	Static for 7 days	2.27	Ye et al. 41
7	Oats hull and black tea	Medusomyces gisevii Sa-12	4.5	30 °C	Static for 12 days	2.20	Skiba et al. 42
8	Cashew apple juice with soybean molasses	Gluconacetobacter xylinus	5.5	30 °C	Static for 7 days	4.50	Souza et al. 43
9	Kitchen waste	Komagataeibacter rhaeticus	6	30 °C	Static for 10 days.	4.76	Li et al. 44
10	Oil palm frond juice	Acetobacter Xylinum 0416	4.5	30 °C	Static for 14 days.	2.88	Mohamad et al. 45
11	Corn stover	Komagataeibacter xylinus	6	30 °C	Static for 120 h.	4.44	Li et al. 46
12	Bamboo dust	Acetobacter xylinus	5	30 °C	2 days dynamic and 5 days static	5.8 ± 0.21	Current study

Different concentrations of chitosan in the bacterial cellulose (BC) synthesis medium influence Acetobacter xylinum growth and BC production through complex interactions. At a low concentration (0.2%), chitosan can enhance BC synthesis by providing a favorable environment that supports bacterial growth and cellulose production. As the concentration increases to 0.6%, chitosan may still support BC production but with reduced efficiency due to potential stress or partial inhibition of bacterial metabolism. At high concentrations (1%), chitosan can significantly hinder bacterial growth and BC synthesis, possibly due to interference with cell wall integrity, nutrient absorption, or medium viscosity as shown in the Supplemental File 1, similar effect observed with silver nanoparticles, another antimicrobial agent. ⁷ Zhang et al. ³³ tested various concentrations of high molecular weight chitosan (0.25%, 0.50%, and 0.75%) in a BC-synthesizing medium, observing up to an 86% reduction in BC production yield with 0.2% chitosan incorporation. High molecular weight chitosan at low concentrations strongly inhibits Acetobacter xylinus growth. ³² In contrast, low molecular weight chitosan at low concentrations only slightly suppresses Acetobacter xylinus growth and the biosynthesis of chitosan-incorporated BC. The 0.2% chitosan-treated BC showed a 3.48% reduction in production yield, while 0.6% chitosan incorporation led to a 21% reduction. Notably, a 1% chitosan-treated BC medium significantly reduced BC production in static culture and drastically decreased sheet thickness. In this study, the BC production yield without chitosan treatment was more than four times higher than the BC yield obtained by Zhang et al., ³³ attributed to the use of agro-residues as additional carbon sources. Agro-based media provide diverse carbon sources and nutrients, which may enhance and simplify the metabolic pathways involved in cellulose production, ³⁴ similar to what has been demonstrated in the presence of additional sugars, organic acids, and amino acids. ³⁵ Additionally, the use of acetic acid in a BC synthesizing medium has been shown to enhance BC production yield, as reported by Yang et al. ³⁶

Analysis of BC and chitosan-treated BC production costs (g/l)

The cost of bacterial cellulose (BC) and chitosan-incorporated BC primarily depends on production yield. The total cost breakdown for BC production includes the cost of raw materials (40%–50%), fermentation (20%–30%), downstream processing (20%–30%), and quality control and other overheads (10%–20%), as reported by Keshk et al. ⁴⁷ A decade ago, the cost of BC was estimated at $142/kg by Keshk ⁴⁷ and Mohite and Patil. ⁴⁸ However, recent data on BC cost analysis has not been published in the last decade.

In this study, the cost of raw materials was calculated based on the current prices provided by the chemical manufacturer Sisco Research Laboratory (SRL) Pvt. Ltd. The costs associated with production and purification of BC were calculated using the cost breakdowns provided by Keshk ⁴⁷ and Mohite and Patil. ⁴⁸ The average production yields obtained in this study were 5.8 ± 0.21 g/l for pure BC, 5.2 ± 0.21 g/l for 0.2% (w/v) chitosan-treated BC, and 4.6 ± 0.21 g/l for 0.6% (w/v) chitosan-treated BC, compared to a yield of 2.2 g/l reported for BC in the Hestrin-Schramm (HS) medium. ¹⁸ The total cost of BC was estimated from the costs of raw materials, production, and purification, as shown in Table 2.

OPEN IN VIEWER

Table 2.

Cost analysis of bacterial cellulose and chitosan incorporated BC in modified medium.

Cost analysis of bacterial cellulose and chitosan incorporated BC
Sr. No	SRL make nutrients	Quantity in gram	HS medium cost in $	Sr. No	Raw material of this study	Quantity in g/ml	Modified medium cost in $
1	D-glucose	20	0.1	1	Reducing sugar	7 g	0.18
2	Yeast extract	5	0.21	2	HS medium cost	-	0.50
3	Sodium phosphate dibasic	2.7	0.05			Sum	0.68/2 l
4	Peptone	5	0.13
5	Citric acid	1.15	0.0069
		Total	0.50/liter
Sr. No.	Bacterial cellulose cost analysis in HS medium			Sr. No.	Bacterial cellulose cost analysis in modified medium
1	Cost of medium	0.5	Cost of BC in $/kg	1	Cost of Medium	0.34	Cost of BC in $/kg
2	Cost of preparation	0.1		2	Cost of preparation	0.1
3	Cost of fermentation	0.2	477.27	3	Cost of fermentation	0.2	153.44
4	Cost of energy consumption	0.1		4	Cost of energy consumption	0.1
5	Cost of purification	0.1		5	Cost of purification	0.1
6	Cost of drying	0.05		6	Cost of Drying	0.05
	Total	1.05/2.2 g			Total	0.89/5.8 g
Sr. No	0.2% chitosan incorporated Bacterial cellulose cost analysis			Sr. No.	0.6% chitosan incorporated Bacterial cellulose cost analysis
1	Cost of 0.2% chitosan content medium	1.04	Cost of BC in $/kg	1	Cost of 0.6% chitosan content medium	2.44	Cost of BC in $/kg
2	Cost of preparation	0.15		2	Cost of preparation	0.15
3	Cost of fermentation	0.25	334.61	3	Cost of fermentation	0.25	682.60
4	Cost of energy consumption	0.15		4	Cost of energy consumption	0.15
5	Cost of purification	0.1		5	Cost of purification	0.1
6	Cost of drying	0.05		6	Cost of drying	0.05
	Total	1.74/5.2 g			Total	3.14/4.6 g

The bold entries in Table 2 illustrate the cost analysis for producing BC across different media, including HS medium, agricultural waste-based modified medium, and chitosan-based modified mediums. These entries emphasize the variations in production costs per kilogram of BC, highlighting the economic impact of substrate choice and chitosan concentration on overall production efficiency. The cost analysis presented in Table 2 demonstrates a significant economic advantage in using a modified medium enriched with agro-residues compared to the conventional Hestrin-Schramm (HS) medium. Specifically, the modified medium reduces the cost of BC production to $153.44 kg, representing a 68% reduction from the $477.27 kg cost associated with the HS medium. This cost efficiency is achieved through the use of sustainable and cost-effective raw materials, thereby promoting waste valorization. However, incorporating chitosan into the BC synthesis medium, particularly at concentrations of 0.2%, 0.6%, and 1% of low molecular weight chitosan, significantly increases production costs. The cost rises to $334.61 kg for 0.2% chitosan and $682.60 kg for 0.6% chitosan. This increase is due to both the added cost of chitosan and its inhibitory effect on the growth of Acetobacter xylinus, which reduces BC yields.

Among the tested concentrations, 0.2% chitosan was found to be optimal, yielding a BC product with physical and chemical properties comparable to pure BC while maintaining a more uniform distribution of chitosan within the BC matrix. However, at a concentration of 0.6%, the chitosan distribution becomes uneven, negatively impacting the overall geometry and mechanical properties of the BC. Moreover, increasing the chitosan concentration to 1% results in a complete cessation of BC membrane production, likely due to a strong interaction between chitosan and Acetobacter xylinus that disrupts the bacterial synthesis process. While post-synthesis chitosan incorporation results in similar costs, it leads to poor distribution and mechanical durability compared to in situ synthesis, where chitosan is directly integrated during BC formation.^12,13 The in situ method ensures a more homogeneous integration of chitosan, enhancing the material’s uniformity and mechanical strength. However, further optimization is needed to balance cost, uniformity, and durability for industrial-scale applications.

SEM analysis

The physical morphology of the bacterial cellulose (BC) membrane primarily depends on the carbon source, incubation conditions, and type of bacterial strains used in its production. ⁴⁹ The SEM images of the pure BC membrane obtained from the modified medium were captured at magnifications of 10,000×, 5000×, and 1000×, as shown in Figure 3(a)–(c). These images reveal that the BC membrane consists of randomly arranged microfibrils forming a three-dimensional network with closely interconnected fibrils. The average microfibril dimension was measured at 5.2 µm, with an average pore size of 3.2 µm. The microfibril geometry of BC is influenced by the bacterial strain, such as Acetobacter xylinus NCIM 2526, and its incubation conditions, as reported by Pandey et al. ⁵⁰ and Mohite and Patil. ⁵¹ The pores are nearly evenly distributed within the three-dimensional structure, enhancing the water-holding capacity of the BC. Water molecules are entrapped within the porous geometry of the BC membrane.^52,53

OPEN IN VIEWER

Figure 3.

SEM image of BC (a–c), 0.2% chitosan incorporated BC (d and e), 0.6% chitosan incorporated BC (f and g) and EDX result of chitosan incorporated BC (h) for 0.2% concentration and (i) for 0.6%.

Similarly, the morphology of BC incorporated with 0.2% chitosan also displays randomly arranged microfibrils in a three-dimensional network with high compactness between BC fibrils due to chitosan treatment, as depicted in Figure 3(d) and (e). The SEM images of 0.2% chitosan-treated BC at 3500× (Figure 3(d)) and 5000× (Figure 3(e)) magnifications show an even distribution of chitosan throughout the BC’s three-dimensional structure. The physical properties of BC are only slightly affected by the lower concentration of low molecular weight chitosan.^32,54 As the concentration of chitosan increases to 0.6%, an uneven distribution of chitosan is observed in SEM images taken at 1000× and 500× magnifications, as shown in Figure 3(f) and (g). The presence of chitosan in the BC membrane was further confirmed by energy-dispersive X-ray (EDX) analysis, which measured the nitrogen content percentage. The nitrogen content was 3.51% in the 0.2% chitosan-treated BC membrane and 8.26% in the 0.6% chitosan-treated BC membrane. The porosity of chitosan-incorporated BC is reduced due to the interaction between chitosan and cellulose polymers during BC biosynthesis, in agreement with Phisalaphong and Jatupaiboon ³² and Zhang et al. ³³

Moisture holding potential

The moisture-holding capacity was found to be 64.1% for pure BC, 62.3% for 0.2% chitosan-treated BC, and 50.5% for 0.6% chitosan-treated BC. The higher moisture-holding capacity of pure BC is attributed to its porous structure, which allows for the entrapment of water molecules. In contrast, the lower moisture-holding potential of chitosan-incorporated BC is due to the replacement of hydroxyl groups with amino groups, leading to a more compact structure, as reported by Ul-Islam et al. ⁵² and Emeka Arikibe et al. ⁵⁵ However, the moisture-holding capacity of 0.2% chitosan-incorporated BC is closer to that of pure BC than the 0.6% chitosan-treated BC, due to the lower concentration of low molecular weight chitosan in the BC synthesizing medium, which results in negligible changes to the physical properties of BC.^32,33 The 0.2% chitosan-incorporated BC shows potential for wound care applications due to its favorable moisture-holding and releasing abilities, which promote wound healing, ⁵⁶ as suggested by Cazón and Vázquez. ¹⁰

Antimicrobial ability of chitosan-treated bacterial cellulose (BC)

The antimicrobial properties of chitosan-incorporated bacterial cellulose (BC) were evaluated using bacterial strains such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The chitosan-incorporated BC was synthesized in a modified medium through an in situ process, where 0.2% low molecular weight chitosan was uniformly distributed throughout the BC membrane, as confirmed by scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) analysis. The antimicrobial activity of the 0.2% and 0.6% chitosan-treated BC was assessed using the disk diffusion method against E. coli and S. aureus, following the methodology characterized by No et al. ⁵⁷

After ten washes, a significant inhibition zone was observed for both 0.2% and 0.6% chitosan-treated BC. In contrast, no inhibition zone was detected for the pure BC control against either S. aureus or E. coli, as shown in the Supplemental File 2. This result confirms the antibacterial activity of chitosan-incorporated BC at both concentrations, whereas pure BC does not exhibit any antibacterial properties.

The diameter of the inhibition zone against S. aureus was recorded as 6.2 ± 0.08 mm for 0.6% chitosan-treated BC and 5.8 ± 0.09 mm for 0.2% chitosan-treated BC. Similarly, for E. coli, the inhibition zone was 5.9 ± 0.06 mm for 0.6% chitosan-treated BC and 4.7 ± 0.09 mm for 0.2% chitosan-treated BC. A more significant reduction in bacterial growth was observed for the Gram-positive S. aureus, likely due to its thicker peptidoglycan layer, which lacks an outer membrane. Chitosan can effectively disrupt the peptidoglycan layer, leading to cell lysis. However, the effectiveness of chitosan can vary depending on the bacterial species and environmental conditions. Generally, Gram-positive bacteria are somewhat more resistant to chitosan than gram-negative bacteria, primarily due to the thicker peptidoglycan layer.^7,10,58

Low molecular weight chitosan is more effective as a bactericidal agent than high molecular weight chitosan because it penetrates the BC network more efficiently. ⁵⁹ The 0.2% chitosan-incorporated BC has sufficient antibacterial potential against both Gram-positive and Gram-negative bacteria and allows for cost-effective BC production in a modified medium. ⁷ While 0.6% chitosan-incorporated BC shows slightly higher bacterial reduction ⁶⁰ than 0.2% chitosan-treated BC, using 0.6% chitosan in the BC synthesis medium leads to a significant reduction in BC production yield and an uneven distribution of chitosan within the BC’s three-dimensional structure, resulting in resource loss and increased cost.^32,33 The cytotoxicity of 0.2% chitosan-treated BC is minimal due to the low molecular weight and low concentration of chitosan used. ³² Therefore, 0.2% chitosan-incorporated BC, a natural polysaccharide, can be effectively used as a biomaterial for bandages and wound dressings.

Solubility of BC

The purity of BC membranes produced in the modified medium was assessed using a chemical solubility test. One gram of BC membrane was dissolved in 20 ml of 60% (v/v) sulfuric acid. After stirring at 300 rpm for 1 h at room temperature, the BC dissolved completely, resulting in a transparent solution with some residual material, as shown in the Supplemental File 2. A small amount of BC degradation occurred during acid treatment, which was collected as residual material. The solubility test not only indicates the purity of the BC but also suggests potential applications in the healthcare sector, as discussed by Pandey et al. ⁵⁰ and Mohite and Patil. ⁵¹

FTIR analysis

The investigation into the physical morphology and moisture-holding capacity of bacterial cellulose (BC) reveals subtle but significant differences between pure BC and BC incorporated with 0.2% chitosan. This variation is primarily attributed to the low concentration of chitosan in the bamboo dust-based medium, as noted by Phisalaphong and Jatupaiboon. ³²

The FTIR spectra for both 0.6% and 0.2% chitosan-incorporated BC (Figure 4(a) and (b)) and pure BC (Figure 4(c)) exhibit notable transmittance bands at similar wavenumbers. The structural similarities between chitosan and cellulose result in overlapping spectral features. For instance, the band at 3336 cm⁻¹ signifies O-H stretching in the glucose units of pure BC (Figure 4(c)), while a comparable band in the chitosan-incorporated samples indicates N-H₂ stretching in glucosamine units. This reflects changes in molecular orientation and the replacement of hydroxyl groups with amino groups in the cellulose crystal structure, as described by Geng et al. ⁶¹

OPEN IN VIEWER

Figure 4.

FTIR spectrum of 0.6% chitosan treated BC membrane (a), 0.2% chitosan treated BC membrane (b), and pure BC membrane (c).

In the FTIR spectra of the chitosan-incorporated BC (Figure 4(a) and (b)), the transmittance bands at 2852 cm⁻¹, characteristic of C-H stretching in BC, exhibit a shift due to the emergence of a new band at 2922 cm⁻¹. This shift is attributed to the incorporation of chitosan into the BC polymer chain, according to Dara et al., ⁶² Pasaribu et al., ⁶³ and Riaz et al. ⁶⁴ The presence of chitosan in bacterial cellulose is further supported by additional transmittance bands at 1745 cm⁻¹, 1650 cm⁻¹, 1560 cm⁻¹, and 1375 cm⁻¹, which are indicative of amino groups. These findings align with those reported by Phisalaphong and Jatupaiboon, ³² Dara et al., ⁶² Pasaribu et al., ⁶³ and Riaz et al. ⁶⁴ Cabañas-Romero et al. ⁶⁵ also observed that the transmittance band of chitosan powder at 1599 cm⁻¹, associated with the amide group, shifts to 1560 cm⁻¹ (Amide-II) in chitosan-incorporated BC, reflecting interactions between chitosan’s amino groups and cellulose’s hydroxyl groups. Similarly, the transmittance bands for pure BC at 1640 cm⁻¹ and 1370 cm⁻¹ shift to 1650 cm⁻¹ (Amide-I) and 1375 cm⁻¹ (Amide-III) in the chitosan-incorporated BC spectra (Figure 4(a)), confirming the presence of amino groups and intermolecular bonding between chitosan and bacterial cellulose. Similar results were previously reported by Pasaribu et al., ⁶³ Riaz et al., ⁶⁴ Cabañas-Romero et al., ⁶⁵ and Ul-Islam et al. ⁶⁶

The FTIR spectrum of pure BC (Figure 4(c)) highlights several characteristic transmittance bands: O-H stretching at 3336 cm⁻¹, as noted by Zhang et al. ⁶⁷ ; C-H stretching at 2900 cm⁻¹, reported by Zhang et al. ⁶⁷ ; O-H bending of absorbed water at 1640 cm⁻¹, as described by de Olyveira et al. ⁶⁸ ; C-H asymmetric deformation at 1427 cm⁻¹, C-H bending at 1370 cm⁻¹, reported by Kruer-Zerhusen et al. ⁶⁹ ; C-O-C stretching at 1157 cm⁻¹, and 1109 cm⁻¹, related to C-O stretching between β-(1→4) D-glucose unit linkages, as indicated by Vazquez et al. ⁷⁰ The band at 1028 cm⁻¹ is attributed to the C-O group of secondary alcohols and ether functions within the bacterial cellulose chain. In the fingerprint region, the band at 896 cm⁻¹ indicates β-(1→4) D-glucose unit linkages, confirming the presence of pure cellulose in the obtained BC, as consistent with the findings of Zhang et al., ⁶⁷ de Olyveira et al., ⁶⁸ Kruer-Zerhusen et al., ⁶⁹ and Vazquez et al. ⁷⁰

The observed shifts in transmittance bands and changes in band intensity, particularly in the fingerprint region, are attributed to the initial treatment with 0.1 N NaOH, used to remove bacterial cells from the BC and the chitosan-incorporated BC samples, as discussed by Phisalaphong and Jatupaiboon. ³² FTIR analysis not only elucidates the presence of functional groups but also enables the assessment of crystalline and amorphous regions within the cellulose matrix. The cellulose crystallinity index (CI) was calculated using the ratio of transmittance/absorbance band areas, such as the 3350/1337 cm⁻¹ or 1427/895 cm⁻¹ peaks. Additionally, the total crystallinity index was derived from the area of the transmittance bands at 1370/2900 cm⁻¹ for pure BC and 1375/2922 cm⁻¹ for chitosan-incorporated BC using Origin-Pro 2024 software. The crystallinity indices for pure bacterial cellulose, 0.2% chitosan-incorporated BC, and 0.6% chitosan-incorporated BC were found to be 71.7%, 67.72%, and 61.6%, respectively. The relatively lower crystallinity index for 0.2% chitosan-incorporated BC is attributed to the formation of strong hydrogen bonds between amino groups in low molecular weight chitosan and the hydroxyl groups in cellulose during in situ BC synthesis. This interaction enhances the bond between chitosan and bacterial cellulose, leading to modifications in the molecular arrangement of cellulose and a reduction in overall crystallinity, as demonstrated by Kim et al. ⁹ and Vazquez et al. ⁷⁰

XRD analysis

Cellulose is classified into various polymorphs, such as cellulose I, II, III, and IV, each characterized by distinct crystal morphologies that can undergo transformation depending on extraction methods and environmental conditions. Cellulose I include two main crystalline forms: cellulose Iα and cellulose Iβ, identified by major diffraction peaks (2θ) at 14.9°, 16.5°, and 22.7°, corresponding to the reflection planes 110, 110, and 200, respectively. ⁷¹ Chitosan, which shares structural similarities with cellulose, exhibits diffraction peaks that are analogous to those of cellulose. ⁹

In this study, the crystal morphology of bacterial cellulose (BC) produced from bamboo waste material was characterized using XRD analysis. The XRD patterns of pure BC, 0.6% chitosan-treated BC, and 0.2% chitosan-treated BC membranes are shown in Figure 5. The XRD pattern of pure BC (Figure 5(a)) reveals three distinct crystalline peaks (2θ) at 14.73°, 16.64°, and 22.59°, corresponding to cellulose Iα and cellulose Iβ. These findings are in agreement with those reported by Dara et al., ⁶² Vazquez et al., ⁷⁰ and Lin and Dufresne. ⁷¹

OPEN IN VIEWER

Figure 5.

XRD graph of pure BC (a), 0.6% chitosan treated BC membrane (b), and 0.2% chitosan treated BC membrane (c).

The XRD patterns of 0.6% chitosan-treated BC (Figure 5(b)) and 0.2% chitosan-treated BC (Figure 5(c)) show three crystalline peaks at 14.68°, 16.43°, and 22.55° for 0.6% chitosan, and at 14.71°, 16.48°, and 22.57° for 0.2% chitosan. These peaks indicate the incorporation of chitosan into the cellulose crystal morphology, which can be attributed to the similar molecular structure of chitosan. However, the intensity of crystalline peaks in chitosan-treated BC is lower compared to pure BC. This reduction is likely due to changes in molecular orientation and the substitution of hydroxyl groups by amino groups in the cellulose crystal structure, as described by Kim et al. ⁹ and Geng et al. ⁶¹

For crystallinity analysis, diffraction peaks of pure BC and chitosan-incorporated BC were selected after baseline correction. A new diffraction peak (2θ) at 19.5°, associated with an amorphous peak, was utilized to determine the crystallinity of pure BC and chitosan-incorporated BC membranes, similar to the approach used by Ul-Islam et al. ⁶⁶ Gaussian function analysis, as detailed by Mohite and Patil, ⁵¹ was employed to determine the area and full width at half maximum (FWHM) of crystalline and amorphous peaks. The crystal sizes and d-spacings were recorded as 7.8 and 0.50 nm for pure BC, 8.4 and 0.51 nm for 0.2% chitosan-treated BC, and 9.2 and 0.51 nm for 0.6% chitosan-treated BC.

The crystallinity of pure BC was measured at 72%, while chitosan-treated BC exhibited reduced crystallinities of 62% for 0.6% chitosan-treated BC and 68% for 0.2% chitosan-treated BC, as shown in Table 3. This reduction in crystallinity upon chitosan incorporation is attributed to the disruption of crystalline regions by chitosan, which intercalates between cellulose chains, altering the molecular orientation and packing of cellulose molecules during in situ synthesis. This observation is consistent with the findings of Kim et al. ⁹ and Vazquez et al. ⁷⁰

OPEN IN VIEWER

Table 3.

X ray diffraction analysis.

Model	Gaussian
Equation	y = y0 + A/(w*sqrt(pi/(4*ln(2)))) *exp(-4*ln(2)*(x-xc)^2/w^2)
Sample ID	2θ	FWHM (full width half maxima)	Crystallite size (10−9)	D-spacing (10−9)	Average crystallite size (10−9)	Average D-spacing (10−9)	Crystallinity %
(a)	14.73	1.12	7.47	0.60
	16.64	0.87	9.63	0.53	7.87	0.50	72
	22.59	1.30	6.51	0.39
(b)	14.68	1.03	8.12	0.60
	16.43	0.70	11.97	0.54	9.19	0.51	62
	22.55	1.13	7.48	0.39
(c)	14.71	1.09	7.67	0.60
	16.48	0.82	10.22	0.54	8.40	0.51	68
	22.57	1.15	7.35	0.39

Depending on the concentration of low molecular weight chitosan, the crystal size of cellulose may increase or decrease. At lower concentrations, chitosan may enhance cellulose chain alignment, resulting in larger crystallites. Conversely, at higher concentrations, excessive intercalation may disrupt crystalline regions, further reducing crystallinity and potentially increasing crystal size due to restricted movement of BC molecular chains. Similar observations were reported by Kim et al. ⁹ and Vazquez et al. ⁷⁰

Mechanical test

The mechanical properties of bacterial cellulose (BC) and 0.2% chitosan-incorporated BC membranes were evaluated using tensile strength and elongation measurements. These samples were derived from an in situ synthesis process utilizing bamboo waste. The average tensile strength and elongation values were recorded from 10 samples for both pure BC and 0.2% chitosan-incorporated BC membranes. The pure BC membrane showed a tensile strength of 1.2 ± 0.04 MPa and an elongation of 8.6 ± 0.03%. In contrast, the 0.2% chitosan-incorporated BC membrane demonstrated a reduced tensile strength of 0.8 ± 0.06 MPa and elongation of 5.3 ± 0.04%. The observed tensile strength of bacterial cellulose and chitosan-incorporated BC can be attributed to the high crystallinity and high production yield, which contribute to increased thickness and strength, as reported by Zhang et al. ³³ According to Pasaribu et al., ⁶³ the reduction in tensile strength noted in the chitosan-incorporated BC membrane is likely due to changes in crystallinity and molecular orientation caused by the substitution of hydroxyl groups with amino groups.

Chitosan, a biopolymer with functional groups such as amino and hydroxyl groups, interacts with the hydroxyl groups in cellulose to form additional hydrogen bonds. These interactions are believed to increase the stiffness of the chitosan-incorporated BC, which is evidenced by the observed decrease in elongation, similarly confirm by Phisalaphong and Jatupaiboon. ³² Despite the reduction in both elongation and tensile strength, the 0.2% chitosan-incorporated BC membrane remains cost-effective and provides adequate mechanical strength. According to Lin et al., ⁶⁰ this makes it a suitable biopolymer for applications in wound care, where its properties can offer beneficial attributes.