Expanding the Operational Window of Consolidated Bioprocessing Hydrogen Production from Non-Detoxified Alkali-Pretreated Tobacco Stalks via Microbial Co-Cultivation

Expanding the Operational Window of Consolidated Bioprocessing Hydrogen Production from Non-Detoxified Alkali-Pretreated Tobacco Stalks via Microbial Co-Cultivation | 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 Expanding the Operational Window of Consolidated Bioprocessing Hydrogen Production from Non-Detoxified Alkali-Pretreated Tobacco Stalks via Microbial Co-Cultivation Ming-Hao Li, Ming-Jun Zhu, Bin-Bin Hu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8915427/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract This study evaluates the feasibility of using non-detoxified alkali-pretreated tobacco stalks for biohydrogen production through separate hydrolysis and fermentation (SHF) and consolidated bioprocessing (CBP). In SHF, Thermoanaerobacterium thermosaccharolyticum MJ2 produced 215.26 ± 49.61 mM hydrogen from non-detoxified enzymatic hydrolysates, showing high tolerance to pretreatment inhibitors. In CBP, while the hydrogen production of Acetivibrio thermocellus DSM1313 alone was inhibited by 90.71% when using non-detoxified stalks, the co-culture of DSM1313 and MJ2 significantly mitigated this inhibitory effect, reaching 91.34% of the yield obtained from detoxified stalks. To further elucidate the detoxification mechanism, the system's tolerance was quantitatively assessed by introducing a gradient of pretreatment liquor (0–100%, v/v). Kinetic analysis using the modified Gompertz model revealed that the co-culture achieved a maximum hydrogen potential of 125.91 ± 0.54 mM under 40% (v/v) pretreatment liquor stress—a 44.27% increase over the control. While a hormetic effect was observed at 20% liquor concentration, a critical threshold was identified at 60%, where the system failed due to the growth arrest of the primary degrader, DSM1313. Collectively, these results demonstrate that microbial co-cultivation significantly expands the operational window of non-detoxified CBP. By providing a higher system-level tolerance threshold to pretreatment-derived inhibitors, this strategy reduces the dependence on intensive detoxification processes, offering a robust approach for cost-effective biohydrogen production. Tobacco stalks Biohydrogen Lignocellulose Separate hydrolysis and fermentation Consolidated Bioprocessing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The rapid development of human society has been accompanied by the continuous consumption of fossil fuels and an increasing demand for energy, leading to growing environmental concerns and challenges to sustainable development. Replacing fossil fuels with renewable energy sources has become an inevitable trend for the future [ 1 ]. Among renewable energy options, biohydrogen stands out due to its high calorific value (141.8 kJ/g), low energy cost, and the absence of greenhouse gas emissions during combustion, making it a highly promising alternative energy source [ 2 , 3 ]. Biohydrogen can be produced through several pathways, among which the conversion of lignocellulosic biomass is particularly noteworthy, owing to its renewability, abundance, low cost, and wide availability. Among various lignocellulosic agricultural residues, tobacco stalks represent one of the most abundant non-food biomass sources worldwide. China, the largest tobacco producer, generates over 2.1 million tons of tobacco stalks annually [ 4 ]. These stalks, the main by-product of tobacco cultivation, are currently disposed of primarily through incineration or landfill, which can cause serious environmental pollution [ 5 ]. Therefore, developing efficient and sustainable strategies to convert tobacco stalks into biohydrogen represents both an environmental necessity and a practical opportunity. Due to the rigid and recalcitrant structure of lignocellulose, it is difficult to degrade and directly utilize by enzymes or microorganisms. Therefore, pretreatment is required to improve the accessibility and bioavailability of the biomass [ 6 ]. However, various by-products, such as phenols, furans, and fatty acids, are typically generated during pretreatment [ 7 ]. In addition, tobacco stalks produce unique toxic compounds—including alkaloids, tar, and benzo[a]pyrene—during pretreatment, which can further inhibit cellulase activity and microbial growth [ 5 ]. As a result, detoxification is commonly applied prior to fermentation, despite its associated drawbacks such as sugar loss, increased water consumption, and added process complexity. Traditional biohydrogen fermentation consists of separate steps: enzyme production, biomass hydrolysis, and hydrogen production, a process known as separate hydrolysis and fermentation (SHF). In contrast, consolidated bioprocessing (CBP) integrates these steps into a single reactor, simplifying operations, reducing capital and substrate costs, shortening processing time, improving hydrogen yield, and lowering contamination risk. Thus, CBP is regarded as the most promising industrial configuration for hydrogen production from cellulosic materials [ 8 ]. Nevertheless, CBP systems are inherently more vulnerable to inhibitory stress, as cellulose depolymerization and microbial hydrogen production are simultaneously affected. In CBP systems, cellulolytic microorganisms capable of efficient cellulose degradation often exhibit limited tolerance to pretreatment-derived inhibitors, whereas non-cellulolytic hydrogen producers may display higher inhibitor resistance but lack the ability to utilize solid substrates directly. This functional trade-off has prompted increasing interest in microbial co-cultivation strategies, which may enhance overall system robustness through metabolic cooperation. However, whether such cooperation can effectively expand the operational window of CBP hydrogen production under non-detoxified conditions remains insufficiently explored. Thermoanaerobacterium thermosaccharolyticum MJ2 is an anaerobic strain isolated from paper sludge, capable of utilizing a wide range of soluble sugars to efficiently produce biohydrogen. Several studies have shown that the Thermoanaerobacterium genus can tolerate and even transform lignocellulosic inhibitors [ 9 – 11 ]. Acetivibrio thermocellus DSM1313 is a thermophilic anaerobic commercial strain known for its high efficiency in direct cellulose degradation and its potential application in CBP-based hydrogen production from lignocellulose [ 12 ]. In this study, alkali-pretreated tobacco stalks were evaluated as feedstocks for hydrogen production under non-detoxified conditions. Enzymatic hydrolysis performance and hydrogen production from hydrolysates were first examined as reference systems, followed by a systematic comparison of monoculture and co-culture CBP configurations under increasing inhibitory stress. By combining experimental observations with kinetic analysis, this work aims to elucidate whether microbial co-cultivation can enhance CBP robustness and expand the feasible operational range for biohydrogen production without detoxification. 2. Materials and methods 2.1. Substrate sources Tobacco stalks were provided by the Songming Niulanjiang Tobacco Planting Centre, Kunming, Yunnan Province. The stalks were crushed in a pulverizer, sieved through a 100-mesh screen, placed in sealed bags, and stored in a cool, dry place. Commercial cellulase Cellic™ Ctec2 was purchased from Novozymes North America Inc. (Franklin, NC, USA). The filter paper activity of the cellulase was 198.49 FPU/mL, measured according to the National Renewable Energy Laboratory protocol LAP-006. 2.2. Tobacco stalks pretreatment Acid pretreatment: Raw tobacco stalks were treated with dilute sulfuric acid (4.0%, w/v) in a 1 L reagent flask at 120 ℃ for 1.5 h, with a solid-to-liquid ratio of 1:10 (g dry weight: mL). The supernatant and solids were separated by centrifugation (8000×g, 5 min). The solid fraction was washed to neutrality with tap water and dried in an oven at 55 ℃ until constant weight, yielding detoxified acid-pretreated tobacco stalks (DACTS). Non-detoxified acid-pretreated tobacco stalks (NACTS) were obtained by directly drying the solids at 55 ℃ until constant weight without washing. Alkali pretreatment: Raw tobacco stalks were treated with NaOH solution (2.0%, w/v) at 90 ℃ for 2 h in a 1 L reagent bottle, using a solid-to-liquid ratio of 1:10. The supernatant and solids were separated by centrifugation. The solids were washed to neutrality with tap water and dried in an oven at 55 ℃ until constant weight to obtain detoxified alkali-pretreated tobacco stalks (DAKTS). Non-detoxified alkali-pretreated tobacco stalks (NAKTS) were obtained by drying directly at 55 ℃ without washing. 2.3. Determination of total phenolic content Total phenolic content was measured using the Folin-Ciocalteu method [ 10 ]. with phloroglucinol dihydrate used as a standard. Briefly, 20 µL of the diluted sample was mixed with 100 µL of Folin-Ciocalteu reagent (Sangon Biotech, China) and incubated at room temperature for 5 min in the dark. Then, 80 µL of 7.5% Na₂CO₃ was added and mixed. After 2 h of incubation at room temperature in the dark, the absorbance was measured at 750 nm using an EnSpire-2300 multimode plate reader (PerkinElmer, USA). 2.4. Hydrolysis of pretreated tobacco stalks by cellulase Pretreated tobacco stalks were suspended in phosphate buffer at solid concentrations of 3%, 5%, 7%, and 10% (w/v). The pH was adjusted to 4.8 ± 0.05. Cellic™ Ctec2 cellulase was added at a dosage of 20 FPU/g substrate, and the mixture was incubated in a shaker at 55 ℃ and 150 rpm for 72 h. 2.5. Determination of reducing sugar for enzyme hydrolysate The 3,5-dinitrosalicylic acid (DNS) reagent was prepared as follows: 19.8 g of NaOH and 10.6 g of 3,5-dinitrosalicylic acid were dissolved in 1416 mL of distilled water with continuous stirring. Subsequently, 306 g of sodium potassium tartrate, 7.6 mL of phenol (melted at 50 ℃), and 8.3 g of sodium metabisulfite were added. For sample measurement, 3 mL of the hydrolysate was titrated with 0.1 M HCl to the phenolphthalein endpoint. Then, 0.3 mL of the titrated sample was transferred into a 5 mL centrifuge tube, followed by the addition of 0.3 mL of DNS reagent. The mixture was incubated in a boiling water bath for 5 min, immediately cooled in an ice-water bath, diluted with 2.4 mL of distilled water, and the absorbance was measured at 540 nm. The enzymolysis efficiency was calculated using the following equation: \(\:Enzymolysis\:efficiency\:\left(\%\right)=\frac{\text{R}\text{e}\text{d}\text{u}\text{c}\text{i}\text{n}\text{g}\:\text{s}\text{u}\text{g}\text{a}\text{r}\text{s}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}\:(\text{g}/\text{L})}{(\text{g}\text{l}\text{u}\text{c}\text{a}\text{n}\:\text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}\text{ %}\:\times\:\:1.11\:+\:\text{x}\text{y}\text{l}\text{a}\text{n}\:\text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}\text{\%}\:\times\:\:1.14)\:\times\:\:\text{S}\text{o}\text{l}\text{i}\text{d}\:\text{l}\text{o}\text{a}\text{d}\text{i}\text{n}\text{g}\text{s}\:(\text{g}/\text{L})}\times\:100\) Eq. (1) 2.6 General Fermentation Procedures All anaerobic fermentations were conducted in 55 mL serum vials with a 20 mL working volume. The initial pH was adjusted to 7.0 ± 0.05 using 5 M NaOH or HCl. Each vial was inoculated with a 10% (v/v) seed culture and incubated at 55 ℃ with orbital shaking at 150 rpm. The medium was DSMZ 122 medium containing (NH 4 ) 2 SO 4 1.3 g/L, MgCl 2 x 6 H 2 O 2.6 g/L, KH 2 PO 4 1.43 g/L, K 2 HPO 4 5.50 g/L, CaCl 2 x 2 H 2 O 0.13 g/L, Na 2 -ß-glycerophosphate x 5 H 2 O 6.00 g/L, FeSO 4 x 7 H 2 O (0.1% w/v in 0.05 M H 2 SO 4 ) 1.10 ml/L, L-Glutathione (reduced) 0.25 g/L, Yeast extract 4.50 g/L, Sodium resazurin (0.1% w/v) 0.50 ml/L. 2.7. Separate Hydrolysis and Fermentation (SHF) The fermentation medium was prepared using enzymatic hydrolysates of tobacco stalks as the substrate. Vials were inoculated with T. thermosaccharolyticum MJ2 and fermented for 24 h to evaluate the impact of pretreatment-derived inhibitors on hydrogen production. 2.8. Hydrogen production from consolidated bioprocessing (CBP). CBP was carried out using alkali-pretreated tobacco stalks as the sole carbon source. For mono-cultures, either A. thermocellus DSM 1313 was used; for co-cultures, A. thermocellus DSM 1313 and T. thermosaccharolyticum MJ2 were inoculated at a 1:1 (v/v) ratio. The fermentation was conducted for 24 h. The initial substrate concentration was set at 2% (w/v), with further evaluation conducted across a range of 2% to 10% (w/v) to determine the operational limits of the system. 2.9. Fermentation procedure under inhibitory stress The alkali pretreatment liquid was collected from the liquid fraction following the alkali pretreatment of tobacco stalks. To investigate the dose-response relationship of inhibitors, a series of fermentation media were prepared by substituting deionized water with the pretreatment liquid in varying volume proportions: 0%, 20%, 40%, 60%, 80%, and 100% (v/v). For all inhibitory groups, detoxified alkali-pretreated tobacco stalks were used as the solid substrate at a loading of 3% (w/v). CBP was conducted in 55 mL serum bottles with a working volume of 20 mL. For the monoculture group, the medium was inoculated with a 10% (v/v) active culture of A. thermocellus DSM 1313. For the co-culture group, a 10% (v/v) inoculum consisting of DSM 1313 and T. thermosaccharolyticum MJ2 (at a ratio of 1:1, v/v) was used. The fermentation was conducted for 72 h. 2.10. Kinetic modeling and data analysis To quantitatively describe the hydrogen production dynamics, the cumulative hydrogen production curves were fitted using the modified Gompertz model: \(\:\text{P(t)}\text{=}{\text{P}}_{\text{m}}\text{×exp}\left\{\text{-exp}\left[\frac{{\text{R}}_{\text{m}}\text{×e}}{{\text{P}}_{\text{m}}}\left(\text{λ-t}\right)\text{+1}\right]\right\}\) (Eq. 2) Where: P(t) is the cumulative hydrogen production (mM) at time t (h); P m is the maximum hydrogen production potential (mM); R m is the maximum hydrogen production rate (mM/h); λ is the lag phase duration (h); e is the mathematical constant (approximately 2.718). The kinetic parameters were estimated via non-linear regression analysis using Origin 2021 software. All experiments were performed in triplicate, and the results are presented as mean ± standard deviation. Statistical significance was analyzed using one-way ANOVA with a confidence level of 95% (p < 0.05). 2.11. Methods of analysis Compositional analysis was conducted following the procedure described by the National Renewable Energy Laboratory (NREL) for the analysis of tobacco stalks and pretreatment supernatant fractions. Cell density in the liquid medium was monitored by measuring the optical density at 600 nm (OD₆₀₀) using a 10 mL serum bottle (GENESYS™ 10S, Thermo Fisher Scientific, USA). The concentrations of oligosaccharides, monosaccharides, acetate, formate, furfural, 5-hydroxymethyl furfural (HMF), and metabolites were determined by high performance liquid chromatography (Waters 2695, USA) under the following conditions: column temperature of 60 ℃, injection rate of 10 µL, flow rate of 0.6 mL/min, analytical column of Bio-Rad Annex HPX-87H with a differential refractive index detector at 40 ℃. Gas chromatography-mass spectrometry (GC-MS) was used to determine the composition of inhibitors in the hydrolysis products of tobacco stalks. The pretreatment was extracted and concentrated by ethyl acetate, and then the treated samples were examined on an InertCap5 capillary column (30 m × 0.25 mm × 0.25 µm) using a gas chromatography-mass spectrometer (QP2010ultra). GC conditions were as follows: carrier gas: He (99. 999%); column flow rate: 1.0 mL/min; shunt mode: no shunt; injection volume: 1.0 µL; inlet temperature: 300 ℃; the initial temperature of the column was 50 ℃, retained for 1 min, and then the programmed temperature was increased to 200 ℃ by 8 ℃/min and kept for 2 min. The column temperature was 50 ℃ at the beginning and kept for 1 min, then the programmed temperature was increased to 200 ℃ at 8 ℃/min and kept for 2 min, then the programmed temperature was increased to 300 ℃ at 8 ℃/min and kept for 2 min; the auxiliary heating zone 2 was opened and the initial temperature was 280 ℃, and the total running time of the method was 29 min. The mass spectrometry conditions were as follows: ion source: EI source; ion source temperature: 230 ℃; quadrupole temperature: 150 ℃; chromatography-mass spectrometry junction temperature: 280 ℃; electron energy: 70 eV; electron multiplier voltage: 1,500 V; scanning mode: full scanning, scanning mass number range is: 20 ~ 550. Hydrogen in the headspace was measured by gas chromatography (Foley 9790 Plus, Foley, China). For hydrogen quantification, the gas from the headspace of sealed serum bottles was withdrawn using a 25 mL gas-tight syringe and immediately injected into the gas chromatograph inlet. The gas mixture was carried by nitrogen through a capillary column (TDX-01, 100 ℃), where separation occurred based on adsorption-desorption interactions. The separated hydrogen was detected using a thermal conductivity detector (TCD) maintained at 80 ℃. The signal was recorded as a chromatogram, and hydrogen yield was calculated based on the peak area. 2.12. Statistical analysis Statistical analysis of the data in this study was performed using the software of SPSS 17.0 (SPSS Inc. Chicago) and Microsoft® Excel. ANOVA and Student's T test were applied to determine significant differences. A p < 0.05 was considered statistically significant. 3. Results and discussion 3.1 Screening of pretreatment strategies for non-detoxified CBP feedstocks To ensure the feasibility of consolidated bioprocessing (CBP) under non-detoxified conditions, the pretreatment strategy must provide substrates with sufficient enzymatic accessibility while avoiding excessive generation of fermentation inhibitors. Therefore, different pretreatment methods were first evaluated as a screening step to identify suitable feedstocks for subsequent CBP experiments, rather than to establish a comprehensive comparison of pretreatment technologies. Lignocellulosic biomasses commonly used in fermentation typically contain 20.2%–49.5% cellulose, 14.6%–43.5% hemicellulose, and 7.3%–21.8% lignin [ 13 – 16 ]. Compared to these commonly used feedstocks, the tobacco stalks used in this study exhibited lower cellulose and hemicellulose contents, but higher lignin content (Table 1 ) [ 17 ]. Therefore, pretreatment is essential to improve their suitability for biorefinery applications. Chemical pretreatment with acid or alkali is among the most effective methods to enhance biomass digestibility [ 6 ]. Both pretreatments effectively increased the glucan content of tobacco stalks, from 29.18% in raw material to 43.87% (alkali) and 47.42% (acid) (Table 1 ). However, they differed markedly in their effects on hemicellulose and inhibitor profiles. Alkali pretreatment selectively removed lignin (from 27.33% to 24.74%) while preserving most hemicellulose (14.15% xylan retained). In contrast, acid pretreatment drastically reduced hemicellulose (xylan dropped to 4.91%) and increased lignin content (32.19%), indicating more extensive sugar degradation. Alkali pretreatment mainly disrupts lignin structures, effectively reducing lignin content in most crop residues with minimal sugar degradation and low formation of furan derivatives. In contrast, dilute acid breaks glycosidic bonds in lignocellulose, dissolving hemicellulose and some lignin, thereby enhancing the release of fermentable sugars and improving biomass accessibility [ 18 ]. Table 1 The compositions of raw and pretreated tobacco stalks Composition Content (%) Raw tobacco stalks Acid pretreated tobacco stalks Alkali pretreated tobacco stalks Glucan 29.18 ± 0.07 47.42 ± 0.20 43.87 ± 0.63 Xylan 14.29 ± 0.24 4.91 ± 0.05 14.15 ± 0.15 Arabinan 1.13 ± 0.13 ND 0.72 ± 0.03 Lignin 27.33 ± 0.61 32.19 ± 0.47 24.74 ± 0.16 Ash 4.85 ± 0.07 5.55 ± 0.02 4.68 ± 0.20 ND: Not detected. Consistent with previous reports [ 19 , 20 ], alkali pretreatment generated almost no furans, whereas acid pretreatment produced substantial furfural (1686.64 mg/kg) (Table 2 ). Both pretreatments released phenolic compounds and trace inhibitors such as vanillin, syringaldehyde, and 2,3′-bipyridine, which are known to inhibit microbial fermentation [ 21 ]. Table 2 Composition of pretreatment inhibitors from tobacco stalks. Composition Content Acid pretreated tobacco stalks (mg/kg) Alkali pretreated tobacco stalks (mg/kg) Acid pretreated supernatant (mg/L) Alkali pretreated supernatant (mg/L) Phenolics 6362.57 ± 53.25 10763.74 ± 397.12 2512.46 ± 111.00 1782.46 ± 67.77 Furfural 1686.64 ± 85.86 ND 451.85 ± 7.80 ND Phenol, 2-methoxy- 92.18 ± 26.58 131.25 ± 3.54 8.03 ± 1.86 14.43 ± 1.47 2-Methoxy-4-vinylphenol 208.36 ± 8.40 121.20 ± 14.53 11.93 ± 2.32 93.77 ± 9.08 Phenol, 2,6-dimethoxy- 58.27 ± 7.04 31.22 ± 1.46 12.55 ± 2.78 9.59 ± 0.88 Vanillin 60.53 ± 49.90 334.44 ± 62.04 15.64 ± 2.02 38.52 ± 1.75 Acetovanillone 13.37 ± 4.72 40.20 ± 13.72 1.60 ± 0.20 5.46 ± 0.47 2,3'-Dipyridyl 36.22 ± 17.67 18.40 ± 5.31 10.36 ± 2.41 2.74 ± 0.58 Syringaldehyde 61.04 ± 32.79 127.86 ± 5.92 28.91 ± 3.22 64.75 ± 5.58 n-Hexadecanoic acid 1300.87 ± 317.20 1554.92 ± 73.11 35.14 ± 1.28 129.93 ± 5.37 Octadecanoic acid 547.07 ± 162.50 522.85 ± 22.80 19.31 ± 1.64 68.48 ± 4.88 ND: Not detected. Enzymatic hydrolysis was performed to evaluate the digestibility of detoxified and non-detoxified substrates from both pretreatments. At 10% solid loading, detoxified alkali-pretreated tobacco stalks (DAKTS) produced 49.61 ± 3.09 g/L of reducing sugars, while non-detoxified alkali-pretreated tobacco stalks (NAKTS) yielded 36.71 ± 1.24 g/L—a 27.97% reduction attributable to inhibitor presence (Fig. 1 ). For acid-pretreated substrates, detoxified samples (DACTS) achieved 25.51 ± 1.56 g/L, whereas non-detoxified samples (NACTS) produced only 17.62 ± 1.18 g/L, representing a 30.93% reduction (Fig. 2 ). Notably, even under non-detoxified conditions, alkali-pretreated substrates outperformed detoxified acid-pretreated substrates in reducing sugar yield, highlighting the inherent advantage of alkali pretreatment in preserving fermentable sugar availability. These results demonstrate that alkali pretreatment consistently confers superior enzymatic digestibility compared to acid pretreatment, regardless of detoxification status. NAKTS: Non-detoxified alkali pretreated tobacco stalks; DAKTS: Detoxified alkali pretreated tobacco stalks Under non-detoxified conditions, alkali-pretreated tobacco stalks exhibited higher enzymatic digestibility than acid-pretreated substrates, as reflected by increased fermentable sugar release during enzymatic hydrolysis. In contrast, acid-pretreated substrates showed lower hydrolysis efficiency, which may be attributed to the presence of residual inhibitory compounds and reduced cellulose accessibility. Given these differences, alkali pretreatment was selected for subsequent fermentation and CBP experiments. Although variations in enzymatic hydrolysis performance were observed between pretreatment methods, it should be noted that enzymatic digestibility alone does not determine downstream fermentation performance, particularly under CBP conditions. Instead, the ability of microorganisms to tolerate pretreatment-derived inhibitors becomes increasingly critical when cellulose depolymerization and fermentation occur simultaneously. Therefore, the selected alkali-pretreated, non-detoxified substrate was used as a representative feedstock for further evaluation of hydrogen production systems. 3.2. Hydrogen production from enzymatic hydrolysates as SHF reference systems To isolate the inhibitory effects of pretreatment-derived compounds from the constraints of cellulose depolymerization, SHF was conducted using enzymatic hydrolysates of pretreated tobacco stalks. In this system, fermentable sugars were readily available, allowing direct assessment of microbial inhibitor tolerance independent of hydrolysis efficiency. Thermoanaerobacterium thermosaccharolyticum MJ2, a thermophilic hydrogen producer previously reported to tolerate lignocellulose-derived inhibitors [ 9 ], was used for SHF. When cultivated on hydrolysates from detoxified (DAKTS) and non-detoxified (NAKTS) alkali-pretreated stalks, MJ2 exhibited comparable hydrogen production at reducing sugar concentrations ≤ 20 g/L (Fig. 3 A). At 20 g/L, hydrogen yields reached 241.19 ± 30.09 mM (DAKTS) and 215.26 ± 49.61 mM (NAKTS), with the 10.75% reduction being statistically insignificant. These values compare favorably with reported hydrogen production from other lignocellulosic residues, such as sugarcane bagasse (121.60 mM) [ 22 , 23 ] and paper sludge (133.68 mM) [ 24 ]. However, at 30 g/L reducing sugars, hydrogen production from NAKTS hydrolysate declined significantly to 165.80 ± 3.17 mM—a 21.32% reduction relative to DAKTS (p < 0.05)—indicating that inhibitor effects become pronounced at higher substrate concentrations. This threshold aligns with common practice in dark fermentation, where sugar concentrations seldom exceed 20 g/L [ 8 , 25 – 27 ]. Analysis of fermentation metabolites showed that acetate and butyrate concentrations increased with sugar concentration in both hydrolysates (Fig. 4 ). Notably, the butyrate-to-acetate ratio was elevated in NAKTS fermentations, consistent with previous observations that phenolic inhibitors like vanillin can shift metabolic pathways [ 28 ]. However, this metabolic shift did not translate into increased hydrogen production, suggesting that the inhibitors primarily affected electron flux rather than overall metabolic activity. After fermentation, the key metabolites associated with hydrogen production—acetate and butyrate—were quantified across different sugar concentrations in the DAKTS and NAKTS hydrolysates. From 0–20 g/L of reducing sugars, both acetate and butyrate concentrations increased with increasing sugar concentration (Fig. 4 A). These SHF results establish a reference for interpreting subsequent CBP performance: while MJ2 exhibits substantial tolerance to pretreatment-derived inhibitors, its hydrogen-producing capacity is compromised at higher inhibitor concentrations. This highlights the need for enhanced stress mitigation strategies in CBP systems, where both hydrolysis and fermentation occur simultaneously. 3.3. Hydrogen production from alkali pretreated tobacco stalks by CBP CBP fermentation was carried out by inoculating A. thermocellus DSM1313 with detoxified (DAKTS) and non-detoxified (NAKTS) alkali-pretreated tobacco stalks (Fig. 5 A). Hydrogen production by DSM1313 using DAKTS at 3%, 5%, 7%, and 10% substrate concentrations was 62.42 ± 11.38 mM, 60.10 ± 5.95 mM, 56.28 ± 2.23 mM, and 61.85 ± 5.68 mM, respectively. When using NAKTS, hydrogen production was 61.77 ± 5.73 mM, 62.47 ± 4.51 mM, 27.64 ± 1.64 mM, and 5.74 ± 2.25 mM at the same substrate concentrations. There was no significant difference in hydrogen production from DAKTS at 3%–10% substrate concentration, indicating that DSM1313 could utilize up to 3% substrate effectively, with no further improvement at higher concentrations. For NAKTS, increasing substrate concentration led to higher inhibitor levels, significantly reducing hydrogen production. At 7% substrate concentration, hydrogen production from NAKTS decreased by 50.88% compared to DAKTS (p < 0.005), and at 10%, it dropped by 90.71% (p < 0.0005) (Fig. 5 C). This suggests that non-detoxification at higher substrate concentrations strongly inhibits hydrogen production in DSM1313 monocultures. Due to the limited efficiency of single-species fermentation, co-culturing cellulolytic bacteria with high-efficiency hydrogen-producing strains can improve substrate utilization and hydrogen yield [ 29 , 30 ]. MJ2 was co-cultured with DSM1313 for CBP fermentation using DAKTS and NAKTS (Fig. 5 B). Hydrogen production from DAKTS was 66.34 ± 2.66 mM, 97.28 ± 7.00 mM, 102.21 ± 1.59 mM, and 93.88 ± 1.10 mM at 3%, 5%, 7%, and 10% substrate concentrations, respectively. Compared with monocultures, co-culture systems significantly increased hydrogen yield at 5%–10% substrate concentrations (p < 0.005). For NAKTS, hydrogen production was 88.22 ± 12.99 mM, 117.78 ± 19.94 mM, 118.77 ± 23.32 mM, and 85.75 ± 3.12 mM, respectively. Hydrogen production increased by 32.97%, 21.07%, and 16.20% at 3%–7% substrate concentrations compared to DAKTS (Fig. 5 C). However, at 10% substrate concentration, hydrogen production decreased by 8.66%. The addition of MJ2 to the CBP system not only increased hydrogen production from NAKTS but also mitigated the inhibitory effects of pretreatment by-products. At 7% substrate concentration, hydrogen production in the NAKTS monoculture system decreased by more than half, while the co-culture system showed a slight increase. Even at 10% substrate concentration, where inhibitors almost completely suppressed hydrogen production in the DSM1313 monoculture, hydrogen yield in the co-culture only decreased by 8.66%. This indicates that the inhibitor load at high solids loading exceeded the intrinsic tolerance threshold of A. thermocellus DSM 1313. In contrast, the MJ2-augmented co-culture exhibited significant system-level robustness, effectively shifting the operational boundary toward higher stress levels. Low concentrations of inhibitors in pretreated tobacco stalks showed a stimulatory effect, while high concentrations exhibited inhibition during co-culture fermentation. According to Zhang et al., the addition of 2 g/L furfural or HMF increased the growth of Clostridium acetobutylicum ATCC 824 by 25% [ 31 ]. Ezeji et al. [ 32 ] also found that Clostridium beijerinckii BA101 growth increased by 13% with furfural or HMF (≤ 2 g/L). Within certain limits, lignocellulosic pretreatment inhibitors may stimulate microbial activity. Jung et al. [ 33 ] reported that E. coli expressing polyhydroxybutyrate synthesis genes accumulated higher biomass and showed enhanced growth and resistance at 15 mM furfural. MJ2 contains polyhydroxybutyrate synthesis-related genes and may have similar resistance. Thus, increased hydrogen production at low substrate concentrations and the decrease at higher concentrations may be attributed to the hormesis effect, enhancing microbial activity at low inhibitor levels [ 34 ]. Moreover, different tolerance levels of the two strains may shift the biomass ratio in favor of MJ2, further improving hydrogen yield in co-culture systems using NAKTS over DAKTS. 3.4. Effects of different strategies on biohydrogen fermentation of tobacco stalks According to Fig. A1, alkali pretreatment retained a large amount of fermentable sugars from 1 kg of waste tobacco stalks, suitable for hydrogen fermentation. After alkali pretreatment without detoxification, SHF fermentation yielded 94.4 L of hydrogen, and CBP fermentation yielded 65.8 L. Washing removed many inhibitors adsorbed onto the tobacco stalks. However, this did not improve hydrogen yield; instead, SHF and CBP fermentation from DAKTS produced 79.6 L and 29.7 L of hydrogen, respectively. The hydrogen yield from tobacco stalks was comparable to that from lignocellulosic feedstocks such as wheat straw, agave bagasse, and sugarcane bagasse [ 9 , 19 , 26 ]. The hydrogen yield from non-detoxified tobacco stalks was higher than that from detoxified samples. Moreover, CBP fermentation offers the advantage of omitting costly cellulase addition, making the process more economical with further optimization. This difference likely reflects the combined effects of sugar loss during washing, residual alkali buffering, and microbial tolerance, rather than the inhibitory removal step alone. 3.5. Assessment of microbial tolerance and synergistic mechanism under inhibitory stress To quantitatively evaluate the dose-response relationship between pretreatment-derived inhibitors and fermentative performance, varying proportions of pretreatment liquid (0–100%, v/v) were introduced into the fermentation system. This approach overcomes the limitation of fixed inhibitor concentrations in dried tobacco stalks, providing a controlled inhibitory gradient. The results demonstrated that the co-culture system possessed superior robustness compared to the A. thermocellus DSM 1313 monoculture. At a 40% (v/v) liquid addition, the co-culture achieved a peak hydrogen yield of 125.91 ± 0.54 mM, representing a 44.27% increase over the inhibitor-free control. Notably, while the hydrogen production of DSM 1313 was severely suppressed at 24 h under 40% liquid stress, the co-culture maintained a production rate comparable to the control. These results suggest that MJ2 contributes to mitigating the inhibitory effects of the pretreatment liquid, which may help maintain hydrogen production performance in the co-culture system. The observed behavior may arise from multiple non-exclusive system-level interactions, including differential tolerance and altered metabolic coupling between consortium members. Interestingly, at a lower concentration (20% v/v), the co-culture exhibited significantly enhanced hydrogen production, whereas DSM 1313 alone still suffered from inhibition. This stimulatory response at low inhibitor load is phenotypically consistent with the hormetic effects reported in other anaerobic fermentation systems [ 34 ]. While the specific molecular triggers of this response were not resolved in this study, the reproducible nature of this phenomenon suggests a complex interaction between sub-inhibitory stress and metabolic flux in the co-culture system. However, hydrogen production was completely arrested when the liquid concentration exceeded 60%. This failure underscores the "bottleneck" of the CBP system: as the primary degrader, the total growth arrest of DSM 1313 under high chemical stress leads to a cessation of sugar supply, which MJ2 cannot overcome despite its own high tolerance. These findings delineate the functional boundaries of the co-culture, emphasizing that system stability is fundamentally anchored to the viability of the primary cellulolytic strain. Table 3 Effects of tobacco stalks alkali pretreatment supernatant liquid on hydrogen production kinetics of CBP by monoculture. Alkali pretreatment supernatant liquid proportion (%) Pm (mM) Rm (mM/h) λ (h) R 2 Control 52.83 ± 0.91 5.17 ± 0.62 6.14 ± 0.51 0.99 20 68.39 ± 1.58 3.33 ± 0.29 6.93 ± 0.71 0.99 40 55.17 ± 1.90 1.53 ± 0.11 18.21 ± 1.23 0.99 60 — — — — 80 — — — — 100 — — — — —: No valid kinetic parameters could be fitted by the model. Although inhibitors like vanillin and syringaldehyde were present in the non-detoxified hydrolysate (Table 4 ), the co-culture of A. thermocellum DSM 1313 and T. thermosaccharolyticum MJ2 maintained high H 2 productivity. This resilience is functionally associated with the inclusion of MJ2 in the consortium, which may alleviate inhibitory stress through metabolic buffering and/or other tolerance-related mechanisms. Previous studies have reported detoxification-related capabilities in Thermoanaerobacterium species; however, in the present study, the enhanced performance is interpreted as a functional tolerance effect rather than confirmed biochemical transformation. Such capabilities may partially explain the improved performance observed in the co-culture; however, direct evidence of inhibitor biotransformation was not obtained in the present study [ 30 ]. Table 4 Effects of tobacco stalks alkali pretreatment supernatant liquid on hydrogen production kinetics of CBP by co-culture. Alkali pretreatment supernatant liquid proportion (%) Pm (mM) Rm (mM/h) λ (h) R 2 Control 86.35 ± 3.72 4.87 ± 0.21 6.53 ± 0.27 0.99 20 123.17 ± 2.61 6.36 ± 0.52 10.38 ± 0.77 0.99 40 125.91 ± 0.54 7.31 ± 0.35 14.93 ± 3.72 0.99 60 80.78 ± 3.72 2.40 ± 0.16 33.20 ± 0.96 0.99 80 — — — — 100 — — — — —: No valid kinetic parameters could be fitted by the model. The sustained hydrogen production under high inhibitory load indicates a robust system-level stress mitigation effect. While direct quantification of inhibitor biotransformation was not conducted, the innate resistance of T. thermosaccharolyticum MJ2 likely provides a metabolic buffer. Elucidating the precise contributions of specific detoxification pathways will require future studies incorporating inhibitor mass balances; however, the current results clearly define the enhanced operational tolerance gained through co-cultivation. 4. Conclusion This study demonstrates that alkali-pretreated tobacco stalks can be effectively utilized for biohydrogen production via both SHF and CBP routes without the need for energy-intensive detoxification. The co-culture of A. thermocellus DSM1313 and T. thermosaccharolyticum MJ2 proved to be a robust strategy, significantly overcoming the inhibitory stress that otherwise crippled monoculture performance. Kinetic analysis revealed that the inclusion of MJ2 significantly enhanced hydrogen production performance under moderate inhibitory stress. This improvement is consistent with an increased system-level tolerance to inhibitory stress and/or favorable microbial interactions within the co-culture system, rather than evidence of direct or complete detoxification of inhibitory compounds. However, the identification of a 60% liquor concentration threshold clarifies that the stability of this synergistic system is ultimately anchored to the physiological limits of the primary degrader. Overall, these findings provide a simplified and sustainable framework for the high-value bioconversion of complex tobacco waste, providing experimental insights relevant to the development and optimization of scalable biorefining strategies. Declarations Conflicts of interest The authors have no conflicts of interest to declare. Acknowledgements This study was supported by the National Natural Science Foundation of China, China [grant no. 52070079], and Yunnan Fundamental Applied Research Project (202001AU070010). Author Contributions M . L . : Investigation, Writing – Original Draft, Validation, Formal Analysis, Data Curation. M . Z . : Writing – Review & Editing, Investigation, Resources, Conceptualization, Methodology, Funding Acquisition. B . H . : Resources, Supervision, Funding Acquisition, Project Administration. All authors read and approved the final manuscript. Conflicts of interest The authors have no conflicts of interest to declare. Appendix A. Supplementary data E-supplementary data of this work can be found in online version of the paper. Data availability The datasets generated during and/or analyzed in this study are available from the Zenodo repository. A private sharing link has been provided for peer review: https://zenodo.org/records/15605353?preview=1&token=eyJhbGciOiJIUzUxMiJ9.eyJpZCI6ImRjYWIwZGMwLWZmZTctNGQ5ZC04NmIzLTMyYzRkOWI5NjE4YiIsImRhdGEiOnt9LCJyYW5kb20iOiI0ZDRhZTAyMWE4ZTRmZmZmOWIwYjE5OWViYmNiMGQwZCJ9.d5ZF4KT9tDPuY4LJndPoj_ncuDDm-1DoVRwdDE0O02gwD4DL0ywGNJRBEbwOtUJaZ6aV12ky-5rY8VDU1NAtiw. The data will be made publicly available upon publication. References Choe C, Cheon S, Kim H, Lim H (2023) Mitigating climate change for negative CO2 emission via syngas methanation: Techno-economic and life-cycle assessments of renewable methane production. 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Supplementary Files Appendixfigure.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 10 Mar, 2026 Reviews received at journal 09 Mar, 2026 Reviewers agreed at journal 26 Feb, 2026 Reviewers invited by journal 23 Feb, 2026 Editor assigned by journal 23 Feb, 2026 Submission checks completed at journal 23 Feb, 2026 First submitted to journal 19 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8915427","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596434511,"identity":"801fc6b5-626b-42f0-8218-18636ecee4e6","order_by":0,"name":"Ming-Hao Li","email":"","orcid":"","institution":"South China University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Ming-Hao","middleName":"","lastName":"Li","suffix":""},{"id":596434513,"identity":"8749a280-1ec5-4f95-bbfc-44412a1e5411","order_by":1,"name":"Ming-Jun Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYFCCA2wMDBVQNg/xWs4AaTbitQDVMraRokW38fizx7zz6uQN7jcwPnjbxiBvTkiL2YED6ca82w4bbjjGwGw4t43BcGcDYS3HpHm3HUgwOMbAJs3bxpBgcICgloNt0rxz6kBa2H8TqeUw0PAGZrAtzERqOcYmOefYYcOZxxKbJeeckzDcQFDLjePPJN7U1MnzHT588MObMht5grYwSMBVMDaAuITUAwF/AxGKRsEoGAWjYGQDAHJxP98gzRSnAAAAAElFTkSuQmCC","orcid":"","institution":"South China University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Ming-Jun","middleName":"","lastName":"Zhu","suffix":""},{"id":596434518,"identity":"2e40bd38-f340-4cfb-a27b-c76af7de0b5a","order_by":2,"name":"Bin-Bin Hu","email":"","orcid":"","institution":"Yunnan Academy of Tobacco Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Bin-Bin","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2026-02-19 08:24:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8915427/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8915427/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103409472,"identity":"df869773-ace3-4da5-91ec-6f4700524bcb","added_by":"auto","created_at":"2026-02-25 10:50:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":621802,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NAKTS on reducing sugar production during enzymatic hydrolysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: 3% substrate concentration；\u003cstrong\u003eb\u003c/strong\u003e: 5% substrate concentration；\u003cstrong\u003ec\u003c/strong\u003e: 7% substrate concentration；\u003cstrong\u003ed\u003c/strong\u003e: 10% substrate concentration\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8915427/v1/58e27c3ad16447b6917044cc.png"},{"id":103506821,"identity":"0ef56b87-a913-48a5-af60-867d6e75d9a6","added_by":"auto","created_at":"2026-02-26 13:39:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":600425,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NACTS on reducing sugar production during enzymatic hydrolysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: 3% substrate concentration; \u003cstrong\u003eb\u003c/strong\u003e: 5% substrate concentration; \u003cstrong\u003ec\u003c/strong\u003e: 7% substrate concentration; \u003cstrong\u003ed\u003c/strong\u003e: 10% substrate concentration\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8915427/v1/d91ad086bccdc7a28767f02c.png"},{"id":103409476,"identity":"26dbe981-64ad-4d84-b4f5-9486a3ae7c9c","added_by":"auto","created_at":"2026-02-25 10:50:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":142739,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of reducing sugar concentration in hydrolysates from tobacco stalks on hydrogen production and growth of \u003cem\u003eT. thermosaccharolyticum\u003c/em\u003e MJ2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: Hydrogen production by MJ2 using DAKTS and NAKTS hydrolysate; \u003cstrong\u003eb\u003c/strong\u003e: OD\u003csub\u003e600\u003c/sub\u003e and pH at the end of fermentation by MJ2 using DAKTS and NAKTS hydrolysate; \u003cstrong\u003ec\u003c/strong\u003e: Hydrogen production by MJ2 using DACTS and NACTS hydrolysate\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8915427/v1/3d5309c3560a743f667168f9.png"},{"id":103409475,"identity":"b8b3e070-6a97-40db-8c41-928febd1ccd2","added_by":"auto","created_at":"2026-02-25 10:50:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":485982,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different reducing sugar concentrations on fermentation metabolites during hydrogen production by MJ2 using alkali-pretreated tobacco stalk hydrolysates\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: Fermentation performance of DAKTS hydrolysates; \u003cstrong\u003eb\u003c/strong\u003e: Fermentation performance of NAKTS hydrolysates. *：\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8915427/v1/0dc2ecb1b336da4dbfb42e48.png"},{"id":103409474,"identity":"b7b18200-1cf2-4822-a9e5-248b71bafd62","added_by":"auto","created_at":"2026-02-25 10:50:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":553667,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of pretreated tobacco stalks without detoxification on hydrogen production by CBP\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e：DSM1313 mono-culture; \u003cstrong\u003eb\u003c/strong\u003e: DSM1313 and MJ2 co-culture; \u003cstrong\u003ec\u003c/strong\u003e: Hydrogen production change rate of mono- and co-culture using DAKTS and NAKTS, respectively. *：\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **：\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005，***：\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0005\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8915427/v1/319419c9dd5d323096df6a99.png"},{"id":103409477,"identity":"64d65dc4-fdff-4142-8372-d41d6b12aa3f","added_by":"auto","created_at":"2026-02-25 10:50:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":646141,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of different alkali pretreatment supernatant liquid proportion on CBP hydrogen production with alkali pretreated tobacco stalks of monoculture and co-culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: Effect on hydrogen production in monoculture; \u003cstrong\u003eb\u003c/strong\u003e: Effect on hydrogen production in co-culture; \u003cstrong\u003ec\u003c/strong\u003e: Effect on monoculture metabolites; \u003cstrong\u003ed\u003c/strong\u003e: Effect on metabolites in co-culture\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8915427/v1/de9f1a32e89ad6dfc6bec28d.png"},{"id":103509890,"identity":"b622557f-564e-49c4-b9d2-dea31a2ce24d","added_by":"auto","created_at":"2026-02-26 14:01:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4162291,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8915427/v1/fda8c801-12fc-48d8-8204-7d1d8e4b78b0.pdf"},{"id":103409478,"identity":"d93794fa-b264-48fa-8808-9c9d011aa1f4","added_by":"auto","created_at":"2026-02-25 10:50:37","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":95440,"visible":true,"origin":"","legend":"","description":"","filename":"Appendixfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-8915427/v1/eb33051590d987706616fb58.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Expanding the Operational Window of Consolidated Bioprocessing Hydrogen Production from Non-Detoxified Alkali-Pretreated Tobacco Stalks via Microbial Co-Cultivation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe rapid development of human society has been accompanied by the continuous consumption of fossil fuels and an increasing demand for energy, leading to growing environmental concerns and challenges to sustainable development. Replacing fossil fuels with renewable energy sources has become an inevitable trend for the future [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among renewable energy options, biohydrogen stands out due to its high calorific value (141.8 kJ/g), low energy cost, and the absence of greenhouse gas emissions during combustion, making it a highly promising alternative energy source [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiohydrogen can be produced through several pathways, among which the conversion of lignocellulosic biomass is particularly noteworthy, owing to its renewability, abundance, low cost, and wide availability. Among various lignocellulosic agricultural residues, tobacco stalks represent one of the most abundant non-food biomass sources worldwide. China, the largest tobacco producer, generates over 2.1\u0026nbsp;million tons of tobacco stalks annually [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These stalks, the main by-product of tobacco cultivation, are currently disposed of primarily through incineration or landfill, which can cause serious environmental pollution [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, developing efficient and sustainable strategies to convert tobacco stalks into biohydrogen represents both an environmental necessity and a practical opportunity.\u003c/p\u003e \u003cp\u003eDue to the rigid and recalcitrant structure of lignocellulose, it is difficult to degrade and directly utilize by enzymes or microorganisms. Therefore, pretreatment is required to improve the accessibility and bioavailability of the biomass [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, various by-products, such as phenols, furans, and fatty acids, are typically generated during pretreatment [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, tobacco stalks produce unique toxic compounds\u0026mdash;including alkaloids, tar, and benzo[a]pyrene\u0026mdash;during pretreatment, which can further inhibit cellulase activity and microbial growth [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. As a result, detoxification is commonly applied prior to fermentation, despite its associated drawbacks such as sugar loss, increased water consumption, and added process complexity.\u003c/p\u003e \u003cp\u003eTraditional biohydrogen fermentation consists of separate steps: enzyme production, biomass hydrolysis, and hydrogen production, a process known as separate hydrolysis and fermentation (SHF). In contrast, consolidated bioprocessing (CBP) integrates these steps into a single reactor, simplifying operations, reducing capital and substrate costs, shortening processing time, improving hydrogen yield, and lowering contamination risk. Thus, CBP is regarded as the most promising industrial configuration for hydrogen production from cellulosic materials [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Nevertheless, CBP systems are inherently more vulnerable to inhibitory stress, as cellulose depolymerization and microbial hydrogen production are simultaneously affected. In CBP systems, cellulolytic microorganisms capable of efficient cellulose degradation often exhibit limited tolerance to pretreatment-derived inhibitors, whereas non-cellulolytic hydrogen producers may display higher inhibitor resistance but lack the ability to utilize solid substrates directly. This functional trade-off has prompted increasing interest in microbial co-cultivation strategies, which may enhance overall system robustness through metabolic cooperation. However, whether such cooperation can effectively expand the operational window of CBP hydrogen production under non-detoxified conditions remains insufficiently explored.\u003c/p\u003e \u003cp\u003e \u003cem\u003eThermoanaerobacterium thermosaccharolyticum\u003c/em\u003e MJ2 is an anaerobic strain isolated from paper sludge, capable of utilizing a wide range of soluble sugars to efficiently produce biohydrogen. Several studies have shown that the \u003cem\u003eThermoanaerobacterium\u003c/em\u003e genus can tolerate and even transform lignocellulosic inhibitors [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. \u003cem\u003eAcetivibrio thermocellus\u003c/em\u003e DSM1313 is a thermophilic anaerobic commercial strain known for its high efficiency in direct cellulose degradation and its potential application in CBP-based hydrogen production from lignocellulose [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, alkali-pretreated tobacco stalks were evaluated as feedstocks for hydrogen production under non-detoxified conditions. Enzymatic hydrolysis performance and hydrogen production from hydrolysates were first examined as reference systems, followed by a systematic comparison of monoculture and co-culture CBP configurations under increasing inhibitory stress. By combining experimental observations with kinetic analysis, this work aims to elucidate whether microbial co-cultivation can enhance CBP robustness and expand the feasible operational range for biohydrogen production without detoxification.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Substrate sources\u003c/h2\u003e \u003cp\u003eTobacco stalks were provided by the Songming Niulanjiang Tobacco Planting Centre, Kunming, Yunnan Province. The stalks were crushed in a pulverizer, sieved through a 100-mesh screen, placed in sealed bags, and stored in a cool, dry place.\u003c/p\u003e \u003cp\u003eCommercial cellulase Cellic\u0026trade; Ctec2 was purchased from Novozymes North America Inc. (Franklin, NC, USA). The filter paper activity of the cellulase was 198.49 FPU/mL, measured according to the National Renewable Energy Laboratory protocol LAP-006.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Tobacco stalks pretreatment\u003c/h2\u003e \u003cp\u003eAcid pretreatment: Raw tobacco stalks were treated with dilute sulfuric acid (4.0%, w/v) in a 1 L reagent flask at 120 ℃ for 1.5 h, with a solid-to-liquid ratio of 1:10 (g dry weight: mL). The supernatant and solids were separated by centrifugation (8000\u0026times;g, 5 min). The solid fraction was washed to neutrality with tap water and dried in an oven at 55 ℃ until constant weight, yielding detoxified acid-pretreated tobacco stalks (DACTS). Non-detoxified acid-pretreated tobacco stalks (NACTS) were obtained by directly drying the solids at 55 ℃ until constant weight without washing.\u003c/p\u003e \u003cp\u003eAlkali pretreatment: Raw tobacco stalks were treated with NaOH solution (2.0%, w/v) at 90 ℃ for 2 h in a 1 L reagent bottle, using a solid-to-liquid ratio of 1:10. The supernatant and solids were separated by centrifugation. The solids were washed to neutrality with tap water and dried in an oven at 55 ℃ until constant weight to obtain detoxified alkali-pretreated tobacco stalks (DAKTS). Non-detoxified alkali-pretreated tobacco stalks (NAKTS) were obtained by drying directly at 55 ℃ without washing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Determination of total phenolic content\u003c/h2\u003e \u003cp\u003eTotal phenolic content was measured using the Folin-Ciocalteu method [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. with phloroglucinol dihydrate used as a standard. Briefly, 20 \u0026micro;L of the diluted sample was mixed with 100 \u0026micro;L of Folin-Ciocalteu reagent (Sangon Biotech, China) and incubated at room temperature for 5 min in the dark. Then, 80 \u0026micro;L of 7.5% Na₂CO₃ was added and mixed. After 2 h of incubation at room temperature in the dark, the absorbance was measured at 750 nm using an EnSpire-2300 multimode plate reader (PerkinElmer, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Hydrolysis of pretreated tobacco stalks by cellulase\u003c/h2\u003e \u003cp\u003ePretreated tobacco stalks were suspended in phosphate buffer at solid concentrations of 3%, 5%, 7%, and 10% (w/v). The pH was adjusted to 4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05. Cellic\u0026trade; Ctec2 cellulase was added at a dosage of 20 FPU/g substrate, and the mixture was incubated in a shaker at 55 ℃ and 150 rpm for 72 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Determination of reducing sugar for enzyme hydrolysate\u003c/h2\u003e \u003cp\u003eThe 3,5-dinitrosalicylic acid (DNS) reagent was prepared as follows: 19.8 g of NaOH and 10.6 g of 3,5-dinitrosalicylic acid were dissolved in 1416 mL of distilled water with continuous stirring. Subsequently, 306 g of sodium potassium tartrate, 7.6 mL of phenol (melted at 50 ℃), and 8.3 g of sodium metabisulfite were added.\u003c/p\u003e \u003cp\u003eFor sample measurement, 3 mL of the hydrolysate was titrated with 0.1 M HCl to the phenolphthalein endpoint. Then, 0.3 mL of the titrated sample was transferred into a 5 mL centrifuge tube, followed by the addition of 0.3 mL of DNS reagent. The mixture was incubated in a boiling water bath for 5 min, immediately cooled in an ice-water bath, diluted with 2.4 mL of distilled water, and the absorbance was measured at 540 nm.\u003c/p\u003e \u003cp\u003eThe enzymolysis efficiency was calculated using the following equation:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:Enzymolysis\\:efficiency\\:\\left(\\%\\right)=\\frac{\\text{R}\\text{e}\\text{d}\\text{u}\\text{c}\\text{i}\\text{n}\\text{g}\\:\\text{s}\\text{u}\\text{g}\\text{a}\\text{r}\\text{s}\\:\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}\\:(\\text{g}/\\text{L})}{(\\text{g}\\text{l}\\text{u}\\text{c}\\text{a}\\text{n}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{e}\\text{n}\\text{t}\\text{ %}\\:\\times\\:\\:1.11\\:+\\:\\text{x}\\text{y}\\text{l}\\text{a}\\text{n}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{e}\\text{n}\\text{t}\\text{\\%}\\:\\times\\:\\:1.14)\\:\\times\\:\\:\\text{S}\\text{o}\\text{l}\\text{i}\\text{d}\\:\\text{l}\\text{o}\\text{a}\\text{d}\\text{i}\\text{n}\\text{g}\\text{s}\\:(\\text{g}/\\text{L})}\\times\\:100\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;(1)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 General Fermentation Procedures\u003c/h2\u003e \u003cp\u003eAll anaerobic fermentations were conducted in 55 mL serum vials with a 20 mL working volume. The initial pH was adjusted to 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 using 5 M NaOH or HCl. Each vial was inoculated with a 10% (v/v) seed culture and incubated at 55 ℃ with orbital shaking at 150 rpm. The medium was DSMZ 122 medium containing (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 1.3 g/L, MgCl\u003csub\u003e2\u003c/sub\u003e x 6 H\u003csub\u003e2\u003c/sub\u003eO 2.6 g/L, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1.43 g/L, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 5.50 g/L, CaCl\u003csub\u003e2\u003c/sub\u003e x 2 H\u003csub\u003e2\u003c/sub\u003eO 0.13 g/L, Na\u003csub\u003e2\u003c/sub\u003e-\u0026szlig;-glycerophosphate x 5 H\u003csub\u003e2\u003c/sub\u003eO 6.00 g/L, FeSO\u003csub\u003e4\u003c/sub\u003e x 7 H\u003csub\u003e2\u003c/sub\u003eO (0.1% w/v in 0.05 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) 1.10 ml/L, L-Glutathione (reduced) 0.25 g/L, Yeast extract 4.50 g/L, Sodium resazurin (0.1% w/v) 0.50 ml/L.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Separate Hydrolysis and Fermentation (SHF)\u003c/h2\u003e \u003cp\u003eThe fermentation medium was prepared using enzymatic hydrolysates of tobacco stalks as the substrate. Vials were inoculated with \u003cem\u003eT. thermosaccharolyticum\u003c/em\u003e MJ2 and fermented for 24 h to evaluate the impact of pretreatment-derived inhibitors on hydrogen production.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Hydrogen production from consolidated bioprocessing (CBP).\u003c/h2\u003e \u003cp\u003eCBP was carried out using alkali-pretreated tobacco stalks as the sole carbon source. For mono-cultures, either \u003cem\u003eA. thermocellus\u003c/em\u003e DSM 1313 was used; for co-cultures, \u003cem\u003eA. thermocellus\u003c/em\u003e DSM 1313 and \u003cem\u003eT. thermosaccharolyticum\u003c/em\u003e MJ2 were inoculated at a 1:1 (v/v) ratio. The fermentation was conducted for 24 h. The initial substrate concentration was set at 2% (w/v), with further evaluation conducted across a range of 2% to 10% (w/v) to determine the operational limits of the system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Fermentation procedure under inhibitory stress\u003c/h2\u003e \u003cp\u003eThe alkali pretreatment liquid was collected from the liquid fraction following the alkali pretreatment of tobacco stalks. To investigate the dose-response relationship of inhibitors, a series of fermentation media were prepared by substituting deionized water with the pretreatment liquid in varying volume proportions: 0%, 20%, 40%, 60%, 80%, and 100% (v/v). For all inhibitory groups, detoxified alkali-pretreated tobacco stalks were used as the solid substrate at a loading of 3% (w/v).\u003c/p\u003e \u003cp\u003eCBP was conducted in 55 mL serum bottles with a working volume of 20 mL. For the monoculture group, the medium was inoculated with a 10% (v/v) active culture of \u003cem\u003eA. thermocellus\u003c/em\u003e DSM 1313. For the co-culture group, a 10% (v/v) inoculum consisting of DSM 1313 and \u003cem\u003eT. thermosaccharolyticum\u003c/em\u003e MJ2 (at a ratio of 1:1, v/v) was used. The fermentation was conducted for 72 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Kinetic modeling and data analysis\u003c/h2\u003e \u003cp\u003eTo quantitatively describe the hydrogen production dynamics, the cumulative hydrogen production curves were fitted using the modified Gompertz model:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\text{P(t)}\\text{=}{\\text{P}}_{\\text{m}}\\text{\u0026times;exp}\\left\\{\\text{-exp}\\left[\\frac{{\\text{R}}_{\\text{m}}\\text{\u0026times;e}}{{\\text{P}}_{\\text{m}}}\\left(\\text{\u0026lambda;-t}\\right)\\text{+1}\\right]\\right\\}\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;2)\u003c/p\u003e \u003cp\u003eWhere: P(t) is the cumulative hydrogen production (mM) at time t (h); P\u003csub\u003em\u003c/sub\u003e is the maximum hydrogen production potential (mM); R\u003csub\u003em\u003c/sub\u003e is the maximum hydrogen production rate (mM/h); λ is the lag phase duration (h); e is the mathematical constant (approximately 2.718).\u003c/p\u003e \u003cp\u003eThe kinetic parameters were estimated via non-linear regression analysis using Origin 2021 software. All experiments were performed in triplicate, and the results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical significance was analyzed using one-way ANOVA with a confidence level of 95% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Methods of analysis\u003c/h2\u003e \u003cp\u003eCompositional analysis was conducted following the procedure described by the National Renewable Energy Laboratory (NREL) for the analysis of tobacco stalks and pretreatment supernatant fractions.\u003c/p\u003e \u003cp\u003eCell density in the liquid medium was monitored by measuring the optical density at 600 nm (OD₆₀₀) using a 10 mL serum bottle (GENESYS\u0026trade; 10S, Thermo Fisher Scientific, USA).\u003c/p\u003e \u003cp\u003eThe concentrations of oligosaccharides, monosaccharides, acetate, formate, furfural, 5-hydroxymethyl furfural (HMF), and metabolites were determined by high performance liquid chromatography (Waters 2695, USA) under the following conditions: column temperature of 60 ℃, injection rate of 10 \u0026micro;L, flow rate of 0.6 mL/min, analytical column of Bio-Rad Annex HPX-87H with a differential refractive index detector at 40 ℃.\u003c/p\u003e \u003cp\u003eGas chromatography-mass spectrometry (GC-MS) was used to determine the composition of inhibitors in the hydrolysis products of tobacco stalks. The pretreatment was extracted and concentrated by ethyl acetate, and then the treated samples were examined on an InertCap5 capillary column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m) using a gas chromatography-mass spectrometer (QP2010ultra). GC conditions were as follows: carrier gas: He (99. 999%); column flow rate: 1.0 mL/min; shunt mode: no shunt; injection volume: 1.0 \u0026micro;L; inlet temperature: 300 ℃; the initial temperature of the column was 50 ℃, retained for 1 min, and then the programmed temperature was increased to 200 ℃ by 8 ℃/min and kept for 2 min. The column temperature was 50 ℃ at the beginning and kept for 1 min, then the programmed temperature was increased to 200 ℃ at 8 ℃/min and kept for 2 min, then the programmed temperature was increased to 300 ℃ at 8 ℃/min and kept for 2 min; the auxiliary heating zone 2 was opened and the initial temperature was 280 ℃, and the total running time of the method was 29 min. The mass spectrometry conditions were as follows: ion source: EI source; ion source temperature: 230 ℃; quadrupole temperature: 150 ℃; chromatography-mass spectrometry junction temperature: 280 ℃; electron energy: 70 eV; electron multiplier voltage: 1,500 V; scanning mode: full scanning, scanning mass number range is: 20\u0026thinsp;~\u0026thinsp;550.\u003c/p\u003e \u003cp\u003eHydrogen in the headspace was measured by gas chromatography (Foley 9790 Plus, Foley, China). For hydrogen quantification, the gas from the headspace of sealed serum bottles was withdrawn using a 25 mL gas-tight syringe and immediately injected into the gas chromatograph inlet. The gas mixture was carried by nitrogen through a capillary column (TDX-01, 100 ℃), where separation occurred based on adsorption-desorption interactions. The separated hydrogen was detected using a thermal conductivity detector (TCD) maintained at 80 ℃. The signal was recorded as a chromatogram, and hydrogen yield was calculated based on the peak area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of the data in this study was performed using the software of SPSS 17.0 (SPSS Inc. Chicago) and Microsoft\u0026reg; Excel. ANOVA and Student's T test were applied to determine significant differences. A \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Screening of pretreatment strategies for non-detoxified CBP feedstocks\u003c/h2\u003e\n \u003cp\u003eTo ensure the feasibility of consolidated bioprocessing (CBP) under non-detoxified conditions, the pretreatment strategy must provide substrates with sufficient enzymatic accessibility while avoiding excessive generation of fermentation inhibitors. Therefore, different pretreatment methods were first evaluated as a screening step to identify suitable feedstocks for subsequent CBP experiments, rather than to establish a comprehensive comparison of pretreatment technologies.\u003c/p\u003e\n \u003cp\u003eLignocellulosic biomasses commonly used in fermentation typically contain 20.2%\u0026ndash;49.5% cellulose, 14.6%\u0026ndash;43.5% hemicellulose, and 7.3%\u0026ndash;21.8% lignin [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. Compared to these commonly used feedstocks, the tobacco stalks used in this study exhibited lower cellulose and hemicellulose contents, but higher lignin content (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, pretreatment is essential to improve their suitability for biorefinery applications.\u003c/p\u003e\n \u003cp\u003eChemical pretreatment with acid or alkali is among the most effective methods to enhance biomass digestibility [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e]. Both pretreatments effectively increased the glucan content of tobacco stalks, from 29.18% in raw material to 43.87% (alkali) and 47.42% (acid) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). However, they differed markedly in their effects on hemicellulose and inhibitor profiles. Alkali pretreatment selectively removed lignin (from 27.33% to 24.74%) while preserving most hemicellulose (14.15% xylan retained). In contrast, acid pretreatment drastically reduced hemicellulose (xylan dropped to 4.91%) and increased lignin content (32.19%), indicating more extensive sugar degradation.\u003c/p\u003e\n \u003cp\u003eAlkali pretreatment mainly disrupts lignin structures, effectively reducing lignin content in most crop residues with minimal sugar degradation and low formation of furan derivatives. In contrast, dilute acid breaks glycosidic bonds in lignocellulose, dissolving hemicellulose and some lignin, thereby enhancing the release of fermentable sugars and improving biomass accessibility [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe compositions of raw and pretreated tobacco stalks\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eComposition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eContent (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRaw tobacco stalks\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAcid pretreated tobacco stalks\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAlkali pretreated tobacco stalks\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlucan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e43.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eXylan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eArabinan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLignin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAsh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eND: Not detected.\u003c/p\u003e\n \u003cp\u003eConsistent with previous reports [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e], alkali pretreatment generated almost no furans, whereas acid pretreatment produced substantial furfural (1686.64 mg/kg) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Both pretreatments released phenolic compounds and trace inhibitors such as vanillin, syringaldehyde, and 2,3\u0026prime;-bipyridine, which are known to inhibit microbial fermentation [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComposition of pretreatment inhibitors from tobacco stalks.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eComposition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"4\" align=\"left\"\u003e\n \u003cp\u003eContent\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAcid pretreated tobacco stalks (mg/kg)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAlkali pretreated tobacco stalks (mg/kg)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAcid pretreated supernatant (mg/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAlkali pretreated supernatant (mg/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhenolics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6362.57\u0026thinsp;\u0026plusmn;\u0026thinsp;53.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10763.74\u0026thinsp;\u0026plusmn;\u0026thinsp;397.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2512.46\u0026thinsp;\u0026plusmn;\u0026thinsp;111.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1782.46\u0026thinsp;\u0026plusmn;\u0026thinsp;67.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFurfural\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1686.64\u0026thinsp;\u0026plusmn;\u0026thinsp;85.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e451.85\u0026thinsp;\u0026plusmn;\u0026thinsp;7.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhenol, 2-methoxy-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e92.18\u0026thinsp;\u0026plusmn;\u0026thinsp;26.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e131.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2-Methoxy-4-vinylphenol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e208.36\u0026thinsp;\u0026plusmn;\u0026thinsp;8.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e121.20\u0026thinsp;\u0026plusmn;\u0026thinsp;14.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.93\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.77\u0026thinsp;\u0026plusmn;\u0026thinsp;9.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhenol, 2,6-dimethoxy-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e58.27\u0026thinsp;\u0026plusmn;\u0026thinsp;7.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.55\u0026thinsp;\u0026plusmn;\u0026thinsp;2.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVanillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60.53\u0026thinsp;\u0026plusmn;\u0026thinsp;49.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e334.44\u0026thinsp;\u0026plusmn;\u0026thinsp;62.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.64\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetovanillone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.37\u0026thinsp;\u0026plusmn;\u0026thinsp;4.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.20\u0026thinsp;\u0026plusmn;\u0026thinsp;13.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2,3\u0026apos;-Dipyridyl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.22\u0026thinsp;\u0026plusmn;\u0026thinsp;17.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.40\u0026thinsp;\u0026plusmn;\u0026thinsp;5.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.36\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSyringaldehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e61.04\u0026thinsp;\u0026plusmn;\u0026thinsp;32.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e127.86\u0026thinsp;\u0026plusmn;\u0026thinsp;5.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.91\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.75\u0026thinsp;\u0026plusmn;\u0026thinsp;5.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en-Hexadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1300.87\u0026thinsp;\u0026plusmn;\u0026thinsp;317.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1554.92\u0026thinsp;\u0026plusmn;\u0026thinsp;73.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e129.93\u0026thinsp;\u0026plusmn;\u0026thinsp;5.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOctadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e547.07\u0026thinsp;\u0026plusmn;\u0026thinsp;162.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e522.85\u0026thinsp;\u0026plusmn;\u0026thinsp;22.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.48\u0026thinsp;\u0026plusmn;\u0026thinsp;4.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eND: Not detected.\u003c/p\u003e\n \u003cp\u003eEnzymatic hydrolysis was performed to evaluate the digestibility of detoxified and non-detoxified substrates from both pretreatments. At 10% solid loading, detoxified alkali-pretreated tobacco stalks (DAKTS) produced 49.61\u0026thinsp;\u0026plusmn;\u0026thinsp;3.09 g/L of reducing sugars, while non-detoxified alkali-pretreated tobacco stalks (NAKTS) yielded 36.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24 g/L\u0026mdash;a 27.97% reduction attributable to inhibitor presence (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). For acid-pretreated substrates, detoxified samples (DACTS) achieved 25.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56 g/L, whereas non-detoxified samples (NACTS) produced only 17.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18 g/L, representing a 30.93% reduction (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Notably, even under non-detoxified conditions, alkali-pretreated substrates outperformed detoxified acid-pretreated substrates in reducing sugar yield, highlighting the inherent advantage of alkali pretreatment in preserving fermentable sugar availability. These results demonstrate that alkali pretreatment consistently confers superior enzymatic digestibility compared to acid pretreatment, regardless of detoxification status.\u003c/p\u003e\n \u003cp\u003eNAKTS: Non-detoxified alkali pretreated tobacco stalks; DAKTS: Detoxified alkali pretreated tobacco stalks\u003c/p\u003e\n \u003cp\u003eUnder non-detoxified conditions, alkali-pretreated tobacco stalks exhibited higher enzymatic digestibility than acid-pretreated substrates, as reflected by increased fermentable sugar release during enzymatic hydrolysis. In contrast, acid-pretreated substrates showed lower hydrolysis efficiency, which may be attributed to the presence of residual inhibitory compounds and reduced cellulose accessibility. Given these differences, alkali pretreatment was selected for subsequent fermentation and CBP experiments.\u003c/p\u003e\n \u003cp\u003eAlthough variations in enzymatic hydrolysis performance were observed between pretreatment methods, it should be noted that enzymatic digestibility alone does not determine downstream fermentation performance, particularly under CBP conditions. Instead, the ability of microorganisms to tolerate pretreatment-derived inhibitors becomes increasingly critical when cellulose depolymerization and fermentation occur simultaneously. Therefore, the selected alkali-pretreated, non-detoxified substrate was used as a representative feedstock for further evaluation of hydrogen production systems.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Hydrogen production from enzymatic hydrolysates as SHF reference systems\u003c/h2\u003e\n \u003cp\u003eTo isolate the inhibitory effects of pretreatment-derived compounds from the constraints of cellulose depolymerization, SHF was conducted using enzymatic hydrolysates of pretreated tobacco stalks. In this system, fermentable sugars were readily available, allowing direct assessment of microbial inhibitor tolerance independent of hydrolysis efficiency.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eThermoanaerobacterium thermosaccharolyticum\u003c/em\u003e MJ2, a thermophilic hydrogen producer previously reported to tolerate lignocellulose-derived inhibitors [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e], was used for SHF. When cultivated on hydrolysates from detoxified (DAKTS) and non-detoxified (NAKTS) alkali-pretreated stalks, MJ2 exhibited comparable hydrogen production at reducing sugar concentrations\u0026thinsp;\u0026le;\u0026thinsp;20 g/L (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). At 20 g/L, hydrogen yields reached 241.19\u0026thinsp;\u0026plusmn;\u0026thinsp;30.09 mM (DAKTS) and 215.26\u0026thinsp;\u0026plusmn;\u0026thinsp;49.61 mM (NAKTS), with the 10.75% reduction being statistically insignificant. These values compare favorably with reported hydrogen production from other lignocellulosic residues, such as sugarcane bagasse (121.60 mM) [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e] and paper sludge (133.68 mM) [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eHowever, at 30 g/L reducing sugars, hydrogen production from NAKTS hydrolysate declined significantly to 165.80\u0026thinsp;\u0026plusmn;\u0026thinsp;3.17 mM\u0026mdash;a 21.32% reduction relative to DAKTS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u0026mdash;indicating that inhibitor effects become pronounced at higher substrate concentrations. This threshold aligns with common practice in dark fermentation, where sugar concentrations seldom exceed 20 g/L [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eAnalysis of fermentation metabolites showed that acetate and butyrate concentrations increased with sugar concentration in both hydrolysates (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Notably, the butyrate-to-acetate ratio was elevated in NAKTS fermentations, consistent with previous observations that phenolic inhibitors like vanillin can shift metabolic pathways [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, this metabolic shift did not translate into increased hydrogen production, suggesting that the inhibitors primarily affected electron flux rather than overall metabolic activity.\u003c/p\u003e\n \u003cp\u003eAfter fermentation, the key metabolites associated with hydrogen production\u0026mdash;acetate and butyrate\u0026mdash;were quantified across different sugar concentrations in the DAKTS and NAKTS hydrolysates. From 0\u0026ndash;20 g/L of reducing sugars, both acetate and butyrate concentrations increased with increasing sugar concentration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eThese SHF results establish a reference for interpreting subsequent CBP performance: while MJ2 exhibits substantial tolerance to pretreatment-derived inhibitors, its hydrogen-producing capacity is compromised at higher inhibitor concentrations. This highlights the need for enhanced stress mitigation strategies in CBP systems, where both hydrolysis and fermentation occur simultaneously.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Hydrogen production from alkali pretreated tobacco stalks by CBP\u003c/h2\u003e\n \u003cp\u003eCBP fermentation was carried out by inoculating \u003cem\u003eA. thermocellus\u003c/em\u003e DSM1313 with detoxified (DAKTS) and non-detoxified (NAKTS) alkali-pretreated tobacco stalks (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Hydrogen production by DSM1313 using DAKTS at 3%, 5%, 7%, and 10% substrate concentrations was 62.42\u0026thinsp;\u0026plusmn;\u0026thinsp;11.38 mM, 60.10\u0026thinsp;\u0026plusmn;\u0026thinsp;5.95 mM, 56.28\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23 mM, and 61.85\u0026thinsp;\u0026plusmn;\u0026thinsp;5.68 mM, respectively. When using NAKTS, hydrogen production was 61.77\u0026thinsp;\u0026plusmn;\u0026thinsp;5.73 mM, 62.47\u0026thinsp;\u0026plusmn;\u0026thinsp;4.51 mM, 27.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64 mM, and 5.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25 mM at the same substrate concentrations. There was no significant difference in hydrogen production from DAKTS at 3%\u0026ndash;10% substrate concentration, indicating that DSM1313 could utilize up to 3% substrate effectively, with no further improvement at higher concentrations. For NAKTS, increasing substrate concentration led to higher inhibitor levels, significantly reducing hydrogen production. At 7% substrate concentration, hydrogen production from NAKTS decreased by 50.88% compared to DAKTS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005), and at 10%, it dropped by 90.71% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). This suggests that non-detoxification at higher substrate concentrations strongly inhibits hydrogen production in DSM1313 monocultures.\u003c/p\u003e\n \u003cp\u003eDue to the limited efficiency of single-species fermentation, co-culturing cellulolytic bacteria with high-efficiency hydrogen-producing strains can improve substrate utilization and hydrogen yield [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. MJ2 was co-cultured with DSM1313 for CBP fermentation using DAKTS and NAKTS (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Hydrogen production from DAKTS was 66.34\u0026thinsp;\u0026plusmn;\u0026thinsp;2.66 mM, 97.28\u0026thinsp;\u0026plusmn;\u0026thinsp;7.00 mM, 102.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59 mM, and 93.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10 mM at 3%, 5%, 7%, and 10% substrate concentrations, respectively. Compared with monocultures, co-culture systems significantly increased hydrogen yield at 5%\u0026ndash;10% substrate concentrations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005). For NAKTS, hydrogen production was 88.22\u0026thinsp;\u0026plusmn;\u0026thinsp;12.99 mM, 117.78\u0026thinsp;\u0026plusmn;\u0026thinsp;19.94 mM, 118.77\u0026thinsp;\u0026plusmn;\u0026thinsp;23.32 mM, and 85.75\u0026thinsp;\u0026plusmn;\u0026thinsp;3.12 mM, respectively. Hydrogen production increased by 32.97%, 21.07%, and 16.20% at 3%\u0026ndash;7% substrate concentrations compared to DAKTS (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). However, at 10% substrate concentration, hydrogen production decreased by 8.66%.\u003c/p\u003e\n \u003cp\u003eThe addition of MJ2 to the CBP system not only increased hydrogen production from NAKTS but also mitigated the inhibitory effects of pretreatment by-products. At 7% substrate concentration, hydrogen production in the NAKTS monoculture system decreased by more than half, while the co-culture system showed a slight increase. Even at 10% substrate concentration, where inhibitors almost completely suppressed hydrogen production in the DSM1313 monoculture, hydrogen yield in the co-culture only decreased by 8.66%. This indicates that the inhibitor load at high solids loading exceeded the intrinsic tolerance threshold of \u003cem\u003eA. thermocellus\u003c/em\u003e DSM 1313. In contrast, the MJ2-augmented co-culture exhibited significant system-level robustness, effectively shifting the operational boundary toward higher stress levels.\u003c/p\u003e\n \u003cp\u003eLow concentrations of inhibitors in pretreated tobacco stalks showed a stimulatory effect, while high concentrations exhibited inhibition during co-culture fermentation. According to Zhang et al., the addition of 2 g/L furfural or HMF increased the growth of \u003cem\u003eClostridium acetobutylicum\u003c/em\u003e ATCC 824 by 25% [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. Ezeji et al. [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e] also found that \u003cem\u003eClostridium beijerinckii\u003c/em\u003e BA101 growth increased by 13% with furfural or HMF (\u0026le;\u0026thinsp;2 g/L). Within certain limits, lignocellulosic pretreatment inhibitors may stimulate microbial activity. Jung et al. [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e] reported that \u003cem\u003eE. coli\u003c/em\u003e expressing polyhydroxybutyrate synthesis genes accumulated higher biomass and showed enhanced growth and resistance at 15 mM furfural. MJ2 contains polyhydroxybutyrate synthesis-related genes and may have similar resistance. Thus, increased hydrogen production at low substrate concentrations and the decrease at higher concentrations may be attributed to the hormesis effect, enhancing microbial activity at low inhibitor levels [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Moreover, different tolerance levels of the two strains may shift the biomass ratio in favor of MJ2, further improving hydrogen yield in co-culture systems using NAKTS over DAKTS.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Effects of different strategies on biohydrogen fermentation of tobacco stalks\u003c/h2\u003e\n \u003cp\u003eAccording to Fig. A1, alkali pretreatment retained a large amount of fermentable sugars from 1 kg of waste tobacco stalks, suitable for hydrogen fermentation. After alkali pretreatment without detoxification, SHF fermentation yielded 94.4 L of hydrogen, and CBP fermentation yielded 65.8 L. Washing removed many inhibitors adsorbed onto the tobacco stalks. However, this did not improve hydrogen yield; instead, SHF and CBP fermentation from DAKTS produced 79.6 L and 29.7 L of hydrogen, respectively. The hydrogen yield from tobacco stalks was comparable to that from lignocellulosic feedstocks such as wheat straw, agave bagasse, and sugarcane bagasse [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. The hydrogen yield from non-detoxified tobacco stalks was higher than that from detoxified samples. Moreover, CBP fermentation offers the advantage of omitting costly cellulase addition, making the process more economical with further optimization. This difference likely reflects the combined effects of sugar loss during washing, residual alkali buffering, and microbial tolerance, rather than the inhibitory removal step alone.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. Assessment of microbial tolerance and synergistic mechanism under inhibitory stress\u003c/h2\u003e\n \u003cp\u003eTo quantitatively evaluate the dose-response relationship between pretreatment-derived inhibitors and fermentative performance, varying proportions of pretreatment liquid (0\u0026ndash;100%, v/v) were introduced into the fermentation system. This approach overcomes the limitation of fixed inhibitor concentrations in dried tobacco stalks, providing a controlled inhibitory gradient.\u003c/p\u003e\n \u003cp\u003eThe results demonstrated that the co-culture system possessed superior robustness compared to the \u003cem\u003eA. thermocellus\u003c/em\u003e DSM 1313 monoculture. At a 40% (v/v) liquid addition, the co-culture achieved a peak hydrogen yield of 125.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 mM, representing a 44.27% increase over the inhibitor-free control. Notably, while the hydrogen production of DSM 1313 was severely suppressed at 24 h under 40% liquid stress, the co-culture maintained a production rate comparable to the control. These results suggest that MJ2 contributes to mitigating the inhibitory effects of the pretreatment liquid, which may help maintain hydrogen production performance in the co-culture system. The observed behavior may arise from multiple non-exclusive system-level interactions, including differential tolerance and altered metabolic coupling between consortium members.\u003c/p\u003e\n \u003cp\u003eInterestingly, at a lower concentration (20% v/v), the co-culture exhibited significantly enhanced hydrogen production, whereas DSM 1313 alone still suffered from inhibition. This stimulatory response at low inhibitor load is phenotypically consistent with the hormetic effects reported in other anaerobic fermentation systems [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. While the specific molecular triggers of this response were not resolved in this study, the reproducible nature of this phenomenon suggests a complex interaction between sub-inhibitory stress and metabolic flux in the co-culture system. However, hydrogen production was completely arrested when the liquid concentration exceeded 60%. This failure underscores the \u0026quot;bottleneck\u0026quot; of the CBP system: as the primary degrader, the total growth arrest of DSM 1313 under high chemical stress leads to a cessation of sugar supply, which MJ2 cannot overcome despite its own high tolerance. These findings delineate the functional boundaries of the co-culture, emphasizing that system stability is fundamentally anchored to the viability of the primary cellulolytic strain.\u0026nbsp;\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of tobacco stalks alkali pretreatment supernatant liquid on hydrogen production kinetics of CBP by monoculture.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAlkali pretreatment supernatant liquid proportion (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePm (mM)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRm (mM/h)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026lambda; (h)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u0026mdash;: No valid kinetic parameters could be fitted by the model.\u003c/p\u003e\n \u003cp\u003eAlthough inhibitors like vanillin and syringaldehyde were present in the non-detoxified hydrolysate (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), the co-culture of \u003cem\u003eA. thermocellum\u003c/em\u003e DSM 1313 and \u003cem\u003eT. thermosaccharolyticum\u003c/em\u003e MJ2 maintained high H\u003csub\u003e2\u003c/sub\u003e productivity. This resilience is functionally associated with the inclusion of MJ2 in the consortium, which may alleviate inhibitory stress through metabolic buffering and/or other tolerance-related mechanisms. Previous studies have reported detoxification-related capabilities in Thermoanaerobacterium species; however, in the present study, the enhanced performance is interpreted as a functional tolerance effect rather than confirmed biochemical transformation. Such capabilities may partially explain the improved performance observed in the co-culture; however, direct evidence of inhibitor biotransformation was not obtained in the present study [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of tobacco stalks alkali pretreatment supernatant liquid on hydrogen production kinetics of CBP by co-culture.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAlkali pretreatment supernatant liquid proportion (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePm (mM)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRm (mM/h)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026lambda; (h)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e123.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.93\u0026thinsp;\u0026plusmn;\u0026thinsp;3.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.78\u0026thinsp;\u0026plusmn;\u0026thinsp;3.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026mdash;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u0026mdash;: No valid kinetic parameters could be fitted by the model.\u003c/p\u003e\n \u003cp\u003eThe sustained hydrogen production under high inhibitory load indicates a robust system-level stress mitigation effect. While direct quantification of inhibitor biotransformation was not conducted, the innate resistance of \u003cem\u003eT. thermosaccharolyticum\u003c/em\u003e MJ2 likely provides a metabolic buffer. Elucidating the precise contributions of specific detoxification pathways will require future studies incorporating inhibitor mass balances; however, the current results clearly define the enhanced operational tolerance gained through co-cultivation.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study demonstrates that alkali-pretreated tobacco stalks can be effectively utilized for biohydrogen production via both SHF and CBP routes without the need for energy-intensive detoxification. The co-culture of \u003cem\u003eA. thermocellus\u003c/em\u003e DSM1313 and \u003cem\u003eT. thermosaccharolyticum\u003c/em\u003e MJ2 proved to be a robust strategy, significantly overcoming the inhibitory stress that otherwise crippled monoculture performance. Kinetic analysis revealed that the inclusion of MJ2 significantly enhanced hydrogen production performance under moderate inhibitory stress. This improvement is consistent with an increased system-level tolerance to inhibitory stress and/or favorable microbial interactions within the co-culture system, rather than evidence of direct or complete detoxification of inhibitory compounds. However, the identification of a 60% liquor concentration threshold clarifies that the stability of this synergistic system is ultimately anchored to the physiological limits of the primary degrader. Overall, these findings provide a simplified and sustainable framework for the high-value bioconversion of complex tobacco waste, providing experimental insights relevant to the development and optimization of scalable biorefining strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China, China [grant no. 52070079], and Yunnan Fundamental Applied Research Project (202001AU070010).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003eL\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e: Investigation, Writing \u0026ndash; Original Draft, Validation, Formal Analysis, Data Curation.\u0026nbsp;\u003cstrong\u003eM\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Z\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e: \u0026nbsp;Writing \u0026ndash; Review \u0026amp; Editing, Investigation, Resources, Conceptualization, Methodology, Funding Acquisition.\u0026nbsp;\u003cstrong\u003eB\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;H\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e: Resources, Supervision, Funding Acquisition, Project Administration. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eConflicts of interest\u003c/h2\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003ch2\u003eAppendix A. Supplementary data\u003c/h2\u003e\n\u003cp\u003eE-supplementary data of this work can be found in online version of the paper.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed in this study are available from the Zenodo repository. A private sharing link has been provided for peer review:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehttps://zenodo.org/records/15605353?preview=1\u0026amp;token=eyJhbGciOiJIUzUxMiJ9.eyJpZCI6ImRjYWIwZGMwLWZmZTctNGQ5ZC04NmIzLTMyYzRkOWI5NjE4YiIsImRhdGEiOnt9LCJyYW5kb20iOiI0ZDRhZTAyMWE4ZTRmZmZmOWIwYjE5OWViYmNiMGQwZCJ9.d5ZF4KT9tDPuY4LJndPoj_ncuDDm-1DoVRwdDE0O02gwD4DL0ywGNJRBEbwOtUJaZ6aV12ky-5rY8VDU1NAtiw. The data will be made publicly available upon publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChoe C, Cheon S, Kim H, Lim H (2023) Mitigating climate change for negative CO2 emission via syngas methanation: Techno-economic and life-cycle assessments of renewable methane production. Renew Sustain Energy Rev 185:113628\u0026ndash;113640. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.rser.2023.113628\u003c/span\u003e\u003cspan address=\"10.1016/j.rser.2023.113628\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDincer I, Acar C (2015) Review and evaluation of hydrogen production methods for better sustainability. 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Process Biochem 109:46\u0026ndash;58. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.procbio.2021.06.021\u003c/span\u003e\u003cspan address=\"10.1016/j.procbio.2021.06.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Tobacco stalks, Biohydrogen, Lignocellulose, Separate hydrolysis and fermentation, Consolidated Bioprocessing","lastPublishedDoi":"10.21203/rs.3.rs-8915427/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8915427/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluates the feasibility of using non-detoxified alkali-pretreated tobacco stalks for biohydrogen production through separate hydrolysis and fermentation (SHF) and consolidated bioprocessing (CBP). In SHF, \u003cem\u003eThermoanaerobacterium thermosaccharolyticum\u003c/em\u003e MJ2 produced 215.26\u0026thinsp;\u0026plusmn;\u0026thinsp;49.61 mM hydrogen from non-detoxified enzymatic hydrolysates, showing high tolerance to pretreatment inhibitors. In CBP, while the hydrogen production of \u003cem\u003eAcetivibrio thermocellus\u003c/em\u003e DSM1313 alone was inhibited by 90.71% when using non-detoxified stalks, the co-culture of DSM1313 and MJ2 significantly mitigated this inhibitory effect, reaching 91.34% of the yield obtained from detoxified stalks. To further elucidate the detoxification mechanism, the system's tolerance was quantitatively assessed by introducing a gradient of pretreatment liquor (0\u0026ndash;100%, v/v). Kinetic analysis using the modified Gompertz model revealed that the co-culture achieved a maximum hydrogen potential of 125.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 mM under 40% (v/v) pretreatment liquor stress\u0026mdash;a 44.27% increase over the control. While a hormetic effect was observed at 20% liquor concentration, a critical threshold was identified at 60%, where the system failed due to the growth arrest of the primary degrader, DSM1313. Collectively, these results demonstrate that microbial co-cultivation significantly expands the operational window of non-detoxified CBP. By providing a higher system-level tolerance threshold to pretreatment-derived inhibitors, this strategy reduces the dependence on intensive detoxification processes, offering a robust approach for cost-effective biohydrogen production.\u003c/p\u003e","manuscriptTitle":"Expanding the Operational Window of Consolidated Bioprocessing Hydrogen Production from Non-Detoxified Alkali-Pretreated Tobacco Stalks via Microbial Co-Cultivation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-25 10:50:31","doi":"10.21203/rs.3.rs-8915427/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-10T11:01:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-09T05:01:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2965467234867642692806077068053367525","date":"2026-02-26T16:40:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-24T02:41:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-23T11:02:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-23T10:43:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioprocess and Biosystems Engineering","date":"2026-02-19T08:12:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"63867a73-ec90-410e-b18c-fcd8a440f76a","owner":[],"postedDate":"February 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-24T10:12:38+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-25 10:50:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8915427","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8915427","identity":"rs-8915427","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}