High Butanol Concentration Research Articles

Continuous Fermentation Coupled with Online Gas Stripping for Effective Biobutanol Production

The main problems with the butanol fermentation process include high cost of grain raw materials, low product concentration and low butanol productivity caused by butanol cytotoxicity. In this study, cassava, a cheap crop, was used as the raw material. A symbiotic system TSH06, which possesses the capability to synthesize butanol under non-strict anaerobic conditions, was used as the fermentation strain. The fermentation performance of TSH06 in a cassava system was investigated. In order to eliminate product inhibition and promote the concentration and productivity of butanol, a strategy of continuous fermentation coupled with online gas stripping was developed. By using the strategy of two-stage continuous fermentation using immobilized cells coupled with online gas stripping, the butanol productivity reached 0.9 g/(L·h); at the same time, a high butanol concentration was achieved, and the concentration of butanol obtained in the condensate reached 71.2 g/L.

Potential of Butanol Production from Thailand Marine Macroalgae Using Clostridium beijerinckii ATCC 10132-Based ABE Fermentation.

The economical bio-butanol-based fermentation process is mainly limited by the high price of first-generation biomass, which is an intensive cost for the pretreatment of second-generation biomass. As third-generation biomass, marine macroalgae could be potentially advantageous for conversion to clean and renewable bio-butanol through acetone-butanol-ethanol (ABE) fermentation. In this study, butanol production from 3 macroalgae species (Gracilaria tenuistipitata, Ulva intestinalis, and Rhizoclonium sp.) by Clostridium beijerinckii ATCC 10132 was assessed comparatively. The enriched C beijerinckii ATCC 10132 inoculum produced a high butanol concentration of 14.07g/L using 60g/L of glucose. Among the three marine seaweed species, G. tenuistipitata exhibited the highest potential for butanol production (1.38g/L). Under the 16 conditions designed using the Taguchi method for low-temperature hydrothermal pretreatment (HTP) of G. tenuistipitata, the maximum reducing sugar yield rate of 57.6% and ABE yield of 19.87% were achieved at a solid to liquid (S/L) ratio of 120, temperature of 110°C, and holding time of 10min (Severity factor, R0 1.29). In addition, pretreated G. tenuistipitata could be converted to 3.1g/L of butanol using low-HTP at an S/L ratio of 50g/L, temperature of 80°C (R0 0.11), and holding time of 5min. This article is protected by copyright. All rights reserved.

Impact on combustion stability, fuel economy and emission compliance with butanol enleanment strategy in gasoline engine – A drop-in fuel approach

In this work, the butanol was investigated at various concentrations with gasoline at three different throttle positions to identify the potential replacement in gasoline engines. Fueling with the butanol blends, the enhanced performance of better fuel saving rate and increased thermal efficiency were observed with the Bu20 and Bu40 blends. Moreover, the increase of butanol ratio displayed a remarkable reduction in the carbon monoxide and carbon dioxide emissions at all the throttle positions as well as the reduction in hydrocarbon was achieved at 80% of the throttle position. However, this reduction was not observed in the oxides of nitrogen emissions at all the throttle positions and the hydrocarbon emissions at low throttle positions. The combustion characteristics such as in-cylinder pressure and heat release rate of Bu20 and Bu40 were witnessed to be much better than gasoline and this effectiveness was not observed with higher butanol concentrations. Also, better stability in the combustion process was observed for all the butanol blends where the CoV of Pmax was within 10% except the Bu0 and Bu100. In conclusion it was identified that the Bu40 would be a most potential replacement and Bu60 can provide almost equivalent output responses to gasoline in a spark ignition engine.

Combining ABE fermentation and anaerobic digestion to treat with lipid extracted algae for enhanced bioenergy production

As a downstream process output, biobutanol can be produced via acetone, butanol, and ethanol (ABE) fermentation from lipid-extracted algae (LEA), but the leftover residue has not been treated for additional value. In current study, LEA were acid hydrolyzed to extract glucose into the hydrolysate, which was then used for ABE fermentation to produce butanol. In the meantime, anaerobic digestion was performed on the hydrolysis residue to produce methane and release nutrients for algae recultivation. To optimize butanol and methane production, several carbon or nitrogen supplements were applied. The results showed that the hydrolysate produced a high butanol concentration of 8.5 g/L with bean cake supplemented, and the residue co-digested with wastepaper had a higher methane production compared to the direct anaerobic digestion of LEA. The causes of the enhanced performances were discussed. The digestates were reused for algae recultivation and were proved to be effective for algae and oil reproduction. The combined process of ABE fermentation and anaerobic digestion was thus proved a promising technique to treat LEA for economic benefit.

Effectiveness of Low-Cost Bioreactors Integrated with a Gas Stripping System for Butanol Fermentation from Sugarcane Molasses by Clostridium beijerinckii

The effectiveness of column bioreactors for butanol fermentation from sugarcane molasses by Clostridium beijerinckii TISTR 1461 was investigated. This fermentation was operated at an initial pH of 6.5 and temperature of 37 °C under anaerobic conditions. A 1-L bubble column bioreactor was used with various gas circulation rates ranging from 0.2 to 1.0 L/min. The highest butanol concentration (PB, 8.72 g/L), productivity (QB, 0.24 g/L∙h) and yield (YB/S, 0.21 g/g) were obtained with a gas circulation of 0.2 L/min. To improve butanol production efficiency, gas-lift column bioreactors with internal and external loops at 0.2 L/min of circulating gas were used. Higher PB (10.50–10.58 g/L), QB (0.29 g/L∙h) and YB/S (0.22–0.23 g/g) values were obtained in gas-lift column bioreactors. These values were similar to those using a more complex 2-L stirred-tank bioreactor (PB, 10.10 g/L; QB, 0.28 g/L h and YB/S, 0.22 g/g). Hence, gas-lift column bioreactors have potential for use as low-cost fermenters instead of stirred-tank bioreactors for butanol fermentation. When the gas-lift column bioreactor with an internal loop was coupled with a gas stripping system, it yielded an enhanced PB and sugar consumption of approximately 9% and 7%, respectively, compared to a system with no gas stripping.

Effect of Endogenous and Exogenous Butyric Acid on Butanol Production From CO by Enriched Clostridia.

Butanol is a potential renewable fuel. To increase the selectivity for butanol during CO fermentation, exogenous acetic acid and ethanol, exogenous butyric acid or endogenous butyric acid from glucose fermentation have been investigated using CO as reducing power, with a highly enriched Clostridium sludge. Addition of 3.2 g/L exogenous butyric acid led to the highest 1.9 g/L butanol concentration with a conversion efficiency of 67%. With exogenous acetate and ethanol supply, the butanol concentration reached 1.6 g/L at the end of the incubation. However, the presence of acetic acid and ethanol favoured butanol production to 2.6 g/L from exogenous butyric acid by the enriched sludge. Finally, exogenous 14 g/L butyric acid yielded the highest butanol production of 3.4 g/L, which was also among the highest butanol concentration from CO/syngas fermentation reported so far. CO addition triggered butanol production from endogenous butyric acid (produced from glucose, Glucose + N2) with as high as 58.6% conversion efficiency and 62.1% butanol yield. However, no efficient butanol production was found from glucose and CO co-fermentation (Glucose + CO), although a similar amount of endogenous butyric acid was produced compared to Glucose + N2. The Clostridium genus occupied a relative abundance as high as 82% from the initial inoculum, while the Clostridia and Bacilli classes were both enriched and dominated in Glucose + N2 and Glucose + CO incubations. This study shows that the supply of butyric acid is a possible strategy for enhancing butanol production by CO fed anaerobic sludge, either via exogenous butyric acid, or via endogenous production by sugar fermentation.

Proteomic Analysis Identifies Dysregulated Proteins in Butanol-Tolerant Gram-Positive Lactobacillus mucosae BR0713-33.

Butanol can be produced biologically through fermentation of lignocellulosic biomass-derived sugars by Gram-positive Clostridium species. For cost-effective production, increased butanol fermentation titers are desired. However, the currently available butanol-fermenting microbes do not tolerate sufficiently high butanol concentrations; thus, new butanol-tolerant strains are desired. One promising strategy is to genetically modify Clostridium species by introducing stress tolerance-associated genes. This study was aimed to seek butanol tolerance genes from other Gram-positive species, which might be better suited than those from Gram-negative E. coli or eukaryotic Saccharomyces cerevisiae. Several butanol-tolerant lactobacilli were reported previously, and Lactobacillus mucosae BR0713–33, which showed the most robust anaerobic growth in 4% butanol, was used here for proteomics analyses. Cellular proteins that responded to 2, 3, and 4% butanol were characterized. Twenty-nine proteins that were identified were dysregulated in response to increased concentrations of butanol in L. mucosae. Seventeen genes involved in coding for stress-tolerant proteins GroES, GroEL, and DnaK and genes involved in substrate utilization, fatty acid metabolism, and nucleotide synthesis were induced by increased butanol, and 12 genes involving energy production (F0F1ATP synthases) and redox balance preservation were repressed by increased butanol. These results can help guide targeted engineering strategies to improve tolerance and production of biobutanol.

Influence of acetic and butyric acid and gas release modes on immobilized Clostridium acetobutylicum (DSMZ 792) in repeated batch experiments

The effects of supplementation of standard media with metabolic intermediates (2 g/L butyric acid, 2 g/L butyric acid + 1.5 g/L acetic acid) on butanol and hydrogen production/yield were evaluated. Fermentations were conducted with immobilized Clostridium acetobutylicum (DSMZ 792). Additionally, the influence of gas release modes (constant gas release vs. interval gas release) was tested in all setups to evaluate butanol and hydrogen production and yields. Fermentations were implemented in repeated batch mode (48 h, 37 °C, 100 rpm). Overpressure conditions had a negative impact on volumetric hydrogen production and hydrogen yield and a positive impact on butanol yield. Supplementation significantly increased the butanol concentrations in constant gas release mode, by 23.6%. When interval gas mode was applied in the supplemented fermentations, the butanol concentrations decreased by 8.4% when butyric acid was fed, and reached similar concentrations when both acids were added. The highest butanol concentration and volumetric butanol production rate of 9.6 ± 0.6 g/L and 0.2 ± 0.0 g/L*h, respectively, were reached with butyric acid supplementation and constant gas release mode. The highest volumetric hydrogen production was reached with the constant gas release mode in fermentations conducted with the addition of both acids, and reached 165 ± 9 mL/L*h.

Bioprocess intensification for isopropanol, butanol and ethanol (IBE) production by fermentation from sugarcane and sweet sorghum juices through a gas stripping-pervaporation recovery process

Butanol and isopropanol are important commodity chemicals with a variety of applications. One of the main obstacles for biobutanol production by IBE (isopropanol–butanol–ethanol) fermentation is the intensive energy consumption for product recovery by conventional distillation due to low butanol titer in fermentation broth caused by butanol toxicity to cells. In the present study, butanol production by batch IBE fermentation coupled to an in situ gas stripping-pervaporation process to recover the butanol is proposed using Clostridium beijerinckii DSM 6423 and a mixture of sugarcane-sweet sorghum juices as substrate. Gas stripping was used to continuously remove butanol from the fermentation broth, followed with pervaporation to further concentrate butanol. The strategy used allows alleviating butanol inhibition and to recuperate a condensate containing high butanol concentration (559 g/L). A kinetic model describing butanol production by IBE fermentation was developed. Kinetic parameters and experimental data were used to estimate the energy consumption of the sugarcane-sweet sorghum IBE production process. It was found that although the IBE production process showed less energy consumption (15%) than the butanol production process by ABE (acetone-butanol-ethanol) fermentation, a substantial improvement is still necessary for the process to be energetically/economically attractive.

Bio-butanol recovery by adsorption/desorption processes

The Acetone-Butanol-Ethanol (ABE) fermentation route to produce chemicals and fuels from renewable resources still suffers from several issues that severely limit the industrial development. In particular, the low butanol concentration in the fermentation broth - due to butanol toxicity to microorganisms – affects the energy sustainability of technologies for butanol recovering. Novel techniques have been investigated in the last years. Among these, adsorption-based technique has been proposed as the most promising one.Present study reports on the assessment of the efficiency of butanol recovery according to adsorption and desorption processes. Tests were carried out using Amberlite XAD-7 as adsorbent material with respect to butanol, ethanol, acetone, and acetic/butyric acids in aqueous solutions. Amberlite XAD-7 was successfully used in fixed-bed column lab apparatus. Two desorption techniques were investigated – thermal drying/desorption and displacement desorption by methanol – to select the best candidate in terms of mass and energy efficiency.This study proved that Amberlite XAD-7 is a potential good adsorbent material to be successfully used in the process of butanol recovery. Amberlite XAD-7 was characterized by high adsorption capacity and selectivity towards butanol. Indeed, the adsorption capacity for acetone, butanol, ethanol, acetic and butyric acid was 17.1, 102.1, 4.2, 14.1 and 21.3 mg/g, respectively when using a solution of 13 g/L of butanol, 5.8 g/L of acetone, 1.6 g/L of ethanol, 6 g/L of acetic acid and 9 g/L of butyric acid. Moreover, adsorbed butanol was recovered as a high butanol concentration solution according to thermal (butanol concentration higher than 800 g/L) and chemical desorption processes (butanol concentration in methanol solution higher than 20 g/L, that is about 1.7 times the concentration in the stream used to saturate the resin).The stability of the performance of Amberlite XAD-7 bed with respect to butanol capture and concentration was provided by carrying out the adsorption–desorption cycle ten times. Butanol recovery was always higher than 97% for both the recovery techniques investigated.The overview of the energy demand for the proposed adsorption/thermal drying/desorption process was reported. The energy demand to concentrate butanol from diluted aqueous mixtures (about 13 g/L) is about 13 MJ/kgB, which is a fraction of the energy content of butanol (36 MJ/kgB).

Determining butanol inhibition kinetics on the growth of Clostridium pasteurianum based on continuous operation and pulse substrate additions

AbstractBACKGROUNDClostridium pasteurianum is a well described strain for the conversion of glycerol into butanol. In general, cell growth kinetics depends on the type of glycerol used and the concentration of butanol in the fermentation broth. However, despite the numerous studies existing on the subject, there is limited information in the literature regarding growth inhibition kinetics due to the cytotoxic effect of butanol on cell growth. This can be attributed to the difficulty of growing cells at high butanol concentration which renders the determination of inhibition kinetics a rather challenging task.RESULTSDuring this study a new approach for the determination of butanol inhibition kinetics on the growth of C. pasteurianum was tested. Specifically, pulses of crude glycerol were applied when steady state was reached during continuous fermentation experiments with increasing butanol concentration in the feed. Combining pulse experiments with batch fermentation at low substrate concentration allowed for accurate determination of the kinetic constants for inhibited growth. This approach also minimized the correlation of the growth constants which often leads to poor identifiability.CONCLUSIONThe proposed experimental approach showed good identifiability of the kinetic parameters for butanol inhibition of the microbial growth and can be proven valuable for the determination of inhibitory effects of highly toxic compounds. © 2019 Society of Chemical Industry

Comparison of mild alkaline and oxidative pretreatment methods for biobutanol production from brewer’s spent grains

In this study, a comparative analysis of different pretreatment strategies on hydrolysis and fermentation of brewers’ spent grain for biobutanol production by C. beijerinckii is presented. Ozone (2.7% w/w, 30 min, 40–60% w/w humidity), sodium hydroxide (1–5 % w/w NaOH, 30 min, 120 ºC, 5–15 % w/w BSG) and hydrogen peroxide (5% w/w H2O2, pH 11.5, 60–180 min, 50 °C, 5–15 % w/w BSG) were the pretreatments analyzed.Ozonolysis was not very effective for either the degradation of lignin or the recovery of fermentable sugars in the enzymatic process under the tested operation conditions. Maximum butanol and ABE concentrations, 5.3± 0.2 g butanol/L and 6.8 ± 0.2 g ABE/L, were observed when low moisture content was used (40% w/w).The peroxide alkaline pretreatment was found to be the most effective for BSG, when it was conducted at 5% BSG for 60 min. Under these conditions, sugar enzymatic yields of 62.8% for glucose and 28.1% for xylose and arabinose, referred to untreated BSG, and product concentrations of 11.0± 0.2 g butanol/L and 13.7 ± 0.2 g ABE/L, were achieved. The alkaline pretreatment at 15% BSG with 1% w/w NaOH also provided high butanol and ABE concentrations (7.3 ± 0.1 g butanol/L and 8.9 ± 0.1 g ABE/L).Both the sodium hydroxide alkaline and peroxide alkaline methods were highly successful as pretreatments of BSG for ABE production, with overall yields of 44.4 g butanol/kg BSG and 54.0 g ABE/kg BSG for NaOH pretreatment (15% BSG, 1% w/w NaOH) and 45.1 g butanol/kg BSG and 56.1 g ABE/kg BSG for H2O2 pretreatment (5% BSG, 60 min).

Energy requirements for the in-situ recovery of biobutanol via gas stripping

Biobutanol has been accepted as the preferred renewable, bio-based additive or replacement for petroleum based fuels. The production of biobutanol has, however, encountered low productivity and titres due to a high toxicity of butanol to the bacteria. This leads to an energy intensive distillation step. Gas stripping has been presented as a method of good potential because of its simplicity and clean operation, with the ability to concentrate the product by several fold. However, these investigations have lacked rigorous analysis of energy requirements, with the energy required at best being estimated using simplified assumptions. A detailed assessment of the energy requirements for gas stripping is presented in this work. This includes a rigorous analysis of the key operating variables of 21 experimental mass balance data sets from 16 investigations in the literature. Higher butanol concentrations in the culture and higher stripping temperatures are shown to be the two most significant parameters influencing the energy efficiency of the process. Gas stripping with distillation consumes 34.2 to 380 MJ/kg butanol and is 41–370% less energy efficient than the use of distillation only for product recovery. Hence an integrated analysis of the production and separation steps is essential to determine overall process benefits.

High-efficient production of biobutanol by a novel Clostridium sp. strain WST with uncontrolled pH strategy

A novel Clostridium sp. strain WST isolated from mangrove sediments demonstrated its unique characteristics of producing high titer of biobutanol from low concentration of substrates via anaerobic fermentation. The strain is able to convert glucose and galactose to high amount of biobutanol up to 16.62 and 12.11 g/L, respectively, and the yields of 0.54 and 0.55 g/g were determined to be much higher than those from the previous reports on Clostridial batch fermentation. Moreover, the inherent strong regulatory system of strain WST also prompts itself to perform the fermentation process without any requirement of pH control. In addition to tolerance of high butanol concentration and negligible production of by-products (e.g., ethanol or acids), this strain has immense potential for the sustainable industry-scale production of biobutanol.

Impact of pH and butyric acid on butanol production during batch fermentation using a new local isolate of Clostridium acetobutylicum YM1.

The effect of pH and butyric acid supplementation on the production of butanol by a new local isolate of Clostridium acetobutylicum YM1 during batch culture fermentation was investigated. The results showed that pH had a significant effect on bacterial growth and butanol yield and productivity. The optimal initial pH that maximized butanol production was pH 6.0 ± 0.2. Controlled pH was found to be unsuitable for butanol production in strain YM1, while the uncontrolled pH condition with an initial pH of 6.0 ± 0.2 was suitable for bacterial growth, butanol yield and productivity. The maximum butanol concentration of 13.5 ± 1.42 g/L was obtained from cultures grown under the uncontrolled pH condition, resulting in a butanol yield (YP/S) and productivity of 0.27 g/g and 0.188 g/L h, respectively. Supplementation of the pH-controlled cultures with 4.0 g/L butyric acid did not improve butanol production; however, supplementation of the uncontrolled pH cultures resulted in high butanol concentrations, yield and productivity (16.50 ± 0.8 g/L, 0.345 g/g and 0.163 g/L h, respectively). pH influenced the activity of NADH-dependent butanol dehydrogenase, with the highest activity obtained under the uncontrolled pH condition. This study revealed that pH is a very important factor in butanol fermentation by C. acetobutylicum YM1.

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.

Genomic and Haplotype Comparison of Butanol Producing Bacteria Based on 16S rDNA

High butanol demand for transportation was support to butanol development. Exploration of butanol producing bacteria using genome comparison and biogeography give role to butanol industrialization. Objective of this research are butanol production, genome comparison and haplotype analysis of butanol producing bacteria from Ranu Pani Lake sediment using 16S rDNA sequences. Highest butanol concentrations were resulted by Paenibacillus polymyxa RP 2.2 isolate (10.34 ± 0.00 g.l -1 ) then Bacillus methylotrophicus RP 3.2 and Bacillus methylotrophicus RP 7.2 isolate (10.11 ± 0.01 g.l -1 and 9.63 ± 0.01 g.l -1 ). Number of bases (T, C, A, G) of group 1 are similar, but different with group 2 . Least G+C content is Clostridium saccharobutylicum Ox29 (51.35%) and highest is Bacillus methylotrophicus RP 7.2. Conserve region (1044 bp) of 16S rDNA higher then variative region (367 bp). The number of 319 bp is PIS whereas single tone as much as 48 bp. There are 17 conserves sequences. All of butanol producing bacterial sequences was clustered to 8 haplotype. Based on source of bacteria, there are three group of haplotype. Group A was isolated from Asia, group B was isolated from America and group C was isolated from Europe.

The role of 1,3-propanediol production in fermentation of glycerol by Clostridium pasteurianum

Waste crude glycerol from biodiesel production can be used to produce biobutanol using Clostridium pasteurianum with the main products being n-butanol, 1,3-propanediol (PDO) and ethanol. There has been much discrepancy and mystery around the cause and effect of process parameters on the product distribution, thus a better understanding of the pathway regulation is required. This study shows that as process pH decreased, the rate of cell growth and CO2 production also decreased, resulting in slower fermentations, increased duration of butanol production and higher butanol concentrations and yields. The production rate of PDO was multi-modal and the role of PDO appears to function in redox homeostasis. The results also showed that C. pasteurianum displayed little biphasic behavior when compared to Clostridia spp. typically used in ABE fermentation due to the alternative glycolysis-independent reductive pathway of PDO production, rendering it suitable for a continuous fermentation process.

Synthesis and design of new hybrid configurations for biobutanol purification

The development of new technologies for biobutanol production by fermentation has resulted in higher butanol concentrations, less by-products and higher volumetric productivities during fermentation. These new technology developments have the potential to provide a production process that is economically viable in comparison to the petrochemical pathway for butanol production. New alternative hybrid configurations based on liquid–liquid extraction and distillation for the biobutanol purification were presented. The alternatives are designed and optimized minimizing two objective functions: the total annual cost (TAC) as an economical index and the eco-indicator 99 as an environmental function. All the new configurations presented reduced the TAC compared to the traditional hybrid configuration, in particular a thermally coupled alternative exhibited a 24.5% reduction of the TAC together with a 11.8% reduction of the environmental indicator. Also intensified sequences represented a promising option in the reduction of the TAC but with some penalty in the eco-indicator.

Enhancing Butanol Production under the Stress Environments of Co-Culturing Clostridium acetobutylicum/Saccharomyces cerevisiae Integrated with Exogenous Butyrate Addition.

In this study, an efficient acetone-butanol-ethanol (ABE) fermentation strategy integrating Clostridium acetobutylicum/Saccharomyces cerevisiae co-culturing system with exogenous butyrate addition, was proposed and experimentally conducted. In solventogenic phase, by adding 0.2 g-DCW/L-broth viable S. cerevisiae cells and 4.0 g/L-broth concentrated butyrate solution into C. acetobutylicum culture broth, final butanol concentration and butanol/acetone ratio in a 7 L anaerobic fermentor reached the highest levels of 15.74 g/L and 2.83 respectively, with the increments of 35% and 43% as compared with those of control. Theoretical and experimental analysis revealed that, the proposed strategy could, 1) extensively induce secretion of amino acids particularly lysine, which are favorable for both C. acetobutylicum survival and butanol synthesis under high butanol concentration environment; 2) enhance the utilization ability of C. acetobutylicum on glucose and over-produce intracellular NADH for butanol synthesis in C. acetobutylicum metabolism simultaneously; 3) direct most of extra consumed glucose into butanol synthesis route. The synergetic actions of effective amino acids assimilation, high rates of substrate consumption and NADH regeneration yielded highest butanol concentration and butanol ratio in C. acetobutylicum under this stress environment. The proposed method supplies an alternative way to improve ABE fermentation performance by traditional fermentation technology.