Co-Cultivation of Komagataeibacter xylinus MS2530 with Different Yeast Strains. Production and Characterization of Bacterial Cellulose Films

1. Introduction

Bacterial cellulose (BC) is a natural polymer that has excellent biocompatibility, high strength, purity, porosity, high water absorption capacity, non-immunogenicity, ease of production and modification [1,2,3]. Plant cellulose mainly consists of cellulose, hemicellulose and lignin [4,5]. The most well-known method of producing cellulose is the extraction of cellulose from plants, but alkaline or acidic treatment is used to re-move lignin and hemicellulose. Unlike plant-based cellulose, BC is synthesized in its pure form, without residues of other plant molecules [6,7,8,9]. Due to its high purity and excellent physical and chemical properties, it is widely used in various fields such as food industry, biomedicine, cosmetology, pharmaceuticals, papermaking, electronic components, textiles and environmental protection. [10,11,12,13].

It is also used to form polymers and bio-base d nanocomposites [14,15,16]. The structure of bacterial and plant cellulose is very similar, but BC has several unique characteristics - high crystallinity, Young's modulus, tensile strength, thermal stability, as well as elas-ticity and porosity [17,18].

The porous structure and large specific surface area increase the water-holding capacity of BC, as well as the ability to form strong bonds with other biomaterials, enzymes, nanoparticles, etc. [19,20]. Its well-organized fibrous network structure allows it to en-capsulate nanoparticles, acting as a matrix [20,21].

BC is synthesized as an exopolysaccharide by aerobic bacteria such as Achromobacter, Alcaligenes, Aerobacter, Agrobacterium, Azotobacter, Komagataeibacter, Pseudomo-nas, etc. [22,23,24]. The efficiency of BC synthesis is high, since it is produced by micro-organisms that have a short growth cycle, fast metabolism, and the ability to reproduce quickly.

The relatively large diversity of cellulose-producing microorganisms and the wide range of cultivation methods provide an excellent opportunity to modify and adjust the properties of the material and find new areas of its application [25]. The most studied BC-synthesizing bacterium belongs to the genus Komagataeibacter.

It is capable of metabolizing a wide range of carbon/nitrogen sources and is a source of BC with higher productivity [25,26,27,28].

BC is obtained by static and dynamic fermentation, depending on the application of the obtained BC. Both methods have their negative sides. Thus, in the case of static fer-mentation, although this mode is the most widely used for obtaining biofilms, there is a problem with the low level of dissolved oxygen in the fermentation medium, the dura-tion of the fermentation process and the massiveness of the cultivation structures. Fermentation in the dynamic mode increases the content of dissolved oxygen in the fermentation medium by shaking or mixing, but in this case there is a possibility of spontaneous mutations in bacterial cells, which can lead to a loss of the ability to synthesize BC.

Considering the uniqueness of BC, its high cost greatly limits its large-scale industrial production, since its production requires a fermentation medium rich in glucose and other nutrients, which increases the price of BC by almost 30%–65% [29,30].

The duration of the fermentation process and the low yield of BC also affect the increase in cost, so the choice of a cheap and accessible medium, as well as a highly effective producer strain are the most important prerequisites for organizing inexpensive large-scale production of BC. Growing strains - producers of BC on various wastes of the agricultural and food industries corresponds to the principles of a green and sustainable economy.

Regardless of the medium used for fermentation (HS or a medium containing agricul-tural or food industry waste), the yield of BC depends on the producer strain and fer-mentation conditions (pH, dissolved oxygen, temperature, process duration, etc.) [31,32,33,34].

Despite the presence of many useful properties, the high cost of BC production limits its use. [30,35]. Studies on reducing the production cost emphasize the use of wastes as carbon or nitrogen sources [36.], therefore, their use as substrates can yield high BC concentrations when the cultivation conditions are optimized. Many wastes, such as wastewater and agro-industrial waste, whey, have been investigated as alternative substrates for BC production.

Significant global environmental and economic challenges have brought to the forefront the affordable and sustainable disposal of various industrial wastes, such as waste from the agro-industry, dairy industry, brewing and soft drink industry, textile mills, micro-algae industry, etc. [37,38,39,40].

Using these industrial wastes for the production of BC helps prevent environ-mental pollution and reduces the costs of industrial waste disposal [41].

Waste from food, agricultural and brewing industries, which are a rich source of car-bon, proteins and microelements, have been investigated as an alternative medium for the production of BC due to their low cost [42,43,44,45,46].

It has now been established that the production of BC from industrial waste has comparable yield, physical, physicochemical, crystalline and mechanical properties compared to standard media. Compared to BC obtained on the HS medium, BC obtained on the spent medium did not have significant changes in the microstructure, physico-chemical structural characteristics, FTIR peaks, crystallinity index. [32,47].

Revin et al. [34] investigated the use of dairy and alcohol industry waste for the cost-effective production of BC using Gluconacetobacter sucrofermentans, which produced up to 5.45 g/L over a three-day cultivation period.

Wu et al. using wastewater from rice wine factories over a seven-day cultivation period of Gluconacetobacter xylinus produced 6.26 g/L BC [48].

Furthermore, using the hydrolysate obtained from waste fiber textiles as a culture medium for BC production, Hong et al. obtained 83% higher yield (10.8 g/L) and 79% higher tensile strength (0.070 MPa) of BC compared to production using glu-cose-based HS medium [49].

Zhao et al. obtained 1.177 g/L BC using fermented wastewater as a substrate, which is much lower than that in HS medium (1.757 g/L) [50]. However, considering the low cost of the production process, the BC yield was sufficient to support large-scale commercial application.

Agriculture in Armenia is one of the main and most developed branches of the economy, therefore, the problem of proper disposal of agricultural waste is very acute for the protection of the environment.

Residual brewer's yeast (BSY) is the second largest by-product of the brewing process [51]. This sludge residue accounts for almost 15% of the total amount of by-products formed during the brewing process [52]. BSY can be used as a nutrient medium for the production of BC, as they are rich in proteins, essential amino acids, polyphenolic compounds and B vitamins (mainly B1, B2, B3, B6, B8) [53]. BSY has been shown to contain a large amount of carbon (45-47% of dry yeast matter) [54], and is also an important source of some valuable saccharides (mono-, di- and oligosaccharides).

In recent years, the co-culture fermentation strategy, which promotes the synthesis of metabolites through microbial interactions, has attracted increasing atten-tion in industrial fermentation production. [55,56]. Scientists have investigated the ef-fects of co-cultivation of

BC-producing strains and other strains, including bacteria and fungi, on BC production and properties. Co-cultivation of Gluconacetobacter xylinus st-60-12 and Lactobacillus mali st-20 resulted in three-fold higher BC production than monoculture of G. xylinus st-60-12, but the BC characteristics were not analyzed. Liu and Catchmark [57] found that compared with the monoculture of Gluconacetobacter hansenii ATCC 23769, the co-culture of G. hansenii ATCC 23769 and Escherichia coli ATCC 700728 resulted in a 10.8% increase in BC yield and improved BC mechanical properties. Hu, Catchmark, and Demirci found that the co-culture of Komagataeibacter hansenii ATCC 23769 and Aureobasidium pullulans ATCC 201253 im-proved BC mechanical properties but did not affect BC production . Notably, different strain combinations had different effects on BC yield and properties [58].

In co-cultivation, different strains are grown simultaneously in the same nu-trient medium, which means that during growth, the strains can metabolically influence each other [59,60].

Several different symbiotic processes of co-cultivation of acetic acid bacteria with strains of yeast and fungi have been described in the literature [61,62,63].

It is assumed that co-cultivation of yeast and acetic acid bacteria can increase the yield of BC, however, studies to support this claim are still limited. The symbiotic mechanism between acetic acid bacteria and yeast is unclear. It is supposed that yeast breaks down sucrose into reducing sugars, which are then metabolized by yeast and acetic acid bacteria into ethanol and organic acids. Glucose, the building block for BC biosynthesis, is also used by the acetic acid bacteria to produce BC.

Our study is aimed at optimizing fermentation media and fermentation conditions for the K. xylinus MS2530 strain. To increase the yield of bacterial cellulose, a co-fermentation method was used with various yeast strains on brewery waste. The use of this method not only led to an increase in the yield of BC, which has a low cost price (no additional waste treatment is required during use, and the duration of the fermentation process is shortened), but also contributed to the utilization of agricultural waste, thereby reducing environmental pollution.

2. Materials and Methods

2.1. Microorganisms

The strain Komagataeibacter xylinus MS2530 (obtained by us using the method of chemical mutagenesis based on the strain Komagataeibacter xylinus VKPM B-12429) was used in the work. The strains of yeasts Kluyveromyces marxianus (Hansen 1888) van der Wait 1971 MDC 10081, Pichia fermentans Lodder 1932 MDC10169, Pichia pastoris MDC 10178, Candida stellata (Kroemer & Krubholz 1931) Myer & Yarrow 1978 MDC 10139, Candida stellata (Kroemer & Krubholz 1931) Myer & Yarrow 1978 MDC 10280, Candida kefyr (Beijerinck 1889) van Uden & Buckley 1970 MDC 10228, Candida kefyr (Beijerinck 1889) van Uden & Buckley 1970 MDC 10035 were provided by the Microbial Depository Center of the SPC “Armbiotechnology”.

2.2. BC Films and BC Modified Films Obtaining and Purification

2.2.1. BC Films and BC Modified Films Obtaining

To obtain the inoculum, the strains were grown in 250- or 500-ml Erlenmeyer flasks containing 100–200 ml of HS medium of the following composition, g/l: glucose – 20.0; peptone – 5.0; yeast extract – 5.0; sodium hydrogen phosphate (Na₂HPO₄) – 2.7; citric acid (C₆H₈O₇) – 1.15, The seed material was grown on a shaker-incubator at 100–250 rpm for 24 h at a temperature of 26–32 °C, pH 4–7.5. The wash from Petri dishes with agar nutrient medium HS (agar – 20.0 g/l) was also used as the inoculum of the strains. The obtained inoculates in the amount of 5–10% of the medium volume were added to flasks containing 50–300 ml of the nutrient medium. The strains were cultured on liquid media HS of various compositions and residual brewer's yeast (BSY) under static conditions for 5-7 days at a temperature of 26–32 °C, pH 4-7.5.

To obtain BC composites by the co-cultivation method, inoculates of the K. xylinus MS2530 strain and yeast strains in the amount of 5 and 10% of the medium volume in the 1:1 ratio was seeded into flasks containing 50-300 ml of HS or BSY nutrient medium. The strains were cultivated under static conditions for 5-7 days at a temperature of 30 °C, pH 5.5

Scheme 1. BC films and BC modified films obtaining, purification, analysis and characterization.

Scheme 1. BC films and BC modified films obtaining, purification, analysis and characterization.

2.2.2. Purification of BC Films and BC Modified Films

After culturing, the BC films and modified BC films were washed with distilled water to remove cells and media components. They were then immersed in 0.5% NaOH at room temperature for 24 hours to completely remove any bacterial cells that might have attached to the BC films. If necessary, the BC films were treated with 0.5% HCl for 24 hours to remove yellowing. To ensure complete removal of the alkali, the films were washed several times with deionized water, leaving the film at neutral pH [20].

2.3. The Yield of BC and BC Productivity Were Calculated as Described by Jacek et al. [64].

The BC yield (g/L) and BC productivity (g/(L⋅day)) for different fermentation processes were calculated using Eqs. (1) and (2):

$$\mathrm{B}\mathrm{C} \mathrm{y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d}(\mathrm{g}/\mathrm{L})={\displaystyle \frac{\mathrm{B}\mathrm{C} \mathrm{w}\mathrm{e}\mathrm{t} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\left(\mathrm{g}\right)}{\mathrm{V}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{u}\mathrm{l}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e} \mathrm{m}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{u}\mathrm{m}\left(\mathrm{L}\right)}}$$

(1)

$$\mathrm{B}\mathrm{C} \mathrm{p}\mathrm{r}\mathrm{o}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{g}/(\mathrm{L}\cdot \mathrm{d}\mathrm{a}\mathrm{y}\left)\right)={\displaystyle \frac{\mathrm{B}\mathrm{C} \mathrm{y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d}(\mathrm{g}/\mathrm{L})}{\mathrm{t}\mathrm{m}-\mathrm{t}\mathrm{o}\left(\mathrm{d}\mathrm{a}\mathrm{y}\right)}}$$

(2)

where t₀ is the start time of the cultivation, and t_m is the time when the maximum production of BC was obtained.

2.4. The Reducing Sugar Content in the Culture Was Determined as Described by Lin et al. [37]

2.5. Water Content and Rehydration Degree of BC Films and BC Modified Films

The purified BC films and BC modified films samples were shaken briefly twice and weighed. The weight of the wet samples was recorded as W₁. The samples were then freeze-dried for 24-48 hours, and the weight of the dry samples was recorded as W₂. Distilled water was then added to the dry samples and incubated for 48 hours at room temperature, weighed again, and recorded as W₀.

Water content and rehydration degree are calculated by the following formula:

$$\mathrm{W}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\left(\mathrm{\%}\right)={\displaystyle \frac{{\mathrm{W}}_1\left(\mathrm{g}\right)-{\mathrm{W}}_2\left(\mathrm{g}\right)}{{\mathrm{W}}_1\left(\mathrm{g}\right)}}\times 100\mathrm{\%}$$

(2)

$$\mathrm{R}\mathrm{e}\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{d}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{e}\mathrm{e}\left(\mathrm{\%}\right)={\displaystyle \frac{{\mathrm{W}}_0\left(\mathrm{g}\right)}{{\mathrm{W}}_1\left(\mathrm{g}\right)}}\times 100\mathrm{\%}$$

(3)

2.6. BC Films and BC Modified Films Characterizations

Prior to the analysis, the BC films and BC modified films films were freeze-dried in an ALPHA 1-2/LD freeze dryer for 24-48 h.

2.6.1. Fourier Transformed Infrared (FT-IR) Spectroscopy

FTIR spectroscopy was used to determine the functional groups and chemical bonds present in the BC films and BC modified films .

A freeze-dried films of BC films and BC modified films were analyzed by IRTracer-100 FTIR Spectrophotometer (Shimadzu, Kyoto, Japan) using KBr prism (7800–350 cm-1) with single reflection, at resolution of 4 cm-1. IR spectral data were collected on the 500–4000 cm-1 range.

2.6.2. Scanning Electron Microscopy (SEM)

The microstructure of the surface of the freeze-dried BC films and BC modified films was analyzed using scanning electron microscopy ((JEOL JCM-7000, JEOL Ltd., Tokyo, Japan). Prior to SEM observation, the freeze-dried samples were fixed and coated with a thin layer of gold nanoparticles under high-vacuum conditions. SEM experiments were imaged at a 10,000 × magnification at an accelerating voltage of 10 kV. Fiber diameters were measured by analyzing SEM images using the ImageJ program. The diameters of 100 individual ultrafine fibers were estimated in 10 fields of SEM in triplicate.

2.7. Mechanical Characterization of BC Films

The tensile strength (MPa) and elongation at break (%) of the BC (10 mm×10 cm) were measured in uniaxial mode by using the Instron bursting machine (model 3365, Norwood, MA, USA). The mechanical properties of each sample were the average values determined for three specimens.

2.8. Statistical Analysis

All experiments were performed in triplicate and are presented as mean ± standard deviation and statistically processed using RStudio software (version 1.4.1106). Student's t-tests were performed to identify significant differences between bacterial nanocellulose synthesized in SH and Residual brewer's yeast (BSY) medium. Data were analyzed using analysis of variance (ANOVA) with Bonferroni's multiple comparison test. Differences were considered statistically significant at p < 0.05.

3. Results

It is known that the yield of BC and its structure depend not only on the producer, cultivation modes (static or dynamic), but also on the fermentation conditions. This paper presents the results of a study of BC synthesis by the strain K. Xylinus MS2530 obtained by us both on media containing various sugars as a carbon source and on brewing waste. The microstructure, physicochemical and mechanical properties of BC and BC composites obtained by co-fermentation with yeast strains were also studied.

3.1. Study of the Influence of Various Factors on BC Biosynthesis During Fermentation

The studies showed that the most important factors influencing the increase in BC yield were the concentration of the added inoculum, temperature, pH and incubation duration (Figure 1).

According to the obtained results (using ANOVA), it was shown that all the above factors had a significant impact on the increase in BC yield, but the greatest effect was exerted by the inoculum concentration in the medium (Figure 2). Similar results were obtained by Volova et al. [20]. As a result of the research, the optimal values ​​were selected for each parameter: temperature 30^oC, pH 5.5, inoculum concentration – 10% and incubation time – 7 days. The maximum yield of BC (wet weight) under optimal parameters was 12.6 g/l; fermentation was carried out in opaque containers with a wide surface (area 600 sm²) containing 500 ml of medium.

3.2. Cultivation of K. xylinus MC2530 Strain Using Different Carbon Sources

The effect of different sugars as a carbon source on the BC yield during cultivation of K. xylinus MS2530 strain was studied. During seven days of cultivation in 500 ml Erlenmeyer flasks at 30 °C and an initial pH of 5.5, a high BC yield was obtained on standard HS medium with glucose (12.6 g/l); almost the same yield was observed on HS media containing sucrose (11.9 g/l) and fructose (11.75 g/l). Maltose and mannose did not support the growth of this strain. Similar results were obtained by Volova et al. [20].

The studies showed that the BC yield depended on the carbon source (Figure 3a, b).

3.3. Use of Brewing Waste as a Cheap and Accessible Medium for Obtaining BC

The most important task for the biotechnological production of BC is the use of industrial waste. This will significantly reduce the cost price and increase the availability of this unique biopolymer, the demand for which is constantly growing. Tons of industrial waste are generated daily, and the use of some of them in the production of BC will make it possible to eliminate or reduce the economic and environmental burden caused by industrial waste. Currently, to reduce the cost of BC biosynthesis, agro-industrial waste is used [6,34,65].

The strain K. xylinus MS2530 obtained by us was grown on HS and BSY media under optimal conditions (t = 30 ^oC, pH 5.5) for 7 days. Comparative analysis showed that on the medium with waste the same film thickness (5 mm) was formed within 5 days, and on the classical medium (HS) a film of similar thickness was formed in 7 days. In addition, the BSY medium was used without sterilization, which significantly reduces the costs of obtaining BC in industrial conditions (Figure 4).

3.4. Co-Cultivation of K. xylinus MS2530 Strain with Yeast Strains

Various modification methods are used for the purpose of functionalization and specific applications of BC biofilms. One of these methods is co-fermentation with various cultures, which not only increases the BC yield, but also changes the microstructure and key physical properties. The co-cultivation method, although simple, is very effective. Co-fermentation was carried out with five yeast strains on HS medium (Figure 5). As can be seen from the figure, co-fermentation increased the BC yield compared to the K. xylinus MS2530 strain. The highest BC yield was observed during co-fermentation of K. xylinus MS2530 with the yeast strain Kluyveromyces marxianus MDC 10081.

Co-fermentation of K. xylinus MS2530 with yeast strains was also carried out on BSY medium (non-sterile) under static fermentation conditions (t = 30 oC, pH 5.5, inoculum volume 10%) for 7 days. As can be seen from Figure 6, on the fifth day of fermentation, the biofilm formed as a result of co-cultivation of the K. xylinus MS2530 strain with the K. marxianus MDC 10081 yeast strain was significantly thicker than the biofilm obtained during fermentation of the K. xylinus MS2530 strain.

Table 1 shows the data on the highest BC yield during co-cultivation of K. xylinus MS2530 with yeast strains compared to the K. xylinus MS2530 strain. The table shows that both on the HS and BSY mediums, the highest BC yield was observed in the case of co-fermentation of the producer strain K. xylinus MS2530 with the K. marxianus MDC 10081 strain.

As can be seen from the data presented in Table 1, the use of BSY as a medium had a positive effect on the BC yield compared to the HS medium.

Thus, using brewing waste as a nutrient source for the production of BC will help to reduce production costs and environmental footprint.

3.5. Study of the Structure and Properties of BC Obtained by the K.xylinus MS2530 Strain, as well as by Co-fermentation of K.xylinus MS2530 with Yeast Strains

All biofilms obtained by fermentation of the K. xylinus MS2530 strain, as well as by co-fermentation with yeast strains on the HS medium, were transparent with a whitish tint, and the films obtained on the BSY medium were distinguished by yellowness, which is associated with the composition of the waste on which they were grown. The color of the obtained yellowish films is removed during the purification process.

The morphological structure of the purified bacterial films was studied using SEM imaging.

The unique properties of BC are determined by both the ultrastructure and the size of the fibers.

As can be seen in Figure 7, the ultrastructure of the films and the thickness of the BC fibers obtained on HS and BSY media, as well as the biofilms obtained by co-fermentation with yeast on similar media, are quite different.

The SEM study proved that as a result of purification, the remains of the culture liquid and bacterial cells were completely removed, which is consistent with the literature data [66].

As can be seen from Figure 7, BC obtained on the BSY medium has a more compact structure, compared to the films obtained on the HS medium. Only during co-fermentation with Pichiya pastoris MDC 10178, the porosity of the BC obtained on the BSY medium increased greatly. This fact requires further research, since this property of BC, which has a lowcost price, can subsequently be used for its application in biomedicine, pharmacology and cosmetology.

All BC films obtained on two different media differed in size and average fiber diameter. Calculations showed that the films obtained on the HS medium had an average fiber diameter of 42 nm, while the films obtained on the BSY medium had a lower diameter of about 38 nm.

The greatest advantage of the co-cultivation method is the ability of the yeast strain to participate in the regulation of the culture medium, including both the components of the medium and the cultivation method [67], thereby modifying the structure and changing the properties of BC for use in specific purposes.

3.6. Determination of the chemical structure of synthesized BC films using FT-IR spectroscopy

FT-IR spectra were studied to determine the chemical structure of the synthesized BC. FT-IR spectra were recorded in the wavenumber range from 500 to 4000 cm⁻¹.

Figure 8 shows the FT-IR spectra for BC synthesized by the K. xylinus strain MS2530, as well as BC modified films obtained by co-fermentation of K. xylinus MS2530 with yeast strains on the classical HS and BSY media (Figure 8 (a, b). The strong absorption band in the spectrum of 3336-3348 cm⁻¹ in all the studied BC films was due to the presence of the hydroxyl group (-OH) , which is consistent with the literature data of Volova et al., Feng et al. [20,68].

The next strong absorption band at 2854-2920 cm⁻¹ was attributed to the presence of C-H stretching vibrations (Figure 8a, b). Such data were obtained by Bai et al. [15]. The absorption band within 1647-1654 cm⁻¹ is due to the presence of the carboxyl (C = O) functional group (Figure 8a,b).

The bands were also observed at 1427-1454 cm⁻¹ (C-H bending vibration), 1315 cm⁻¹ (C-H bond bending vibration). The peak that appeared at 1056 cm⁻¹ is a characteristic peak of the stretching vibration of C-O-C and C-O bonds of cellulose (Figure 8 a,b), which correlates with the data of Zhantlessova et al. and Feng et al. [68,69]. The absorption band at 898-902 cm⁻¹ corresponds to the first carbon atom involved in the formation of the β-glycosidic bond. The analysis of the FT-IR band data is largely consistent with the literature data [20].

The observed shifts of some absorption bands in BC obtained by the K. xylinus MS2530 strain on HS and BSY media indicate a change in the O-H and C-H ratios, which is also consistent with literature data [68]. This is especially noticeable in BC synthesized by cofermentation of K. xylinus MS2530 with Pichiya pastoris MDC 10178 on BSY medium (BC-BSY) (Figure 8b). This correlates with the SEM.

The presence of shifts in some absorption bands in BC suggests the possibility of a significant influence of co-fermentation and the use of BSY as a culture medium.

It is also important to note that the use of waste for the production of BC will significantly ease the burden on waste disposal, thereby promoting the emergence of green industry, and will also reduce environmental pollution [70].

3.7. Physicomechanical Parameters of BC Obtained from the K.xylinusMS2530 Producer Strain

Mechanical properties are an important factor in assessing the applicability of BC as wound dressings, tissue engineering and other biomedical fields.The mechanical and surface properties of BC are primarily determined by the BC producer strain, fermentation conditions, microstructure and drying method.

Tensile strength significantly depends on the moisture content of the BC sample.

Thus, the BC films directly studied after fermentation and purification, with a humidity of almost 100%, had a Young's modulus of 9.86±0.29 MPa, a tensile strength of 0.69±0.31 MPa and an elongation at break of 4.95±1.39%. For films with a humidity of 50–55%, the Young's modulus was 45.7±1.02 MPa, respectively, the tensile strength of lyophilized samples is very low.

Table 2 presents the physical properties of BC samples from K. xylinus MS2530 in the form of a hydrogel and after lyophilization drying.

The results obtained correlate with the results obtained by Volova et al. [20].

4. Conclusions

The synthesis of BC obtained (as a result of chemical mutagenesis) by the acetic acid bacteria strain K.xylinus MS2530 on media containing various carbohydrate sources was studied. The strain is capable of assimilating various sugars, but the highest yield of BC was observed on the HS medium with glucose. As a result of improving the technology for obtaining BC in laboratory conditions (optimization of the fermentation medium composition, cultivation conditions), it was possible to significantly reduce the fermentation time from 14 to 5-7 days. The effect of the amount of inoculum introduced on the yield of BC was also demonstrated. Based on the high cost and low productivity of BC, waste from the brewing industry was used as a cheaper and more accessible medium, which was used without additional processing, which also significantly reduces the cost of BC. As a result, the yield of BC was almost doubled. In addition, the disposal of waste from the agro-industrial complex leads to a decrease in environmental pollution. To further increase the yield and improve the most important properties of BC, co-fermentation of the K.xylinus MS2530 strain with various yeast strains was used.

As a result of symbiotic growth of BC, the yield increased by almost 3.5 times in some variants, and by almost 5 times on the BSY medium. The resulting biofilms and biofilms obtained as a result of co-fermentation were studied using SEM, FTIR, etc. According to FTIR, all the obtained biofilms had characteristic peaks of typical functional groups of cellulose. Studies of BC samples and biofilms obtained as a result of co-fermentation using the SEM method showed significant changes in the ultrastructure, which will allow them to be used for various purposes and industries