INTRODUCTION:

Cellulose is the predominant biopolymer that occurs naturally and is found in a wide range of creatures, such as animals, plants, and certain microorganisms. The innovative biopolymer has gained widespread application due to its remarkable mechanical qualities, beneficial environmental impact, low density, biodegradability, and abundant availability from bioresources.

Although cellulose offers numerous advantages, it is limited in its usage due to its high-water absorption capacity and inadequate interfacial adhesion ¹. Cellulose is recognized as the major component of plant biomass than lignin and hemicellulose. It is extensively utilized in the textile and food sectors. Bacterial cellulose (BC) is a variant form of cellulose that is synthesized by bacteria ².

Bacterial cellulose is an additional polysaccharide synthesised by different bacterial species, including Acetobacter (now known as Gluconacetobacter), Rhizobium, Sarcina, and Agrobacterium³. On the other hand, natural polysaccharide types were recorded in several studies of biological activity, medical and pharmaceutical applications^4-6.BC is a polysaccharide made up of glucose units that are linked together through β-(1-4)-glycosidic linkages, resulting in the formation of glucan chains with the chemical formula [(C₆H₁₀O₅)n]^7-8, The gelatinous mat at the air-liquid interface during acetic acid fermentation was initially observed by Brown in 1886⁹ originating from Acetobacterxylinus^10-11.

Gluconoacetobacter hansenii biosynthesis of BC is mediated by a 9217 kb long operon that consists of four subunits; namely A, B, C, and D, preceded by two more genes reportedly essential for cellulose synthesis; cmcax that encodes endo-β-1,4-glucanase enzyme which hydrolyses BC and improves its synthesis, and ccpAx that is believed to play an important role in extracellular transport of BC ¹². The successful production of BC in the E. coli (DH5α) platform for the first time and compare the BC productivity with the typically used E. coli BL21 (DE3) expression strain ¹³.

Microbial cellulose is regarded as a primary source of cellulose, often produced by bacteria through synthesis. Due to its exceptional purity and unique physicochemical properties, this substance is extensively utilized in many industries, serving as a thickening agent and food stabiliser¹⁴, electric conductors ¹⁵, artificial skin ¹⁶, food packaging ¹⁷, artificial blood vessels or tissue engineering ¹⁸, biomaterial for manufacturing cosmetics ¹⁹, and preparation of optically transparent films ²⁰.In recent decades, as the awareness of environmental sustainability has grown, there has been a rising demand for "going green". This has led to a search for new biobased materials that offer both high performance and affordability. BC has multiple uses in environmentally friendly composite materials ²¹.The productivity of BC must be addressed to ensure its economic viability.

Thus, it is crucial to improve the production of BC by using several strategies for process optimisation. Various research has concentrated on improving the elements of culture and medium parameters to improve the production of BC ²². The One Variable-at-a-Time (OVAT) strategy is a traditional approach employed to optimise BC. Statistical experimental designs, commonly referred to as statistical optimisation, provide a more reliable technique to optimising processes and have shown to be more efficient than the OVAT method ²³. Statistical experimental designs provide a systematic and efficient method for conducting experiments to achieve certain objectives. They enable the examination of numerous parameters simultaneously. Statistical optimisation enables rapid evaluation of extensive experimental accuracy and domains to assess the influence of each component and their interconnections. The Taguchi and Plackett–Burman Designs are effective methods for optimising agricultural conditions ²⁴. The primary benefit of PBD is its capacity to optimize experimental design, resulting in a cost-effective and more efficient procedure by minimising the number of tests required and minimizing material expenses. The Taguchi and Plackett–Burman Designs can be employed to determine the optimal amounts of culture conditions that have been identified as the most significant while minimizing the number of tests required^25-27. Recently, statistical designs have been used to determine the most crucial characteristics that affect BC synthesis, as well as the ideal quantities of these important ingredients ²⁸.

Bacterial cellulose acquired new activity in nano form and also when conjugated with nanoparticles ²⁹. Studies confirmed also that biosynthesized materials are usually safer and more selective, especially in nano form^30-31.

This work extensively illustrated the impact of culture conditions on the production of bacterial cellulose (BC) by Gluconacetobacter xylinus NRRL B-469, using experimental design methodologies. The Taguchi Designs and Plackett–Burman were mostly used to screen many components, including Mannitol, Fructose, Starch, Sucrose, Glucose, Molasses, Yeast, Peptone, CSL, Citric acid, Na₂HPO₄, volume, incubation period, pH, and temperature to optimize the important parameters. In addition, XRD, SEM, and FTIR analyses were employed to analyse the characteristics of BC.

MATERIAL AND METHOD:

Microorganism:

The Gluconacetobacter xylinus NRRL B-469 strain utilized in this investigation was acquired from the Agricultural Research Service (ARS) Culture Collection and acknowledged as a producer of bacterial cellulose (BC). The culture was preserved on Mannitol liquid slants, moved, and kept at 4°C in the refrigerator for subsequent analysis.

Production of BC on mannitol medium:

The composition of the Mannitol medium is as follows (g/L): A solution containing mannitol (25.0g), yeast extract (5.0g), and peptone (3.0g) was made. The solution was then autoclaved at 121°C for 20 minutes and inoculated with Gluconacetobacter xylinus NRRL B-469. The mixture was incubated under static conditions at 30°C for 6 days ^32-33.

Treatment of sugar cane molasses:

The sugar cane molasses utilized in this investigation was provided by a local company, namely the Sugar and Integrated Industries Corporation, Egypt. The concentrated sugar cane molasses was mixed with distilled water at a ratio of 2 parts molasses to 1 part water by weight/volume. The mixture was then acidified to a pH of 3.0 using 6 M H₂SO₄. Subsequently, the molasses was subjected to a temperature of 60◦C for 1 hour. Subsequently, the pH was modified to pH 1.0 and consistently heated at a temperature of 6◦C for 2 hours. The molasses solution underwent centrifugation at a force of 6000 times the acceleration due to gravity for 20 minutes to isolate solid solids. Before sterilizing the molasses, the solution was brought to a pH of 7.0 by adding 10 M NaOH. The treatment was referred to as the H₂SO₄–heat treatment, and the liquid portion was called H₂SO₄–heat-treated molasses.

Investigation of several factors influencing BC production using the Plackett-Burman design:

The Plackett-Burman design was utilized to determine the key elements that have a substantial impact on the manufacturing of BC. The Plackett-Burman design is capable of efficiently screening a large number of variables and effectively identifying non-effective factors by examining the impacts of each variable without the need for a large number of tests ^24,34-35. The Plackett-Burman design was employed in this experiment to assess the significance of multiple factors on BC production. Gluconacetobacter xylinus NRRL B-469 Eleven specific variables (Mannitol, Fructose, Starch, Sucrose, Glucose, H₂SO₄–heat-treated molasses, Yeast, Peptone, Corn Steep Liquor (CSL), Citric acid, and Na₂HPO₄) were utilised and examined in 32 experiments to assess the effectiveness and precision of the Plackett-Burman design in optimising the quantity of bacterial cellulose (BC) under the conditions of pH 6, volume of 50 mL, incubation period of 6 days, and temperature of 30^oC. The independent variable was manipulated at two levels: the greatest level (1) and the lowest level (-1), as shown in Table 1. The Minitab programme (version 18, developed by Minitab Inc. at State College, PA, USA) was utilized. The studies were carried out using 250 ml Erlenmeyer flasks, each containing 50 ml of medium. The experiments were conducted three times and the mean value was computed. The BC production was determined by measuring the dry weight of each run. The Plackett-Burman screening design relies on the first-order model equation 1 as follow: Y = β₀ + Σ β_iX_i (1)

In this model, Y represents the response variable, which is the dry weight of BC in grammes per litre. β₀ is the intercept of the model, βi is the estimate of the variable, and X_i is the variable itself. The importance of variables was assessed by computing the p-value using standard regression analysis.

Table 1: Factors utilized to determine the medium composition in the Plackett-Burman design

Serial	Factors	Level 1	Level 2
1	A: Mannitol (g/L)	0	25
2	B: Fructose (g/L)	0	20
3	C: Starch (g/L)	0	20
4	D: Sucrose (g/L)	0	20
5	E: Glucose (g/L)	0	20
6	F: Molasses (mL)	0	110
7	G: Yeast (g/L)	0	5
8	H: Peptone (g/L)	0	5
9	J: CSL (ml)	0	80
10	K: Citric acid (g/L)	0	1.15
11	L: Na₂HPO₄(g/L)	0	2.7

The orthogonal array:

An L-27 (3⁹) standard orthogonal array was created to investigate nine factors: Mannitol, H₂SO₄–heat-treated molasses, CSL, Citric acid, Na₂HPO₄, Volume, Incubation period, PH, and Temperature. These factors were chosen based on the findings of a previously conducted Plackett-Burman design. The L-27 symbolic array of the experimental matrix denotes a set of 27 experimental trials, each with a layout of 3⁹. The three tiers of the nine factors were designated as levels 1, 2, and 3, as shown in Table 2. The total degrees of freedom (df) for the OA L-27 set was 26, which is calculated by subtracting one from the number of runs. Experiments were conducted by setting specific levels for the elements involved, and the interaction between several factors was examined by crossing them ^35-36.

Table (2): The Taguchi design was used to determine the optimal values of selected fermentation variables for maximizing the weight of BC.

Serial No.	Factors	Levels 1	Level 2	Level 3
1	A: Mannitol (g/L)	20	25	30
2	B: H₂SO₄–heat-treated molasses (mL)	70	110	200
3	C: CSL (mL)	40	80	120
4	D: Citric acid (g/L)	0.5	1.15	2.5
5	E: Na₂HPO₄(g/L)	1.0	2.7	5
6	F: Volume (mL)	25	50	100
7	G: Incubation period (hours)	72	144	246
8	H: PH	5	6	8
9	J: Temperature (^oC)	25	30	35

The purification of BC:

According to Atykyan et al.; 2020 with small modifications the obtained pellicle was collected and subsequently rinsed three times with distilled water to eliminate any remnants of the growing media³⁷. Afterwards, the BC pellicle was immersed in a 0.5% NaOH solution for 24 hours. This was done to eliminate any microbiological contaminants and other impurities that may have been absorbed into the BC membranes. Subsequently, the pellicle was rinsed with distilled water 3-4 times until the pH of the rinsed liquid reached a neutral level. Next, the purified BC was subjected to air drying at room temperature for the duration of one night, following which the weight of the BC in its dry state was obtained.

Analysis of BC membrane properties:

Scanning Electron Microscope (SEM):

The morphological composition of the BC membrane was analysed through observation using a scanning electron microscope (JEOLJSM 6360 LA, Japan). The dried BC membrane was prepared for SEM imaging by mounting it and applying a thin layer of gold nanoparticles as a coating. The SEM experiment was performed using an accelerated voltage of 15 kilovolts (kV) and a magnification of 5000 times (X).

Fourier Transform Infrared (FTIR) Spectroscopy:

FTIR Spectroscopy was employed to examine the functional groups and chemical bonds present in the dried BC membrane. The BC FTIR spectra were analysed using modified protocols based on the methodology described by Atykyan et al.; in 2020³⁷. The dehydrated BC pellicle sheet was cut to dimensions of 5 mm×5 mm in preparation for transmission measurements using an FTIR spectrometer (Nicolet 6700, Thermo Scientific TM, Waltham, MA, USA). The spectra were obtained within the spectral range of 4000 - 400 cm^-1, with a resolution of 6 cm^-1.

X-ray diffractometry (XRD):

The crystallinity of the dried BC membrane was assessed by analysing XRD patterns obtained from an X-ray. The X-ray diffraction analysis was performed following the methodology of Bandyopadhyay et al. (2018) with certain alterations³⁸. The X-ray diffraction patterns of BC sheets were obtained using a diffractometer (Rigaku, Mini Flex II, Tokyo, Japan) with an operating voltage of 40 kV and a current of 40 mA. The diffractograms were obtained by measuring the diffraction angles from 0 to 60 degrees on a 2θ scale, with a precision of 0.02 degrees for each step.

RESULTS AND DISCUSSION:

Measurement of BC production in Mannitol Medium using Gluconacetobacter xylinus NRRL B-469:

To assess the production capability of Gluconacetobacter xylinus NRRL B-469 in terms of BC (bacterial cellulose), the dry weight of BC production was examined under static culture conditions using a conventional mannitol medium. Figure 1 illustrates the BC that occurs at different stages, such as production in culture media, harvesting, purification, and drying.

(A) (B)

Figure 1: BC generated by Gluconacetobacter xylinus NRRL B-469, developed. Purification (A) and the optimized medium (B).

Utilizing multifactorial statistical methods to optimize bacterial cellulose production:

1- Plackett-Burman design of experiment.:

The Plackett-Burman design is a widely utilised method for screening the parameters that influence the generation of bioactive chemicals.Bilgi et al.; 2016 and Hegde et al.; 2013utilized this methodology to identify the most influential factors that improve the output of BC while reducing production costs^39-40. Typically, the factors that have the greatest impact on enhancing BC production differ depending on the strain of bacteria and the content of the growth medium. A resolution IV, 2-level Plackett-Burman design was used to assess the impact of 11 independent factors on the production of BC by Gluconacetobacter xylinus NRRL B-469. The experiment consisted of 32 runs, and the results, including the generated runs and the average production in grammes per litre for each run, are summarised in Table 3. The Gluconacetobacter xylinus NRRL B-469 strain exhibited significant variability in the production of BC across the 32 runs of the Plackett-Burman design experiment. The range of variance was between 1.5 and 9.5 g/L BC. The highest BC yield, measuring 9.5 g/l, was achieved during run 2. This run involved using Mannitol at 25 g/L, H₂SO₄–heat-treated molasses at a volume of 110 ml, CSL at a volume of 80 ml, Citric acid at 1.15 g/L, and Na₂HPO₄ at 2.7 g/L. The fermentation process took place at a temperature of 30^oC for 144 hours. The lowest yield of BC, which was 1.5 g/l, was achieved during run 6. Run 6 involved using a mixture of mannitol (25 g/l), starch (20 g/l), yeast (5 g/l), peptone (5 g/l), and citric acid (1.15 g/l). The fermentation process was carried out at a temperature of 30^oC for 144 hours. In their study,Hegde et al. (2013) found that the optimal parameters for producing BC by Gluconacetobacter persimmonis were glucose, yeast extract, and peptone while using a standard HS medium⁴⁰. Also, Zeng et al. (2011) and Wang et al. (2018) identified the key factors that significantly influence the synthesis of bacterial cellulose (BC) by Acetobacter xylinum BPR 2001^41-42. These factors include the concentration of maple syrup, the duration of incubation, the size of the inoculum, and the speed of shaking. The researchers used a fructose-based medium for their experiments. Furthermore, in their study,Bilgi et al.; 2016found that the protein concentration, incubation length, and inoculum size were the most influential factors in the generation of BC by Gluconacetobacter xylinus³⁷. They used the Carob-haricot bean medium for their experiments.

A- Statistical significance and analysis of variance (ANOVA) for the Plackett-Burman design:

To assess the importance of the model, the adjusted R2, the coefficients R2, and the anticipated R2 were computed. The estimated R2 values were determined to be 84.52%, suggesting that the model can effectively account for the data in the Plackett-Burman design. The modified R2 and Predicted R2 values were 76.00% and 60.36%, respectively, indicating that the models utilized effectively explain the collected data, as reported by Othman et al. in 2017⁴³.An analysis of variance was conducted to identify the significant factors influencing BC production. The probability of error associated with each variable is indicated by the p-value. A p-value less than 0.05 indicates a significant effect on the model, while a p-value greater than 0.05 indicates an insignificant effect. The ANOVA results from the Plackett-Burman design are summarized in Table 4. The study revealed that Mannitol, Sucrose, H₂SO₄-heat-treated molasses, CSL, and the interaction among mannitol, H₂SO₄-heat-treated molasses, and CSL had a significant impact (p-value < 0.05) on BC production. Conversely, the remaining factors and their interactions exhibited p-values > 0.05. The Pareto chart in Figure 2 confirms that factors F (H₂SO₄–heat-treated molasses), J (CSL), and A (Mannitol) have significant effects on BC production compared to the other tested factors (B Fructose, C Starch, D Sucrose, E Glucose, G Yeast, H Peptone, K Citric acid, and L Na₂HPO₄). These results are supported by the standardized effects.

Table 3: Factors used to Determine the Medium Composition in Plackett-Burman Design for the production of BC by Gluconacetobacterxylinus NRRL B-469

Run No.	Factors No.												Response
Run No.	A: Mannitol (g/L)	B: Fructose (g/L)	C: Starch (g/L)	D: Sucrose (g/L)	E: Glucose (g/L)	F: H₂SO₄–heat-treated molasses (mL)	G: Yeast (g/L)	H: Peptone (g/L)	J: CSL (mL)	K: Citric acid (g/L)	L: Na₂HPO₄ (g/L)	Mean BC (g/l)
1	0	0	0	0	0	0	0	0	0	0	0	0
2	25	0	0	0	0	110	0	0	80	1.15	2.7	9.5
3	0	20	0	0	0	110	5	0	0	0	2.7	3.5
4	25	20	0	0	0	0	5	0	80	1.15	2.7	6.5
5	0	0	20	0	0	110	5	5	80	0	0	9
6	25	0	20	0	0	0	5	5	0	1.15	0	1.5
7	0	20	20	0	0	0	0	5	80	0	2.7	2
8	25	20	20	0	0	110	0	5	0	1.15	2.7	6.5
9	0	0	0	20	0	0	5	5	80	1.15	2.7	3.5
10	25	0	0	20	0	110	5	5	0	0	2.7	7
11	0	20	0	20	0	110	0	5	80	1.15	0	8.5
12	25	20	0	20	0	0	0	5	0	0	0	4.5
13	0	0	20	20	0	110	0	0	0	1.15	2.7	6.5
14	25	0	20	20	0	0	0	0	80	0	2.7	5.5
15	0	20	20	20	0	0	5	0	0	1.15	0	2.5
16	25	20	20	20	0	110	5	0	80	0	0	9.5
17	0	0	0	0	20	0	0	5	0	1.15	2.7	3.5
18	25	0	0	0	20	110	0	5	80	0	2.7	9.5
19	0	20	0	0	20	110	5	5	0	1.15	0	2.5
20	25	20	0	0	20	0	5	5	80	0	0	6.5
21	0	0	20	0	20	110	5	0	80	1.15	2.7	8.5
22	25	0	20	0	20	0	5	0	0	0	2.7	3.5
23	0	20	20	0	20	0	0	0	80	1.15	0	1.5
24	25	20	20	0	20	110	0	0	0	0	0	6.5
25	0	0	0	20	20	0	5	0	80	0	0	2.5
26	25	0	0	20	20	110	5	0	0	1.15	0	7.5
27	0	20	0	20	20	110	0	0	80	0	2.7	9
28	25	20	0	20	20	0	0	0	0	1.15	2.7	4.5
29	0	0	20	20	20	110	0	5	0	0	0	6.5
30	25	0	20	20	20	0	0	5	80	1.15	0	6.5
31	0	20	20	20	20	0	5	5	0	0	2.7	3.5
32	25	20	20	20	20	110	5	5	80	1.15	2.7	9.5

Table 4: ANOVA Analysis of Variance of Plackett-Burman Design for Media Composition

Source	DF	Adj SS	Adj MS	F-Value	P-Value
Model	19	226.210	11.906	8.42	0.000
Linear	11	205.522	18.684	13.21	0.000
Mannitol (g/L)	1	31.008	31.008	21.93	0.001
Fructose(g/L)	1	0.383	0.383	0.27	0.612
Starch(g/L)	1	0.008	0.008	0.01	0.942
Sucrose(g/L)	1	8.508	8.508	6.02	0.030
Glucose(g/L)	1	0.945	0.945	0.67	0.430
Molasses(ml)	1	118.195	118.195	83.59	0.000
Yeast(g/L)	1	0.383	0.383	0.27	0.612
Peptone(g/L)	1	0.383	0.383	0.27	0.612
CSL(ml)	1	43.945	43.945	31.08	0.000
Citric acid(g/L)	1	0.008	0.008	0.01	0.942
Na2HPO4(g/L)	1	1.758	1.758	1.24	0.287
2-Way Interactions	7	12.180	1.740	1.23	0.359
Mannitol (g/L) *Glucose(g/L)	1	0.070	0.070	0.05	0.827
Mannitol (g/L) *Molasses(ml)	1	2.258	2.258	1.60	0.230
Mannitol (g/L) *CSL (ml)	1	0.945	0.945	0.67	0.429
Fructose(g/L) *Peptone(g/L)	1	0.383	0.383	0.27	0.612
Starch(g/L) *Peptone(g/L)	1	0.070	0.070	0.05	0.827
Molasses(ml)*CSL (ml)	1	7.508	7.508	5.31	0.040
Yeast(g/L)*Peptone(g/L)	1	0.945	0.945	0.67	0.430
3-Way Interactions	1	8.508	8.508	6.02	0.030
Mannitol (g/L) Molasses(ml)CSL (ml)	1	8.508	8.508	6.02	0.030
Error	12	16.969	1.414
Total	31	243.179

Table 5: Experimental setup for maximization of the BC weight using Taguchi’s L₂₇ (3⁹) orthogonal array designfor the production of BC by GluconacetobacterxylinusNRRL B-469

Run No.	A: Mannitol (g/L)	B: H₂SO₄–heat-treated molasses (ml)	C: CSL (mL)	D: Citric acid (g/L)	E: Na₂HPO₄ (g/L)	F: Volume (mL)	G: Incubation period (h)	H: PH	J: Temp ̊C	Mean BC (g/L)	Predicted values of BC (g/L)
1	20	70	40	0.5	1	25	72	5	25	2.5	3.3
2	20	70	40	0.5	2.7	50	144	6	30	3	2.316
3	20	70	40	0.5	5	100	246	8	35	0.75	0.55
4	20	110	80	1.15	1	25	72	6	30	5.5	5.3
5	20	110	80	1.15	2.7	50	144	8	35	0.8	1.68
6	20	110	80	1.15	5	100	246	5	25	15.5	14.81
7	20	200	120	2.5	1	25	72	8	35	0.55	0
8	20	200	120	2.5	2.7	50	144	5	25	7.5	7.3
9	20	200	120	2.5	5	100	246	6	30	11	11.883
10	25	70	80	2.5	1	50	246	5	30	9.5	9.8
11	25	70	80	2.5	2.7	100	72	6	35	6.5	6.1833
12	25	70	80	2.5	5	25	144	8	25	0.8	0.816
13	25	110	120	0.5	1	50	246	6	35	16.5	16.51
14	25	110	120	0.5	2.7	100	72	8	25	0.6	0.9
15	25	110	120	0.5	5	25	144	5	30	17	16.68
16	25	200	40	1.15	1	50	246	8	25	0.75	0.433
17	25	200	40	1.15	2.7	100	72	5	30	6	6.01
18	25	200	40	1.15	5	25	144	6	35	8.5	8.8
19	30	70	120	1.15	1	100	144	5	35	7	7.66
20	30	70	120	1.15	2.7	25	246	6	25	5	4.9
21	30	70	120	1.15	5	50	72	8	30	0.5	0
22	30	110	40	2.5	1	100	144	6	25	8.5	7.93
23	30	110	40	2.5	2.7	25	246	8	30	0.85	1.51
24	30	110	40	2.5	5	50	72	5	35	4.5	4.4
25	30	200	80	0.5	1	100	144	8	30	0.8	0.7
26	30	200	80	0.5	2.7	25	246	5	35	6.5	5.93
27	30	200	80	0.5	5	50	72	6	25	4	4.66

Table 6: Response table for the SNR

Level	Mannitol (g/L)	H₂SO₄–heat-treated molasses (ml)	CSL (ml)	Citric acid (g/L)	Na₂HPO₄ (g/L)	Volume (ml)	Incubation period (h)	PH	Temp ̊ C
1	9.293	8.005	8.399	9.331	10.152	9.611	6.776	17.075	9.212
2	12.094	12.271	10.596	10.178	8.903	9.302	11.005	16.418	10.478
3	8.835	9.945	11.226	10.712	11.166	11.308	12.440	-3.271	10.531
Delta	3.258	4.266	2.826	1.381	2.263	2.007	5.664	20.346	1.319
Rank	4	3	5	8	6	7	2	1	9

*Larger S/N ratio is better, Bold values are the highest S/N ratio values

Figure 3: Experimental results mean compared to the model-predicted values for each run

4 5

Figure 4; and 5: Main effects plot for SNR for the BC production; Main effects plot for SNR for the interaction between the incubation period and H₂SO₄–heat-treated molasses for the BC production

Table 7: Analysis of Variance for SN Ratios

Source	DF	Seq SS	Adj SS	Adj MS	F	P
Mannitol (g/L)	2	56.02	56.02	28.01	15.64	0.013
Molasses (ml)	2	82.12	82.12	41.06	22.93	0.006
CSL (ml)	2	39.63	39.63	19.82	11.06	0.023
Citric acid (g/L)	2	8.73	8.73	4.36	2.44	0.203
Na 2 HPO 4 (g/L)	2	23.13	23.13	11.57	6.46	0.056
Volume (ml)	2	21.01	21.01	10.50	5.86	0.065
Incubation period (h)	2	156.06	156.06	78.03	43.56	0.002
PH	2	2406.13	2406.13	1203.07	671.70	0.000
Temp ̊C	2	10.03	10.03	5.02	2.80	0.174
Molasses(ml)*Incubation period (h)	4	17.65	17.65	4.41	2.46	0.202
Residual Error	4	7.16	7.16	1.79
Total	26	2827.68

Table 8. Response table for the means

Level	Mannitol (g/L)	H₂SO₄–heat-treated molasses (mL)	CSL (ml)	Citric acid(g/L)	Na₂HPO₄ (g/L)	Volume (ml)	Incubation period (h)	PH	Temp ̊C
1	5.2333	3.9500	3.9278	5.7389	5.7333	5.2444	3.4056	8.4444	5.0167
2	7.3500	7.7500	5.5444	5.5056	4.0833	5.2278	5.9889	7.6111	6.0167
3	4.1833	5.0667	7.2944	5.5222	6.9500	6.2944	7.3722	0.7111	5.7333
Delta	3.1667	3.8000	3.3667	0.2333	2.8667	1.0667	3.9667	7.7333	1.0000
Rank	5	3	4	9	6	7	2	1	8

6 7

Figure 6 and 7. Main effects plot for means of the BC Production; Main effects plot for Means for the interaction between the incubation period (h) and H₂SO₄–heat-treated molasses for the BC production

Table 9. Analysis of Variance for Means

Source	DF	Seq SS	Adj SS	Adj MS	F	P
Mannitol (g/L)	2	46.832	46.832	23.416	13.86	0.016
H₂SO₄–heat-treated molasses (ml)	2	68.662	68.662	34.331	20.31	0.008
CSL (ml)	2	51.032	51.032	25.516	15.10	0.014
Citric acid (g/L)	2	0.305	0.305	0.153	0.09	0.916
Na₂HPO₄ (g/L)	2	37.262	37.262	18.631	11.02	0.024
Volume (ml)	2	6.722	6.722	3.361	1.99	0.251
Incubation period (h)	2	72.965	72.965	36.482	21.59	0.007
PH	2	324.327	324.327	162.163	95.95	0.000
Temp ̊C	2	4.782	4.782	2.391	1.41	0.343
Molasses (ml)* Incubation period (h)	4	31.420	31.420	7.855	4.65	0.083
Residual Error	4	6.760	6.760	1.690
Total	26	651.067

C- Model significance and ANOVA for the means:

The model's fitness was assessed by calculating R² and Adjusted R², which yielded values of 98.96% and 93.25% respectively. These values indicate the high level of fitness and dependability of the model. An ANOVA study was conducted to determine the relevance of factors influencing the means of BC. Table 9 presents the ANOVA results for the components and their interactions. The analysis revealed that Mannitol, H2SO4–heat-treated molasses, CSL, Na₂HPO₄, incubation duration, and pH all have a statistically significant effect (p-Value < 0.05) on the BC means.

D- Regression model:

To accurately match the experimental data and determine the parallel components of the model, a regression model was conducted at a 95% confidence level, using the optimal lambda value (estimated lambda = -0.0653093, rounded lambda = 0). The regression equation is defined as follows:

Ln (Response g/L) = 4.75 + 0.0060 Mannitol (g/L) + 0.0077 Temp ̊C + 0.00155 H₂SO₄–heat treated molasses + 0.00467 CSL (ml) + 0.063 Citric acid (g/L) + 0.0444 Na₂HPO₄(g/L) + 0.00190 Volume (ml) + 0.00368 Incubation period (h) - 0.8325 PH - 0.000006 Molasses*Incubation period (h) (4)

The regression model predicts a yield of 18.04 g/L under the optimal conditions determined through means optimisation using Taguchi design. This prediction is very close to Taguchi's prediction of 18.07 g/L and significantly higher than the experimental result of 17 g/L. These findings confirm the accuracy of the regression model.

Characterization of bacterial cellulose:

Scanning electron microscopy:

Figure 8 illustrates the properties of the BC pellicle that were produced in an optimised medium with the use of Plackett-Burman and Taguchi Design. The BC membrane derived from Gluconacetobacterxylinus NRRL B-469 exhibits a uniformly dispersed, homogenous tightly packed network of randomly oriented cellulose ribbons, comparable to those often observed in BC obtained from the seam strain ^47-⁴⁸. The distinctive arrangement implied that certain boundary requirements could be advantageous for some applications. In addition, BC displays fibrils that are slightly narrower and have a more compact structure, with measurements ranging from 73.9 to 161.0 nm. These qualities are similar to BC produced from other waste materials used as feedstock ^49,50. Therefore, the size of BCs is mostly determined by the cultural conditions rather than the origin of the feedstock in a static society.

Figure 8: Scanning electron microscope of BC produced in optimization medium by Gluconacetobacter xylinus NRRL B-469

XRD analysis:

The XRD pattern of the dried BC obtained after incubation for 144 hours in the optimization medium showed three relatively intense peaks at 2θ angles of 14.72°, 17.01°, and 22.78°. These peaks corresponded to the (100), (010), and (110) crystallographic planes, respectively, as determined by triclinic indexation (Figure 9). These findings are consistent with the results of other investigations ^51-53. Additionally, BC derived from GluconacetobacterxylinusNRRL B-469 exhibits the characteristic structure of cellulose I, with both amorphous and crystalline sections. This discovery is consistent with prior research on the topic ⁵⁴. The increased crystallinity of the BC membrane produced from Gluconacetobacter xylinus NRRL B-469 is evident. As per the methodology described by certain writers^55-57-65, the crystallinity index (CI) is determined by dividing the intensity of the main peak by the count numbers of the neighbouring minima.

FT-IR analysis:

Specific functional groups were identified in the BC membrane via FT-IR analysis, which also disclosed its chemical composition ⁵⁵. The Gluconacetobacter xylinus NRRL B-469 derived BC membrane Fourier-transform infrared (FT-IR) spectra were examined in Figure 10 over the wavenumber range of 500 to 4000 cm^-1. The prominent absorption band observed at 3345.2 cm^–1 in the BC spectrum is attributable due to the hydroxyl group (OH) being present in type I cellulose. This band is significant for understanding the hydrogen-bonding patterns, which align with certain literature sources [Lin et al.; 2016, Gündüz and Aşık, 2018, and Lazarini, 2018], The intense absorption peak at 2896.8 cm^–1 was also associated with the stretching of CH bonds, which is consistent with findings from previous research^{52,
54, 58-65}. Due to the presence of the carboxyl functional group (C=O), the cellulose absorption spectra display a prominent peak at 1638 cm⁻¹⁵⁸. The band at 1389.6 cm⁻¹ corresponds to the asymmetric angular deformation of C-H bonds. In secondary and primary alcohols, the elongation of C-O-C and C-O-H bonds is associated with the spectral range between 1055.6 cm⁻¹ and 1066.9 cm⁻¹, respectively. The Fourier-transform infrared (FT-IR) characteristics of bacterial cellulose (BC) derived from Gluconacetobacter xylinus NRRL B-469 in this investigation align with previous research employing the same strain but under varying circumstances ^52-53.

Figure 9: and 10: XRD analysis of BC membrane obtained from Gluconacetobacterxylinus NRRL B-469; FT-IR spectrum of BC membrane obtained from GluconacetobacterxylinusNRRL B-469.

CONCLUSIONS:

The utilization of experimental techniques, such as the Taguchi method and Plackett–Burman design, can serve as a valuable means to enhance the synthesis of bacterial cellulose pellicle. The modified Mannitol medium could be a good formula for cultivating Gluconacetobacter xylinus NRRL B-469 in order to produce the film. An enlarged vessel basal area can lead to a higher production rate of BC in static culture. The productivity of Gluconacetobacter xylinus NRRL B-469 BC was enhanced through the utilization of Plackett-Burman and Taguchi designs, resulting in a yield of 18.04 g/l under the most favourable production circumstances. The solution consists of 25 g/L of mannitol, 110 mL of heat-processed molasses treated with sulfuric acid, 120 mL of CSL (corn steep liquor), 0.5 g/L of citric acid, 5 g/L of Na₂HPO₄, with a total volume of 100 mL. The incubation duration is 246 hours, with a pH of 5 and a temperature of 30°C. The characterisation of dried BC revealed a unique structure, suggesting that it could be a promising material for specific applications.

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Received on 28.05.2024 Revised on 17.09.2024

Accepted on 11.11.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):6050-6062.

DOI: 10.52711/0974-360X.2024.00918

S	R-sq	R-sq(adj)	R-sq(pred)
1.18913	93.02%	81.97%	50.38%