Butanol Toxicity Research Articles

Bioconversion of barley straw and corn stover to butanol (a biofuel) in integrated fermentation and simultaneous product recovery bioreactors

In these studies concentrated sugar solutions of barley straw and corn stover hydrolysates were fermented using Clostridium beijerinckii P260 with simultaneous product recovery and compared with the performance of a control glucose batch fermentation process. The control glucose batch fermentation resulted in the production of 23.25gL−1 ABE from 55.7gL−1 glucose solution resulting in an ABE productivity and yield of 0.33gL−1h−1 and 0.42, respectively. The control reactor (I) was started with 62.5gL−1 initial glucose and the culture left 6.8gL−1 unused sugar due to butanol toxicity resulting in incomplete sugar utilization. Barley straw (BS) hydrolysate sugars (90.3gL−1) resulted in the production of 47.20gL−1 ABE with a productivity of 0.60gL−1h−1 and a yield of 0.42. Fermentation of corn stover (CS) hydrolysate sugars (93.1gL−1) produced 50.14gL−1 ABE with a yield of 0.43 and a productivity of 0.70gL−1h−1. These productivities are 182–212% higher than the control run. The culture was able to use 99.4–100% sugars (CS & BS respectively) present in these hydrolysates and improve productivities which were possible due to simultaneous product removal. Use of >100gL−1 hydrolysate sugars was not considered as it would have been toxic to the culture in the integrated (simultaneous fermentation and recovery) process.

MFI zeolite as adsorbent for selective recovery of hydrocarbons from ABE fermentation broths

1-Butanol and butyric acid are two interesting compounds that may be produced by acetone, butanol, and ethanol fermentation using e.g. Clostridium acetobutylicum. The main drawback, restricting the commercialization potential of this process, is the toxicity of butanol for the cell culture resulting in low concentrations of this compound in the broth. To make this process economically viable, an efficient recovery process has to be developed. In this work, a hydrophobic MFI type zeolite with high silica to alumina ratio was evaluated as adsorbent for the recovery of butanol and butyric acid from model solutions. Dual component adsorption experiments revealed that both butanol and butyric acid showed a high affinity for the hydrophobic MFI zeolite when adsorbed from aqueous model solutions. Multicomponent adsorption experiments using model solutions, mimicking real fermentation broths, revealed that the adsorbent was very selective to the target compounds. Further, the adsorption of butyric and acetic acid was found to be pH dependent with high adsorption below, and low adsorption above, the respective pKa values of the acids. Thermal desorption of butanol from MFI type zeolite was also studied and a suitable desorption temperature was identified.

Enhanced butanol production in Clostridium acetobutylicum ATCC 824 by double overexpression of 6-phosphofructokinase and pyruvate kinase genes

This study elucidated the importance of two critical enzymes in the regulation of butanol production in Clostridium acetobutylicum ATCC 824. Overexpression of both the 6-phosphofructokinase (pfkA) and pyruvate kinase (pykA) genes increased intracellular concentrations of ATP and NADH and also resistance to butanol toxicity. Marked increases of butanol and ethanol production, but not acetone, were also observed in batch fermentation. The butanol and ethanol concentrations were 29.4 and 85.5% higher, respectively, in the fermentation by double-overexpressed C. acetobutylicum ATCC 824/pfkA+pykA than the wild-type strain. Furthermore, when fed-batch fermentation using glucose was carried out, the butanol and total solvent (acetone, butanol, and ethanol) concentrations reached as high as 19.12 and 28.02g/L, respectively. The reason for improved butanol formation was attributed to the enhanced NADH and ATP concentrations and increased tolerance to butanol in the double-overexpressed strain.

Butanol production from wood pulping hydrolysate in an integrated fermentation–gas stripping process

Wood pulping hydrolysate (WPH) containing mainly xylose and glucose as a potential substrate for acetone-butanol-ethanol (ABE) fermentation was studied. Due to the inhibitors present in the hydrolysate, several dilution levels and detoxification treatments, including overliming, activated charcoal adsorption, and resin adsorption, were evaluated for their effectiveness in relieving the inhibition on fermentation. Detoxification using resin and evaporation was found to be the most effective method in reducing the toxicity of WPH. ABE production in batch fermentation by Clostridium beijerinckii increased 68%, from 6.73 g/L in the non-treated and non-diluted WPH to 11.35 g/L in the resin treated WPH. With gas stripping for in situ product removal, ABE production from WPH increased to 17.73 g/L, demonstrating that gas stripping was effective in alleviating butanol toxicity by selectively separating butanol from the fermentation broth, which greatly improved solvents production and sugar conversion in the fermentation.

Production of butanol from glucose and xylose with immobilized cells of Clostridium acetobutylicum

Pretreated cotton towels were used as carriers to immobilize Clostridium acetobutylicum CGMCC 5234 cells for butanol or ABE production from glucose and xylose. Results showed that cell immobilization was a promising method to increase butanol concentration, yield and productivity regardless of the sugar sources compared with cell suspension. In this study, a high butanol concentration of 10.02 g/L with a yield of 0.20 g/g was obtained from 60 g/L xylose with 9.9 g/L residual xylose using immobilized cells compared with 8.48 g/L butanol and a yield of 0.141 g/g with 20.2 g/L residual xylose from 60 g/L xylose using suspended cells. In mixed-sugar fermentation (30 g/L glucose plus 30 g/L xylose), the immobilized cultures produced 11.1 g/L butanol with a yield of 0.190 g/g, which were 28.3% higher than with suspended cells (8.65 g/L) during which 30 g/L glucose was utilized completely using both immobilized and suspended cells while 3.46 and 13.1 g/L xylose maintained untilized for immobilized and suspended cells, respectively. Based on the results, we speculated that immobilized cells showed enhanced tolerance to butanol toxicity and the cultures preferred glucose to xylose during ABE fermentation. Moreover, the cultures showed obvious difference when grown between high initial concentrations of glucose and those of xylose. Repeated-batch fermentations from glucose with immobilized cells showed better long-term stability than from xylose. At last, the morphologies of free and immobilized cells adsorbed on pretreated cotton towels during the growth cycle were examined by SEM.

Enhancement of butanol tolerance and butanol yield in Clostridium acetobutylicum mutant NT642 obtained by nitrogen ion beam implantation

As a promising alternative biofuel, biobutanol can be produced through acetone/butanol/ethanol (ABE) fermentation. Currently, ABE fermentation is still a small-scale industry due to its low production and high input cost. Moreover, butanol toxicity to the Clostridium fermentation host limits the accumulation of butanol in the fermentation broth. The wild-type Clostridium acetobutylicum D64 can only produce about 13 g butanol/L and tolerates less than 2% (v/v) butanol. To improve the tolerance of C. acetobutylicum D64 for enhancing the production of butanol, nitrogen ion beam implantation was employed and finally five mutants with enhanced butanol tolerance were obtained. Among these, the most butanol tolerant mutant C. acetobutylicum NT642 can tolerate above 3% (v/v) butanol while the wide-type strain can only withstand 2% (v/v). In batch fermentation, the production of butanol and ABE yield of C. acetobutylicum NT642 was 15.4 g/L and 22.3 g/L, respectively, which were both higher than those of its parental strain and the other mutants using corn or cassava as substrate. Enhancing butanol tolerance is a great precondition for obtaining a hyper-yield producer. Nitrogen ion beam implantation could be a promising biotechnology to improve butanol tolerance and production of the host strain C. acetobutylicum.

Screening and characteristics of a butanol-tolerant strain and butanol production from enzymatic hydrolysate of NaOH-pretreated corn stover

As a gasoline substitute, butanol has advantages over traditional fuel ethanol in terms of energy density and hydroscopicity. However, solvent production appeared limited by butanol toxicity. The strain of Clostridium acetobutylicum was subjected to mutation by mutagen of N-methyl-N'-nitro-N-nitrosoguanidine for 0.5h. Screening of mutants was done according to the individual resistance to butanol. A selected butanol-resistant mutant, strain 206, produced 50% higher solvent concentrations than the wild-type strain when 60g glucose/l was employed as substrate. The strain was also able to produce solvents of 23.47g/l in 80g/l glucose P2 medium after 70h fermentation, including 5.41g acetone/l, 15.05g butanol/l and 3.02g ethanol/l, resulting in an ABE yield and productivity of 0.32g/g and 0.34g/(lh). Subsequently, Acetone-butanol-ethanol (ABE) production from enzymatic hydrolysate of NaOH-pretreated corn stover was investigated in this study. An ABE yield of 0.41 and a productivity of 0.21g/(lh) was obtained, compared to the yield of 0.33 and the productivity of 0.20g/(lh) in the control medium containing 52.47 mixed sugars. However, it is important to note that although strain 206 was able to utilize all the glucose rapidly in the hydrolysate, only 32.9% xylose in the hydrolysate was used after fermentation stopped compared to 91.4% xylose in the control medium. Strain 206 was shown to be a robust strain for ABE production from lignocellulosic materials and has a great potential for industrial application.

Integrated bioprocess for long-term continuous cultivation of Clostridium acetobutylicum coupled to pervaporation with PDMS composite membranes

A continuous cultivation of Clostridium acetobutylicum ATCC 824 is described using a two-stage design to mimic the two phases of batch culture growth of the organism. A hydrophobic pervaporation unit was coupled to the second fermentor containing the highest solvent titers. This in situ product recovery technology efficiently decreased butanol toxicity in the fermentor while the permeate was enriched to 57–195gL−1 total solvents depending on the solvent concentrations in the fermentor. By the alleviation of product inhibition, the glucose concentration could be increased from 60 to 126gL−1 while the productivity increased concomitantly from 0.13 to 0.30gL−1h−1. The continuous fermentation was conducted for 1172h during which the pervaporation was coupled to the second fermentor for 475h with an average flux of 367gm−2h−1. The energy consumption was calculated for a 2wt.% n-butanol fermentation broth and compared with the conventional process.

Assessment of in situ butanol recovery by vacuum during acetone butanol ethanol (ABE) fermentation

AbstractBACKGROUND: Butanol fermentation is product limiting owing to butanol toxicity to microbial cells. Butanol (boiling point: 118 °C) boils at a higher temperature than water (boiling point: 100 °C) and application of vacuum technology to integrated acetone–butanol–ethanol (ABE) fermentation and recovery may have been ignored because of direct comparison of boiling points of water and butanol. This research investigated simultaneous ABE fermentation using Clostridium beijerinckii 8052 and in situ butanol recovery by vacuum. To facilitate ABE mass transfer and recovery at fermentation temperature, batch fermentation was conducted in triplicate at 35 °C in a 14 L bioreactor connected in series with a condensation system and vacuum pump.RESULTS: Concentration of ABE in the recovered stream was greater than that in the fermentation broth (from 15.7 g L−1 up to 33 g L−1). Integration of the vacuum with the bioreactor resulted in enhanced ABE productivity by 100% and complete utilization of glucose as opposed to a significant amount of residual glucose in the control batch fermentation.CONCLUSION: This research demonstrated that vacuum fermentation technology can be used for in situ butanol recovery during ABE fermentation and that C. beijerinckii 8052 can tolerate vacuum conditions, with no negative effect on cell growth and ABE production. Copyright © 2011 Society of Chemical Industry

Toxicity of butanol and/or aldehyde by-products arising from its incomplete catalytic degradation in human epithelial lung cells (BEAS-2B)

Toxicity of butanol and/or aldehyde by-products arising from its incomplete catalytic degradation in human epithelial lung cells (BEAS-2B)

Energy Requirements for Butanol Recovery Using the Flash Fermentation Technology

Acetone–butanol–ethanol (ABE) facilities have traditionally presented unattractive economics because of the large energy consumption during recovery of the products from a dilute fermentation broth (∼13 g/L butanol). This problem results from the high toxicity of butanol to microorganisms that catalyze its production. Flash fermentation is a continuous fermentation system with integrated product recovery. The bioreactor is operated at atmospheric pressure and the broth is circulated in a closed loop to a vacuum chamber where ABE is continuously boiled off at 37 °C and condensed afterward. With this technology the beer achieved a concentration of butanol as high as 30–37 g/L. This paper studies the energy requirements for butanol recovery using the flash fermentation technology and its effect on the energy consumption by the downstream distillation system. Compressors are used to remove the vapors from the flash tank, thus maintaining the desired vacuum. The heat recovery technique of vapor recompression i...

Bioproduction of butanol in bioreactors: New insights from simultaneous in situ butanol recovery to eliminate product toxicity

Simultaneous acetone butanol ethanol (ABE) fermentation by Clostridium beijerinckii P260 and in situ product recovery was investigated using a vacuum process operated in two modes: continuous and intermittent. Integrated batch fermentations and ABE recovery were conducted at 37 °C using a 14-L bioreactor (7.0 L fermentation volume) containing initial substrate (glucose) concentration of 60 g/L. The bioreactor was connected in series with a condensation system and vacuum pump. Vacuum was applied continuously or intermittently with 1.5 h vacuum sessions separated by 4, 6, and 8 h intervals. A control ABE fermentation experiment was characterized by incomplete glucose utilization due to butanol toxicity to C. beijerinckii P260, while fermentation coupled with in situ recovery by both continuous and intermittent vacuum modes resulted in complete utilization of glucose, greater productivity, improved cell growth, and concentrated recovered ABE stream. These results demonstrate that vacuum technology can be applied to integrated ABE fermentation and recovery even though the boiling point of butanol is greater than that of water.

Comparative analysis on the membrane proteome of Clostridium acetobutylicum wild type strain and its butanol-tolerant mutant

The solventogenic bacterium Clostridium acetobutylicum is the most important species of Clostridium used in the fermentation industry. However, the intolerance to butanol hampers the efficient production of solvents. Butanol toxicity has been attributed to the chaotropic effect on the cell membrane, but the knowledge on the effect of butanol on membrane associated proteins is quite limited. Using 2-DE combined with MALDI-TOF MS/MS and 1-DE integrated with LC-MS/MS, 341 proteins in the membrane fractions of cell lysate were identified, thus establishing the first comprehensive membrane proteome of C. acetobutylicum. The identified proteins are mainly involved in transport, cellular membrane/wall machinery, formation of surface coat and flagella, and energy metabolism. Comparative analysis on the membrane proteomes of the wild type strain DSM 1731 and its butanol-tolerant mutant Rh8 revealed 73 differentially expressed proteins. Hierarchical clustering analysis suggested that mutant Rh8 may have evolved a more stabilized membrane structure, and have developed a cost-efficient energy metabolism strategy, to cope with the butanol challenge. This comparative membrane proteomics study, together with our previous published work on comparative cytoplasmic proteomics, allows us to obtain a systemic understanding of the effect of butanol on cellular physiology of C. acetobutylicum.

Engineering butanol‐tolerance in escherichia coli with artificial transcription factor libraries

Escherichia coli has been explored as a host for butanol production because of its many advantages such as a fast growth and easy genetic manipulation. Butanol toxicity, however, is a major concern in the biobutanol production with E. coli. In particular, E. coli growth is severely inhibited by butanol, being almost completely stopped by 1% (vol/vol) butanol. Here we developed a new method to increase the butanol-tolerance of E. coli with artificial transcription factor (ATF) libraries which consist of zinc finger (ZF) DNA-binding proteins and an E. coli cyclic AMP receptor protein (CRP). Using these ATFs, we selected a butanol-tolerant E. coli which can tolerate up to 1.5% (vol/vol) butanol, with a concomitant increase in heat resistance. We also identified genes of E. coli that are associated with the butanol-tolerance. These results show that E. coli can be engineered as a promising host for high-yield butanol production.

Aerobic biodegradation of butanol and diesel oil blends

This work aimed to evaluate the aerobic biodegradation of butanol/diesel oil blends (5, 10, 15, 20%, v/v) in comparison to the biodiesel/diesel oil blend (20%, v/v). Respirometric experiments simulating the contamination of natural environments (soil and water from a river) were carried out in biometer flasks (250 mL) used to measure microbial carbon dioxide (CO 2) production. The automated turbidimeter Bioscreen C was used to follow the growth of Pseudomonas aeruginosa LBI on butanol/diesel oil blends. A redox indicator (2,6-dichlorophenol indophenol - DCPIP) test was used to evaluate the capability of four inocula to biodegrade the blends with 20% (v/v). The experiment which simulated the soil contamination demonstrated that butanol is less biodegradable than diesel oil, and for this reason the increase in the portion of butanol in the butanol/diesel blend from 5 to 20% had negative effects on biodegradation. While in soil the biodiesel/diesel blend was more easily biodegraded than the butanol/diesel blend, in water this order was the inverse. The insoluble fuels (diesel and biodiesel) were poorly biodegraded in water and the biodegradation of the butanol/diesel blend was favored by the water solubilization of the butanol, which enhances the bioavailability of this compound. On the other hand, initial concentrations of butanol in the water higher than 10 mL L -1 inhibited the cell growth of the tested microorganisms. Thus, butanol toxicity presumably had a significant effect on the degree of biodegradation of the fuel blends.

Production of n-butanol from concentrated sugar maple hemicellulosic hydrolysate by Clostridia acetobutylicum ATCC824

Using fermentation to replace chemical processes in the production of acetone and butanol depends largely on the availability of inexpensive and abundant raw materials and efficient conversion of these materials to solvents. In this study solvent production of Clostridium acetobutylicum ATCC824 from nano-membrane concentrated hemicellulosic hydrolysate was investigated. Alkali pretreatment methods were applied to improve fermentability of nano-membrane concentrated hemicellulosic hydrolysate and solvent production by ATCC824. Results demonstrated that though nanofiltration could remove nearly all small molecular organic acids (acetic acid, formic acid), furfural and HMF, the resulting hydrolysate found to be still inhibiting solvent production of C. acetobutylicum. Solid particles separated from filtering hydrolysate were found not toxic to cells when xylose or glucose was used as carbon resource. Overliming treatment can significantly improve the ultimate butanol concentration to 7 g l−1 from 0.8 g l−1. Providing cells with more carbon source at the final stage of fermentation was found to have no impact on butanol production, but acetic acid and butyric acid production were found to increase significantly. The reasons leading to low solvent yield at later fermentation stages is not cell degeneration, but the toxicity of butanol and inhibitors remaining in the hydrolysate.

O.010 Blood, cerebrospinal fluid and brain dynamics in communicating hydrocephalus

The energy crisis, depletion of oil reserves, and global climate changes are pressing problems of developed societies. One possibility to counteract that is microbial production of butanol, a promising new fuel and alternative to many petrochemical reagents. However, the high butanol toxicity to all known microbial species is the main obstacle to its industrial implementation. The present state of the art review aims to expound the recent advances in modern omics approaches to resolving this insurmountable to date problem of low butanol tolerance. Genomics, transcriptomics, and proteomics show that butanol tolerance is a complex phenomenon affecting multiple genes and their expression. Efflux pumps, stress and multidrug response, membrane transport, and redox-related genes are indicated as being most important during butanol challenge, in addition to fine-tuning of global regulators of transcription (Spo0A, GntR), which may further improve tolerance. Lipidomics shows that the alterations in membrane composition (saturated lipids and plasmalogen increase) are very much species-specific and butanol-related. Glycomics discloses the pleiotropic effect of CcpA, the role of alternative sugar transport, and the production of exopolysaccharides as alternative routes to overcoming butanol stress. Unfortunately, the strain that simultaneously syntheses and tolerates butanol in concentrations that allow its commercialization has not yet been discovered or produced. Omics insight will allow the purposeful increase of butanol tolerance in natural and engineered producers and the effective heterologous expression of synthetic butanol pathways in strains hereditary butanol-resistant up to 3.2 - 4.9% (w/v). Future breakthrough can be achieved by a detailed study of the membrane proteome, of which 21% are proteins with unknown functions.

Butanol, ‘a superior biofuel’ production from agricultural residues (renewable biomass): recent progress in technology

AbstractThis article reviews bioconversion of plant materials such as wheat straw (WS), corn stover (CS), barley straw (BS), and switchgrass (SG) to butanol and process technology that converts these materials into this superior biofuel. Successful fermentation of low‒value WS makes butanol fermentation economically attractive. Simultaneous hydrolysis, fermentation, and product recovery has been successfully performed in a single reactor using WS and C. beijerinckii P260. Research on the production of butanol from other agricultural residues including CS, BS, and SG has steadily progressed. Use of several product‒recovery technologies such as liquid‒liquid extraction, gas stripping, perstraction, and pervaporation has been successfully applied in laboratory‒scale bioreactors. It is expected that these recovery technologies will play a major role in commercialization of this fermentation. By employing in line/in situ product‒recovery systems during fermentation, butanol toxicity to the culture has been drastically reduced. In addition to the use of low‒cost plant materials for the production of this biofuel, process integration is expected to play a major role in the economics of this product. © 2008 Society of Chemical Industry and John Wiley & Sons, Ltd

Bioproduction of butanol from biomass: from genes to bioreactors

Butanol is produced chemically using either the oxo process starting from propylene (with H2 and CO over a rhodium catalyst) or the aldol process starting from acetaldehyde. The key problems associated with the bioproduction of butanol are the cost of substrate and butanol toxicity/inhibition of the fermenting microorganisms, resulting in a low butanol titer in the fermentation broth. Recent interest in the production of biobutanol from biomass has led to the re-examination of acetone-butanol-ethanol (ABE) fermentation, including strategies for reducing or eliminating butanol toxicity to the culture and for manipulating the culture to achieve better product specificity and yield. Advances in integrated fermentation and in situ product removal processes have resulted in a dramatic reduction of process streams, reduced butanol toxicity to the fermenting microorganisms, improved substrate utilization, and overall improved bioreactor performance.

Maintenance of ΔpH by a butanol-tolerant mutant of Clostridium beijerinckii

The isolation of Clostridium beijerinckii mutants that are more tolerant of butanol than the wild-type offered the opportunity to investigate whether the membrane activities which are required for maintaining the transmembrane DeltapH (the difference in pH between the cellular interior and exterior) are sensitive targets of butanol toxicity. The DeltapH was measured by the accumulation of [14C]benzoate using late-exponential-phase cells which were suspended in citrate/phosphate buffer at pH 5 (to maximize the DeltapH component of the protonmotive force) and supplemented with glucose and Mg2+. The DeltapH of the butanol-tolerant tolerant mutant, strain BR54, of C. beijerinckii NCIMB 8052 was found to be significantly more tolerant of added butanol than the wild-type. Thus, in potassium citrate/phosphate buffer the mutant cells maintained a DeltapH of 1.4 when butanol was added to a concentration of 1.5 % (w/v), while the wild-type DeltapH was reduced to 0.1. The DeltapH of both strains was completely dissipated with 1.75 % butanol, an effect attributed to a chaotropic effect on the membrane phospholipids. Similar results were obtained in sodium citrate/phosphate buffer. In the absence of added Mg2+, the DeltapH of the mutant decreased in both sodium and potassium citrate/phosphate buffer, but more rapidly in the former. Interestingly, the addition of butanol at low concentrations (0.8 %) prevented this DeltapH dissipation, but only in cells suspended in sodium citrate/phosphate buffer, and not in potassium citrate/phosphate buffer. In wild-type cells the decrease in DeltapH occurred more slowly than in the mutant, and sparing of the DeltapH by 0.8 % butanol was less pronounced. The authors interpret these data to mean that the DeltapH is dissipated in the absence of Mg2+ by a Na+- or K+-linked process, possibly by a Na+/H+ or a K+/H+ antiporter, and that the former is inhibited by butanol. Apparently, butanol can selectively affect a membrane-associated function at concentrations lower than required for the complete dissipation of transmembrane ion gradients. Additionally, since the butanol-tolerant mutant BR54 is deficient in the ability to detoxify methylglyoxal (MG) and contains higher levels of MG than the wild-type, the higher Na+/H+ antiporter activity of the mutant may be due to the greater degree of protein glycation by MG in the mutant cells. The mechanism of butanol tolerance may be an indirect result of the elevated glycation of cell proteins in the mutant strain. Analysis of membrane protein fractions revealed that mutant cells contained significantly lower levels of unmodified arginine residues than those of the wild-type cells, and that unmodified arginine residues of the wild-type were decreased by exposure of the growing cells to added MG.