Saccharoperbutylacetonicum N1-4 Research Articles

Biobutanol production from rice bran and de-oiled rice bran by Clostridium saccharoperbutylacetonicum N1-4

Rice bran (RB) and de-oiled rice bran (DRB) have been treated and used as the carbon source in acetone-butanol-ethanol (ABE) production using Clostridium saccharoperbutylacetonicum N1-4. The results showed that pretreated DRB produced more ABE than pretreated RB. Dilute sulfuric acid was the most suitable treatment method among the various pretreatment methods that were applied. The highest ABE obtained was 12.13 g/L, including 7.72 g/L of biobutanol, from sulfuric acid. The enzymatic hydrolysate of DRB (ESADRB), when treated with XAD-4 resin, resulted in an ABE productivity and yield of 0.1 g/L h and 0.44 g/g, respectively. The results also showed that the choice of pretreatment method for RB and DRB is an important factor in butanol production.

Fermentation of sago starch to biobutanol in a batch culture using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

Acetone–butanol–ethanol (ABE) was produced in Clostridium saccharoperbutylacetonicum N1-4 using sago starch as a substrate, and the effects of nutrient supplementation on ABE production were studied. Using sago starch as a substrate led to higher butanol production in C. saccharoperbutylacetonicum N1-4 when compared to other starchy materials as substrates. TYA medium supplemented with sago starch led to enhanced ABE and butanol production; however, P2 medium improved ABE production more than TYA medium. The highest ABE and butanol production was achieved with P2 medium supplemented with 70 g/L of sago starch. Using these fermentation conditions, ABE and butanol were produced at 16.65 and 9.83 g/L, respectively, and productivity was 0.15 g/L h for ABE and 0.09 g/L h for butanol. Using P2 medium supplemented with either 50 g/L enzymatically hydrolyzed sago starch or 70 g/L sago starch without hydrolysis, 9.81 and 9.83 g/L butanol were produced, respectively. These results reveal the importance of media formulation in the biobutanol fermentation of sago starch by C. saccharoperbutylacetonicum N1-4.

Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4

We investigated butanol production from crystalline cellulose by cocultured cellulolytic Clostridium thermocellum and the butanol-producing strain, Clostridium saccharoperbutylacetonicum (strain N1-4). Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by cocultured Clostridium acetobutylicum and Clostridium beijerinckii than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system.

Development of high-speed and highly efficient butanol production systems from butyric acid with high density of living cells of Clostridium saccharoperbutylacetonicum

Living cells are alive and have the butanol-producing ability but not much proliferation under nitrogen source-limited condition. We investigated various butanol production systems with high density of living cells of Clostridium saccharoperbutylacetonicum N1-4 supplemented with methyl viologen (MV) as an electron carrier and nutrient dosing for activity regeneration. In continuous butanol production with high density of living cells, butanol yield was drastically increased from 0.365 C-mol/C-mol with growing cells to 0.528 C-mol/C-mol at a dilution rate of 0.85 h⁻¹, being increased with the butanol to total solvent ratio. This yield was increased to 0.591 C-mol/C-mol by adding 0.01 mM MV. MV addition increased not only butanol yield but also butanol concentration and productivity as compared to those without MV addition. However, living cells lost their activity with incubation time, which lowered the operational stability of the system. Therefore, to maintain constant stability, activity regeneration was carried out with high density of living cells and MV. This system produced butanol at high concentration (9.40 g l⁻¹) and productivity (7.99 g l⁻¹ h⁻¹) for approximately 100 h with maintenance of considerably high yield of butanol (0.686 C-mol/C-mol). Thus, we established a high-speed and highly efficient butanol production system.

Bioconversion of Butyric Acid to Butanol by Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) in a Limited Nutrient Medium

This study was designed to investigate the ability of Clostridium saccharoperbutylacetonicum N1-4 to produce butanol in a limited nutrient medium using mixtures of glucose and butyric acid as substrates. Specific combinations of glucose and butyric acid were found to influence the enhancement and retardation of butanol production as well as the reduction and modulation of the number of bacterial cells. Increasing the butyric acid concentration leads to the inhibition of bacterial growth, whereas the presence of (0–5 g/L) butyric acid and (0–10 g/L) glucose enhances the butanol production. The combination of 5 g/L butyric acid with 5 and 10 g/L of glucose was found to be the most suitable, but the use of glucose at concentrations greater than 10 g/L shifted the optimal butyric acid concentrations to 10 and 15 g/L for maximum butanol production signifying the requirement of a specific combination of glucose and butyric acid for enhanced butanol production in the fermentation process. C. saccharoperbutylacetonicum N1-4 demonstrated the ability to produce butanol in the absence of glucose, but no acetone or ethanol was produced under these conditions, reflecting the nature of the pathways involved in the production of butanol using only butyric acid. Ten grams per litre of butyric acid was found able to produce 13 g/L of butanol in the presence of 20 g/L of glucose, and 0.7 g/L butanol was produced in the absence of glucose. This study indicates the importance of the glucose to butyric acid ratio to the enhancement of butanol production.

Applications of the Box-Wilson Design Model for Bio-hydrogen Production using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

Box-Wilson design (BWD) model was applied to determine the optimum values of influencing parameters in anaerobic fermentation to produce hydrogen using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). The main focus of the study was to find the optimal relationship between the hydrogen yield and three variables including initial substrate concentration, initial medium pH and reaction temperature. Microbial growth kinetic parameters for hydrogen production under anaerobic conditions were determined using the Monod model with incorporation of a substrate inhibition term. The values of micro(max) (maximum specific growth rate) and K, (saturation constant) were 0.398 h(-1) and 5.509 g L(-1), respectively, using glucose as the substrate. The experimental substrate and biomass-concentration profiles were in good agreement with those obtained by the kinetic-model predictions. By varying the conditions of the initial substrate concentration (1-40 g L(-1)), reaction temperature (25-40 degrees C) and initial medium pH (4-8), the model predicted a maximum hydrogen yield of 3.24 mol H2 (mol glucose)(-1). The experimental data collected utilising this design was successfully fitted to a second-order polynomial model. An optimum operating condition of 10 g L(-1) initial substrate concentration, 37 degrees C reaction temperature and 6.0 +/- 0.2 initial medium pH gave 80% of the predicted maximum yield of hydrogen where as the experimental yield obtained in this study was 77.75% exhibiting a close accuracy between estimated and experimental values. This is the first report to predict bio-hydrogen yield by applying Box-Wilson Design in anaerobic fermentation while optimizing the effects of environmental factors prevailing there by investigating the effects of environmental factors.

Efficient conversion of lactic acid to butanol with pH-stat continuous lactic acid and glucose feeding method by Clostridium saccharoperbutylacetonicum

In order to achieve high butanol production by Clostridium saccharoperbutylacetonicum N1-4, the effect of lactic acid on acetone-butanol-ethanol fermentation and several fed-batch cultures in which lactic acid is fed have been investigated. When a medium containing 20 g/l glucose was supplemented with 5 g/l of closely racemic lactic acid, both the concentration and yield of butanol increased; however, supplementation with more than 10 g/l lactic acid did not increase the butanol concentration. It was found that when fed a mixture of lactic acid and glucose, the final concentration of butanol produced by a fed-batch culture was greater than that produced by a batch culture. In addition, a pH-controlled fed-batch culture resulted in not only acceleration of lactic acid consumption but also a further increase in butanol production. Finally, we obtained 15.5 g/l butanol at a production rate of 1.76 g/l/h using a fed-batch culture with a pH-stat continuous lactic acid and glucose feeding method. To confirm whether lactic acid was converted to butanol by the N1-4 strain, we performed gas chromatography-mass spectroscopy (GC-MS) analysis of butanol produced by a batch culture during fermentation in a medium containing [1,2,3-(13)C(3)] lactic acid as the initial substrate. The results of the GC-MS analysis confirmed the bioconversion of lactic acid to butanol.

Bio-Hydrogen Production using a Two-Stage Fermentation Process

A two-stage fermentation process consisting of dark and photo-fermentation periods was carried out in a batch reactor. In the first stage, glucose was fermented in the dark stage using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564; CSN1-4) to produce acetate, CO2 and H2. The acetate produced in the first stage is fermented to H2 and CO2 by Rhodobacter sphaeroides NCIMB 8253 for further hydrogen production in the second, illuminated stage. The yield of hydrogen in the first stage was about 3.10 mol H2 (mol glucose)(-1) at a glucose concentration of 10 g L(-1), pH 6 +/- 0.2 and 37 degrees C and the second stage yield was about 1.10-1.25 mol H2 (mol acetic acid)(-1) at pH 6.8 +/- 0.2 and 32 degrees C, without removal of the Clostridium CSN1-4. The overall yield of hydrogen in the two-stage process, with glucose as the main substrate was higher than single-stage fermentation.

Production of Acetone–Butanol–Ethanol (ABE) in Direct Fermentation of Cassava by Clostridium saccharoperbutylacetonicum N1-4

In this work, acetone-butanol-ethanol (ABE) fermentation characteristics of cassava starch and cassava chips when using Clostridium saccharoperbutylacetonicum N1-4 was presented. The obtained results in batch mode using a 1-L fermenter showed that C. saccharoperbutylacetonicum N1-4 was a hyperamylolytic strain and capable of producing solvents efficiently from cassava starch and cassava chips, which was comparable to when glucose was used. Batch fermentation of cassava starch and cassava chips resulted in 21.0 and 19.4 g/L of total solvent as compared with 24.2 g/L of total solvent when using glucose. Solvent productivity in fermentation of cassava starch was from 42% to 63% higher than that obtained in fermentation using corn and sago starches in the same condition. In fermentation of cassava starch and cassava chips, maximum butanol concentration was 16.9 and 15.5 g/L, respectively. Solvent yield and butanol yield (based on potential glucose) was 0.33 and 0.41, respectively, for fermentation of cassava starch and 0.30 and 0.38, respectively for fermentation using cassava chips.

Effect of Environmental Parameters on hydrogen Production using Clostridium Saccharoperbutylacetonicum N1-4(ATCC 13564)

Problem statement: Hydrogen gas production by Clostridium can be improved by several ways through media formulation, or suitable environment condition. This study was carried out to investigate the environmental factors effects on hydrogen production using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). Approach: The environmental factor studied includes initial substrate concentration, initial medium pH, temperature, sparging nitrogen and addition of Fe2+. Results: The result showed that the best yield of hydrogen produced (YP/S) was 3.10 moL (moL glucose)-1 when an initial glucose concentration was 10 g L-1, initial pH 6.0±0.2 at temperature 37°C. The volume of hydrogen produced was decreased when higher initial glucose concentration was applied. The yield of hydrogen increased when Fe2+ added to medium at concentration of 25 mg L−1. The yield and growth were further increased by sparging with nitrogen gas. Conclusion: It was observed that the best condition for highest hydrogen yield when initial pH 6.0±0.2 at 37°C and enhanced by adding ferrous sulfate in anaerobic process.

Effect of Environmental Parameters on hydrogen Production using <i>Clostridium Saccharoperbutylacetonicum</i> N1-4(ATCC 13564)

Problem statement: Hydrogen gas production by Clostridium can be improved by several ways through media formulation, or suitable environment condition. This study was carried out to investigate the environmental factors effects on hydrogen production using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). Approach: The environmental factor studied includes initial substrate concentration, initial medium pH, temperature, sparging nitrogen and addition of Fe2+. Results: The result showed that the best yield of hydrogen produced (YP/S) was 3.10 moL (moL glucose)-1 when an initial glucose concentration was 10 g L-1, initial pH 6.0±0.2 at temperature 37°C. The volume of hydrogen produced was decreased when higher initial glucose concentration was applied. The yield of hydrogen increased when Fe2+ added to medium at concentration of 25 mg L−1. The yield and growth were further increased by sparging with nitrogen gas. Conclusion: It was observed that the best condition for highest hydrogen yield when initial pH 6.0±0.2 at 37°C and enhanced by adding ferrous sulfate in anaerobic process.

Hydrogen production using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

Fermentative hydrogen production was carried out using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). This work investigates the effects of initial substrate concentration, initial medium pH, and temperature. The hydrogen yield was about 3.1 mol (mol glucose) −1 when starting with an initial glucose concentration of 10 gl −1 and initial a pH of 6.0 ± 0.2 at a temperature of 37 °C. The volume of hydrogen produced decreased when higher initial glucose concentrations were applied. The most suitable conditions for hydrogen production in a batch reactor were observed at initial pH 6.0 ± 0.2 and 37 °C.

Kinetic study of substrate dependency for higher butanol production in acetone–butanol–ethanol fermentation

A kinetic simulation model of acetone–butanol–ethanol (ABE) fermentation of xylose (Model XYL) was proposed by substituting Embden–Meyerhof–Parnas (EMP) pathway equations in the glucose model (Model GLC) by pentose phosphate (PP) pathway equations of xylose utilization. We estimated the equation parameters of the PP pathway and set other equation parameters to the same as those in Model GLC, by which Model XYL exhibited an r 2 value of 0.901 between the experimental time course of metabolites with initial xylose concentrations ranging from 40.7 to 292 mM and the calculated values. The results with the developed model suggested that Clostridium saccharoperbutylacetonicum N1-4 has a robust metabolic network in acid- and solvent-producing pathways. Furthermore, sensitivity analysis revealed that slow substrate utilization would be effective for higher butanol production; this coincided with the experimental results. Therefore, we consider the proposed model to be one of the best kinetic simulation candidates describing the dynamic metabolite behavior in ABE production.

Novel high-efficient butanol production from butyrate by non-growing Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) with methyl viologen

Non-growing Clostridium saccharoperbutylacetonicum N1-4 hardly produced butanol from only butyrate. As adding glucose to the medium, butyrate utilization and butanol production were stimulated. Addition of 0.1 mM methyl viologen as electron carrier resulted in the highest yield of butanol of 0.671 mol/mol to butyrate and glucose.

Bioconversion of industrial wastewater from palm oil processing to butanol by Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

The feasibility of utilizing enzymatic hydrolysates of separator sludge, a palm oil mill effluent, and sago starch in the acetone–butanol–ethanol fermentation by Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) was investigated. The sludge hydrolysate was used as a growth medium substitute, a fermentation substrate, and as a source of nitrogen and micronutrients. Butanol produced, 3.50 g l −1, was the same when the sludge hydrolysate was used as a second (refreshing) growth medium. As a substrate, initial nitrogen gas sparging shortened the lag phase which then enhanced butanol production. Fermentation without pH control enhanced butanol productivity whereas controlled fermentation at pH 5.5 and 5.8 progressively favored acid production while redox dyes, methyl and benzyl viologen, neutral red and methylene blue, had no effect on solvent production. As a source of nitrogen and micronutrients in sago starch hydrolysate, the final butanol concentration, 10.4 g l −1, was comparable to that in glucose and sago starch hydrolysate media supplemented with tryptone–yeast–acetate (TYA) medium components medium, although productivity was low. Cell growth, butanol and total solvent productivity in TYA-supplemented sago starch hydrolysate and glucose media were the same.

Kinetic modeling and sensitivity analysis of acetone–butanol–ethanol production

A kinetic simulation model of metabolic pathways that describes the dynamic behaviors of metabolites in acetone-butanol-ethanol (ABE) production by Clostridium saccharoperbutylacetonicum N1-4 was proposed using a novel simulator WinBEST-KIT. This model was validated by comparing with experimental time-course data of metabolites in batch cultures over a wide range of initial glucose concentrations (36.1-295 mM). By introducing substrate inhibition, product inhibition of butanol, activation of butyrate and considering the cessation of metabolic reactions in the case of insufficiency of energy after glucose exhaustion, the revised model showed 0.901 of squared correlation coefficient (r(2)) between experimental time-course of metabolites and calculated ones. Thus, the final revised model is assumed to be one of the best candidates for kinetic simulation describing dynamic behavior of metabolites in ABE production. Sensitivity analysis revealed that 5% increase in reaction of reverse pathway of butyrate production (R(17)) and 5% decrease in reaction of CoA transferase for butyrate (R(15)) highly contribute to high production of butanol. These system analyses should be effective in the elucidation which pathway is metabolic bottleneck for high production of butanol.

Production of Acetone, Butanol and Ethanol (ABE) by Clostridium saccharoperbutylacetonicum N1-4 with Different Immobilization Systems

Production of Acetone, Butanol and Ethanol (ABE) by Clostridium saccharoperbutylacetonicum N1-4 with Different Immobilization Systems

High production of acetone–butanol–ethanol with high cell density culture by cell-recycling and bleeding

A continuous acetone–butanol–ethanol (ABE) production system with high cell density obtained by cell-recycling of Clostridium saccharoperbutylacetonicum N1-4 has been studied. In conventional continuous culture of ABE without cell-recycling, the cell concentration was below 5.2 g l −1 and the maximum ABE productivity was only 1.85 g l −1 h −1 at a dilution rate of 0.20 h −1. To obtain a high cell density at a faster rate, we concentrated the solventogenic cells of the broth 10 times by membrane filtration and were able to obtain approximately 20 g l −1 of active cells after only 12 h of cultivation. Continuous culture with cell-recycling was then started, and the cell concentration increased gradually through cultivation to a value greater than 100 g l −1. The maximum ABE productivity of 11.0 g l −1 h −1 was obtained at a dilution rate of 0.85 h −1. However, a cell concentration greater than 100 g l −1 resulted in heavy bubbling and broth outflow, which made it impossible to carry out continuous culture. Therefore, to maintain a stable cell concentration, cell-bleeding was performed together with cell-recycling. At dilution rates of 0.11 h −1 and above for cell-bleeding, continuous culture with cell-recycling could be operated for more than 200 h without strain degeneration and the overall volumetric ABE productivity of 7.55 g l −1 h −1 was achieved at an ABE concentration of 8.58 g l −1.

Utilization of excess sludge by acetone–butanol–ethanol fermentation employing Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

Clostridium saccharoperbutylacetonicum N1-4 could not grow or produce butanol in excess sludge medium. However, adding glucose to the excess sludge medium resulted in a specific growth rate and butanol productivity of 0.29 h(-1) and 0.55 g/l/h, respectively, and the final butanol production reached 9.3 g/l. Since the content of suspended solids in medium reduced to less than 50% of the initial content during acetone-butanol-ethanol (ABE) fermentation, the sludge was quantitatively decreased by this fermentation employing this strain.

High Butanol Production by Clostridium saccharoperbutylacetonicum N1-4 in Fed-Batch Culture with pH-Stat Continuous Butyric Acid and Glucose Feeding Method

A pH-stat fed-batch culture by feeding butyric acid and glucose has been studied in an acetone–butanol–ethanol (ABE) fermentation using Clostridium saccharoperbutylacetonicum N1-4. The specific butanol production rate increased from 0.10 g-butanol/g-cells/h with no feeding of butyric acid to 0.42 g-butanol/g-cells/h with 5.0 g/ l butyric acid. The pH value in broth decreases with butyric acid production during acidogenesis, and then butyric acid reutilization and butanol production result in a pH increase during solventogensis. The pH-stat fed-batch culture was performed to maintain a constant pH and butyric acid concentration in the culture broth, but feeding only butyric acid could not support butyric acid utilization and butanol production. Subsequently, when a mixture of butyric acid and glucose was fed, butyric acid was utilized and butanol was produced. To investigate the effect of the feeding ratio of butyric acid to glucose (B/G ratio), several B/G ratio solutions were fed. The maximum butanol production was 16 g/ l and the residual glucose concentration in broth was very low at a B/G ratio of 1.4. Moreover, yields of butanol in relation to cell mass and glucose utilization were 54% and 72% higher in pH-stat fed-batch culture with butyric acid than that of conventional batch culture, respectively.