Acetone-butanol-ethanol Production Research Articles

Butanol production from hexoses and pentoses by fermentation of Clostridium acetobutylicum

The present paper reports the characterization of ABE (acetone-butanol-ethanol) production by Clostridium acetobutylicum DSM 792 for sugars representative of hydrolysed lignocellulosic biomass (glucose, mannose, arabinose, xylose). The attention was focused on: the selection of an optimal medium for the simultaneous conversion of the investigated sugars; the assessment of interference-synergistic effects during the fermentation of mixtures of the investigated sugars.The synthetic medium was optimised in terms of nutritional factors: the KH2PO4–K2HPO4 concentration was increased up to 5 g/L; the MgSO4 concentration was increased up to 2 g/L; the MnSO4 concentration was increased up to 0.1 g/L; the FeSO4 concentration ranged between 0.002 and 0.01 g/L); the CaCO3 concentration was increased up to 10 g/L. The optimal concentration of the investigated factors was assessed and it varied from one sugar to another.The batch fermentations of a mixture of the four sugars highlighted their synergistic effects. Once set the initial concentration of the sugars (60 g/L), the butanol and solvent concentration increased up to 14.6 and 20.6 g/L, respectively, when the four sugars were present.

Feasibility of acetone–butanol–ethanol (ABE) fermentation from Amorphophallus konjac waste by Clostridium acetobutylicum ATCC 824

Using Amorphophallus konjac waste as a source of fermentable carbohydrate, the feasibility of acetone–butanol–ethanol (ABE) fermentation by Clostridium acetobutylicum ATCC 824 was investigated in this study. Two distinct combination strategies for the enzymatic hydrolysis and fermentation, simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF), for konjac waste fermentation were compared. β-Mannanase was added to facilitate the hydrolysis of glucomannan and to increase the fermentability of the substrate. The ABE concentration (6.64g/L) from SSF was less than that from SHF; the result from the control experiment was 4.34g/L of ABE. SHF obtained a higher production of total ABE solvents (10.95g/L) with 7.10g/L of butanol at a productivity rate of 0.30g/L/h. The effect of the bioactive alkaloid abundant in the konjac waste on ABE fermentation was evaluated. The results indicated that the alkaloid affected the ABE fermentation results when using C. acetobutylicum, and the effective extraction of alkaloids can help to increase the butanol concentration and shorten the fermentation period. Therefore, the utilization of Amorphophallus konjac waste (a low-cost agricultural resource) in ABE fermentation provides an alternative to increase the economic viability of ABE production.

Improvement of butanol production from a hardwood hemicelluloses hydrolysate by combined sugar concentration and phenols removal

The feasibility of using hardwood hemicellulosic pre-hydrolysate recovered from a dissolving pulping process for Acetone–Butanol–Ethanol (ABE) fermentation has been investigated. Dilutions and detoxification methods based on flocculation and nanofiltration were tested to determine the inhibitory concentration of phenolic compounds and to evaluate the efficiency of inhibitors removal on fermentation. Flocculation carried out with ferric sulfate was the most effective method for removal of phenolics (56%) and acetic acid (80%). The impact on fermentation was significant, with an ABE production of 6.40g/L and 4.25g/L when using flocculation or combined nanofiltration/flocculation respectively, as compared to a non-significant production for the untreated hydrolysate. By decreasing the toxicity effect of inhibitors, this study reports for the first time that the use of these techniques is efficient to increase the inhibitory concentration threshold of phenols, from 0.3g/L in untreated hydrolysate, to 1.1g/L in flocculated and in nanofiltrated and flocculated hydrolysates.

Acetone–butanol–ethanol production in a novel continuous flow system

This study investigates the potential of using a novel integrated biohydrogen reactor clarifier system (IBRCS) for acetone–butanol–ethanol (ABE) production using a mixed culture at different organic loading rates (OLRs). The results of this study showed that using a setting tank after the fermenter and recycle the settled biomass to the fermenter is a practical option to achieve high biomass concentration in the fermenter and thus sustainable ABE fermentation in continuous mode. The average ABE concentrations of 2.3, 7.0, and 14.6gABE/L which were corresponding to ABE production rates of 0.4, 1.4, and 2.8gABE/Lreactorh were achieved at OLRs of 21, 64, and 128gCOD/Lreactord, respectively. The main volatile fatty acids components in the effluent were acetic, propionic, and butyric acids. Acetic acid was the predominant component in the OLR-1, while butyric acid was the predominant acid in OLRs 2 and 3.

Efficient acetone–butanol–ethanol production by Clostridium beijerinckii from sugar beet pulp

Sugar beet pulp (SBP) has been investigated as a promising feedstock for ABE fermentation by Clostridium beijerinckii. Although lignin content in SBP is low, a pretreatment is needed to enhance enzymatic hydrolysis and fermentation yields. Autohydrolysis at pH 4 has been selected as the best pretreatment for SBP in terms of sugars release and acetone and butanol production. The best overall sugars release yields from raw SBP ranged from 66.2% to 70.6% for this pretreatment. The highest ABE yield achieved was 0.4g/g (5.1g/L of acetone and 6.6g/L butanol) and 143.2g ABE/kg SBP (62.3g acetone and 80.9g butanol) were obtained when pretreated SBP was enzymatically hydrolyzed at 7.5% (w/w) solid loading. Higher solid loadings (10%) offered higher acetone and butanol titers (5.8g/L of acetone and 7.8g/L butanol). All the experiments were carried out under not-controlling pH conditions reaching about 5.3 in the final samples.

Enhanced sugar production from pretreated barley straw by additive xylanase and surfactants in enzymatic hydrolysis for acetone–butanol–ethanol fermentation

This study aims to improve enzymatic sugar production from dilute sulfuric acid-pretreated barley straw for acetone–butanol–ethanol (ABE) fermentation. The effects of additive xylanase and surfactants (polyethylene glycol [PEG] and Tween) in an enzymatic reaction system on straw hydrolysis yields were investigated. By combined application of 2g/100g dry-matter (DM) xylanase and PEG 4000, the glucose yield was increased from 53.2% to 86.9% and the xylose yield was increased from 36.2% to 70.2%, which were considerably higher than results obtained with xylanase or surfactant alone. The ABE fermentation of enzymatic hydrolysate produced 10.8g/L ABE, in which 7.9g/L was butanol. The enhanced sugar production increased the ABE yield from 93.8 to 135.0g/kg pretreated straw. The combined application of xylanase and surfactants has a large potential to improve sugar production from barley straw pretreated with a mild acid and that the hydrolysate showed good fermentability in ABE production.

Kinetic study of butanol production from various sugars by Clostridium acetobutylicum using a dynamic model

This paper presents a kinetic dynamic model of acetone–butanol–ethanol production by Clostridium acetobutylicum DSM 792 developed with the biochemical networks simulator COPASI. This model is an evolution of previous models described in the literature, updated by including various mono-, di-, hexose and pentose sugars: glucose, mannose, fructose, sucrose, lactose, xylose and arabinose. The kinetic relationships of uptake of substrate, butanol production, cell growth and cell death are also included.The batch fermentation tests were carried out at an initial sugar concentration ranging from 5 to 100g/L. The data from the batch tests were used to assess the kinetic parameters of the model. This model gave satisfactory results for each sugar, both in terms of simulation of fermentation – the square correlation coefficient of metabolite concentrations, calculated by comparing experiments and simulations, ranged between 0.87 and 0.925 – and of comparison with the models reported in the literature.The effects of mono-, di-, hexose and pentose sugars on the growth and production of metabolites, including acids and solvents, were reviewed according to the proposed model. The low fermentation performance measured for xylose and lactose were interpreted taking into account the sugar uptake, the acid production and the hydrolysis path.

Block copolymer pervaporation membrane for in situ product removal during acetone–butanol–ethanol fermentation

We address two major challenges facing commercialization of acetone–butanol–ethanol (ABE) fermentation: product inhibition and low productivity. We studied a polystyrene-b-polydimethylsiloxane-b-polystyrene (SDS) triblock copolymer membrane for selective removal of butanol from aqueous solutions by pervaporation. The SDS membrane exhibited higher permeabilities than a commercially available cross-linked polydimethylsiloxane (PDMS) membrane. Both types of pervaporation membrane were also used for in situ product removal of ABE biofuels in Clostridium acetobutylicum fermentations operated in a semi-continuous mode. Membrane performance and its effect on the fermentation process were assessed by measuring flux, OD600 and concentrations of different components in the fermenter as a function of time. Volumetric ABE productivity increased from 0.45g/(Lh) in simple batch fermentation to 0.66g/(Lh) in the case of pervaporative-fermentation with the PDMS membrane. A further increase in productivity to 0.94g/(Lh) was obtained in the case of pervaporative-fermentation with the SDS membrane. Overall, total ABE production improved by a factor of three, viable fermentation time increased by a factor of two, and cell density increased by a factor of 2.5 upon applying SDS membrane pervaporation, relative to the batch process.

Allopurinol-mediated lignocellulose-derived microbial inhibitor tolerance by Clostridium beijerinckii during acetone-butanol-ethanol (ABE) fermentation.

In addition to glucans, xylans, and arabinans, lignocellulosic biomass hydrolysates contain significant levels of nonsugar components that are toxic to the microbes that are typically used to convert biomass to biofuels and chemicals. To enhance the tolerance of acetone-butanol-ethanol (ABE)-generating Clostridium beijerinckii NCIMB 8052 to these lignocellulose-derived microbial inhibitory compounds (LDMICs; e.g., furfural), we have been examining different metabolic perturbation strategies to increase the cellular reductant pools and thereby facilitate detoxification of LDMICs. As part of these efforts, we evaluated the effect of allopurinol, an inhibitor of NAD(P)H-generating xanthine dehydrogenase (XDH), on C. beijerinckii grown in furfural-supplemented medium and found that it unexpectedly increased the rate of detoxification of furfural by 1.4-fold and promoted growth, butanol, and ABE production by 1.2-, 2.5-, and 2-fold, respectively. Since NAD(P)H/NAD(P)(+) levels in C. beijerinckii were largely unchanged upon allopurinol treatment, we postulated and validated a possible basis in DNA repair to account for the solventogenic gains with allopurinol. Following the observation that supplementation of allopurinol in the C. beijerinckii growth media mitigates the toxic effects of nalidixic acid, a DNA-damaging antibiotic, we found that allopurinol elicited 2.4- and 6.7-fold increase in the messenger RNA (mRNA) levels of xanthine and hypoxanthine phosphoribosyltransferases, key purine-salvage enzymes. Consistent with this finding, addition of inosine (a precursor of hypoxanthine) and xanthine led to 1.4- and 1.7-fold increase in butanol production in furfural-challenged cultures of C. beijerinckii. Taken together, our results provide a purine salvage-based rationale for the unanticipated effect of allopurinol in improving furfural tolerance of the ABE-fermenting C. beijerinckii.

Acetone–butanol–ethanol production from substandard and surplus dates by Egyptian native Clostridium strains

One hundred and seven mesophilic isolates of Clostridium were isolated from agricultural soils cultivated with different plants in Assuit Governorate, Egypt. Eighty isolates (out of 107) showed the ability to produce ABE (Acetone, butanol and ethanol) on T6 medium ranging from 0.036 to 31.89 g/L. The highest numbers of ABE producing isolates were obtained from soil samples of potato contributing 27 isolates, followed by 18 isolates from wheat and 10 isolates from onion. On the other hand, there were three native isolates that produced ABE more than those produced by the reference isolate Clostridium acetobutylicum ATCC 824 (11.543 g/L). The three isolates were identified based on phenotypic and gene encoding 16S rRNA as Clostridium beijerinckii ASU10 (KF372577), Clostridium chauvoei ASU55 (KF372580) and Clostridium roseum ASU58 (KF372581). The highest ABE level from substandard and surplus dates was produced by C. beijerinckii ASU10 (24.07 g/L) comprising butanol 67.15% (16.16 g/L), acetone 30.73% (7.4 g/L) and ethanol 2.12% (0.51 g/L), while C. roseum ASU58 and C. chauvoei ASU55 produced ABE contributing 20.20 and 13.79 g/L, respectively. ABE production by C. acetobutylicum ATCC 824 was 15.01 g/L. This study proved that the native strains C. beijerinckii ASU10 and C. roseum ASU58 have high competitive efficacy on ABE production from economical substrate as substandard and surplus date fruits. Additionally, using this substrate without any nutritional components is considered to be a commercial substrate for desired ABE production.

Optimizing Acid Hydrolysis of Jerusalem Artichoke-Derived Inulin for Fermentative Butanol Production

In this study, a central composite design and response surface methodology were used to study the effect of various hydrolysis variables (temperature, pH, and time) on the acid hydrolysis of Jerusalem artichoke-derived inulin using three different mineral acids (HCl, H2SO4, and H3PO4). Numerical optimization was used to maximize the sugar yield of Jerusalem artichoke powder within the experimental range for each of the mentioned acid. The influence of each acid on the formation of hydroxymethylfurfural (HMF; a known by-product and inhibitor for fermentative organisms) was also investigated. H2SO4 was found to have a better potential for sugar yields compared to two other acids (HCl and H3PO4) since it can hydrolyze the highest amount of inulin (98.5 %) under optimal conditions (temperature of 97 °C, pH of 2.0, and time period of 35 min) without producing inhibiting HMF concentrations. The sulfuric hydrolysate of Jerusalem artichoke was fermented via solventogenic clostridia to acetone-butanol-ethanol (ABE). An ABE yield of 0.31 g g−1 and an overall fermentation productivity of 0.25 g l−1 h−1 were obtained, indicating the suitability of this feedstock for fermentative ABE production.

Acetone–butanol–ethanol production using pH control strategy and immobilized cells in an integrated fermentation–pervaporation process

The effects of pH and cell immobilization on acetone–butanol–ethanol (ABE) production coupled with a pervaporation membrane have been investigated using Clostridium acetobutylicum XY16. The solvent productivity of 0.29g/L/h was obtained using a two-stage controlled-pH in the coupled process, which was 11% higher than a previous process without pH control, nevertheless, membrane was still fouled. Furthermore, butanol in the fermenter remained below the critical concentration using a larger membrane, solvent productivity achieved 0.38g/L/h. However, these methods could not prevent membrane fouling, the average permeation flux and butanol separation factor decreased by 41.6% and 31.3%. Then, sugarcane bagasse was used as a cell immobilization carrier and applied to the coupled process; many cells aggregated and adsorbed on sugarcane bagasse surface, the dry cell weight (DCW) in broth was maintained at 1.1g/L. Membrane fouling was relieved, the pervaporation maintained a steady state for more than 90h, the average permeation flux was 0.676kg/m2/h, which only decreased by 13.9% compared to that in the initial stage, and the butanol separation factor remained stable. A maximum solvent productivity of 0.62g/L/h was also obtained. Thus, the pH control and cell immobilization are efficient methods for high-intensity ABE production with in situ recovery.

Feasibility of acetone–butanol–ethanol fermentation from eucalyptus hydrolysate without nutrients supplementation

The economic feasibility of acetone–butanol–ethanol (ABE) fermentation is greatly affected by the type of raw material used. The easy availability of eucalyptus from marginal environments is an alternative feedstock for use as raw material to reduce the production cost. In this study, hydrolyzed eucalyptus was used for ABE production without any nutrients supplementation. Increasing the solid concentration in the eucalyptus slurry from 6.7% (w-dry matter/v) to 25% led to an increase in the initial glucose concentration from 33.7g/L to 86.7g/L after enzymatic hydrolysis. Dosed cellulases not only hydrolyzed cellulose but also supplied nitrogen source for ABE producing strain. However, ABE production from the obtained hydrolysate decreased when the solid concentration was increased to more than 10%. The maximum ABE of 12.3g/L was obtained at 10% solid concentration, with an initial glucose concentration of approximately 40g/L. In addition, the fermentation capability of eucalyptus hydrolysate was found to be improved by diluting the hydrolysate, which prevented inhibition by substrate and fermentation inhibitors. Finally, ABE concentration was improved to 13.1g/L by diluting the hydrolysate from the initial solid concentration of 25% to an initial glucose concentration of 45g/L, which resulted in ABE productivity of 0.109g/L/h and ABE yield of 0.413g/g. Thus, the high ABE production from eucalyptus makes it a potential feedstock for biofuel production.

Co-fermentation of hemicellulose and starch from barley straw and grain for efficient pentoses utilization in acetone–butanol–ethanol production

This study aims to efficiently use hemicellulose-based biomass for ABE (acetone–butanol–ethanol) production by co-fermentation with starch-based biomass. Two processes were investigated: (I) co-fermentation of sugars derived from hemicellulose and starch in a mixture of barley straw and grain that was pretreated with dilute acid; (II) co-fermentation of straw hemicellulosic hydrolysate and gelatinized grain slurry in which the straw was pretreated with dilute acid. The two processes produced 11.3 and 13.5g/L ABE that contains 7.4 and 7.8g/L butanol, respectively. In process I, pretreatment with 1.0% H2SO4 resulted in better ABE fermentability than with 1.5% H2SO4, but only 19% of pentoses were consumed. In process II, 95% of pentoses were utilized even in the hemicellulosic hydrolysate pretreated with more severe condition (1.5% H2SO4). The results suggest that process II is more favorable for hemicellulosic biomass utilization, and it is also attractive for sustainable biofuel production due to great biomass availability.

Iron effect on the fermentative metabolism of Clostridium acetobutylicum ATCC 824 using cheese whey as substrate

Butanol is considered a superior liquid fuel that can replace gasoline in internal combustion engines. It is produced by acetone-butanol-ethanol (ABE) fermentation using various species of solventogenic clostridia. Performance of ABE fermentation process is severely limited mostly by high cost of substrate, substrate inhibition and low solvent tolerance; leading to low product concentrations, low productivity, low yield, and difficulty in controlling culture metabolism. In order to decrease the cost per substrate and exploit a waste generated by dairy industry, this study proposes using cheese whey as substrate for ABE fermentation. It was observed that the addition of an iron source was strictly necessary for the cheese whey to be a viable substrate because this metal is needed to produce ferredoxin, a key protein in the fermentative metabolism of Clostridium acetobutylicum serving as a temporary electron acceptor. Lack of iron in the cheese whey impedes ferredoxin synthesis and therefore, restricts pyruvate-ferredoxin oxidoreductase activity leading to the production of lactic acid instead of acetone, butanol and ethanol. Moreover, the addition of FeSO4 notably improved ABE production performance by increasing butanol content (7.13 ± 1.53 g/L) by 65% compared to that of FeCl3 (4.32 ± 0.94 g/L) under the same fermentation conditions.

Feasibility of producing butanol from industrial starchy food wastes

To evaluate the feasibility of using industrial starchy food wastes as alternative feedstocks for fermentative production of butanol, 34 different food waste samples were obtained from 10 major Ohio food manufacturing companies. Of the food waste samples, 8 were characterized for acetone butanol ethanol (ABE) production. The food waste samples were characterized for moisture, total solids, ash, nitrogen, total organic carbon (TOC), pH, calorific value, minerals and starch content. Results showed that the pH of inedible dough and breadings was in the acceptable substrate pH range (pH 6.2–7.4) for culturing solventogenic Clostridium species, whereas the pH of batter liquid was 4.5, hence, adjusted to pH 6.5 with NH4OH prior to fermentation. Further, batch fermentations by Clostridium beijerinckii NCIMB 8052 using inedible dough, breadings, and batter liquid as substrates generated the following maximum ABE concentrations: 14.4, 14.8 and 15.1g/L ABE, respectively, which is comparable to the glucose control. Besides, the ABE yield from inedible dough and batter liquid was over 2% better than that of the glucose control. Collectively, these results demonstrate that industrial starchy food waste is a viable alternative substrate for butanol production.

Microbial inhibitors: formation and effects on acetone-butanol-ethanol fermentation of lignocellulosic biomass.

Biobutanol is a promising biofuel due to the close resemblance of its fuel properties to gasoline, and it is produced via acetone-butanol-ethanol (ABE) fermentation using Clostridium species. However, lignin in the crystalline structure of the lignin-cellulose-hemicellulose biomass complex is not readily consumed by the Clostridium; thus, pretreatment is required to degrade this complex. During pretreatment, some fractions of cellulose and hemicellulose are converted into fermentable sugars, which are further converted to ABE. However, a major setback resulting from common pretreatment processes is the formation of sugar and lignin degradation compounds, including weak acids, furan derivatives, and phenolic compounds, which have inhibitory effects on the Clostridium. In addition, butanol concentration above 13 g/L in the fermentation broth is itself toxic to most Clostridium strain(s). This review summarizes the current state-of-the-art knowledge on the formation of microbial inhibitors during the most common lignocellulosic biomass pretreatment processes. Metabolic effects of inhibitors and their impacts on ABE production, as well as potential solutions for reducing inhibitor formation, such as optimizing pretreatment process parameters, using inhibitor tolerant strain(s) with high butanol yield ability, continuously recovering butanol during ABE fermentation, and adopting consolidated bioprocessing, are also discussed.

Acetone–butanol–ethanol production by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) methods using acorns and wood chips of Quercus acutissima as a carbon source

We investigated the production of acetone–butanol–ethanol (ABE) from acorns (starchy material) and wood chips (cellulosic material) of Quercus acutissima through separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) by Clostridium acetobutylicum NBRC13948. With acorns, 15.45g/L of ABE was obtained in 96h of fermentation time from the enzymatic hydrolyzate of acorns by SHF. From the SSF, it was found that ABE (16.70g/L, 120h) was produced without enzyme addition, given that C. acetobutylicum NBRC 13948 possesses an amylolytic enzyme. With wood chips, steam explosion was used for pretreatment. In the SHF, when the enzymatic hydrolyzate of residue of water and methanol extraction after pretreatment was used as the substrate, the maximum ABE concentration, 15.29g/L, was obtained in 120h. From the SSF, 13.41g/L of ABE was obtained in 144h, and it was determined that sufficient enzyme loading was 7.8mg of protein to 1g of dry substrate.

Optimizing enzymatic hydrolysis of inulin from Jerusalem artichoke tubers for fermentative butanol production

In this study, a central composite design and response surface methodology were used to study the effect of various enzymatic hydrolysis variables (temperature, pH, substrate concentration and enzyme loading) on the enzymatic hydrolysis of Jerusalem artichoke-derived inulin. It was found that a quadratic model was able to predict inulin conversion as a function of all four investigated factors. The model was confirmed through additional experiments and via analysis of variance (ANOVA). Subsequently, numerical optimization was used to maximize the inulin conversion (94.5%) of Jerusalem artichoke powder within the experimental range (temperature of 48 °C, pH of 4.8, substrate concentration of 60 g l−1, and enzyme loading of 10unitsgsubstrate−1 for 24 h). The enzymatic hydrolyzate of Jerusalem artichoke was fermented via solventogenic clostridia to acetone–butanol–ethanol (ABE). An ABE yield of 0.33gSolventgsugar−1 and an overall fermentation productivity of 0.25 g l−1 h−1 were obtained indicating the suitability of this feedstock for fermentative ABE production.

A novel strategy for sequential co-culture of Clostridium thermocellum and Clostridium beijerinckii to produce solvents from alkali extracted corn cobs

Increasing concern on fossil fuels depletion and global environmental deterioration has recently renewed interest in butanol production from lignocellulose by acetone–butanol–ethanol (ABE) fermentation. Unfortunately, there was barely microorganism possessing the compound ability of lignocellulose decomposition and solvents production natively. Comparing with complicated genetic and metabolic engineering strategy to engineer native cellulolytic or solventogenic bacteria, mixed culture is a convenient and feasible approach for ABE fermentation by consolidated bioprocessing (CBP) from lignocellulose. But few communities of microorganisms succeeded in producing butanol of high titer or productivity without adding butyrate. Here a novel strategy for sequential co-culture of Clostridium thermocellum ATCC 27405 and Clostridium beijerinckii NCIMB 8052 was proposed to produce solvents efficiently in one pot reaction with alkali extracted corn cobs (AECC), a low-cost renewable feedstock, as the sole carbon source. In this strategy, soluble sugars accumulation by C. thermocellum hydrolyzing AECC was considered to be paramount for the CBP and was promoted considerably by contrast with previous co-culture studies. Under the combinatorial optimal culture parameters for sugars accumulation and ABE production, the CBP decomposed 88.9gL−1 of AECC and manufactured ABE 19.9gL−1 (acetone 3.96, butanol 10.9 and ethanol 5.04gL−1) in 200h without feeding butyrate.