{"paper_id":"20cf8607-419e-4a89-9dd0-97479f7aab0e","body_text":"Synthesis, Chemical, Morphological and Thermal Characterization Yeast Immobilized Bacterial Cellulose and It Use in Color Removal | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis, Chemical, Morphological and Thermal Characterization Yeast Immobilized Bacterial Cellulose and It Use in Color Removal Filiz BORAN This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1768385/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bacterial cellulose is a biomaterial and can be used for many applications such as medical products and food ingredients with its superior properties such as being pure, high crystallinity and durability. It can also be used as an as an immobilization support. It is a good and practical support for yeast immobilization. The present study aimed to immobilize yeast on lyophilized bacterial cellulose produced by Gluconacetobacter xylinus B759 in cheese whey medium as a cheap and natural culture medium. Optimum conditions such as cultivation time, carbon source, temperature, pH and nitrogen sources for bacterial cellulose production in cheese whey medium were determined. The highest bacterial cellulose yield obtained under optimized conditions was 13.18 g/L. Saccharomyces cerevisiae cells were immobilized on purified and lyophilized 0.5 cm pure bacterial cellulose. Bacterial cellulose and yeast-immobilized bacterial cellulose structures were characterized by different instrumental techniques. Furthermore, Reactive Blue 171 dye decolorization ability of immobilized yeast was tested. The structure of bacterial cellulose showed very suitable morphology and chemical structure for different biotechnological applications. The use of a cheap and natural medium is very important in terms of production conditions, as it is an environmentally friendly application in terms of biotechnology. Bacterial cellulose Cheese whey Dye decolorization Immobilization Yeast Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Cellulose, the most abundant biopolymer in nature, is mostly found in plants but some bacteria can also produce this polymer (Lynd et al. 2002 ). Plant cellulose is not pure and it is associated with lignin, pectin, hemicellulose and other substances. Therefore, additional purification processes are required to obtain pure cellulose (Cacicedo et al. 2016 ; Islam et al. 2017 ). Various chemicals must be used during the purification process of plant cellulose. These chemicals cause environmental pollution and also damage the structure of natural cellulose (Çakar et al. 2014 ). On the other hand, bacterial cellulose (BC) is pure and therefore, there is no need for hard chemicals to obtain the BC in pure form. Many bacterial species belonging to genera of Gluconacetobacter ( formerly Acetobacter), Achromobacter, Agrobacterium, Aerobacter, Azotobacter, Rhizobium, Pseudomonas, Sarcina, Salmonella, Enterobacter, Escherichia and etc. are reported as BC producers (Hungund et al. 2013 ; Huang et al. 2014 ; Ullah et al. 2016 , 2017 ). Gram-negative bacterium Acetobacter xylinum (now known as Gluconacetobacter xylinus or Komagataeibacter xylinus ) could synthesize abundant cellulose (Karahan et al. 2011 ; Żywicka et al. 2019 ). G. xylinus is the most studied bacterium in BC production due to its high BC production capacity. The effect of different sugars and other compounds are tested as carbon sources to induce the BC production (Gama et al. 2012; Ullah et al. 2016 ). BC obtained from this species is one of the most preferred classes of biopolymer recently (Ross et al. 1991 ; Żywicka et al. 2019 ). It has important superior properties such as high purity, high crystallinity, a high degree of polymerization, a nano-structured work, excellent mechanical strength, large surface area, higher porosity, a high water holding capacity and also good biocompatibility and biodegradability (Hungund et al. 2013 ; Yamanaka et al. 1989 ; Bagewadi et al. 2020 ). These superior properties make it as an interesting material in the important areas such as medicine, food and cosmetics etc. (Buruaga-Ramiro et al. 2020 ; Fernandes et al. 2020 ; Hungund et al. 2013 ). The usability of BC as an immobilization agent for many industrially important microorganisms such as yeasts is being tested and these studies reveal that it can be an effective immobilization agent. Saccharomyces cerevisiae is the most preferred and studied model organism in molecular and cell biology and in many industrial processes (Żywicka et al. 2016 , 2019 ; Johnson and Echavarri-Erasun 2011 ). BC is commonly produced in Hestrin-Schramm (HS) medium (Çakar et al. 2014 ). However, this is an expensive medium (Ghozali et al. 2021 ). The cost of the production is an important problem affecting industrial use of BC (Çoban and Biyik 2011 ). The use of agro-industrial wastes or by-products as the growth media can make the industrial production process more economical (Shi et al 2014 ). Therefore, cheese whey can be used as an inexpensive and natural media (Battad-Bernardo et al. 2004 ). Thus, the industrial scale production of the BC, which becomes difficult due to its high cost, can also be facilitated by using cheaper substrates (Carreira et al. 2011 ; Çoban and Biyik 2011 ). Cheese whey, by-product of cheese production, is a natural medium with high nutritional content (Revin et al. 2018 ). It contains various compounds such as lactose, soluble proteins, lipids, mineral salts, lactic acid, citric acid, non-protein nitrogen compounds (urea and uric acid) and B group vitamins (Siso 1996 ; Prazeres et al. 2012 ). As I stated before, low cost and high efficiency BC production is very important in industrial applications. This can be achieved both by using inexpensive industrial wastes/raw materials and by optimizing the conditions and the factors such as incubation time, temperature, pH, carbon and nitrogen sources (Shi et al. 2014 ; Azerado et al. 2019). In this study, BC production ability of G. xylinus B759 in the cheese whey medium obtained from local cheese factory in Malatya, Türkiye was investigated. The effect of different cultivation parameters such as incubation time, temperature, pH and carbon and nitrogen sources on BC production efficiency of this strain was also tested. Obtained BC and yeast-immobilized BC were characterized. The usability of this BC as an immobilization support of Saccharomyces cerevisiae has also been studied. In addition, the dye decolourization ability of yeast-immobilized BC was determined. Materials And Methods Bacterial strain used for BC production Gluconacetobacter xylinus B759 was used as the bacterium for production of bacterial cellulose (BC). This bacteria was provided by Prof. Dr. Aynur Gül Karahan Çakmakçı. This bacterium was grown on the Hestrin-Schramm (HS) agar plates containing (g/L) glucose, 20; peptone, 5; yeast extract, 5; Na 2 HPO 4 , 2,7; citric acid, 1,15; agar, 15 at 30°C for 10 days under static condition and then stored at 4°C. It was subcultivated every 3–4 weeks. Culture medium and BC production The inoculum was prepared in 50 mL Hestrin-Schramm (HS) broth consisted of (g/L) glucose, 20; peptone, 5; yeast extract, 5; Na 2 HPO 4 , 2,7; citric acid, 1,15 (Hestrin and Schramm, 1954). This medium was inoculated with G. xylinus and then, the culture was incubated at 30°C for 10 days under static conditions. After incubation, 1 mL of G. xylinus liquid culture transferred into 50 mL of HS medium for obtaining stock inoculum culture. Cheese whey was chosen as the main culture medium for BC production. This natural medium was purchased from a local cheese factory (Dutpınar, Malatya/Turkiye) and it was filtered before using. Erlenmeyer flasks containing 40 mL of cheese whey were autoclaved at 121°C for 20 min. Then, the sterilized media were inoculated with G. xylinus stock inoculum culture at the rate of 2% and they were incubated at 30°C for 10 days under static conditions. After incubation, BC pellicles were filtered and washed several times with distilled water. Then, they were treated with 0.1 N NaOH at 75°C for 2 hours. After that, they were washed with distilled water a few times until neutralization. These purified and filtered pellicles were dried at 40°C for 15 min and then they were lyophilized for obtaining lyophilized BC. All lyophilized samples were weighed and the dry weight of the BC product obtained was expressed as grams per liter (g/L). All experiments were performed in three replicates. Optimization of BC production The culture conditions such as incubation time, temperature, pH, carbon source and nitrogen source are important parameters to achieve the highest BC production efficiency. To test the effect of the incubation time on BC production, G. xylinus inoculated cheese whey media were incubated for 3, 5, 7, 10 and 15 days at 30°C. After the optimal incubation time was determined, the cultures were incubated at 25, 28, 30, 32 and 35°C for 10 days for testing the optimal temperature for BC production. pH is also an another important parameter for BC production. Therefore, the ranges of pH 3.0–9.0 were also tested for BC formation. To this end, the initial pHs of the cheese whey media were adjusted to pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 and after inoculation, they were incubated at 30°C for 10 days. Glucose, sucrose and fructose were used as additional carbon sources and each were separately added into 40 mL cheese whey media at the concentrations of 0.5, 1.0 and 2.0 g. The prepared media were inoculated with bacterium and these cultures were incubated at 30°C for 10 days. The effect of yeast extract and pepton (as nitrogen sources) was also tested at the concentrations of 0.1, 0.2 and 0.4 g. Characterization of BC samples Scanning electron microscope (SEM), Fourier transform infrared spectrometer (FTIR) and X-ray diffraction (XRD) analyzes of BC pellicles were determined at Inonu University Scientific and Technological Research Center Laboratories, Malatya / Türkiye. Within the scope of the study, chemical characterizations of the produced cellulose structures were carried out using Perkin Elmer spectrum two model FTIR spectrophotometer. FTIR analyses of BC samples were scanned with a working sensitivity of 4 cm − 1 in the range of 400–4000 cm − 1 . ATR mode was used as the operating mode. For the determination of the crystallinity level of the obtained BC structures, Rigaku brand X-ray spectrophotometer was used for X-ray analysis. The obtained cellulosic films were measured in the range of 2–80 θ, with a measurement sensitivity of 0.2 θ. The surface properties and surface morphologies of the cellulose structures were examined by AFM and SEM analyses. In AFM analysis, 40 µm x 40 µm scanning area was performed using non-contact mode. AFM samples were monitored at room temperature and in Faraday cages using the Park system XE-100 AFM device. Leo-Evo 40 model scanning electron microscope was used for SEM analysis. Before the samples were analysed, they were coated with a Baltec brand sputter with 20 m Au/Pd layer. Shimadzu brand thermal analysis system was used to examine the thermal properties of prepared cellulose samples. Shimadzu TGA-50 model analyser was used for thermal stability measurements. Shimadzu DTA 50 was used to determine the decay energies. Heating rate of 10°C/min was preferred in the measurement performed against the reference material of aluminium oxide. All thermal analyses were performed in a static air atmosphere and in platinum cuvettes using 10 mg of sample. Yeast Immobilization Procedure A small amount of solid S. cerevisiae culture was inoculated into 50 mL/250 mL flask Sabouraud dextrose broth (SDB) medium and the culture was incubated at 30°C and 150 rpm for 48 hours. Lyophilized BC samples cut in 0.5 cm sizes were added into 50 mL SDB medium and it was autoclaved at 121°C for 20 minutes. Then, 1 mL from S. cerevisiae culture was inoculated into the medium containing BC samples and the culture was incubated at 30°C and 150 rpm for 48 h. At the end of the incubation, S. cerevisiae immobilized BC samples was obtained. Dye decolorization activity of immobilized S. cerevisiae The decolorization activity of immobilized yeast against Chrocion Blue H-ERD (Reactive Blue 171, RB 171) was studied. RB171 dye was used at 200 mg/L concentration. Various pieces of S. cerevisiae immobilized BC (3, 6 and 12) were transferred into 3 mL dye solution and they were incubated at 30°C under static and agitated (150 rpm) conditions for 24 h. Dye decolorization was determined spectrophotometrically (Shimadzu-UV-1601, UV/Visible) as a relative decrease of absorbance for RB171 dye at its maximum absorbance wavelength (615 nm). All experiments were performed in three replicates and dye decolorization percentages were calculated with SPSS 15.0 package program. Results And Discussion Production of BC Cheese whey is a by-product produced during cheese production and it contains lactose and other carbohydrates (glucose, galactose, lactose, and arabinose). In addition, whey has high biological value because it contains various proteins, amino acids, vitamins and organic acids (Revin et al 2018 ). Because cheese whey is a simple, inexpensive and abundant medium, it can be used as the basal culture medium for BC production. Therefore, in this study, BC was produced in cheese whey medium. The obtained BC pellicles were purified, weighed after lyophilization and its yield was calculated as g/L. In Fig. 1 (a-c), the steps of BC production in cheese whey medium could be seen. Optimization of Culture Conditions for BC Production The optimization of culture conditions is very important for production of high amount of BC pellicles. Different factors such as incubation time, temperature, pH, and carbon and nitrogen sources were tested in order to obtain the highest yield of BC. Firstly, the effect of incubation time on BC production was determined. As shown Fig. 2 a, the maximum yield was obtained at the 10th day. While 1.59 g/L BC pellicle was obtained at the 3th day, it was 3.16 g/L for 10 days of incubation at 30°C, statically. Type and amount of carbon and nitrogen sources in the culture medium are also effective on the BC production (Szymańska-Chargot et al, 2011 ). The carbon source is an essential component that enhances cell growth and metabolism during BC synthesis (Yim et al, 2017 ). Therefore, glucose, fructose and sucrose were added into cheese whey media at different concentrations (0.5, 1.0, 2.0 g/40 mL) for testing their possible effect on BC production and cultures were incubated at 30°C for 10 days (Fig. 2 b). Glucose addition positively affected the BC production activity of this bacterium. Fructose addition also induced the BC production but sucrose addition resulted in low BC production. The highest BC amount was obtained from the medium containing of 0.5 g glucose per 40 mL cheese whey as 5.62 g/L (Fig. 2 b). Therefore, 0.5 g glucose in 40 mL medium was determined as the best carbon amount for BC production. Mikkelsen et al. ( 2009 ) aimed to increase the BC cellulose yield of Gluconacetobacter xylinus strain ATCC 53524 by modifying the HS medium. For this purpose, HS media were prepared with the addition of different inducers and the BC yield was calculated at 48 and 96 hours. While the highest BC yield was 1.89 g/L at 48th hour in HS medium with an initial pH of 5.0 to which glucose was added, this value was determined as 3.10 g/L at 96th hour. Rangaswamy et al. (2005), on the other hand, tested the effect of different carbon sources on BC yield, in HS medium. It was reported that Gluconacetobacter sp produces approximately 1.35 g/L BC in HS medium containing 2% (w/v) glucose. In the study of Trovatti et al ( 2011 ) in which BC production was tested with Gluconacetobacter sacchari in HS medium containing different carbon sources such as glucose, sucrose, and fructose, the highest BC efficiency was obtained at the end of 96 hours in HS medium with glucose (2.7 g/L). Temperature is also an important factor for high product formation (Fernandes et al, 2020 ). In order to determine the effect of temperature for BC production, different temperature values at the range of 25–35°C were tested. As shown in Fig. 2 c, the optimum temperature for BC production was 30°C and the BC value obtained at this temperature was 7.63 g/L. Revin et al. ( 2018 ) tested the BC production of Gluconacetobacter sucrofermentans B-11267 in cheese whey medium (without pH adjustment) and they determined the highest BC yield as 5.45 g/L after 3 days of incubation at 28°C and 250 rpm. The maximum BC production of Acetobacter pasteurianus RSV-4 (MTCC 25117) in the whey medium was 5.6 g/L at 30°C after 8 days incubation (Kumar et al. 2021 ). On the other hand, Carreira et al. ( 2011 ) reported very low BC production 0.08 g/L in the whey medium. Initial pH is an important factor for BC production. Therefore, pHs of the cheese whey media containing 0.5 g glucose were adjusted to the range of pH 3.0–9.0 and the effect pH on BC production was tested (Fig. 2 d). As the pH increased, the amount of BC obtained also increased. As shown Fig. 2 d the highest BC amount was 13.18 g/L at pH 7.0. No BC formation observed at pH 3.0. and pH 9.0. Jozala et al. ( 2015 ) used various culture media such as HS, rotten fruit and milk whey for BC production. In their study, they tested the effect of pH on BC yield and obtained optimum BC yields at different pH values between 3.2 and 5.4 according to the culture medium they used. Nitrogen constitutes 8–14% of the dry weight of bacteria and is the main component of proteins required for cell metabolism (Chawla et al, 2009 ). However, nitrogen sources did not show any positive effect on BC production activity of the bacterium (Fig. 2 e). The optimal medium and the optimum culture conditions for high amount of BC production was determined as 10 days incubation time, 30°C temperature, 7.0 pH and 0.5 g glucose in 40 mL cheese whey medium. Immobilization of yeast cells on BC BC can be a good support for cell immobilization thanks to its high crystallinity, high water holding capacity, porous structure, better mechanical properties and biocompatibility (Żywicka et al 2019 ). As a support, BC can be used for the immobilization of various enzymes, as well as for the immobilization of industrially important microorganisms such as yeasts (Żywicka et al 2016 ; Żywicka et al 2019 ). Therefore, in this study, BC produced by G. xylinus in cheese whey medium under optimized conditions was used as a support for the immobilization of S. cerevisiae and Fig. 3 shows the microscopic images of lyophilized BC without yeast and S. cerevisiae immobilized BC samples stained with the simple staining method. Chemical, morphological and thermal characterization of pure and yeast-immobilized BC samples Figure 4 shows the Scanning electron micrographs (SEM) of the pure cellulose samples obtained from the cheese whey medium. SEM analysis of BC was performed with lyophilized BC pellicle under 20000 × and 40000 × magnifications. From the SEM images, it could be seen that the BC pellicle had a reticulated structure. The average fibril diameter of the pure cellulose sample obtained from the cheese whey medium was measured as 123.7 nm. Revin et al. ( 2018 ) reported the width of the microfibrils obtained from the whey medium as 100–180 nm. Furthermore, yeast cells immobilized on cellulose were also proven by SEM images (Fig. 5 ). Structural characterizations of pure cellulose and yeast-immobilized cellulose samples were determined by Fourier transformed infrared spectrophotometer. Obtained infrared spectra were given in Fig. 6 comparatively. In the spectrum of pure cellulose structure, a wide H bonds band of free -OH group on the cellulose units was seen in the range of 3000–3600 cm − 1 . Aliphatic C-H peaks in cellulose units were observed in the range of 2830–2950 cm − 1 . Main chain C-C stretching vibration was observed at 1580 cm − 1 . The etheric C-O-C stretching vibration in the cellulose structures was detected as a severe peak at 1057 cm − 1 . In addition, CH stretching vibration at 888 cm − 1 and CH out-of-plane bending vibration at 559 cm − 1 confirmed the obtained structure. Figure 6 shows the FTIR spectrum of the yeast-immobilized cellulose samples. Along with the binding of yeast structures, on this spectrum, a sharp peak originating from protein structures was observed at approximately 1750 cm − 1 . In addition, the effect of yeast structures on the spectrum was observed on the aliphatic methyl and H bonds peaks. Methyl bond strength increased, but H bond strength decreased. The peaks were observed more broadly. All these findings prove that the desired structure was obtained. The crystallinity of the obtained cellulose structures The degree of crystallinity of the obtained cellulose structures and the effects of yeast structures on the crystal system were examined with X-ray spectra. Obtained X-ray spectra were given in Fig. 7 . When the X-ray spectrum of the pure BC structure was examined, although it has an amorphous appearance, the crystalline peaks were clearly seen in the structure. Due to the cellulose structure, the 100 and 110 peaks, which are the main cellulose peaks, were clearly seen, especially at 15° and 22° 2θ values (Leal et al 2021 ; Salari et al 2019 ). In addition, a wide band originating from amorphous regions was observed at 19.48°. On the other hand, in the X-ray spectrum obtained from the cellulose sample with yeast structure, a more amorphous image appeared in which the crystalline structures were partially lost. This is due to the yeast covering the crystalline regions on the surface, and the change in this spectrum proves the existence of the yeast structure. In order to visualize the yeast layers on the surface in more detail, AFM images of pure cellulose and yeast-immobilized cellulose surfaces were obtained at different magnifications (Fig. 8 ). When the AFM images of the pure cellulose structure were examined, the cellulose fiber structures were clearly seen. Fibers generally showed regular fiber structures in the 100–200 nm range. The surface was quite cavitated surface roughness varies between 50–80 nm. Yeast arrested on these surfaces was clearly selected on the surface in the form of pyramidal cones. Especially at high magnifications, the surface roughness up to 150 nm was due to the yeast structures on the surface. Yeasts were seen attached in zones of approximately 10 µm, especially on fiber structures. DTA and TGA analyses were performed to determine the effect of yeast structures attached to the surface on the thermal properties of cellulose fiber structures. The obtained TGA thermograms were given in Fig. 9 . According to Fig. 9 , thermal degradation of the pure cellulose structure occurred as a 2-stage weight loss. First weight loss started around 200°C. This weight loss due to the deterioration of the cellulose main chain structure was approximately 70% of the weight loss value and ends at around 380°C. The second weight loss was in the range of about 380–600°C. This weight loss was around 18% and is due to carbonization. When yeast is arrested on this structure, the decomposition temperature of the obtained structure decreases. The onset of degradation was observed around 160°C. First weight loss was around 160–350°C. Degradation of proteins and cellulosic unit in yeast structure was observed together. A second weight loss was observed around 350–420°C. This is due to the degradation of aromatic structures in the yeast structure. At around 420–530°C, carbonization of the cellulose structure was observed. DTA thermograms were taken to confirm the TGA findings (Fig. 10 ). In these thermograms, two main exotherm regions were seen in the cellulose fiber structure. The first exotherm appeared as a broad band around 300–400°C. The second exotherm was in the form of an exotherm region with many peaks around 400–590°C. When yeast was included in the structure, 3 basic exotherm zones were seen. The first exotherm zone started at 268°C and ended at 388°C. The second exotherm zone started at 388°C and ended at about 450°C. The last exotherm was observed between 450–540°C. The second exotherm is seen only in the yeast-retained structure and is due to the yeast structure. Also, the first degradation peak energy decreased from 1.56 kJ/g to 1.19 kJ/g in the yeast-retained structures. In addition, the initial decomposition temperature decreased by about 50°C. All these changes prove the existence of yeast in the structure. Dye decolorization with immobilized S. cerevisiae was immobilized on lyophilized BC pieces and their RB 171 dye decolorization activity were tested. For this aim, various amounts of yeast-immobilized BC samples (3, 6 and 12 pieces of BC samples in 0.5 cm sizes) were incubated in RB 171 dye solutions at 30° C for 24 h under static and also agitated conditions. As shown in Fig. 11 a, RB 171 dye decolorization activities of these 3 pieces, 6 pieces, and 12 pieces yeast-immobilized BC samples were 1%, 12%, and 25% and 12%, 24%, and 35% under static and agitated conditions after 24 h, respectively. Figure 11 b and 11 c show the photographs of the RB 171 dye solutions incubated with yeast-immobilized BC samples under static and agitated conditions. Conclusion The results of the study showed that Gluconacetobacter xylinus B759 has a high BC production potantial in cheese whey medium. Using cheap and a natural medium is an economic, effective and environmentally friendly method for production of BC. The BC which produced in cheese whey medium was detected as a good support for yeast immobilization. The chemical, morphological and thermal analyses indicated that the yeast cells can be successfully immobilized on this BC. The decolorization results showed that this immobilized yeast could be used to decolorize the textile dyes. The decolorization of dyes by immobilized yeast on BC may be an effective and promising solution to solve the environmental pollution problem, with such a cheap, efficient and environmental friendly application. Declarations Ethics approval and consent to participate The manuscript includes results obtained from this research study and it has not been published elsewhere and it has not been submitted simultaneously for publication elsewhere. A single study did not be split up into several parts to increase the quantity of submissions. All elements of this submission are in compliance with the journal publishing ethics policy. This study did not involve any experiments on human subjects or animals. Consent for publication Not applicable. Availability of data and materials Not applicable. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author’s contribution Conceptualization, Experimental stage, Methodology, Formal analysis, Writing - original draft-review and editing, Investigation Acknowledgements This study was supported by Inonu University Scientific Research Projects Coordination Unit (Project No: FBA-2019-1797). Conflict of interest: The authors declare that they have no confict of interest. References Azeredo HMC, Barud H, Farinas CS, Vasconcellos VM, Claro AM (2019) Bacterial cellulose as a raw material for food and food packaging applications. Front. Sustain. Food Syst. 3:7. https://doi.org/10.3389/fsufs.2019.00007 Bagewadi ZK, Bhavikatti JS, Muddapur UM, Yaraguppi DA (2020) Statistical optimization and characterization of bacterial cellulose produced by isolated thermophilic Bacillus licheniformis strain ZBT2. 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Saudi J Biol Sci 24: 314–319. http://dx.doi.org/10.1016/j.sjbs.2015.09.031 Kumar V, Sharma DK, Sandhu PP, Jadaun J, Sangwan RS, Yadav SK (2021) Sustainable process for the production of cellulose by an Acetobacter pasteurianus RSV-4 (MTCC 25117) on whey medium Cellulose (2021) 28:103–116. https://doi.org/10.1007/s10570-020-03519-6 Leal ANR, Limal ACA, Azevedo MGFA, Santos DKDN, Zaidan LEMC, Lima VF, Filho IJC (2021) Removal of Remazol Black B dye using bacterial cellulose as an adsorbent. Sci Plena 17: 034201. https://doi.org/10.14808/sci.plena.2021.034201 Lynd LR, Weimer PJ, Zyl, W.H, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66 (3): 506–577. https://doi.org/10.1128/MMBR.66.3.506-577.2002 Mikkelsen D, Flanagan BM, Dykes GA, Gidley MJ (2009) Influence of different carbon sources on bacterial cellulose production by Gluconacetobacter xylinus strain ATCC 53524. J Appl Microbiol 107: 576–583. https://doi.org/10.1111/j.1365-2672.2009.04226.x Mohite BV, Patil SV (2014) A novel biomaterial: bacterial cellulose and its new era applications Biotechnol Appl Biochem 61(2): 101–110. https://doi.org/10.1002/bab.1148 Prazeres AR, Carvalho F, Rivas J (2012) Cheese whey management: A review J Environ Manage 110: 48–68. https://doi.org/10.1016/j.jenvman.2012.05.018 Rangaswamy BE, Vanitha KP, Hungund BS (2015) Microbial cellulose production from bacteria isolated from rotten fruit. Int J Polym Sci 2015(2) 1–8. https://doi.org/10.1155/2015/280784 Revin V, Liyaskina E, Nazarkina M, Bogatyreva A, Shchankin M (2018) Cost-effective production of bacterial cellulose using acidic food industry by-products. Braz J Microbiol 49. 151–159. https://doi.org/10.1016/j.bjm.2017.12.012 Ross P, Mayer R, Benziman M (1991) Cellulose biosynthesis and function in bacteria. Microbiol Rev 55(1): 35–58. https://doi.org/10.1128/mr.55.1.35-58.1991 Salari M, Khiabani MS, Mokarram RR, Ghanbarzadeh B, Kafil HS (2019) Preparation and characterization of cellulose nanocrystals from bacterial cellulose produced in sugar beet molasses and cheese whey media. Int J Biol Macromol 122: 280–288. https://doi.org/10.1016/j.ijbiomac.2018.10.136 Shi Z, Zhang Y, Phillips GO, Yang G (2014) Utilization of bacterial cellulose in food. Food Hydrocoll 35: 539–545. https://doi.org/10.1016/j.foodhyd.2013.07.012 Siso MIG (1996) The biotechnological utilization of cheese whey: A review. Bioresour Technol 57: 1–11. https://doi.org/10.1016/0960-8524(96)00036-3 Szymańska-Chargot M, Cybulska J, Zdunek A (2011) Sensing the structural differences in cellulose from apple and bacterial cell wall materials by Raman and FT-IR spectroscopy. Sensors 11: 5543–5560. https://doi.org/10.3390/s110605543 Trovatti E, Serafim LS, Freire CSR et al (2011) Gluconacetobacter sacchari : an efficient bacterial cellulose cell-factory. Carbohydr Polym 86:1417–1420. https://doi.org/10. 1016/j.carbpol.2011.06.046 Ullah H, Santos HA, Khan T (2016) Applications of bacterial cellulose in food, cosmetics and drug delivery. Cellulose 23: 2291–2314. https://doi.org/10.1007/s10570-016-0986-y Ullah H, Badshah M, Mäkilä E, Salonen J, Shahbazi MA, Santos HA, Khan T (2017) Fabrication, characterization and evaluation of bacterial cellulose-based capsule shells for oral drug delivery. Cellulose 24: 1445–1454. https://doi.org/10.1007/s10570-017-1202-4 Yamanaka S, Watanabe K, Kitamura N, Iguchi M, Mitsuhashi S, Nishi Y, Uryu M (1989) The structure and mechanical properties of sheets prepared from bacterial cellulose. J Mater Sci 24: 3141–3145. https://doi.org/10.1007/BF01139032 Yim SM, Song JE, Kim HR (2017) Production and characterization of bacterial cellulose fabrics by nitrogen sources of tea and carbon sources of sugar. Process Biochem 59: 26–36. http://dx.doi.org/10.1016/j.procbio.2016.07.001 Żywicka A, Peitler D, Rakoczy R, Junka AF, Fijalkowski K (2016) Wet and dry forms of bacterial cellulose synthetized by different strains of Gluconacetobacter xylinus as carriers for yeast immobilization. Appl Biochem Biotechnol 180:805–816. https://doi.org/10.1007/s12010-016-2134-4 Żywicka A, Banach A, Junka AF, Drozd R, Fijalkowski K (2019) Bacterial cellulose as a support for yeast immobilization – Correlation between carrier properties and process efficiency J Biotechnol 291: 1–6. https://doi.org/10.1016/j.jbiotec.2018.12.010 Additional Declarations No competing interests reported. Supplementary Files graphicalabstract.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-1768385\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":116333202,\"identity\":\"2489ac3a-1978-46ff-b912-70f56556508b\",\"order_by\":0,\"name\":\"Filiz BORAN\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBAC9mbmhgMMDAlAJvOBA1BBA7xaeA4zwrSwJSBpScCj5QBjAwNEBQ/ccAJa2BkbD/5gSEvcPiPn4+GCijuJDezN2yQYf9zDrYUZ6DAJhpzEOTdyNxyeceZZYgPPsTIJhoRinFrsQVoMGCoSZ0gAtfC2HU5skMgxA2rB7TKwLQlgLTkPIFrk3xCh5QDQYUAtDFBbeAhrOdhgkGY8g+eZwWGeM4eN23jSii0S0vBo4T98+OOPimTZGezJjz/zVByW7Wc/vPHGBxvcWiAAObrZQAQhDaNgFIyCUTAK8AMAb4FT0fxNNNQAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Inonu University\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Filiz\",\"middleName\":\"\",\"lastName\":\"BORAN\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2022-06-17 11:14:18\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-1768385/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-1768385/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":23141338,\"identity\":\"028f9b99-1f9f-407a-a5d9-e27829ff5e44\",\"added_by\":\"auto\",\"created_at\":\"2022-06-27 19:21:14\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":222257,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eBC production steps: a- BC pellicle obtained after production in cheese whey medium, b- Pure BC pellicle, c- Lyophilized and dry BC pellicle\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1768385/v1/d66f6abb7adb71190706daf9.png\"},{\"id\":23141337,\"identity\":\"a1ddb093-33aa-4532-9353-5c59e04e1715\",\"added_by\":\"auto\",\"created_at\":\"2022-06-27 19:21:14\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":122318,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe effect of incubation time (a), carbon sources (b), temperature (c), pH (d) and nitrogen sources (e) on BC production\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1768385/v1/668e92d1dda18320f4007efb.png\"},{\"id\":23141339,\"identity\":\"38c9a075-11ef-4873-9fe2-738d8af33fc7\",\"added_by\":\"auto\",\"created_at\":\"2022-06-27 19:21:15\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":179357,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMicroscopic images of BC samples with and without yeast: a- Lyophilized BC sample and b- \\u003cem\\u003eS. cerevisiae\\u003c/em\\u003e immobilized lyophilized BC sample\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1768385/v1/6a2400181c32b881fedc7a75.png\"},{\"id\":23141341,\"identity\":\"4ce2181e-7999-40bb-8878-114a1d315f01\",\"added_by\":\"auto\",\"created_at\":\"2022-06-27 19:21:15\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":444526,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eScanning electron micrographs of the pure cellulose sample: a- (20,000 ×) and b- (40,000 ×)\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1768385/v1/33e4cd0d91948a8b4efbde32.png\"},{\"id\":23141343,\"identity\":\"3ed064ae-8815-42fb-8ec4-dd6e3d32d308\",\"added_by\":\"auto\",\"created_at\":\"2022-06-27 19:21:15\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":485535,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eScanning electron micrographs of the immobilized yeast cells on pure cellulose sample, in different magnifications\\u003cem\\u003e.\\u003c/em\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1768385/v1/87863e0d31305017e79bc795.png\"},{\"id\":23141340,\"identity\":\"fdd88d96-bbdd-4c31-8626-89f16157325a\",\"added_by\":\"auto\",\"created_at\":\"2022-06-27 19:21:15\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":89383,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFTIR spectra of pure cellulose and yeast-immobilized cellulose structures\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1768385/v1/890a763a2b659ac8d3b16c7d.png\"},{\"id\":23141346,\"identity\":\"9de3dada-ef97-42dc-9f00-3e447f43e3ab\",\"added_by\":\"auto\",\"created_at\":\"2022-06-27 19:21:15\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":70812,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eX-ray spectra of pure cellulose and yeast-immobilized cellulose structures\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1768385/v1/a8f46b036ce6dc7a2a43f4f6.png\"},{\"id\":23141345,\"identity\":\"0e90ab5e-57eb-42f1-962c-426a339e1f63\",\"added_by\":\"auto\",\"created_at\":\"2022-06-27 19:21:15\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3350521,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAFM structures of cellulose fiber (a) and yeast-immobilized cellulose (b) structures with different magnification\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1768385/v1/d0fdb4b83570f919f29ccabd.png\"},{\"id\":23141335,\"identity\":\"6d440ce6-9141-4b9d-8476-086789e543db\",\"added_by\":\"auto\",\"created_at\":\"2022-06-27 19:21:14\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":74057,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTGA thermograms of pure cellulose and yeast-immobilized cellulose structures\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1768385/v1/3752c1e90d90f6ac167b9678.png\"},{\"id\":23141344,\"identity\":\"6cc2c7a5-7f67-41bc-9c52-fc85e3261322\",\"added_by\":\"auto\",\"created_at\":\"2022-06-27 19:21:15\",\"extension\":\"png\",\"order_by\":10,\"title\":\"Figure 10\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":24906,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDTA thermograms of pure cellulose and yeast-immobilized cellulose structures\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure10.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1768385/v1/266889c9de1b2c19b1c2a149.png\"},{\"id\":23141342,\"identity\":\"06d78e24-f916-4787-8b7c-9a86cc8f51b7\",\"added_by\":\"auto\",\"created_at\":\"2022-06-27 19:21:15\",\"extension\":\"png\",\"order_by\":11,\"title\":\"Figure 11\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":138221,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDecolorization of RB 171 by yeast immobilized on different amount of BC samples (3, 6, 12 pieces) under static and agitated conditions (a), Photographs of the decolorized Reactive Blue 171 dye solutions (from left to right: untreated textile dye solution, dye solutions decolorized with 3, 6 and 12 pieces of yeast-immobilized BC samples) under static (b) and agitated (c) conditions\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure11.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1768385/v1/7b3d7465a2eb397dbca5610a.png\"},{\"id\":24205557,\"identity\":\"fdecfa5a-d3b7-41d9-ad2f-04961e32d6f5\",\"added_by\":\"auto\",\"created_at\":\"2022-07-22 16:44:25\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2577526,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1768385/v1/ee3b7ee0-f392-4adb-8167-e91ee16dfb0b.pdf\"},{\"id\":23141336,\"identity\":\"3dadaf1c-71b8-42c9-9722-a73e8d9ea331\",\"added_by\":\"auto\",\"created_at\":\"2022-06-27 19:21:14\",\"extension\":\"png\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":476936,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"graphicalabstract.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1768385/v1/da50aa18ce8d86fe78f9e964.png\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Synthesis, Chemical, Morphological and Thermal Characterization Yeast Immobilized Bacterial Cellulose and It Use in Color Removal\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eCellulose, the most abundant biopolymer in nature, is mostly found in plants but some bacteria can also produce this polymer (Lynd et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2002\\u003c/span\\u003e). Plant cellulose is not pure and it is associated with lignin, pectin, hemicellulose and other substances. Therefore, additional purification processes are required to obtain pure cellulose (Cacicedo et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Islam et al. \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Various chemicals must be used during the purification process of plant cellulose. These chemicals cause environmental pollution and also damage the structure of natural cellulose (\\u0026Ccedil;akar et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). On the other hand, bacterial cellulose (BC) is pure and therefore, there is no need for hard chemicals to obtain the BC in pure form. Many bacterial species belonging to genera of \\u003cem\\u003eGluconacetobacter (\\u003c/em\\u003eformerly \\u003cem\\u003eAcetobacter), Achromobacter, Agrobacterium, Aerobacter, Azotobacter, Rhizobium, Pseudomonas, Sarcina, Salmonella, Enterobacter, Escherichia\\u003c/em\\u003e and \\u003cem\\u003eetc.\\u003c/em\\u003e are reported as BC producers (Hungund et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e; Huang et al. \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Ullah et al. \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Gram-negative bacterium \\u003cem\\u003eAcetobacter xylinum\\u003c/em\\u003e (now known as \\u003cem\\u003eGluconacetobacter xylinus or Komagataeibacter xylinus\\u003c/em\\u003e) could synthesize abundant cellulose (Karahan et al. \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e; Żywicka et al. \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). \\u003cem\\u003eG. xylinus\\u003c/em\\u003e is the most studied bacterium in BC production due to its high BC production capacity. The effect of different sugars and other compounds are tested as carbon sources to induce the BC production (Gama et al. 2012; Ullah et al. \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). BC obtained from this species is one of the most preferred classes of biopolymer recently (Ross et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e1991\\u003c/span\\u003e; Żywicka et al. \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). It has important superior properties such as high purity, high crystallinity, a high degree of polymerization, a nano-structured work, excellent mechanical strength, large surface area, higher porosity, a high water holding capacity and also good biocompatibility and biodegradability (Hungund et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e; Yamanaka et al. \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e1989\\u003c/span\\u003e; Bagewadi et al. \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). These superior properties make it as an interesting material in the important areas such as medicine, food and cosmetics etc. (Buruaga-Ramiro et al. \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Fernandes et al. \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Hungund et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e). The usability of BC as an immobilization agent for many industrially important microorganisms such as yeasts is being tested and these studies reveal that it can be an effective immobilization agent. \\u003cem\\u003eSaccharomyces cerevisiae\\u003c/em\\u003e is the most preferred and studied model organism in molecular and cell biology and in many industrial processes (Żywicka et al. \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Johnson and Echavarri-Erasun \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eBC is commonly produced in Hestrin-Schramm (HS) medium (\\u0026Ccedil;akar et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). However, this is an expensive medium (Ghozali et al. \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). The cost of the production is an important problem affecting industrial use of BC (\\u0026Ccedil;oban and Biyik \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). The use of agro-industrial wastes or by-products as the growth media can make the industrial production process more economical (Shi et al \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). Therefore, cheese whey can be used as an inexpensive and natural media (Battad-Bernardo et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). Thus, the industrial scale production of the BC, which becomes difficult due to its high cost, can also be facilitated by using cheaper substrates (Carreira et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e; \\u0026Ccedil;oban and Biyik \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). Cheese whey, by-product of cheese production, is a natural medium with high nutritional content (Revin et al. \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). It contains various compounds such as lactose, soluble proteins, lipids, mineral salts, lactic acid, citric acid, non-protein nitrogen compounds (urea and uric acid) and B group vitamins (Siso \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e1996\\u003c/span\\u003e; Prazeres et al. \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). As I stated before, low cost and high efficiency BC production is very important in industrial applications. This can be achieved both by using inexpensive industrial wastes/raw materials and by optimizing the conditions and the factors such as incubation time, temperature, pH, carbon and nitrogen sources (Shi et al. \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Azerado et al. 2019).\\u003c/p\\u003e \\u003cp\\u003eIn this study, BC production ability of \\u003cem\\u003eG. xylinus\\u003c/em\\u003e B759 in the cheese whey medium obtained from local cheese factory in Malatya, T\\u0026uuml;rkiye was investigated. The effect of different cultivation parameters such as incubation time, temperature, pH and carbon and nitrogen sources on BC production efficiency of this strain was also tested. Obtained BC and yeast-immobilized BC were characterized. The usability of this BC as an immobilization support of \\u003cem\\u003eSaccharomyces cerevisiae\\u003c/em\\u003e has also been studied. In addition, the dye decolourization ability of yeast-immobilized BC was determined.\\u003c/p\\u003e\"},{\"header\":\"Materials And Methods\",\"content\":\"\\u003cp\\u003eBacterial strain used for BC production\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eGluconacetobacter xylinus\\u003c/em\\u003e B759 was used as the bacterium for production of bacterial cellulose (BC). This bacteria was provided by Prof. Dr. Aynur G\\u0026uuml;l Karahan \\u0026Ccedil;akmak\\u0026ccedil;ı. This bacterium was grown on the Hestrin-Schramm (HS) agar plates containing (g/L) glucose, 20; peptone, 5; yeast extract, 5; Na\\u003csub\\u003e2\\u003c/sub\\u003eHPO\\u003csub\\u003e4\\u003c/sub\\u003e, 2,7; citric acid, 1,15; agar, 15 at 30\\u0026deg;C for 10 days under static condition and then stored at 4\\u0026deg;C. It was subcultivated every 3\\u0026ndash;4 weeks.\\u003c/p\\u003e \\u003cp\\u003eCulture medium and BC production\\u003c/p\\u003e \\u003cp\\u003eThe inoculum was prepared in 50 mL Hestrin-Schramm (HS) broth consisted of (g/L) glucose, 20; peptone, 5; yeast extract, 5; Na\\u003csub\\u003e2\\u003c/sub\\u003eHPO\\u003csub\\u003e4\\u003c/sub\\u003e, 2,7; citric acid, 1,15 (Hestrin and Schramm, 1954). This medium was inoculated with \\u003cem\\u003eG. xylinus\\u003c/em\\u003e and then, the culture was incubated at 30\\u0026deg;C for 10 days under static conditions. After incubation, 1 mL of \\u003cem\\u003eG. xylinus\\u003c/em\\u003e liquid culture transferred into 50 mL of HS medium for obtaining stock inoculum culture.\\u003c/p\\u003e \\u003cp\\u003eCheese whey was chosen as the main culture medium for BC production. This natural medium was purchased from a local cheese factory (Dutpınar, Malatya/Turkiye) and it was filtered before using. Erlenmeyer flasks containing 40 mL of cheese whey were autoclaved at 121\\u0026deg;C for 20 min. Then, the sterilized media were inoculated with \\u003cem\\u003eG. xylinus\\u003c/em\\u003e stock inoculum culture at the rate of 2% and they were incubated at 30\\u0026deg;C for 10 days under static conditions. After incubation, BC pellicles were filtered and washed several times with distilled water. Then, they were treated with 0.1 N NaOH at 75\\u0026deg;C for 2 hours. After that, they were washed with distilled water a few times until neutralization. These purified and filtered pellicles were dried at 40\\u0026deg;C for 15 min and then they were lyophilized for obtaining lyophilized BC. All lyophilized samples were weighed and the dry weight of the BC product obtained was expressed as grams per liter (g/L). All experiments were performed in three replicates.\\u003c/p\\u003e \\u003cp\\u003eOptimization of BC production\\u003c/p\\u003e \\u003cp\\u003eThe culture conditions such as incubation time, temperature, pH, carbon source and nitrogen source are important parameters to achieve the highest BC production efficiency. To test the effect of the incubation time on BC production, \\u003cem\\u003eG. xylinus\\u003c/em\\u003e inoculated cheese whey media were incubated for 3, 5, 7, 10 and 15 days at 30\\u0026deg;C. After the optimal incubation time was determined, the cultures were incubated at 25, 28, 30, 32 and 35\\u0026deg;C for 10 days for testing the optimal temperature for BC production. pH is also an another important parameter for BC production. Therefore, the ranges of pH 3.0\\u0026ndash;9.0 were also tested for BC formation. To this end, the initial pHs of the cheese whey media were adjusted to pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 and after inoculation, they were incubated at 30\\u0026deg;C for 10 days. Glucose, sucrose and fructose were used as additional carbon sources and each were separately added into 40 mL cheese whey media at the concentrations of 0.5, 1.0 and 2.0 g. The prepared media were inoculated with bacterium and these cultures were incubated at 30\\u0026deg;C for 10 days. The effect of yeast extract and pepton (as nitrogen sources) was also tested at the concentrations of 0.1, 0.2 and 0.4 g.\\u003c/p\\u003e \\u003cp\\u003eCharacterization of BC samples\\u003c/p\\u003e \\u003cp\\u003eScanning electron microscope (SEM), Fourier transform infrared spectrometer (FTIR) and X-ray diffraction (XRD) analyzes of BC pellicles were determined at Inonu University Scientific and Technological Research Center Laboratories, Malatya\\u003cem\\u003e/\\u003c/em\\u003eT\\u0026uuml;rkiye. Within the scope of the study, chemical characterizations of the produced cellulose structures were carried out using Perkin Elmer spectrum two model FTIR spectrophotometer. FTIR analyses of BC samples were scanned with a working sensitivity of 4 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e in the range of 400\\u0026ndash;4000 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. ATR mode was used as the operating mode.\\u003c/p\\u003e \\u003cp\\u003eFor the determination of the crystallinity level of the obtained BC structures, Rigaku brand X-ray spectrophotometer was used for X-ray analysis. The obtained cellulosic films were measured in the range of 2\\u0026ndash;80 θ, with a measurement sensitivity of 0.2 θ.\\u003c/p\\u003e \\u003cp\\u003eThe surface properties and surface morphologies of the cellulose structures were examined by AFM and SEM analyses. In AFM analysis, 40 \\u0026micro;m x 40 \\u0026micro;m scanning area was performed using non-contact mode. AFM samples were monitored at room temperature and in Faraday cages using the Park system XE-100 AFM device. Leo-Evo 40 model scanning electron microscope was used for SEM analysis. Before the samples were analysed, they were coated with a Baltec brand sputter with 20 m Au/Pd layer.\\u003c/p\\u003e \\u003cp\\u003eShimadzu brand thermal analysis system was used to examine the thermal properties of prepared cellulose samples. Shimadzu TGA-50 model analyser was used for thermal stability measurements. Shimadzu DTA 50 was used to determine the decay energies. Heating rate of 10\\u0026deg;C/min was preferred in the measurement performed against the reference material of aluminium oxide. All thermal analyses were performed in a static air atmosphere and in platinum cuvettes using 10 mg of sample.\\u003cdiv class=\\\"BlockQuote\\\"\\u003e\\u003cp\\u003eYeast Immobilization Procedure\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/p\\u003e \\u003cp\\u003eA small amount of solid \\u003cem\\u003eS. cerevisiae\\u003c/em\\u003e culture was inoculated into 50 mL/250 mL flask Sabouraud dextrose broth (SDB) medium and the culture was incubated at 30\\u0026deg;C and 150 rpm for 48 hours. Lyophilized BC samples cut in 0.5 cm sizes were added into 50 mL SDB medium and it was autoclaved at 121\\u0026deg;C for 20 minutes. Then, 1 mL from \\u003cem\\u003eS. cerevisiae\\u003c/em\\u003e culture was inoculated into the medium containing BC samples and the culture was incubated at 30\\u0026deg;C and 150 rpm for 48 h. At the end of the incubation, \\u003cem\\u003eS. cerevisiae\\u003c/em\\u003e immobilized BC samples was obtained.\\u003c/p\\u003e \\u003cp\\u003eDye decolorization activity of immobilized \\u003cem\\u003eS. cerevisiae\\u003c/em\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe decolorization activity of immobilized yeast against Chrocion Blue H-ERD (Reactive Blue 171, RB 171) was studied. RB171 dye was used at 200 mg/L concentration. Various pieces of \\u003cem\\u003eS. cerevisiae\\u003c/em\\u003e immobilized BC (3, 6 and 12) were transferred into 3 mL dye solution and they were incubated at 30\\u0026deg;C under static and agitated (150 rpm) conditions for 24 h. Dye decolorization was determined spectrophotometrically (Shimadzu-UV-1601, UV/Visible) as a relative decrease of absorbance for RB171 dye at its maximum absorbance wavelength (615 nm). All experiments were performed in three replicates and dye decolorization percentages were calculated with SPSS 15.0 package program.\\u003c/p\\u003e\"},{\"header\":\"Results And Discussion\",\"content\":\"\\u003cp\\u003eProduction of BC\\u003c/p\\u003e \\u003cp\\u003eCheese whey is a by-product produced during cheese production and it contains lactose and other carbohydrates (glucose, galactose, lactose, and arabinose). In addition, whey has high biological value because it contains various proteins, amino acids, vitamins and organic acids (Revin et al \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Because cheese whey is a simple, inexpensive and abundant medium, it can be used as the basal culture medium for BC production. Therefore, in this study, BC was produced in cheese whey medium. The obtained BC pellicles were purified, weighed after lyophilization and its yield was calculated as g/L. In Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e (a-c), the steps of BC production in cheese whey medium could be seen.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eOptimization of Culture Conditions for BC Production\\u003c/p\\u003e \\u003cp\\u003eThe optimization of culture conditions is very important for production of high amount of BC pellicles. Different factors such as incubation time, temperature, pH, and carbon and nitrogen sources were tested in order to obtain the highest yield of BC. Firstly, the effect of incubation time on BC production was determined. As shown Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea, the maximum yield was obtained at the 10th day. While 1.59 g/L BC pellicle was obtained at the 3th day, it was 3.16 g/L for 10 days of incubation at 30\\u0026deg;C, statically.\\u003c/p\\u003e \\u003cp\\u003eType and amount of carbon and nitrogen sources in the culture medium are also effective on the BC production (Szymańska-Chargot et al, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). The carbon source is an essential component that enhances cell growth and metabolism during BC synthesis (Yim et al, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Therefore, glucose, fructose and sucrose were added into cheese whey media at different concentrations (0.5, 1.0, 2.0 g/40 mL) for testing their possible effect on BC production and cultures were incubated at 30\\u0026deg;C for 10 days (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb). Glucose addition positively affected the BC production activity of this bacterium. Fructose addition also induced the BC production but sucrose addition resulted in low BC production. The highest BC amount was obtained from the medium containing of 0.5 g glucose per 40 mL cheese whey as 5.62 g/L (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb). Therefore, 0.5 g glucose in 40 mL medium was determined as the best carbon amount for BC production. Mikkelsen et al. (\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e) aimed to increase the BC cellulose yield of \\u003cem\\u003eGluconacetobacter xylinus\\u003c/em\\u003e strain ATCC 53524 by modifying the HS medium. For this purpose, HS media were prepared with the addition of different inducers and the BC yield was calculated at 48 and 96 hours. While the highest BC yield was 1.89 g/L at 48th hour in HS medium with an initial pH of 5.0 to which glucose was added, this value was determined as 3.10 g/L at 96th hour. Rangaswamy et al. (2005), on the other hand, tested the effect of different carbon sources on BC yield, in HS medium. It was reported that \\u003cem\\u003eGluconacetobacter\\u003c/em\\u003e sp produces approximately 1.35 g/L BC in HS medium containing 2% (w/v) glucose. In the study of Trovatti et al (\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e) in which BC production was tested with \\u003cem\\u003eGluconacetobacter sacchari\\u003c/em\\u003e in HS medium containing different carbon sources such as glucose, sucrose, and fructose, the highest BC efficiency was obtained at the end of 96 hours in HS medium with glucose (2.7 g/L).\\u003c/p\\u003e \\u003cp\\u003eTemperature is also an important factor for high product formation (Fernandes et al, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). In order to determine the effect of temperature for BC production, different temperature values at the range of 25\\u0026ndash;35\\u0026deg;C were tested. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec, the optimum temperature for BC production was 30\\u0026deg;C and the BC value obtained at this temperature was 7.63 g/L. Revin et al. (\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e) tested the BC production of \\u003cem\\u003eGluconacetobacter sucrofermentans\\u003c/em\\u003e B-11267 in cheese whey medium (without pH adjustment) and they determined the highest BC yield as 5.45 g/L after 3 days of incubation at 28\\u0026deg;C and 250 rpm. The maximum BC production of \\u003cem\\u003eAcetobacter pasteurianus\\u003c/em\\u003e RSV-4 (MTCC 25117) in the whey medium was 5.6 g/L at 30\\u0026deg;C after 8 days incubation (Kumar et al. \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). On the other hand, Carreira et al. (\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e) reported very low BC production 0.08 g/L in the whey medium.\\u003c/p\\u003e \\u003cp\\u003eInitial pH is an important factor for BC production. Therefore, pHs of the cheese whey media containing 0.5 g glucose were adjusted to the range of pH 3.0\\u0026ndash;9.0 and the effect pH on BC production was tested (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ed). As the pH increased, the amount of BC obtained also increased. As shown Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ed the highest BC amount was 13.18 g/L at pH 7.0. No BC formation observed at pH 3.0. and pH 9.0. Jozala et al. (\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e) used various culture media such as HS, rotten fruit and milk whey for BC production. In their study, they tested the effect of pH on BC yield and obtained optimum BC yields at different pH values between 3.2 and 5.4 according to the culture medium they used.\\u003c/p\\u003e \\u003cp\\u003eNitrogen constitutes 8\\u0026ndash;14% of the dry weight of bacteria and is the main component of proteins required for cell metabolism (Chawla et al, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). However, nitrogen sources did not show any positive effect on BC production activity of the bacterium (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ee).\\u003c/p\\u003e \\u003cp\\u003eThe optimal medium and the optimum culture conditions for high amount of BC production was determined as 10 days incubation time, 30\\u0026deg;C temperature, 7.0 pH and 0.5 g glucose in 40 mL cheese whey medium.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eImmobilization of yeast cells on BC\\u003c/p\\u003e \\u003cp\\u003eBC can be a good support for cell immobilization thanks to its high crystallinity, high water holding capacity, porous structure, better mechanical properties and biocompatibility (Żywicka et al \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). As a support, BC can be used for the immobilization of various enzymes, as well as for the immobilization of industrially important microorganisms such as yeasts (Żywicka et al \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Żywicka et al \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Therefore, in this study, BC produced by \\u003cem\\u003eG. xylinus\\u003c/em\\u003e in cheese whey medium under optimized conditions was used as a support for the immobilization of \\u003cem\\u003eS. cerevisiae\\u003c/em\\u003e and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e shows the microscopic images of lyophilized BC without yeast and \\u003cem\\u003eS. cerevisiae\\u003c/em\\u003e immobilized BC samples stained with the simple staining method.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eChemical, morphological and thermal characterization of pure and yeast-immobilized BC samples\\u003c/p\\u003e \\u003cp\\u003eFigure \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e shows the Scanning electron micrographs (SEM) of the pure cellulose samples obtained from the cheese whey medium. SEM analysis of BC was performed with lyophilized BC pellicle under 20000 \\u0026times; and 40000 \\u0026times; magnifications. From the SEM images, it could be seen that the BC pellicle had a reticulated structure. The average fibril diameter of the pure cellulose sample obtained from the cheese whey medium was measured as 123.7 nm. Revin et al. (\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e) reported the width of the microfibrils obtained from the whey medium as 100\\u0026ndash;180 nm. Furthermore, yeast cells immobilized on cellulose were also proven by SEM images (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eStructural characterizations of pure cellulose and yeast-immobilized cellulose samples were determined by Fourier transformed infrared spectrophotometer.\\u003c/p\\u003e \\u003cp\\u003eObtained infrared spectra were given in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e comparatively. In the spectrum of pure cellulose structure, a wide H bonds band of free -OH group on the cellulose units was seen in the range of 3000\\u0026ndash;3600 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. Aliphatic C-H peaks in cellulose units were observed in the range of 2830\\u0026ndash;2950 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. Main chain C-C stretching vibration was observed at 1580 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. The etheric C-O-C stretching vibration in the cellulose structures was detected as a severe peak at 1057 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. In addition, CH stretching vibration at 888 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e and CH out-of-plane bending vibration at 559 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e confirmed the obtained structure.\\u003c/p\\u003e \\u003cp\\u003eFigure \\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e shows the FTIR spectrum of the yeast-immobilized cellulose samples. Along with the binding of yeast structures, on this spectrum, a sharp peak originating from protein structures was observed at approximately 1750 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. In addition, the effect of yeast structures on the spectrum was observed on the aliphatic methyl and H bonds peaks. Methyl bond strength increased, but H bond strength decreased. The peaks were observed more broadly. All these findings prove that the desired structure was obtained.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe crystallinity of the obtained cellulose structures\\u003c/p\\u003e \\u003cp\\u003eThe degree of crystallinity of the obtained cellulose structures and the effects of yeast structures on the crystal system were examined with X-ray spectra. Obtained X-ray spectra were given in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e. When the X-ray spectrum of the pure BC structure was examined, although it has an amorphous appearance, the crystalline peaks were clearly seen in the structure. Due to the cellulose structure, the 100 and 110 peaks, which are the main cellulose peaks, were clearly seen, especially at 15\\u0026deg; and 22\\u0026deg; 2θ values (Leal et al \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e; Salari et al \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). In addition, a wide band originating from amorphous regions was observed at 19.48\\u0026deg;. On the other hand, in the X-ray spectrum obtained from the cellulose sample with yeast structure, a more amorphous image appeared in which the crystalline structures were partially lost. This is due to the yeast covering the crystalline regions on the surface, and the change in this spectrum proves the existence of the yeast structure.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn order to visualize the yeast layers on the surface in more detail, AFM images of pure cellulose and yeast-immobilized cellulose surfaces were obtained at different magnifications (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e). When the AFM images of the pure cellulose structure were examined, the cellulose fiber structures were clearly seen. Fibers generally showed regular fiber structures in the 100\\u0026ndash;200 nm range. The surface was quite cavitated surface roughness varies between 50\\u0026ndash;80 nm. Yeast arrested on these surfaces was clearly selected on the surface in the form of pyramidal cones. Especially at high magnifications, the surface roughness up to 150 nm was due to the yeast structures on the surface. Yeasts were seen attached in zones of approximately 10 \\u0026micro;m, especially on fiber structures.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eDTA and TGA analyses were performed to determine the effect of yeast structures attached to the surface on the thermal properties of cellulose fiber structures. The obtained TGA thermograms were given in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003e. According to Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003e, thermal degradation of the pure cellulose structure occurred as a 2-stage weight loss. First weight loss started around 200\\u0026deg;C. This weight loss due to the deterioration of the cellulose main chain structure was approximately 70% of the weight loss value and ends at around 380\\u0026deg;C. The second weight loss was in the range of about 380\\u0026ndash;600\\u0026deg;C. This weight loss was around 18% and is due to carbonization. When yeast is arrested on this structure, the decomposition temperature of the obtained structure decreases. The onset of degradation was observed around 160\\u0026deg;C. First weight loss was around 160\\u0026ndash;350\\u0026deg;C. Degradation of proteins and cellulosic unit in yeast structure was observed together. A second weight loss was observed around 350\\u0026ndash;420\\u0026deg;C. This is due to the degradation of aromatic structures in the yeast structure. At around 420\\u0026ndash;530\\u0026deg;C, carbonization of the cellulose structure was observed.\\u003c/p\\u003e \\u003cp\\u003eDTA thermograms were taken to confirm the TGA findings (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003e). In these thermograms, two main exotherm regions were seen in the cellulose fiber structure. The first exotherm appeared as a broad band around 300\\u0026ndash;400\\u0026deg;C. The second exotherm was in the form of an exotherm region with many peaks around 400\\u0026ndash;590\\u0026deg;C. When yeast was included in the structure, 3 basic exotherm zones were seen. The first exotherm zone started at 268\\u0026deg;C and ended at 388\\u0026deg;C. The second exotherm zone started at 388\\u0026deg;C and ended at about 450\\u0026deg;C. The last exotherm was observed between 450\\u0026ndash;540\\u0026deg;C. The second exotherm is seen only in the yeast-retained structure and is due to the yeast structure. Also, the first degradation peak energy decreased from 1.56 kJ/g to 1.19 kJ/g in the yeast-retained structures. In addition, the initial decomposition temperature decreased by about 50\\u0026deg;C. All these changes prove the existence of yeast in the structure.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eDye decolorization with immobilized \\u003cem\\u003eS. cerevisiae\\u003c/em\\u003e\\u003c/p\\u003e \\u003cp\\u003ewas immobilized on lyophilized BC pieces and their RB 171 dye decolorization activity were tested. For this aim, various amounts of yeast-immobilized BC samples (3, 6 and 12 pieces of BC samples in 0.5 cm sizes) were incubated in RB 171 dye solutions at 30\\u0026deg; C for 24 h under static and also agitated conditions. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig11\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003ea, RB 171 dye decolorization activities of these 3 pieces, 6 pieces, and 12 pieces yeast-immobilized BC samples were 1%, 12%, and 25% and 12%, 24%, and 35% under static and agitated conditions after 24 h, respectively. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig11\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003eb and \\u003cspan refid=\\\"Fig11\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003ec show the photographs of the RB 171 dye solutions incubated with yeast-immobilized BC samples under static and agitated conditions.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eThe results of the study showed that \\u003cem\\u003eGluconacetobacter xylinus\\u003c/em\\u003e B759 has a high BC production potantial in cheese whey medium. Using cheap and a natural medium is an economic, effective and environmentally friendly method for production of BC. The BC which produced in cheese whey medium was detected as a good support for yeast immobilization. The chemical, morphological and thermal analyses indicated that the yeast cells can be successfully immobilized on this BC. The decolorization results showed that this immobilized yeast could be used to decolorize the textile dyes. The decolorization of dyes by immobilized yeast on BC may be an effective and promising solution to solve the environmental pollution problem, with such a cheap, efficient and environmental friendly application.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe manuscript includes results obtained from this research study and it has not been published elsewhere and it has not been submitted simultaneously for publication elsewhere. A single study did not be split up into several parts to increase the quantity of submissions. All elements of this submission are in compliance with the journal publishing ethics policy. This study did not involve any experiments on human subjects or animals.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials \\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding \\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor\\u0026rsquo;s contribution \\u003c/strong\\u003eConceptualization, Experimental stage, Methodology, Formal analysis, Writing - original draft-review and editing, Investigation\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements \\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was supported by Inonu University Scientific Research Projects Coordination Unit (Project No: FBA-2019-1797).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest: \\u003c/strong\\u003eThe authors declare that they have no confict of interest.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAzeredo HMC, Barud H, Farinas CS, Vasconcellos VM, Claro AM (2019) Bacterial cellulose as a raw material for food and food packaging applications. 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Appl Biochem Biotechnol 180:805\\u0026ndash;816. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1007/s12010-016-2134-4\\u003c/span\\u003e\\u003cspan address=\\\"10.1007/s12010-016-2134-4\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eŻywicka A, Banach A, Junka AF, Drozd R, Fijalkowski K (2019) Bacterial cellulose as a support for yeast immobilization \\u0026ndash; Correlation between carrier properties and process efficiency J Biotechnol 291: 1\\u0026ndash;6. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.jbiotec.2018.12.010\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.jbiotec.2018.12.010\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Bacterial cellulose, Cheese whey, Dye decolorization, Immobilization, Yeast\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-1768385/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-1768385/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eBacterial cellulose is a biomaterial and can be used for many applications such as medical products and food ingredients with its superior properties such as being pure, high crystallinity and durability. It can also be used as an as an immobilization support. It is a good and practical support for yeast immobilization.\\u003c/p\\u003e \\u003cp\\u003eThe present study aimed to immobilize yeast on lyophilized bacterial cellulose produced by \\u003cem\\u003eGluconacetobacter xylinus\\u003c/em\\u003e B759 in cheese whey medium as a cheap and natural culture medium. Optimum conditions such as cultivation time, carbon source, temperature, pH and nitrogen sources for bacterial cellulose production in cheese whey medium were determined. The highest bacterial cellulose yield obtained under optimized conditions was 13.18 g/L. \\u003cem\\u003eSaccharomyces cerevisiae\\u003c/em\\u003e cells were immobilized on purified and lyophilized 0.5 cm pure bacterial cellulose. Bacterial cellulose and yeast-immobilized bacterial cellulose structures were characterized by different instrumental techniques. Furthermore, Reactive Blue 171 dye decolorization ability of immobilized yeast was tested. The structure of bacterial cellulose showed very suitable morphology and chemical structure for different biotechnological applications. The use of a cheap and natural medium is very important in terms of production conditions, as it is an environmentally friendly application in terms of biotechnology.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Synthesis, Chemical, Morphological and Thermal Characterization Yeast Immobilized Bacterial Cellulose and It Use in Color Removal\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2022-06-27 19:21:11\",\"doi\":\"10.21203/rs.3.rs-1768385/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"8b203c21-2082-490b-97eb-a2edc327f55c\",\"owner\":[],\"postedDate\":\"June 27th, 2022\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2022-07-22T16:44:21+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2022-06-27 19:21:11\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-1768385\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-1768385\",\"identity\":\"rs-1768385\",\"version\":[\"v1\"]},\"buildId\":\"WrCJVZZCHTDjtuVLN7oU0\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}