Improving thermal fermentation and production of Bacillus subtilis from lignocellulosic hydrolysate by glycerol protection

Improving thermal fermentation and production of Bacillus subtilis from lignocellulosic hydrolysate by glycerol protection | 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 Improving thermal fermentation and production of Bacillus subtilis from lignocellulosic hydrolysate by glycerol protection Biying Guo, Liang Wu, Xia Hua, Dylan Liu, Yong Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7459279/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bacillus subtilis prebiotic is a promising alternative to antibiotics, offering advantages in animal wellbeing and the utilization of abundant raw materials. To align with the enzyme hydrolysis processing temperature of lignocellulose materials, typically 45–50 ℃, it is essential to enhance the temperature tolerance of Bacillus subtilis as much as possible. Based on the comparison of varied protectants, a glycerol-protecting method was established to effectively improve the thermal fermentation performance. Comprehensive evaluation metrics, such as cell dry weight and viable bacterial count, were used to determine the optimal fermentation conditions for B. subtilis at 50 ℃, which included 70 g/L of glucose substrate and 1% v/v glycerol protection. To validate the practicality of this approach, the glycerol protection fermentation was further applied in lignocellulose hydrolysate, resulting in a yield of 22.43 g/L of B. subtilis , which is an increase of 20.9%, corresponding to a yield of 0.43 g/g. This study presents a feasible and systematic approach to develop thermal fermentation and production of Bacillus subtilis prebiotic from lignocellulose biorefinery. Bacillus subtilis Thermal fermentation Glycerol-protecting fermentation Lignocellulosic hydrolysate Systematic adaptability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Bacillus subtilis , a gram-positive bacterium, is renowned for its robustness and versatility in industrial biotechnology. Its capabilities include producing a range of enzymes, antibiotics, and bioactive compounds (Drejer et al., 2018 ; Hara et al., 2014 ). B. subtilis is a typical probiotic bacillus capable of forming endogenous stress-resistant spores under adverse conditions (Khatri et al., 2016 ). Unlike non-bacillus probiotics such as bifidobacterium and lactobacillus, B. subtilis exhibits superior resistance to extreme conditions, including high temperatures, acids, and alkalis (Elshaghabee et al., 2017 ). This resilience underpins the growing interest in high-temperature fermentation as a strategy to enhance the production efficiency of B. subtilis -derived bioproducts (Ren et al., 2022 ). High-temperature fermentation, typically conducted above 37°C, confers several benefits: accelerated metabolic rates, a reduced risk of microbial contamination, and enhanced substrate utilization efficiency (Tiwari et al., 2017 ). These advantages position B. subtilis as an ideal candidate for processes that require elevated fermentation temperatures (Zeldes et al., 2015 ). Under high-temperature conditions, B. subtilis undergoes physiological adaptations to maintain cellular homeostasis and metabolic activity. These adaptations include changes in membrane fluidity, heat shock protein expression, and enzyme kinetics (Isticato et al., 2020 ; Zhang and Gou, 2014 ). The utility of B. subtilis in high-temperature fermentation settings extends across various industries, including pharmaceuticals, food processing, and biofuel production (Miljaković et al., 2020 ). Examples include the production of thermostable enzymes, antimicrobial peptides, and biodegradable plastics (Song et al., 2016 ). Despite its potential, the high-temperature fermentation of B. subtilis presents challenges such as metabolic burden, product inhibition, and the energy demands of cooling processes (Li et al., 2014 ). Industrial-scale fermentation at high temperature can adversely affect the growth and metabolism of the microorganisms, thus impacting the yield and quality of the product (Kumar et al., 2021 ). Consequently, developing strategies to enhance temperature resistance is crucial for expanding the industrial applications of B. subtilis . Although genetic engineering advances have facilitated the creation of tailored B. subtilis strains with improved thermotolerance and productivity, their instability has hindered large-scale industrial or commercial production (Put et al., 2024 ). High-temperature fermentation protectants are designed to enhance the survival and functionality of microorganisms under extreme conditions(Oliveira et al., 2014 ). These protectants help reduce oxidative damage, enhance cell membrane stability, or modulate cellular metabolic pathways (Shemesh and Chai, 2013 ). Research in high-temperature fermentation protectants aims to improve the efficiency and robustness of fermentation processes in various industries, such as food, beverage, and biofuel production (Yang et al., 2024 ). At present, some common protective agents include glycerol, glutamic acid, histidine, polyethylene glycol (PEG) and betaine, they improve the survival rate and productivity of B. subtilis at high temperatures by maintaining the osmotic pressure of the cells and protecting the stability of intracellular proteins (Bashir et al., 2014 ). This paper explores the potential of using protective agents to prepare probiotic B. subtilis from lignocellulosic biomass and discusses how these optimization strategies could be applied to the high-temperature cultivation of other microorganisms. 2. Materials and methods 2.1 Chemicals and reagents B. subtilis CICC1073 was purchased from the China Center of Industrial Culture Collection (CICC). YPD medium, glycerol, glutamic acid, histidine, PEG, betaine, and other chemicals used in of the B. subtilis broth were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. All other chemicals were of analytical grade and commercially available. 2.2 Corncob pretreatment with xylonic acid Air-dried corncobs were obtained from Dongtai (China). Corncobs were crushed and sieved to a particle size of 40–80 mesh. The main constitutes were glucan (35.24%), xylan (30.70%), arabinose (3.76%), and lignin (20.28%) as determined by the standard analytical method of the National Renewable Energy Laboratory (Templeton et al., 2010 ). Corncob powder was pretreated with 5 wt% xylonic acid (XA) at 143 ℃ for 75 min at a solid-to-liquid ratio of 1:10 in a 100 mL stainless steel tube within a hot-oil bath (Guo et al., 2020 ). After cooling to room temperature in water, the pretreated corncobs were centrifuged at 6700 ×g for 10 minutes to separate the liquid and solid fractions. The crude solution was treated with electrodialysis to recover and recycle XA, following the method described by Liu et.al.(2021). 2.3 Enzymatic hydrolysis The pretreated solid was washed with 20 times its weight in water and neutralized with sodium hydroxide to a pH of 4.80, containing 56.86% glucan and 14.80% xylan. Enzymatic hydrolysis was conducted at 50 ℃ and 150 rpm for 48 h in a 250 mL shaking flask containing a 50 mL slurry. The total solid content was 5% (w/v) solid with 20 FPIU of cellulase activity loading (SAE0020, Sigma, Shanghai, China) per gram of glucan. The enzymatic hydrolysate was finally harvested by centrifuging at 5000 ×g for 10 min to remove the residual solid. 2. 4 Culture and fermentation of Bacillus subtilis B. subtilis CICC10732 was maintained on an agar plate at 4 ℃ and activated in YPD medium at 30 ℃ for 24 h on a shaker with 200 rpm. The activated cells were inoculated at 2% (v/v) into 50 mL of sterilized fermentation media containing 20 g/L of corn steep powder (C116026, Aladdin, Shanghai, China) as a source of nitrogen and vitamins in a 250 mL Erlenmeyer flask. Bacterial culture and fermentation were run at temperatures ranging from 30 ℃ to 50 ℃ at 200 rpm. Sample of 0.5-1.0 mL of the fermentation mixture were taken periodically for analysis. 2.5 Cell optimal density and dry weight determination The cell density was measured at a 600 nm wavelength light using a UV-Vis spectrophotometer (Spectrumlab752s, Shanghai, China). Samples ranging from 0.5 to 1.0 mL WERE washed and diluted to an absorbance value of 0.7-1.0. The dry cell weight of B. subtilis was calculated using the optimal absorption density according to Eq. (1): Y (g) = 0.80113X − 0.06183 (R 2 = 0.992; where Y is the dry cell weight of B. subtilis and X is the OD 600 nm value). The cell yield was calculated based on the decrease in saccharide weight according to the Eq. (2): Y (g/g) = the increase in dry cell weight / decrease in saccharide weight. 2.6 Viable count - spreading method A specific volume of bacterial suspension was placed in the counting cell of the bacterial counting plate and examined under a microscope. Subsequently, the bacterial count in the sample was calculated by multiplying the bacterial count in the counting chamber scale by the corresponding dilution factor. 2.7Orthogonal experiments and analysis The high temperature fermentation conditions of B. subtilis were optimized using glycerol concentration, bacterial concentration, and glucose concentration as evaluation indicators. According to the orthogonal design table of L 16 (4 3 ), three factors, glycerol concentration (A), glucose concentration (B), and bacterial concentration (C), were selected for a three-factor four-level orthogonal experiment. All factors and levels are listed in Table 1 . Orthogonal design software Latin 3.1.1.9 was used to perform the combination of factor levels and data analysis. Table 1 ( A: glycerol concentration, B: glucose concentration, C: bacterial concentration) Level Factors A B C 1 1% 10 0.1 2 2% 30 0.5 3 3% 50 1 4 4% 70 2 2.8Analytical methods of chemical contents A 0.5–1.0 mL sample was centrifuged at 6700×g for 5 min to obtain the supernatant, which was then diluted with deionized water, centrifuged again at 6700×g for 5 min, and filtered through a 0.22 µm membrane for content analysis. The precipitate was washed twice with a 0.9% (w/w) NaCl solution and analyzed for cell density and dry weight. Monosaccharides and cellobiose were analyzed using high-performance liquid chromatography (HPLC, Agilent 1260, USA) equipped with a Bio-Rad Aminex HPX-87H column at 55 ℃, using a 5 mmol/L H 2 SO 4 mobile phase at a flow rate of 0.6 mL/min. The sample injection volume was 10 µL. XOS components were determined using a high-performance anion exchange chromatograph (HPAEC, Dionex ICS-5000, USA) equipped with a Dionex CarboPac PA200 analytical column (Lian et al., 2020 ). The samples were gradient eluted with a mobile phase of 0.1 M NaOH and 0.5 M NaOAc at 0.3 mL/min and 30 ℃ (Xu et al., 2013 ). 3. Result and discussion 3.1 Screening of protectants for high-temperature fermentation of B. subtilis Changes in colony morphology are macroscopic manifestations of the biological strategies adopted by microorganisms to resist stresses such as starvation, antibacterial and osmotic pressure (Sousa et al., 2013 ). To evaluate the viability of B. subtilis at high temperature, the bacterium was cultured under various temperature conditions, and the number of live bacteria on glucose agar plates was observed, as shown in Fig. 1 . The results indicated significant changes in the colony morphology of B. subtilis under different temperature conditions. At 30°C, the colonies were full and highly opaque, indicating robust growth. As the temperature increased, the opacity of the B. subtilis colonies on the agar plate decreased, and the colony diameter became smaller. Growth was significantly inhibited above 45°C, although survive was still possible at 50°C for a certain period. This indicates that B. subtilis possesses some degree of high temperature resistance, but excessively high temperatures will have a negative impact on its growth. Isticato et al. found that spores produced by B. subtilis at 42°C contained more dipicolinic acid and were more resistant to heat or lysozyme treatment(Isticato et al., 2020 ). To explore the effects of various additives on the fermentation of B. subtilis at 50°C, 1 g/L of betaine, glycerol, PEG, glutamic acid, and histidine were added to the culture medium. These additives can stabilize the cell membrane structure under high temperature conditions to a certain extent, thereby protecting the cells from damage. Boch et al. showed that betaine is an effective osmoprotectant in B. subtilis under high osmotic pressure environments, although it could not be increased by de novo synthesis (Boch et al., 1994 ). However, when choline (a precursor for betaine) is present in the culture medium, the bacteria can synthesize betaine. The growth ability on glucose agar plates was observed at 6 h and 12 h of fermentation, with results shown in Fig. 2 . At 6 h, the high temperature resistance of B. subtilis was significantly improved in the presence of betaine, glycerol, PEG and histidine, with notably improved morphology compared to colonies without protective agents. However, the culture medium with glutamate added showed a significant inhibitory effect, likely due to the sharp drop in pH caused by glutamate addition. In an acidic environment, B. subtilis differentiates into spores that are extremely resistant to potentially damaging conditions (Barney and Austin, 2017 ). Cells grown at low pH adapt to acidic media and induce a pronounced acid-tolerant acceptance response (Thomassin et al., 2006 ). By 12 h, the protective effect of glutamate became slightly evident, while, the protective effects of betaine, PEG and histidine gradually diminished, and glycerol continued to provide good protection. 3.2 Effect of glycerol concentration on fermentation of B. subtilis at 50°C Various concentrations of glycerol (0% v/v, 0.1% v/v, 0.5% v/v, 1% v/v, 1.5% v/v, and 2% v/v) were added to the culture medium, and viable bacterial counts were observed on glucose agar plates at intervals up to 30 h, with results shown in Fig. 3 . The experimental results showed that when the fermentation was carried out for 3 h, there was no significant difference between the control group (no glycerol added) and the experimental group (adding 0.1% v/v to 2% v/v glycerol), and the average number of viable bacteria was 17×10 7 CFU/mL. However, at 6 h, the protective effect of glycerol was evident; the number of viable bacteria in the blank control group was 90×10 7 CFU/mL, while the number of viable bacteria in the group with only 0.1% v/v glycerol added reached 298×10 7 CFU/mL. As the reaction progresses, the high temperature protection effect of glycerol is positively correlated with its concentration within a certain period of time. When the reaction is carried out for 9 h, the protective effect of 0.1% v/v glycerol gradually disappeared. After 12 h, 0.1% v/v-0.5% v/v glycerol had almost no surviving colonies at a dilution factor of 10 7 , the same as the control group. When glycerol concentration increased to 1% v/v to 1.5% v/v, bacteria still survived even after 30 h, and the highest number of viable bacteria reached 49×10 7 CFU/mL. This indicates that within this concentration range, glycerol offers the best high temperature protection effect on B. subtilis . However, beyond this range, its protective effect diminished. Wang et al. studied the growth characteristics of the mutant B. subtilis M270I and the parent B. subtilis 168Δup in a medium with glycerol as the sole carbon source, finding that glycerol was converted to the glycolytic intermediate dihydroxyacetone phosphate, which at high concentrations can be converted to the toxic metabolite methylglyoxal (Wang et al., 2022 ). The experimental results showed that when the glycerol concentration was 2.0% v/v, its protective effect was consistent with that of the control group after 12 h, that is, there were almost no live colonies at the same dilution gradient. This may be due to the toxic effect of excessive glycerol concentration on the cells, or the decrease in dissolved oxygen in the culture medium, thereby inhibiting cell growth. Further observation of the effect of glycerol concentration on cell dry weight and glucose contribution rate of high-temperature fermentation of B. subtilis revealed that changes in glycerol concentration also had a significant impact on these two indicators. As shown in Fig. 4 , when the glycerol concentration reached 1.5% v/v and 2.0% v/v, the maximum dry cell weight of B. subtilis increased significantly, achieving 16.5 g/L, while the dry cell weight of other concentration groups ranged between 13–15 g/L. However, the group with 1.5% v/v glycerol concentration showed the highest glucose contribution rate, achieving 0.84 g/g. In comparison, the increases for the 0.1% v/v, 0.5% v/v, 1.0% v/v, and 2.0% v/v groups were 425%, 320%, 147%, and 29%, respectively. These results indicate that the protective effect of glycerol on the high-temperature fermentation of B. subtilis is most significant when the glycerol concentration ranges from 1.0–1.5% v/v. 3.3 Orthogonal experimental design of glycerol high temperature protection Based on the specific factors and levels outlined in Table 1 , an orthogonal experimental table was constructed and presented as Table 2 . This table details the orthogonal experiment for the high-temperature fermentation of glucose and glycerol by B. subtilis , highlighting variations in the maximum cell dry weight of B. subtilis ranging from 16.20 to 23.10 g/L. Concurrently, the count of viable bacteria was sustained between 45.67×10 8 CFU/mL and 240×10 8 CFU/mL. Under different ratios of glucose and glycerol, the ratio of viable bacteria to cell dry weight was different. This shows that high-temperature fermentation of B. subtilis spores selectively responds to different ratios of glucose and glycerol, indicating the necessity to optimize fermentation conditions to refine the B. subtilis fermentation process. The orthogonal experimental results reveal that at a 1% glycerol concentration, the count of viable B. subtilis bacteria progressively increases with the glucose concentration, yet the cell dry weight does not exhibit a proportional relationship with glucose levels. Specifically, under combination 13, the viable cell count peaked at 240×10 8 CFU/mL, with a glycerol concentration of 1%, glucose at 70 g/L, and an initial cell concentration of 2 OD. Conversely, the maximum cell dry weight recorded was 23.10 g/L under combination 7, where the glycerol concentration was 3%, the glucose concentration stood at 30 g/L, and the initial bacterial concentration was 2 OD. This pattern demonstrates that lower glycerol/glucose ratios correspond with higher viable bacteria counts, whereas higher ratios favor increased cell dry weight. However, in order to obtain more accurate optimal process conditions for high-temperature fermentation of B. subtilis , a range analysis was performed based on the orthogonal test results. the range analysis results of each index are shown in Table 3 . Table 2 Orthogonal experiment table L 16 (4 3 ) (X represents the cell dry weight (g/L), Y represents the number of viable bacteria (×10 8 CFU/mL) Level A B C X Y 1 1% 10 0.1 16.37 ± 0.74 65.00 ± 1.00 2 2% 10 0.5 16.34 ± 0.40 74.00 ± 1.00 3 3% 10 1.0 16.88 ± 0.40 80.00 ± 1.53 4 4% 10 2.0 12.58 ± 0.27 57.67 ± 1.00 5 1% 30 0.5 22.40 ± 0.52 82.00 ± 1.00 6 2% 30 0.1 18.24 ± 0.25 73.67 ± 1.53 7 3% 30 2.0 23.10 ± 0.19 60.33 ± 1.53 8 4% 30 1.0 21.38 ± 0.23 80.33 ± 2.08 9 1% 50 1.0 16.98 ± 0.21 127.67 ± 1.53 10 2% 50 2.0 19.55 ± 0.57 45.67 ± 0.58 11 3% 50 0.1 16.20 ± 0.50 61.00 ± 1.00 12 4% 50 0.5 16.90 ± 0.34 52.67 ± 1.53 13 1% 70 2.0 16.79 ± 0.52 240 ± 0.64 14 2% 70 1.0 20.67 ± 0.21 172 ± 0.53 15 3% 70 0.5 17.39 ± 0.17 77.67 ± 1.52 16 4% 70 0.1 19.91 ± 0.14 57.33 ± 0.48 Table 3 Range Analysis Evaluation index K A B C X K i1X 22.83 19.7 22.3 K i2X 23.5 26.89 22.95 K i3X 21.63 21.79 22.46 K i4X 23.65 23.23 23.9 R ix 2.02 7.19 1.6 Y K i1Y 130 69.25 70.75 K i2Y 89 73.75 64.25 K i3Y 58.5 71.5 110 K i3Y 72.5 135.5 105 R iY 71.5 66.25 45.75 When assessing cell dry weight, glycerol concentration and initial bacterial concentration appeared to exert minimal influence, suggesting that cell growth under high temperatures primarily depends on sugar availability. In contrast, when the number of viable bacteria was used as the evaluation index, glycerol concentration significantly influenced outcomes, followed by glucose concentration, with initial bacterial concentration having a minor impact. This shows that the protective effect of glycerol on cells is extremely important in high-temperature fermentation. Therefore, as far as the high-temperature fermentation process of B. subtilis is concerned, the initial cell concentration is not the main parameter. When the glycerol concentration was 1% v/v, the glucose concentration was 70 g/L and the initial bacterial concentration was 1 OD, the cell dry weight and the number of viable bacteria were both at a high level. Therefore, this combination was selected as the optimal process condition for high-temperature fermentation of B. subtilis . 3.4 High temperature fermentation of B. subtilis with enzyme hydrolysate Under the optimal process conditions optimized by orthogonal experiment, high temperature fermentation was carried out for 48 h, and a glycerol-free control group was set up. After fermentation for 48 h in the centrifugal sugar solution of corncob cellulose hydrolysis containing 52.36 g/L glucose, the sugar utilization rate reached 76.09%. The cell concentration and yield of B. subtilis reached 22.43 g/L and 0.43 g/g, respectively. Compared with the controls, the cell yield increased by 20.86% while the sugar utilization rate decreased by 14.12%. Compared with the fermentation at 30°C, the glucose utilization rate of B. subtilis increased by 2 times at the medium temperature of 50°C, while the cell yield decreased by 46.58%. This result shows that under the protection of glycerol, B. subtilis could utilize glucose and proliferate cells at a medium temperature of 50°C. Figure 5 shows the effect of glycerol on the high temperature fermentation of B. subtilis in corncob hydrolysate. At the initial 6 h of fermentation, the cell dry weight of the two groups was similar, about 13.0 g/L. However, in terms of viable bacterial count, the glycerol group was significantly higher than the control group, achieving 111.5×10 7 CFU/mL, which was almost twice that of the control group (56×10 7 CFU/mL). This indicates that glycerol plays an protective role in high temperature fermentation. As the fermentation progressed, the difference in cell dry weight gradually emerged. At the same time, the number of viable bacteria also increased to 199.5×10 7 CFU/mL, which was 64.20% higher than that of the control group. It is worth noting that at 24 h, the cell dry weight of the two groups reached the maximum value, which was 22.43 g/L and 17.84 g/L, respectively. Thereafter, the cell dry weight remained essentially unchanged. However, the number of viable bacteria in both groups began to decrease during the fermentation process. although the number of viable bacteria in the glycerol group was always higher than that in the control group, at 48 h, the difference between the two groups was not significant, and the number of viable bacteria in the glycerol group was even slightly lower. This may be because the presence of glycerol leads to a decrease in dissolved oxygen in the subsequent fermentation reaction, thereby accelerating cell death. Arjes et al. reported population heterogeneity observed in oxygen-depleted cultures of B. subtilis 3610 , in which 90% of cells died and 10% remained viable (Arjes et al., 2020 ). Therefore, it is recommended that the reaction time of adding glycerol during fermentation under high temperature conditions should be controlled within 24 h. The enzymatic hydrolysate of the corncob experiment in this section was obtained from the solid residue obtained after xylose pretreatment. The enzymatic hydrolysate contained 56.10 g/L glucose, 10.01 g/L xylose, 5.81 g/L arabinose and 1.89 g/L cellobiose. Figure 6 a shows the effect of glycerol on sugar consumption by B. subtilis in corncob hydrolysate. As shown in the figure, during the fermentation process, the sugar consumption rate of the glycerol group was significantly higher than that of the control group, with the glucose consumption rates in the first 12 h being 3.22 g/L/h and 2.97 g/L/h, respectively. This further confirms the promoting effect of glycerol on the growth of bacillus subtilis during high temperature fermentation. However, as the reaction progressed, the rate of glucose consumption in the glycerol group gradually slowed down. This may be related to the decrease in dissolved oxygen, which leads to a decrease in the number of living cells and a weakened ability to consume glycogen. Based on the above, the protective effect of glycerol on cells at different temperatures (30 ℃ and 40 ℃) was further explored. The experimental results are shown in Fig. 6 b. The results indicated that at 50 ℃, the cell yield and viable cell count reached 0.43 g/g and 199.5×107 CFU/mL, respectively, after 48 hours of fermentation with 1% v/v glycerol added. When the temperature dropped to 40 ℃, the cell yield and viable cell count increased by 74.4% and 18.8% to 0.75 g/g and 237×107 CFU/mL, respectively. When the temperature further decreased to 30 ℃, the cell yield and viable cell count increased by 81.4% and 26.8%, respectively. According to the experimental data and considering the temperature during the subsequent simultaneous saccharification and enzymatic hydrolysis process, adding 1% glycerol at 40 ℃ was found to be the most effective for the high-temperature fermentation of Bacillus subtilis. Conclusion In conclusion, high-temperature fermentation of B. subtilis represents a promising approach for improving bioprocess efficiency and expanding the biotechnological applications of this versatile bacterium. Continued research efforts are essential to harnessing the full potential of B. subtilis in high-temperature industrial fermentations. By exploring novel protectants like glycerol and optimizing fermentation conditions, the overall productivity and yield of microbial fermentation processes conducted at elevated temperatures were enhanced. This research provides crucial technical support for improving the fermentation performance of B. subtilis , especially in bio-refineries. Declarations Ethical approval Our research does not involve any human or animal subjects and therefore does not require ethical approval from relevant authorities. Consent to participate Our research does not involve any human or animal subjects and therefore does not require ethical approval from relevant authorities. Consent to publish All authors approved the final manuscript and the submission to this journal. Author Contributions All authors contributed to the conception and design of the study. Biying Guo and Liang Wu were responsible for the specific investigation work; Biying Guo and Yong Xu completed data collation and verification; Biying Guo, Liang Wu, and Yong Xu jointly completed the formal analysis. The initial draft was written by Biying Guo, and Xia Hua, Dylan Liu, and Yong Xu reviewed and revised the manuscript. Yong Xu was responsible for project guidance, funding acquisition, and overall supervision. All authors read and approved the final manuscript. Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding: The research was supported by the National Key R&D Program of China (2022YFD1300903). Availability of data and materials: The datasets generated during and analyzed during the current study are publicly available. References Arjes, H.A., Vo, L., Dunn, C.M., Willis, L., DeRosa, C.A., Fraser, C.L., Kearns, D.B., Huang, K.C., 2020. Biosurfactant-Mediated Membrane Depolarization Maintains Viability during Oxygen Depletion in Bacillus subtilis. Current Biology 30, 1011-1022.e6. https://doi.org/10.1016/j.cub.2020.01.073 Barney, B.L., Austin, D.E., 2017. Dynamics of rebounding Bacillus subtilis spores determined using image-charge detection. J Biol Phys 43, 481–492. https://doi.org/10.1007/s10867-017-9464-5 Bashir, A., Hoffmann, T., Smits, S.H.J., Bremer, E., 2014. 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Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals. Front. Microbiol. 6. https://doi.org/10.3389/fmicb.2015.01209 Zhang, T., Gou, Q., 2014. Minimal Wave Speed of Bacterial Colony Model with Saturated Functional Response. Abstract and Applied Analysis 2014, 1–9. https://doi.org/10.1155/2014/510671 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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09:14:44","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":122440,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7459279/v1/d6bcf583b30f072fa2ab076b.html"},{"id":92069217,"identity":"df41cde1-b5e8-47f7-a4cd-158b5e4c8a89","added_by":"auto","created_at":"2025-09-24 09:30:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":61280,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of temperature on the fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459279/v1/d0a94eb87915afbd60ab9692.jpg"},{"id":92067689,"identity":"b712dee9-ec06-4e03-9f4d-2bfefcc59ba6","added_by":"auto","created_at":"2025-09-24 09:14:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122759,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different additives on high temperature fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459279/v1/21766ee13e69dba64ff8acce.jpg"},{"id":92067690,"identity":"43f619a9-1003-4707-9ec4-f3fbaa9be711","added_by":"auto","created_at":"2025-09-24 09:14:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":80377,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of glycerol on the number of viable bacteria in high-temperature fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459279/v1/ee491616ba7e4e72fcb07736.jpg"},{"id":92067691,"identity":"b8cef76a-6233-41bc-a26f-854700f386b7","added_by":"auto","created_at":"2025-09-24 09:14:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":75334,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of glycerol on sugar conversion in high temperature fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459279/v1/6c2e8ad61d48eac6b70202b6.jpg"},{"id":92067694,"identity":"f73599ff-ef4f-4e2e-b192-4104c64d9676","added_by":"auto","created_at":"2025-09-24 09:14:44","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":79923,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of glycerol on the fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e in corncob hydrolysate\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459279/v1/7c4dbce63465125b2be68656.jpg"},{"id":92069218,"identity":"e445e99a-82f9-49bf-b484-c2d4d9a31c53","added_by":"auto","created_at":"2025-09-24 09:30:44","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":84874,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Effect of glycerol on consumption of sugar by \u003cem\u003eB. subtilis\u003c/em\u003e in enzymatic solution of corncob; (b) Effect of different temperatures on the protective effect of glycerol\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7459279/v1/deea65e43475f1a1f243271c.jpg"},{"id":92465297,"identity":"54bc4a1f-fa4e-417c-99e9-7998634669dc","added_by":"auto","created_at":"2025-09-30 05:14:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1346427,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7459279/v1/886e3118-bb69-4a9e-8981-a7056c4782be.pdf"}],"financialInterests":"","formattedTitle":"Improving thermal fermentation and production of Bacillus subtilis from lignocellulosic hydrolysate by glycerol protection","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e, a gram-positive bacterium, is renowned for its robustness and versatility in industrial biotechnology. Its capabilities include producing a range of enzymes, antibiotics, and bioactive compounds (Drejer et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hara et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003eB. subtilis\u003c/em\u003e is a typical probiotic bacillus capable of forming endogenous stress-resistant spores under adverse conditions (Khatri et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Unlike non-bacillus probiotics such as bifidobacterium and lactobacillus, \u003cem\u003eB. subtilis\u003c/em\u003e exhibits superior resistance to extreme conditions, including high temperatures, acids, and alkalis (Elshaghabee et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This resilience underpins the growing interest in high-temperature fermentation as a strategy to enhance the production efficiency of \u003cem\u003eB. subtilis\u003c/em\u003e -derived bioproducts (Ren et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHigh-temperature fermentation, typically conducted above 37\u0026deg;C, confers several benefits: accelerated metabolic rates, a reduced risk of microbial contamination, and enhanced substrate utilization efficiency (Tiwari et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These advantages position \u003cem\u003eB. subtilis\u003c/em\u003e as an ideal candidate for processes that require elevated fermentation temperatures (Zeldes et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Under high-temperature conditions, \u003cem\u003eB. subtilis\u003c/em\u003e undergoes physiological adaptations to maintain cellular homeostasis and metabolic activity. These adaptations include changes in membrane fluidity, heat shock protein expression, and enzyme kinetics (Isticato et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang and Gou, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The utility of \u003cem\u003eB. subtilis\u003c/em\u003e in high-temperature fermentation settings extends across various industries, including pharmaceuticals, food processing, and biofuel production (Miljaković et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Examples include the production of thermostable enzymes, antimicrobial peptides, and biodegradable plastics (Song et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite its potential, the high-temperature fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e presents challenges such as metabolic burden, product inhibition, and the energy demands of cooling processes (Li et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Industrial-scale fermentation at high temperature can adversely affect the growth and metabolism of the microorganisms, thus impacting the yield and quality of the product (Kumar et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, developing strategies to enhance temperature resistance is crucial for expanding the industrial applications of \u003cem\u003eB. subtilis\u003c/em\u003e. Although genetic engineering advances have facilitated the creation of tailored \u003cem\u003eB. subtilis\u003c/em\u003e strains with improved thermotolerance and productivity, their instability has hindered large-scale industrial or commercial production (Put et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHigh-temperature fermentation protectants are designed to enhance the survival and functionality of microorganisms under extreme conditions(Oliveira et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These protectants help reduce oxidative damage, enhance cell membrane stability, or modulate cellular metabolic pathways (Shemesh and Chai, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Research in high-temperature fermentation protectants aims to improve the efficiency and robustness of fermentation processes in various industries, such as food, beverage, and biofuel production (Yang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). At present, some common protective agents include glycerol, glutamic acid, histidine, polyethylene glycol (PEG) and betaine, they improve the survival rate and productivity of \u003cem\u003eB. subtilis\u003c/em\u003e at high temperatures by maintaining the osmotic pressure of the cells and protecting the stability of intracellular proteins (Bashir et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This paper explores the potential of using protective agents to prepare probiotic \u003cem\u003eB. subtilis\u003c/em\u003e from lignocellulosic biomass and discusses how these optimization strategies could be applied to the high-temperature cultivation of other microorganisms.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Chemicals and reagents\u003c/h2\u003e\u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e CICC1073 was purchased from the China Center of Industrial Culture Collection (CICC). YPD medium, glycerol, glutamic acid, histidine, PEG, betaine, and other chemicals used in of the \u003cem\u003eB. subtilis\u003c/em\u003e broth were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. All other chemicals were of analytical grade and commercially available.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Corncob pretreatment with xylonic acid\u003c/h2\u003e\u003cp\u003eAir-dried corncobs were obtained from Dongtai (China). Corncobs were crushed and sieved to a particle size of 40\u0026ndash;80 mesh. The main constitutes were glucan (35.24%), xylan (30.70%), arabinose (3.76%), and lignin (20.28%) as determined by the standard analytical method of the National Renewable Energy Laboratory (Templeton et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCorncob powder was pretreated with 5 wt% xylonic acid (XA) at 143 ℃ for 75 min at a solid-to-liquid ratio of 1:10 in a 100 mL stainless steel tube within a hot-oil bath (Guo et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). After cooling to room temperature in water, the pretreated corncobs were centrifuged at 6700 \u0026times;g for 10 minutes to separate the liquid and solid fractions. The crude solution was treated with electrodialysis to recover and recycle XA, following the method described by Liu et.al.(2021).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Enzymatic hydrolysis\u003c/h2\u003e\u003cp\u003eThe pretreated solid was washed with 20 times its weight in water and neutralized with sodium hydroxide to a pH of 4.80, containing 56.86% glucan and 14.80% xylan. Enzymatic hydrolysis was conducted at 50 ℃ and 150 rpm for 48 h in a 250 mL shaking flask containing a 50 mL slurry. The total solid content was 5% (w/v) solid with 20 FPIU of cellulase activity loading (SAE0020, Sigma, Shanghai, China) per gram of glucan. The enzymatic hydrolysate was finally harvested by centrifuging at 5000 \u0026times;g for 10 min to remove the residual solid.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.\u003cem\u003e4 Culture and fermentation of Bacillus subtilis\u003c/em\u003e\u003c/h2\u003e\u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e CICC10732 was maintained on an agar plate at 4 ℃ and activated in YPD medium at 30 ℃ for 24 h on a shaker with 200 rpm. The activated cells were inoculated at 2% (v/v) into 50 mL of sterilized fermentation media containing 20 g/L of corn steep powder (C116026, Aladdin, Shanghai, China) as a source of nitrogen and vitamins in a 250 mL Erlenmeyer flask. Bacterial culture and fermentation were run at temperatures ranging from 30 ℃ to 50 ℃ at 200 rpm. Sample of 0.5-1.0 mL of the fermentation mixture were taken periodically for analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Cell optimal density and dry weight determination\u003c/h2\u003e\u003cp\u003eThe cell density was measured at a 600 nm wavelength light using a UV-Vis spectrophotometer (Spectrumlab752s, Shanghai, China). Samples ranging from 0.5 to 1.0 mL WERE washed and diluted to an absorbance value of 0.7-1.0.\u003c/p\u003e\u003cp\u003eThe dry cell weight of \u003cem\u003eB. subtilis\u003c/em\u003e was calculated using the optimal absorption density according to Eq.\u0026nbsp;(1):\u003c/p\u003e\u003cp\u003eY (g)\u0026thinsp;=\u0026thinsp;0.80113X\u0026thinsp;\u0026minus;\u0026thinsp;0.06183 (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.992; where Y is the dry cell weight of \u003cem\u003eB. subtilis\u003c/em\u003e and X is the OD\u003csub\u003e600 nm\u003c/sub\u003e value).\u003c/p\u003e\u003cp\u003eThe cell yield was calculated based on the decrease in saccharide weight according to the Eq.\u0026nbsp;(2):\u003c/p\u003e\u003cp\u003eY (g/g)\u0026thinsp;=\u0026thinsp;the increase in dry cell weight / decrease in saccharide weight.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Viable count - spreading method\u003c/h2\u003e\u003cp\u003eA specific volume of bacterial suspension was placed in the counting cell of the bacterial counting plate and examined under a microscope. Subsequently, the bacterial count in the sample was calculated by multiplying the bacterial count in the counting chamber scale by the corresponding dilution factor.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7Orthogonal experiments and analysis\u003c/h2\u003e\u003cp\u003eThe high temperature fermentation conditions of \u003cem\u003eB. subtilis\u003c/em\u003e were optimized using glycerol concentration, bacterial concentration, and glucose concentration as evaluation indicators. According to the orthogonal design table of L\u003csub\u003e16\u003c/sub\u003e (4\u003csup\u003e3\u003c/sup\u003e), three factors, glycerol concentration (A), glucose concentration (B), and bacterial concentration (C), were selected for a three-factor four-level orthogonal experiment. All factors and levels are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Orthogonal design software Latin 3.1.1.9 was used to perform the combination of factor levels and data analysis.\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\u003e\u003cb\u003e(\u003c/b\u003eA: glycerol concentration, B: glucose concentration, C: bacterial concentration)\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=\"char\" char=\".\" 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\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLevel\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eFactors\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8Analytical methods of chemical contents\u003c/h2\u003e\u003cp\u003eA 0.5\u0026ndash;1.0 mL sample was centrifuged at 6700\u0026times;g for 5 min to obtain the supernatant, which was then diluted with deionized water, centrifuged again at 6700\u0026times;g for 5 min, and filtered through a 0.22 \u0026micro;m membrane for content analysis. The precipitate was washed twice with a 0.9% (w/w) NaCl solution and analyzed for cell density and dry weight. Monosaccharides and cellobiose were analyzed using high-performance liquid chromatography (HPLC, Agilent 1260, USA) equipped with a Bio-Rad Aminex HPX-87H column at 55 ℃, using a 5 mmol/L H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e mobile phase at a flow rate of 0.6 mL/min. The sample injection volume was 10 \u0026micro;L. XOS components were determined using a high-performance anion exchange chromatograph (HPAEC, Dionex ICS-5000, USA) equipped with a Dionex CarboPac PA200 analytical column (Lian et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The samples were gradient eluted with a mobile phase of 0.1 M NaOH and 0.5 M NaOAc at 0.3 mL/min and 30 ℃ (Xu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Result and discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Screening of protectants for high-temperature fermentation of B. subtilis\u003c/h2\u003e\u003cp\u003eChanges in colony morphology are macroscopic manifestations of the biological strategies adopted by microorganisms to resist stresses such as starvation, antibacterial and osmotic pressure (Sousa et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). To evaluate the viability of \u003cem\u003eB. subtilis\u003c/em\u003e at high temperature, the bacterium was cultured under various temperature conditions, and the number of live bacteria on glucose agar plates was observed, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The results indicated significant changes in the colony morphology of \u003cem\u003eB. subtilis\u003c/em\u003e under different temperature conditions. At 30\u0026deg;C, the colonies were full and highly opaque, indicating robust growth. As the temperature increased, the opacity of the \u003cem\u003eB. subtilis\u003c/em\u003e colonies on the agar plate decreased, and the colony diameter became smaller. Growth was significantly inhibited above 45\u0026deg;C, although survive was still possible at 50\u0026deg;C for a certain period. This indicates that \u003cem\u003eB. subtilis\u003c/em\u003e possesses some degree of high temperature resistance, but excessively high temperatures will have a negative impact on its growth. Isticato et al. found that spores produced by \u003cem\u003eB. subtilis\u003c/em\u003e at 42\u0026deg;C contained more dipicolinic acid and were more resistant to heat or lysozyme treatment(Isticato et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo explore the effects of various additives on the fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e at 50\u0026deg;C, 1 g/L of betaine, glycerol, PEG, glutamic acid, and histidine were added to the culture medium. These additives can stabilize the cell membrane structure under high temperature conditions to a certain extent, thereby protecting the cells from damage. Boch et al. showed that betaine is an effective osmoprotectant in \u003cem\u003eB. subtilis\u003c/em\u003e under high osmotic pressure environments, although it could not be increased by de novo synthesis (Boch et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). However, when choline (a precursor for betaine) is present in the culture medium, the bacteria can synthesize betaine. The growth ability on glucose agar plates was observed at 6 h and 12 h of fermentation, with results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. At 6 h, the high temperature resistance of \u003cem\u003eB. subtilis\u003c/em\u003e was significantly improved in the presence of betaine, glycerol, PEG and histidine, with notably improved morphology compared to colonies without protective agents. However, the culture medium with glutamate added showed a significant inhibitory effect, likely due to the sharp drop in pH caused by glutamate addition. In an acidic environment, \u003cem\u003eB. subtilis\u003c/em\u003e differentiates into spores that are extremely resistant to potentially damaging conditions (Barney and Austin, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Cells grown at low pH adapt to acidic media and induce a pronounced acid-tolerant acceptance response (Thomassin et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). By 12 h, the protective effect of glutamate became slightly evident, while, the protective effects of betaine, PEG and histidine gradually diminished, and glycerol continued to provide good protection.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Effect of glycerol concentration on fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e at 50\u0026deg;C\u003c/h2\u003e\u003cp\u003eVarious concentrations of glycerol (0% v/v, 0.1% v/v, 0.5% v/v, 1% v/v, 1.5% v/v, and 2% v/v) were added to the culture medium, and viable bacterial counts were observed on glucose agar plates at intervals up to 30 h, with results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The experimental results showed that when the fermentation was carried out for 3 h, there was no significant difference between the control group (no glycerol added) and the experimental group (adding 0.1% v/v to 2% v/v glycerol), and the average number of viable bacteria was 17\u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU/mL. However, at 6 h, the protective effect of glycerol was evident; the number of viable bacteria in the blank control group was 90\u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU/mL, while the number of viable bacteria in the group with only 0.1% v/v glycerol added reached 298\u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU/mL. As the reaction progresses, the high temperature protection effect of glycerol is positively correlated with its concentration within a certain period of time. When the reaction is carried out for 9 h, the protective effect of 0.1% v/v glycerol gradually disappeared. After 12 h, 0.1% v/v-0.5% v/v glycerol had almost no surviving colonies at a dilution factor of 10\u003csup\u003e7\u003c/sup\u003e, the same as the control group. When glycerol concentration increased to 1% v/v to 1.5% v/v, bacteria still survived even after 30 h, and the highest number of viable bacteria reached 49\u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU/mL. This indicates that within this concentration range, glycerol offers the best high temperature protection effect on \u003cem\u003eB. subtilis\u003c/em\u003e. However, beyond this range, its protective effect diminished. Wang et al. studied the growth characteristics of the mutant \u003cem\u003eB. subtilis\u003c/em\u003e M270I and the parent \u003cem\u003eB. subtilis\u003c/em\u003e 168Δup in a medium with glycerol as the sole carbon source, finding that glycerol was converted to the glycolytic intermediate dihydroxyacetone phosphate, which at high concentrations can be converted to the toxic metabolite methylglyoxal (Wang et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The experimental results showed that when the glycerol concentration was 2.0% v/v, its protective effect was consistent with that of the control group after 12 h, that is, there were almost no live colonies at the same dilution gradient. This may be due to the toxic effect of excessive glycerol concentration on the cells, or the decrease in dissolved oxygen in the culture medium, thereby inhibiting cell growth.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurther observation of the effect of glycerol concentration on cell dry weight and glucose contribution rate of high-temperature fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e revealed that changes in glycerol concentration also had a significant impact on these two indicators. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, when the glycerol concentration reached 1.5% v/v and 2.0% v/v, the maximum dry cell weight of \u003cem\u003eB. subtilis\u003c/em\u003e increased significantly, achieving 16.5 g/L, while the dry cell weight of other concentration groups ranged between 13\u0026ndash;15 g/L. However, the group with 1.5% v/v glycerol concentration showed the highest glucose contribution rate, achieving 0.84 g/g. In comparison, the increases for the 0.1% v/v, 0.5% v/v, 1.0% v/v, and 2.0% v/v groups were 425%, 320%, 147%, and 29%, respectively. These results indicate that the protective effect of glycerol on the high-temperature fermentation of B. subtilis is most significant when the glycerol concentration ranges from 1.0\u0026ndash;1.5% v/v.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Orthogonal experimental design of glycerol high temperature protection\u003c/h2\u003e\u003cp\u003eBased on the specific factors and levels outlined in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, an orthogonal experimental table was constructed and presented as Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. This table details the orthogonal experiment for the high-temperature fermentation of glucose and glycerol by \u003cem\u003eB. subtilis\u003c/em\u003e, highlighting variations in the maximum cell dry weight of \u003cem\u003eB. subtilis\u003c/em\u003e ranging from 16.20 to 23.10 g/L. Concurrently, the count of viable bacteria was sustained between 45.67\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL and 240\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL. Under different ratios of glucose and glycerol, the ratio of viable bacteria to cell dry weight was different. This shows that high-temperature fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e spores selectively responds to different ratios of glucose and glycerol, indicating the necessity to optimize fermentation conditions to refine the \u003cem\u003eB. subtilis\u003c/em\u003e fermentation process. The orthogonal experimental results reveal that at a 1% glycerol concentration, the count of viable \u003cem\u003eB. subtilis\u003c/em\u003e bacteria progressively increases with the glucose concentration, yet the cell dry weight does not exhibit a proportional relationship with glucose levels. Specifically, under combination 13, the viable cell count peaked at 240\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL, with a glycerol concentration of 1%, glucose at 70 g/L, and an initial cell concentration of 2 OD. Conversely, the maximum cell dry weight recorded was 23.10 g/L under combination 7, where the glycerol concentration was 3%, the glucose concentration stood at 30 g/L, and the initial bacterial concentration was 2 OD. This pattern demonstrates that lower glycerol/glucose ratios correspond with higher viable bacteria counts, whereas higher ratios favor increased cell dry weight. However, in order to obtain more accurate optimal process conditions for high-temperature fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e, a range analysis was performed based on the orthogonal test results. the range analysis results of each index are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\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\u003eOrthogonal experiment table L\u003csub\u003e16\u003c/sub\u003e(4\u003csup\u003e3\u003c/sup\u003e) (X represents the cell dry weight (g/L), Y represents the number of viable bacteria (\u0026times;10\u003csup\u003e8\u003c/sup\u003eCFU/mL)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLevel\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e16.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e65.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e16.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e74.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e16.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e80.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e12.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e57.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e22.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e82.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e18.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e73.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e23.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e60.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e21.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e80.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e16.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e127.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e19.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e45.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e16.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e61.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e16.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e52.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e16.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e240\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e20.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e172\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e17.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e77.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e19.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e57.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\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\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\u003eRange Analysis\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=\"char\" char=\".\" 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\u003cp\u003eEvaluation index\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003csub\u003ei1X\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e22.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003csub\u003ei2X\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e22.95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003csub\u003ei3X\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e22.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003csub\u003ei4X\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e23.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\u003csub\u003eix\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003csub\u003ei1Y\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e69.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e70.75\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003csub\u003ei2Y\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e73.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e64.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003csub\u003ei3Y\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e58.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e71.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e110\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003csub\u003ei3Y\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e72.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e135.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e105\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR\u003csub\u003eiY\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e71.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e66.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e45.75\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\u003eWhen assessing cell dry weight, glycerol concentration and initial bacterial concentration appeared to exert minimal influence, suggesting that cell growth under high temperatures primarily depends on sugar availability. In contrast, when the number of viable bacteria was used as the evaluation index, glycerol concentration significantly influenced outcomes, followed by glucose concentration, with initial bacterial concentration having a minor impact. This shows that the protective effect of glycerol on cells is extremely important in high-temperature fermentation. Therefore, as far as the high-temperature fermentation process of \u003cem\u003eB. subtilis\u003c/em\u003e is concerned, the initial cell concentration is not the main parameter. When the glycerol concentration was 1% v/v, the glucose concentration was 70 g/L and the initial bacterial concentration was 1 OD, the cell dry weight and the number of viable bacteria were both at a high level. Therefore, this combination was selected as the optimal process condition for high-temperature fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4 High temperature fermentation of B. subtilis with enzyme hydrolysate\u003c/h2\u003e\u003cp\u003eUnder the optimal process conditions optimized by orthogonal experiment, high temperature fermentation was carried out for 48 h, and a glycerol-free control group was set up. After fermentation for 48 h in the centrifugal sugar solution of corncob cellulose hydrolysis containing 52.36 g/L glucose, the sugar utilization rate reached 76.09%. The cell concentration and yield of \u003cem\u003eB. subtilis\u003c/em\u003e reached 22.43 g/L and 0.43 g/g, respectively. Compared with the controls, the cell yield increased by 20.86% while the sugar utilization rate decreased by 14.12%. Compared with the fermentation at 30\u0026deg;C, the glucose utilization rate of \u003cem\u003eB. subtilis\u003c/em\u003e increased by 2 times at the medium temperature of 50\u0026deg;C, while the cell yield decreased by 46.58%. This result shows that under the protection of glycerol, \u003cem\u003eB. subtilis\u003c/em\u003e could utilize glucose and proliferate cells at a medium temperature of 50\u0026deg;C.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the effect of glycerol on the high temperature fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e in corncob hydrolysate. At the initial 6 h of fermentation, the cell dry weight of the two groups was similar, about 13.0 g/L. However, in terms of viable bacterial count, the glycerol group was significantly higher than the control group, achieving 111.5\u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU/mL, which was almost twice that of the control group (56\u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU/mL). This indicates that glycerol plays an protective role in high temperature fermentation. As the fermentation progressed, the difference in cell dry weight gradually emerged. At the same time, the number of viable bacteria also increased to 199.5\u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU/mL, which was 64.20% higher than that of the control group. It is worth noting that at 24 h, the cell dry weight of the two groups reached the maximum value, which was 22.43 g/L and 17.84 g/L, respectively. Thereafter, the cell dry weight remained essentially unchanged. However, the number of viable bacteria in both groups began to decrease during the fermentation process. although the number of viable bacteria in the glycerol group was always higher than that in the control group, at 48 h, the difference between the two groups was not significant, and the number of viable bacteria in the glycerol group was even slightly lower. This may be because the presence of glycerol leads to a decrease in dissolved oxygen in the subsequent fermentation reaction, thereby accelerating cell death. Arjes et al. reported population heterogeneity observed in oxygen-depleted cultures of \u003cem\u003eB. subtilis 3610\u003c/em\u003e, in which 90% of cells died and 10% remained viable (Arjes et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, it is recommended that the reaction time of adding glycerol during fermentation under high temperature conditions should be controlled within 24 h.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe enzymatic hydrolysate of the corncob experiment in this section was obtained from the solid residue obtained after xylose pretreatment. The enzymatic hydrolysate contained 56.10 g/L glucose, 10.01 g/L xylose, 5.81 g/L arabinose and 1.89 g/L cellobiose. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea shows the effect of glycerol on sugar consumption by \u003cem\u003eB. subtilis\u003c/em\u003e in corncob hydrolysate. As shown in the figure, during the fermentation process, the sugar consumption rate of the glycerol group was significantly higher than that of the control group, with the glucose consumption rates in the first 12 h being 3.22 g/L/h and 2.97 g/L/h, respectively. This further confirms the promoting effect of glycerol on the growth of bacillus subtilis during high temperature fermentation. However, as the reaction progressed, the rate of glucose consumption in the glycerol group gradually slowed down. This may be related to the decrease in dissolved oxygen, which leads to a decrease in the number of living cells and a weakened ability to consume glycogen. Based on the above, the protective effect of glycerol on cells at different temperatures (30 ℃ and 40 ℃) was further explored. The experimental results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb. The results indicated that at 50 ℃, the cell yield and viable cell count reached 0.43 g/g and 199.5\u0026times;107 CFU/mL, respectively, after 48 hours of fermentation with 1% v/v glycerol added. When the temperature dropped to 40 ℃, the cell yield and viable cell count increased by 74.4% and 18.8% to 0.75 g/g and 237\u0026times;107 CFU/mL, respectively. When the temperature further decreased to 30 ℃, the cell yield and viable cell count increased by 81.4% and 26.8%, respectively. According to the experimental data and considering the temperature during the subsequent simultaneous saccharification and enzymatic hydrolysis process, adding 1% glycerol at 40 ℃ was found to be the most effective for the high-temperature fermentation of Bacillus subtilis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, high-temperature fermentation of \u003cem\u003eB. subtilis\u003c/em\u003e represents a promising approach for improving bioprocess efficiency and expanding the biotechnological applications of this versatile bacterium. Continued research efforts are essential to harnessing the full potential of \u003cem\u003eB. subtilis\u003c/em\u003e in high-temperature industrial fermentations. By exploring novel protectants like glycerol and optimizing fermentation conditions, the overall productivity and yield of microbial fermentation processes conducted at elevated temperatures were enhanced. This research provides crucial technical support for improving the fermentation performance of \u003cem\u003eB. subtilis\u003c/em\u003e, especially in bio-refineries.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur research does not involve any human or animal subjects and therefore does not require ethical approval from relevant authorities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur research does not involve any human or animal subjects and therefore does not require ethical approval from relevant authorities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors approved the final manuscript and the submission to this journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the conception and design of the study. Biying Guo and Liang Wu were responsible for the specific investigation work; Biying Guo and Yong Xu completed data collation and verification; Biying Guo, Liang Wu, and Yong Xu jointly completed the formal analysis. The initial draft was written by Biying Guo, and Xia Hua, Dylan Liu, and Yong Xu reviewed and revised the manuscript. Yong Xu was responsible for project guidance, funding acquisition, and overall supervision. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThe research was supported by the National Key R\u0026amp;D Program of China (2022YFD1300903).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe datasets generated during and analyzed during the current study are publicly available.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArjes, H.A., Vo, L., Dunn, C.M., Willis, L., DeRosa, C.A., Fraser, C.L., Kearns, D.B., Huang, K.C., 2020. Biosurfactant-Mediated Membrane Depolarization Maintains Viability during Oxygen Depletion in Bacillus subtilis. Current Biology 30, 1011-1022.e6. https://doi.org/10.1016/j.cub.2020.01.073\u003c/li\u003e\n\u003cli\u003eBarney, B.L., Austin, D.E., 2017. Dynamics of rebounding Bacillus subtilis spores determined using image-charge detection. J Biol Phys 43, 481\u0026ndash;492. https://doi.org/10.1007/s10867-017-9464-5\u003c/li\u003e\n\u003cli\u003eBashir, A., Hoffmann, T., Smits, S.H.J., Bremer, E., 2014. Dimethylglycine Provides Salt and Temperature Stress Protection to Bacillus subtilis. Appl Environ Microbiol 80, 2773\u0026ndash;2785. https://doi.org/10.1128/AEM.00078-14\u003c/li\u003e\n\u003cli\u003eBoch, J., Kempf, B., Bremer, E., 1994. Osmoregulation in Bacillus subtilis: synthesis of the osmoprotectant glycine betaine from exogenously provided choline. J Bacteriol 176, 5364\u0026ndash;5371. https://doi.org/10.1128/jb.176.17.5364-5371.1994\u003c/li\u003e\n\u003cli\u003eDrejer, E.B., Hakv\u0026aring;g, S., Irla, M., Brautaset, T., 2018. Genetic Tools and Techniques for Recombinant Expression in Thermophilic Bacillaceae. Microorganisms 6, 42. https://doi.org/10.3390/microorganisms6020042\u003c/li\u003e\n\u003cli\u003eElshaghabee, F.M.F., Rokana, N., Gulhane, R.D., Sharma, C., Panwar, H., 2017. Bacillus As Potential Probiotics: Status, Concerns, and Future Perspectives. Front. Microbiol. 8, 1490. https://doi.org/10.3389/fmicb.2017.01490\u003c/li\u003e\n\u003cli\u003eGuo, J., Cao, R., Huang, K., Xu, Y., 2020. Comparison of selective acidolysis of xylan and enzymatic hydrolysability of cellulose in various lignocellulosic materials by a novel xylonic acid catalysis method. Bioresource Technology 304, 122943. https://doi.org/10.1016/j.biortech.2020.122943\u003c/li\u003e\n\u003cli\u003eHara, K.Y., Araki, M., Okai, N., Wakai, S., Hasunuma, T., Kondo, A., 2014. Development of bio-based fine chemicalproduction through synthetic bioengineering. Microbial Cell Factories 13, 173. https://doi.org/10.1186/s12934-014-0173-5\u003c/li\u003e\n\u003cli\u003eIsticato, R., Lanzilli, M., Petrillo, C., Donadio, G., Baccigalupi, L., Ricca, E., 2020. \u003cem\u003eBacillus subtilis\u003c/em\u003e builds structurally and functionally different spores in response to the temperature of growth. Environmental Microbiology 22, 170\u0026ndash;182. https://doi.org/10.1111/1462-2920.14835\u003c/li\u003e\n\u003cli\u003eKhatri, I., Sharma, S., Ramya, T.N.C., Subramanian, S., 2016. Complete Genomes of Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, Two Phylogenetically Distinct Probiotics. PLoS ONE 11, e0156745. https://doi.org/10.1371/journal.pone.0156745\u003c/li\u003e\n\u003cli\u003eKumar, V., Ahluwalia, V., Saran, S., Kumar, J., Patel, A.K., Singhania, R.R., 2021. Recent developments on solid-state fermentation for production of microbial secondary metabolites: Challenges and solutions. 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Abstract and Applied Analysis 2014, 1\u0026ndash;9. https://doi.org/10.1155/2014/510671\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bacillus subtilis, Thermal fermentation, Glycerol-protecting fermentation, Lignocellulosic hydrolysate, Systematic adaptability","lastPublishedDoi":"10.21203/rs.3.rs-7459279/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7459279/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e prebiotic is a promising alternative to antibiotics, offering advantages in animal wellbeing and the utilization of abundant raw materials. To align with the enzyme hydrolysis processing temperature of lignocellulose materials, typically 45\u0026ndash;50 ℃, it is essential to enhance the temperature tolerance of \u003cem\u003eBacillus subtilis\u003c/em\u003e as much as possible. Based on the comparison of varied protectants, a glycerol-protecting method was established to effectively improve the thermal fermentation performance. Comprehensive evaluation metrics, such as cell dry weight and viable bacterial count, were used to determine the optimal fermentation conditions for \u003cem\u003eB. subtilis\u003c/em\u003e at 50 ℃, which included 70 g/L of glucose substrate and 1% v/v glycerol protection. To validate the practicality of this approach, the glycerol protection fermentation was further applied in lignocellulose hydrolysate, resulting in a yield of 22.43 g/L of \u003cem\u003eB. subtilis\u003c/em\u003e, which is an increase of 20.9%, corresponding to a yield of 0.43 g/g. This study presents a feasible and systematic approach to develop thermal fermentation and production of \u003cem\u003eBacillus subtilis\u003c/em\u003e prebiotic from lignocellulose biorefinery.\u003c/p\u003e","manuscriptTitle":"Improving thermal fermentation and production of Bacillus subtilis from lignocellulosic hydrolysate by glycerol protection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 09:14:39","doi":"10.21203/rs.3.rs-7459279/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8e6a5f64-5fd3-421d-a310-1fbc53a13a4e","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-30T05:06:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-24 09:14:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7459279","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7459279","identity":"rs-7459279","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}