Physical and Chemical Degradation of Bacterial Cellulose via Rearranging Fibers

Physical and Chemical Degradation of Bacterial Cellulose via Rearranging Fibers | 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 Physical and Chemical Degradation of Bacterial Cellulose via Rearranging Fibers Fulya Şahin, Neslihan Kayra, Ali Özhan Aytekin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4205627/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 (BC) has unique properties such as high tensile strength, high crystallinity, and high purity. Fiber length of BC causes different attributes. Therefore, degradation of BC has been studied deeply. In this study, fibers of BC were rearranged via DMAc-LiCl solvent and BC was degraded in wet state. Two different degradation methods, milling with liquid nitrogen and autoclave treatment, were applied. Degraded BCs were characterized by FTIR, TEM, AFM, TGA, and XRD. The solvent helps to align the fibers, making it more crystalline. Degraded BCs had lower crystalline ratio than BC, because of increasing hydrogen bonding during degradation in wet state. Degradation with autoclave made two different degraded BCs as nanofibrils and spherical nanocrystals with no pretreatment and solvent pretreatment, respectively. The nanofibril lengths were between 312 – 700 nm depending on applied method and spherical nanocrystal size was 56 nm. The rearrangement via solvent causes an important difference in degradation of BC. Nanofibrils and nanocrystals can be obtained, depending on the rearrangement of fibers before degradation process. Bacterial cellulose nanocrystal Bacterial cellulose nanofiber Liquid nitrogen milling Sulfuric acid degradation Fiber arrangement Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights Degradation of bacterial cellulose is successfully achieved at wet state of polymer. Low exposure time of solvent asissted to change fibers position for degradation. Freezing by liquid nitrogen and milling sustain nanofibril formation. Sulfuric acid treatment of rearranged fibers resulted in spherical nanocrystals. Rearrangement of fibers at wet state will help to obtain desired nanoparticle form and sizes. Introduction Bacterial cellulose (BC) is a unique polysaccharide that has significant properties such as high tensile strength, high crystallinity, and high purity. Because of these properties, BC has been reported to be used in different areas. The changing fiber lengths of BC have significantly different uses. Therefore, various cellulose degradation processes have been presented. They are based on chemical such as sulfuric acid [ 1 – 4 ], hydrochloric acid [ 5 ], and enzymes [ 6 ] or physical as sonication [ 7 ], hydrothermal explosion [ 8 , 9 ], and high-pressure homogenization [ 10 ]. Different kinds of solvents have been used for changing fiber formations to obtain new and/or unique features [ 11 – 15 ]. The one of the most studied solvents is dimethyl acetamide-LiCl solution (DMAc-LiCl). The highest concentration of LiCl is 8% (w/v) in DMAc. The solvent penetrates the BC fibers, and it causes swelling in solvent. The swelling effect make transparent BC. Because fibers are rearranged and positioned as parallel [ 11 , 12 , 14 ]. The extraction of cellulose and preparation of cellulose nanofibers were initially performed from plants and their wastes [ 8 , 16 ]. The harsh conditions such as acid with hydrothermal process as for explosion of lignin and hemicellulose structures are commonly used. Later, acid-based degradations of bacterial cellulose studies were proposed [ 3 ]. The investigation of degradation was based on different concentrations of sulfuric acid and/or hydrochloric acid, incubation time, acid/polymer weight ratio, and temperature. Commonly, around 50% concentrations of sulfuric acid, 25–40°C, and 30:1 w/w of acid/polymer ratio were suggested [ 2 , 3 , 5 , 17 ]. However, incubation time from 2 h to 69 h yielded a wide range of crystallinity values. The resulting products, bacterial cellulose nanocrystals (BCNCs) or bacterial cellulose nanofibers (BCNFs), can be obtained by these degradation processes. However, there is a main problem in these degradation processes that the desired polymer length cannot be obtained by these processes. The degradation process is highly affected by the quality of starting material. Even if different researchers use BC, the quality parameters such as density, tensile strength, and crystallinity as are different. Therefore, proposed degradation methods can only be repeated if previously used for BC. To understand the effect of degradation process on BC, various characterization methods were used as Fourier-transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM), Atomic force microscopy (AFM), Scanning electron microscope (SEM), and Thermogravimetric analysis (TGA) methods. It is very difficult to get significant data and/or observations from SEM analysis, because of uniformly disturbed fibers of BC and degraded BCs. Some of the researchers use AFM to measure fiber sizes, but TEM images are clearer than AFM. Therefore, TEM is useful for measuring the sizes of fibers recently. FTIR is essential for understanding the intra- and inter-molecular bonding interactions. To define these interactions, different measures of crystallinity such as TCI, LOI, and HBI have been proposed. The values of the formulations are proportional to crystallinity degree. However, the exact value of crystallinity should be calculated by XRD method. Thermal behavior of BC and degraded BCs were significantly different. Hence, TGA analysis is essential for characterization of compounds. The study aimed to investigate the effect of physical and chemical degradation methods. To control the fiber length and shape, fibers were previously rearranged by solvent of DMAc-LiCl. The rearrangement of fibers is based on increasing the crystallinity of polymer (Vasconcelos et al. 2017). After rearrangement of BC fibers, degradation of BC was performed with milling with liquid nitrogen or sulfuric acid treatment. The compounds were analyzed with FTIR, TEM, AFM, TGA, and XRD. Materials and Method Production of Bacterial Cellulose Komagataeibacter xylinus FC01 strain was inoculated in M1A05P5 medium that was composed of glucose 10 g/L, yeast extract 10 g/L, peptone 7g/L, acetic acid 1.5 g/L and ethanol 5 g/L at pH 5.0. BC was produced at room temperature under static culture and at the end of seventh day of production, BC was collected from the surface of medium. The impurities were removed by NaOH (10%, w/v) at 80°C for 1h. Then, pellicles were consecutively incubated with distilled water, ethanol (70%, v/v), and acetone for 24h, respectively. Treatments of Bacterial Cellulose Pellicles Solvent Pre-Treatments BC pellicles were pre-treated with 2%, 4%, and 8% (w/v) LiCl-Dimethylacetamide (N,N Dimethylacetamide DMAc) (Sigma-Aldrich) solution for 2 days at room temperature. After that, they were washed with ethanol (70%, v/v) and distilled water. Degradation with Sulfuric Acid Pre-treated samples were filtered using cellulose nitrate filter (0.2 µm). Residues on the filter were collected with NaOH (40%, w/v) solution and washed with distilled water. They were collected by centrifugation at 8000 rpm for 20 min 3 times. They were filtrated again with cellulose nitrate filter (0.2 µm). The concentration of wet residues was adjusted to 30% (w/v) and they were degraded with 40% or 65% (v/v) sulfuric acid solution for autoclave conditions (121°C, 15 min.) or 2 days at room temperature, respectively [ 3 , 8 ]. After the termination of treatment with addition of water, samples were centrifuged at 12000 rpm for 15 min 3 times. Degradation with Liquid Nitrogen Samples were ground in rice mortar for 15 min after the addition of excess amount of liquid nitrogen (LN). Characterization of Degraded BC Transmission Electron Microscopy (TEM) Suspensions of samples were sonicated for 1 min and deposited on TEM copper grid covered with carbon film (Agar Scientific, Holey Carbon Film on 200 Mesh Copper Grid) and held for 3 min. Excess liquid was carefully removed by filter paper and samples were stained with 2% uranyl acetate solution (TED PELLA 19485) to enhance microscopic resolution. After drying of samples, Jeol 2100 Plus TEM with an acceleration voltage of 120 kV was used for imaging. Atomic Force Microscopy (AFM) Sample suspensions were sonicated for 30 sec and dropped on glass slide surface. After drying of samples at room temperature, they were analyzed in contact mode using Si cantilever with a scanning rate of 1.0 Hz by AFM (Park Systems XE 100). XEI image processing program was used for examining the AFM images. Fourier Transform Infrared Spectroscopy (FT-IR) Samples were dried for overnight and mixed with KBr. Samples were analyzed with Thermo Scientific NicoletTM iS10T with ATR. The spectra were recorded with 64 scans in the ranges of 4000–550 cm − 1 and a spectral resolution of 1 cm − 1 [ 18 ]. Thermogravimetric Analysis (TGA) Determination of thermal stability of produced bacterial cellulose samples was achieved by thermogravimetric analysis by using Perkin Elmer TGA 8000 device. Dried bacterial cellulose samples were used for this analysis and analysis was performed with the temperature range of 25°C to 550°C at 20°C/min. X-Ray Diffraction Analysis (XRD) The samples were dried and pattern were obtained by Shimadzu XRD-6000 with Cu X-ray tube (λ = 1.5405 Å). The measuring angle was 5–50° with the step size of 0.02°. Crystallinity index (CrI) was calculated by using Eq. ( 1 ); $$CrI= \left(1-\frac{{I}_{am}}{{I}_{200}}\right)\times 100$$ 1 I am is the amorphous region around 16° and I 200 is the maximum peak at crystalline region around 22.5° [ 19 ]. Crystallite size (CS) was calculated by using Eq. ( 2 ); $$CS=\frac{k \times \lambda }{B \times cos\theta }$$ 2 K is a dimensionless number that was assumed as 0.94 and λ is the wavelength of X-ray as 1.5405 Å. B is the full width half maximum at θ (Bragg’s angle, radian) [ 20 ]. B was calculated by peak deconvolution by Gaussian using Origin Pro 2021 software [ 21 , 22 ]. Software All measurements were performed at least in triplicate, and the results are reported as the mean ± standard deviation. Statistical analysis was conducted using Microsoft Excel. Statistical significance was considered when the p-value was < 0.05 in all the analyses. FTIR plots with baseline corrections, the derivations of TGA data with their plots, and XRD analysis were done by Origin Pro 2021 (Originlab, USA). After visualization of degraded BC by TEM, the sizes of fibrils and crystals were measured by ImageJ (NIH, USA). Results and Discussion Degradation performance of the treatments The degradation of BC was performed under different conditions that were based on observation of the effect of rearranging fibers of BC with control groups (Table 1 ). During the dissolution of LiCl in DMAc, a chemical reaction takes place involving DMAc, Li + ions, hydroxyl groups of bacterial cellulose, and Cl − ions. The presence of Cl − ions has a significant impact on the molecular interactions within the system. Specifically, Cl − ions interact with the hydroxyl groups of bacterial cellulose, leading to the breaking of hydrogen bonds. As a result, the bacterial cellulose fibers undergo dispersion and separation. This phenomenon is attributed to the disruptive effect of Cl − ions on the hydrogen bonding network, facilitating the solvation, and restructuring of bacterial cellulose fibers within the DMAc-LiCl solvent system ([ 23 ]. The sets of BCNF3, BCNF4, and BCNF5 were not degraded by the low concentrations of DMAc-LiCl and liquid nitrogen treatment (Supplementary material). After treatments, BC sheets were aggregated and unhomogenized forms in undegraded sets. BCNF1 was the control set from reference studies, while BCNF2 was the control set of observation of the differences between liquid nitrogen and acid treatments. Table 1 Degradation sets of this study Set name Pretreatment step Degradation step Degradation observation BCNF1 - S65-R + BCNF2 - S40-A + BCNF3 - LN n.a. BCNF4 L2-R LN n.a. BCNF5 L4-R LN n.a. BCNF6 L8-R LN + BCNF7 L8-R S40-A + L2, L4, and L8: LiCl concentration at 2, 4, and 8% (g/100mL) in DMAc solvent, S40: 40% sulfuric acid, S65: 65% sulfuric acid, R: room temperature, A: autoclave, LN: grinding after freezing with liquid nitrogen, n.a: not applicable TEM analysis TEM images of degraded BCs as BCNF1, BCNF2, BCNF6, and BCNF7, are shown in Fig. 1. Fibrils can be seen clearly in BCNF1, BCNF2, and BCNF6, while BCNF7 seemed like spherical-like nanocrystal. Milling with liquid nitrogen did not sustain effective degradation of BC. However, when the BC fibers were rearranged with DMAc-LiCl solution at 8%, fine and uniform fibers were obtained. The effect of rearranging process can be seen in BCNF2 and BCNF7. Even though the main degradation process was the same, resulting forms of BC were completely different as nanofiber in BCNF2 and nanocrystal in BCNF7. The sizes of degraded BCs were shown in Fig. 2. BCNF7 was spherical-like nanocrystal and average diameter was 56 ± 11 nm. The fibers of BCNF1, BCNF2, and BCNF6 were in various lengths and widths. The smallest length and widths were obtained in BCNF1 set that was 312 ± 35 nm and 9 ± 1 nm, respectively. The mild concentration with harsh treatment condition of BCNF2 showed 491 ± 28 nm and 12 ± 1 nm, respectively. The longest fiber was obtained from BCNF6 set as 700 nm ± 12 nm. The width was 13 ± 1 nm that was almost the same as BCNF2. Vasconcelos et al., (2017) measured the length and width of fibers after degradation. They obtained 622 nm and 1322 nm length fibers from 50% sulfuric acid for 1 h and mixture of sulfuric acid and hydrochloric acid treatment, respectively [ 24 ] Martinez-Sanz et al., (2011) increased the degradation time from 2 h to 48 h with sulfuric acid that gave decreasing of fiber lengths from 1450 nm to 469 nm, respectively [ 3 ]. The higher sulfuric acid concentration of BCNF1 than sample of Martinez-Sanz et al., (2011) at the same time of degradation gave low fiber length as 312 nm that was expected result, because of the strength of sulfuric acid. Doan & Chiang (2022) used 55% sulfuric acid with different ratios of acid/polymer. They found that elevating ratio of acid/polymer sustain spherical-like nanocrystal formation while rod-like nanofibrils were obtained by low ratio of it. The sizes of spherical-like nanocrystal and rod-like fibers were 14 nm and around 50 nm, respectively [ 4 ]. Chen et al., (2017) used enzymes and grinding for obtaining CNFs from plants. They obtained around 760 nm length and 9 nm diameter CNFs. The process was expensive because of using enzymes and even the Authors claimed the controlled degradation of cellulose, there was no any reaction kinetics and/or correlation [ 25 ]. The researcher’s starting material was microcrystalline cellulose that commonly has lower polymerization degree and fiber length compared to bacterial cellulose. Therefore, they could get low sizes of nanocrystals. AFM Analysis AFM images were shown in Fig. 3. Fine fibers were obtained in set of BCNF1. The long-time incubation in BCNF1 sustained homogenized fibers. Although preparation time of BCNF2 is shorter than BCNF1, the autoclave treatment caused aggregations. Milling with liquid nitrogen also caused aggregations of fibers. Because of the lackness of uniform milling process, this result was overseen and acceptable. However, homogenization seems higher in rearranged and milled with liquid nitrogen sample (BCNF6) than autoclave treated sample (BCNF2). Spherical-like nanocrystals showed highly homogenized particles as shown in Fig. 3d. FTIR Analysis The spectrum patterns of BCNF2 and BCNF7 are similar (Fig. 4). The degradation process of autoclave treatment caused the same pattern. Therefore, rearrangement of fibrils before degradation had no effect on molecular interactions. The bands at around 3340 cm − 1 are related to main hydrogen bonds for 3-OH … O5. All degraded BCs showed high intensity for hydroxyl groups, except BCNF1. As can be seen in Fig. 3a, BCNF1 fibrils dispersed more uniformly than other degraded BCs. The result supports the low intensity for hydrogen bonds, because of low interaction between each fibril. C-H stretching vibration at 2900 cm − 1 can be observed in all degraded BCs. The intensity was the highest in BCNF2 and BCNF7. There was a slight peak on BCNF1 sample, while the peak was sharp on BCNF6. On the other side, C-OH out of plane bending is related to around 670 cm − 1 . The C-OH bending of BCNF1 is less than other degraded BCs. The bands of absorption spectra through 1200 cm − 1 -1000 cm − 1 indicate that samples have inter- and intra-molecular bonding. All degraded BCs showed CH 2 symmetric bending, C-C and C-C-OH stretching, and C-O-C glycoside bonds. The one of the characteristic bands at 1373 cm − 1 related with C-H bending, O-H bending and CH 2 wagging. The ratio of this band to band at 2900 cm − 1 gives Total Crystallinity Index (TCI). CH 2 scissoring vibrations at 1427 cm − 1 is another characteristic band for crystallinity of cellulose. The amorphous region of BC can be observed at 895 cm − 1 . The ratio of these bands gives Lateral Order Index (LOI). Hydrogen Bond Index (HBI) is the ratio O-H stretches at 3350 cm − 1 to O-H in plane bending vibrations at 1330 cm − 1 . TCI, LOI, and HBI values of BC and degraded BCs were shown in Table 2 . TCI and LOI values were lower, while HBI values were higher in degraded BCs than BC. According to the crystallinity indices of these items, all degraded BCs had lower crystallinity degrees. However, because of low fiber length, crystallinity must be higher in degraded BCs than BC. The reason for this is the structure of the starting material. The difference of literature is that we used BC in wet state. Therefore, fibril strength and chemical bonds were preserved. During the degradation of BCs, each fibrils make new hydrogen bonds, and they cause amorphous structure. Table 2 Crystallinity degree and investigations of BC and degraded BCs via XRD and FTIR. Crystallinity by XRD (%) Crystallite Size (Å) TCI (1373/2900) LOI (1427/895) HBI (3350/1337) BC 47.1 0.157 0.95 5.88 0.28 BCNF1 45.5 0.070 0.41 0.44 20.52 BCNF2 64.7 0.035 0.48 1.31 10.14 BCNF6 68.2 0.515 0.74 3.54 13.64 BCNF7 95.5 0.636 0.56 1.47 8.13 Anwar et al., (2021) performed sulfuric acid treatment on bacterial cellulose and crystallinity degree rose from 74–89% [ 1 ]. Vasconcelos et al., (2017) used sulfuric acid and hydrochloric acid with different treatment times and concentrations. Most of the degradation sets showed increasing crystallinity degree. However, there was only one set that was 65% (w/w) sulfuric acid at room temperature for 2 h had low crystallinity degree [ 26 ]. This data can also be supported by Martinez-Sanz et al., (2011). They compared 55% sulfuric acid degradation at 2 h and 48 h and crystallinity degree was slightly lower in 2 h degradation than untreated BC while 48 h degradation increased the crystallinity degree to 90% from 79% [ 3 ]. Yan et al., (2017) also used 50% (w/w) sulfuric acid at 40°C for 3 h, but crystallinity degree was elevated from 75–90% [ 2 ]. Even in physical degradations, crystallinity degree was decreasing as Tsalagkas et al., (2016) showed the effect of sonication. TCI, LOI, and HBI values were also higher than untreated BC [ 7 ]. XRD Analysis XRD analysis is essential to understand the amorphous structure of cellulose. Crystallinity degree and crystallite size of polymers were shown in Table 2 . Low crystallinity value was observed in untreated BC, according to literature [ 27 – 31 ]. BCNF1 showed almost the same crystallinity as untreated BC. However, sulfuric acid with autoclave treatment, BCNF2, had a significant effect on crystallinity. It elevated 17.6% based on untreated BC. Solvent treatment with liquid nitrogen degradation also increased crystallinity to 68.2%. The highest crystallinity, 95.5%, was observed at BCNF7. Sulfuric acid treatments decreased the crystallite size at higher than 2- and 4-fold for BCNF1 and BCNF2, respectively. However, the crystallite sizes of solvent treated samples were around 3.5- and 4-fold higher than untreated BC. Cellulose types of the polymer are strongly changing by the application. XRD spectra were shown in Fig. 5. Characteristic peaks of cellulose I have 14.7° (1–10), 16.6° (110), 22.7° (200), and 34.5° (004). Untreated BC had all characteristic peaks of cellulose I. At the sets of sulfuric acid treatments, BCNF1 and BCNF2, the peaks at 20° and 22.7° of untreated BC merged at 22° that is characteristic peak of cellulose II. Solvent treated samples, BCNF6 and BCNF7, still had two peaks but they were at 20° and 22°. In addition to that there were strong peaks at around 12.4° and 26.7° of BCNF6 and BCNF7. These peaks are essential for cellulose II [ 27 , 32 , 33 ]. TGA Analysis Nanofibrils of BCs as BCNF1, BCNF2, and BCNF6 had almost same degradation pattern (Fig. 6a). Weight loss of them was higher than BC as between 3–5 times (Table 3 ). Spherical nanocrystal, BCNF7, had three degradation temperatures and the highest one was 488.7°C. The second degradation temperatures of nanofibrils were between 303–309°C (Fig. 6b). However, the weight loss of nanofibrils were completely different. Nanofibrils showed two degradation steps while nanocrystal had three degradation steps. Sulfated fibers were depolymerized firstly, and continuous degradation was occurred in crystallite fractions in nanofibrils [ 28 , 29 ]. However, nanocrystals were depolymerized three times and nanocrystals were obtained at the last degradation step. Table 3 Onset temperature, degradation temperature, and weight loss at this temperature of BC and BCNFs. Sample 1st degradation temp. Weight loss at 1st degradation 2nd degradation temp. Weight loss at 2nd degradation 3rd degradation temp. Weight loss at 3rd degradation BC 128.1 15.6 381.7 71.4 BCNF1 148.8 61.3 304.7 19.7 BCNF2 124.5 48.2 309.1 49.4 BCNF6 200.0 80.5 303.6 14.4 BCNF7 87.9 60.1 276.6 12.4 488.7 19.3 Conclusion Rearranging fiber positions in BC helps efficient degradation even using milling with liquid nitrogen. Depending on the degradation method, desired nanoparticle forms like nanofiber or nanocrystal can be obtained by using previously rearranged fibers. Milling with liquid nitrogen was not useful to obtain nanoparticles. However, when it is combined with rearrangement process, nanofibers were obtained by milling with liquid nitrogen. In addition to that rearrangement helps to obtain spherical-like nanocrystals. The low concentration of LiCl had no effect on degradation of BC. BC was not dried before degradation in this study while it is different from literature. It also causes BC to be in amorphous form after degradation. Declarations Ethics approval and consent to participate Not applicable Consent for publication We have consent from the responsible authorities at the Yeditepe University where the work has been carried out. Availability of data and materials The results/data/figures in this manuscript have not been published elsewhere, nor are they under consideration (from you or one of your Contributing Authors) by another publisher. Competing interests I declare that the authors have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author contributions A.O.A prepare the concept of the study and made calculations of analysis, and wrote the main manuscript, F.S and N.K. performed experiments and collected data. 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Morais, F.K. Andrade, M. de S.M. de Souza Filho, M. de F. Rosa, Bacterial cellulose nanocrystals produced under different hydrolysis conditions: Properties and morphological features, Carbohydr Polym 155 (2017) 425–431. https://doi.org/10.1016/j.carbpol.2016.08.090. S. Naduparambath, J. T.V., V. Shaniba, S. M.P., A.K. Balan, E. Purushothaman, Isolation and characterisation of cellulose nanocrystals from sago seed shells, Carbohydr Polym 180 (2018) 13–20. https://doi.org/10.1016/j.carbpol.2017.09.088. J. Araki, S. Kuga, Effect of trace electrolyte on liquid crystal type of cellulose microcrystals, Langmuir 17 (2001) 4493–4496. https://doi.org/10.1021/la0102455. S. Julien, E. Chornet, R.P. Overend, Influence of acid pretreatment (H2SO4, HCl, HNO3) on reaction selectivity in the vacuum pyrolysis of cellulose, J Anal Appl Pyrolysis 27 (1993) 25–43. https://doi.org/10.1016/0165-2370(93)80020-Z. T.K.Q. Doan, K.Y. Chiang, Characteristics and kinetics study of spherical cellulose nanocrystal extracted from cotton cloth waste by acid hydrolysis, Sustainable Environment Research 32 (2022). https://doi.org/10.1186/s42834-022-00136-9. H. Yan, X. Chen, H. Song, J. Li, Y. Feng, Z. Shi, X. Wang, Q. Lin, Synthesis of bacterial cellulose and bacterial cellulose nanocrystals for their applications in the stabilization of olive oil pickering emulsion, Food Hydrocoll 72 (2017) 127–135. https://doi.org/10.1016/j.foodhyd.2017.05.044. A.D. French, Idealized powder diffraction patterns for cellulose polymorphs, Cellulose 21 (2014) 885–896. https://doi.org/10.1007/s10570-013-0030-4. Y. Meng, C.I. Contescu, P. Liu, S. Wang, S.H. Lee, J. Guo, T.M. Young, Understanding the local structure of disordered carbons from cellulose and lignin, Wood Sci Technol 55 (2021) 587–606. https://doi.org/10.1007/s00226-021-01286-6. Additional Declarations No competing interests reported. Supplementary Files floatimage1.png Graphical abstract SUPPLEMENTARYMATERIALCellulose.docx 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-4205627","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":288531306,"identity":"21f38d57-e12f-4d5b-afab-2405cd1824a2","order_by":0,"name":"Fulya Şahin","email":"","orcid":"","institution":"Yeditepe University","correspondingAuthor":false,"prefix":"","firstName":"Fulya","middleName":"","lastName":"Şahin","suffix":""},{"id":288531307,"identity":"8e53fcc0-237e-4c10-af23-776ff604e39d","order_by":1,"name":"Neslihan Kayra","email":"","orcid":"","institution":"Yeditepe University","correspondingAuthor":false,"prefix":"","firstName":"Neslihan","middleName":"","lastName":"Kayra","suffix":""},{"id":288531308,"identity":"223dd237-6973-45ea-8419-ed887d8281d6","order_by":2,"name":"Ali Özhan Aytekin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIie3PPQrCMBiA4YRAXVKzJmTwCpWCICqeRRxcFDyASKUQl+hcJ2/hXgJO/hwgo+BcEURUxNaf0bajYN4hX4bvIQQAk+knw58JPRDF0yrkJDQhMEgIyk8AQM97FiFkHR77AzUkWIld/bIoFRGA0aH7nTBv0+bBUlE2GY3d3lSXBQKIzRbfiRNKh2NLUWcLBe9JDWNiITuNKOxe8V3RZkKqUjezyRJXuC3iV+yR4OCsW5mESVyp2dMOC1ahYBNPtwWCfupfCFm5Gp9qhMjOnp5vujEf+2F0SCHv/NeA4nl6mftxw/e85Vk2mUymf+sB6MBK2U1kHVMAAAAASUVORK5CYII=","orcid":"","institution":"Gozen Corp","correspondingAuthor":true,"prefix":"","firstName":"Ali","middleName":"Özhan","lastName":"Aytekin","suffix":""}],"badges":[],"createdAt":"2024-04-02 09:46:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4205627/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4205627/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54316187,"identity":"bdd288f4-1384-431c-b487-6a089edd08cf","added_by":"auto","created_at":"2024-04-08 17:48:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":326259,"visible":true,"origin":"","legend":"\u003cp\u003eTEM A) BCNF1, B) BCNF2, C) BCNF6, D) BCNF7\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4205627/v1/dad9646921b3c7be5f42d934.png"},{"id":54316184,"identity":"d5ff96eb-820f-4b07-b5cb-a012af61fee1","added_by":"auto","created_at":"2024-04-08 17:48:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13262,"visible":true,"origin":"","legend":"\u003cp\u003eThe sizes of degraded BC’s as a) length and b) width\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4205627/v1/0a27dcb4b671984f39fc4a31.png"},{"id":54316190,"identity":"d34ac5a0-54c9-4d83-97da-97a4d95b12fa","added_by":"auto","created_at":"2024-04-08 17:48:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":548942,"visible":true,"origin":"","legend":"\u003cp\u003eAFM images of a) BCNF1, b) BCNF2, c) BCNF6, and d) BCNF7\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4205627/v1/12ad53d7bbc21ae170118384.png"},{"id":54316188,"identity":"0e4dab51-3aa0-4027-856c-7951b43be521","added_by":"auto","created_at":"2024-04-08 17:48:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":76667,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of BCNF1, BCNF2, BCNF6, and BCNF7.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4205627/v1/8913acd92a5679111267be3a.png"},{"id":54316185,"identity":"9dc8f4a9-3a24-489f-af2d-522531472818","added_by":"auto","created_at":"2024-04-08 17:48:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":88069,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4205627/v1/ead0e8e0c7e7f8dd5b3aab2a.png"},{"id":55264446,"identity":"befb0f85-35e1-43ff-b8c4-73754251bada","added_by":"auto","created_at":"2024-04-25 01:43:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1620184,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4205627/v1/b2ed7de6-dd67-4cba-a6ea-d05b6a4b9814.pdf"},{"id":54316189,"identity":"d4f90d5d-2294-46d9-97cc-b788260a8431","added_by":"auto","created_at":"2024-04-08 17:48:27","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":218253,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4205627/v1/d09093d0396b775e2d00330b.png"},{"id":54316186,"identity":"f8eb505d-89a8-4388-a194-8bf127e4fb8a","added_by":"auto","created_at":"2024-04-08 17:48:26","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":620233,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYMATERIALCellulose.docx","url":"https://assets-eu.researchsquare.com/files/rs-4205627/v1/642599b5912aa38c45908752.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Physical and Chemical Degradation of Bacterial Cellulose via Rearranging Fibers","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eDegradation of bacterial cellulose is successfully achieved at wet state of polymer.\u003c/li\u003e\n \u003cli\u003eLow exposure time of solvent asissted to change fibers position for degradation.\u003c/li\u003e\n \u003cli\u003eFreezing by liquid nitrogen and milling sustain nanofibril formation.\u003c/li\u003e\n \u003cli\u003eSulfuric acid treatment of rearranged fibers resulted in spherical nanocrystals.\u003c/li\u003e\n \u003cli\u003eRearrangement of fibers at wet state will help to obtain desired nanoparticle form and sizes.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eBacterial cellulose (BC) is a unique polysaccharide that has significant properties such as high tensile strength, high crystallinity, and high purity. Because of these properties, BC has been reported to be used in different areas.\u003c/p\u003e \u003cp\u003eThe changing fiber lengths of BC have significantly different uses. Therefore, various cellulose degradation processes have been presented. They are based on chemical such as sulfuric acid [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], hydrochloric acid [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and enzymes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] or physical as sonication [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], hydrothermal explosion [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and high-pressure homogenization [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferent kinds of solvents have been used for changing fiber formations to obtain new and/or unique features [\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The one of the most studied solvents is dimethyl acetamide-LiCl solution (DMAc-LiCl). The highest concentration of LiCl is 8% (w/v) in DMAc. The solvent penetrates the BC fibers, and it causes swelling in solvent. The swelling effect make transparent BC. Because fibers are rearranged and positioned as parallel [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe extraction of cellulose and preparation of cellulose nanofibers were initially performed from plants and their wastes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The harsh conditions such as acid with hydrothermal process as for explosion of lignin and hemicellulose structures are commonly used. Later, acid-based degradations of bacterial cellulose studies were proposed [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The investigation of degradation was based on different concentrations of sulfuric acid and/or hydrochloric acid, incubation time, acid/polymer weight ratio, and temperature. Commonly, around 50% concentrations of sulfuric acid, 25\u0026ndash;40\u0026deg;C, and 30:1 w/w of acid/polymer ratio were suggested [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, incubation time from 2 h to 69 h yielded a wide range of crystallinity values.\u003c/p\u003e \u003cp\u003eThe resulting products, bacterial cellulose nanocrystals (BCNCs) or bacterial cellulose nanofibers (BCNFs), can be obtained by these degradation processes. However, there is a main problem in these degradation processes that the desired polymer length cannot be obtained by these processes. The degradation process is highly affected by the quality of starting material. Even if different researchers use BC, the quality parameters such as density, tensile strength, and crystallinity as are different. Therefore, proposed degradation methods can only be repeated if previously used for BC.\u003c/p\u003e \u003cp\u003eTo understand the effect of degradation process on BC, various characterization methods were used as Fourier-transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM), Atomic force microscopy (AFM), Scanning electron microscope (SEM), and Thermogravimetric analysis (TGA) methods. It is very difficult to get significant data and/or observations from SEM analysis, because of uniformly disturbed fibers of BC and degraded BCs. Some of the researchers use AFM to measure fiber sizes, but TEM images are clearer than AFM. Therefore, TEM is useful for measuring the sizes of fibers recently. FTIR is essential for understanding the intra- and inter-molecular bonding interactions. To define these interactions, different measures of crystallinity such as TCI, LOI, and HBI have been proposed. The values of the formulations are proportional to crystallinity degree. However, the exact value of crystallinity should be calculated by XRD method. Thermal behavior of BC and degraded BCs were significantly different. Hence, TGA analysis is essential for characterization of compounds.\u003c/p\u003e \u003cp\u003eThe study aimed to investigate the effect of physical and chemical degradation methods. To control the fiber length and shape, fibers were previously rearranged by solvent of DMAc-LiCl. The rearrangement of fibers is based on increasing the crystallinity of polymer (Vasconcelos et al. 2017). After rearrangement of BC fibers, degradation of BC was performed with milling with liquid nitrogen or sulfuric acid treatment. The compounds were analyzed with FTIR, TEM, AFM, TGA, and XRD.\u003c/p\u003e"},{"header":"Materials and Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProduction of Bacterial Cellulose\u003c/h2\u003e \u003cp\u003e \u003cem\u003eKomagataeibacter xylinus\u003c/em\u003e FC01 strain was inoculated in M1A05P5 medium that was composed of glucose 10 g/L, yeast extract 10 g/L, peptone 7g/L, acetic acid 1.5 g/L and ethanol 5 g/L at pH 5.0. BC was produced at room temperature under static culture and at the end of seventh day of production, BC was collected from the surface of medium. The impurities were removed by NaOH (10%, w/v) at 80\u0026deg;C for 1h. Then, pellicles were consecutively incubated with distilled water, ethanol (70%, v/v), and acetone for 24h, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTreatments of Bacterial Cellulose Pellicles\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eSolvent Pre-Treatments\u003c/h2\u003e \u003cp\u003eBC pellicles were pre-treated with 2%, 4%, and 8% (w/v) LiCl-Dimethylacetamide (N,N Dimethylacetamide DMAc) (Sigma-Aldrich) solution for 2 days at room temperature. After that, they were washed with ethanol (70%, v/v) and distilled water.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDegradation with Sulfuric Acid\u003c/h2\u003e \u003cp\u003ePre-treated samples were filtered using cellulose nitrate filter (0.2 \u0026micro;m). Residues on the filter were collected with NaOH (40%, w/v) solution and washed with distilled water. They were collected by centrifugation at 8000 rpm for 20 min 3 times. They were filtrated again with cellulose nitrate filter (0.2 \u0026micro;m). The concentration of wet residues was adjusted to 30% (w/v) and they were degraded with 40% or 65% (v/v) sulfuric acid solution for autoclave conditions (121\u0026deg;C, 15 min.) or 2 days at room temperature, respectively [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. After the termination of treatment with addition of water, samples were centrifuged at 12000 rpm for 15 min 3 times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDegradation with Liquid Nitrogen\u003c/h2\u003e \u003cp\u003eSamples were ground in rice mortar for 15 min after the addition of excess amount of liquid nitrogen (LN).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of Degraded BC\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eTransmission Electron Microscopy (TEM)\u003c/h2\u003e \u003cp\u003eSuspensions of samples were sonicated for 1 min and deposited on TEM copper grid covered with carbon film (Agar Scientific, Holey Carbon Film on 200 Mesh Copper Grid) and held for 3 min. Excess liquid was carefully removed by filter paper and samples were stained with 2% uranyl acetate solution (TED PELLA 19485) to enhance microscopic resolution. After drying of samples, Jeol 2100 Plus TEM with an acceleration voltage of 120 kV was used for imaging.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAtomic Force Microscopy (AFM)\u003c/h2\u003e \u003cp\u003eSample suspensions were sonicated for 30 sec and dropped on glass slide surface. After drying of samples at room temperature, they were analyzed in contact mode using Si cantilever with a scanning rate of 1.0 Hz by AFM (Park Systems XE 100). XEI image processing program was used for examining the AFM images.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFourier Transform Infrared Spectroscopy (FT-IR)\u003c/h2\u003e \u003cp\u003eSamples were dried for overnight and mixed with KBr. Samples were analyzed with Thermo Scientific NicoletTM iS10T with ATR. The spectra were recorded with 64 scans in the ranges of 4000\u0026ndash;550 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a spectral resolution of 1 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThermogravimetric Analysis (TGA)\u003c/h2\u003e \u003cp\u003eDetermination of thermal stability of produced bacterial cellulose samples was achieved by thermogravimetric analysis by using Perkin Elmer TGA 8000 device. Dried bacterial cellulose samples were used for this analysis and analysis was performed with the temperature range of 25\u0026deg;C to 550\u0026deg;C at 20\u0026deg;C/min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eX-Ray Diffraction Analysis (XRD)\u003c/h2\u003e \u003cp\u003eThe samples were dried and pattern were obtained by Shimadzu XRD-6000 with Cu X-ray tube (λ\u0026thinsp;=\u0026thinsp;1.5405 \u0026Aring;). The measuring angle was 5\u0026ndash;50\u0026deg; with the step size of 0.02\u0026deg;. Crystallinity index (CrI) was calculated by using Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e);\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$CrI= \\left(1-\\frac{{I}_{am}}{{I}_{200}}\\right)\\times 100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eI\u003c/em\u003e \u003csub\u003e \u003cem\u003eam\u003c/em\u003e \u003c/sub\u003e is the amorphous region around 16\u0026deg; and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003e200\u003c/em\u003e\u003c/sub\u003e is the maximum peak at crystalline region around 22.5\u0026deg; [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Crystallite size (CS) was calculated by using Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e);\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$CS=\\frac{k \\times \\lambda }{B \\times cos\\theta }$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eK is a dimensionless number that was assumed as 0.94 and λ is the wavelength of X-ray as 1.5405 \u0026Aring;. B is the full width half maximum at \u003cem\u003eθ\u003c/em\u003e (Bragg\u0026rsquo;s angle, radian) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. B was calculated by peak deconvolution by Gaussian using Origin Pro 2021 software [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSoftware\u003c/h2\u003e \u003cp\u003eAll measurements were performed at least in triplicate, and the results are reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analysis was conducted using Microsoft Excel. Statistical significance was considered when the p-value was \u0026lt;\u0026thinsp;0.05 in all the analyses. FTIR plots with baseline corrections, the derivations of TGA data with their plots, and XRD analysis were done by Origin Pro 2021 (Originlab, USA). After visualization of degraded BC by TEM, the sizes of fibrils and crystals were measured by ImageJ (NIH, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDegradation performance of the treatments\u003c/h2\u003e \u003cp\u003eThe degradation of BC was performed under different conditions that were based on observation of the effect of rearranging fibers of BC with control groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). During the dissolution of LiCl in DMAc, a chemical reaction takes place involving DMAc, Li\u003csup\u003e+\u003c/sup\u003e ions, hydroxyl groups of bacterial cellulose, and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions. The presence of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions has a significant impact on the molecular interactions within the system. Specifically, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions interact with the hydroxyl groups of bacterial cellulose, leading to the breaking of hydrogen bonds. As a result, the bacterial cellulose fibers undergo dispersion and separation. This phenomenon is attributed to the disruptive effect of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions on the hydrogen bonding network, facilitating the solvation, and restructuring of bacterial cellulose fibers within the DMAc-LiCl solvent system ([\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The sets of BCNF3, BCNF4, and BCNF5 were not degraded by the low concentrations of DMAc-LiCl and liquid nitrogen treatment (Supplementary material). After treatments, BC sheets were aggregated and unhomogenized forms in undegraded sets. BCNF1 was the control set from reference studies, while BCNF2 was the control set of observation of the differences between liquid nitrogen and acid treatments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDegradation sets of this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSet name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePretreatment step\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDegradation step\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDegradation observation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS65-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS40-A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL2-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL4-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL8-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL8-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS40-A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eL2, L4, and L8: LiCl concentration at 2, 4, and 8% (g/100mL) in DMAc solvent, S40: 40% sulfuric acid, S65: 65% sulfuric acid, R: room temperature, A: autoclave, LN: grinding after freezing with liquid nitrogen, n.a: not applicable\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTEM analysis\u003c/h2\u003e \u003cp\u003eTEM images of degraded BCs as BCNF1, BCNF2, BCNF6, and BCNF7, are shown in Fig.\u0026nbsp;1. Fibrils can be seen clearly in BCNF1, BCNF2, and BCNF6, while BCNF7 seemed like spherical-like nanocrystal. Milling with liquid nitrogen did not sustain effective degradation of BC. However, when the BC fibers were rearranged with DMAc-LiCl solution at 8%, fine and uniform fibers were obtained. The effect of rearranging process can be seen in BCNF2 and BCNF7. Even though the main degradation process was the same, resulting forms of BC were completely different as nanofiber in BCNF2 and nanocrystal in BCNF7.\u003c/p\u003e \u003cp\u003eThe sizes of degraded BCs were shown in Fig.\u0026nbsp;2. BCNF7 was spherical-like nanocrystal and average diameter was 56\u0026thinsp;\u0026plusmn;\u0026thinsp;11 nm. The fibers of BCNF1, BCNF2, and BCNF6 were in various lengths and widths. The smallest length and widths were obtained in BCNF1 set that was 312\u0026thinsp;\u0026plusmn;\u0026thinsp;35 nm and 9\u0026thinsp;\u0026plusmn;\u0026thinsp;1 nm, respectively. The mild concentration with harsh treatment condition of BCNF2 showed 491\u0026thinsp;\u0026plusmn;\u0026thinsp;28 nm and 12\u0026thinsp;\u0026plusmn;\u0026thinsp;1 nm, respectively. The longest fiber was obtained from BCNF6 set as 700 nm\u0026thinsp;\u0026plusmn;\u0026thinsp;12 nm. The width was 13\u0026thinsp;\u0026plusmn;\u0026thinsp;1 nm that was almost the same as BCNF2. Vasconcelos et al., (2017) measured the length and width of fibers after degradation. They obtained 622 nm and 1322 nm length fibers from 50% sulfuric acid for 1 h and mixture of sulfuric acid and hydrochloric acid treatment, respectively [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] Martinez-Sanz et al., (2011) increased the degradation time from 2 h to 48 h with sulfuric acid that gave decreasing of fiber lengths from 1450 nm to 469 nm, respectively [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The higher sulfuric acid concentration of BCNF1 than sample of Martinez-Sanz et al., (2011) at the same time of degradation gave low fiber length as 312 nm that was expected result, because of the strength of sulfuric acid. Doan \u0026amp; Chiang (2022) used 55% sulfuric acid with different ratios of acid/polymer. They found that elevating ratio of acid/polymer sustain spherical-like nanocrystal formation while rod-like nanofibrils were obtained by low ratio of it. The sizes of spherical-like nanocrystal and rod-like fibers were 14 nm and around 50 nm, respectively [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Chen et al., (2017) used enzymes and grinding for obtaining CNFs from plants. They obtained around 760 nm length and 9 nm diameter CNFs. The process was expensive because of using enzymes and even the Authors claimed the controlled degradation of cellulose, there was no any reaction kinetics and/or correlation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The researcher\u0026rsquo;s starting material was microcrystalline cellulose that commonly has lower polymerization degree and fiber length compared to bacterial cellulose. Therefore, they could get low sizes of nanocrystals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAFM Analysis\u003c/h2\u003e \u003cp\u003eAFM images were shown in Fig.\u0026nbsp;3. Fine fibers were obtained in set of BCNF1. The long-time incubation in BCNF1 sustained homogenized fibers. Although preparation time of BCNF2 is shorter than BCNF1, the autoclave treatment caused aggregations. Milling with liquid nitrogen also caused aggregations of fibers. Because of the lackness of uniform milling process, this result was overseen and acceptable. However, homogenization seems higher in rearranged and milled with liquid nitrogen sample (BCNF6) than autoclave treated sample (BCNF2). Spherical-like nanocrystals showed highly homogenized particles as shown in Fig.\u0026nbsp;3d.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFTIR Analysis\u003c/h2\u003e \u003cp\u003eThe spectrum patterns of BCNF2 and BCNF7 are similar (Fig.\u0026nbsp;4). The degradation process of autoclave treatment caused the same pattern. Therefore, rearrangement of fibrils before degradation had no effect on molecular interactions. The bands at around 3340 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are related to main hydrogen bonds for 3-OH\u003csup\u003e\u0026hellip;\u003c/sup\u003eO5. All degraded BCs showed high intensity for hydroxyl groups, except BCNF1. As can be seen in Fig.\u0026nbsp;3a, BCNF1 fibrils dispersed more uniformly than other degraded BCs. The result supports the low intensity for hydrogen bonds, because of low interaction between each fibril. C-H stretching vibration at 2900 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be observed in all degraded BCs. The intensity was the highest in BCNF2 and BCNF7. There was a slight peak on BCNF1 sample, while the peak was sharp on BCNF6. On the other side, C-OH out of plane bending is related to around 670 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The C-OH bending of BCNF1 is less than other degraded BCs. The bands of absorption spectra through 1200 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e-1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicate that samples have inter- and intra-molecular bonding. All degraded BCs showed CH\u003csub\u003e2\u003c/sub\u003e symmetric bending, C-C and C-C-OH stretching, and C-O-C glycoside bonds.\u003c/p\u003e \u003cp\u003eThe one of the characteristic bands at 1373 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e related with C-H bending, O-H bending and CH\u003csub\u003e2\u003c/sub\u003e wagging. The ratio of this band to band at 2900 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e gives Total Crystallinity Index (TCI). CH\u003csub\u003e2\u003c/sub\u003e scissoring vibrations at 1427 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is another characteristic band for crystallinity of cellulose. The amorphous region of BC can be observed at 895 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The ratio of these bands gives Lateral Order Index (LOI). Hydrogen Bond Index (HBI) is the ratio O-H stretches at 3350 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to O-H in plane bending vibrations at 1330 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTCI, LOI, and HBI values of BC and degraded BCs were shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. TCI and LOI values were lower, while HBI values were higher in degraded BCs than BC. According to the crystallinity indices of these items, all degraded BCs had lower crystallinity degrees. However, because of low fiber length, crystallinity must be higher in degraded BCs than BC. The reason for this is the structure of the starting material. The difference of literature is that we used BC in wet state. Therefore, fibril strength and chemical bonds were preserved. During the degradation of BCs, each fibrils make new hydrogen bonds, and they cause amorphous structure.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCrystallinity degree and investigations of BC and degraded BCs via XRD and FTIR.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCrystallinity by XRD (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCrystallite Size (\u0026Aring;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTCI (1373/2900)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLOI (1427/895)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHBI (3350/1337)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.070\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e64.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e68.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.515\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e13.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e95.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAnwar et al., (2021) performed sulfuric acid treatment on bacterial cellulose and crystallinity degree rose from 74\u0026ndash;89% [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Vasconcelos et al., (2017) used sulfuric acid and hydrochloric acid with different treatment times and concentrations. Most of the degradation sets showed increasing crystallinity degree. However, there was only one set that was 65% (w/w) sulfuric acid at room temperature for 2 h had low crystallinity degree [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This data can also be supported by Martinez-Sanz et al., (2011). They compared 55% sulfuric acid degradation at 2 h and 48 h and crystallinity degree was slightly lower in 2 h degradation than untreated BC while 48 h degradation increased the crystallinity degree to 90% from 79% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Yan et al., (2017) also used 50% (w/w) sulfuric acid at 40\u0026deg;C for 3 h, but crystallinity degree was elevated from 75\u0026ndash;90% [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Even in physical degradations, crystallinity degree was decreasing as Tsalagkas et al., (2016) showed the effect of sonication. TCI, LOI, and HBI values were also higher than untreated BC [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eXRD Analysis\u003c/h2\u003e \u003cp\u003eXRD analysis is essential to understand the amorphous structure of cellulose. Crystallinity degree and crystallite size of polymers were shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Low crystallinity value was observed in untreated BC, according to literature [\u003cspan additionalcitationids=\"CR28 CR29 CR30\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. BCNF1 showed almost the same crystallinity as untreated BC. However, sulfuric acid with autoclave treatment, BCNF2, had a significant effect on crystallinity. It elevated 17.6% based on untreated BC. Solvent treatment with liquid nitrogen degradation also increased crystallinity to 68.2%. The highest crystallinity, 95.5%, was observed at BCNF7. Sulfuric acid treatments decreased the crystallite size at higher than 2- and 4-fold for BCNF1 and BCNF2, respectively. However, the crystallite sizes of solvent treated samples were around 3.5- and 4-fold higher than untreated BC.\u003c/p\u003e \u003cp\u003eCellulose types of the polymer are strongly changing by the application. XRD spectra were shown in Fig.\u0026nbsp;5. Characteristic peaks of cellulose I have 14.7\u0026deg; (1\u0026ndash;10), 16.6\u0026deg; (110), 22.7\u0026deg; (200), and 34.5\u0026deg; (004). Untreated BC had all characteristic peaks of cellulose I. At the sets of sulfuric acid treatments, BCNF1 and BCNF2, the peaks at 20\u0026deg; and 22.7\u0026deg; of untreated BC merged at 22\u0026deg; that is characteristic peak of cellulose II. Solvent treated samples, BCNF6 and BCNF7, still had two peaks but they were at 20\u0026deg; and 22\u0026deg;. In addition to that there were strong peaks at around 12.4\u0026deg; and 26.7\u0026deg; of BCNF6 and BCNF7. These peaks are essential for cellulose II [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eTGA Analysis\u003c/h2\u003e \u003cp\u003eNanofibrils of BCs as BCNF1, BCNF2, and BCNF6 had almost same degradation pattern (Fig.\u0026nbsp;6a). Weight loss of them was higher than BC as between 3\u0026ndash;5 times (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Spherical nanocrystal, BCNF7, had three degradation temperatures and the highest one was 488.7\u0026deg;C. The second degradation temperatures of nanofibrils were between 303\u0026ndash;309\u0026deg;C (Fig.\u0026nbsp;6b). However, the weight loss of nanofibrils were completely different. Nanofibrils showed two degradation steps while nanocrystal had three degradation steps. Sulfated fibers were depolymerized firstly, and continuous degradation was occurred in crystallite fractions in nanofibrils [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, nanocrystals were depolymerized three times and nanocrystals were obtained at the last degradation step.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOnset temperature, degradation temperature, and weight loss at this temperature of BC and BCNFs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1st degradation temp.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeight loss at 1st degradation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2nd degradation temp.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWeight loss at 2nd degradation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3rd degradation temp.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWeight loss at 3rd degradation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e128.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e381.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e71.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e148.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e61.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e304.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e124.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e309.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e49.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e200.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e80.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e303.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCNF7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e87.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e276.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e488.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e19.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eRearranging fiber positions in BC helps efficient degradation even using milling with liquid nitrogen. Depending on the degradation method, desired nanoparticle forms like nanofiber or nanocrystal can be obtained by using previously rearranged fibers. Milling with liquid nitrogen was not useful to obtain nanoparticles. However, when it is combined with rearrangement process, nanofibers were obtained by milling with liquid nitrogen. In addition to that rearrangement helps to obtain spherical-like nanocrystals. The low concentration of LiCl had no effect on degradation of BC. BC was not dried before degradation in this study while it is different from literature. It also causes BC to be in amorphous form after degradation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have consent from the responsible authorities at the Yeditepe University where the work has been carried out.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results/data/figures in this manuscript have not been published elsewhere, nor are they under consideration (from you or one of your Contributing Authors) by another publisher.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI declare that the authors have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.O.A prepare the concept of the study and made calculations of analysis, and wrote the main manuscript, F.S and N.K. performed experiments and collected data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge use of the services and Turkish Light Source (Turkish DeLight) X-ray facility at University of Health Sciences Türkiye, Experimental Medicine Research and Application Center (SBU-DETUAM).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eB. Anwar, B. Bundjali, Y. Sunarya, I.M. Arcana, Properties of Bacterial Cellulose and Its Nanocrystalline Obtained from Pineapple Peel Waste Juice, Fibers and Polymers 22 (2021) 1228\u0026ndash;1236. https://doi.org/10.1007/s12221-021-0765-8.\u003c/li\u003e\n\u003cli\u003eH. Yan, X. Chen, H. Song, J. Li, Y. Feng, Z. Shi, X. Wang, Q. Lin, Synthesis of bacterial cellulose and bacterial cellulose nanocrystals for their applications in the stabilization of olive oil pickering emulsion, Food Hydrocoll 72 (2017) 127\u0026ndash;135. https://doi.org/10.1016/j.foodhyd.2017.05.044.\u003c/li\u003e\n\u003cli\u003eM. Mart\u0026iacute;nez-Sanz, A. Lopez-Rubio, J.M. Lagaron, Optimization of the nanofabrication by acid hydrolysis of bacterial cellulose nanowhiskers, Carbohydr Polym 85 (2011) 228\u0026ndash;236. https://doi.org/10.1016/j.carbpol.2011.02.021.\u003c/li\u003e\n\u003cli\u003eT.K.Q. Doan, K.Y. Chiang, Characteristics and kinetics study of spherical cellulose nanocrystal extracted from cotton cloth waste by acid hydrolysis, Sustainable Environment Research 32 (2022). https://doi.org/10.1186/s42834-022-00136-9.\u003c/li\u003e\n\u003cli\u003eN.F. Vasconcelos, J.P.A. Feitosa, F.M.P. da Gama, J.P.S. Morais, F.K. Andrade, M. de S.M. de Souza Filho, M. de F. Rosa, Bacterial cellulose nanocrystals produced under different hydrolysis conditions: Properties and morphological features, Carbohydr Polym 155 (2017) 425\u0026ndash;431. https://doi.org/10.1016/j.carbpol.2016.08.090.\u003c/li\u003e\n\u003cli\u003eN. Kruer-Zerhusen, B. Cantero-Tubilla, D.B. Wilson, Characterization of cellulose crystallinity after enzymatic treatment using Fourier transform infrared spectroscopy (FTIR), Cellulose 25 (2018) 37\u0026ndash;48. https://doi.org/10.1007/s10570-017-1542-0.\u003c/li\u003e\n\u003cli\u003eD. Tsalagkas, R. Lagaňa, I. Poljan\u0026scaron;ek, P. Oven, L. Csoka, Fabrication of bacterial cellulose thin films self-assembled from sonochemically prepared nanofibrils and its characterization, Ultrason Sonochem 28 (2016) 136\u0026ndash;143. https://doi.org/10.1016/j.ultsonch.2015.07.010.\u003c/li\u003e\n\u003cli\u003eB. Deepa, E. Abraham, B.M. Cherian, A. Bismarck, J.J. Blaker, L.A. Pothan, A.L. Leao, S.F. de Souza, M. Kottaisamy, Structure, morphology and thermal characteristics of banana nano fibers obtained by steam explosion, Bioresour Technol 102 (2011) 1988\u0026ndash;1997. https://doi.org/10.1016/j.biortech.2010.09.030.\u003c/li\u003e\n\u003cli\u003eB.M. Cherian, A.L. Le\u0026atilde;o, S.F. de Souza, S. Thomas, L.A. Pothan, M. Kottaisamy, Isolation of nanocellulose from pineapple leaf fibres by steam explosion, Carbohydr Polym 81 (2010) 720\u0026ndash;725. https://doi.org/10.1016/j.carbpol.2010.03.046.\u003c/li\u003e\n\u003cli\u003eD. Lin, R. Li, P. Lopez-Sanchez, Z. 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Young, Understanding the local structure of disordered carbons from cellulose and lignin, Wood Sci Technol 55 (2021) 587\u0026ndash;606. https://doi.org/10.1007/s00226-021-01286-6.\u003c/li\u003e\n\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":"[email protected]","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 nanocrystal, Bacterial cellulose nanofiber, Liquid nitrogen milling, Sulfuric acid degradation, Fiber arrangement","lastPublishedDoi":"10.21203/rs.3.rs-4205627/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4205627/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacterial cellulose (BC) has unique properties such as high tensile strength, high crystallinity, and high purity. Fiber length of BC causes different attributes. Therefore, degradation of BC has been studied deeply. In this study, fibers of BC were rearranged via DMAc-LiCl solvent and BC was degraded in wet state. Two different degradation methods, milling with liquid nitrogen and autoclave treatment, were applied. Degraded BCs were characterized by FTIR, TEM, AFM, TGA, and XRD. The solvent helps to align the fibers, making it more crystalline. Degraded BCs had lower crystalline ratio than BC, because of increasing hydrogen bonding during degradation in wet state. Degradation with autoclave made two different degraded BCs as nanofibrils and spherical nanocrystals with no pretreatment and solvent pretreatment, respectively. The nanofibril lengths were between 312 – 700 nm depending on applied method and spherical nanocrystal size was 56 nm. The rearrangement via solvent causes an important difference in degradation of BC. Nanofibrils and nanocrystals can be obtained, depending on the rearrangement of fibers before degradation process.\u003c/p\u003e","manuscriptTitle":"Physical and Chemical Degradation of Bacterial Cellulose via Rearranging Fibers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-08 17:48:22","doi":"10.21203/rs.3.rs-4205627/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","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":"6b840919-06ad-4e16-8492-5dd6bd0b6038","owner":[],"postedDate":"April 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-22T06:17:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-08 17:48:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4205627","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4205627","identity":"rs-4205627","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}