Specific Butanol Research Articles

Biosynthesis of NPs CuS/Cu2S and self-assembly with C. beijerinckii for improving lignocellulosic butanol production in staged butyrate-butanol fermentation process

A staged butyrate-butanol fermentation process (SBBFP) has been developed to efficiently produce biobutanol from renewable lignocellulose. The SBBFP combines the fermentation of bioaugmented degradation consortium for butyrate (BDCB) and butanol production of Clostridium beijerinckii, but is still challenged by the lower specific butanol yield (SBY). In this study, a novel technology for synthesizing semiconductor nanoparticles (NPs) and their self-assembly with Clostridium beijerinckii was proposed to enhance the SBY. Results showed that supplement of 0.1 g/L CuSO4 or 0.2 g/L ZnSO4 were more favorable for the formation of butyrate and NPs (CuS/Cu2S or ZnS) in the acidogenic fermentation process of BDCB. Butyrate production achieved 19–21 g/L in the fed-batch fermentation mode from NaOH-pretreated rice straw. Only CuS/Cu2S can be assembled with NPs@C. beijerinckii hybrid system, and a butanol production of 14.6 g/L was obtained using acidogenic fermentation broth and saccharide liquid from rice straw as the co-substrates, with the SBY of 0.17 g/g initial pretreated rice straw. The enhanced butanol synthesis was likely attributed to the upgraded intracellular NADH/NAD+ and specific activity of enzyme CoA-transferase (ctfAB) in C. beijerinckii. The research work provides an efficient approach for biofuels and biochemical production from lignocellulose, along with the extended application of biological nanotechnology.

Formation and multiple catalysis of copper-cysteine complex enhance butanol fermentation from rice straw

Butanol production from rice straw via Acetone-Butanol-Ethanol (ABE) fermentation has been considered a sustainable and cost-effective approach to cope with energy crisis and environmental issues. However, a number of factors, particularly low butanol production and energy-intensive separation, impede its industrial application. In this study, Cu2+, Zn2+, Fe2+, and Ni2+ were individually induced into the ABE fermentation system of Clostridium beijerinckii NCIMB 8052 with butyric acid and sugars both from rice straw as co-substrates to increase the specific butanol yield. The results demonstrated that the additional 0.20 g/L Cu2+ (i.e. 1.25 mmol/L) significantly increased SBY and butanol/acetone to 0.194 ± 0.006 g/g rice straw and 3.90 ± 0.21, respectively, compared to those of 0.126 ± 0.004 g/g rice straw and 2.61 ± 0.17 in control system. The improved butanol production was attributed to the formation of Cu2+-cysteine/Cu+-cysteine complex, which enhanced the adsorption of butyrate from rice straw by upgrading the activity of key enzyme (ctfAB) and NADH/NAD+. The study provides a reinforcement strategy for efficient lignocellulosic butanol production.

Butanol production by Clostridium acetobutylicumATCC 824 using electro‐fermentation in culture medium supplemented with butyrate and neutral red

AbstractBACKGROUNDButanol is a versatile chemical that can be used as fuel, solvent or intermediate in chemical synthesis. Several strategies have been used to increase the availability of reducing power in Clostridium sp. with the aim of maximizing the production of butanol, the use of electron carriers being one of the most effective. Moreover, electrons from an external power source can be transferred to cells through electron carriers such neutral red, or directly reducing NAD+. From a previous metabolic analysis, we hypothesized that adding butyrate to the culture medium and using an external electron source as a co‐reducing agent, along with glucose‐derived reducing power (electro‐fermentation), would result in an increase in the specific butanol productivity.RESULTSButyrate caused a decrease in the maximum concentration of butanol reached at the concentration used (6 g L−1). The supplementation of neutral red to the culture medium by itself and along with electro‐fermentation were effective in stimulating the production of butanol. The mean specific rate of butanol production obtained for electro‐fermentation in medium supplemented with butyrate and neutral red was 307% and 156% higher than that recorded using medium supplemented with butyrate, or butyrate and neutral red, respectively, this value being even 113% higher than that recorded for the non‐supplemented medium.CONCLUSIONSThe presence of butyrate and absence of neutral red in the electro‐fermentation were detrimental for the microorganism. The electro‐fermentation in medium supplemented with neutral red led to an increase in both volumetric and specific productivities of butanol. © 2021 Society of Chemical Industry (SCI).

Effect of changes in continuous carboxylate feeding on the specific production rate of butanol using Clostridium saccharoperbutylacetonicum

Substrate variability in multi-feedstock biorefineries has implications for the stability of downstream bioprocesses. Here, we studied potential effects of fluctuating feed rates and pH on substrate uptake and butanol production by Clostridium saccharoperbutylacetonicum during continuous co-feeding with butyric and acetic acid. Monitoring the fermentation extensively and at high frequency, enabled us to perform irregular fraction experimental designs. The total acid feed rate and the ratio of butyric acid to acetic acid in the feed were found to be significant factors in their uptake by the culture. Furthermore, to maximize the specific butanol production rate, glucose may not be limited and butyric acid should be supplied at a rate of 7.5 mmol L−1 h−1. Surprisingly, pH played a role only indirectly, in its effect on process stability. Obtained results facilitate the control of feed rates based on physiological descriptors, which will be a critical factor in the establishment of multi-feedstock biorefineries.

Enhanced coproduction of hydrogen and butanol from rice straw by a novel two-stage fermentation process

Lignocellulosic biomass has been increasingly used for biofuel production, such as biohydrogen and biobutanol. However, the enzymes used in hydrolysis result in a large increase in production cost. To reduce the enzyme involvement in lignocellulosic butanol, a novel two-stage fermentation process was constructed by combining a Carboxylate Platform and acetone-butanol-ethanol (ABE) fermentation. First, without enzyme involvement, lignocellulose was converted to volatile fatty acids (VFAs) using the Carboxylate Platform. Subsequently, the VFAs were used as feedstock to generate butanol by ABE fermentation of Clostridium beijerinckii NCIMB 8052. Fed with 20 g/L pretreated rice straw, the acidogenic fermentation produced 660 mL/L hydrogen and 6.87 g/L butyric acid, with a total VFAs of 9.52 g/L in the supernatant of fermentation culture (SFC). With 40% rice straw hydrolysate and 60% SFC as co-substrate, hydrogen and butanol productions of 5897 mL/L and 13.8 g/L, respectively, were obtained in the ABE fermentation. Mass balance and energy generation analysis showed that the specific butanol and energy yields from rice straw reached 230 g/kg and 9633.7 kJ/kg, respectively. Additionally, the carbon-flow distribution analysis indicated that the SFC supplement decreased the metabolic flux of acetone producing pathway and increased the metabolic flux of butanol producing pathway. The novel two-stage fermentation process was a more effective approach for energy generation from lignocellulose that avoided external addition of commercial butyric acid and reduced enzyme involvement for fermentation product.

Progress and perspectives on improving butanol tolerance.

Fermentative production of butanol for use as a biofuel or chemical feedstock is regarded as a promising renewable technology that reduces greenhouse gas emissions and has the potential to become a substitute for non-sustainable chemical production route. However, butanol toxicity to the producing microbes remains a barrier to achieving sufficiently high titers for cost-effective butanol fermentation and recovery. Investigations of the external stress of high butanol concentration on butanol-producing microbial strains will aid in developing improved microbes with increased tolerance to butanol. With currently available molecular tool boxes, researchers have aimed to address and understand how butanol affects different microbes. This review will cover the individual organism's inherent responses to surrounding butanol levels, and the collective efforts by researchers to improve production and tolerance. The specific microorganisms discussed here include the native butanol producer Clostridium species, the fermentation industrial model Saccharomyces cerevisiae and the photosynthetic cyanobacteria, the genetic engineering workhorse Escherichia coli, and also the butanol-tolerant lactic acid bacteria that utilize diverse substrates. The discussion will help to understand the physiology of butanol resistance and to identify specific butanol tolerance genes that will lead to informed genetic engineering strategies for new strain development.

High acetone–butanol–ethanol production in pH-stat co-feeding of acetate and glucose

We previously reported the metabolic analysis of butanol and acetone production from exogenous acetate by (13)C tracer experiments (Gao etal., RSC Adv., 5, 8486-8495, 2015). To clarify the influence of acetate on acetone-butanol-ethanol (ABE) production, we first performed an enzyme assay in Clostridium saccharoperbutylacetonicum N1-4. Acetate addition was found to drastically increase the activities of key enzymes involved in the acetate uptake (phosphate acetyltransferase and CoA transferase), acetone formation (acetoacetate decarboxylase), and butanol formation (butanol dehydrogenase) pathways. Subsequently, supplementation of acetate during acidogenesis and early solventogenesis resulted in a significant increase in ABE production. To establish an efficient ABE production system using acetate as a co-substrate, several shot strategies were investigated in batch culture. Batch cultures with two substrate shots without pH control produced 14.20g/L butanol and 23.27g/L ABE with a maximum specific butanol production rate of 0.26g/(gh). Furthermore, pH-controlled (at pH 5.5) batch cultures with two substrate shots resulted in not only improved acetate consumption but also a further increase in ABE production. Finally, we obtained 15.13g/L butanol and 24.37g/L ABE at the high specific butanol production rate of 0.34g/(gh) using pH-stat co-feeding method. Thus, in this study, we established a high ABE production system using glucose and acetate as co-substrates in a pH-stat co-feeding system with C.saccharoperbutylacetonicum N1-4.

Continuous production of n-butanol by Clostridium pasteurianum DSM 525 using suspended and surface-immobilized cells

For n-butanol production by Clostridium pasteurianum DSM 525, a modified reinforced Clostridium medium was used, where glucose was alternated with glycerol and two kinds of continuous fermentation were tested using suspended and surface immobilized cells on corn stover pieces. A steady state, with butanol productivity of 4.2g/Lh, was reached during the packed-bed continuous fermentation at a dilution rate of 0.44h−1. The average n-butanol concentration, yield and the ratio of n-butanol/liquid by-products were 10.4g/L, 33 % and 2.5, respectively. Unexpectedly, during continuous fermentation with suspended cells, at a dilution rate of 0.01h−1, steady-state was not achieved and regular oscillations occurred in all measured variables, i.e. concentrations of glycerol, products and the number of cells stained with the fluorescent dyes carboxy fluorescein diacetate and propidium iodide. A possible explanation for oscillatory/steady-state behavior of suspended/surface-attached cells, respectively, may be specific butanol toxicity (toxicity per cell), which was higher/lower in respective cases, and which might be caused by lower/higher cell numbers respectively in both systems.

Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production.

BackgroundThere is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products.ResultsAn n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin–Benson–Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden–Meyerhof–Parnas and a reduced butanol ATP demand.ConclusionThese results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0355-9) contains supplementary material, which is available to authorized users.

Continuous xylose fermentation by Clostridium acetobutylicum – Assessment of solventogenic kinetics

This work deals with the specific butanol production rate of Clostridium acetobutylicum using xylose – a relevant fraction of lignocellulosic feedstock for biofuel production – as carbon source. The tests were carried out in a CSTR equipped with a microfiltration unit. The dilution rate (D) ranged between 0.02 and 0.22h−1 and the ratio R between the permeate stream rate and the stream fed to the reactor ranged between 14% and 88%. The biomass present in the broth was identified as a heterogeneous cell population consisting of: acidogenic cells, solventogenic cells and spores. The results were processed to assess the concentration of acidogenic cells, solventogenic cells and spores. The specific butanol production rate was also assessed. The max butanol productivity was 1.3gL−1h−1 at D=0.17h−1 and R=30%. A comparison between the results reported in a previous work carried out with lactose was made.

Continuous lactose fermentation by Clostridium acetobutylicum – Assessment of solventogenic kinetics

This work reports the results of a series of tests on the specific butanol production rate by Clostridium acetobutylicum continuous cultures. The tests were carried out using lactose as carbon source to mimic cheese-whey. A continuous stirred tank reactor equipped with a microfiltration unit was used. The dilution rate (D) ranged between 0.02 and 0.15h−1 and the ratio R of the permeate stream rate to the stream fed to the reactor ranged between 14% and 95%. For each set of D and R values, the continuous cultures were characterized in terms of concentration of cells, acids and solvents. Results were processed to assess the concentration of acidogenic cells, solventogenic cells, spores and the specific butanol production rate. The max butanol productivity was 0.5gL−1h−1 at D=0.1h−1 and R=95%. The butanol productivity referred to solventogenic cells was expressed as a function of concentration of lactose, acids and butanol.

Evaluation of recycling the effluent of hydrogen fermentation for biobutanol production: Kinetic study with butyrate and sucrose concentrations

Butyrate in the effluent of hydrogen-producing bioreactor is a potential feed for biobutanol production. For recycling butyrate, this study investigated the kinetics of biobutanol production by Clostridium beijerinckii NRRL B592 from different paired concentrations of butyrate and sucrose in a series of batch reactors. Results show that the lag time of butanol production increased with higher concentration of either sucrose or butyrate. In regression analyses, the maximum specific butanol production potential of 6.49gg−1 of dry cell was projected for 31.9gL−1 sucrose and 1.3gL−1 butyrate, and the maximum specific butanol production rate of 0.87gd−1g−1 of dry cell was predicted for 25.0gL−1 sucrose and 2.6gL−1 butyrate. The specific butanol production potential will decrease if more butyrate is added to the reactor. However, both sucrose and butyrate concentrations are weighted equally on the specific butanol production rate. This observation also is true on butanol yield. The maximum butanol yield of 0.49molmol−1 was projected for 25.0gL−1 sucrose and 2.3gL−1 butyrate. In addition, a confirmation study found butanol yield increased from 0.2 to 0.3molmol−1 when butyrate addition increased from 0 to 1gL−1 under low sugar concentration (3.8gL−1 sucrose). The existence of butyrate increases the activity of biobutanol production and reduces the fermentable sugar concentration needed for acetone–butanol–ethanol fermentation.

A comparison of three pH control methods for revealing effects of undissociated butyric acid on specific butanol production rate in batch fermentation of Clostridium acetobutylicum

pH control has been essential for butanol production with Clostridium acetobutylicum. However, it is not very clear at what pH level the acid crash will occur, at what pH level butanol production will be dominant, and at what pH level butyric acid production will be prevailing. Furthermore, contradictory results have been reported about required acidic conditions for initiation of solventogenesis. In this study, with the aim of further understanding the role of undissociated butyric acid in butanol production, we investigated the correlation between undissociated butyric acid concentration and specific butanol production rate in batch fermentation of Clostridium acetobutylicum by comparing three pH control approaches: NaOH neutralization (at 12, 24 or 36 h), CaCO3 supplementation (2, 5, or 8 g/l) and NaOAc buffering (pH 4.6, 5.0 or 5.6). By neutralizing the fermentation pH to ~5.0 at different time, we observed that neutralization should take place at the beginning of exponential phase (12 h), and otherwise resulting in lower concentrations of undissociated butyric acid, cell biomass and final butanol. CaCO3 supplementation extended cell growth to 36 h and resulted in higher butyrate yield under 8 g/L of CaCO3. In the NaOAc buffering, the highest specific butanol rate (0.58 h−1) was associated with the highest undissociated butyric acid (1.92 g/L). The linear correlation of the undissociated butyric acid with the specific butanol production rates suggested the undissociated butyric acid could be the major driving force for butanol production.

Adaptation of lactic acid bacteria to butanol

Butanol can be produced biologically through fermentation of various substrates by Gram-positive Clostridium species. However, to profitably produce butanol at industrial scales, new microbial biocatalysts with increased tolerance to butanol are needed. In this study we report the isolation and selection of microbes to butanol. Ten strains, including Lactobacillus mucosae strains BR0605-3, BR0605-B15, BR0713-18, BR0713-20, BR0713-30, BR0713-33, Lactobacillus amylovorus strain NE-L 0206-19, Pediococcus parvulus NE-L 0206-31, Lactobacillus crispatus NE-L 0206-47, and Weissella confusa BR0216-18 were found capable of growth in 3–4% butanol after long term adaptation. These strains can be used to study tolerance mechanisms as well as identifying specific butanol stress response genes for development of butanol tolerant microbes.

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.

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.

Characterization of recombinant strains of theClostridium acetobutylicum butyrate kinase inactivation mutant: Need for new phenomenological models for solventogenesis and butanol inhibition?

Two metabolic engineering tools, namely gene inactivation and gene overexpression, were employed to examine the effects of two genetic modifications on the fermentation characteristics of Clostridium acetobutylicum. Inactivation of the butyrate kinase gene (buk) was examined using strain PJC4BK, while the combined effect of buk inactivation and overexpression of the aad gene-encoding the alcohol aldehyde dehydrogense (AAD) used in butanol formation-was examined using strain PJC4BK(pTAAD). The two strains were characterized in controlled pH > or = 5.0 fermentations, and by a recently enhanced method of metabolic flux analysis. Strain PJC4BK was previously genetically characterized, and fermentation experiments at pH > or = 5.5 demonstrated good, but not exceptional, solvent-production capabilities. Here, we show that this strain is a solvent superproducer in pH > or = 5.0 fermentations producing 225 mM (16.7 g/L) of butanol, 76 mM of acetone (4.4 g/L), and 57 mM (2.6 g/L) of ethanol. Strain PJC4BK(pTAAD) produced similar amounts of butanol and acetone but 98 mM (4.5 g/L) of ethanol. Both strains overcame the 180 mM (13 g/L) butanol toxicity limit, without any selection for butanol tolerance. Work with strain PJC4BK(pTAAD) is the first reported use of dual antibiotic selection in C. acetobutylicum. One antibiotic was used for selection of strain PJC4BK while the second antibiotic selected for the pTAAD presence. Overexpression of aad from pTAAD resulted in increased ethanol production but did not increase butanol titers, thus indicating that AAD did not limit butanol production under these fermentation conditions. Metabolic flux analysis showed a decrease in butyrate formation fluxes by up to 75% and an increase in acetate formation fluxes of up to 100% during early growth. The mean specific butanol and ethanol formation fluxes increased significantly in these recombinant strains, up to 300% and 400%, respectively. Onset of solvent production occurred during the exponential-growth phase when the culture optical density was very low and when total and undissociated butyric acid levels were <1 mM. Butyrate levels were low throughout all fermentations, never exceeding 20 mM. Thus, threshold butyrate concentrations are not necessary for solvent production in these stains, suggesting the need for a new phenomenological model to explain solvent formation.

Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: Need for new phenomenological models for solventogenesis and butanol inhibition?

Two metabolic engineering tools, namely gene inactivation and gene overexpression, were employed to examine the effects of two genetic modifications on the fermentation characteristics of Clostridium acetobutylicum. Inactivation of the butyrate kinase gene (buk) was examined using strain PJC4BK, while the combined effect of buk inactivation and overexpression of the aad gene—encoding the alcohol aldehyde dehydrogense (AAD) used in butanol formation—was examined using strain PJC4BK(pTAAD). The two strains were characterized in controlled pH ≥ 5.0 fermentations, and by a recently enhanced method of metabolic flux analysis. Strain PJC4BK was previously genetically characterized, and fermentation experiments at pH ≥ 5.5 demonstrated good, but not exceptional, solvent-production capabilities. Here, we show that this strain is a solvent superproducer in pH ≥ 5.0 fermentations producing 225 mM (16.7 g/L) of butanol, 76 mM of acetone (4.4 g/L), and 57 mM (2.6 g/L) of ethanol. Strain PJC4BK(pTAAD) produced similar amounts of butanol and acetone but 98 mM (4.5 g/L) of ethanol. Both strains overcame the 180 mM (13 g/L) butanol toxicity limit, without any selection for butanol tolerance. Work with strain PJC4BK(pTAAD) is the first reported use of dual antibiotic selection in C. acetobutylicum. One antibiotic was used for selection of strain PJC4BK while the second antibiotic selected for the pTAAD presence. Overexpression of aad from pTAAD resulted in increased ethanol production but did not increase butanol titers, thus indicating that AAD did not limit butanol production under these fermentation conditions. Metabolic flux analysis showed a decrease in butyrate formation fluxes by up to 75% and an increase in acetate formation fluxes of up to 100% during early growth. The mean specific butanol and ethanol formation fluxes increased significantly in these recombinant strains, up to 300% and 400%, respectively. Onset of solvent production occurred during the exponential-growth phase when the culture optical density was very low and when total and undissociated butyric acid levels were <1 mM. Butyrate levels were low throughout all fermentations, never exceeding 20 mM. Thus, threshold butyrate concentrations are not necessary for solvent production in these stains, suggesting the need for a new phenomenological model to explain solvent formation. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 1–11, 2000.

Continuous butanol/isopropanol fermentation in down‐flow column reactor coupled with pervaporation using supported liquid membrane

Continuous butanol/isopropanol fermentation with immobilized Clostridium isopropylicum was performed in a downflow column reactor using molasses as the substrate. In order to prevent product inhibition and at the same time obtain high concentration of the products, the column reactor was coupled with a pervaporation module using a supported liquid membrane. The liquid membrane was prepared with oleyl alcohol nontoxic to the microorganism. In comparison with the continuous fermentation without product removal, the specific butanol production rate was 2 times higher. The butanol concentration in the permeate was 230 kg/m(3), which was about 50 times higher than that in the culture broth. A numerical investigation suggested a further increase in the productivity by improving the module construction.

NADH fluorscence in a carbon‐limited fermentation

The continuous flow acetone-butanol fermentation conducted at lowered inlet feed sugar concentrations and at a constant dilution rate D =0.075 h(-1) demonstrated a significant decreases in the availability of the cell population "reduction energy" (F/X), resulting in an exclusive accumulation of intermediate acids under those conditions. The cultures resumed its solvent production activity when the inlet sugar concentration in the feed stream was increased from 20 to 40 g/L at the same low growth/dilution rates. A linear correlation between the culture reduction content (F/X) and the specific butanol rate (q(B)) was observed under the present conditions, indicating the necessity of the NADH availability for the increased solvent production.