In situ biobutanol recovery from clostridial fermentations: a critical review

Butanol is a precursor of many industrial chemicals, and a fuel that is more energetic, safer and easier to handle than ethanol. Fermentative biobutanol can be produced using renewable carbon sources such as agro-industrial residues and lignocellulosic biomass. Solventogenic clostridia are known as the most preeminent biobutanol producers. However, until now, solvent production through the fermentative routes is still not economically competitive compared to the petrochemical approaches, because the butanol is toxic to their own producer bacteria, and thus, the production capability is limited by the butanol tolerance of producing cells. In order to relieve butanol toxicity to the cells and improve the butanol production, many recovery strategies (either in situ or downstream of the fermentation) have been attempted by many researchers and varied success has been achieved. In this article, we summarize in situ recovery techniques that have been applied to butanol production through Clostridium fermentation, including liquid–liquid extraction, perstraction, reactive extraction, adsorption, pervaporation, vacuum fermentation, flash fermentation and gas stripping. We offer a prospective and an opinion about the past, present and the future of these techniques, such as the application of advanced membrane technology and use of recent extractants, including polymer solutions and ionic liquids, as well as the application of these techniques to assist the in situ synthesis of butanol derivatives.     

Recent advances and state-of-the-art strategies in strain and process engineering for biobutanol production by Clostridium acetobutylicum

Butanol as an advanced biofuel has gained great attention due to its environmental benefits and superior properties compared to ethanol. However, the cost of biobutanol production via conventional acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is not economically competitive, which has hampered its industrial application. The strain performance and downstream process greatly impact the economics of biobutanol production. Although various engineered strains with carefully orchestrated metabolic and sporulation-specific pathways have been developed, none of them is ideal for industrial biobutanol production. For further strain improvement, it is necessary to develop advanced genome editing tools and a deep understanding of cellular functioning of genes in metabolic and regulatory pathways. Processes with integrated product recovery can increase fermentation productivity by continuously removing inhibitory products while generating butanol (ABE) in a concentrated solution. In this review, we provide an overview of recent advances in C. acetobutylicum strain engineering and process development focusing on in situ product recovery. With deep understanding of systematic cellular bioinformatics, the exploration of state-of-the-art genome editing tools such as CRISPR-Cas for targeted gene knock-out and knock-in would play a vital role in Clostridium cell engineering for biobutanol production. Developing advanced hybrid separation processes for in situ butanol recovery, which will be discussed with a detailed comparison of advantages and disadvantages of various recovery techniques, is also imperative to the economical development of biobutanol.     

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.     

Fermentative production of butanol--the industrial perspective

A sustainable bacterial fermentation route to produce biobutanol is poised for re-commercialization. Today, biobutanol can compete with synthetic butanol in the chemical market. Biobutanol is also a superior biofuel and, in longer term, can make an important contribution towards the demand for next generation biofuels. There is scope to improve the conventional fermentation process with solventogenic clostridia and drive down the production cost of 1-butanol by deploying recent advances in biotechnology and engineering. This review describes re-commercialization efforts and highlights developments in feedstock utilization, microbial strain development and fermentation process development, all of which significantly impact production costs.     

Prospective and development of butanol as an advanced biofuel

Butanol has been acknowledged as an advanced biofuel, but its production through acetone–butanol–ethanol (ABE) fermentation by clostridia is still not economically competitive, due to low butanol yield and titer. In this article, update progress in butanol production is reviewed. Low price and sustainable feedstocks such as lignocellulosic residues and dedicated energy crops are needed for butanol production at large scale to save feedstock cost, but processes are more complicated, compared to those established for ABE fermentation from sugar- and starch-based feedstocks. While rational designs targeting individual genes, enzymes or pathways are effective for improving butanol yield, global and systems strategies are more reasonable for engineering strains with stress tolerance controlled by multigenes. Compared to solvent-producing clostridia, engineering heterologous species such as Escherichia coli and Saccharomyces cerevisiae with butanol pathway might be a solution for eliminating the formation of major byproducts acetone and ethanol so that butanol yield can be improved significantly. Although batch fermentation has been practiced for butanol production in industry, continuous operation is more productive for large scale production of butanol as a biofuel, but a single chemostat bioreactor cannot achieve this goal for the biphasic ABE fermentation, and tanks-in-series systems should be optimized for alternative feedstocks and new strains. Moreover, energy saving is limited for the distillation system, even total solvents in the fermentation broth are increased significantly, since solvents are distilled to ~ 40% by the beer stripper, and more than 95% water is removed with the stillage without phase change, even with conventional distillation systems, needless to say that advanced chemical engineering technologies can distil solvents up to ~ 90% with the beer stripper, and the multistage pressure columns can well balance energy consumption for solvent fraction. Indeed, an increase in butanol titer with ABE fermentation can significantly save energy consumption for medium sterilization and stillage treatment, since concentrated medium can be used, and consequently total mass flow with production systems can be reduced. As for various in situ butanol removal technologies, their energy efficiency, capital investment and contamination risk to the fermentation process need to be evaluated carefully.     

Fermentative butanol production by Clostridia

Butanol is an aliphatic saturated alcohol having the molecular formula of C(4)H(9)OH. Butanol can be used as an intermediate in chemical synthesis and as a solvent for a wide variety of chemical and textile industry applications. Moreover, butanol has been considered as a potential fuel or fuel additive. Biological production of butanol (with acetone and ethanol) was one of the largest industrial fermentation processes early in the 20th century. However, fermentative production of butanol had lost its competitiveness by 1960s due to increasing substrate costs and the advent of more efficient petrochemical processes. Recently, increasing demand for the use of renewable resources as feedstock for the production of chemicals combined with advances in biotechnology through omics, systems biology, metabolic engineering and innovative process developments is generating a renewed interest in fermentative butanol production. This article reviews biotechnological production of butanol by clostridia and some relevant fermentation and downstream processes. The strategies for strain improvement by metabolic engineering and further requirements to make fermentative butanol production a successful industrial process are also discussed.     

Metabolic pathways of clostridia for producing butanol

Worldwide demand for energy has been the impetus for research to produce alcohol biofuels from renewable resources. This review focuses on the biosynthesis of butanol, which is regarded to be superior to ethanol as a fuel. Although acetone/butanol fermentation is one of the oldest large-scale fermentation processes, butanol yield by anaerobic fermentation remains sub-optimal. Metabolic engineering provides a means for fermentation improvements. Consequently, a comprehensive assessment of the intermediary enzymes involved in butanol formation from carbohydrates by the saccharolytic bacterium, Clostridium acetobutylicum and other closely allied clostridia was performed to provide guidelines for potentially enhancing butanol productivity. The activity of the enzymes, their regulation and contribution to the metabolic pathways was reviewed. Published kinetic data for each important enzymatic reaction were assessed. For most enzymatic reactions, the systematic investigation of the kinetic data and the properties of the enzymes led to the development of rate equations that were able to describe activity as the function of the substrates, products, and allosteric effectors.     

Metabolic engineering strategies for butanol production in Escherichia coli

The global market of butanol is increasing due to its growing applications as solvent, flavoring agent, and chemical precursor of several other compounds. Recently, the superior properties of n-butanol as a biofuel over ethanol have stimulated even more interest. (Bio)butanol is natively produced together with ethanol and acetone by Clostridium species through acetone-butanol-ethanol fermentation, at noncompetitive, low titers compared to petrochemical production. Different butanol production pathways have been expressed in Escherichia coli, a more accessible host compared to Clostridium species, to improve butanol titers and rates. The bioproduction of butanol is here reviewed from a historical and theoretical perspective. All tested rational metabolic engineering strategies in E. coli to increase butanol titers are reviewed: manipulation of central carbon metabolism, elimination of competing pathways, cofactor balancing, development of new pathways, expression of homologous enzymes, consumption of different substrates, and molecular biology strategies. The progress in the field of metabolic modeling and pathway generation algorithms and their potential application to butanol production are also summarized here. The main goals are to gather all the strategies, evaluate the respective progress obtained, identify, and exploit the outstanding challenges.     

Integrated butanol recovery for an advanced biofuel: current state and prospects

Butanol has recently gained increasing interest due to escalating prices in petroleum fuels and concerns on the energy crisis. However, the butanol production cost with conventional acetone–butanol–ethanol fermentation by Clostridium spp. was higher than that of petrochemical processes due to the low butanol titer, yield, and productivity in bioprocesses. In particular, a low butanol titer usually leads to an extremely high recovery cost. Conventional biobutanol recovery by distillation is an energy-intensive process, which has largely restricted the economic production of biobutanol. This article thus reviews the latest studies on butanol recovery techniques including gas stripping, liquid–liquid extraction, adsorption, and membrane-based techniques, which can be used for in situ recovery of inhibitory products to enhance butanol production. The productivity of the fermentation system is improved efficiently using the in situ recovery technology; however, the recovered butanol titer remains low due to the limitations from each one of these recovery technologies, especially when the feed butanol concentration is lower than 1 % (w/v). Therefore, several innovative multi-stage hybrid processes have been proposed and are discussed in this review. These hybrid processes including two-stage gas stripping and multi-stage pervaporation have high butanol selectivity, considerably higher energy and production efficiency, and should outperform the conventional processes using single separation step or method. The development of these new integrated processes will give a momentum for the sustainable production of industrial biobutanol.     

Achievements and perspectives to overcome the poor solvent resistance in acetone and butanol-producing microorganisms

Anaerobic bacteria such as the solventogenic clostridia can ferment a wide range of carbon sources (e.g., glucose, galactose, cellobiose, mannose, xylose, and arabinose) to produce carboxylic acids (acetic and butyric) and solvents such as acetone, butanol, and ethanol (ABE). The fermentation process typically proceeds in two phases (acidogenic and solventogenic) in a batch mode. Poor solvent resistance by the solventogenic clostridia and other fermenting microorganisms is a major limiting factor in the profitability of ABE production by fermentation. The toxic effect of solvents, especially butanol, limits the concentration of these solvents in the fermentation broth, limiting solvent yields and adding to the cost of solvent recovery from dilute solutions. The accepted dogma is that toxicity in the ABE fermentation is due to chaotropic effects of butanol on the cell membranes of the fermenting microorganisms, which poses a challenge for the biotechnological whole-cell bio-production of butanol. This mini-review is focused on (1) the effects of solvents on inhibition of cell metabolism (nutrient transport, ion transport, and energy metabolism); (2) cell membrane fluidity, death, and solvent tolerance associated with the ability of cells to tolerate high concentrations of solvents without significant loss of cell function; and (3) strategies for overcoming poor solvent resistance in acetone and butanol-producing microorganisms.     

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.     

Thiolase engineering for enhanced butanol production in Clostridium acetobutylicum

Biosynthetic thiolases catalyze the condensation of two molecules acetyl‐CoA to acetoacetyl‐CoA and represent key enzymes for carbon–carbon bond forming metabolic pathways. An important biotechnological example of such a pathway is the clostridial n‐butanol production, comprising various natural constraints that limit titer, yield, and productivity. In this study, the thiolase of Clostridium acetobutylicum, the model organism for solventogenic clostridia, was specifically engineered for reduced sensitivity towards its physiological inhibitor coenzyme A (CoA‐SH). A high‐throughput screening assay in 96‐well microtiter plates was developed employing Escherichia coli as host cells for expression of a mutant thiolase gene library. Screening of this library resulted in the identification of a thiolase derivative with significantly increased activity in the presence of free CoA‐SH. This optimized thiolase comprised three amino acid substitutions (R133G, H156N, G222V) and its gene was expressed in C. acetobutylicum ATCC 824 to assess the effect of reduced CoA‐SH sensitivity on solvent production. In addition to a clearly delayed ethanol and acetone formation, the ethanol and butanol titers were increased by 46% and 18%, respectively, while the final acetone concentrations were similar to the vector control strain. These results demonstrate that thiolase engineering constitutes a suitable methodology applicable to improve clostridial butanol production, but other biosynthetic pathways involving thiolase‐mediated carbon flux limitations might also be subjected to this new metabolic engineering approach. Biotechnol. Bioeng. 2013; 110: 887–897. © 2012 Wiley Periodicals, Inc.     

丙酮丁醇梭菌的基因编辑工具及代谢工程改造

丙酮丁醇梭菌作为极具潜力的新型生物燃料丁醇的生产菌,受到各国研究学者的广泛关注。通过丙酮丁醇梭菌(ABE)发酵生产丁醇,由于生产成本高,限制了其工业化应用。随着基因组学和分子生物学的快速发展,适用于丙酮丁醇的基因编辑工具不断发展并应用于提高菌株的发酵性能。本文对丙酮丁醇梭菌基因编辑工具和代谢工程改造取得的进展进行综述。     

Biobutanol production in a Clostridium acetobutylicum biofilm reactor integrated with simultaneous product recovery by adsorption

Background

Clostridium acetobutylicum can propagate on fibrous matrices and form biofilms that have improved butanol tolerance and a high fermentation rate and can be repeatedly used. Previously, a novel macroporous resin, KA-I, was synthesized in our laboratory and was demonstrated to be a good adsorbent with high selectivity and capacity for butanol recovery from a model solution. Based on these results, we aimed to develop a process integrating a biofilm reactor with simultaneous product recovery using the KA-I resin to maximize the production efficiency of biobutanol.

Results

KA-I showed great affinity for butanol and butyrate and could selectively enhance acetoin production at the expense of acetone during the fermentation. The biofilm reactor exhibited high productivity with considerably low broth turbidity during repeated batch fermentations. By maintaining the butanol level above 6.5 g/L in the biofilm reactor, butyrate adsorption by the KA-I resin was effectively reduced. Co-adsorption of acetone by the resin improved the fermentation performance. By redox modulation with methyl viologen (MV), the butanol-acetone ratio and the total product yield increased. An equivalent solvent titer of 96.5 to 130.7 g/L was achieved with a productivity of 1.0 to 1.5 g?·?L^-1?·?h^-1. The solvent concentration and productivity increased by 4 to 6-fold and 3 to 5-fold, respectively, compared to traditional batch fermentation using planktonic culture.

Conclusions

Compared to the conventional process, the integrated process dramatically improved the productivity and reduced the energy consumption as well as water usage in biobutanol production. While genetic engineering focuses on strain improvement to enhance butanol production, process development can fully exploit the productivity of a strain and maximize the production efficiency.     

Economical challenges to microbial producers of butanol: feedstock, butanol ratio and titer

Butanol is an important solvent and transport fuel additive, and can be produced by microbial fermentation. Attempts to generate a superior microbial producer of butanol have been made through different metabolic engineering strategies. However, to date, butanol bio-production is still not economically competitive compared to petrochemical-derived production because of its major drawbacks, such as, high cost of the feedstocks, low butanol concentration in the fermentation broth and the co-production of low-value by-products acetone and ethanol. Here we analyze the main bottlenecks in microbial butanol production and summarize relevant advances from recently reported studies. Further needs and directions for developing real industrially applicable strains in butanol production are also discussed.     

丁醇基因在大肠杆菌中表达的现状与展望

随着化石能源过度开采带来的能源短缺与环境恶化,丁醇凭借着其优越的理化性质成为了最具潜力的绿色燃料之一。近几年微生物在生物能源生产研究中受到广泛关注,主要集中在梭菌丁醇合成途径的异源表达。目前利用大肠杆菌产丁醇的产量已经接近产丁醇的天然菌株的产量。然而,大肠杆菌产丁醇仍存在很多限制性因素。主要从乙酰辅酶A依赖途径评述大肠杆菌生产丁醇的限制因素,并讨论提高丁醇产量需要解决的问题。     

New insights and novel developments in clostridial acetone/butanol/isopropanol fermentation

Clostridial acetone/butanol fermentation used to rank second only to ethanol fermentation by yeast in its scale of production and thus is one of the largest biotechnological processes known. Its decline since about 1950 has been caused by increasing substrate costs and the availability of much cheaper feedstocks for chemical solvent synthesis by the petrochemical industry. The so-called oil crisis in 1973 led to renewed interest in novel fermentation and product recovery technologies as well as in the metabolism and genetics of the bacterial species involved. As a consequence, almost all of the enzymes leading to solvent formation are known, their genes have been sequenced (in fact, Clostridium acetobutylicum has been recently included in the microbial genome sequencing project), the regulatory mechanisms controlling solventogenesis have begun to emerge and recombinant DNA techniques have been developed for these clostridia to construct specific production strains. In parallel, cheap agricultural-waste-based feedstocks have been exploited for their potential as novel substrates, continuous culture methods have been successfully established and new on-line product recovery technologies are now available, such as gas stripping, liquid/liquid extraction, and membrane-based methods. In combination with these achievements, a reintroduction of acetone/butanol fermentation on an industrial scale seems to be economically feasible, a view that is supported by a new pilot plant in Austria recently coming into operation. Received: 18 December 1997 / Received revision: 27 January 1998 / Accepted: 27 January 1998     

Recent advances on conversion and co-production of acetone-butanol-ethanol into high value-added bioproducts

Butanol is an important bulk chemical and has been regarded as an advanced biofuel. Large-scale production of butanol has been applied for more than 100 years, but its production through acetone–butanol–ethanol (ABE) fermentation process by solventogenic Clostridium species is still not economically viable due to the low butanol titer and yield caused by the toxicity of butanol and a by-product, such as acetone. Renewed interest in biobutanol as a biofuel has spurred technological advances to strain modification and fermentation process design. Especially, with the development of interdisciplinary processes, the sole product or even the mixture of ABE produced through ABE fermentation process can be further used as platform chemicals for high value added product production through enzymatic or chemical catalysis. This review aims to comprehensively summarize the most recent advances on the conversion of acetone, butanol and ABE mixture into various products, such as isopropanol, butyl-butyrate and higher-molecular mass alkanes. Additionally, co-production of other value added products with ABE was also discussed.     

Improving the <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> butanol fermentation by engineering the strain for co-production of riboflavin

Solvent-producing clostridia are well known for their capacity to use a wide variety of renewable biomass and agricultural waste materials for biobutanol production. To investigate the possibility of co-production of a high value chemical during biobutanol production, the Clostridium acetobutylicum riboflavin operon ribGBAH was over-expressed in C. acetobutylicum on Escherichia coli–Clostridium shuttle vector pJIR750. Constructs that either maintained the original C. acetobutylicum translational start codon or modified the start codons of ribG and ribB from TTG to ATG were designed. Riboflavin was successfully produced in both E. coli and C. acetobutylicum using these plasmids, and riboflavin could accumulate up to 27 mg/l in Clostridium culture. Furthermore, the C. acetobutylicum purine pathway was modified by over-expression of the Clostridium purF gene, which encodes the enzyme PRPP amidotransferase. The function of the plasmid pJaF bearing C. acetobutylicum purF was verified by its ability to complement an E. coli purF mutation. However, co-production of riboflavin with biobutanol by use of the purF over-expression plasmid was not improved under the experimental conditions examined. Further rational mutation of the purF gene was conducted by replacement of amino acid codons D302 V and K325Q to make it similar to the feedback-resistant enzymes of other species. However, the co-expression of ribGBAH and purFC in C. acetobutylicum also did not improve riboflavin production. By buffering the culture pH, C. acetobutylicum ATCC 824(pJpGN) could accumulate more than 70 mg/l riboflavin while producing 190 mM butanol in static cultures. Riboflavin production was shown to exert no effect on solvent production at these levels.