Mixed hexose and pentose sugars induce species-variable bacterial cellulose production by Komagataeibacter spp.

Mixed hexose and pentose sugars induce species-variable bacterial cellulose production by Komagataeibacter spp. | 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 Mixed hexose and pentose sugars induce species-variable bacterial cellulose production by Komagataeibacter spp. Moyinoluwa Oreoluwa Akintunde, Bukola Christianah Adebayo-Tayo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6681004/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Low-cost substrates and agricultural wastes for bacterial cellulose (BC) production have gained significant attention in recent years because of their potential to increase yield and reduce production costs. Diverse bacterial species exhibit heterogeneous metabolic profiles and substrate utilization patterns during BC biosynthesis when cultivated on these substrates. The aim of this study was to mimic the hexose and pentose composition of low-cost substrates to increase the BC yield. This study investigated the substrate utilization patterns of two Komagataeibacter species during BC production on mixed carbon substrates. Both strains used in this study utilized mixed hexose and pentose sugars as carbon sources for BC production, with varying consumption patterns and BC yields. Komagataeibacter sp. CCUG73629 efficiently utilized multiple sugars, with the highest BC yield recorded in the glucose-cellobiose medium (M4). The highest BC yield of Komagataeibacter sp. CCUG73630 was recorded in medium containing glucose as the sole carbon source. The BC produced had functional groups associated with cellulose, well-defined diffraction peaks, and densely interwoven fiber structures. The maximum degree of crystallinity (67.5%) was recorded for BC produced by Komagataeibacter sp. CCUG73630 in a glucose-arabinose-xylose medium (M1). Owing to their unique metabolic profiles, each Komagataeibacter species demonstrates different substrate utilization patterns. This study revealed the complexity, variation, unique metabolism, and strain-specific nature of bacterial BC production using mixed hexose and pentose sugars as carbon sources. Thus, this study contributes to the development of efficient and economical methods for producing BC from alternative substrates. Komagataeibacter Bacterial cellulose Mixed carbon sources Degree of crystallinity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Bacterial cellulose (BC) is a polymeric material of microbial origin. It is a macromolecule with a wide range of applications [ 1 ]. With the rise in environmental challenges associated with the production and application of synthetic polymers, natural materials that can replace fossil-based polymers are constantly being developed. BC is an eco-friendly biomaterial that can replace fossil-based materials. Although BC has the same structure as plant cellulose and is composed of \(\:\beta\:\) -D-glucopyranose units linked by \(\:\beta\:\) -1,4 glycosidic bonds [ 2 ], it is a purer form of cellulose. The BC polymer is characterized by a high degree of polymerization and crystallinity with a unique fibre network of micro- or nanosize, which increases its surface-to-volume ratio, a unique feature over other cellulose source [ 3 ]. BC is produced as an extracellular polymer by diverse groups of microorganisms, with acetic acid bacteria being the most promising. Bacteria of the genera Acetobacter, Komagataeibacter, Rhizobium, Pseudomonas, Agrobacterium, Sarcina and others have the ability to produce BC [ 4 ]. Komagataeibacter species such as K. europaeus, K. hansenii, K. rhaeticus, and K. xylinus are among the best producers of BC and can utilize different carbon sources [ 5 ]. Several factors contribute to the efficient production of BC, including the carbon source and pH of the cultivation media. The carbon source is one of the most extensively studied components of the growth medium, as it significantly affects the BC yield [ 1 ]. BC-producing bacteria vary in their ability to assimilate and utilize different carbon sources [ 6 ]. However, carbon sources contribute to the high cost of producing BC, which has led to research on alternative low-cost substrates. Several works have focused on the use of low-cost, available wastes as substrates for BC production. Low-cost carbon sources such as juices, peels and pomaces of orange, pineapple, and paws have been previously studied for BC production [ 7 , 8 , 9 , 10 ]. Agricultural residues such as sugarcane bagasse, corn cobs, corn stalks, rice straw, and durian shells have also been used for BC production [ 11 , 12 , 9 , 13 ]. These agricultural residues are mostly lignocellulosic materials, which, when pretreated, contain significant quantities of both hexose sugars and pentose sugars, such as xylose, arabinose, and glucose, which are major carbon sources. BC-producing bacteria consume these sugars at different rates and utilize them for BC, resulting in different BC yields. The objective of this study was to mimic the hexose and pentose sugar composition of low-cost substrates, assuming that a substrate is made up of at least one hexose sugar and one pentose sugar for BC production, which will provide better insight into the pattern of sugar consumption by strains during BC production. In this study, some hexose and pentose sugars were combined at different ratios as carbon sources for BC production by Komagataeibacter spp., and the rates of individual sugar consumption and the effects of mixed carbon sources on BC yield and crystallinity were studied. Methods Bacterial Cellulose (BC) Production Two Komagataeibacter strains previously isolated by Akintunde et al. [ 13 ], identified as Komagataeibacter sp. CCUG73629 and Komagataeibacter sp. CCUG73630, were sub-cultured on Hestrin-Schramm (HS) media (composed of yeast extract, glucose, peptone, citric acid, disodium hydrogen phosphate, and agar‒agar) and incubated at 30 \(\:℃\) for 3–5 days [ 14 ]. Colonies from the agar plates were transferred into HS broth and incubated for 3 days at 30 \(\:℃\) to develop the seed inoculum for BC production. Effect of a mixed carbon source on BC production To study the effects of mixed carbon sources on BC production, the two Komagataeibacter strains were cultivated in HS media supplemented with different sugars as the carbon source. The media composition is shown in Table 1 . The effects of the media composition on the BC yield, final pH of the fermentation media and substrate consumption by the isolates were determined. The pH of the media was adjusted to 6.0. After sterilization, 5 mL of the seed inoculum was inoculated into 100 mL of the fermentation media and incubated statically for 16 days at 30°C. After 16 days of fermentation, the quantity of BC produced, the amount of substrate consumed and the final pH of the media were determined. Table 1 Media composition with different sugar combinations used as carbon sources for BC production. Sugars M1 + HS components M2 + HS components M3 + HS components M4 + HS components M5 + HS components M6 + HS components Glucose + + + + + + Arabinose + + + Xylose + + + + Cellobiose + + Mannose + Galactose + Key: M1: Medium 1, M2: Medium 2, M3: Medium 3, M4: Medium 4, M5: Medium 5, M6: Medium 6 BC purification, pH and substrate consumption determination The BC produced after fermentation was harvested by picking with tweezers. BC purification was performed by boiling for 1 h at 80°C in 1 M NaOH. Thereafter, the BC was washed with distilled water until it reached pH 7 and allowed to air dry until a constant weight was achieved. The dry weight of BC (grams per liter of fermentation media (g/L)) was measured via an analytical weighing balance (Kern PFB precision balance, Sigma Aldrich). The percentage BC yield was determined in relation to the amount of carbon source used for production. The BC yield was calculated as follows: BC yield (%) = \(\:\frac{BC\:dry\:weight\:\left(g\right)}{Carbon\:source\:used\:\left(g\right)\:}\times\:100\) The final pH of the fermentation media after BC production was measured with a pH meter. The rate of substrate consumption was determined via high-performance liquid chromatography (HPLC) (Walters Corporation, Milford, USA) by measuring the concentrations of various sugar components in the fermentation media. One milliliter of the fermentation media was pipetted into Eppendorf tubes and centrifuged for 10 minutes at 7000 rpm. A 0.2 µm filter was used for filtration of the supernatant into HPLC vials. A hydrogen base ion-exchange column (Aminex HPX-87H, Bio-Rad, Hercules, USA) that works at 60°C, with 5 mM H 2 SO 4 solution as the eluent, flowing at 0.6 mL/min, was used for the detection and quantification of sugars. The substrate consumption was calculated as follows: Substrate consumption (%)= \(\:\frac{Initial\:total\:sugar\:-Final\:total\:sugar}{Final\:Total\:sugar}\:\times\:100\:\) Scanning Electron Microscopy (SEM) The morphology of the BC produced by the bacterial strains was determined using SEM. Dried BC films were placed on a carbon tape, thereafter coated with gold using LICA EM ACE 600 (Leica, Germany). The gold coated samples were observed with a scanning electron microscope SEM LEO Gemini 1525 (Carl Zeiss, Germany). Fourier Transform Infrared (FTIR) Spectroscopy FTIR was performed via an FTIR spectrometer (Shimadzu model, Germany). The BC samples were analysed by placing the dried film on a diamond accessory. With an accumulation of 128 scans, the absorbance was measured in the wavenumber range of 4000 − 500 cm − 1 . X-ray Diffraction (XRD) XRD was performed to determine the degree of crystallinity of the BC. X-ray diffraction analysis was performed by measuring the diffraction pattern of the BC sheet via a diffractometer (Siefert Sun XRD 3003) operating at 40 kV and 40 mA. The diffractograms were taken from 0 to 60° on a 2θ scale with a step size of 0.02°. The percentage crystallinity and amorphous content of the BC were determined. Statistical analysis All the experiments were carried out in duplicate, and the values are presented as the means ± SDs. All the data were statistically analysed via MINITAB 17. The data were evaluated via One-Way Analysis of Variance (ANOVA). Results Effect of the media composition on the BC yield, final pH and total substrate consumption The effects of the media composition on the BC yield, final pH and substrate consumption of the fermentation media by Komagataeibacter sp. CCUG73629 and Komagataeibacter sp. CCUG73630 are shown in Tables 2 and 3 . The BC yield and final pH of the media enriched with Komagataeibacter sp. CCUG73629 ranged from 100–283.3% and 3.7–4.2, respectively. The highest yield was recorded in M4, whereas the lowest BC yield was observed in M6. The highest pH was observed in M2, and the lowest was observed in M6. For Komagataeibacter sp. CCUG73630, the highest BC yield and final pH of the media ranged from 43.5–100.0% and 3.5–4.0, respectively. The highest yield was recorded in M6, whereas the lowest yield was recorded in M3. The highest pH was observed in M2, and the lowest was observed in M5. During BC production, the highest total substrate consumption of 81.5% was observed in M3 (glucose, mannose, xylose, galactose and arabinose) by Komagataeibacter sp. CCUG73629, whereas in Komagataeibacter sp. CCUG73630, a total substrate consumption of 97.2% was observed in M6 (glucose). However, in M5 (glucose and xylose), 82.6% of the substrate was consumed by Komagataeibacter sp. CCUG73630. Table 2 Effects of media composition on BC yield, substrate consumption and pH by Komagataeibacter sp. CCUG73629. Mixed carbon Media BC yield (%) Total Substrate consumption (%) pH M1 211.0 ± 1.41 c 75.2 ± 1.10 b 3.8 ± 0.01 c M2 228.3 ± 1.06 b 55.4 ± 1.08 d 4.2 ± 0.01 a M3 119.5 ± 0.71 e 81.5 ± 1.59 a 4.0 ± 0.28 b M4 283.3 ± 1.06 a 62.6 ± 1.27 c 3.8 ± 0.01 c M5 176.5 ± 2.12 d 77.1 ± 1.28 ab 3.8 ± 0.01 c M6 100.0 ± 0.00 f 56.4 ± 1.12 d 3.7 ± 0.01 d Keywords M1 − Glucose + Arabinose + Xylose; M2 – Glucose + Cellobiose + Xylose + Arabinose; M3 − Glucose + Mannose + Xylose + Galactose + Arabinose; M4 − Glucose + Cellobiose; M5 – Glucose + Xylose; M6 – Glucose . Table 3 Effects of media composition on BC yield, substrate consumption and pH by Komagataeibacter sp. CCUG73630 Mixed carbon Media BC yield (%) Total Substrate consumption (%) pH M1 68.8 ± 0.17 e 76.9 ± 0.01 c 3.7 ± 0.03 c M2 77.8 ± 0.28 d 64.9 ± 0.16 d 4.0 ± 0.03 a M3 43.5 ± 0.27 f 60.0 ± 0.27 e 3.8 ± 0.01 b M4 91.0 ± 0.03 b 76.3 ± 0.22 c 3.6 ± 0.03 c M5 85.1 ± 0.02 c 82.6 ± 0.40 b 3.5 ± 0.01 d M6 100.0 ± 0.00 a 97.2 ± 0.12 a 3.7 ± 0.01 c Keywords M1 − Glucose + Arabinose + Xylose; M2 – Glucose + Cellobiose + Xylose + Arabinose; M3 − Glucose + Mannose + Xylose + Galactose + Arabinose; M4 − Glucose + Cellobiose; M5 – Glucose + Xylose; M6 – Glucose . Substrate consumption of Komagataeibacter sp. CCUG73629 and Komagataeibacter sp. CCUG73630 during BC production in mixed carbon sources. The pattern of substrate consumption by Komagataeibacter sp. CCUG73629 and Komagataeibacter sp. CCUG73630 in the mixed carbon source medium during BC production is shown in Fig. 1 a-f. During BC production by Komagataeibacter sp. CCUG73629 in M1, the consumption of glucose, arabinose and xylose ranged from 18.0–1.9 g/L, 3.5–1.9 g/L and 6.4–3.3, respectively. By day 16, 89% of the glucose had been consumed. During BC production by Komagataeibacter sp. CCUG73630 in M1, the consumption of glucose, arabinose and xylose ranged from 18–1.3 g/L, 3.5–1.9 g/L and 6.5–3.2 g/L, respectively. By day 16, 92% of the glucose and 50% of the xylose were consumed. In M2, during BC production by Komagataeibacter sp. CCUG73629, the consumption of glucose, cellobiose, xylose and arabinose ranged from 14–1.7 g/L, 7.4–7.1 g/L, 4–1.9 g/L and 2.5–1.5 g/L, respectively. After 16 days, 87% of the glucose and 3% of the cellobiose were consumed. During BC production by Komagataeibacter sp. CCUG73630 in M2, the consumption of glucose, cellobiose, xylose and arabinose ranged from 14–1.3 g/L, 8–5.8 g/L, 3.9–1.6 g/L and 3–1.9 g/L, respectively. By the 16th day, 91% of the glucose, 27% of the cellobiose and 58% of the xylose were consumed. During BC production via M3 and Komagataeibacter sp. CCUG73629, the consumption of glucose, mannose, xylose, galactose and arabinose ranged from 10.8–0.9 g/L, 1.4–0.3 g/L, 6.4–1.4 g/L, 3.5–0.9 g/L and 5.5–5.5 g/L, respectively. After 16 days, 91% of the glucose and over 70% of the other sugars were consumed. During BC production by Komagataeibacter sp. CCUG73630, glucose, mannose, xylose, galactose and arabinose consumption ranged from 10.8–0.8 g/L, 1.4–0.9 g/L, 6.5–3.2 g/L, 3.5–2.9 g/L and 5.5–3.3 g/L, respectively. After 16 days, 92% of the glucose, 50% of the xylose and 18% of the galactose were consumed. With the use of M4 during BC production by Komagataeibacter sp. CCUG73629, the consumption of glucose and cellobiose ranged from 21.6–4.3 g/L and 8.1–6.9 g/L, respectively. During production by Komagataeibacter sp. CCUG73630, the consumption of glucose and cellobiose ranged from 21.7–2.3 g/L and 8.0–2.8 g/L, respectively. After 16 days, Komagataeibacter sp. CCUG73629 consumed 79% of the glucose and 15% of the cellobiose, whereas Komagataeibacter sp. CCUG73630 consumed 89% of the glucose and 39% of the cellobiose. During BC production with M5, Komagataeibacter sp. CCUG73629, the consumption of glucose and xylose ranged from 21.3–2.4 g/L and 6.2–3.5 g/L, respectively, whereas the consumption of glucose and xylose by Komagataeibacter sp. CCUG73630 ranged from 21.4–1.8 g/L and 6.2–3.0 g/L, respectively. After 16 days of BC production, 88% of the glucose and 43% of the xylose were consumed by Komagataeibacter sp. CCUG73629, whereas 91% of the glucose and 50% of the xylose were consumed by Komagataeibacter sp. CCUG73630. Glucose consumption in M6 ranged from 28 − 12 g/L and 28.4–0.7 g/L by Komagataeibacter sp. CCUG73629 and Komagataeibacter sp. CCUG73630 during BC production. By the 16th day, Komagataeibacter sp. CCUG73629 consumed 57%, while Komagataeibacter sp. CCUG73630 consumed 97% of glucose. Characterization of BC Produced with Mixed Carbon Sources The chemical structures of the BCs produced from Komagataeibacter sp. CCUG73629 and Komagataeibacter sp. CCUG73630 in mixed carbon media are shown in the FTIR spectra in Figs. 2 and 3 . The BCs produced by both strains were similar, with slight differences in peak positions and absorbances. The broad peaks at approximately 3338 and 3340 cm − 1 at both strains correspond to O–H stretching vibrations. The peaks at 2891 and 2895 cm − 1 indicate C–H group stretching. The peaks at approximately 1641 and 1651 cm − 1 correspond to O–H bending of the absorbed water. The peaks at 1427 and 1429 cm − 1 are associated with CH 2 bending. The peaks at 1163 and 1161 cm-1 indicate asymmetrical C-O-C stretching. The peaks at approximately 1053 and 1055 cm − 1 indicate vibrations of C-C, C-OH and C-H rings and side groups. The peak at approximately 896 cm − 1 at both strains corresponds to CH vibrations. The morphology of the BC produced by Komagataeibacter sp. CCUG73629 and Komagataeibacter sp. CCUG73630 in M1, M4 and M6 is shown in Fig. 4 a-f. The BC micrograh showed a dense network of oriented fibers produced by both strains. The BC produced by Komagataeibacter sp. CCUG73629 in M1 and M4 was more compact and denser, with the fibres tightly aggregated, while the BC fibers prodcued by Komagataeibacter sp. CCUG73630 in M1 and M4 densely interwoven. In M6, both strainsproduced similar BC fibers that were densely interwoven. The X-ray diffractograms of the BCs produced in M1, M4 and M6 presented two distinct characteristic peaks around 16.8 \(\:^\circ\:\) and 22.7 \(\:^\circ\:\) (Fig. 5 ). The degree of crystallinity and amorphous content of the BC varied with different carbon sources (Table 4 ). In M1, BC produced by Komagataeibacter sp. CCUG73630 had a greater degree of crystallinity (67.5%) than BC produced by Komagataeibacter sp. CCUG73629 (58.6%). However, the M6 BC produced by Komagataeibacter sp. CCUG73629 had a relatively high degree of crystallinity (66.7%). Table 4 Degree of crystallinity and amorphous content of BC produced in mixed carbon media Komagataeibacter sp. CCUG73629 Komagataeibacter sp. CCUG73630 BC produced in Mixed carbon media Degree of Crystallinity (%) Amorphous content (wt. %) Degree of Crystallinity (%) Amorphous content (wt. %) BC_M1 58.6 41.4 67.5 32.5 BC_M4 60.2 39.8 61.2 38.8 BC_M6 66.7 33.2 58.1 41.9 Keywords M1 − Glucose + Arabinose + Xylose; M4 − Glucose + Cellobiose; M6 – Glucose . Discussion The effect of media composition on BC production provides insight into how bacteria can utilize mixed hexose and pentose as a carbon source for production. Growth media containing different sources of nutrients are important for determining the yield and characteristic properties of BC produced by different bacterial species. The carbon source is vital for BC production, as yield depends on the availability and quality of the carbon source [ 15 ]. During BC synthesis (Fig. 6 ), hexose sugars are directly metabolized via glycolysis to produce glucose-6-phosphate, a precursor for UDP-glucose, for BC synthesis, which is energy efficient, enabling a relatively high BC yield. However, pentose sugars enter the pentose phosphate pathway, requiring gluconeogenesis to generate BC precursors and consuming more ATP, which may reduce BC production efficiency [ 16 ]. Komagataeibacter species among the acetic acid bacteria are widely studied for BC production because they achieve considerable BC yields and metabolize different carbon sources [ 17 , 5 ]. The total substrate consumption did not significantly affect the BC yield. During BC production by Komagataeibacter sp. CCUG73629, the medium that contained glucose as the sole source of carbon least supported BC production, which could indicate that the bacterial cells thrived better in media containing more than one carbon source. This may be a result of catabolite repression in single sugar media due to the relatively high concentration of a single sugar (glucose), which can inhibit enzymatic reactions involved in sensing endogenous levels of sugars and can be avoided when mixed sugars are utilized [ 18 , 19 ]. Zhong et al. [ 20 ] suggested that the production of BC may be efficient by adding a combination of several carbon sources, as they reported increased BC production in media containing glucose, glycerol and fructose as mixed carbon sources. When Komagataeibacter sp. CCUG73630 was used for BC production, the glucose-only medium supported the highest BC yield, which means that a single carbon source was more efficient for BC production than a mixed carbon source was. Komagataeibacter sp. CCUG73630 also consumed more glucose than did Komagataeibacter sp. CCUG73629, indicating that Komagaetibacter sp. CCUG73630 preferred glucose as the sole carbon source for BC production. With glucose as the carbon source, both strains presented similar yields of BC, although glucose consumption was lower in Komagataeibacter sp. CCUG73629. During BC production, glucose can be used as a precursor for the assembly of glucose units into cellulose [ 21 ]. The medium containing glucose and cellobiose as the carbon source supported the highest BC production by Komagataeibacter sp. CCUG73629, with only 15% of cellobiose consumed, whereas M4 had the second-best BC yield by Komagataeibacter . sp. CCUG73630, 39% of the cellobiose was consumed. These findings emphasize the metabolic versatility of the individual strains in the medium, indicating that cellobiose may have the ability to activate BC production even though consumption was low, making cellobiose an inducer of BC production. Qi et al. [ 22 ] reported that after the 2nd day of fermentation, when little cellobiose was consumed, Gluconacetobacter xylinus could not further consume cellobiose during BC production. The medium with the most diverse sugar mixture containing glucose, mannose, xylose, galactose and arabinose resulted in the lowest BC production for both Komagateibacter sp. CCUG73629 and Komagataeibacter sp. CCUG73630, with both having similar total substrate consumption. However, when Dahman et al. [ 19 ] used G. xylinus ATCC700178, greater production was recorded for glucose, mannose, xylose, galactose and arabinose mixtures. The pH of the media after BC production for 16 days by both strains did not have any significant effect on BC yield since the deviation from the optimum pH was low [ 19 ]. The carbon sources used for BC production affect its properties, such as the crystallinity index [ 21 ]. The mixed carbon sources influenced the degree of crystallinity and amorphous content of the BC produced by both strains. The X-ray diffractograms of the BCs produced by both strains in the mixed carbon media were similar, with slight differences in intensity. The characteristic peaks were assigned to the (110) and (200) crystallographic planes, which corresponds to the cellulose I structure [ 23 ]. The crystallinity of BC varied with the strain producing it and the medium. However, the crystallinity of BC produced by both strains in M4 was not significantly different. Chen et al. [ 24 ] reported that the crystallinity of BC produced in single sugar media (galactose, xylose and mannose) was greater than that of BC produced in mixed sugar media; however, the yield of BC was lower in single sugar media. The morphology of the BC produced by both strains showed only slight differences. The compact, densely interwoven structure of the BC fibres was similar to those in the study of Arooj et al. [ 25 ] who reported similar effect for BC produced by Komagataeibacter sp. LMG 18909 and FXV3 in glucose and glycerol with 3.0% ethanol. There was no significant difference in the chemical structure of BC. The functional groups present were similar with those reported by Akintunde et al. [ 13 ] during BC production in agricultural residue. The FTIR spectra of both strains in mixed carbon media indicated that all the peaks were consistent with previous characterization of bacterial cellulose [ 26 , 27 ]. In conclusion, this study demonstrated the complexity and strain-specific nature of BC production via the use of mixed carbon sources, which stems from differences in metabolic pathways, energy efficiency and regulatory mechanisms. The strains utilized the carbon sources in the media simultaneously at varying rates, with glucose being prioritized for initial consumption at the highest rate, followed by slower consumption of other sugars in the medium. Efforts to improve BC production with alternative carbon sources, including agricultural wastes and feedstocks such as sugarcane bagasse, corncob, wheat straw, and rice straw, are sustainable. The hydrolysate of these materials contains sugar mixtures of glucose, arabinose, xylose, galactose, mannose, and cellobiose. Therefore, the combination of sugars from this study highlights the consumption pattern of such sugars and their effects on BC yield and some properties, which is essential for the optimization of BC production for future BC applications. Declarations The authors have no competing interests to declare that are relevant to the content of this article Funding No funding was received for conducting this study Competing interests The authors have no competing interests to declare that are relevant to the content of this article Data Availability Statement The authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. Ethics approval Not Applicable Consent Not applicable Author Contribution "MOA designed the study, performed formal analysis and investigation. MOA wrote the main manuscript text . OMA edited the manuscript and prepared figure 6. BCA edited the manuscript and supervised the study. All authors reviewed the manuscript." 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Zhong C, Zhang GC, Liu M, Zheng XT, Han PP, Jia SR (2013) Metabolic flux analyses of Gluconacetobacter xylinus for bacterial cellulose production. Appl Microbiol Biotechnol 97(14):6189–6199. Gorgieva S, Trček J (2019) Bacterial Cellulose: Production, Modification and Perspectives in Biomedical Applications. Nanomat (Basel) 9(10):1352. Qi GX, Luo MT, Huang C, Guo H, Chen XF, Xiong L, Wang B, Lin X, Peng F, Chen XD (2017) Comparison of bacterial cellulose production by Gluconacetobacter xylinus on bagasse acid and enzymatic hydrolysates. J Appl Polym Sci 134:45066. Muhammad H, Alburae NA, Salam MA, Badshah M, Khan T, Abo-Aba SEM (2024) Identification of Cellulose Producing Bacterial Strains-An Eco-friendly and Cost-effective Approach. J Pure Appl Microbiol 18(1):483-499 Chen G, Wu G, Chen L, Wang W, Hong FF, Jönsson LJ (2019) Comparison of productivity and quality of bacterial nanocellulose synthesized using culture media based on seven sugars from biomass. Microb Biotechnol 12(4):677-687. Fatima A, Ortiz-Albo P, Neves LA, Nascimento FX, Crespo JG (2023) Biosynthesis and characterization of bacterial cellulose membranes presenting relevant characteristics for air/gas filtration. J Membr Sci 674:121509. Lu H, Jiang X (2014) Structure and properties of bacterial cellulose produced using a trickling bed reactor. Appl Biochem Biotechnol 172:3844-3861. Drosos A, Kordopati G, Anastasopoulos C, Zafeiropoulos J, Koutinas A, Kanellaki M (2024) Comparative study and characterization of water-treated bacterial cellulose produced by solid or liquid inoculum of Komagateibacter sucrofermentans . Cellul 31:1-29. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 06 Aug, 2025 Reviews received at journal 09 Jul, 2025 Reviews received at journal 01 Jul, 2025 Reviews received at journal 23 Jun, 2025 Reviewers agreed at journal 20 Jun, 2025 Reviewers agreed at journal 19 Jun, 2025 Reviewers agreed at journal 17 Jun, 2025 Reviewers agreed at journal 17 Jun, 2025 Reviewers invited by journal 17 Jun, 2025 Editor assigned by journal 21 May, 2025 Submission checks completed at journal 21 May, 2025 First submitted to journal 16 May, 2025 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. 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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-6681004","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472478046,"identity":"3bbdd563-4686-4df0-8beb-048b1367351f","order_by":0,"name":"Moyinoluwa Oreoluwa Akintunde","email":"data:image/png;base64,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","orcid":"","institution":"Aalen University of Applies Sciences","correspondingAuthor":true,"prefix":"","firstName":"Moyinoluwa","middleName":"Oreoluwa","lastName":"Akintunde","suffix":""},{"id":472478047,"identity":"2b0c5ea0-645f-47e5-ac20-21bc881aceb9","order_by":1,"name":"Bukola Christianah Adebayo-Tayo","email":"","orcid":"","institution":"University of Ibadan","correspondingAuthor":false,"prefix":"","firstName":"Bukola","middleName":"Christianah","lastName":"Adebayo-Tayo","suffix":""},{"id":472478048,"identity":"b0841d80-b5bf-437f-8709-aff610b3a31f","order_by":2,"name":"Obinna Markraphael Ajunwa","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Obinna","middleName":"Markraphael","lastName":"Ajunwa","suffix":""}],"badges":[],"createdAt":"2025-05-16 13:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6681004/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6681004/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84889534,"identity":"3215af22-3dae-4989-b3b7-103d4c45d910","added_by":"auto","created_at":"2025-06-18 12:27:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":76054,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea-f Consumption patterns of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKomagataeibacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esp. CCUG73629 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKomagataeibacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp. CCUG73630 in mixed carbon media: (a) M1, (b) M2, (c) M3, (d) M4, (e) M5, and (f) M6 during BC production\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6681004/v1/aeb20a326c5294a00531e64a.png"},{"id":84889946,"identity":"84164843-7d46-4a01-a714-db40571ad2e9","added_by":"auto","created_at":"2025-06-18 12:35:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":59478,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTIR spectra of BC produced by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKomagataeibacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esp. CCUG73629 in mixed carbon media\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6681004/v1/980dddd2e0fe1473ff7c922f.png"},{"id":84889536,"identity":"9d587eed-8d74-4521-bbec-29af862d280b","added_by":"auto","created_at":"2025-06-18 12:27:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60192,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTIR spectraof BC produced by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKomagataeibacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp. CCUG73630 in mixedcarbon media\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6681004/v1/3bc89fd24278ccb8f3514360.png"},{"id":84889947,"identity":"85d49c39-ff48-4ace-a712-79e0593b59f2","added_by":"auto","created_at":"2025-06-18 12:35:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":430916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicrograph of BC produced by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKomagataeibacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp. CCUG73629 (a-c) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKomagataeibacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp. CCUG73630 (d-f) in M1, M4 and M6 at 15,000x magnification\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6681004/v1/4a9a6777509baa50c9af939d.png"},{"id":84889541,"identity":"857f24f0-e526-4293-9d46-de5d6d3d57ea","added_by":"auto","created_at":"2025-06-18 12:27:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eX-ray diffractogram of BC produced by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKomagataeibacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esp. CCUG73629 (a-c) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKomagataeibacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sp. CCUG73630 (d-f) in M1, M4 and M6\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6681004/v1/f33223ebacefed9ec2fba69c.png"},{"id":84889540,"identity":"d0f92c1d-0fdd-4aed-8f02-dc93f09f3aaf","added_by":"auto","created_at":"2025-06-18 12:27:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":120159,"visible":true,"origin":"","legend":"\u003cp\u003eGeneralised schematic representation of hexose and pentose metabolism for BC production\u003c/p\u003e\n\u003cp\u003eCreated in BioRender. Ajunwa, O. (2025) https://BioRender.com/ejgy96y\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6681004/v1/f4d39336e67212ba2e282626.png"},{"id":84891071,"identity":"085f1c8d-af7a-4f98-81ab-49b77888db3e","added_by":"auto","created_at":"2025-06-18 12:51:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1907069,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6681004/v1/bd81aae2-3140-44de-bc33-c781e18ad32a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mixed hexose and pentose sugars induce species-variable bacterial cellulose production by Komagataeibacter spp.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBacterial cellulose (BC) is a polymeric material of microbial origin. It is a macromolecule with a wide range of applications [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. With the rise in environmental challenges associated with the production and application of synthetic polymers, natural materials that can replace fossil-based polymers are constantly being developed. BC is an eco-friendly biomaterial that can replace fossil-based materials. Although BC has the same structure as plant cellulose and is composed of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e-D-glucopyranose units linked by \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e-1,4 glycosidic bonds [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], it is a purer form of cellulose. The BC polymer is characterized by a high degree of polymerization and crystallinity with a unique fibre network of micro- or nanosize, which increases its surface-to-volume ratio, a unique feature over other cellulose source [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. BC is produced as an extracellular polymer by diverse groups of microorganisms, with acetic acid bacteria being the most promising. Bacteria of the genera \u003cem\u003eAcetobacter, Komagataeibacter, Rhizobium, Pseudomonas, Agrobacterium, Sarcina\u003c/em\u003e and others have the ability to produce BC [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. \u003cem\u003eKomagataeibacter\u003c/em\u003e species such as \u003cem\u003eK. europaeus, K. hansenii, K. rhaeticus, and K. xylinus\u003c/em\u003e are among the best producers of BC and can utilize different carbon sources [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral factors contribute to the efficient production of BC, including the carbon source and pH of the cultivation media. The carbon source is one of the most extensively studied components of the growth medium, as it significantly affects the BC yield [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. BC-producing bacteria vary in their ability to assimilate and utilize different carbon sources [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, carbon sources contribute to the high cost of producing BC, which has led to research on alternative low-cost substrates. Several works have focused on the use of low-cost, available wastes as substrates for BC production. Low-cost carbon sources such as juices, peels and pomaces of orange, pineapple, and paws have been previously studied for BC production [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Agricultural residues such as sugarcane bagasse, corn cobs, corn stalks, rice straw, and durian shells have also been used for BC production [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These agricultural residues are mostly lignocellulosic materials, which, when pretreated, contain significant quantities of both hexose sugars and pentose sugars, such as xylose, arabinose, and glucose, which are major carbon sources. BC-producing bacteria consume these sugars at different rates and utilize them for BC, resulting in different BC yields. The objective of this study was to mimic the hexose and pentose sugar composition of low-cost substrates, assuming that a substrate is made up of at least one hexose sugar and one pentose sugar for BC production, which will provide better insight into the pattern of sugar consumption by strains during BC production. In this study, some hexose and pentose sugars were combined at different ratios as carbon sources for BC production by \u003cem\u003eKomagataeibacter\u003c/em\u003e spp., and the rates of individual sugar consumption and the effects of mixed carbon sources on BC yield and crystallinity were studied.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial Cellulose (BC) Production\u003c/h2\u003e \u003cp\u003eTwo \u003cem\u003eKomagataeibacter\u003c/em\u003e strains previously isolated by Akintunde et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], identified as \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 and \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630, were sub-cultured on Hestrin-Schramm (HS) media (composed of yeast extract, glucose, peptone, citric acid, disodium hydrogen phosphate, and agar‒agar) and incubated at 30\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e for 3\u0026ndash;5 days [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Colonies from the agar plates were transferred into HS broth and incubated for 3 days at 30\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e to develop the seed inoculum for BC production.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEffect of a mixed carbon source on BC production\u003c/h3\u003e\n\u003cp\u003eTo study the effects of mixed carbon sources on BC production, the two \u003cem\u003eKomagataeibacter\u003c/em\u003e strains were cultivated in HS media supplemented with different sugars as the carbon source. The media composition is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The effects of the media composition on the BC yield, final pH of the fermentation media and substrate consumption by the isolates were determined. The pH of the media was adjusted to 6.0. After sterilization, 5 mL of the seed inoculum was inoculated into 100 mL of the fermentation media and incubated statically for 16 days at 30\u0026deg;C. After 16 days of fermentation, the quantity of BC produced, the amount of substrate consumed and the final pH of the media were determined.\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\u003eMedia composition with different sugar combinations used as carbon sources for BC production.\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=\"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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSugars\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM1\u0026thinsp;+\u0026thinsp;HS components\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM2\u0026thinsp;+\u0026thinsp;HS components\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM3\u0026thinsp;+\u0026thinsp;HS components\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eM4\u0026thinsp;+\u0026thinsp;HS components\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eM5\u0026thinsp;+\u0026thinsp;HS components\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eM6\u0026thinsp;+\u0026thinsp;HS components\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose\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\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArabinose\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\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\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\u003eXylose\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\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \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\u003eCellobiose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\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\u003eMannose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\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\u003eGalactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\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 \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eKey: M1: Medium 1, M2: Medium 2, M3: Medium 3, M4: Medium 4, M5: Medium 5, M6: Medium 6\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003eBC purification, pH and substrate consumption determination\u003c/p\u003e \u003cp\u003eThe BC produced after fermentation was harvested by picking with tweezers. BC purification was performed by boiling for 1 h at 80\u0026deg;C in 1 M NaOH. Thereafter, the BC was washed with distilled water until it reached pH 7 and allowed to air dry until a constant weight was achieved. The dry weight of BC (grams per liter of fermentation media (g/L)) was measured via an analytical weighing balance (Kern PFB precision balance, Sigma Aldrich). The percentage BC yield was determined in relation to the amount of carbon source used for production. The BC yield was calculated as follows:\u003c/p\u003e \u003cp\u003eBC yield (%) =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{BC\\:dry\\:weight\\:\\left(g\\right)}{Carbon\\:source\\:used\\:\\left(g\\right)\\:}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe final pH of the fermentation media after BC production was measured with a pH meter. The rate of substrate consumption was determined via high-performance liquid chromatography (HPLC) (Walters Corporation, Milford, USA) by measuring the concentrations of various sugar components in the fermentation media. One milliliter of the fermentation media was pipetted into Eppendorf tubes and centrifuged for 10 minutes at 7000 rpm. A 0.2 \u0026micro;m filter was used for filtration of the supernatant into HPLC vials. A hydrogen base ion-exchange column (Aminex HPX-87H, Bio-Rad, Hercules, USA) that works at 60\u0026deg;C, with 5 mM H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution as the eluent, flowing at 0.6 mL/min, was used for the detection and quantification of sugars. The substrate consumption was calculated as follows:\u003c/p\u003e \u003cp\u003eSubstrate consumption (%)=\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Initial\\:total\\:sugar\\:-Final\\:total\\:sugar}{Final\\:Total\\:sugar}\\:\\times\\:100\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ch3\u003eScanning Electron Microscopy (SEM)\u003c/h3\u003e\n\u003cp\u003eThe morphology of the BC produced by the bacterial strains was determined using SEM. Dried BC films were placed on a carbon tape, thereafter coated with gold using LICA EM ACE 600 (Leica, Germany). The gold coated samples were observed with a scanning electron microscope SEM LEO Gemini 1525 (Carl Zeiss, Germany).\u003c/p\u003e\n\u003ch3\u003eFourier Transform Infrared (FTIR) Spectroscopy\u003c/h3\u003e\n\u003cp\u003eFTIR was performed via an FTIR spectrometer (Shimadzu model, Germany). The BC samples were analysed by placing the dried film on a diamond accessory. With an accumulation of 128 scans, the absorbance was measured in the wavenumber range of 4000\u0026thinsp;\u0026minus;\u0026thinsp;500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eX-ray Diffraction (XRD)\u003c/h3\u003e\n\u003cp\u003eXRD was performed to determine the degree of crystallinity of the BC. X-ray diffraction analysis was performed by measuring the diffraction pattern of the BC sheet via a diffractometer (Siefert Sun XRD 3003) operating at 40 kV and 40 mA. The diffractograms were taken from 0 to 60\u0026deg; on a 2θ scale with a step size of 0.02\u0026deg;. The percentage crystallinity and amorphous content of the BC were determined.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the experiments were carried out in duplicate, and the values are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs. All the data were statistically analysed via MINITAB 17. The data were evaluated via One-Way Analysis of Variance (ANOVA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEffect of the media composition on the BC yield, final pH and total substrate consumption\u003c/h2\u003e \u003cp\u003eThe effects of the media composition on the BC yield, final pH and substrate consumption of the fermentation media by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 and \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 are shown in Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The BC yield and final pH of the media enriched with \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 ranged from 100\u0026ndash;283.3% and 3.7\u0026ndash;4.2, respectively. The highest yield was recorded in M4, whereas the lowest BC yield was observed in M6. The highest pH was observed in M2, and the lowest was observed in M6. For \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630, the highest BC yield and final pH of the media ranged from 43.5\u0026ndash;100.0% and 3.5\u0026ndash;4.0, respectively. The highest yield was recorded in M6, whereas the lowest yield was recorded in M3. The highest pH was observed in M2, and the lowest was observed in M5. During BC production, the highest total substrate consumption of 81.5% was observed in M3 (glucose, mannose, xylose, galactose and arabinose) by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629, whereas in \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630, a total substrate consumption of 97.2% was observed in M6 (glucose). However, in M5 (glucose and xylose), 82.6% of the substrate was consumed by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630.\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\u003eEffects of media composition on BC yield, substrate consumption and pH by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629.\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\u003eMixed carbon Media\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBC yield (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal Substrate consumption (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e211.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e228.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e55.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003c/b\u003e\u003csup\u003e\u003cb\u003ed\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e119.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e81.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e283.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e176.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/b\u003e\u003csup\u003e\u003cb\u003ef\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e56.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003c/b\u003e\u003csup\u003e\u003cb\u003ed\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ed\u003c/sup\u003e\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\u003e \u003cstrong\u003e\u003csup\u003eKeywords\u003c/sup\u003e\u003c/strong\u003e \u003cp\u003e \u003csup\u003eM1 \u0026minus; Glucose + Arabinose + Xylose; M2 \u0026ndash; Glucose + Cellobiose + Xylose + Arabinose; M3 \u0026minus; Glucose + Mannose + Xylose + Galactose + Arabinose; M4 \u0026minus; Glucose + Cellobiose; M5 \u0026ndash; Glucose + Xylose; M6 \u0026ndash; Glucose\u003c/sup\u003e.\u003c/p\u003e \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\u003eEffects of media composition on BC yield, substrate consumption and pH by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630\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\u003eMixed carbon Media\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBC yield (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal Substrate consumption (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e91.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e97.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\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\u003e \u003cstrong\u003e\u003csup\u003eKeywords\u003c/sup\u003e\u003c/strong\u003e \u003cp\u003e \u003csup\u003eM1 \u0026minus; Glucose + Arabinose + Xylose; M2 \u0026ndash; Glucose + Cellobiose + Xylose + Arabinose; M3 \u0026minus; Glucose + Mannose + Xylose + Galactose + Arabinose; M4 \u0026minus; Glucose + Cellobiose; M5 \u0026ndash; Glucose + Xylose; M6 \u0026ndash; Glucose\u003c/sup\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSubstrate consumption of\u003c/b\u003e \u003cb\u003eKomagataeibacter\u003c/b\u003e \u003cb\u003esp. CCUG73629 and\u003c/b\u003e \u003cb\u003eKomagataeibacter\u003c/b\u003e \u003cb\u003esp. CCUG73630 during BC production in mixed carbon sources.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe pattern of substrate consumption by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 and \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 in the mixed carbon source medium during BC production is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-f. During BC production by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 in M1, the consumption of glucose, arabinose and xylose ranged from 18.0\u0026ndash;1.9 g/L, 3.5\u0026ndash;1.9 g/L and 6.4\u0026ndash;3.3, respectively. By day 16, 89% of the glucose had been consumed. During BC production by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 in M1, the consumption of glucose, arabinose and xylose ranged from 18\u0026ndash;1.3 g/L, 3.5\u0026ndash;1.9 g/L and 6.5\u0026ndash;3.2 g/L, respectively. By day 16, 92% of the glucose and 50% of the xylose were consumed.\u003c/p\u003e \u003cp\u003eIn M2, during BC production by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629, the consumption of glucose, cellobiose, xylose and arabinose ranged from 14\u0026ndash;1.7 g/L, 7.4\u0026ndash;7.1 g/L, 4\u0026ndash;1.9 g/L and 2.5\u0026ndash;1.5 g/L, respectively. After 16 days, 87% of the glucose and 3% of the cellobiose were consumed. During BC production by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 in M2, the consumption of glucose, cellobiose, xylose and arabinose ranged from 14\u0026ndash;1.3 g/L, 8\u0026ndash;5.8 g/L, 3.9\u0026ndash;1.6 g/L and 3\u0026ndash;1.9 g/L, respectively. By the 16th day, 91% of the glucose, 27% of the cellobiose and 58% of the xylose were consumed.\u003c/p\u003e \u003cp\u003eDuring BC production via M3 and \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629, the consumption of glucose, mannose, xylose, galactose and arabinose ranged from 10.8\u0026ndash;0.9 g/L, 1.4\u0026ndash;0.3 g/L, 6.4\u0026ndash;1.4 g/L, 3.5\u0026ndash;0.9 g/L and 5.5\u0026ndash;5.5 g/L, respectively. After 16 days, 91% of the glucose and over 70% of the other sugars were consumed. During BC production by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630, glucose, mannose, xylose, galactose and arabinose consumption ranged from 10.8\u0026ndash;0.8 g/L, 1.4\u0026ndash;0.9 g/L, 6.5\u0026ndash;3.2 g/L, 3.5\u0026ndash;2.9 g/L and 5.5\u0026ndash;3.3 g/L, respectively. After 16 days, 92% of the glucose, 50% of the xylose and 18% of the galactose were consumed.\u003c/p\u003e \u003cp\u003eWith the use of M4 during BC production by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629, the consumption of glucose and cellobiose ranged from 21.6\u0026ndash;4.3 g/L and 8.1\u0026ndash;6.9 g/L, respectively. During production by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630, the consumption of glucose and cellobiose ranged from 21.7\u0026ndash;2.3 g/L and 8.0\u0026ndash;2.8 g/L, respectively. After 16 days, \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 consumed 79% of the glucose and 15% of the cellobiose, whereas \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 consumed 89% of the glucose and 39% of the cellobiose.\u003c/p\u003e \u003cp\u003eDuring BC production with M5, \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629, the consumption of glucose and xylose ranged from 21.3\u0026ndash;2.4 g/L and 6.2\u0026ndash;3.5 g/L, respectively, whereas the consumption of glucose and xylose by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 ranged from 21.4\u0026ndash;1.8 g/L and 6.2\u0026ndash;3.0 g/L, respectively. After 16 days of BC production, 88% of the glucose and 43% of the xylose were consumed by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629, whereas 91% of the glucose and 50% of the xylose were consumed by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630.\u003c/p\u003e \u003cp\u003eGlucose consumption in M6 ranged from 28\u0026thinsp;\u0026minus;\u0026thinsp;12 g/L and 28.4\u0026ndash;0.7 g/L by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 and \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 during BC production. By the 16th day, \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 consumed 57%, while \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 consumed 97% of glucose.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of BC Produced with Mixed Carbon Sources\u003c/h2\u003e \u003cp\u003eThe chemical structures of the BCs produced from \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 and \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 in mixed carbon media are shown in the FTIR spectra in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The BCs produced by both strains were similar, with slight differences in peak positions and absorbances. The broad peaks at approximately 3338 and 3340 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at both strains correspond to O\u0026ndash;H stretching vibrations. The peaks at 2891 and 2895 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicate C\u0026ndash;H group stretching. The peaks at approximately 1641 and 1651 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to O\u0026ndash;H bending of the absorbed water. The peaks at 1427 and 1429 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are associated with CH\u003csub\u003e2\u003c/sub\u003e bending. The peaks at 1163 and 1161 cm-1 indicate asymmetrical C-O-C stretching. The peaks at approximately 1053 and 1055 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicate vibrations of C-C, C-OH and C-H rings and side groups. The peak at approximately 896 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at both strains corresponds to CH vibrations.\u003c/p\u003e \u003cp\u003eThe morphology of the BC produced by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 and \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 in M1, M4 and M6 is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-f. The BC micrograh showed a dense network of oriented fibers produced by both strains. The BC produced by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 in M1 and M4 was more compact and denser, with the fibres tightly aggregated, while the BC fibers prodcued by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 in M1 and M4 densely interwoven. In M6, both strainsproduced similar BC fibers that were densely interwoven.\u003c/p\u003e \u003cp\u003eThe X-ray diffractograms of the BCs produced in M1, M4 and M6 presented two distinct characteristic peaks around 16.8\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:^\\circ\\:\\)\u003c/span\u003e\u003c/span\u003e and 22.7\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:^\\circ\\:\\)\u003c/span\u003e\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The degree of crystallinity and amorphous content of the BC varied with different carbon sources (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In M1, BC produced by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 had a greater degree of crystallinity (67.5%) than BC produced by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 (58.6%). However, the M6 BC produced by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 had a relatively high degree of crystallinity (66.7%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDegree of crystallinity and amorphous content of BC produced in mixed carbon media\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBC produced in Mixed carbon media\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDegree of Crystallinity (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmorphous content (wt. %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDegree of Crystallinity (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmorphous content (wt. %)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBC_M1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e67.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBC_M4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBC_M6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e66.7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.9\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\u003e \u003cstrong\u003e\u003csup\u003eKeywords\u003c/sup\u003e\u003c/strong\u003e \u003cp\u003e \u003csup\u003eM1 \u0026minus; Glucose + Arabinose + Xylose; M4 \u0026minus; Glucose + Cellobiose; M6 \u0026ndash; Glucose\u003c/sup\u003e.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe effect of media composition on BC production provides insight into how bacteria can utilize mixed hexose and pentose as a carbon source for production. Growth media containing different sources of nutrients are important for determining the yield and characteristic properties of BC produced by different bacterial species. The carbon source is vital for BC production, as yield depends on the availability and quality of the carbon source [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. During BC synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), hexose sugars are directly metabolized via glycolysis to produce glucose-6-phosphate, a precursor for UDP-glucose, for BC synthesis, which is energy efficient, enabling a relatively high BC yield. However, pentose sugars enter the pentose phosphate pathway, requiring gluconeogenesis to generate BC precursors and consuming more ATP, which may reduce BC production efficiency [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. \u003cem\u003eKomagataeibacter\u003c/em\u003e species among the acetic acid bacteria are widely studied for BC production because they achieve considerable BC yields and metabolize different carbon sources [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The total substrate consumption did not significantly affect the BC yield. During BC production by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629, the medium that contained glucose as the sole source of carbon least supported BC production, which could indicate that the bacterial cells thrived better in media containing more than one carbon source. This may be a result of catabolite repression in single sugar media due to the relatively high concentration of a single sugar (glucose), which can inhibit enzymatic reactions involved in sensing endogenous levels of sugars and can be avoided when mixed sugars are utilized [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Zhong et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] suggested that the production of BC may be efficient by adding a combination of several carbon sources, as they reported increased BC production in media containing glucose, glycerol and fructose as mixed carbon sources. When \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 was used for BC production, the glucose-only medium supported the highest BC yield, which means that a single carbon source was more efficient for BC production than a mixed carbon source was. \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 also consumed more glucose than did \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629, indicating that \u003cem\u003eKomagaetibacter\u003c/em\u003e sp. CCUG73630 preferred glucose as the sole carbon source for BC production. With glucose as the carbon source, both strains presented similar yields of BC, although glucose consumption was lower in \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629. During BC production, glucose can be used as a precursor for the assembly of glucose units into cellulose [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe medium containing glucose and cellobiose as the carbon source supported the highest BC production by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629, with only 15% of cellobiose consumed, whereas M4 had the second-best BC yield by \u003cem\u003eKomagataeibacter\u003c/em\u003e. sp. CCUG73630, 39% of the cellobiose was consumed. These findings emphasize the metabolic versatility of the individual strains in the medium, indicating that cellobiose may have the ability to activate BC production even though consumption was low, making cellobiose an inducer of BC production. Qi et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] reported that after the 2nd day of fermentation, when little cellobiose was consumed, \u003cem\u003eGluconacetobacter xylinus\u003c/em\u003e could not further consume cellobiose during BC production.\u003c/p\u003e \u003cp\u003eThe medium with the most diverse sugar mixture containing glucose, mannose, xylose, galactose and arabinose resulted in the lowest BC production for both \u003cem\u003eKomagateibacter\u003c/em\u003e sp. CCUG73629 and \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630, with both having similar total substrate consumption. However, when Dahman et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] used \u003cem\u003eG. xylinus\u003c/em\u003e ATCC700178, greater production was recorded for glucose, mannose, xylose, galactose and arabinose mixtures. The pH of the media after BC production for 16 days by both strains did not have any significant effect on BC yield since the deviation from the optimum pH was low [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe carbon sources used for BC production affect its properties, such as the crystallinity index [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The mixed carbon sources influenced the degree of crystallinity and amorphous content of the BC produced by both strains. The X-ray diffractograms of the BCs produced by both strains in the mixed carbon media were similar, with slight differences in intensity. The characteristic peaks were assigned to the (110) and (200) crystallographic planes, which corresponds to the cellulose I structure [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The crystallinity of BC varied with the strain producing it and the medium. However, the crystallinity of BC produced by both strains in M4 was not significantly different. Chen et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] reported that the crystallinity of BC produced in single sugar media (galactose, xylose and mannose) was greater than that of BC produced in mixed sugar media; however, the yield of BC was lower in single sugar media. The morphology of the BC produced by both strains showed only slight differences. The compact, densely interwoven structure of the BC fibres was similar to those in the study of Arooj et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] who reported similar effect for BC produced by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. LMG 18909 and FXV3 in glucose and glycerol with 3.0% ethanol. There was no significant difference in the chemical structure of BC. The functional groups present were similar with those reported by Akintunde et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] during BC production in agricultural residue. The FTIR spectra of both strains in mixed carbon media indicated that all the peaks were consistent with previous characterization of bacterial cellulose [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrated the complexity and strain-specific nature of BC production via the use of mixed carbon sources, which stems from differences in metabolic pathways, energy efficiency and regulatory mechanisms. The strains utilized the carbon sources in the media simultaneously at varying rates, with glucose being prioritized for initial consumption at the highest rate, followed by slower consumption of other sugars in the medium. Efforts to improve BC production with alternative carbon sources, including agricultural wastes and feedstocks such as sugarcane bagasse, corncob, wheat straw, and rice straw, are sustainable. The hydrolysate of these materials contains sugar mixtures of glucose, arabinose, xylose, galactose, mannose, and cellobiose. Therefore, the combination of sugars from this study highlights the consumption pattern of such sugars and their effects on BC yield and some properties, which is essential for the optimization of BC production for future BC applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNo funding was received for conducting this study\u003c/p\u003e \u003cp\u003eCompeting interests\u003c/p\u003e \u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article\u003c/p\u003e \u003cp\u003eData Availability Statement\u003c/p\u003e \u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request.\u003c/p\u003e \u003cp\u003eEthics approval\u003c/p\u003e \u003cp\u003eNot Applicable\u003c/p\u003e \u003cp\u003eConsent\u003c/p\u003e \u003cp\u003eNot applicable\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e\"MOA designed the study, performed formal analysis and investigation. MOA wrote the main manuscript text . OMA edited the manuscript and prepared figure 6. BCA edited the manuscript and supervised the study. All authors reviewed the manuscript.\"\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePotočnik V, Gorgieva S, Trček J (2023) From Nature to Lab: Sustainable Bacterial Cellulose Production and Modification with Synthetic Biology. 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Appl Biochem Biotechnol 162:1647-1659.\u003c/li\u003e\n\u003cli\u003eZhong C, Zhang GC, Liu M, Zheng XT, Han PP, Jia SR (2013) Metabolic flux analyses of \u003cem\u003eGluconacetobacter xylinus\u003c/em\u003e for bacterial cellulose production. Appl Microbiol Biotechnol 97(14):6189\u0026ndash;6199.\u003c/li\u003e\n\u003cli\u003eGorgieva S, Trček J (2019) Bacterial Cellulose: Production, Modification and Perspectives in Biomedical Applications. Nanomat (Basel) 9(10):1352.\u003c/li\u003e\n\u003cli\u003eQi GX, Luo MT, Huang C, Guo H, Chen XF, Xiong L, Wang B, Lin X, Peng F, Chen XD (2017) Comparison of bacterial cellulose production by \u003cem\u003eGluconacetobacter xylinus\u003c/em\u003e on bagasse acid and enzymatic hydrolysates. J Appl Polym Sci 134:45066.\u003c/li\u003e\n\u003cli\u003eMuhammad H, Alburae NA, Salam MA, Badshah M, Khan T, Abo-Aba SEM (2024) Identification of Cellulose Producing Bacterial Strains-An Eco-friendly and Cost-effective Approach. J Pure Appl Microbiol 18(1):483-499\u003c/li\u003e\n\u003cli\u003eChen G, Wu G, Chen L, Wang W, Hong FF, J\u0026ouml;nsson LJ (2019) Comparison of productivity and quality of bacterial nanocellulose synthesized using culture media based on seven sugars from biomass. Microb Biotechnol 12(4):677-687.\u003c/li\u003e\n\u003cli\u003eFatima A, Ortiz-Albo P, Neves LA, Nascimento FX, Crespo JG (2023) Biosynthesis and characterization of bacterial cellulose membranes presenting relevant characteristics for air/gas filtration. J Membr Sci 674:121509. \u003c/li\u003e\n\u003cli\u003eLu H, Jiang X (2014) Structure and properties of bacterial cellulose produced using a trickling bed reactor. Appl Biochem Biotechnol 172:3844-3861.\u003c/li\u003e\n\u003cli\u003eDrosos A, Kordopati G, Anastasopoulos C, Zafeiropoulos J, Koutinas A, Kanellaki M (2024) Comparative study and characterization of water-treated bacterial cellulose produced by solid or liquid inoculum of \u003cem\u003eKomagateibacter sucrofermentans\u003c/em\u003e. Cellul 31:1-29.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Komagataeibacter, Bacterial cellulose, Mixed carbon sources, Degree of crystallinity","lastPublishedDoi":"10.21203/rs.3.rs-6681004/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6681004/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLow-cost substrates and agricultural wastes for bacterial cellulose (BC) production have gained significant attention in recent years because of their potential to increase yield and reduce production costs. Diverse bacterial species exhibit heterogeneous metabolic profiles and substrate utilization patterns during BC biosynthesis when cultivated on these substrates. The aim of this study was to mimic the hexose and pentose composition of low-cost substrates to increase the BC yield. This study investigated the substrate utilization patterns of two \u003cem\u003eKomagataeibacter\u003c/em\u003e species during BC production on mixed carbon substrates. Both strains used in this study utilized mixed hexose and pentose sugars as carbon sources for BC production, with varying consumption patterns and BC yields. \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73629 efficiently utilized multiple sugars, with the highest BC yield recorded in the glucose-cellobiose medium (M4). The highest BC yield of \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 was recorded in medium containing glucose as the sole carbon source. The BC produced had functional groups associated with cellulose, well-defined diffraction peaks, and densely interwoven fiber structures. The maximum degree of crystallinity (67.5%) was recorded for BC produced by \u003cem\u003eKomagataeibacter\u003c/em\u003e sp. CCUG73630 in a glucose-arabinose-xylose medium (M1). Owing to their unique metabolic profiles, each \u003cem\u003eKomagataeibacter\u003c/em\u003e species demonstrates different substrate utilization patterns. This study revealed the complexity, variation, unique metabolism, and strain-specific nature of bacterial BC production using mixed hexose and pentose sugars as carbon sources. Thus, this study contributes to the development of efficient and economical methods for producing BC from alternative substrates.\u003c/p\u003e","manuscriptTitle":"Mixed hexose and pentose sugars induce species-variable bacterial cellulose production by Komagataeibacter spp.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 12:27:45","doi":"10.21203/rs.3.rs-6681004/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-07T01:54:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-09T09:09:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-01T20:12:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-23T17:01:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283146071935209594539790054336271517807","date":"2025-06-20T16:29:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289627253652415888677899570089098055022","date":"2025-06-19T09:14:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21118238677532044214544426615147994014","date":"2025-06-17T09:54:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324020314752859344343324624548850314186","date":"2025-06-17T04:11:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-17T04:09:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-21T08:47:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-21T08:28:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioprocess and Biosystems Engineering","date":"2025-05-16T12:55:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bbcfa307-f8f2-48e7-bb83-d1597c2fdc48","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-25T01:23:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-18 12:27:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6681004","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6681004","identity":"rs-6681004","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}