Butanol Concentration Research Articles

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.

Solvent production from rice straw by a co-culture of Clostridium acetobutylicum and Saccharomyces cerevisiae: effect of pH control

One of the challenges in biofuel production from lignocellulosic wastes is to improve its conversion to solvents; therefore, new strategies to enhance xylose uptake are required due to be the secondary abundant sugar. In this context, a novel fermentation strategy integrating a co-culture of Clostridium acetobutylicum and Saccharomyces cerevisiae with pH control was developed. Initially, two different buffers, ammonium acetate and calcium carbonate, were tested under pHmin > 4.8 by fermenting 60 g L−1 of glucose with the C. acetobutylicum monoculture. Ammonium acetate was selected for fermenting media as butanol production was increased from 9.8 to 10.9 g L−1 over the calcium carbonate test. Comparing with the spontaneous acetone-butanol-ethanol (ABE) fermentation with C. acetobutylicum when no xylose consumption was observed, xylose consumption was efficiently increased by controlling pHmin > 4.8. The xylose consumption was > 47% either by using a 45:15 g L−1 glucose:xylose mixture or with rice straw (RS) hydrolysate. Clostridium monoculture using RS hydrolysate and pHmin > 4.8 produced a butanol (ABE) concentration of 6.5 (9.5) g L−1. While it increased to 7.0 (13.1) g L−1 when the co-culture with S. cerevisiae was used using same pH regulation strategy mainly due to ethanol increase up to 2.7 g L−1. Moreover, the xylose uptake doubled to 94% due to amino-acid secretion by yeast. Overall, this combined strategy was a very effective method for promoting sugar consumption and ABE solvent production from lignocellulosic waste.

Dynamical Anomaly of Aqueous Amphiphilic Solutions: Connection to Solution H-Bond Fluctuation Dynamics?

We have investigated the possible connection between “dynamical anomaly” observed in time-resolved fluorescence measurements of reactive and nonreactive solute-centered relaxation dynamics in aqueous binary mixtures of different amphiphiles and the solution intra- and interspecies H-bond fluctuation dynamics. Earlier studies have connected the anomalous thermodynamic properties of binary mixtures at very low amphiphile concentrations to the structural distortion of water. This is termed as “structural anomaly.” Interestingly, the abrupt changes in the composition-dependent average rates of solute relaxation dynamics occur at amphiphile mole fractions approximately twice as large as those where structural anomalies appear. We have investigated this anomalous solution dynamical aspect by considering (water + tertiary butanol) as a model system and performed molecular dynamics simulations at several tertiary butanol (TBA) concentrations covering the extremely dilute to the moderately concentrated regimes. The “dynamical anomaly” has been followed via monitoring the composition dependence of the intra- and interspecies H-bond fluctuations and reorientational relaxations of TBA and water molecules. Solution structural aspects have been followed via examining the tetrahedral order parameter, radial and spatial distribution functions, numbers of H bonds per water and TBA molecules, and the respective populations participating in H-bond formation. Our simulations reveal abrupt changes in the H-bond fluctuations and reorientational dynamics and tetrahedral order parameter at amphiphile concentrations differing approximately by a factor of 2 and corroborates well with the steady-state and the time-resolved spectroscopic measurements. This work therefore explains, following a uniform and cogent manner, both the experimentally observed structural and dynamical anomalies in microscopic terms.

A novel integrated fermentation/recovery system for butanol production by Clostridium acetobutylicum

Intensive butanol production was carried out by immobilized cells of Clostridium acetobutylicum in a novel continuous fermentation system. The fermentation system consisted of four packed bed biofilm reactors (PBBR) connected in series. The novelty of the proposed system is: i) the use of a novel stagewise simulated moving bed (SMB) design/operational mode; ii) the integration of the reaction and the product recovery sections. The SMB operational mode was accomplished by equipping the PBBR with manifolds and time-actuated switching valves. PBBR integration with the product recovery section was also tested. An adsorption column was operated between the 3rd and 4th reactor of the series.The tuning of the PBBR system overall dilution rate (DOV) allowed to maximize: i) sugar conversion (DOV <0.150 h−1); ii) butanol concentration (0.650 h−1<DOV< 0.900 h−1); iii) butanol productivity (13.0 g/Lh at DOV = 0.900 h−1).The PBBR system was upgraded by integrating it with an in-line adsorption column and it guaranteed a twofold increase in butanol productivity (up to 22 g/Lh) and an average butanol concentration close to 24.0 g/L.

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.

Modeling fixed bed bioreactors for isopropanol and butanol production using Clostridium beijerinckii DSM 6423 immobilized on polyurethane foams

Isopropanol-butanol fermentation carried out by C. beijerinckii can be improved by immobilizing cells in a fixed bed bioreactor. In this study, a fixed bed bioreactor was developed using polyurethane foams as a solid support for biofilm growth. The dilution rate was increased over time to follow biofilm development while avoiding solvent accumulation within the bioreactor. A phenomenological model was designed and successfully described batch and continuous fixed bed bioreactor dynamics. The parameters regarding biofilm detachment and attachment were estimated using a bioreactor design allowing biofilm sampling during a continuous fermentation. This last experiment was operated during 550 h and reached a maximum productivity of 1.5 g.L −1.h −1. The model prediction capacity was tested by describing a continuous fermentation in a fixed bed bioreactor column over 800 h. This latter bioreactor enabled a maximum productivity of 2.5 g.L −1 h −1 in 650 h of continuous fermentation. A high solvent concentration (13.5 g.L −1) with a butanol concentration close to the toxicity threshold (7.5 g.L −1) was obtained during the fermentation process. The process enhanced the isopropanol butanol fermentation productivity. The model properly described the batch fermentation and the immobilized cells fermentation. However, a 20% difference between experimental and modeled data was observed. Therefore, the model should be improved to better described such fermentation systems.

High butanol/acetone ratio featured ABE production using mixture of glucose and waste Pichia pastoris medium-based butyrate fermentation supernatant.

In this study, butanol (ABE) fermentations were implemented in a 7 L anaerobic fermentor, by directly using the mixture of glucose solution with the corn/waste Pichia pastoris medium-based butyrate fermentation supernatants (BFS II) as the co-substrate, followed by consecutively feeding of the BFS and concentrated glucose solution. When compared with the major index of ABE fermentation using 150g/L corn-based medium, butanol concentration could be maintained at high level of 12.7-12.8g/L, butanol/acetone (B/A) largely increased from ~ 2.0 to 4.4-5.0, butanol yield on total carbon sources increased from 0.32-0.34 to 0.39-0.41 (mol base) with a higher butyrate/glucose consumption ratio of 37%-53%. Efficient utilization of butyrate, SO42-, amino acids, oligosaccharides, etc. in BFS II and the intracellular NADH contributed to the ABE fermentation performance improvement. The proposed strategy could be considered as the second utilization of waste Pichia pastoris, which could save raw materials/operating costs, fully use the oligosaccharides/SO42- in BFS II to relieve the working loads in downstream waste water treatment process, and increase fermentation products diversity/flexibility to deal with the varied marketing prices and requirements.

PROMOTION OF COBALT-CONTAINING HYDROTALCITE MATERIALS WITH POTASSIUM, CERIA, AND ZIRCONIA FOR CO2 SORPTION-ASSISTED REFORMING OF BUTANOL TO H2

Sorption-enhanced steam butanol reforming (SESBR) is capable of producing pure hydrogen (H2). In CO2 sorption-enhanced (or sorption-assisted) reaction processes, the application of hybrid bifunctional materials consisting of a reforming catalyst and CO2 sorbent is encouraging. In this work, a novel material (CoMgHTlc, here denoted by HM1) containing cobalt catalyst and Mg-based hydrotalcite was synthesized using the coprecipitation method. HM1 was then promoted with K, Zr, or Ce using incipient wet impregnation to yield K-HM1, Zr-HM1, and Ce-HM1, respectively. SESBR was investigated in a fixed-bed reactor between 723 and 923 K. Gas hourly space velocity (GHSV) was changed between 3000 and 6000 mL/g h. Butanol concentration in the feed was varied to provide steam-to-carbon ratios (S/C) ranging from 3 to 7.5 mol/mol. All materials were stable and exhibited a high pre-breakthrough period. At 873 K, H2 concentration of the product stream was 97.3 mol % over Ce-HM1; the CO2-sorption capacity was 1.43 mol CO2/kg sorbent.

Bioprocess intensification for acetone-butanol-ethanol fermentation from brewer's spent grain: Fed-batch strategies coupled with in-situ gas stripping

A fed-batch ABE fermentation process coupled with in-situ gas-stripping to mitigate butanol toxicity was investigated. Two feeding strategies were compared: pulses of sugars and continuous feeding of the liquid released in the dilute acid pretreatment of brewer's spent grain. The concentrations of butanol (13.2 g L-1 total and 50 g L-1 average in the condensates for glucose pulse feeding) were higher than those obtained under batch conditions, showing that in-situ gas-stripping can relieve butanol toxicity. The continuous feeding of the fed-batch reactor produced similar butanol concentrations (10.2 g L-1 total and 65 g L-1 in the condensates), more stable concentrations of solvents in the condensates and enhanced monosaccharides uptake (99.1%) in comparison with the pulse feeding strategy. The efficient utilization of the enzymatic and pretreatment hydrolysates in the same fed-batch reactor is an integrated approach that could reduce capital and operating costs. The mathematical model proposed showed good performance to predict concentrations in the fermentation broth.

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).

Influence of nickel on butanol production by Clostridium beijerinckii using hydrolyzate from green coconut shell

The improvement of biotechnological processes capable of transforming agro-industrial waste into products with high added value has stood out in the area of renewable energies, promoting positive impacts to the environment. Thus, the present work evaluated the influence of nickel on the conversion of fermentable sugars, present in the green coconut shell hydrolyzate (GCSH), into butanol and other products. Fermentation assays were performed at 37 °C, starting with 19.4 g.L-1 of sugars and 1.0 g.L-1 of inoculum (C. beijerinckii). The GCSH was supplemented with tryptone, yeast extract, ammonium acetate, minerals and phosphate buffer. Two conditions were tested: with and without addition of nickel. Concentrations of sugars (glucose and xylose), intermediate products (organic acids), acetone, butanol, and ethanol were determined by high performance liquid chromatography (HPLC). The results show that the butanol production was higher from GCSH without addition of nickel, reaching a concentration of 2.14 g.L-1 of butanol. Therefore, the presence of nickel in the hydrolyzate was not favorable in the production of butanol under the studied process conditions.

The mechanism of high butanol acetone ratio in ABE fermentation with fern root as substrate

The butanol acetone ratio (B/A ratio) is a critical index to evaluate the process of ABE fermentation. In this article, fern root starch with yeast extract added (FY) was used as substrate for ABE fermentation. The final B/A ratio in a 5 L anaerobic fermentor reached the highest levels of 3.42, which signified an increment of 41% compared with corn. The mechanisms for the high B/A ratio of FY substrate were as follows: 1) the weakened organic acid circuit resulted in decreased acetone synthesis; 2) the NADH level of butanol synthesis was high; 3) coA transferase activity was low and butanol dehydrogenase activity was high. The final butanol concentration and substrate utilization were examined in a 5 L anaerobic fermentor, and the results validated the feasibility of ABE fermentation with FY as substrate.

Home-made cider brandy (Calvados): analysis of distillation fractions by gas chromatography-mass spectrometry method

In this study, the distillation fractions of cider brandy produced illegally in Lithuania using gas chromatographic-mass spectrometry method were investigated. The conditions of gas chromatography-mass spectrometry were firstly optimized. A qualitative analysis of three distillation fractions was performed and estimated. The first fraction (initial) of distillation products was collected for 0.5 h, the second one (intermediate) was collected for the next 1.5 h and the third (main) fraction for the next 5 h. A method of the quantitative analysis of different volatile analytes such as acetaldehyde, methanol, ethanol, 1-propanol, isobutanol, 1-butanol, isoamyl alcohol and benzyl alcohol in the distillation products was also developed. The concentration of ethanol decreased significantly in the main fraction in comparison with those of the initial and intermediate fractions. The concentration of 1-propanol, isobutanol, 1-butanol and isoamyl alcohol in the last fraction decreased drastically as well.

Waste-to-energy: biobutanol production from cellulosic residue of sweet potato by Clostridia acetobutylicum

Biobutanol has been emerging as a renewable replacement to overcome fossil fuel limit due to its attractive qualities. The waste resources utilization is an economically feasible and eco-friendly way to produce biobutanol. In this work, the potential of butanol fermentation with sweet potato residue (SPR) obtained from ethanol fermentation was evaluated. To assist sugar release for butanol production by Clostridia acetobutylicum, a pretreatment approach with dilute sulfuric acid was established via response surface methodology. The maximum butanol concentration of 3.77 g/L was obtained at the optimal conditions of pretreatment at 1% (v/v) dilute sulfuric acid, and 115℃ for 30 min. Detoxifying the hydrolysate with XAD-4 resin significantly enhanced butanol fermentation performance, while adding nutritional supplements had no significant influence. The stability of the fermentation process was verified by the 2 L-scale of fermentation, resulting in 7.96 g/L of butanol, 13.42 g/L h of Acetone-Butanol-Ethanol (ABE), and 0.34 g/g of ABE yield, respectively. This study demonstrates a bio-refinery strategy of conversion from waste biomass to energy, confirms the feasibility of utilizing the cost saving SPR as feedstock to produce butanol through fermentation, and provides a novel approach for minimization of environmental pollution.

Enhancing Biobutanol Production from biomass willow by pre-removal of water extracts or bark

Aiming to understand the importance of debarking on the controlled utilization of phenolic-rich willow biomass, biobutanol was produced from it by using Clostridium acetobutylicum. Acid-catalysed steam explosion and enzymatic hydrolysis (EH) were investigated before the acetone-butanol-ethanol (ABE) fermentation. The hydrolysable sugar yield and ABE fermentation efficiency were found to decline progressively from willow wood (WW) to HWE WB (hot water extracted willow biomass), WB (willow biomass) and the WW + HWE (willow wood plus the artificial willow bark water extracts), indicating that the pre-removal of water extracts or the bark can significantly improve ABE yield. Notably, the ABE productivity of WW achieved 12.7 g/L at the solvent yield of 31%, and the butanol concentration (i.e. 8.5 g/L) generated by WW is relatively high among the reported lignocellulosic-derived biomass. Additionally, it is hypothesized that under acidic conditions and high temperatures the fructose present in willow water extracts form hydroxymethylfurfural during steam explosion, which then spontaneously condenses with phenolic substances of willow bark to form a solid furanic precipitate. The formed furanic precipitates play inhibitory role in the enzymatic hydrolysis and are thereby deleterious to the ABE fermentation.

Synthetic co-culture of autotrophic Clostridium carboxidivorans and chain elongating Clostridium kluyveri monitored by flow cytometry.

SummarySyngas fermentation with acetogens is known to produce mainly acetate and ethanol efficiently. Co‐cultures with chain elongating bacteria making use of these products are a promising approach to produce longer‐chain alcohols. Synthetic co‐cultures with identical initial cell concentrations of Clostridium carboxidivorans and Clostridium kluyveri were studied in batch‐operated stirred‐tank bioreactors with continuous CO/CO2‐gassing and monitoring of the cell counts of both clostridia by flow cytometry after fluorescence in situ hybridization (FISH‐FC). At 800 mbar CO, chain elongation activity was observed at pH 6.0, although growth of C. kluyveri was restricted. Organic acids produced by C. kluyveri were reduced by C. carboxidivorans to the corresponding alcohols butanol and hexanol. This resulted in a threefold increase in final butanol concentration and enabled hexanol production compared with a mono‐culture of C. carboxidivorans. At 100 mbar CO, growth of C. kluyveri was improved; however, the capacity of C. carboxidivorans to form alcohols was reduced. Because of the accumulation of organic acids, a constant decay of C. carboxidivorans was observed. The measurement of individual cell concentrations in co‐culture established in this study may serve as an effective tool for knowledge‐based identification of optimum process conditions for enhanced formation of longer‐chain alcohols by clostridial co‐cultures.

Selective butanol production from carbon monoxide by an enriched anaerobic culture

An anaerobic mixed culture able to grow on pure carbon monoxide (CO) as well as syngas (CO, CO2 and H2), that produced unusual high concentrations of butanol, was enriched in a bioreactor with intermittent CO gas feeding. At pH 6.2, it mainly produced acids, generally acetic and butyric acid. After adaptation, under stress conditions of CO exposure at a partial pressure of 1.8 bar and low pH (e.g., 5.7), the enrichment accumulated ethanol, but also high amounts of butanol, up to 6.8 g/L, never reported before, with a high butanol/butyric acid molar ratio of 12.6, highlighting the high level of acid to alcohol conversion. At the end of the assay, both the acetic acid and ethanol concentrations decreased, with concomitant butyric acid production, suggesting C2 to C4 acid bioconversion, though this was not a dominant bioconversion process. The reverse reaction of ethanol oxidation to acetic acid was observed in the presence of CO2 produced during CO fermentation. Interestingly, butanol oxidation with simultaneous butyric acid production occurred upon production of CO2 from CO, which has to the best of our knowledge never been reported. Although the sludge inoculum contained a few known solventogenic Clostridia, the relative taxonomic abundance of the enriched sludge was diverse in Clostridia and Bacilli classes, containing known solventogens, e.g., Clostridium ljungdhalii, Clostridium ragsdalei and Clostridium coskatii, confirming their efficient enrichment. The relative abundance of unassigned Clostridium species amounted to 27% with presumably novel ethanol/butanol producers.

Fed-batch simultaneous saccharification and fermentation including in-situ recovery for enhanced butanol production from rice straw

This paper describes a study of fed-batch SSFR (simultaneous saccharification, fermentation and recovery) for butanol production from alkaline-pretreated rice straw (RS) in a 2-L stirred tank reactor. The initial solid (9.2% w/v) and enzyme (19.9 FPU g-dw-1) loadings were previously optimized by 50-mL batch SSF assays. Maximum butanol concentration of 24.80 g L-1 was obtained after three biomass feedings that doubled the RS load (18.4% w/v). Butanol productivity (0.344 g L-1h−1) also increased two-fold in comparison with batch SSF without recovery (0.170 g L-1h−1). Although fed-batch SSFR was able to operate with a single initial enzyme dosage, an extra dosage of nutrients was required with the biomass additions to achieve this high productivity. The study showed that SSFR can efficiently improve butanol production from a lignocellulosic biomass accompanied by the efficient use of the enzyme.

Unsteady conjugate model of fluid mechanics and mass transfer for butanol pervaporation process by non-porous membrane

A diffusion-convective mass transfer mathematical conjugated model of butanol recovery from a re-circulating aqueous solution by pervaporation using a polydimethylsiloxane (PDMS) membrane was developed based on computational fluid dynamics. Discretized continuity, linear momentum, and convective-diffusion mass transfer equations were solved by an in-house code based on the finite volume method for a laminar flow with a flat attached non-porous membrane. In addition, a novel numerical procedure to determine the butanol diffusivity in the PDMS membrane was developed based on the solution of the conjugate unsteady convection-diffusion model that included five coupled partial differential equations. The evolution of the distribution of the velocity profile in the fluid flow and the transient variation of butanol concentration in the feed solution and inside the PDMS membrane were determined. Variations in time and space of the dimensionless mass flux along the length of the pervaporation module were calculated from the conjugate convection-diffusion mass transfer model. The numerical predictions of the butanol mass flux in the feed solution exhibited good agreement with the experimental results with an error smaller than 3%. Understanding the mechanisms of mass and fluid transport phenomena by computational modeling can be helpful for improving the design of pervaporation processes.

Combining acetone-butanol-ethanol production and methyl orange decolorization in wastewater by fermentation with solid food waste as substrate

Simultaneously producing acetone-butanol-ethanol (ABE) and decolorizing Methyl orange (MO) by fermentation with solid food waste (SFW) was studied. During the batch fermentation with MO concentration from 100 mgL−1 to 1000 mgL−1, carbon source utilization efficiency was above 93% with yields of acetone, butanol and ethanol being 0.13 gg-1, 0.23 gg-1 and 0.04 gg-1, respectively. During the repeated fermentation with the bagasse as the cell immobilization material, carbon source utilization efficiencies were all above 90% in the first 5 cycles fermentation. The maximum butanol and ABE concentration were 12.8 gL-1 and 22.1 gL-1, respectively. The solvent yield of acetone, butanol and ethanol were around 0.13 gg-1, 0.25 gg-1 and 0.06 gg-1, respectively. The MO decolorization had no negative effect on the ABE production and the decolorization efficiencies were all above 99% during batch fermentation or repeated fermentation with electrons transferred from intracellular to extracellular by mediator with the help of azo reductase to decolorize MO.