N-butanol Production Research Articles

Performance of Saccharomyces cerevisiae strains against the application of adaptive laboratory evolution strategies for butanol tolerance

Although the industrial production of butanol has been carried out for decades by bacteria of the Clostridium species, recent studies have shown the use of the yeast Saccharomyces cerevisiae as a promising alternative. While the production of n-butanol by this yeast is still very far from its tolerability (up to 2% butanol), the improvement in the tolerance can lead to an increase in butanol production. The aim of the present work was to evaluate the adaptive capacity of the laboratory strain X2180-1B and the Brazilian ethanol-producing strain CAT-1 when submitted to two strategies of adaptive laboratory Evolution (ALE) in butanol. The strains were submitted, in parallel, to ALE with successive passages or with UV irradiation, using 1% butanol as selection pressure. Despite initially showing greater tolerance to butanol, the CAT-1 strain did not show great improvements after being submitted to ALE. Already the laboratory strain X2180-1B showed an incredible increase in butanol tolerance, starting from a condition of inability to grow in 1% butanol, to the capacity to grow in this same condition. With emphasis on the X2180_n100#28 isolated colony that presented the highest maximum specific growth rate among all isolated colonies, we believe that this colony has good potential to be used as a model yeast for understanding the mechanisms that involve tolerance to alcohols and other inhibitory compounds.

Techno-Economic Assessment of Electromicrobial Production of n-Butanol from Air-Captured CO2.

Electromicrobial production (EMP), where electrochemically generated substrates (e.g., H2) are used as energy sources for microbial processes, has garnered significant interest as a method of producing fuels and other value-added chemicals from CO2. Combining these processes with direct air capture (DAC) has the potential to enable a truly circular carbon economy. Here, we analyze the economics of a hypothetical system that combines adsorbent-based DAC with EMP to produce n-butanol, a potential replacement for fossil fuels. First-principles-based modeling is used to predict the performance of the DAC and bioprocess components. A process model is then developed to map material and energy flows, and a techno-economic assessment is performed to determine the minimum fuel selling price. Beyond assessing a specific set of conditions, this analytical framework provides a tool to reveal potential pathways toward the economic viability of this process. We show that an EMP system utilizing an engineered knallgas bacterium can achieve butanol production costs of <$6/gal ($1.58/L) if a set of optimistic assumptions can be realized.

Effect of Nb as dopant of hydrotalcite catalysts during the ethanol condensation to optimize n-Butanol production

The present study optimizes Cu-supported catalysts on hydrotalcite for the synthesis of n-butanol whose interest is in its use as biofuel, due to its superior performance in comparison with other short-chain alcohols. The catalytic performance was improved by adding Nb to the catalytic formulation. In this way, several Cu-Nb catalysts supported on hydrotalcites (Mg/Al) with different Nb loadings were synthesized by incipient wetness impregnation and characterized by H2−temperature-programmed reduction, X-ray diffraction, N2 physisorption, CO2− and NH3−thermo-programmed desorption and X-ray photoelectron spectroscopy. It was shown that the incorporation of Nb promoted a suitable balance between the acid-basic and redox sites, modulating the Cu0/Cu+ dehydrogenation/hydrogenation properties for the n-BuOH production. The synthesis of multiple value-added products (n-BuOH, acetaldehyde, ethyl acetate, and 1-hexanol) was achieved in both the liquid− (at 180 ºC) and gas−phase (150−350 ºC), where it was demonstrated that both the n-BuOH selectivity and ethanol conversion improved with the Nb addition. The performance of the catalysts under time on stream and their recycling is also described.

Towards the effective distillation sequences for the purification of acetone-butanol-ethanol from the condensate of gas stripping-vapor permeation system

Gas stripping-vapor permeation (GSVP) is an attractive method for in situ acetone-butanol-ethanol (ABE) separation from fermentation broth to obtain renewable biological n-butanol. However, the downstream distillation systems for effective purification of ABE from the upstream GSVP process has not been developed. In this study, focusing on the generation of high purify A-B-E solvents as the final products, the downstream distillation systems for GSVP condensate purification were investigated. According to literature survey, four candidate distillation sequences were selected and optimized by steady-state simulation, followed by cross-over comparisons of energy requirements, techno-economics and environmental influences. The results indicate that the Scenario 3 that containing three individual columns and one decanter exhibited the lowest sensitivity to the key factors, lowest total annual cost (6.483 × 106 $/yr) and CO2 emission (3707.23 kg/h) in a scheme with 30 kt/yr n-butanol production among the four candidate distillation sequences. This study provides a valuable reference for the development of downstream ABE separation and purification processes in future works.

The selective ethanol Guerbet condensation over alkali metal-doped sepiolite

In this study, alkali metal-doped sepiolite based catalysts were developed for the up-grading of ethanol through the Guerbet reaction mechanism. The catalysts were prepared by wetness impregnation with aqueous solution containing alkali metal precursors salts over calcined sepiolite, and finally activated at 500ºC in air. The catalysts have been characterized by XRD, SEM-EDS, TGA-DSC, N2-adsorption. In addition, TPD-CO2 and TPD-NH3 were used to determine the acid and basic characteristics of catalysts. The catalysts were tested into a quartz continuous gas-flow reaction system working at atmospheric pressure to investigate the effects of metal loading (wt%), the nature of the alkali metals and of the main reaction parameters (e.g., reaction temperature, weight hourly space velocity), on the production of n-butanol starting from ethanol. The highest yield (18%) was obtained at 400 ºC with the catalyst consisting in sepiolite, calcined at 500ºC, impregnated with 7 wt% of Cs. This study proves that ad hoc modified natural sepiolites with alkali metals are effective catalysts for the Guerbet upgrading of ethanol.

Current progress on engineering microbial strains and consortia for production of cellulosic butanol through consolidated bioprocessing.

In the last decades, fermentative production of n-butanol has regained substantial interest mainly owing to its use as drop-in-fuel. The use of lignocellulose as an alternative to traditional acetone-butanol-ethanol fermentation feedstocks (starchy biomass and molasses) can significantly increase the economic competitiveness of biobutanol over production from non-renewable sources (petroleum). However, the low cost of lignocellulose is offset by its high recalcitrance to biodegradation which generally requires chemical-physical pre-treatment and multiple bioreactor-based processes. The development of consolidated processing (i.e., single-pot fermentation) can dramatically reduce lignocellulose fermentation costs and promote its industrial application. Here, strategies for developing microbial strains and consortia that feature both efficient (hemi)cellulose depolymerization and butanol production will be depicted, that is, rational metabolic engineering of native (hemi)cellulolytic or native butanol-producing or other suitable microorganisms; protoplast fusion of (hemi)cellulolytic and butanol-producing strains; and co-culture of (hemi)cellulolytic and butanol-producing microbes. Irrespective of the fermentation feedstock, biobutanol production is inherently limited by the severe toxicity of this solvent that challenges process economic viability. Hence, an overview of strategies for developing butanol hypertolerant strains will be provided.

Molecular characterization of the missing electron pathways for butanol synthesis in Clostridium acetobutylicum

Clostridium acetobutylicum is a promising biocatalyst for the renewable production of n-butanol. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it has previously demonstrated that the activities of both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase, whose encoding genes remain unknown, are necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purify, identify and partially characterize the proteins responsible for both activities and demonstrate the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrate the yield of butanol formation is limited by the level of expression of CA_C0764, the ferredoxin-NADP+ reductase encoding gene and the bcd operon, encoding a ferredoxin-NAD+ reductase. The integration of these enzymes into metabolic engineering strategies introduces opportunities for developing a homobutanologenic C. acetobutylicum strain.

Active and stable Cu doped NiMgAlO catalysts for upgrading ethanol to n-butanol

Upgrading ethanol to n-butanol is an attractive way for renewable n-butanol production. Herein, Cu was selected to modify NiMgAlO catalysts for improving ethanol conversion and n-butanol selectivity. Over the optimized 2%Cu-NiMgAlO catalyst, ethanol conversion and n-butanol selectivity were enhanced to 30.0% and 64.2%, respectively, in 200 h time on stream at 523 K. According to physicochemical characterizations and theoretical calculations, the key role of multiple active sites in this reaction was extensively investigated. The plate-like structure of hydrotalcite was maintained over 2%Cu-NiMgAlO catalysts, with an average Ni particle size of ca. 5.4 nm. The presence of Cu species created CuNi alloy sites and Lewis acid-base pairs, and increased hydrogen transfer and condensation reactions, resulting in elevated ethanol conversion and n-butanol selectivity. Additionally, CuNi alloy had a strong interaction with CuNiMgAl oxides, forming homogeneous boundary due to their close ionic radius and lattice matching, and afforded the long time stability in the ethanol to n-butanol reaction.

Enhancing biofuels production by engineering the actin cytoskeleton in Saccharomyces cerevisiae

Saccharomyces cerevisiae is widely employed as a cell factory for the production of biofuels. However, product toxicity has hindered improvements in biofuel production. Here, we engineer the actin cytoskeleton in S. cerevisiae to increase both the cell growth and production of n-butanol and medium-chain fatty acids. Actin cable tortuosity is regulated using an n-butanol responsive promoter-based autonomous bidirectional signal conditioner in S. cerevisiae. The budding index is increased by 14.0%, resulting in the highest n-butanol titer of 1674.3 mg L−1. Moreover, actin patch density is fine-tuned using a medium-chain fatty acid responsive promoter-based autonomous bidirectional signal conditioner. The intracellular pH is stabilized at 6.4, yielding the highest medium-chain fatty acids titer of 692.3 mg L−1 in yeast extract peptone dextrose medium. Engineering the actin cytoskeleton in S. cerevisiae can efficiently alleviate biofuels toxicity and enhance biofuels production.

Glucose/Xylose Co-Fermenting Saccharomyces cerevisiae Increases the Production of Acetyl-CoA Derived n-Butanol From Lignocellulosic Biomass.

Efficient xylose catabolism in engineered Saccharomyces cerevisiae enables more economical lignocellulosic biorefinery with improved production yields per unit of biomass. Yet, the product profile of glucose/xylose co-fermenting S. cerevisiae is mainly limited to bioethanol and a few other chemicals. Here, we introduced an n-butanol-biosynthesis pathway into a glucose/xylose co-fermenting S. cerevisiae strain (XUSEA) to evaluate its potential on the production of acetyl-CoA derived products. Higher n-butanol production of glucose/xylose co-fermenting strain was explained by the transcriptomic landscape, which revealed strongly increased acetyl-CoA and NADPH pools when compared to a glucose fermenting wild-type strain. The acetate supplementation expected to support acetyl-CoA pool further increased n-butanol production, which was also validated during the fermentation of lignocellulosic hydrolysates containing acetate. Our findings imply the feasibility of lignocellulosic biorefinery for producing fuels and chemicals derived from a key intermediate of acetyl-CoA through glucose/xylose co-fermentation.

Potentials of bio-butanol conversion to valuable products

Abstract In the last decade, there was observed a growing demand for both n-butanol as a potential fuel or fuel additive, and propylene as the only raw material for production of alcohol and other more bulky propylene chemical derivatives with faster growing outputs (polymers, propylene oxide, and acrylic acid). The predictable oilfield depletion and the European Green Deal adoption stimulated interest in alternative processes for n-butanol production, especially those involving bio-based materials. Their commercialization will promote additional market penetration of n-butanol for its application as a basic chemical. We analyze briefly the current status of two most advanced bio-based processes, i.e. ethanol–to-n-butanol and acetone–butanol–ethanol (ABE) fermentation. In the second part of the review, studies of n-butanol and ABE conversion to valuable products are considered with an emphasis on the most perspective catalytic systems and variants of the future processes realization.

N-Butanol production by Rhodopseudomonas palustris TIE-1

Anthropogenic carbon dioxide (CO2) release in the atmosphere from fossil fuel combustion has inspired scientists to study CO2 to biofuel conversion. Oxygenic phototrophs such as cyanobacteria have been used to produce biofuels using CO2. However, oxygen generation during oxygenic photosynthesis adversely affects biofuel production efficiency. To produce n-butanol (biofuel) from CO2, here we introduce an n-butanol biosynthesis pathway into an anoxygenic (non-oxygen evolving) photoautotroph, Rhodopseudomonas palustris TIE-1 (TIE-1). Using different carbon, nitrogen, and electron sources, we achieve n-butanol production in wild-type TIE-1 and mutants lacking electron-consuming (nitrogen-fixing) or acetyl-CoA-consuming (polyhydroxybutyrate and glycogen synthesis) pathways. The mutant lacking the nitrogen-fixing pathway produce the highest n-butanol. Coupled with novel hybrid bioelectrochemical platforms, this mutant produces n-butanol using CO2, solar panel-generated electricity, and light with high electrical energy conversion efficiency. Overall, this approach showcases TIE-1 as an attractive microbial chassis for carbon-neutral n-butanol bioproduction using sustainable, renewable, and abundant resources.

Recent progress on n-butanol production by lactic acid bacteria.

n-Butanol is an essential chemical intermediate produced through microbial fermentation. However, its toxicity to microbial cells has limited its production to a great extent. The anaerobe lactic acid bacteria (LAB) are the most resistant to n-butanol, so it should be the first choice for improving n-butanol production. The present article aims to review the following aspects of n-butanol production by LAB: (1) the tolerance of LAB to n-butanol, including its tolerance level and potential tolerance mechanisms; (2) genome editing tools in the n-butanol-resistant LAB; (3) methods of LAB modification for n-butanol production and the production levels after modification. This review will provide a theoretical basis for further research on n-butanol production by LAB.

Recent Progress and Trends in the Development of Microbial Biofuels from Solid Waste—A Review

This review covers the recent progress in the design and application of microbial biofuels, assessing the advancement of genetic engineering undertakings and their marketability, and lignocellulosic biomass pretreatment issues. Municipal solid waste (MSW) is a promising sustainable biofuel feedstock due to its high content of lignocellulosic fiber. In this review, we compared the production of fatty alcohols, alkanes, and n-butanol from residual biogenic waste and the environmental/economic parameters to that of conventional biofuels. New synthetic biology tools can be used to engineer fermentation pathways within micro-organisms to produce long-chain alcohols, isoprenoids, long-chain fatty acids, and esters, along with alkanes, as substitutes to petroleum-derived fuels. Biotechnological advances have struggled to address problems with bioethanol, such as lower energy density compared to gasoline and high corrosive and hygroscopic qualities that restrict its application in present infrastructure. Biofuels derived from the organic fraction of municipal solid waste (OFMSW) may have less environmental impacts compared to traditional fuel production, with the added benefit of lower production costs. Unfortunately, current advanced biofuel production suffers low production rates, which hinders commercial scaling-up efforts. Microbial-produced biofuels can address low productivity while increasing the spectrum of produced bioenergy molecules.

Enhanced sucrose fermentation by introduction of heterologous sucrose transporter and invertase into Clostridium beijerinckii for acetone-butanol-ethanol production.

A heterologous pathway for sucrose transport and metabolism was introduced into Clostridium beijerinckii to improve sucrose use for n-butanol production. The combined expression of StSUT1, encoding a sucrose transporter from potato (Solanum tuberosum), and SUC2, encoding a sucrose invertase from Saccharomyces cerevisiae, remarkably enhanced n-butanol production. With sucrose, sugarcane molasses and sugarcane juice as substrates, the C. beijerinckii strain harbouring StSUT1 and SUC2 increased acetone–butanol–ethanol production by 38.7%, 22.3% and 52.8%, respectively, compared with the wild-type strain. This is the first report to demonstrate enhanced sucrose fermentation due to the heterologous expression of a sucrose transporter and invertase in Clostridium. The metabolic engineering strategy used in this study can be widely applied in other microorganisms to enhance the production of high-value compounds from sucrose-based biomass.

Clostridium cellulovorans Proteomic Responses to Butanol Stress.

Combination of butanol-hyperproducing and hypertolerant phenotypes is essential for developing microbial strains suitable for industrial production of bio-butanol, one of the most promising liquid biofuels. Clostridium cellulovorans is among the microbial strains with the highest potential for direct production of n-butanol from lignocellulosic wastes, a process that would significantly reduce the cost of bio-butanol. However, butanol exhibits higher toxicity compared to ethanol and C. cellulovorans tolerance to this solvent is low. In the present investigation, comparative gel-free proteomics was used to study the response of C. cellulovorans to butanol challenge and understand the tolerance mechanisms activated in this condition. Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH-MS) analysis allowed identification and quantification of differentially expressed soluble proteins. The study data are available via ProteomeXchange with the identifier PXD024183. The most important response concerned modulation of protein biosynthesis, folding and degradation. Coherent with previous studies on other bacteria, several heat shock proteins (HSPs), involved in protein quality control, were up-regulated such as the chaperones GroES (Cpn10), Hsp90, and DnaJ. Globally, our data indicate that protein biosynthesis is reduced, likely not to overload HSPs. Several additional metabolic adaptations were triggered by butanol exposure such as the up-regulation of V- and F-type ATPases (involved in ATP synthesis/generation of proton motive force), enzymes involved in amino acid (e.g., arginine, lysine, methionine, and branched chain amino acids) biosynthesis and proteins involved in cell envelope re-arrangement (e.g., the products of Clocel_4136, Clocel_4137, Clocel_4144, Clocel_4162 and Clocel_4352, involved in the biosynthesis of saturated fatty acids) and a redistribution of carbon flux through fermentative pathways (acetate and formate yields were increased and decreased, respectively). Based on these experimental findings, several potential gene targets for metabolic engineering strategies aimed at improving butanol tolerance in C. cellulovorans are suggested. This includes overexpression of HSPs (e.g., GroES, Hsp90, DnaJ, ClpC), RNA chaperone Hfq, V- and F-type ATPases and a number of genes whose function in C. cellulovorans is currently unknown.

Control of n-Butanol Induced Lipidome Adaptations in E. coli.

The versatile compound n-butanol is one of the most promising biofuels for use in existing internal combustion engines, contributing to a smooth transition towards a clean energy society. Furthermore, n-butanol is a valuable resource to produce more complex molecules such as bioplastics. Microbial production of n-butanol from waste materials is hampered by the biotoxicity of n-butanol as it interferes with the proper functioning of lipid membranes. In this study we perform a large-scale investigation of the complete lipid-related enzyme machinery and its response to exposure to a sublethal concentration of n-butanol. We profiled, in triplicate, the growth characteristics and phospholipidomes of 116 different genetic constructs of E. coli, both in the presence and absence of 0.5% n-butanol (v/v). This led to the identification of 230 lipid species and subsequently to the reconstruction of the network of metabolites, enzymes and lipid properties driving the homeostasis of the E. coli lipidome. We were able to identify key lipids and biochemical pathways leading to altered n-butanol tolerance. The data led to new conceptual insights into the bacterial lipid metabolism which are discussed.

Engineering Clostridium cellulovorans for highly selective n-butanol production from cellulose in consolidated bioprocessing.

Cellulosic n-butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans, a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adhE2 from C. acetobutylicum for n-butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl-CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adhE2 would enhance n-butanol production. In this study, thiolase (thlACA ) from C. acetobutylicum and 3-hydroxybutyryl-CoA dehydrogenase (hbdCT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adhE2 to increase the flux from acetyl-CoA to butyryl-CoA. In addition, ferredoxin-NAD(P)+ oxidoreductase (fnr), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl-CoA, which resulted in increased production of butyrate and butanol. The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n-butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adhE2-fnrCA -thlACA -hbdCT produced n-butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n-butanol production from cellulosic biomass in consolidated bioprocessing.

Changes in freshwater sediment microbial populations during fermentation of crude glycerol

Background: This work studied how the exposure to an unusual substrate forced a change in microbial populations during anaerobic fermentation of crude glycerol, a by-product of biodiesel production, with freshwater sediment used as an inoculum. Results: The microbial associations almost completely (99.9%) utilized the glycerol contained in crude glycerol 6 g L-1 within four days, releasing gases, organic acids (acetic, butyric) and alcohols (ethanol, n-butanol) under anaerobic conditions. In comparison with control medium without glycerol, adding crude glycerol to the medium increased the amount of ethanol and n-butanol production and it was not significantly affected by incubation temperature (28°C or 37°C), nor incubation time (4 or 8 d), but it resulted in reduced amount of butyric acid. Higher volume of gas was produced at 37°C despite the fact that the overall bacterial count was smaller than the one measured at 20°C. Main microbial phyla of the inoculum were Actinobacteria, Proteobacteria and Firmicutes. During fermentation, significant changes were observed and Firmicutes, especially Clostridium spp., began to dominate, and the number of Actinobacteria and Gammaproteobacteria decreased accordingly. Concentration of Archaea decreased, especially in medium with crude glycerol. These changes were confirmed both by culturing and culture-independent (concentration of 16S rDNA) methods. Conclusions: Crude glycerol led to the adaptation of freshwater sediment microbial populations to this substrate. Changes of microbial community were a result of a community adaptation to a new source of carbon. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi;}

Reassessment of the role of CaCO3 in n-butanol production from pretreated lignocellulosic biomass by Clostridium acetobutylicum

In this study, the role of CaCO3 in n-butanol production was further investigated using corn straw hydrolysate (CSH) media by Clostridium acetobutylicum CICC 8016. CaCO3 addition stimulated sugars utilization and butanol production. Further study showed that calcium salts addition to CSH media led to the increase in Ca2+ concentration both intracellularly and extracellularly. Interestingly, without calcium salts addition, intracellular Ca2+ concentration in the synthetic P2 medium was much higher than that in the CSH medium despite the lower extracellular Ca2+ concentrations in the P2 medium. These results indicated that without additional calcium salts, Ca2+ uptake by C. acetobutylicum CICC 8016 in the CSH medium may be inhibited by non-sugar biomass degradation compounds, such as furans, phenolics and organic acids. Comparative proteomics analysis results showed that most enzymes involved in glycolysis, redox balance and amino acids metabolism were up-regulated with CaCO3 addition. This study provides further insights into the role of CaCO3 in n-butanol production using real biomass hydrolysate.