Production of acetone–butanol–ethanol (ABE) in a continuous flow bioreactor using degermed corn and Clostridium beijerinckii

An examination of the sustainability of the long-term cultivation of C. beijerinckii BA101 in degermed corn/saccharified degermed corn based P2 medium has been described in this work. It was found that long-term continuous cultivation of C. beijerinckii BA101 in a degermed corn based medium was not possible due to the instability of the gelatinized degermed corn starch during storage often called “retrogradation”. Using this substrate, continuous ABE fermentation was run for 228 h, before the fermentation turned acidogenic. However continuous fermentations of saccharified degermed corn with normal and half P2 medium nutrients were successful. In saccharified degermed corn continuous fermentation, ABE concentration up to 14.28 g/L was achieved at a dilution rate of 0.03 h⁻¹. This work demonstrated that byproduct (germ/oil, corn fiber) credit can be obtained by fermenting saccharified degermed corn in continuous flow bioreactors. Additionally significant savings can be achieved by supplementing with half of normal P2 medium nutrients.     

Direct fermentation of gelatinized sago starch to acetone–butanol–ethanol by Clostridium acetobutylicum

Direct fermentation of gelatinized sago starch into solvent (acetone–butanol–ethanol) by Clostridium acetobutylicum P262 was studied using a 250 ml Schott bottle anaerobic fermentation system. Total solvent production from fermentation using 30 g sago starch/l (11.03g/l) was comparable to fermentation using corn starch and about 2-fold higher than fermentation using potato or tapioca starch. At the range of sago starch concentration investigated (10–80 g/l), the highest total solvent production (18.82 g/l) was obtained at 50 g/l. The use of a mixture of organic and inorganic nitrogen source (yeast extract + NH₄NO₃) enhanced growth of C. acetobutylicum, starch hydrolysis and solvent production (24.47 g/l) compared to the use of yeast extract alone. This gave the yield based on sugar consumed of 0.45 g/g. Result from this study also showed that the individual concentrations of nitrogen and carbon influenced solvent production to a greater extent than did carbon to nitrogen (C/N) ratio.     

Targeted mutagenesis of the Clostridium acetobutylicum acetone–butanol–ethanol fermentation pathway

The production of the chemical solvents acetone and butanol by the bacterium Clostridium acetobutylicum was one of the first large-scale industrial processes to be developed, and in the first part of the last century ranked second in importance only to ethanol production. After a steep decline in its industrial use, there has been a recent resurgence of interest in the acetone–butanol–ethanol (ABE) fermentation process, with a particular emphasis on butanol production. In order to generate strains suitable for efficient use on an industrial scale, metabolic engineering is required to alter the AB ratio in favour of butanol, and eradicate the production of unwanted products of fermentation. Using ClosTron technology, a large-scale targeted mutagenesis in C. acetobutylicum ATCC 824 was carried out, generating a set of 10 mutants, defective in alcohol/aldehyde dehydrogenases 1 and 2 (adhE1, adhE2), butanol dehydrogenases A and B (bdhA, bdhB), phosphotransbutyrylase (ptb), acetate kinase (ack), acetoacetate decarboxylase (adc), CoA transferase (ctfA/ctfB), and a previously uncharacterised putative alcohol dehydrogenase (CAP0059). However, inactivation of the main hydrogenase (hydA) and thiolase (thl) could not be achieved. Constructing such a series of mutants is paramount for the acquisition of information on the mechanism of solvent production in this organism, and the subsequent development of industrial solvent producing strains. Unexpectedly, bdhA and bdhB mutants did not affect solvent production, whereas inactivation of the previously uncharacterised gene CAP0059 resulted in increased acetone, butanol, and ethanol formation. Other mutants showed predicted phenotypes, including a lack of acetone formation (adc, ctfA, and ctfB mutants), an inability to take up acids (ctfA and ctfB mutants), and a much reduced acetate formation (ack mutant). The adhE1 mutant in particular produced very little solvents, demonstrating that this gene was indeed the main contributor to ethanol and butanol formation under the standard batch culture conditions employed in this study. All phenotypic changes observed could be reversed by genetic complementation, with exception of those seen for the ptb mutant. This mutant produced around 100 mM ethanol, no acetone and very little (7 mM) butanol. The genome of the ptb mutant was therefore re-sequenced, together with its parent strain (ATCC 824 wild type), and shown to possess a frameshift mutation in the thl gene, which perfectly explained the observed phenotype. This finding reinforces the need for mutant complementation and Southern Blot analysis (to confirm single ClosTron insertions), which should be obligatory in all further ClosTron applications.     

Promotion of the Clostridium acetobutylicum ATCC 824 growth and acetone–butanol–ethanol fermentation by flavonoids

An unexpected promotion effect of Ginkgo leaf on the growth of Clostridium acetobutylicum ATCC 824 and acetone–butanol–ethanol (ABE) fermentation was investigated. Component analysis of Ginkgo leaf was carried out and flavonoids were determined as the potential key metabolites. Then the flavonoids feeding experiments were carried out. Results showed that addition of only 10 mg/L flavonoids to the fermentation broth can promote butanol and ABE titre up to 14.5 and 17.8 g/L after 5 days of fermentation, that is, 74 and 68 % higher than the control. A 2.2-fold biomass also has been achieved. Furthermore, by employing such novel founding, we easily exploited flavonoids from soybean and some agriculture wastes as the wide-distributed and economic feasible ABE fermentation promoter. The mechanism of the above effects was investigated from the perspective of oxidation–reduction potential. This work opens a new way in the efforts to increase the titer of butanol.     

Acetone–butanol–ethanol (ABE) fermentation using Clostridium acetobutylicum XY16 and in situ recovery by PDMS/ceramic composite membrane

PDMS/ceramic composite membrane was directly integrated with acetone-butanol-ethanol (ABE) fermentation using Clostridium acetobutylicum XY16 at 37 °C and in situ removing ABE from fermentation broth. The membrane was integrated with batch fermentation, and approximately 46 % solvent was extracted. The solvent in permeates was 118 g/L, and solvent productivity was 0.303 g/(L/h), which was approximately 33 % higher compared with the batch fermentation without in situ recovery. The fed-batch fermentation with in situ recovery by pervaporation continued for more than 200 h, 61 % solvent was extracted, and the solvent in penetration was 96.2 g/L. The total flux ranged from 0.338 to 0.847 kg/(m(2)/h) and the separation factor of butanol ranged from 5.1 to 27.1 in this process. The membrane was fouled by the active fermentation broth, nevertheless the separation performances were partially recovered by offline membrane cleaning, and the solvent productivity was increased to 0.252 g/(L/h), which was 19 % higher compared with that in situ recovery process without membrane cleaning.     

Mechanistic simulation of batch acetone–butanol–ethanol (ABE) fermentation with in situ gas stripping using Aspen Plus™

Bioprocess and Biosystems Engineering - Process simulations of batch fermentations with in situ product separation traditionally decouple these interdependent steps by simulating a separate...     

Continuous acetone–butanol–ethanol production by corn stalk immobilized cells

Corn stalk was used as a support to immobilize Clostridia beijerinckii ATCC 55025 in the fermentation process of acetone, butanol, and ethanol production. The effect of the dilution rate on solvent production was examined in a steady-state 20-day continuous flow operation. The maximum total solvent concentration of 8.99 g l⁻¹ was obtained at a dilution rate of 0.2 h⁻¹. Increasing the dilution rate between 0.2 and 1.0 h⁻¹ resulted in an increased solvent productivity, and the highest solvent productivity was obtained at 5.06 g l⁻¹ h⁻¹ with a dilution rate of 1 h⁻¹. The maximum solvent yield from glucose of 0.32 g g⁻¹ was observed at 0.25 h⁻¹. The cell adsorption and morphology change during the growth on corn stalk support were examined by the SEM.     

Acetone–butanol–ethanol fermentation of corn stover by Clostridium species: present status and future perspectives

Sustainable vehicle fuel is indispensable in future due to worldwide depletion of fossil fuel reserve, oil price fluctuation and environmental degradation. Microbial production of butanol from renewable biomass could be one of the possible options. Renewable biomass such as corn stover has no food deficiency issues and is also cheaper in most of the agricultural based countries. Thus it can effectively solve the existing issue of substrate cost. In the last 30 years, a few of Clostridium strains have been successfully implemented for biobutanol fermentation. However, the commercial production is hindered due to their poor tolerance to butanol and inhibitors. Metabolic engineering of Clostridia strains is essential to solve above problems and ultimately enhance the solvent production. An effective and efficient pretreatment of raw material as well as optimization of fermentation condition could be another option. Furthermore, biological approaches may be useful to optimize both the host and pathways to maximize butanol production. In this context, this paper reviews the existing Clostridium strains and their ability to produce butanol particularly from corn stover. This study also highlights possible fermentation pathways and biological approaches that may be useful to optimize fermentation pathways. Moreover, challenges and future perspectives are also discussed.     

Ionic liquid-based in situ product removal design exemplified for an acetone–butanol–ethanol fermentation

Selecting an appropriate separation technique is essential for the application of in situ product removal (ISPR) technology in biological processes. In this work, a three-stage systematic design method is proposed as a guide to integrate ionic liquid (IL)-based separation techniques into ISPR. This design method combines the selection of a suitable ISPR processing scheme, the optimal design of an IL-based liquid–liquid extraction (LLE) system followed by process simulation and evaluation. As a proof of concept, results for a conventional acetone–butanol–ethanol fermentation are presented (40,000 ton/year butanol production). In this application, ILs tetradecyl(trihexyl)phosphonium tetracyanoborate ([TDPh][TCB]) and tetraoctylammonium 2-methyl-1-naphthoate ([TOA] [MNaph]) are identified as the optimal solvents from computer-aided IL design (CAILD) method and reported experimental data, respectively. The dynamic simulation results for the fermentation process show that, the productivity of IL-based in situ (fed-batch) process and in situ (batch) process is around 2.7 and 1.8fold that of base case. Additionally, the IL-based in situ (fed-batch) process and in situ (batch) process also have significant energy savings (79.6% and 77.6%) when compared to the base case.     

Simultaneous acetone–butanol–ethanol fermentation,gas stripping,and full-cell-catalyzed esterification for effective production of butyl oleate

Bioprocess and Biosystems Engineering - In this study, aiming to improve the economic feasibility of acetone–butanol–ethanol (ABE) fermentation process, generate valuable products and...     

Recent progress on industrial fermentative production of acetone–butanol–ethanol by Clostridium acetobutylicum in China

China is one of the few countries, which maintained the fermentative acetone–butanol–ethanol (ABE) production for several decades. Until the end of the last century, the ABE fermentation from grain was operated in a few industrial scale plants. Due to the strong competition from the petrochemical industries, the fermentative ABE production lost its position in the 1990s, when all the solvent fermentation plants in China were closed. Under the current circumstances of concern about energy limitations and environmental pollution, new opportunities have emerged for the traditional ABE fermentation industry since it could again be potentially competitive with chemical synthesis. From 2006, several ABE fermentation plants in China have resumed production. The total solvent (acetone, butanol, and ethanol) production capacity from ten plants reached 210,000 tons, and the total solvent production is expected to be extended to 1,000,000 tons (based on the available data as of Sept. 2008). This article reviews current work in strain development, the continuous fermentation process, solvent recovery, and economic evaluation of ABE process in China. Challenges for an economically competitive ABE process in the future are also discussed.     

Combining chemical flocculation and bacterial co-culture of Cupriavidus taiwanensis and Ureibacillus thermosphaericus to detoxify a hardwood hemicelluloses hydrolysate and enable acetone–butanol–ethanol fermentation leading to butanol

Butanol, a fuel with better characteristics than ethanol, can be produced via acetone–butanol–ethanol (ABE) fermentation using lignocellulosic biomass as a carbon source. However, many inhibitors present in the hydrolysate limit the yield of the fermentation process. In this work, a detoxification technology combining flocculation and biodetoxification within a bacterial co-culture composed of Ureibacillus thermosphaericus and Cupriavidus taiwanensis is presented for the first time. Co-culture-based strategies to detoxify filtered and unfiltered hydrolysates have been investigated. The best results of detoxification were obtained for a two-step approach combining flocculation to biodetoxification. This sequential process led to a final phenolic compounds concentration of 1.4 g/L, a value close to the minimum inhibitory level observed for flocculated hydrolysate (1.1 g/L). The generated hydrolysate was then fermented with Clostridium acetobutylicum ATCC 824 for 120 h. A final butanol production of 8 g/L was obtained, although the detoxified hydrolysate was diluted to reach 0.3 g/L of phenolics to ensure noninhibitory conditions. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2753, 2019.     

Continuous bio-catalytic conversion of sugar mixture to acetone–butanol–ethanol by immobilized <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> DSM 792

Continuous production of acetone, n-butanol, and ethanol (ABE) was carried out using immobilized cells of Clostridium acetobutylicum DSM 792 using glucose and sugar mixture as a substrate. Among various lignocellulosic materials screened as a support matrix, coconut fibers and wood pulp fibers were found to be promising in batch experiments. With a motive of promoting wood-based bio-refinery concept, wood pulp was used as a cell holding material. Glucose and sugar mixture (glucose, mannose, galactose, arabinose, and xylose) comparable to lignocellulose hydrolysate was used as a substrate for continuous production of ABE. We report the best solvent productivity among wild-type strains using column reactor. The maximum total solvent concentration of 14.32 g L⁻¹ was obtained at a dilution rate of 0.22 h⁻¹ with glucose as a substrate compared to 12.64 g L⁻¹ at 0.5 h⁻¹ dilution rate with sugar mixture. The maximum solvent productivity (13.66 g L⁻¹ h⁻¹) was obtained at a dilution rate of 1.9 h⁻¹ with glucose as a substrate whereas solvent productivity (12.14 g L⁻¹ h⁻¹) was obtained at a dilution rate of 1.5 h⁻¹ with sugar mixture. The immobilized column reactor with wood pulp can become an efficient technology to be integrated with existing pulp mills to convert them into wood-based bio-refineries.     

Microbial production of a biofuel (acetone–butanol–ethanol) in a continuous bioreactor: impact of bleed and simultaneous product removal

Acetone butanol ethanol (ABE) was produced in an integrated continuous one-stage fermentation and gas stripping product recovery system using Clostridium beijerinckii BA101 and fermentation gases (CO₂ and H₂). In this system, the bioreactor was fed with a concentrated sugar solution (250–500 g L^?1 glucose). The bioreactor was bled semi-continuously to avoid accumulation of inhibitory chemicals and products. The continuous system was operated for 504 h (21 days) after which the fermentation was intentionally terminated. The bioreactor produced 461.3 g ABE from 1,125.0 g total sugar in 1 L culture volume as compared to a control batch process in which 18.4 g ABE was produced from 47.3 g sugar. These results demonstrate that ABE fermentation can be operated in an integrated continuous one-stage fermentation and product recovery system for a long period of time, if butanol and other microbial metabolites in the bioreactor are kept below threshold of toxicity.     

Acetone–butanol–ethanol production from corn stover pretreated by alkaline twin-screw extrusion pretreatment

In this study, the alkaline twin-screw extrusion pretreated corn stover was subjected to enzymatic hydrolysis after washing. The impact of solid loading and enzyme dose on enzymatic hydrolysis was investigated. It was found that 68.2 g/L of total fermentable sugar could be obtained after enzymatic hydrolysis with the solid loading of 10 %, while the highest sugar recovery of 91.07 % was achieved when the solid loading was 2 % with the cellulase dose of 24 FPU/g substrate. Subsequently, the hydrolyzate was fermented by Clostridium acetobutylicum ATCC 824. The acetone–butanol–ethanol (ABE) production of the hydrolyzate was compared with the glucose, xylose and simulated hydrolyzate medium which have the same reducing sugar concentration. It was shown that 7.1 g/L butanol and 11.2 g/L ABE could be produced after 72 h fermentation for the hydrolyzate obtained from enzymatic hydrolysis with 6 % solid loading. This is comparable to the glucose and simulated hydrozate medium, and the overall ABE yield could reach 0.112 g/g raw corn stover.     

Genome Shuffling of Clostridium acetobutylicum CICC 8012 for Improved Production of Acetone–Butanol–Ethanol (ABE)

Genome shuffling was applied to increase ABE production of the strict anaerobe C. acetobutylicum CICC 8012. By using physical and chemical mutagenesis, strains with superior streptomycin sulfate, 2-deoxy-D-glucose and butanol tolerance levels were isolated. These strains were used for genome shuffling. The best performing strain F2-GA was screened after two rounds of genome shuffling. With 55 g glucose/l as carbon source, F2-GA produced 22.21 g ABE/l in 72 h and ABE yield reached 0.42 g/g which was about 34.53 % improvement compared to the wild type. Fermentation parameters and gene expression of several key enzymes in ABE metabolic pathways were varied significantly between F2-GA and the wild type. These results demonstrated the potential use of genome shuffling to microbial breeding which were difficult to deal with traditional methods.     

Production of poly(β-hydroxybutyric acid) and exopolysaccharide by Azotobacter beijerinckii WDN-01

The physico-chemical factors influencing the production of poly(-hydroxybutyric acid) [PHB] and exopolysaccharide (EPS) by a yellow pigmented Azotobacter beijerinckii strain WDN-01 were investigated. Under N-free condition with excess carbon, PHB accumulation attained its maximum at the late exponential phase followed by a sharp decline while EPS production was more or less parallel with growth. Polymer synthesis, however, was carbon-source-specific, the highest yield of PHB (2.73 g/l) and EPS (1.5 g/l) was obtained with 3% (w/v) glucose and mannitol respectively. Organic N-sources enhanced PHB production significantly, but inorganic nitrogenous compounds were inhibitory to both PHB and EPS synthesis. At optimum K₂HPO₄ concentration, the polymer yield was attributed to biomass yield. Oxygen-limiting conditions, irrespective of carbon sources favoured production of PHB and EPS.