Stirring the Hydrogen and Butanol production from Enset Fiber via Simultaneous Saccharification and Fermentation (SSF) process

Stirring the Hydrogen and Butanol production from Enset Fiber via Simultaneous Saccharification and Fermentation (SSF) process | 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 Stirring the Hydrogen and Butanol production from Enset Fiber via Simultaneous Saccharification and Fermentation (SSF) process Nebyat Seid, Lea Wießner, Habibu Aliyu, Anke Neumann This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3836946/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Replacing fossil fuels with biofuels such as hydrogen and butanol has the potential to reduce carbon emissions and improve the sustainability of the energy system. Enset fiber is a promising feedstock to produce these biofuels. Clostridium saccharoperbutylacetonicum DSM 14923 can produce hydrogen and butanol simultaneously; however, to achieve the best results, each product requires different fermentation conditions. This study aimed to optimize process parameters for the simultaneous saccharification and fermentation (SSF) process to produce hydrogen and butanol from Enset fiber, and to investigate the impact of pH control in the SSF process in the bioreactor. Results The SSF process in bottles resulted in a elevated butanol concentration of 11.36 g/L with a yield of 0.23 g/g and a productivity of 0.16 g/L − 1 h − 1 at the optimal process parameters of 5% (w/v) substrate loading, 16 FPU/g cellulase loading, and 100 rpm agitation speed from pretreated Enset fiber. A comparable result was observed in the bioreactor during the pH-uncontrolled SSF process; however, the butanol productivity decreased to 0.095 g/L − 1 h − 1 . Furthermore, the pre-hydrolysis simultaneous saccharification and fermentation (PSSF) process in the bioreactor with a 7% (w/v) substrate loading led to the highest butanol concentration and productivity of 12.84 g/L and 0.104 g/L − 1 h − 1 respectively. By further optimizing the SSF process, we achieved 442.57 mmol of hydrogen with a yield of 198.27 mL/g-Enset fiber in the bioreactor under the optimal conditions of temperature 37°C, initial pH 8.0 and atmospheric pressure. Conclusion In this study, a scalable process for both products was developed to achieve similar results to bottle-scale experiments, indicating potential applicability on an industrial scale. This is the first study to combine C. saccharoperbutylacetonicum and Enset fiber for the SSF process and contributes to the development of sustainable energy sources. Hydrogen Butanol Enset fiber C. saccharoperbutylacetonicum SSF PSSF Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background The impacts of human-driven global warming, such as the melting of sea ice, rising sea levels, unpredictable heat waves, wildfires, and intense rainfall, are getting worse day by day. Although some countries have made progress in adapting their climate action plans to achieve net-zero emissions, the situation remains challenging [ 1 , 2 ]. According to the International Energy Agency (IEA) report (2022), CO 2 emissions increased by 6% from 2020 to 2021, the highest emission level recorded for a single year. This increase in emissions clearly indicates that the world is far from achieving the goal of limiting warming to 1.5°C, as set by the United Nations [ 2 , 3 ]. The goal can be achieved by shifting global energy consumption from fossil fuels to biofuels such as hydrogen and butanol, which can potentially reduce carbon emissions and establish a more sustainable energy system [ 4 ]. In recent years, hydrogen has become increasingly popular as an energy carrier for fuelling vehicles, generating electricity, and various industrial applications [ 5 , 6 ]. The high energy content of hydrogen (140 MJ/kg) and the fact that its combustion produces only water as a byproduct make it an ideal clean fuel for various applications [ 7 ]. Similarly, butanol is a highly versatile fuel that can be blended with gasoline in high proportions, used in existing engines, and easily transported through current pipeline infrastructure. This versatility arises from its excellent properties, such as higher energy density (29 MJ/L), lower vapor pressure (0.53 kPa), lower hygroscopicity, and lower volatility than ethanol [ 8 , 9 ]. Several studies have shown that biological processes can successfully produce butanol and hydrogen. However, there are still numerous challenges that need to be addressed to make this process competitive with other processes. These includes substrate availability [ 10 ], low production yield [ 5 , 11 ], scalability, high processing cost [ 12 , 13 ], and maintaining optimal fermentation conditions [ 14 , 15 ]. Lignocellulosic biomass is readily available and economically viable worldwide and can be obtained from agricultural residues, industrial wastes, forest residues, and municipal wastes [ 10 ]. It is composed of cellulose, hemicellulose, and lignin, which are the three components of plant cell walls. These components can be broken down using enzymes and other chemicals, allowing subsequent utilisation by microorganism as a substrate for biofuel production, such as hydrogen and butanol [ 16 ]. Enset fiber is Ethiopian agricultural residue obtained from the Enset plant. A large amount of Enset fiber goes to waste after the traditional Enset food processing in Ethiopia, which feeds more than 20 million people [ 17 ]. Enset contains 67.1% cellulose, 15.6% hemicellulose, and 5.1% lignin, which makes Enset fiber a promising substrate for biofuel production due to its high cellulose and very low lignin content [ 18 ]. A recent study conducted by Seid N. et al. [ 18 ] examined the potential of Enset fiber for butanol production using the separate hydrolysis and fermentation (SHF) method compared with various other Enset plant residues. The results revealed that Enset fiber holds significant promise as a viable resource for butanol production and has a higher hydrolysis conversion efficiency than other Enset plant residues. However, the main challenges for the SHF process were substrate inhibition, low butanol yield and productivity, the complexity of operational procedures requiring multiple distinct steps, and the economic viability of the process [ 19 , 20 ]. Several studies showed that butanol can be produced by combining pretreated biomass, enzymes, and Clostridia species in a single bioreactor. This process is called simultaneous saccharification and fermentation (SSF), enables the simultaneous occurrence of sugar release and fermentation [ 10 , 21 , 22 ]. Compared with the SHF process, SSF has many advantages, including minimising substrate inhibition, reducing operating costs, the overall risk of contamination, and maximising butanol productivity and yield [ 23 ]. However, the SSF process faces several challenges that limit its industrial application. These include the different optimal operating conditions for the saccharification process and acetone-butanol-ethanol (ABE) fermentation, which depend on the type of substrate and microorganism used. Therefore, finding a balance between the two processes is challenging and may affect the efficiency and productivity of butanol production [ 24 , 25 ]. ABE fermentation consists of two distinct phases: acidogenic and solventogenic phases. During the acetogenic phase, the strain undergoing exponential growth produces hydrogen, as well as acids such as acetic acid and butyric acid. Due to the accumulation of these acids, cell growth begins to slow down, and the metabolism of the strain shifts into the solventogenic phase. In this phase, the strain produces solvents such as acetone, butanol, and ethanol as a survival mechanism [ 5 , 26 ]. Clostridium saccharoperbutylacetonicum DSM 14923 is well known for producing butanol and hydrogen as a sugar fermentation product, making it ideal for biofuel production [ 27 ]. The strain is versatile, capable of using a variety of substrates, resistant to solvents, and can thrive in harsh environments [ 28 ]. Studies showed that several factors, including temperature, pH, electron flow and reducing equivalents, play a significant role in C. saccharoperbutylacetonicum fermentation, influencing the amount of hydrogen and butanol [ 27 , 29 – 31 ]. These factors contribute to a competitive relationship between the two products. Therefore, optimal fermentation conditions are crucial for maximising both hydrogen and butanol production. This study aimed to optimize the process parameters for producing hydrogen and butanol from Enset fiber using the SSF process. We examined the impact of controlling pH on the SSF process from Enset fiber. Finally, we attempted to maximize butanol productivity through the PSSF process at a high substrate loading with Enset fiber. It is worth noting that this report is the first study on the simultaneous production of hydrogen and butanol from Enset fiber using the SSF process. Results Effect of SSF process parameters on butanol production This work investigates process parameters to improve butanol production from Enset fiber using the SSF process. Important parameters, such as agitation speed, cellulase loading and substrate loading were selected and optimized. The preliminary results showed that C. saccharoperbutylacetonicum fermentation produced metabolites only between temperatures of 28°C and 37°C; however, no metabolites were detected above 37°C. Unless otherwise stated, all subsequent experiments were carried out at a constant temperature of 30 °C and an initial pH of 6.8. Table 1 shows the effect of agitation speed on butanol production in the SSF process and glucose production in the saccharification process (control experiment) from Enset fiber. After 72 hours of the SSF process, a maximum butanol concentration of 11.48 g/L was produced at an agitation speed of 100 rpm. At this speed, the strain consumed almost all glucose, resulting in a butanol yield of 0.24 g/g and productivity of 0.16 g/L − 1 h − 1 . However, at 130 rpm, butanol production was lower (10.33 g/L), and 9.48 g/L glucose remained unconsumed. The saccharification process showed that the enzyme released more glucose (44.57 g/L) at 130 rpm than at other speeds after 72 hours. Table 1 Effect of agitation speed on butanol production in the SSF process and glucose production in the saccharification process in bottles a, b Agitation speed (rpm) SSF process Saccharification process Final glucose concentration (g/L) Butanol concentration (g/L) Butanol yield (g/g) Butanol productivity (g/L − 1 h − 1 ) Glucose released (g/L) Maximum glucose production rate (g/L − 1 h − 1 ) 0 7.93 ± 1.12 9.64 ± 0.97 0.19 0.13 37.13 ± 0.14 2.93 100 0.24 ± 0.17 11.48 ± 0.22 0.23 0.16 38.94 ± 1.99 3.29 130 9.48 ± 1.36 10.33 ± 0.08 0.21 0.14 44.57 ± 1.01 2.96 a All calculations accounted for 2.5 g Enset fiber in 50 ml medium at 72 hours fermentation period; b Values are means from triplicate bottles. In addition, the effects of cellulase loading on butanol production from Enset fiber were investigated. Table 2 presents the effects of different cellulase loadings on butanol production in the SSF process and glucose production in the saccharification process from Enset fiber. The result showed no significant difference in butanol production in the SSF process between 16 and 24 FPU/g, while 9 FPU/g cellulase loading resulted in a slightly lower butanol production of 10.73 g/L. The saccharification process released a higher glucose concentration of 47.47 g/L at 30 FPU/g cellulase loading, but the SSF process showed the lowest butanol concentration of 9.6 g/L and a yield of 0.19 g/g. Table 2 Effect of cellulase loading on butanol production in the SSF process and glucose production in the saccharification process in bottles a, b Cellulase loading (FPU/g) SSF process Saccharification process Final glucose concentration (g/L) Butanol concentration (g/L) Butanol yield (g/g) Butanol productivity (g/L − 1 h − 1 ) Glucose released (g/L) Maximum glucose production rate (g/L − 1 h − 1 ) 9 3.73 ± 0.34 10.73 ± 0.29 0.21 0.15 29.67 ± 0.33 1.11 16 4.99 ± 0.18 11.23 ± 1.28 0.22 0.16 39.47 ± 0.62 1.85 24 0.24 ± 0.19 11.48 ± 0.22 0.23 0.16 38.94 ± 1.99 3.29 30 6.82 ± 1.42 9.60 ± 0.59 0.19 0.13 47.47 ± 0.75 3.21 a All calculations accounted for 2.5 g Enset fiber in 50 ml medium at 72 hours fermentation period; b Values are means from triplicate bottles. The effects of substrate loading with Enset fiber on butanol production in the SSF process were also examined. Table 3 shows how substrate loading affects butanol production in the SSF process and glucose production in the saccharification process from Enset fiber. The results showed that the same butanol concentration of 11 g/L was observed at both 5 and 7% (w/v) substrate loading of Enset fiber. However, at 7% (w/v) substrate loading, the butanol yield was much lower (0.16 g/g), and 12.41 g/L of glucose remained unfermented compared to other substrate loadings. On the other hand, higher butanol yields were achieved at 2 and 3% (w/v) substrate loadings despite lower butanol concentration and productivity. In the saccharification process, the highest glucose concentration (44.74 g/L) was released at a substrate loading of 7% (w/v), but the glucose production rate was slower than other substrate loadings. Table 3 Effect of substrate loading on biobutanol production in the SSF process and glucose production in the saccharification process in bottles a, b Substrate loading (% (w/v) SSF process Saccharification process Final glucose concentration (g/L) Butanol concentration (g/L) Butanol yield (g/g) Butanol productivity (g/L − 1 h − 1 ) Glucose released (g/L) Maximum glucose production rate (g/L − 1 h − 1 ) 2 0.01 ± 0.00 5.23 ± 0.14 0.26 0.07 16.02 ± 1.05 1.87 3 0.01 ± 0.00 7.96 ± 0.17 0.27 0.11 25.44 ± 1.54 1.83 5 4.99 ± 0.18 11.23 ± 1.28 0.22 0.16 39.47 ± 0.62 1.85 7 12.42 ± 0.43 11.06 ± 0.09 0.16 0.15 44.74 ± 1.78 1.64 a All calculations accounted for different substrate loading of Enset fiber in 50 ml medium at 72 hours fermentation period; b Values are means from triplicate bottles. Overall, at the end of fermentation, the validation experiment confirmed that under the optimum process parameters, including 5% (w/v) substrate loading, 16 FPU/g cellulase loading, and 100 rpm agitation speed, the SSF process from Enset fiber resulted in a maximum butanol concentration of 11.36 g/L, with a corresponding yield of 0.23 g/g and a productivity of 0.16 g/L − 1 h − 1 . In addition, 4.33 g/L acetone and 0.71 g/L ethanol were produced, while 1.77 g/L glucose, 0.41 g/L acetic acid, and 1.08 g/L butyric acid remained unconsumed (Fig. 1 A). The validation experiment in the saccharification process also showed an almost similar glucose concentration (37.4 g/L) after 72 hours. However, the maximum glucose production rate of this experiment was slightly faster than the previous one at 2.34 g/L − 1 h − 1 (Fig. 1 B). The impact of controlled and uncontrolled pH values in the SSF process on butanol production This study aimed to establish a small-scale SSF process for butanol production from Enset fiber in a 2.5 L bioreactor and to investigate the influence of pH control on the process. All fermentations were carried out under the optimal conditions of 5% (w/v) substrate loading, 16 FPU/g cellulase loading, a temperature of 30°C, and an initial pH of 6.8, as determined from the previous bottle experiments, except for the stirrer speed, which was 150 rpm. The SSF process was scaled up in a 2.5 L stirred tank reactor with a pressure relief valve that maintained the hydrogen-rich gas in the medium. The bioreactor pressure reached an absolute pressure of 1.55 bar in about 5 hours; after that, the valve opened and regulated the pressure, while the excess gas was released through BlueVCount and collected in a gas bag. The pressure dropped to 1.52 bar after 24 hours of fermentation and remained constant until 54 hours. Then, the pressure fluctuated between 1.23 and 1.48 bar until the end of fermentation. The influence of pH control on butanol production in the SSF process was examined using the same bioreactor setup, and the results were compared with those of pH-uncontrolled fermentation. Figure 2 A &B shows the metabolites and pH profiles in the SSF process from Enset fiber with the pH-controlled and pH-uncontrolled fermentations. The pH value in both fermentations decreased from 6.8 to 5.0 within 8 hours of inoculation. In the pH-controlled fermentation, the base pump was turned on after 8 hours to keep the pH above 5.0; however, after 19 hours, no addition of NaOH was observed. Within 19 hours, the pH of this fermentation reached a peak pH of 5.96 and then decreased to 5.53 after 68 hours. Afterwards, it fluctuated between 5.51 and 5.60. In contrast, the pH-uncontrolled fermentation showed a slight increase in pH to 5.29 after 19 hours and a gradual decrease to 4.83 after 68 hours, where it remained constant until the end of the fermentation. In addition, a comparison was made between the pH-controlled and pH-uncontrolled SSF processes regarding their effects on liquid metabolites. In both fermentations, as shown in Fig. 2 A&B, the enzyme released a maximum glucose concentration of about 8 g/L after 8 hours, which was similar to the time when the pH was lowered to 5.0. Following that, a higher glucose concentration was observed in the pH-uncontrolled fermentation until the end of the fermentation than in the pH-controlled fermentation. Similarly, other sugars such as xylose, mannose and galactose were detected in higher concentrations in the pH-uncontrolled fermentation than in pH-controlled. In both fermentations, the strain began producing butanol around 19 hours. However, the pH-uncontrolled fermentation had a faster production rate of 0.23 g/L − 1 h − 1 than the pH-controlled one. It also had a higher butanol concentration of 10.54 g/L after 72 hours compared to 8.67 g/L in the pH-controlled fermentation. At the end of fermentation, butanol concentration was increased to 11.35 g/L, yielding 0.23 g/g in the pH-uncontrolled fermentation. However the productivity decreased to 0.095 g/L − 1 h − 1 compared to a fermentation time of 72 hours. Likewise, in the pH-controlled fermentation, the butanol concentration increased to 9.86 g/L, yielding 0.20 g/g and productivity of 0.082 g/L − 1 h − 1 . In addition, both fermentations produced other solvents, such as acetone and ethanol. However, the difference in acetone production between the pH-controlled and pH-uncontrolled fermentations was insignificant at 3.61 g/L and 3.00 g/L, respectively, and ethanol was detected in small amounts in both fermentation, which was lower than 0.5 g/L. The acid compositions, such as acetic acid and butyric acid, also differed in both fermentations. Acetic acid and butyric acid levels were higher in the pH-controlled fermentation than in the pH-uncontrolled fermentation. As with butanol production, hydrogen production showed the opposite trend when comparing pH-controlled and pH-uncontrolled fermentations. A higher amount of hydrogen was observed in the pH-controlled fermentation at 308.02 mmol than in the pH-uncontrolled fermentation at 246.47 mmol. PSSF process at high substrate loading with Enset fiber The purpose of this study was to maximize butanol productivity using the PSSF process at high substrate loading with Enset fiber. This experiment was done in a bioreactor with pH-controlled fermentation similar to the experiment described above. Figure 3 shows the metabolites produced in the PSSF process at a substrate loading of 7% (w/v) using Enset fiber as a carbon source. The prehydrolysis step lasted for 2 hours, followed by a 1 hour transition period to adjust the temperature and initial pH value, and the enzyme hydrolysed the Enset fiber into 12.53 g/L of glucose and 2.66 g/L of other sugars, such as xylose, mannose, and galactose. In addition, we observed that the Enset fiber was partially degraded, and the medium was homogenized at the end of this process. After inoculation with C. saccharoperbutylacetonicum , the PSSF process achieved a faster butanol production rate of 0.21 g/L − 1 h − 1 than the pH-controlled SSF process but a slightly similar rate to the pH-uncontrolled SSF process. However, the butanol concentration after 72 hours was higher at 11.04 g/L in the PSSF process than in the SSF process in both pH-controlled and uncontrolled fermentation. At the end of fermentation, the butanol concentration and productivity in the PSSF process were increased to 12.84 g/L and 0.104 g/L − 1 h − 1 , respectively. Although the butanol concentration and productivity were higher in the PSSF process than in the SSF process, the butanol yield of 0.18 (g/g) was significantly lower than in the SSF process. Furthermore, the PSSF process produced a higher concentration of acetone (5.64 g/L) and a higher amount of hydrogen (378.69 mmol) than the SSF process. However, at the end of the fermentation, we found that 4.51 g/L glucose, 2.58 g/L acetic acid and 1.37 g/L butyric acid remained unused. Effect of SSF process parameter on hydrogen production The objective of this study was to examine the influence of SSF process parameters on hydrogen production from Enset fiber. The key process parameters affecting hydrogen production were selected and optimized, including temperature, initial pH, and hydrogen partial pressure. At the same time, other factors were kept constants for all experiments based on results from previous bottle experiments. Figure 4 shows the effect of temperature on hydrogen production from Enset fiber in the SSF process. After 44 hours of incubation, at both 35°C and 37°C, hydrogen production reached a maximum of 15.75 mmol and then slightly declined at both temperatures. At the end of fermentation, hydrogen production was lower at 30°C (12.03 mmol) than at other temperatures. However, the butanol yield and productivity were much higher at 30°C, with 0.23 g/g and 0.17 g/L − 1 h − 1 , respectively. In contrast, at 37°C, the butanol yield was lower at 0.17 g/g, while the hydrogen yield was higher at 133.50 mL/g-Enset fiber compared to other temperatures (see Additional File 1: Table S1 ). In addition, the effects of initial pH value on hydrogen production from Enset fiber were investigated. Figure 5 A shows the pH changes during the SSF process with Enset fiber at different initial pH values. The initial pH of 6.0, 7.0, and 8.0 showed similar pH trends, decreasing at 8 hours, and increasing to about 6.23 after 24 hours. At the end of fermentation, they had nearly the same pH of 6.01, except for the initial pH of 6.0, which was slightly lower at 5.76. In contrast, the initial pH of 5.0 and 9.0 showed similar pH trends, dropping below 5.0 after 8 hours and remaining low until the end of fermentation. Similarly, the initial pH values affected the amount of hydrogen produced by the SSF process, as shown in Fig. 5 B. After 44 hours of fermentation, the highest hydrogen production was observed at 16.32 mmol at initial pH values of 7.0 and 8.0. However, the initial pH of 8.0 had a slightly faster rate of 0.53 mmol/h than the others. At the end of fermentation, a similar hydrogen yield (139 mL/g-Enset fiber) was observed at all initial pH values of 6.0, 7.0 and 8.0. The lowest amounts of hydrogen were found at both initial pH of 5.0 and 9.0, with 1.74 mmol and 5.33 mmol, respectively. Similar to the amount of hydrogen, there was no significant difference in butanol yield (0.18 g/g-Enset fiber) between the initial pH of 6.0, 7.0, and 8.0 at the end of fermentation. In contrast, at the end of fermentation, the lowest butanol yield was observed at initial pH of 5.0 and 9.0; however, a significantly higher glucose concentration of 38 g/L and 30 g/L were observed at an initial pH of 5.0 and 9.0, respectively (see Additional File 1: Table S2). The effects of hydrogen partial pressure on hydrogen production in the SSF process from Enset fiber were also investigated. Figure 6 A & B shows the hydrogen partial pressure and hydrogen production in the SSF process from Enset fiber under different gas release strategies. In both experiments, where gas was released into 1-L and 2-L bottles, the hydrogen partial pressure was lower compared to the control experiment without gas released. However, the gas released into the 1-L bottle resulted in a slightly lower hydrogen partial pressure than that released into the 2-L bottle (Fig. 6 A). Similarly, at the end of fermentation, there was a slight difference in hydrogen production of 17.24 mmol and 18.86 mmol between the fermentation, where the gas was released into 1-L and 2-L bottles, respectively. However, a smaller amount of hydrogen (15.52 mmol) was observed in the control experiment (Fig. 6 B). Furthermore, it was found that there was little difference in butanol production between all experiments (see Additional File 1: Table S3). Overall, at the optimum process parameters, including a temperature of 37°C, an initial pH of 8.0, and the lowest hydrogen partial pressure, a maximum hydrogen yield of 168.99 mL/g-Enset fiber was achieved in the SSF process from Enset fiber. Hydrogen production in the SSF process at atmospheric pressure This study aimed to develop a small-scale SSF process for hydrogen production from Enset fiber in a 2.5 L bioreactor. This was achieved by using optimal process parameters, including temperature (37°C), initial pH (8.0), and atmospheric pressure as determined in bottle experiments. The SSF process was scaled up using a 2.5 L stirred tank reactor operated at atmospheric pressure and without a pH control method. Initially, the outlet tube was closed for 4 hours to maintain an anaerobic environment in the bioreactor. During this period, the strain produced its gas, and the pressure was increased to an absolute pressure of 1.3 bar. Following this, the bioreactor outlet was connected to the BlueVCount, which released gases into the gas bag, thereby reducing the pressure to the atmospheric level of 1 bar. Figure 7 shows the metabolites and pH profiles in the SSF process from Enset fiber at atmospheric pressure. The result of this study was compared to a similar SSF process, except that the SSF was performed at 30°C, with an initial pH of 6.8, without pH control and under overpressure (see Fig. 2 B). Unless otherwise stated, this was the reference process for the comparison. After inoculation, hydrogen gas production was started in 4 hours in both fermentations, and the amount was similar at 3.64 mmol. However, after that, the hydrogen production rate in the SSF process at 37°C was faster, with a maximum production rate of 8.38 mmol/h, than in the SSF process at 30°C with 6.56 mmol/h. Further, after 72 hours, a higher amount of hydrogen was found at 428.53 mmol in the SSF process at 37°C than in the SSF process at 30°C with 239.14 mmol. However, hydrogen production did not change significantly when the fermentation time was extended to 120 hours. Both fermentations showed a slight increase in hydrogen production with 442.57 mmol and 246.47 mmol for the SSF processes at 37°C and 30°C, respectively, resulting in relatively low hydrogen productivity 1.65 mLg − 1 h − 1 and 0.92 mLg − 1 h − 1 , respectively. In addition, a higher hydrogen yield of 198.27 mL/g-Enset fiber was achieved at the end of fermentation in the SSF process at 37°C compared to the yield of 110.42 mL/g-Enset fiber at 30°C. Furthermore, the pH profiles and liquid metabolites of the SSF processes showed different results at different temperatures and initial pH values ​​(Fig. 7 and Fig. 2 B). The pH of both fermentations decreased to 5.0 in the first 8 hours of fermentation and then increased slightly above 5.0 in the next 19 hours. After that, the pH value in the SSF process at 37°C remained above 5.0 and fluctuated between 5.5 and 5.76, while the pH value in the SSF process at 30°C dropped again for about 44 hours and varied between 4.82 and 4.96. At the end of fermentation, the SSF process at 37°C had a higher pH of 5.6 than the SSF process at 30°C, which had a lower pH of 4.89. Similarly, the glucose concentration released by the enzyme after 8 hours of fermentation was the same in both SSF processes (8.2 g/L). However, after 19 hours, the SSF process at 37°C reached a maximum glucose concentration of 15.54 g/L, while the SSF process at 30°C did not increase further. At the end of fermentation, the strain consumed almost all of the glucose in the SSF process at 37°C, while 1.77 g/L glucose remained unused at 30°C. Moreover, both SSF processes had a maximum of 4.25 g/L of other sugars, such as xylose, mannose and galactose, which were also consumed by the end of fermentation. At the end of fermentation, the liquid metabolites such as butanol and acetone also differed depending on the temperature and initial pH value. The SSF process at 30°C produced more butanol (11.35 g/L) than the SSF process at 37°C (8.92 g/L). However, the opposite was observed for acetone, which was higher in the SSF process at 37°C (6.04 g/L) than the SSF process at 30°C (3.00 g/L). In addition, the SSF process at 37°C contained more acetic acid and less butyric acid than the SSF process at 30°C. Discussions Maximizing butanol production from Enset fiber using the SSF process The SSF process is a widely recognized method for enhancing product yield, minimizing equipment and energy costs, and offering the advantage of faster processing time compared to conventional methods [ 20 ]. However, one of the primary challenges in maximizing butanol production in the SSF process using C. saccharoperbutylacetonicum is the large temperature difference required for ABE fermentation and saccharification, typically between 30°C and 50°C [ 32 ]. The cellulase enzymes require higher temperatures of 40−50 °C to efficiently degrade lignocellulosic biomass [ 33 ], while the strain prefers lower temperatures of 30 °C for optimal butanol production [ 28 ]. This makes it challenging to choose an optimal temperature for the SSF process to maximize butanol production. In this study, we observed that C. saccharoperbutylacetonicum was able to grow and produce butanol from pretreated Enset fiber using the SSF process within a temperature range of 28 to 37°C, and the maximum butanol production was observed at 30°C. This could be due to the structural properties of Enset fiber, which has a high cellulose content (67.1%) and a low lignin content (5.1%), making it a promising feedstock for the SSF process [ 18 ]. Lignin is a natural polymer that acts as a barrier to prevent the enzymatic hydrolysis of cellulose and hemicellulose. It can be removed by various pretreatment methods such as dilute acid pretreatment, dilute alkali pretreatment, steam explosion, and liquid hot water; however, depending on the biomass and the specific type of pretreatment, the pretreatment methods can lead to lignin degradation. The resulting lignin degradation products, such as aromatic alcohols, phenolic compounds, furan derivatives, and organic acids, act as soluble inhibitors that can affect enzyme activity and strain growth [ 34 , 35 ]. Previous studies have shown that the SSF process is affected by inhibitory compounds resulting from the degradation of lignin in alkali-pretreated switchgrass [ 21 ] and acid-pretreated corn stover [ 36 ], which inhibits the clostridial strain. However, alkali-pretreated Enset fiber did not generate any inhibitory compounds, according to a study [ 18 ]. In this study, the effects of other process parameters, such as agitation speed, cellulase loading and substrate loading on the SSF process were further optimized while maintaining a constant temperature (30 °C) and initial pH (6.8), which were found to be the optimal parameters for ABE fermentation using C. saccharoperbutylacetonicum [ 28 ]. Mixing the SSF medium improves the mass transfer between biomass, enzyme, microorganism, and nutrients in the SSF process. However, agitation speed affects the ABE fermentation and saccharification process differently. The bottle experiments in the current study revealed that the SSF process produced the highest butanol at an agitation speed of 100 rpm. However, higher rotation reduced the butanol concentration by 10% in the SSF process. On the other hand, the saccharification process benefited from high rotation; more glucose was produced at higher agitation speeds, possibly due to the enhanced interaction between the enzyme and the Enset fiber. The results of this study were similar to those of Al-Shorgani N. et al. [ 37 ], who investigated the effects of agitation on butanol production in ABE fermentation using the same strain as in this study. They found that 100 rpm was the optimal agitation speed for maximum butanol production and at higher speeds, butanol production decreased. Maintaining an optimal cellulase loading during the SSF process is essential for maximizing the butanol production and ensuring the overall cost-effectiveness of the process. In this study, it was observed that there was only a 4% difference in butanol yield between the cellulase loadings of 16 and 24 FPU/g, despite a 33% increase in the cellulase loading. For this reason, a cellulase loading of 16 FPU/g was selected as an optimal point for the SSF process from Enset fiber. Study shows that enzymes contribute to 40% of the total cost of biofuel production from lignocellulosic biomass [ 38 ] and reducing cellulase loading is economically beneficial when the yield loss is less than 6−7% [ 32 ]. Further increasing cellulase loading to 30 FPU/g in the SSF process resulted in a 16% decrease in butanol production. This could be due to the inhibitory effect of excessive cellulase on the strain [ 39 ]. On the other hand, in the saccharification process, higher cellulase loading was beneficial for releasing more glucose from Enset fiber. Compared to the previous study, this result was similar to the research conducted by Md R. et al. [ 25 ], which investigated the effect of cellulase loading in the SSF process using the C. acetobutylicum strain and reported a reduction in butanol production from pretreated oil palm empty fruit bunch as the cellulase loading increased from 20 to 30 FPU/g. One of the challenges in butanol production from lignocellulosic biomass using the SSF process is balancing the required fermentable sugars and substrate loading, which can lead to mass transfer limitations. This study revealed that even at low substrate loading of 2% and 3% (w/v) Enset fiber, the SSF process achieved a maximum butanol yield of 0.26 (g/g) and 0.27 (g/g), respectively. In addition, 80−84% of Enset fiber was converted into glucose during the saccharification process. These proved that the SSF and saccharification processes using Enset fiber were efficient, despite the reduced enzyme activity due to the low process temperature. However, the butanol productivity was significantly lower at low substrate loading than in fermentations with high substrate loading. Butanol productivity was maximized by 45% when the substrate loading increased from 3–5% (w/v) of Enset fiber. However, further increases in substrate loading to 7% (w/v) showed no significant difference; instead, the butanol yield decreased by 27%, and 12.42 g/L glucose remained unconsumed. This might be due to poor mixing caused by high solid and low water content, leading to a mass transfer problem in the SSF process [ 16 ]. A similar challenge was identified in a study by Guan W. et al. [ 21 ], which focused on butanol production from kraft paper mill sludge using the SSF process; they found that the slurry became too viscous, limiting mass transfer at substrate loadings of 6.3% and higher. Several studies suggest that the PSSF process can solve the mixing problem during the initial stages of fermentation in the SSF process [ 14 , 16 , 40 ]. Table 4 compares the butanol production from different biomass and Clostridial strains using the SSF process in bottle experiments. Compared with other studies, Enset fiber had the highest butanol concentration (11.36 g/L), yield (0.23 g/g), and productivity (0.16 g/L − 1 h − 1 ) among rice straw, oil palm empty fruit bunch, and avicel at optimal process parameters. However, wheat straw had a higher butanol concentration (12.64 g/L) than Enset fiber, as reported by Qi G. et al. [ 22 ], while Enset fiber had a higher butanol yield and productivity. To our knowledge, no studies have yet reported butanol production in the SSF process using C. saccharoperbutylacetonicum . Compared to the SHF method described in our previous study [ 18 ], which used the same strain and biomass with separate fermentation and saccharification steps, the butanol concentration was 9.9 g/L, which was lower than the current study. Furthermore, the SSF process enhanced butanol yield by 20% and productivity by 21% compared to the SHF process. This could be because the SSF process can avoid substrate inhibition by releasing the sugar gradually rather than all at once, as in the SHF process. Table 4 Comparison of butanol production from different biomass using the SSF process in bottle experiments Biomass Pretreatment method Strain Process parameters Butanol titer (g/L) Butanol yield (g/g biomass) Butanol productivity (g/L − 1 h − 1 ) Ref. Enset fiber 2% (w/v) NaOH C. saccharo-perbutyl-acetonicum DSM 14923 5% (w/v) substrate loading, Cellic CTec2 (16 FPU/g-substrate), initial pH = 6.8, 30°C, 100 rpm, 72 h 11.36 0.23 0.16 This study a Rice straw Microwave-assisted hydrothermo- lysis C. beijerinckii DSM 6422 9% (w/v) substrate loading, Cellic CTec2 (12 FPU/g-glucan, initial pH = 6.4, 37°C, 150 rpm, 48 h 5.5 0.06 0.11 [ 10 ] Oil palm empty fruit bunch 2% (w/v) NaOH C. aceto-butylicum ATCC 824 5% (w/v) substrate loading, acremonium cellulase (15 FPU/g-substrate), initial pH = 5.5, 35°C, 150 rpm, 120 h 3.97 0.08 0.03 [ 25 ] Avicel None C. aceto-butylicum ATCC-824 5.8% (w/v) substrate loading, Cellic CTec2 (20 FPU/g-glucan), initial pH = 6.7, 36°C, 150 rpm, 120 h 9.5 0.16 0.08 [ 21 ] Wheat straw 10% (w/v) Ammonium sulfite C. aceto-butylicum ATCC 824 9% (w/v) substrate loading, Cellulase (5 FPU/g-substrate) & xylanase (10 IU/g-substrate), initial pH = 5.0, 37°C, 150 rpm, 144 h 12.64 0.14 0.088 [ 22 ] a All calculations accounted for different substrate loading of Enset fiber in 50 ml medium at 72 hours fermentation period. Scaling up butanol production from Enset fiber To develop butanol production from Enset fiber at an industrial scale, a scale-up process that matches the bottle-scale experiment performance is essential. This study successfully established a small-scale SSF process from pretreated Enset fiber using a 2.5 L stirred tank bioreactor by maintaining a slight overpressure inside the bioreactor. In this study, the bioreactor fermentation using the pH-uncontrolled method showed a comparable butanol production result as the bottle experiment under similar optimal process parameters, except for the stirrer speed in the bioreactor. In both experiments, there was only a small difference in butanol concentration of 7.8% after 72 hours. However, after 120 hours, a similar butanol concentration of 11.35 g/L was observed with a yield of 0.23 g/g. These comparable results were achieved due to the utilization of a partially similar configuration when setting up the bioreactor system, which included maintaining the hydrogen-rich gas in the bioreactor. Several studies showed that maintaining hydrogen gas during fermentation is beneficial for maximizing biobutanol production in clostridial strains. Brosseau J. et al. [ 41 ] observed that C. saccharoperbutylacetonicum requires hydrogen gas to produce butanol, and when the hydrogen gas reaches a certain level, the bacteria reduce their growth rate and start producing butanol. Similarly, in this study, the highest hydrogen production rate was observed during the initial cell growth phase of C. saccharoperbutylacetonicum , which remained faster until 24 hours. Afterwards, the rate of hydrogen gas production slowed down, and the strain started producing butanol and entered the solventogenic phase. Furthermore, a separate study conducted by Stein U. et al. [ 42 ], focusing on the effect of pressure on ABE fermentation using C. acetobutylicum , concluded that butanol production could be enhanced through overpressure fermentation compared to lower pressure. In addition, this study investigated the influence of controlled and uncontrolled pH values on butanol production from Enset fiber in the SSF process. The results showed that controlling the pH above 5.0 was not essential to achieve optimal butanol production in the SSF process from Enset fiber. In this study, butanol concentration decreased by 18% in pH-controlled fermentation compared to pH-uncontrolled fermentation, this could be due to the influence of pH value on the efficiency of the cellulase enzyme. Previous research has shown that the optimal pH range for commercial cellulase is typically between 4.8 and 5.5 [ 33 , 43 ]. However, in this study, during the SSF process in the pH-controlled fermentation, there were fluctuations in the pH values after 8 hours, which ranged between 5.5 to 5.96. The primary cause of these fluctuations was the substantial addition of NaOH. When the base pump started adding NaOH to the medium, challenges arose due to the high solid content of the medium and inadequate mixing, impacting the accurate operation of the bioreactor's pH sensor. As a result, the base pump introduced an excessive amount of NaOH, resulting in an elevated pH level. The observed deviation from the optimal pH range reduced enzyme activity, ultimately limiting the release of glucose during pH-controlled fermentation compared to pH-uncontrolled fermentation. In contrast, the pH values ​​in the pH-uncontrolled fermentation after 8 hours were between 4.8 and 5.29, which was in the optimal pH range for cellulase enzyme activity. This pH range resulted from the two-phase fermentation of the Clostridia strain. Initially, the strain produced acids such as acetic acid and butyric acid, leading to a pH drop to 4.8 during the first phase. Subsequently, the strain transitioned to the solventogenic phase, producing butanol, acetone, and a small amount of ethanol, causing the pH to rose to 5.29 [ 44 ]. Numerous studies have used pH control methods for ABE fermentation to prevent the occurrence of an acid crash, which is often triggered by the presence of inhibitory compounds in the fermentation medium resulting from pretreatment of the lignocellulosic biomass [ 45 ]; however, in this study, there were no inhibitory compounds that could have led to an acid crash. The pH is crucial for SSF process, keeping the pH low after the start of the solventogenic phase might increase butanol production rate and yield but also comes with the costs for pH control. In this study, the major challenges observed in the bioreactor SSF process were inadequate mixing and reduced butanol productivity. To address these issues, an experiment was conducted using the PSSF method with a 7% (w/v) substrate loading of Enset fiber. The PSSF method alleviated the mass transfer problem by prehydrolysing for 2–3 hours before inoculation. This was because the SSF process had poor mixing from the start of fermentation until the Enset fiber was saccharified by the enzyme [ 46 ]. Furthermore, the butanol concentration and productivity were increased by 30% and 27%, respectively, compared to the SSF process with pH control method. This might be due to the initial glucose concentration at the end of the prehydrolysis step, which could facilitate the initial growth of the C. saccharoperbutylacetonicum strain [ 16 ], and the high substrate loading might have contributed to maximizing the butanol concentration. Studies showed that high substrate loading is crucial for large-scale operations to minimize overall production costs and maximize productivity [ 47 ]; by increasing the substrate loading from 5% (w/v) to 8% (w/v) of biomass, the production cost can be reduced by 19%, as this can lower the volume of downstream processing equipment and the energy consumption that is related to the dilution level [ 48 ]. Compared to previous PSSF studies with different biomass and Clostridial strains, the highest butanol concentration and yield were observed in Enset fiber than in sugarcane straw [ 49 ], corn stover [ 14 ] and napier grass [ 40 ]. However, the butanol productivity of this study was lower than that of Dong J. et al. [ 16 ], who performed the PSSF process using C. saccharobutylicum from corn stover at an SSF temperature of 37 °C. This could be due to the lower working temperature of the SSF process in this study, which slowed down enzyme activity. To enhance the butanol productivity of this study, utilizing a strain with high-temperature tolerance [ 50 ] or cold-active cellulase [ 51 ] may enable an effective PSSF process from Enset fiber. Hydrogen production from Enset fiber using SSF process Producing hydrogen and butanol from affordable materials such as Enset fiber is essential to ensure the sustainability of the biofuel industry. The choice between butanol or hydrogen as a biofuel depends on application, cost considerations, and environmental sustainability [ 52 , 53 ]. In this study, butanol and hydrogen were simultaneously produced by the SSF process from Enset fiber using C. saccharoperbutylacetonicum . However, the fermentation conditions must be adjusted separately to achieve maximum yields for both products. This is because fermentation conditions affect cell metabolism and can potentially shift the metabolic pathway in favor of hydrogen or butanol production [ 30 ]. In addition, it has been shown that the amount of hydrogen and butanol produced in the Clostridia strain is influenced by factors such as electron flow and the balance of reducing equivalents in the metabolic pathway, leading to a competitive relationship between the two products [ 29 ]. Temperature is a key factor in the SSF process, as it influences both the growth of the cells and the enzyme activity in their metabolism. The results indicated that higher temperatures (35 and 37°C) improved hydrogen production compared to lower temperature (30°C); however, butanol production decreased with increasing temperature in the SSF process using C. saccharoperbutylacetonicum . A possible explanation could be that the cells grew faster at the higher temperature in the initial growth phase [ 5 ], which coincided with the highest hydrogen production rate in the acidogenic phase observed in this study. The result of this study was similar to those of Alalayah W. et al. [ 27 ] and Yadav S. et al. [ 5 ], who investigated the influence of temperature on hydrogen production in ABE fermentation using C. saccharoperbutylacetonicum and found the highest hydrogen values at 37°C in both studies. Another factor that affected hydrogen production in the SSF process was the initial pH value of the medium, which was influenced by the buffers in the medium and the metabolites in the cell growth phase. As C. saccharoperbutylacetonicum initiated growth, it began by producing acetic acid, butyric acid, and hydrogen. The buildup of organic acids resulted in a decrease in the pH of the medium and created stressful conditions for cell growth. Subsequently, the culture converted these acids into solvents such as butanol, acetone, and ethanol as it entered the stationary phase, increasing in pH value. Under optimal conditions favorable for cell growth, these phase changes represent the natural pathway for ABE fermentation [ 44 ]. However, in the presence of inhibitors or unfavorable conditions, the cells struggle to survive, leading to increased acid production and eventual cell death [ 54 ]. In this study, the pH profile of the SSF process at initial pH of 6.0, 7.0, and 8.0 followed a similar phase change and showed no significant difference in hydrogen and butanol production. However, at initial pH of 5.0 and 9.0, the strain could not enter the solventogenic phase, indicating that lower and higher initial pH values inhibited the cell. A study using the same medium and strain for ABE fermentation as in this study found that hydrogen production was maximized in the initial pH range of 7.0-8.5 [ 55 ]. In contrast, other studies suggested an optimal initial pH of 6.5, and beyond this point, hydrogen production decreased, although they used a different medium [ 26 , 27 ]. In addition, this study examined the effect of hydrogen partial pressure on hydrogen production and found that lower hydrogen partial pressure was beneficial for maximizing hydrogen production. The results of bottle experiments showed that hydrogen production was increased by 21.5% at lower hydrogen partial pressure compared to higher values. To validate the bottle experiment results and maximize hydrogen production, a scale up SSF process without a pH control system was implemented at the optimal process parameters with a temperature of 37°C, an initial pH of 8.0 and under atmospheric pressure. The results confirmed that hydrogen production increased by 79.7% compared to the SSF process (pH-uncontrolled) at 30°C, with an initial pH of 6.8 and under overpressure. However, butanol production was reduced by 21.4% at optimum process conditions for hydrogen production. Several studies have shown that hydrogen partial pressure is an essential factor in maximizing hydrogen production; it could also potentially inhibit cell growth and limit the thermodynamics of the process, depending on the microorganisms involved [ 31 , 42 ]. Electrons play a crucial role in the Clostridial metabolic pathways, and the strain can release excess electrons as hydrogen gas to balance the nicotinamide adenine dinucleotide (NADH/NAD + ) ratio. However, when the hydrogen gas levels are high, it becomes more difficult for the bacteria to continue producing more hydrogen gas; this means the bacteria have too many electrons and insufficient energy, which can stop their growth and activity. As a consequence of this electron backlog, the strain utilises alternative pathways in which electrons are transferred from NADH to ferredoxin, which can serve as an electron carrier to reduce NADP + to NADPH, which facilitates butanol production [ 29 , 56 , 57 ]. For this reason, removing hydrogen gas from the medium might help the strain to produce more hydrogen. This study obtained a similar hydrogen yield of 170 mL/gCOD in the bottle experiment as the previous study by Singh V. et al. [ 7 ], which used starchy wastewater and acid-treated water hyacinth as substrates with a similar strain. However, a higher hydrogen yield of 198.27 mL/g-Enset fiber from alkali-pretreated Enset fiber was achieved in the bioreactor experiment at optimal process parameters. This was also higher than the hydrogen yield of 68 ml/g-biomass from steam-exploded straw using the SSF process with C. butyricum AS1.209 strain, reported by Li D. et al. [ 58 ]. Conclusions This study showed the potential of Enset fiber for hydrogen and butanol production in the SSF process. By optimizing the SSF process with C. saccharoperbutylacetonicum , we achieved high yields and concentrations of both biofuels under different conditions. Furthermore, a scalable process for both products was developed to achieve similar results to bottle-scale experiments, indicating potential applicability on an industrial scale. This is the first study to use this strain and substrate combination for the SSF process, and it contributes to the development of sustainable energy sources. However, more research on the 5L, 10L bioreactors and pilot scale processes, along with a feasibility study, is required to make industrial-scale butanol production feasible using the SSF process from Enset fiber. Materials and methods Enset fiber preparation Enset fiber was obtained from a privately owned Enset plantation in Wolkite, Ethiopia. It was ground to a particle size of 2 mm and then pretreated with 2% (w/v) NaOH according to the methods previously described by Seid N. et al. [ 18 ]. Bacterial strain and culture medium Clostridium saccharoperbutylacetonicum DSM 14923 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). To prepare the inoculum, glycerol stock of the strain (1 mL) was added to 50 mL of anaerobic TGY medium, and the culture was incubated at 30 °C until the optical density (OD 600 ) reached 1.0−1.2. TGY medium contained 30 g/L tryptone, 20 g/L glucose, 10 g/L yeast extract, and 0.4 g/L cysteine-HCl·H 2 O. The main fermentation medium comprises 1% (v/v) P2 stock solutions, which was prepared separately, buffer stock solution (50 g/L KH 2 PO 4 , 50 g/L K 2 HPO 4 , and 220 g/L CH 3 COONH 4 ), mineral stock solution (20 g/L MgSO 4 ·7H 2 O, 1 g/L MnSO 4 ·H 2 O, 1 g/L FeSO 4 ·7H 2 O, and 1 g/L NaCl), and vitamin stock solution (0.1 g/L para-aminobenzoic acid, 0.1 g/L thiamin, and 0.001 g/L biotin) [ 28 ]. All stock solutions were filter-sterilized and anaerobized using N 2 gas. Preparation of SSF medium from pretreated Enset fiber for the bottle experiment To prepare the SSF medium, different amounts of pretreated Enset fiber were added as the sole carbon source into 250 mL Duran pressure plus bottles, each containing a total volume of 50 mL of medium. Subsequently, 1 g/L of yeast extract, 1 mL of 1 g/L resazurin and 0.4 g/L of cysteine-HCl·H 2 O were added, and the initial pH was adjusted to different pH values. The bottles were sealed using a rubber stopper and an aluminum cap, anaerobized with N 2 gas, and autoclaved. Following autoclaving, 1% (v/v) of each P2 stock solutions and 5 mL cellulase-water mixed solution (Cellic CTec2) (Sigma-Aldrich Chemie GmbH, Hamburg, Germany) at varying cellulase loading were added. The bottles were inoculated with 5% (v/v) active C. saccharoperbutylacetonicum culture and incubated in Infors Thermotron incubators (Infors AG, Bottmingen, Switzerland) for 72 hours at different temperatures and agitation speeds. In each experiment, the pressure inside the bottles was measured with a GMH 3100 Series manometer (Greisinger, Mainz, Germany), and samples of 3 mL of gas and 1.5 mL of liquid were collected using needles and syringes. In addition, the saccharification experiment was conducted as a control, and the medium was prepared following the procedure outlined by Seid N. et al. [ 18 ]. Influence of SSF process parameters on butanol and hydrogen production in the bottle experiment Preliminary experiments were carried out to find out which temperature ranges were suitable for the cultivation of C. saccharoperbutylacetonicum in the SSF process using Enset fiber at various temperatures, including 28, 30, 33, 35, 37, 40, 45, and 50°C. It was performed with a substrate loading of 5% (w/v), which was 2.5 g pretreated Enset fiber (50 g/L), a cellulase loading of 24 FPU/g, an initial pH of 6.8 and an agitation speed of 130 rpm. The influences of agitation speed, cellulase loading and substrate loading on butanol production in the SSF process were investigated at a constant temperature of 30 °C and an initial pH of 6.8. Initially, the effects of agitation were tested at different speeds (0, 100, 130 rpm) while maintaining a constant substrate loading of 5% (w/v) and a cellulase loading of 24 FPU/g. After determining an optimal agitation speed (100 rpm), experiments were conducted to examine the effect of different cellulase loadings (9, 16, 24, 30 FPU/g) while maintaining a constant substrate loading of 5% (w/v). Subsequently, the effects of substrate loading on butanol production were tested at different substrate loadings of Enset fiber (2, 3, 5, 7% (w/v)) while maintaining constant agitation (100 rpm) and cellulase loading (16 FPU/g). Finally, a validation experiment was performed under optimal conditions with an agitation speed of 100 rpm, a cellulase loading of 16 FPU/g and a substrate loading of 5% (w/v). As a control experiment, optimization of the saccharification process was performed in a similar manner to determine how changes in process parameters affect the efficiency of enzymatic hydrolysis. In addition, the influence of temperature, initial pH, and hydrogen partial pressure on hydrogen production in the SSF process were examined under optimal process parameters, which included an agitation speed of 100 rpm, a cellulase loading of 16 FPU/g, and a substrate loading of 5% (w/v) Enset fiber. First, to study the effects of temperature on hydrogen production, bottles were placed in incubators set at various temperatures (30, 35, 37 °C) while maintaining a constant initial pH of 6.8 in closed bottles. Following the selection of the best temperature (37°C) for hydrogen production, the effects of initial pH were examined by adjusting the SSF medium to the initial pH (5.0, 6.0, 7.0, 8.0, and 9.0) while maintaining a constant temperature. Afterwards, the effects of hydrogen partial pressure were determined at a constant temperature of 37 °C and an initial pH of 8.0. The following experimental setup was established to investigate the effect of hydrogen partial pressure on hydrogen production, the 250 mL bottles containing the fermentation medium were connected to empty bottles (anaerobized 1-L and 2-L bottles) by tube. This setup allowed the hydrogen-rich gas produced by fermentation to flow from the smaller bottles to the larger ones. All experiments were performed in triplicate and lasted 72 hours. Bioreactor setup for the SSF process from pretreated Enset fiber A 2.5 L bioreactor (Minifors, Infors HT, Bottmingen, Switzerland) with two six-bladed Rushton turbine impellers was used for the SSF process. The impellers had a diameter of 4.6 cm and a width of 1.1 cm and positioned 6.0 cm apart on the stirrer. For the SSF medium preparation in the bioreactor, 50 g of pretreated Enset fiber with a substrate loading of 5% (w/v) was added to the bioreactor as the sole carbon source, and the medium had a total volume of 1 liter. The bioreactor was filled with liquids containing 1 g/L yeast extract and 1 mL of 1 g/L resazurin and autoclaved. After autoclaving, all tubes and the bioreactor were sealed airtight and anaerobized overnight using filter-sterilized N 2 gas while being mixed at a stirring speed of 500 rpm. After anaerobization, the bioreactor was supplemented with 1% (v/v) of each P2 stock solution and 0.4 g/L of cysteine-HCl·H 2 O using needles and syringes. For the main fermentation, the temperature was adjusted to the desired level, and the stirring speed was reduced to 150 rpm. Subsequently, the N 2 gas flow was stopped, 10 mL of a cellulase-water mixed solution (Cellic CTec2) with a cellulase loading of 16 FPU/g was added to the bioreactor and inoculated with 5% (v/v) active C. saccharoperbutylacetonicum culture. 5 mL gas and 2 mL liquid samples were collected and analyzed. The bioreactor setup varied depending on the type of fermentation. For butanol production, fermentations were performed at a temperature of 30 °C, with an initial pH of 6.8 and under overpressure, with the gases partially kept in the bioreactor. The bioreactor outlet tube was connected to the pressure relief valve (V07, IMI Norgren, Lichfield, United Kingdom) and set to an absolute pressure of 1.55 bar. After the pressure reached 1.55 bar, the valve was opened, and the gases produced were passed through BlueVCount (BlueSens gas sensor GmbH, Herten, Germany) to measure the volume of gases accumulated in the gas bags. In addition, for the pH-controlled experiment, the pH of the medium was controlled at pH ≥ 5.0 by using the bioreactor's base pump containing 4 M NaOH. For hydrogen production, fermentation was performed at 37 °C, with an initial pH of 8.0 and under atmospheric pressure, releasing the gas from the bioreactor. In this fermentation, the bioreactor outlet tube was directly connected to the BlueVCount and accumulated in the gas bags. All experiments were incubated for 120 hours and performed in duplicate. PSSF process from pretreated Enset fiber in the bioreactor The PSSF medium was prepared similarly to the SSF medium except for the substrate loading, which was 7% (w/v) of pretreated Enset fiber (70 g/L), and operating conditions. Before inoculation with an active preculture of C. saccharoperbutylacetonicum , the media was hydrolyzed for 2 hours. The prehydrolysis experiment was started by adjusting the pH to 5.0 and the temperature to 40 °C. Then, the cellulase enzyme was added at 16 FPU/g cellulase loading and stirred at 500 rpm. Afterwards, the temperature and stirrer speed were minimized to 30 °C and 150 rpm, respectively, and the initial pH was adjusted to 6.8, then inoculated with 5% (v/v) active preculture of C. saccharoperbutylacetonicum . During the fermentation, the pH was controlled with 4M NaOH to pH ≥ 5.0 and the pressure to 1.5 bar. The experiment was incubated for 120 hours and performed in duplicate. Analytical methods The liquid samples were analysed following the method described by Seid N. et al. [ 18 ]. The gas pressure developed during fermentation was measured using a manometer, and the gas composition was determined using the Micro GC Fusion® gas analyzer (Inficon, Bad Ragaz, Switzerland) equipped with PLOT and WCOT columns and utilizing Argon and Helium as carrier gases [ 59 ]. The volume of gas generated from the bioreactor was measured using BlueVCount. The total moles of the gas were calculated by summing the moles of gas within the bioreactor and the moles of gas passing through the BlueVCount. Declarations Acknowledgments The authors express gratitude to the technical team at the Institute of Process Engineering in Life Sciences 2: Electrobiotechnology, Karlsruhe Institute of Technology, for their valuable assistance. Additionally, the authors thank to Daniel Richter for his contribution to milling the Enset fiber required for the experiments. Author contributions Conceptualization, N.S.; methodology, N.S., H.A. and A.N.; formal analysis, N.S.; investigation, N.S. and L.W.; resources, N.S. and A.N.; data curation, N.S.; writing-original draft preparation, N.S.; writing-review and editing, A.N. and H.A.; visualization, N.S.; supervision, A.N.; project administration, A.N.; funding acquisition, N.S. and A.N.; All authors read and approved the final manuscript. Funding This research was funded by the DAAD-EECBP Homegrown Ph.D. Scholarship Program 2019, and the APC was funded by the Karlsruhe Institute of Technology (KIT) Publication Fund. Availability of data and materials Not applicable. Ethics approval and consent to participate. Not applicable. Consent for publication Not applicable. Competing interests The authors declare no conflict of interest. References Rajak, J. A Preliminary Review on Impact of Climate change and our Environment with Reference to Global Warming. Int. J. Environ. Sci. 2021 , 10 , 11–14. 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Supplementary Files SupplementarymaterialSeid20240105.docx Additional file 1: Table S1. Effect of temperature on hydrogen and biobutanol production in the SSF process from Enset fiber in bottles. Table S2. Effect of initial pH on hydrogen and biobutanol production in the SSF process from Enset fiber in bottles. Table S3. Effect of hydrogen partial pressure on hydrogen and biobutanol production in the SSF process from Enset fiber in bottles. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-3836946","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":265558392,"identity":"7580180f-69af-47a0-8d9d-f65fc0a8004f","order_by":0,"name":"Nebyat Seid","email":"","orcid":"","institution":"Karlsruhe Institute of Technology (KIT)","correspondingAuthor":false,"prefix":"","firstName":"Nebyat","middleName":"","lastName":"Seid","suffix":""},{"id":265558393,"identity":"a0281706-60e6-4e9b-8d9f-e119bab0d915","order_by":1,"name":"Lea Wießner","email":"","orcid":"","institution":"Karlsruhe Institute of Technology (KIT)","correspondingAuthor":false,"prefix":"","firstName":"Lea","middleName":"","lastName":"Wießner","suffix":""},{"id":265558394,"identity":"00edfce9-c743-4b3f-b0f6-ed7bb30e8161","order_by":2,"name":"Habibu Aliyu","email":"","orcid":"","institution":"Karlsruhe Institute of Technology (KIT)","correspondingAuthor":false,"prefix":"","firstName":"Habibu","middleName":"","lastName":"Aliyu","suffix":""},{"id":265558395,"identity":"b962efbc-4f87-4d1c-9722-21a50c79b1d4","order_by":3,"name":"Anke Neumann","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIie3PMQrCMBSA4QTBLoWuT4r2ChWhdBDPYhDSpbgIji5CXDyA4BkcXIJjoJCp4voGB8G1Q926aevkYtTNIf+SBPKRF0Jstr+s/bIvFTRL+D2hGwXwG2m5inwm3lawCz0M2W511NdhHi+Is5RgInDWWUhzzmQ+TQYp1oO5em4kBBMBVGRMqjTy07ImkEZGEmCyqqi4M3kqIj9uSFCYSYhcEyoUk1i/QprBwDWTPvIJMDEZSCyizjqHjnD5LDaRHvJ+eROjrjzVX6j0wvOcbI8m8mz8emi/uWSz2Wy2H3oAY61H769JVQgAAAAASUVORK5CYII=","orcid":"","institution":"Karlsruhe Institute of Technology (KIT)","correspondingAuthor":true,"prefix":"","firstName":"Anke","middleName":"","lastName":"Neumann","suffix":""}],"badges":[],"createdAt":"2024-01-05 10:15:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3836946/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3836946/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49351382,"identity":"4fb6087c-8000-4321-a58b-3c2d382f8aec","added_by":"auto","created_at":"2024-01-09 06:35:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":137687,"visible":true,"origin":"","legend":"\u003cp\u003eValidation experiment at 5% substrate loading, 16 FPU/g cellulase loading, and 100 rpm (A) SSF process, (B) saccharification process. All values are means from triplicate bottles.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3836946/v1/38e517c94f2ac74556949f38.png"},{"id":49351663,"identity":"7cbc34d9-cc5f-4d45-bc1a-0f09d165afa2","added_by":"auto","created_at":"2024-01-09 06:43:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":258445,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolites in the SSF process from Enset fiber with (A) pH-controlled and (B) pH-uncontrolled. All values are means from duplicate fermentations in the bioreactor.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3836946/v1/7cca73fab69f0da3326802b4.png"},{"id":49351662,"identity":"36dcecc2-d939-4e97-a675-9a1d8fe70cc2","added_by":"auto","created_at":"2024-01-09 06:43:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":358291,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolites in the PSSF process at high substrate loading with Enset fiber. All values are means from duplicate fermentations in the bioreactor.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3836946/v1/dfc852c9aaac5a8d61dd2de8.png"},{"id":49351383,"identity":"0488ab29-852a-4dca-9391-c8f06234ed6c","added_by":"auto","created_at":"2024-01-09 06:35:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74260,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of temperature on hydrogen production in the SSF process. All values are means from triplicate bottles.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3836946/v1/864a972723bc5b9669ba6147.png"},{"id":49351387,"identity":"0c4a6da7-1d2a-4898-89bc-387bbbb85714","added_by":"auto","created_at":"2024-01-09 06:35:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":167138,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of initial pH values on hydrogen production in the SSF process from Enset fiber (A) pH profile and (B) hydrogen production. All values are means from triplicate bottles.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3836946/v1/7e9f79e4c220d288ba87b9e9.png"},{"id":49351664,"identity":"94ee75ba-dfd2-425f-bc83-f90e70ee5bc2","added_by":"auto","created_at":"2024-01-09 06:43:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":144639,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of gas release strategy in the SSF process from Enset fiber (A) hydrogen partial pressure (B) hydrogen production. All values are means from triplicate bottles.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3836946/v1/70ee6dacaf0691a61248a099.png"},{"id":49351385,"identity":"f57098c2-e2d4-427a-9acb-a87f35a7eec5","added_by":"auto","created_at":"2024-01-09 06:35:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":166551,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolites in the SSF process from the Enset fiber SSF process at atmospheric pressure. All values are means from duplicate fermentations in the bioreactor.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3836946/v1/5d1cc05b80525929c6bf34cc.png"},{"id":52661975,"identity":"9a452528-f65d-48a7-90a1-429c62265e2e","added_by":"auto","created_at":"2024-03-14 08:06:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2130027,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3836946/v1/14107149-d9d5-41ef-aa00-c439ed9c31d0.pdf"},{"id":49352364,"identity":"aa33d5a2-fddc-495d-8e46-523590a32f85","added_by":"auto","created_at":"2024-01-09 06:51:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":22271,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1\u003c/strong\u003e: \u003cstrong\u003eTable S1\u003c/strong\u003e. Effect of temperature on hydrogen and biobutanol production in the SSF process from Enset fiber in bottles. \u003cstrong\u003eTable S2\u003c/strong\u003e. Effect of initial pH on hydrogen and biobutanol production in the SSF process from Enset fiber in bottles. \u003cstrong\u003eTable S3\u003c/strong\u003e. Effect of hydrogen partial pressure on hydrogen and biobutanol production in the SSF process from Enset fiber in bottles.\u003c/p\u003e","description":"","filename":"SupplementarymaterialSeid20240105.docx","url":"https://assets-eu.researchsquare.com/files/rs-3836946/v1/35b9b198989e456928ae0b73.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Stirring the Hydrogen and Butanol production from Enset Fiber via Simultaneous Saccharification and Fermentation (SSF) process","fulltext":[{"header":"Background","content":"\u003cp\u003eThe impacts of human-driven global warming, such as the melting of sea ice, rising sea levels, unpredictable heat waves, wildfires, and intense rainfall, are getting worse day by day. Although some countries have made progress in adapting their climate action plans to achieve net-zero emissions, the situation remains challenging [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to the International Energy Agency (IEA) report (2022), CO\u003csub\u003e2\u003c/sub\u003e emissions increased by 6% from 2020 to 2021, the highest emission level recorded for a single year. This increase in emissions clearly indicates that the world is far from achieving the goal of limiting warming to 1.5\u0026deg;C, as set by the United Nations [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The goal can be achieved by shifting global energy consumption from fossil fuels to biofuels such as hydrogen and butanol, which can potentially reduce carbon emissions and establish a more sustainable energy system [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In recent years, hydrogen has become increasingly popular as an energy carrier for fuelling vehicles, generating electricity, and various industrial applications [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The high energy content of hydrogen (140 MJ/kg) and the fact that its combustion produces only water as a byproduct make it an ideal clean fuel for various applications [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Similarly, butanol is a highly versatile fuel that can be blended with gasoline in high proportions, used in existing engines, and easily transported through current pipeline infrastructure. This versatility arises from its excellent properties, such as higher energy density (29 MJ/L), lower vapor pressure (0.53 kPa), lower hygroscopicity, and lower volatility than ethanol [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Several studies have shown that biological processes can successfully produce butanol and hydrogen. However, there are still numerous challenges that need to be addressed to make this process competitive with other processes. These includes substrate availability [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], low production yield [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], scalability, high processing cost [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and maintaining optimal fermentation conditions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLignocellulosic biomass is readily available and economically viable worldwide and can be obtained from agricultural residues, industrial wastes, forest residues, and municipal wastes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It is composed of cellulose, hemicellulose, and lignin, which are the three components of plant cell walls. These components can be broken down using enzymes and other chemicals, allowing subsequent utilisation by microorganism as a substrate for biofuel production, such as hydrogen and butanol [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Enset fiber is Ethiopian agricultural residue obtained from the Enset plant. A large amount of Enset fiber goes to waste after the traditional Enset food processing in Ethiopia, which feeds more than 20\u0026nbsp;million people [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Enset contains 67.1% cellulose, 15.6% hemicellulose, and 5.1% lignin, which makes Enset fiber a promising substrate for biofuel production due to its high cellulose and very low lignin content [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A recent study conducted by Seid N. et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] examined the potential of Enset fiber for butanol production using the separate hydrolysis and fermentation (SHF) method compared with various other Enset plant residues. The results revealed that Enset fiber holds significant promise as a viable resource for butanol production and has a higher hydrolysis conversion efficiency than other Enset plant residues. However, the main challenges for the SHF process were substrate inhibition, low butanol yield and productivity, the complexity of operational procedures requiring multiple distinct steps, and the economic viability of the process [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral studies showed that butanol can be produced by combining pretreated biomass, enzymes, and \u003cem\u003eClostridia\u003c/em\u003e species in a single bioreactor. This process is called simultaneous saccharification and fermentation (SSF), enables the simultaneous occurrence of sugar release and fermentation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Compared with the SHF process, SSF has many advantages, including minimising substrate inhibition, reducing operating costs, the overall risk of contamination, and maximising butanol productivity and yield [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, the SSF process faces several challenges that limit its industrial application. These include the different optimal operating conditions for the saccharification process and acetone-butanol-ethanol (ABE) fermentation, which depend on the type of substrate and microorganism used. Therefore, finding a balance between the two processes is challenging and may affect the efficiency and productivity of butanol production [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. ABE fermentation consists of two distinct phases: acidogenic and solventogenic phases. During the acetogenic phase, the strain undergoing exponential growth produces hydrogen, as well as acids such as acetic acid and butyric acid. Due to the accumulation of these acids, cell growth begins to slow down, and the metabolism of the strain shifts into the solventogenic phase. In this phase, the strain produces solvents such as acetone, butanol, and ethanol as a survival mechanism [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. \u003cem\u003eClostridium saccharoperbutylacetonicum\u003c/em\u003e DSM 14923 is well known for producing butanol and hydrogen as a sugar fermentation product, making it ideal for biofuel production [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The strain is versatile, capable of using a variety of substrates, resistant to solvents, and can thrive in harsh environments [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Studies showed that several factors, including temperature, pH, electron flow and reducing equivalents, play a significant role in \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e fermentation, influencing the amount of hydrogen and butanol [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These factors contribute to a competitive relationship between the two products. Therefore, optimal fermentation conditions are crucial for maximising both hydrogen and butanol production. This study aimed to optimize the process parameters for producing hydrogen and butanol from Enset fiber using the SSF process. We examined the impact of controlling pH on the SSF process from Enset fiber. Finally, we attempted to maximize butanol productivity through the PSSF process at a high substrate loading with Enset fiber. It is worth noting that this report is the first study on the simultaneous production of hydrogen and butanol from Enset fiber using the SSF process.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEffect of SSF process parameters on butanol production\u003c/h2\u003e \u003cp\u003eThis work investigates process parameters to improve butanol production from Enset fiber using the SSF process. Important parameters, such as agitation speed, cellulase loading and substrate loading were selected and optimized. The preliminary results showed that \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e fermentation produced metabolites only between temperatures of 28\u0026deg;C and 37\u0026deg;C; however, no metabolites were detected above 37\u0026deg;C. Unless otherwise stated, all subsequent experiments were carried out at a constant temperature of 30 \u0026deg;C and an initial pH of 6.8. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the effect of agitation speed on butanol production in the SSF process and glucose production in the saccharification process (control experiment) from Enset fiber. After 72 hours of the SSF process, a maximum butanol concentration of 11.48 g/L was produced at an agitation speed of 100 rpm. At this speed, the strain consumed almost all glucose, resulting in a butanol yield of 0.24 g/g and productivity of 0.16 g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. However, at 130 rpm, butanol production was lower (10.33 g/L), and 9.48 g/L glucose remained unconsumed. The saccharification process showed that the enzyme released more glucose (44.57 g/L) at 130 rpm than at other speeds after 72 hours.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of agitation speed on butanol production in the SSF process and glucose production in the saccharification process in bottles \u003csup\u003ea, b\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAgitation speed (rpm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eSSF process\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eSaccharification process\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFinal glucose concentration (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eButanol concentration (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eButanol yield (g/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eButanol productivity (g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGlucose released (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMaximum glucose production rate (g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e37.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e38.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e10.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e44.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e All calculations accounted for 2.5 g Enset fiber in 50 ml medium at 72 hours fermentation period; \u003csup\u003eb\u003c/sup\u003e Values are means from triplicate bottles.\u003c/p\u003e \u003cp\u003eIn addition, the effects of cellulase loading on butanol production from Enset fiber were investigated. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e presents the effects of different cellulase loadings on butanol production in the SSF process and glucose production in the saccharification process from Enset fiber. The result showed no significant difference in butanol production in the SSF process between 16 and 24 FPU/g, while 9 FPU/g cellulase loading resulted in a slightly lower butanol production of 10.73 g/L. The saccharification process released a higher glucose concentration of 47.47 g/L at 30 FPU/g cellulase loading, but the SSF process showed the lowest butanol concentration of 9.6 g/L and a yield of 0.19 g/g.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of cellulase loading on butanol production in the SSF process and glucose production in the saccharification process in bottles \u003csup\u003ea, b\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCellulase loading (FPU/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eSSF process\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eSaccharification process\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFinal glucose concentration (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eButanol concentration (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eButanol yield (g/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eButanol productivity (g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGlucose released (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMaximum glucose production rate (g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e10.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e29.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e39.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e38.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e47.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e All calculations accounted for 2.5 g Enset fiber in 50 ml medium at 72 hours fermentation period; \u003csup\u003eb\u003c/sup\u003e Values are means from triplicate bottles.\u003c/p\u003e \u003cp\u003eThe effects of substrate loading with Enset fiber on butanol production in the SSF process were also examined. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows how substrate loading affects butanol production in the SSF process and glucose production in the saccharification process from Enset fiber. The results showed that the same butanol concentration of 11 g/L was observed at both 5 and 7% (w/v) substrate loading of Enset fiber. However, at 7% (w/v) substrate loading, the butanol yield was much lower (0.16 g/g), and 12.41 g/L of glucose remained unfermented compared to other substrate loadings. On the other hand, higher butanol yields were achieved at 2 and 3% (w/v) substrate loadings despite lower butanol concentration and productivity. In the saccharification process, the highest glucose concentration (44.74 g/L) was released at a substrate loading of 7% (w/v), but the glucose production rate was slower than other substrate loadings.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of substrate loading on biobutanol production in the SSF process and glucose production in the saccharification process in bottles \u003csup\u003ea, b\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSubstrate loading (% (w/v)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eSSF process\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eSaccharification process\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFinal glucose concentration (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eButanol concentration (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eButanol yield (g/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eButanol productivity (g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGlucose released (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMaximum glucose production rate (g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e16.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e25.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e39.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e44.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e All calculations accounted for different substrate loading of Enset fiber in 50 ml medium at 72 hours fermentation period; \u003csup\u003eb\u003c/sup\u003e Values are means from triplicate bottles.\u003c/p\u003e \u003cp\u003eOverall, at the end of fermentation, the validation experiment confirmed that under the optimum process parameters, including 5% (w/v) substrate loading, 16 FPU/g cellulase loading, and 100 rpm agitation speed, the SSF process from Enset fiber resulted in a maximum butanol concentration of 11.36 g/L, with a corresponding yield of 0.23 g/g and a productivity of 0.16 g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In addition, 4.33 g/L acetone and 0.71 g/L ethanol were produced, while 1.77 g/L glucose, 0.41 g/L acetic acid, and 1.08 g/L butyric acid remained unconsumed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The validation experiment in the saccharification process also showed an almost similar glucose concentration (37.4 g/L) after 72 hours. However, the maximum glucose production rate of this experiment was slightly faster than the previous one at 2.34 g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eThe impact of controlled and uncontrolled pH values in the SSF process on butanol production\u003c/h2\u003e \u003cp\u003eThis study aimed to establish a small-scale SSF process for butanol production from Enset fiber in a 2.5 L bioreactor and to investigate the influence of pH control on the process. All fermentations were carried out under the optimal conditions of 5% (w/v) substrate loading, 16 FPU/g cellulase loading, a temperature of 30\u0026deg;C, and an initial pH of 6.8, as determined from the previous bottle experiments, except for the stirrer speed, which was 150 rpm. The SSF process was scaled up in a 2.5 L stirred tank reactor with a pressure relief valve that maintained the hydrogen-rich gas in the medium. The bioreactor pressure reached an absolute pressure of 1.55 bar in about 5 hours; after that, the valve opened and regulated the pressure, while the excess gas was released through BlueVCount and collected in a gas bag. The pressure dropped to 1.52 bar after 24 hours of fermentation and remained constant until 54 hours. Then, the pressure fluctuated between 1.23 and 1.48 bar until the end of fermentation. The influence of pH control on butanol production in the SSF process was examined using the same bioreactor setup, and the results were compared with those of pH-uncontrolled fermentation. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA \u0026amp;B shows the metabolites and pH profiles in the SSF process from Enset fiber with the pH-controlled and pH-uncontrolled fermentations. The pH value in both fermentations decreased from 6.8 to 5.0 within 8 hours of inoculation. In the pH-controlled fermentation, the base pump was turned on after 8 hours to keep the pH above 5.0; however, after 19 hours, no addition of NaOH was observed. Within 19 hours, the pH of this fermentation reached a peak pH of 5.96 and then decreased to 5.53 after 68 hours. Afterwards, it fluctuated between 5.51 and 5.60. In contrast, the pH-uncontrolled fermentation showed a slight increase in pH to 5.29 after 19 hours and a gradual decrease to 4.83 after 68 hours, where it remained constant until the end of the fermentation.\u003c/p\u003e \u003cp\u003eIn addition, a comparison was made between the pH-controlled and pH-uncontrolled SSF processes regarding their effects on liquid metabolites. In both fermentations, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026amp;B, the enzyme released a maximum glucose concentration of about 8 g/L after 8 hours, which was similar to the time when the pH was lowered to 5.0. Following that, a higher glucose concentration was observed in the pH-uncontrolled fermentation until the end of the fermentation than in the pH-controlled fermentation. Similarly, other sugars such as xylose, mannose and galactose were detected in higher concentrations in the pH-uncontrolled fermentation than in pH-controlled. In both fermentations, the strain began producing butanol around 19 hours. However, the pH-uncontrolled fermentation had a faster production rate of 0.23 g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e than the pH-controlled one. It also had a higher butanol concentration of 10.54 g/L after 72 hours compared to 8.67 g/L in the pH-controlled fermentation. At the end of fermentation, butanol concentration was increased to 11.35 g/L, yielding 0.23 g/g in the pH-uncontrolled fermentation. However the productivity decreased to 0.095 g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e compared to a fermentation time of 72 hours. Likewise, in the pH-controlled fermentation, the butanol concentration increased to 9.86 g/L, yielding 0.20 g/g and productivity of 0.082 g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In addition, both fermentations produced other solvents, such as acetone and ethanol. However, the difference in acetone production between the pH-controlled and pH-uncontrolled fermentations was insignificant at 3.61 g/L and 3.00 g/L, respectively, and ethanol was detected in small amounts in both fermentation, which was lower than 0.5 g/L. The acid compositions, such as acetic acid and butyric acid, also differed in both fermentations. Acetic acid and butyric acid levels were higher in the pH-controlled fermentation than in the pH-uncontrolled fermentation. As with butanol production, hydrogen production showed the opposite trend when comparing pH-controlled and pH-uncontrolled fermentations. A higher amount of hydrogen was observed in the pH-controlled fermentation at 308.02 mmol than in the pH-uncontrolled fermentation at 246.47 mmol.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePSSF process at high substrate loading with Enset fiber\u003c/h2\u003e \u003cp\u003eThe purpose of this study was to maximize butanol productivity using the PSSF process at high substrate loading with Enset fiber. This experiment was done in a bioreactor with pH-controlled fermentation similar to the experiment described above. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the metabolites produced in the PSSF process at a substrate loading of 7% (w/v) using Enset fiber as a carbon source. The prehydrolysis step lasted for 2 hours, followed by a 1 hour transition period to adjust the temperature and initial pH value, and the enzyme hydrolysed the Enset fiber into 12.53 g/L of glucose and 2.66 g/L of other sugars, such as xylose, mannose, and galactose. In addition, we observed that the Enset fiber was partially degraded, and the medium was homogenized at the end of this process. After inoculation with \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e, the PSSF process achieved a faster butanol production rate of 0.21 g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e than the pH-controlled SSF process but a slightly similar rate to the pH-uncontrolled SSF process. However, the butanol concentration after 72 hours was higher at 11.04 g/L in the PSSF process than in the SSF process in both pH-controlled and uncontrolled fermentation. At the end of fermentation, the butanol concentration and productivity in the PSSF process were increased to 12.84 g/L and 0.104 g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Although the butanol concentration and productivity were higher in the PSSF process than in the SSF process, the butanol yield of 0.18 (g/g) was significantly lower than in the SSF process. Furthermore, the PSSF process produced a higher concentration of acetone (5.64 g/L) and a higher amount of hydrogen (378.69 mmol) than the SSF process. However, at the end of the fermentation, we found that 4.51 g/L glucose, 2.58 g/L acetic acid and 1.37 g/L butyric acid remained unused.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEffect of SSF process parameter on hydrogen production\u003c/h2\u003e \u003cp\u003eThe objective of this study was to examine the influence of SSF process parameters on hydrogen production from Enset fiber. The key process parameters affecting hydrogen production were selected and optimized, including temperature, initial pH, and hydrogen partial pressure. At the same time, other factors were kept constants for all experiments based on results from previous bottle experiments. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the effect of temperature on hydrogen production from Enset fiber in the SSF process. After 44 hours of incubation, at both 35\u0026deg;C and 37\u0026deg;C, hydrogen production reached a maximum of 15.75 mmol and then slightly declined at both temperatures. At the end of fermentation, hydrogen production was lower at 30\u0026deg;C (12.03 mmol) than at other temperatures. However, the butanol yield and productivity were much higher at 30\u0026deg;C, with 0.23 g/g and 0.17 g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. In contrast, at 37\u0026deg;C, the butanol yield was lower at 0.17 g/g, while the hydrogen yield was higher at 133.50 mL/g-Enset fiber compared to other temperatures (see Additional File 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the effects of initial pH value on hydrogen production from Enset fiber were investigated. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows the pH changes during the SSF process with Enset fiber at different initial pH values. The initial pH of 6.0, 7.0, and 8.0 showed similar pH trends, decreasing at 8 hours, and increasing to about 6.23 after 24 hours. At the end of fermentation, they had nearly the same pH of 6.01, except for the initial pH of 6.0, which was slightly lower at 5.76. In contrast, the initial pH of 5.0 and 9.0 showed similar pH trends, dropping below 5.0 after 8 hours and remaining low until the end of fermentation. Similarly, the initial pH values affected the amount of hydrogen produced by the SSF process, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. After 44 hours of fermentation, the highest hydrogen production was observed at 16.32 mmol at initial pH values of 7.0 and 8.0. However, the initial pH of 8.0 had a slightly faster rate of 0.53 mmol/h than the others. At the end of fermentation, a similar hydrogen yield (139 mL/g-Enset fiber) was observed at all initial pH values of 6.0, 7.0 and 8.0. The lowest amounts of hydrogen were found at both initial pH of 5.0 and 9.0, with 1.74 mmol and 5.33 mmol, respectively. Similar to the amount of hydrogen, there was no significant difference in butanol yield (0.18 g/g-Enset fiber) between the initial pH of 6.0, 7.0, and 8.0 at the end of fermentation. In contrast, at the end of fermentation, the lowest butanol yield was observed at initial pH of 5.0 and 9.0; however, a significantly higher glucose concentration of 38 g/L and 30 g/L were observed at an initial pH of 5.0 and 9.0, respectively (see Additional File 1: Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effects of hydrogen partial pressure on hydrogen production in the SSF process from Enset fiber were also investigated. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA \u0026amp; B shows the hydrogen partial pressure and hydrogen production in the SSF process from Enset fiber under different gas release strategies. In both experiments, where gas was released into 1-L and 2-L bottles, the hydrogen partial pressure was lower compared to the control experiment without gas released. However, the gas released into the 1-L bottle resulted in a slightly lower hydrogen partial pressure than that released into the 2-L bottle (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Similarly, at the end of fermentation, there was a slight difference in hydrogen production of 17.24 mmol and 18.86 mmol between the fermentation, where the gas was released into 1-L and 2-L bottles, respectively. However, a smaller amount of hydrogen (15.52 mmol) was observed in the control experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Furthermore, it was found that there was little difference in butanol production between all experiments (see Additional File 1: Table S3). Overall, at the optimum process parameters, including a temperature of 37\u0026deg;C, an initial pH of 8.0, and the lowest hydrogen partial pressure, a maximum hydrogen yield of 168.99 mL/g-Enset fiber was achieved in the SSF process from Enset fiber.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHydrogen production in the SSF process at atmospheric pressure\u003c/h2\u003e \u003cp\u003eThis study aimed to develop a small-scale SSF process for hydrogen production from Enset fiber in a 2.5 L bioreactor. This was achieved by using optimal process parameters, including temperature (37\u0026deg;C), initial pH (8.0), and atmospheric pressure as determined in bottle experiments. The SSF process was scaled up using a 2.5 L stirred tank reactor operated at atmospheric pressure and without a pH control method. Initially, the outlet tube was closed for 4 hours to maintain an anaerobic environment in the bioreactor. During this period, the strain produced its gas, and the pressure was increased to an absolute pressure of 1.3 bar. Following this, the bioreactor outlet was connected to the BlueVCount, which released gases into the gas bag, thereby reducing the pressure to the atmospheric level of 1 bar. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the metabolites and pH profiles in the SSF process from Enset fiber at atmospheric pressure. The result of this study was compared to a similar SSF process, except that the SSF was performed at 30\u0026deg;C, with an initial pH of 6.8, without pH control and under overpressure (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Unless otherwise stated, this was the reference process for the comparison. After inoculation, hydrogen gas production was started in 4 hours in both fermentations, and the amount was similar at 3.64 mmol. However, after that, the hydrogen production rate in the SSF process at 37\u0026deg;C was faster, with a maximum production rate of 8.38 mmol/h, than in the SSF process at 30\u0026deg;C with 6.56 mmol/h. Further, after 72 hours, a higher amount of hydrogen was found at 428.53 mmol in the SSF process at 37\u0026deg;C than in the SSF process at 30\u0026deg;C with 239.14 mmol. However, hydrogen production did not change significantly when the fermentation time was extended to 120 hours. Both fermentations showed a slight increase in hydrogen production with 442.57 mmol and 246.47 mmol for the SSF processes at 37\u0026deg;C and 30\u0026deg;C, respectively, resulting in relatively low hydrogen productivity 1.65 mLg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.92 mLg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. In addition, a higher hydrogen yield of 198.27 mL/g-Enset fiber was achieved at the end of fermentation in the SSF process at 37\u0026deg;C compared to the yield of 110.42 mL/g-Enset fiber at 30\u0026deg;C.\u003c/p\u003e \u003cp\u003eFurthermore, the pH profiles and liquid metabolites of the SSF processes showed different results at different temperatures and initial pH values ​​(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The pH of both fermentations decreased to 5.0 in the first 8 hours of fermentation and then increased slightly above 5.0 in the next 19 hours. After that, the pH value in the SSF process at 37\u0026deg;C remained above 5.0 and fluctuated between 5.5 and 5.76, while the pH value in the SSF process at 30\u0026deg;C dropped again for about 44 hours and varied between 4.82 and 4.96. At the end of fermentation, the SSF process at 37\u0026deg;C had a higher pH of 5.6 than the SSF process at 30\u0026deg;C, which had a lower pH of 4.89. Similarly, the glucose concentration released by the enzyme after 8 hours of fermentation was the same in both SSF processes (8.2 g/L). However, after 19 hours, the SSF process at 37\u0026deg;C reached a maximum glucose concentration of 15.54 g/L, while the SSF process at 30\u0026deg;C did not increase further. At the end of fermentation, the strain consumed almost all of the glucose in the SSF process at 37\u0026deg;C, while 1.77 g/L glucose remained unused at 30\u0026deg;C. Moreover, both SSF processes had a maximum of 4.25 g/L of other sugars, such as xylose, mannose and galactose, which were also consumed by the end of fermentation. At the end of fermentation, the liquid metabolites such as butanol and acetone also differed depending on the temperature and initial pH value. The SSF process at 30\u0026deg;C produced more butanol (11.35 g/L) than the SSF process at 37\u0026deg;C (8.92 g/L). However, the opposite was observed for acetone, which was higher in the SSF process at 37\u0026deg;C (6.04 g/L) than the SSF process at 30\u0026deg;C (3.00 g/L). In addition, the SSF process at 37\u0026deg;C contained more acetic acid and less butyric acid than the SSF process at 30\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussions","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMaximizing butanol production from Enset fiber using the SSF process\u003c/h2\u003e \u003cp\u003eThe SSF process is a widely recognized method for enhancing product yield, minimizing equipment and energy costs, and offering the advantage of faster processing time compared to conventional methods [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, one of the primary challenges in maximizing butanol production in the SSF process using \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e is the large temperature difference required for ABE fermentation and saccharification, typically between 30\u0026deg;C and 50\u0026deg;C [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The cellulase enzymes require higher temperatures of 40\u0026minus;50 \u0026deg;C to efficiently degrade lignocellulosic biomass [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], while the strain prefers lower temperatures of 30 \u0026deg;C for optimal butanol production [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This makes it challenging to choose an optimal temperature for the SSF process to maximize butanol production. In this study, we observed that \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e was able to grow and produce butanol from pretreated Enset fiber using the SSF process within a temperature range of 28 to 37\u0026deg;C, and the maximum butanol production was observed at 30\u0026deg;C. This could be due to the structural properties of Enset fiber, which has a high cellulose content (67.1%) and a low lignin content (5.1%), making it a promising feedstock for the SSF process [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Lignin is a natural polymer that acts as a barrier to prevent the enzymatic hydrolysis of cellulose and hemicellulose. It can be removed by various pretreatment methods such as dilute acid pretreatment, dilute alkali pretreatment, steam explosion, and liquid hot water; however, depending on the biomass and the specific type of pretreatment, the pretreatment methods can lead to lignin degradation. The resulting lignin degradation products, such as aromatic alcohols, phenolic compounds, furan derivatives, and organic acids, act as soluble inhibitors that can affect enzyme activity and strain growth [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Previous studies have shown that the SSF process is affected by inhibitory compounds resulting from the degradation of lignin in alkali-pretreated switchgrass [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and acid-pretreated corn stover [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], which inhibits the \u003cem\u003eclostridial\u003c/em\u003e strain. However, alkali-pretreated Enset fiber did not generate any inhibitory compounds, according to a study [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, the effects of other process parameters, such as agitation speed, cellulase loading and substrate loading on the SSF process were further optimized while maintaining a constant temperature (30 \u0026deg;C) and initial pH (6.8), which were found to be the optimal parameters for ABE fermentation using \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Mixing the SSF medium improves the mass transfer between biomass, enzyme, microorganism, and nutrients in the SSF process. However, agitation speed affects the ABE fermentation and saccharification process differently. The bottle experiments in the current study revealed that the SSF process produced the highest butanol at an agitation speed of 100 rpm. However, higher rotation reduced the butanol concentration by 10% in the SSF process. On the other hand, the saccharification process benefited from high rotation; more glucose was produced at higher agitation speeds, possibly due to the enhanced interaction between the enzyme and the Enset fiber. The results of this study were similar to those of Al-Shorgani N. et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], who investigated the effects of agitation on butanol production in ABE fermentation using the same strain as in this study. They found that 100 rpm was the optimal agitation speed for maximum butanol production and at higher speeds, butanol production decreased.\u003c/p\u003e \u003cp\u003eMaintaining an optimal cellulase loading during the SSF process is essential for maximizing the butanol production and ensuring the overall cost-effectiveness of the process. In this study, it was observed that there was only a 4% difference in butanol yield between the cellulase loadings of 16 and 24 FPU/g, despite a 33% increase in the cellulase loading. For this reason, a cellulase loading of 16 FPU/g was selected as an optimal point for the SSF process from Enset fiber. Study shows that enzymes contribute to 40% of the total cost of biofuel production from lignocellulosic biomass [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and reducing cellulase loading is economically beneficial when the yield loss is less than 6\u0026minus;7% [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Further increasing cellulase loading to 30 FPU/g in the SSF process resulted in a 16% decrease in butanol production. This could be due to the inhibitory effect of excessive cellulase on the strain [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. On the other hand, in the saccharification process, higher cellulase loading was beneficial for releasing more glucose from Enset fiber. Compared to the previous study, this result was similar to the research conducted by Md R. et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which investigated the effect of cellulase loading in the SSF process using the \u003cem\u003eC. acetobutylicum\u003c/em\u003e strain and reported a reduction in butanol production from pretreated oil palm empty fruit bunch as the cellulase loading increased from 20 to 30 FPU/g.\u003c/p\u003e \u003cp\u003eOne of the challenges in butanol production from lignocellulosic biomass using the SSF process is balancing the required fermentable sugars and substrate loading, which can lead to mass transfer limitations. This study revealed that even at low substrate loading of 2% and 3% (w/v) Enset fiber, the SSF process achieved a maximum butanol yield of 0.26 (g/g) and 0.27 (g/g), respectively. In addition, 80\u0026minus;84% of Enset fiber was converted into glucose during the saccharification process. These proved that the SSF and saccharification processes using Enset fiber were efficient, despite the reduced enzyme activity due to the low process temperature. However, the butanol productivity was significantly lower at low substrate loading than in fermentations with high substrate loading. Butanol productivity was maximized by 45% when the substrate loading increased from 3\u0026ndash;5% (w/v) of Enset fiber. However, further increases in substrate loading to 7% (w/v) showed no significant difference; instead, the butanol yield decreased by 27%, and 12.42 g/L glucose remained unconsumed. This might be due to poor mixing caused by high solid and low water content, leading to a mass transfer problem in the SSF process [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A similar challenge was identified in a study by Guan W. et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], which focused on butanol production from kraft paper mill sludge using the SSF process; they found that the slurry became too viscous, limiting mass transfer at substrate loadings of 6.3% and higher. Several studies suggest that the PSSF process can solve the mixing problem during the initial stages of fermentation in the SSF process [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e compares the butanol production from different biomass and \u003cem\u003eClostridial\u003c/em\u003e strains using the SSF process in bottle experiments. Compared with other studies, Enset fiber had the highest butanol concentration (11.36 g/L), yield (0.23 g/g), and productivity (0.16 g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) among rice straw, oil palm empty fruit bunch, and avicel at optimal process parameters. However, wheat straw had a higher butanol concentration (12.64 g/L) than Enset fiber, as reported by Qi G. et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], while Enset fiber had a higher butanol yield and productivity. To our knowledge, no studies have yet reported butanol production in the SSF process using \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e. Compared to the SHF method described in our previous study [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], which used the same strain and biomass with separate fermentation and saccharification steps, the butanol concentration was 9.9 g/L, which was lower than the current study. Furthermore, the SSF process enhanced butanol yield by 20% and productivity by 21% compared to the SHF process. This could be because the SSF process can avoid substrate inhibition by releasing the sugar gradually rather than all at once, as in the SHF process.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of butanol production from different biomass using the SSF process in bottle experiments\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiomass\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePretreatment method\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProcess parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eButanol titer (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eButanol yield (g/g biomass)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eButanol productivity (g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRef.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnset fiber\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% (w/v) NaOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC. saccharo-perbutyl-acetonicum\u003c/em\u003e DSM 14923\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5% (w/v) substrate loading, Cellic CTec2 (16 FPU/g-substrate), initial pH\u0026thinsp;=\u0026thinsp;6.8, \u003c/p\u003e \u003cp\u003e30\u0026deg;C, 100 rpm, 72 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eThis study \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRice straw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMicrowave-assisted hydrothermo-\u003c/p\u003e \u003cp\u003elysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC. beijerinckii\u003c/em\u003e DSM 6422\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9% (w/v) substrate loading, Cellic CTec2 \u003c/p\u003e \u003cp\u003e(12 FPU/g-glucan, initial pH\u0026thinsp;=\u0026thinsp;6.4, 37\u0026deg;C, 150 rpm, 48 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOil palm empty fruit bunch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% (w/v) NaOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC. aceto-butylicum\u003c/em\u003e ATCC 824\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5% (w/v) substrate loading, acremonium cellulase (15 FPU/g-substrate), initial pH\u0026thinsp;=\u0026thinsp;5.5, 35\u0026deg;C, 150 rpm, 120 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvicel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC. aceto-butylicum\u003c/em\u003e ATCC-824\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.8% (w/v) substrate loading, Cellic CTec2 (20 FPU/g-glucan), initial pH\u0026thinsp;=\u0026thinsp;6.7, 36\u0026deg;C, 150 rpm, 120 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWheat straw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10% (w/v) Ammonium sulfite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC. aceto-butylicum\u003c/em\u003e\u003c/p\u003e \u003cp\u003eATCC 824\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9% (w/v) substrate loading, Cellulase (5 FPU/g-substrate) \u0026amp; xylanase (10 IU/g-substrate), initial pH\u0026thinsp;=\u0026thinsp;5.0, 37\u0026deg;C, 150 rpm, 144 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.088\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e All calculations accounted for different substrate loading of Enset fiber in 50 ml medium at 72 hours fermentation period.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eScaling up butanol production from Enset fiber\u003c/h3\u003e\n\u003cp\u003eTo develop butanol production from Enset fiber at an industrial scale, a scale-up process that matches the bottle-scale experiment performance is essential. This study successfully established a small-scale SSF process from pretreated Enset fiber using a 2.5 L stirred tank bioreactor by maintaining a slight overpressure inside the bioreactor. In this study, the bioreactor fermentation using the pH-uncontrolled method showed a comparable butanol production result as the bottle experiment under similar optimal process parameters, except for the stirrer speed in the bioreactor. In both experiments, there was only a small difference in butanol concentration of 7.8% after 72 hours. However, after 120 hours, a similar butanol concentration of 11.35 g/L was observed with a yield of 0.23 g/g. These comparable results were achieved due to the utilization of a partially similar configuration when setting up the bioreactor system, which included maintaining the hydrogen-rich gas in the bioreactor. Several studies showed that maintaining hydrogen gas during fermentation is beneficial for maximizing biobutanol production in \u003cem\u003eclostridial\u003c/em\u003e strains. Brosseau J. et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] observed that \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e requires hydrogen gas to produce butanol, and when the hydrogen gas reaches a certain level, the bacteria reduce their growth rate and start producing butanol. Similarly, in this study, the highest hydrogen production rate was observed during the initial cell growth phase of \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e, which remained faster until 24 hours. Afterwards, the rate of hydrogen gas production slowed down, and the strain started producing butanol and entered the solventogenic phase. Furthermore, a separate study conducted by Stein U. et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], focusing on the effect of pressure on ABE fermentation using \u003cem\u003eC. acetobutylicum\u003c/em\u003e, concluded that butanol production could be enhanced through overpressure fermentation compared to lower pressure.\u003c/p\u003e \u003cp\u003eIn addition, this study investigated the influence of controlled and uncontrolled pH values on butanol production from Enset fiber in the SSF process. The results showed that controlling the pH above 5.0 was not essential to achieve optimal butanol production in the SSF process from Enset fiber. In this study, butanol concentration decreased by 18% in pH-controlled fermentation compared to pH-uncontrolled fermentation, this could be due to the influence of pH value on the efficiency of the cellulase enzyme. Previous research has shown that the optimal pH range for commercial cellulase is typically between 4.8 and 5.5 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. However, in this study, during the SSF process in the pH-controlled fermentation, there were fluctuations in the pH values after 8 hours, which ranged between 5.5 to 5.96. The primary cause of these fluctuations was the substantial addition of NaOH. When the base pump started adding NaOH to the medium, challenges arose due to the high solid content of the medium and inadequate mixing, impacting the accurate operation of the bioreactor's pH sensor. As a result, the base pump introduced an excessive amount of NaOH, resulting in an elevated pH level. The observed deviation from the optimal pH range reduced enzyme activity, ultimately limiting the release of glucose during pH-controlled fermentation compared to pH-uncontrolled fermentation. In contrast, the pH values ​​in the pH-uncontrolled fermentation after 8 hours were between 4.8 and 5.29, which was in the optimal pH range for cellulase enzyme activity. This pH range resulted from the two-phase fermentation of the \u003cem\u003eClostridia\u003c/em\u003e strain. Initially, the strain produced acids such as acetic acid and butyric acid, leading to a pH drop to 4.8 during the first phase. Subsequently, the strain transitioned to the solventogenic phase, producing butanol, acetone, and a small amount of ethanol, causing the pH to rose to 5.29 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Numerous studies have used pH control methods for ABE fermentation to prevent the occurrence of an acid crash, which is often triggered by the presence of inhibitory compounds in the fermentation medium resulting from pretreatment of the lignocellulosic biomass [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]; however, in this study, there were no inhibitory compounds that could have led to an acid crash. The pH is crucial for SSF process, keeping the pH low after the start of the solventogenic phase might increase butanol production rate and yield but also comes with the costs for pH control.\u003c/p\u003e \u003cp\u003eIn this study, the major challenges observed in the bioreactor SSF process were inadequate mixing and reduced butanol productivity. To address these issues, an experiment was conducted using the PSSF method with a 7% (w/v) substrate loading of Enset fiber. The PSSF method alleviated the mass transfer problem by prehydrolysing for 2\u0026ndash;3 hours before inoculation. This was because the SSF process had poor mixing from the start of fermentation until the Enset fiber was saccharified by the enzyme [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Furthermore, the butanol concentration and productivity were increased by 30% and 27%, respectively, compared to the SSF process with pH control method. This might be due to the initial glucose concentration at the end of the prehydrolysis step, which could facilitate the initial growth of the \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e strain [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and the high substrate loading might have contributed to maximizing the butanol concentration. Studies showed that high substrate loading is crucial for large-scale operations to minimize overall production costs and maximize productivity [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]; by increasing the substrate loading from 5% (w/v) to 8% (w/v) of biomass, the production cost can be reduced by 19%, as this can lower the volume of downstream processing equipment and the energy consumption that is related to the dilution level [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Compared to previous PSSF studies with different biomass and \u003cem\u003eClostridial\u003c/em\u003e strains, the highest butanol concentration and yield were observed in Enset fiber than in sugarcane straw [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], corn stover [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and napier grass [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, the butanol productivity of this study was lower than that of Dong J. et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], who performed the PSSF process using \u003cem\u003eC. saccharobutylicum\u003c/em\u003e from corn stover at an SSF temperature of 37 \u0026deg;C. This could be due to the lower working temperature of the SSF process in this study, which slowed down enzyme activity. To enhance the butanol productivity of this study, utilizing a strain with high-temperature tolerance [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] or cold-active cellulase [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] may enable an effective PSSF process from Enset fiber.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHydrogen production from Enset fiber using SSF process\u003c/h2\u003e \u003cp\u003eProducing hydrogen and butanol from affordable materials such as Enset fiber is essential to ensure the sustainability of the biofuel industry. The choice between butanol or hydrogen as a biofuel depends on application, cost considerations, and environmental sustainability [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In this study, butanol and hydrogen were simultaneously produced by the SSF process from Enset fiber using \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e. However, the fermentation conditions must be adjusted separately to achieve maximum yields for both products. This is because fermentation conditions affect cell metabolism and can potentially shift the metabolic pathway in favor of hydrogen or butanol production [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In addition, it has been shown that the amount of hydrogen and butanol produced in the \u003cem\u003eClostridia\u003c/em\u003e strain is influenced by factors such as electron flow and the balance of reducing equivalents in the metabolic pathway, leading to a competitive relationship between the two products [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTemperature is a key factor in the SSF process, as it influences both the growth of the cells and the enzyme activity in their metabolism. The results indicated that higher temperatures (35 and 37\u0026deg;C) improved hydrogen production compared to lower temperature (30\u0026deg;C); however, butanol production decreased with increasing temperature in the SSF process using \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e. A possible explanation could be that the cells grew faster at the higher temperature in the initial growth phase [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], which coincided with the highest hydrogen production rate in the acidogenic phase observed in this study. The result of this study was similar to those of Alalayah W. et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and Yadav S. et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], who investigated the influence of temperature on hydrogen production in ABE fermentation using \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e and found the highest hydrogen values at 37\u0026deg;C in both studies.\u003c/p\u003e \u003cp\u003eAnother factor that affected hydrogen production in the SSF process was the initial pH value of the medium, which was influenced by the buffers in the medium and the metabolites in the cell growth phase. As \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e initiated growth, it began by producing acetic acid, butyric acid, and hydrogen. The buildup of organic acids resulted in a decrease in the pH of the medium and created stressful conditions for cell growth. Subsequently, the culture converted these acids into solvents such as butanol, acetone, and ethanol as it entered the stationary phase, increasing in pH value. Under optimal conditions favorable for cell growth, these phase changes represent the natural pathway for ABE fermentation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, in the presence of inhibitors or unfavorable conditions, the cells struggle to survive, leading to increased acid production and eventual cell death [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In this study, the pH profile of the SSF process at initial pH of 6.0, 7.0, and 8.0 followed a similar phase change and showed no significant difference in hydrogen and butanol production. However, at initial pH of 5.0 and 9.0, the strain could not enter the solventogenic phase, indicating that lower and higher initial pH values inhibited the cell. A study using the same medium and strain for ABE fermentation as in this study found that hydrogen production was maximized in the initial pH range of 7.0-8.5 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In contrast, other studies suggested an optimal initial pH of 6.5, and beyond this point, hydrogen production decreased, although they used a different medium [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, this study examined the effect of hydrogen partial pressure on hydrogen production and found that lower hydrogen partial pressure was beneficial for maximizing hydrogen production. The results of bottle experiments showed that hydrogen production was increased by 21.5% at lower hydrogen partial pressure compared to higher values. To validate the bottle experiment results and maximize hydrogen production, a scale up SSF process without a pH control system was implemented at the optimal process parameters with a temperature of 37\u0026deg;C, an initial pH of 8.0 and under atmospheric pressure. The results confirmed that hydrogen production increased by 79.7% compared to the SSF process (pH-uncontrolled) at 30\u0026deg;C, with an initial pH of 6.8 and under overpressure. However, butanol production was reduced by 21.4% at optimum process conditions for hydrogen production. Several studies have shown that hydrogen partial pressure is an essential factor in maximizing hydrogen production; it could also potentially inhibit cell growth and limit the thermodynamics of the process, depending on the microorganisms involved [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Electrons play a crucial role in the \u003cem\u003eClostridial\u003c/em\u003e metabolic pathways, and the strain can release excess electrons as hydrogen gas to balance the nicotinamide adenine dinucleotide (NADH/NAD\u003csup\u003e+\u003c/sup\u003e) ratio. However, when the hydrogen gas levels are high, it becomes more difficult for the bacteria to continue producing more hydrogen gas; this means the bacteria have too many electrons and insufficient energy, which can stop their growth and activity. As a consequence of this electron backlog, the strain utilises alternative pathways in which electrons are transferred from NADH to ferredoxin, which can serve as an electron carrier to reduce NADP\u003csup\u003e+\u003c/sup\u003e to NADPH, which facilitates butanol production [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. For this reason, removing hydrogen gas from the medium might help the strain to produce more hydrogen.\u003c/p\u003e \u003cp\u003eThis study obtained a similar hydrogen yield of 170 mL/gCOD in the bottle experiment as the previous study by Singh V. et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], which used starchy wastewater and acid-treated water hyacinth as substrates with a similar strain. However, a higher hydrogen yield of 198.27 mL/g-Enset fiber from alkali-pretreated Enset fiber was achieved in the bioreactor experiment at optimal process parameters. This was also higher than the hydrogen yield of 68 ml/g-biomass from steam-exploded straw using the SSF process with \u003cem\u003eC. butyricum\u003c/em\u003e AS1.209 strain, reported by Li D. et al. [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study showed the potential of Enset fiber for hydrogen and butanol production in the SSF process. By optimizing the SSF process with \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e, we achieved high yields and concentrations of both biofuels under different conditions. Furthermore, a scalable process for both products was developed to achieve similar results to bottle-scale experiments, indicating potential applicability on an industrial scale. This is the first study to use this strain and substrate combination for the SSF process, and it contributes to the development of sustainable energy sources. However, more research on the 5L, 10L bioreactors and pilot scale processes, along with a feasibility study, is required to make industrial-scale butanol production feasible using the SSF process from Enset fiber.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEnset fiber preparation\u003c/h2\u003e \u003cp\u003eEnset fiber was obtained from a privately owned Enset plantation in Wolkite, Ethiopia. It was ground to a particle size of 2 mm and then pretreated with 2% (w/v) NaOH according to the methods previously described by Seid N. et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strain and culture medium\u003c/h2\u003e \u003cp\u003e \u003cem\u003eClostridium saccharoperbutylacetonicum\u003c/em\u003e DSM 14923 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). To prepare the inoculum, glycerol stock of the strain (1 mL) was added to 50 mL of anaerobic TGY medium, and the culture was incubated at 30 \u0026deg;C until the optical density (OD\u003csub\u003e600\u003c/sub\u003e) reached 1.0\u0026minus;1.2. TGY medium contained 30 g/L tryptone, 20 g/L glucose, 10 g/L yeast extract, and 0.4 g/L cysteine-HCl\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO. The main fermentation medium comprises 1% (v/v) P2 stock solutions, which was prepared separately, buffer stock solution (50 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 50 g/L K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, and 220 g/L CH\u003csub\u003e3\u003c/sub\u003eCOONH\u003csub\u003e4\u003c/sub\u003e), mineral stock solution (20 g/L MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 1 g/L MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO, 1 g/L FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, and 1 g/L NaCl), and vitamin stock solution (0.1 g/L para-aminobenzoic acid, 0.1 g/L thiamin, and 0.001 g/L biotin) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. All stock solutions were filter-sterilized and anaerobized using N\u003csub\u003e2\u003c/sub\u003e gas.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of SSF medium from pretreated Enset fiber for the bottle experiment\u003c/h2\u003e \u003cp\u003eTo prepare the SSF medium, different amounts of pretreated Enset fiber were added as the sole carbon source into 250 mL Duran pressure plus bottles, each containing a total volume of 50 mL of medium. Subsequently, 1 g/L of yeast extract, 1 mL of 1 g/L resazurin and 0.4 g/L of cysteine-HCl\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO were added, and the initial pH was adjusted to different pH values. The bottles were sealed using a rubber stopper and an aluminum cap, anaerobized with N\u003csub\u003e2\u003c/sub\u003e gas, and autoclaved. Following autoclaving, 1% (v/v) of each P2 stock solutions and 5 mL cellulase-water mixed solution (Cellic CTec2) (Sigma-Aldrich Chemie GmbH, Hamburg, Germany) at varying cellulase loading were added. The bottles were inoculated with 5% (v/v) active \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e culture and incubated in Infors Thermotron incubators (Infors AG, Bottmingen, Switzerland) for 72 hours at different temperatures and agitation speeds. In each experiment, the pressure inside the bottles was measured with a GMH 3100 Series manometer (Greisinger, Mainz, Germany), and samples of 3 mL of gas and 1.5 mL of liquid were collected using needles and syringes. In addition, the saccharification experiment was conducted as a control, and the medium was prepared following the procedure outlined by Seid N. et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of SSF process parameters on butanol and hydrogen production in the bottle experiment\u003c/h2\u003e \u003cp\u003ePreliminary experiments were carried out to find out which temperature ranges were suitable for the cultivation of \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e in the SSF process using Enset fiber at various temperatures, including 28, 30, 33, 35, 37, 40, 45, and 50\u0026deg;C. It was performed with a substrate loading of 5% (w/v), which was 2.5 g pretreated Enset fiber (50 g/L), a cellulase loading of 24 FPU/g, an initial pH of 6.8 and an agitation speed of 130 rpm.\u003c/p\u003e \u003cp\u003eThe influences of agitation speed, cellulase loading and substrate loading on butanol production in the SSF process were investigated at a constant temperature of 30 \u0026deg;C and an initial pH of 6.8. Initially, the effects of agitation were tested at different speeds (0, 100, 130 rpm) while maintaining a constant substrate loading of 5% (w/v) and a cellulase loading of 24 FPU/g. After determining an optimal agitation speed (100 rpm), experiments were conducted to examine the effect of different cellulase loadings (9, 16, 24, 30 FPU/g) while maintaining a constant substrate loading of 5% (w/v). Subsequently, the effects of substrate loading on butanol production were tested at different substrate loadings of Enset fiber (2, 3, 5, 7% (w/v)) while maintaining constant agitation (100 rpm) and cellulase loading (16 FPU/g). Finally, a validation experiment was performed under optimal conditions with an agitation speed of 100 rpm, a cellulase loading of 16 FPU/g and a substrate loading of 5% (w/v). As a control experiment, optimization of the saccharification process was performed in a similar manner to determine how changes in process parameters affect the efficiency of enzymatic hydrolysis.\u003c/p\u003e \u003cp\u003eIn addition, the influence of temperature, initial pH, and hydrogen partial pressure on hydrogen production in the SSF process were examined under optimal process parameters, which included an agitation speed of 100 rpm, a cellulase loading of 16 FPU/g, and a substrate loading of 5% (w/v) Enset fiber. First, to study the effects of temperature on hydrogen production, bottles were placed in incubators set at various temperatures (30, 35, 37 \u0026deg;C) while maintaining a constant initial pH of 6.8 in closed bottles. Following the selection of the best temperature (37\u0026deg;C) for hydrogen production, the effects of initial pH were examined by adjusting the SSF medium to the initial pH (5.0, 6.0, 7.0, 8.0, and 9.0) while maintaining a constant temperature. Afterwards, the effects of hydrogen partial pressure were determined at a constant temperature of 37 \u0026deg;C and an initial pH of 8.0. The following experimental setup was established to investigate the effect of hydrogen partial pressure on hydrogen production, the 250 mL bottles containing the fermentation medium were connected to empty bottles (anaerobized 1-L and 2-L bottles) by tube. This setup allowed the hydrogen-rich gas produced by fermentation to flow from the smaller bottles to the larger ones. All experiments were performed in triplicate and lasted 72 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBioreactor setup for the SSF process from pretreated Enset fiber\u003c/h2\u003e \u003cp\u003eA 2.5 L bioreactor (Minifors, Infors HT, Bottmingen, Switzerland) with two six-bladed Rushton turbine impellers was used for the SSF process. The impellers had a diameter of 4.6 cm and a width of 1.1 cm and positioned 6.0 cm apart on the stirrer. For the SSF medium preparation in the bioreactor, 50 g of pretreated Enset fiber with a substrate loading of 5% (w/v) was added to the bioreactor as the sole carbon source, and the medium had a total volume of 1 liter. The bioreactor was filled with liquids containing 1 g/L yeast extract and 1 mL of 1 g/L resazurin and autoclaved. After autoclaving, all tubes and the bioreactor were sealed airtight and anaerobized overnight using filter-sterilized N\u003csub\u003e2\u003c/sub\u003e gas while being mixed at a stirring speed of 500 rpm. After anaerobization, the bioreactor was supplemented with 1% (v/v) of each P2 stock solution and 0.4 g/L of cysteine-HCl\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO using needles and syringes. For the main fermentation, the temperature was adjusted to the desired level, and the stirring speed was reduced to 150 rpm. Subsequently, the N\u003csub\u003e2\u003c/sub\u003e gas flow was stopped, 10 mL of a cellulase-water mixed solution (Cellic CTec2) with a cellulase loading of 16 FPU/g was added to the bioreactor and inoculated with 5% (v/v) active \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e culture. 5 mL gas and 2 mL liquid samples were collected and analyzed.\u003c/p\u003e \u003cp\u003eThe bioreactor setup varied depending on the type of fermentation. For butanol production, fermentations were performed at a temperature of 30 \u0026deg;C, with an initial pH of 6.8 and under overpressure, with the gases partially kept in the bioreactor. The bioreactor outlet tube was connected to the pressure relief valve (V07, IMI Norgren, Lichfield, United Kingdom) and set to an absolute pressure of 1.55 bar. After the pressure reached 1.55 bar, the valve was opened, and the gases produced were passed through BlueVCount (BlueSens gas sensor GmbH, Herten, Germany) to measure the volume of gases accumulated in the gas bags. In addition, for the pH-controlled experiment, the pH of the medium was controlled at pH\u0026thinsp;\u0026ge;\u0026thinsp;5.0 by using the bioreactor's base pump containing 4 M NaOH. For hydrogen production, fermentation was performed at 37 \u0026deg;C, with an initial pH of 8.0 and under atmospheric pressure, releasing the gas from the bioreactor. In this fermentation, the bioreactor outlet tube was directly connected to the BlueVCount and accumulated in the gas bags. All experiments were incubated for 120 hours and performed in duplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePSSF process from pretreated Enset fiber in the bioreactor\u003c/h2\u003e \u003cp\u003eThe PSSF medium was prepared similarly to the SSF medium except for the substrate loading, which was 7% (w/v) of pretreated Enset fiber (70 g/L), and operating conditions. Before inoculation with an active preculture of \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e, the media was hydrolyzed for 2 hours. The prehydrolysis experiment was started by adjusting the pH to 5.0 and the temperature to 40 \u0026deg;C. Then, the cellulase enzyme was added at 16 FPU/g cellulase loading and stirred at 500 rpm. Afterwards, the temperature and stirrer speed were minimized to 30 \u0026deg;C and 150 rpm, respectively, and the initial pH was adjusted to 6.8, then inoculated with 5% (v/v) active preculture of \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e. During the fermentation, the pH was controlled with 4M NaOH to pH\u0026thinsp;\u0026ge;\u0026thinsp;5.0 and the pressure to 1.5 bar. The experiment was incubated for 120 hours and performed in duplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAnalytical methods\u003c/h2\u003e \u003cp\u003eThe liquid samples were analysed following the method described by Seid N. et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The gas pressure developed during fermentation was measured using a manometer, and the gas composition was determined using the Micro GC Fusion\u0026reg; gas analyzer (Inficon, Bad Ragaz, Switzerland) equipped with PLOT and WCOT columns and utilizing Argon and Helium as carrier gases [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The volume of gas generated from the bioreactor was measured using BlueVCount. The total moles of the gas were calculated by summing the moles of gas within the bioreactor and the moles of gas passing through the BlueVCount.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express gratitude to the technical team at the Institute of Process Engineering in Life Sciences 2: Electrobiotechnology, Karlsruhe Institute of Technology, for their valuable assistance. Additionally, the authors thank to Daniel Richter for his contribution to milling the Enset fiber required for the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization,\u0026nbsp;N.S.; methodology,\u0026nbsp;N.S., H.A. and\u0026nbsp;A.N.; formal\u0026nbsp;analysis, N.S.; investigation, N.S.\u0026nbsp;and L.W.; resources, N.S. and A.N.; data curation, N.S.; writing-original\u0026nbsp;draft preparation, N.S.; writing-review and editing, A.N.\u0026nbsp;and H.A.; visualization,\u0026nbsp;N.S.; supervision, A.N.; project administration, A.N.; funding acquisition, N.S. and A.N.;\u0026nbsp;All authors read and approved the final\u0026nbsp;manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the DAAD-EECBP Homegrown Ph.D. Scholarship Program 2019, and the APC was funded by the Karlsruhe Institute of Technology (KIT) Publication Fund.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRajak, J. 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Cell Fact.\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, 1\u0026ndash;12.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hydrogen, Butanol, Enset fiber, C. saccharoperbutylacetonicum, SSF, PSSF","lastPublishedDoi":"10.21203/rs.3.rs-3836946/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3836946/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eReplacing fossil fuels with biofuels such as hydrogen and butanol has the potential to reduce carbon emissions and improve the sustainability of the energy system. Enset fiber is a promising feedstock to produce these biofuels. \u003cem\u003eClostridium saccharoperbutylacetonicum\u003c/em\u003e DSM 14923 can produce hydrogen and butanol simultaneously; however, to achieve the best results, each product requires different fermentation conditions. This study aimed to optimize process parameters for the simultaneous saccharification and fermentation (SSF) process to produce hydrogen and butanol from Enset fiber, and to investigate the impact of pH control in the SSF process in the bioreactor.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe SSF process in bottles resulted in a elevated butanol concentration of 11.36 g/L with a yield of 0.23 g/g and a productivity of 0.16 g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at the optimal process parameters of 5% (w/v) substrate loading, 16 FPU/g cellulase loading, and 100 rpm agitation speed from pretreated Enset fiber. A comparable result was observed in the bioreactor during the pH-uncontrolled SSF process; however, the butanol productivity decreased to 0.095 g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Furthermore, the pre-hydrolysis simultaneous saccharification and fermentation (PSSF) process in the bioreactor with a 7% (w/v) substrate loading led to the highest butanol concentration and productivity of 12.84 g/L and 0.104 g/L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e respectively. By further optimizing the SSF process, we achieved 442.57 mmol of hydrogen with a yield of 198.27 mL/g-Enset fiber in the bioreactor under the optimal conditions of temperature 37\u0026deg;C, initial pH 8.0 and atmospheric pressure.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn this study, a scalable process for both products was developed to achieve similar results to bottle-scale experiments, indicating potential applicability on an industrial scale. This is the first study to combine \u003cem\u003eC. saccharoperbutylacetonicum\u003c/em\u003e and Enset fiber for the SSF process and contributes to the development of sustainable energy sources.\u003c/p\u003e","manuscriptTitle":"Stirring the Hydrogen and Butanol production from Enset Fiber via Simultaneous Saccharification and Fermentation (SSF) process","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-09 06:35:26","doi":"10.21203/rs.3.rs-3836946/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"02f4a203-1b83-48aa-91e6-e0bef6ce5d31","owner":[],"postedDate":"January 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-14T07:58:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-09 06:35:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3836946","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3836946","identity":"rs-3836946","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}