Advances and Developments in Biobutanol Production

By Juan Gabriel Segovia-Hernandez and Eduardo Sanchez-Ramirez

Advances and Developments in Biobutanol Production is a comprehensive reference on the production and purification of biobutanol, from the fundamentals to the latest advances. Focusing on selection of biomass, choice of pretreatments, biochemistry and design of fermentation, purification and biofuel application, the book also provides details on biorefinery design, lifecycle analysis, and offers perspectives on future developments. Through detailed analysis, chapters show readers how to overcome the challenges associated with the correct selection of raw material and adequate biomass pretreatment, the selection of microorganisms for fermenting biomass sugars, the purification of effluent coming from fermentation, and the high energy demands of production.

Solutions are supported by step-by-step guidance on methodologies and processes, with lab and industry-scale case studies providing real-world examples of their implementation. This book provides readers with a unique and comprehensive reference on the production of biobutanol for biofuel that will be of interest to graduates, researchers and professionals involved in bioenergy and renewable energy.

• Presents a holistic approach to the production and purification of biobutanol and its use as the high-value bioproduct
• Provides solutions to the major challenges and bottlenecks in biobutanol production, including feedstock, pretreatment, purification, fermentation, high energy demand and recover costs
• Offers step-by-step guidance on processes and procedures and describes their applications alongside real-world case studies

• Language: English
• Publisher: Elsevier Science
• Release date: Nov 19, 2022
• ISBN: 9780323998055

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Preface

During the last decades, there has been an increasing interest for renewable energy sources because of the problems associated with global warming, climate change, and volatile oil supply. Further environmental concerns have resulted in governmental actions in order to establish a significant energy independence and to promote environmental friendly fuels. In addition, several researches are focused on decreasing the CO2 emissions and the reduction of the dependency on fossil fuels, especially oil, due to environmental as well as geopolitical reasons. Nowadays, there are several biofuels that can be produced from biomass through fermentation of lignocellulose such as biobutanol. Several properties of butanol, such as higher energy density, lower steam pressure, less flammability, and hydrophobicity, are leading to a growing interest in this biofuel. Particularly, in the industry there is an intensive interest in the use of biobutanol. Butanol is used as solvent, hydraulic fluid, detergent, antibiotic; however, it may also be used as fuel.

The earliest commercial production of biobutanol is reported to start in the late nineteenth century after Louis Pasteur devised Acetone-Butanol-Ethanol oxidative pathway from biological matter (fermentation). With the increasing demand and emergence of low-cost petroleum-based butanol, biobutanol synthesis was considered redundant. The resurgence of butanol of biological origin aligns with a reduction in alarming levels of greenhouse gas emissions and mitigation of global climate change. Biobutanol is an alternative to conventional transportation fuels. The benefits of biobutanol include (a) higher energy content—biobutanol's energy content is relatively high among gasoline alternatives; (b) biobutanol's energy density is 10%–20% lower than gasoline's energy density; (c) lower vapor pressure—when compared to ethanol, biobutanol has a lower vapor pressure, which means lower volatility and evaporative emissions; (d) increased energy security—biobutanol can be produced domestically from a variety of feedstocks; (e) fewer emissions are generated with the use of biobutanol compared with petroleum fuels; (f) more transport options—biobutanol is immiscible with water, meaning that it may be able to be transported in pipelines to reduce transport costs. Beyond the direct application of biobutanol as fuel, there are some works that place it as a component that can be used as a platform for products with higher added value.

This book highlights the importance of production and purification of biobutanol and discusses the required interdisciplinary approach to accomplish it. Authors outline novel processes and current challenges in the production of this important biofuel at different levels. This book presents an overview of important ideas addressed within methodologies proposed for sustainable processes in the production of biobutanol.

1: Bio-butanol production: scope, significance, and applications

Hamed Kazemi Shariat Panahi ¹ , ² , Mona Dehhaghi ¹ , ² , Gilles J. Guillemin ¹ , Christopher Chukwudi Okonkwo ³ , James E. Kinder ⁴ , ⁵ , and Thaddeus Chukwuemeka Ezeji ³       ¹ Neuroinflammation Group, Department of Biomedical Sciences, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW, Australia      ² Department of Microbial Biotechnology, School of Biology and Centre of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, Tehran, Iran      ³ Department of Animal Sciences, Ohio State Agricultural Research and Development Center (OARDC), The Ohio State University, Wooster, OH, United States      ⁴ College of Food, Agricultural, and Environmental Sciences, Department of Animal Sciences, The Ohio State University, Columbus, OH, United States      ⁵Institute for Future Farming Systems, School of Health Medical and Applied Sciences, CQUniversity, Rockhampton, QLD, Australia

Abstract

Butanol is a colorless flammable four-carbon alcohol with a characteristic banana-like odor that is currently used as an industrial solvent in the manufacturing of plasticizers, butylamines, butyl acetate, butyl acrylate and methacrylate, glycol ethers and amino resins. In addition, butanol has some properties that make it a viable transportation fuel. These properties include a low flash point, hydrophilicity, vapor pressure and volatility, large energy content per unit mass, and miscibility with hydrocarbon-based fuels. Some of these properties make butanol less corrosive and compatible with the existing oil infrastructure. As interest in environmentally compatible fuels intensifies, butanol is one of the chemicals that have gained attention of scientists and engineers as a potential replacement for gasoline. Meanwhile, butanol fermentation, popularly known as acetone-butanol-ethanol (ABE) fermentation, has a rich history that dates to Louis Pasteur, who in 1861 observed that bacteria can produce butanol. The ABE fermentation is plagued with less-than-optimal butanol titers in the bioreactor due to the inhibitory effect of butanol on fermenting microorganisms. The cost of butanol recovery from the fermentation broth, however, is the second largest contributor to the production cost, the first being the substrate cost. Development of knowledge-based strategies to resolve complex issues related to substrate compatibility and butanol toxicity to butanol-producing microorganisms is evolving. Consequently, in this chapter, there is elaboration on the scope of butanol-producing microorganisms, butanol production from food- and nonfood-based feedstock, technology innovations to advance butanol fermentation, and significance and applications of butanol and butanol derivatives.

Keywords

Butanol; Downstream processing; Jet fuel; Lignocellulosic biomass; Microalgae; Syngas

1.1. Introduction

The costs, environmental impacts, and nonrenewable nature of crude oil are some motivating reasons behind the search for green liquid biofuels. Political instability in some of the oil-producing countries with potential ripple effects on energy insecurity is another factor that motivates the leaders in oil-dependent countries to search for alternative renewable energy sources for their fuel needs (Ezeji et al., 2004a,b; Kazemi Shariat Panahi et al., 2019). Bioethanol is currently used in the transportation sector to reduce net carbon dioxide emissions and to a lesser extent, to address unpredictable increases in the oil. While the bioethanol–gasoline blend with the bioethanol content as large as 85% can power flexible fuel vehicles (Panahi et al., 2019), it is mostly utilized as a gasoline extender (octane booster and gasohol) with markedly lesser bioethanol–gasoline blend ratios, 10:90 (v/v) in the United States and 24:76 (v/v) in Brazil (Panahi et al., 2019, 2020a,b). Biobutanol, however, has excellent fuel characteristics as it has a large energy content per unit mass, has a lesser vapor pressure and flash point, and is more miscible with gasoline and diesel than does ethanol. Biobutanol can be used at a greater blend ratio with gasoline or even at a 100% concentration in car engines with little to no engine modification. The lesser butanol titer in the bioreactor during acetone-butanol-ethanol (ABE) fermentation, which is 8–18 times less than that of ethanol produced by yeast fermentation, however, is a major limiting factor for the use of biobutanol as a biofuel (Panahi et al., 2019).

1.2. History of butanol production

Fermentation of sugar- or starch-containing fruits and vegetables to alcoholic beverage may be regarded as the oldest, largest in scale, and most famous biotechnology process in the human history. In 1861, Louis Pasteur made the first observation that bacteria can produce butanol. In 1905, Austrian biochemist, Franz Schardinger, made a similar observation that bacteria can produce acetone (Dürre et al., 1992). The isolation of Clostridium acetobutylicum by Chaim Weizmann in 1912 led to the discovery of an anaerobic process known today as acetone butanol (AB) or acetone-butanol-ethanol (ABE) fermentation (Jones and Woods 1986; Dürre 1998; Zverlov et al., 2006). Industrial production of ABE started in 1916, during World War I, and acetone was the target product because of the production of the smokeless nitrocellulose explosive, cordite. For this purpose, large-scale industrial ABE fermentation plants were built in Canada and the United States. Following the increased demand for butanol, a group of American businessmen affiliated with the Commercial Solvent Corporation obtained a license for the Chaim Weizmann ABE fermentation process (US Patent 1315585, 1919) at which there was the initiation of butanol production in Terre Haute, Indiana, USA, in 1920. The Commercial Solvent Corporation operated another ABE fermentation plant in Peoria, Illinois, where there was the use of corn as feedstock. The plant started with thirty-two 50,000-gallon fermenters in 1923, and there were an additional 96 fermenters built by the end of 1927 (Gabriel, 1928). In the 1930s, fermentative butanol production became attractive due to the utilization for production of butyl paints and lacquers (protective coatings). Consequently, many ABE plants were established in other countries including the former USSR, Japan, India, Australia, and South Africa (Jones and Woods 1986). Nonetheless, very little is known about the industrial ABE fermentation process in the former USSR and the first ABE industrial plant in which there was use of the Weizmann process that was constructed between 1929 and 1935 (Zverlov et al., 2006). As an aviation fuel and a fuel extender for aircraft, there are some reports indicating there was use of butanol in some Japanese fighter planes as fuel during World War II (Schwarz et al., 2007).

In 1945, while as much as ∼67% of the global butanol demand was met using fermentation procedures in the United States (Jones and Woods, 1986, Lee et al., 2008), there were other large production facilities including Kyowa Hakko, Japan, and National Chemical Products in Germiston, South Africa. In the early 1960s, ABE fermentation was discontinued in the United States due to unfavorable economic conditions brought about by competition with the petrochemical industry. Meanwhile, in South Africa, the use of traditional ABE fermentation continued well into mid-1980s when the last plant closed. Apart from the competition from the petrochemical-derived butanol, the other factor that detracted from further development of industrial ABE fermentation plants was frequent bacteriophage and acid-tolerant bacterial contaminations. Although the Russian Dokshukino, Evremovo, and Grosnyi plants continued to be profitable after the 1960s because the plants were producing 400–600μgL −¹ vitamin B12 as a by-product and the fermenting microbes were resistant to bacteriophage infections, profits later decreased when the fermentation process started to be less because of lactic acid bacteria contamination that resulted in decreased ABE yield and shorter semi-continuous fermentation cycle. Eventually, there was closure of most of the butanol research laboratories and production facilities with only a few Russian plants in Dokshukino, Grosny, and Talitsk continuing to produce butanol into the late 1980s before there was also closure of these plants (Jones and Woods, 1986; Zverlov et al., 2006, Lee et al., 2008).

In China, there was a different industrial ABE production trend, and the first ABE plant was established in the early 1950s by the Shanghai Solvent Plant in Pudong, Shanghai, which quickly expanded (Chiao and Sun, 2007). There were approximately 30 ABE plants in China that were operational at some point in time in which there was production of as much as 170,000 tons of ABE annually, which continued to operate until 2004 (Chiao and Sun, 2007) mainly due to the availability of inexpensive grain. The opening of Chinese market to the international community, however, resulted in an increased grain price and production cost, and biobutanol production was not competitive with that of petrochemical production (Chiao and Sun, 2007). To meet the increasing global demand for butanol, biological processes were substituted with chemical processes, i.e., crotonaldehyde hydrogenation, oxo synthesis (hydroformylation), and Reppe synthesis (Panahi et al., 2019).

Overall, microbial production of butanol and acetone decreased rapidly due to a lack of sustainable substrate, large fermentation costs, decreasing crude oil price, and the availability of less expensive petroleum-derived substrates for chemical synthesis of butanol. The interest in butanol fermentation revived because of the predicted oil crisis and harmful effects of greenhouse gas (GHG) generation from fossil fuels. The potential use of butanol as a biofuel was markedly enhanced in 2005 when David Ramey drove his unmodified 1992 Buick car across the US fueled solely by butanol (Dürre, 2007). Subsequently, in 2007, BP and DuPont announced a joint venture, approved by the European Commission, to develop and commercialize the biological synthesis of butanol. This joint venture also included development of cost-effective technologies to produce butanol from lignocellulosic biomass (Comyns, 2007). Although substantial progress has been made in biobutanol production research, more research is needed in microbiology, biochemistry, and fermentation to effectively increase the butanol yield and productivity and process efficiency to the extent where there is the capacity to decrease production cost of biobutanol. Current research in biobutanol production is multdisciplinary, with there being a combination of microbiology, molecular biology, process and chemical engineering, and economics utilized to develop hyper-butanol producing microbial strains with concomitant reduction of sterilization and downstream processing costs, with there being the feasible economics of production.

1.3. Microbial species and strains commonly used for butanol fermentation

1.3.1. Native butanol producing microorganisms

Native butanol producing microorganisms are typically solventogenic Clostridium species (Table 1.1). This group of microorganisms, which belong to the family of Bacillaceae, are Gram-positive anaerobic bacteria with peritrichous flagella that result in the capacity for being very motile. The vegetative cells of solventogenic Clostridium species are rod-shaped, and environmental factors may result in there being variable shapes of cells of this species. These cells produce heat-resistant oval or spherical endospores. The diameter of the endospores is typically larger than that of the vegetative cell. Solventogenic Clostridium species have a unique physiology during batch fermentation because the cells of these species can undergo a two-phase fermentation process (i.e., acidogenesis and solventogenesis) sometimes referred to as biphasic fermentation to produce three major products (i.e., solvents: acetone, butanol, and ethanol; organic acids: acetic and butyric acid; and gases: CO2 and H2). Acetone, a major product of ABE fermentation by solventogenic Clostridium species, was previously thought to be produced only by the decarboxylation of acetoacetate into acetone and CO2 by acetoacetate decarboxylase (aadc) enzyme encoded by the acetoacetate decarboxylase (adc) gene. In 2011, however, Han and collaborators described the scope of robust nonenzymatic pathway for acetone formation by C. beijerinckii, and as a result, there was a new framework to consider while formulating strategies to increase the butanol to acetone ratio during ABE fermentation (Han et al., 2011). Approximately, 40 solventogenic Clostridium species and strains are available in public culture collections, and this large pool of strains has a wide range of genetic traits for the utilization of alternative substrates, phage resistance, and solvent-producing capacity (Zverlov et al., 2006). In solventogenic Clostridium species, following glycolysis, the butanol production pathway is initiated with the condensation of two acetyl-CoA to form acetoacetyl-CoA, and thiolase is responsible for the catalysis (Fig. 1.1).

Table 1.1

Acetoacetyl-CoA can then be converted into acetone and 1-butanol by a series of dehydration and reduction reactions. To form 1-butanol, acetoacetyl-CoA is reduced into butyryl-CoA with 3-hydroxybutyryl-CoA and crotonyl-CoA being the intermediates (Fig. 1.1). The reduction of crotonyl-CoA into butyryl-CoA is catalyzed by an electron-bifurcating enzyme complex, butyryl-CoA dehydrogenase (Bcd)—an electron-transferring flavoprotein (Etf). The functions of Bcd/Etf complex are NADH dependent for coupling the endergonic reduction of ferredoxin with the exergonic reduction of crotonyl-CoA (Buckel and Thauer, 2013; Chen and Liao, 2016). The pathway is completed by a bifunctional alcohol/aldehyde dehydrogenase (butyraldehyde- and butanol-dehydrogenase), reducing butyryl-CoA into 1-butanol using butyraldehyde as the intermediate. Notably, native solventogenic Clostridium species produce acids (i.e., acetate, butyrate) during the acidogenic growth phase that results in ATP generation when there is an NADH-abundant condition, and the acids are reassimilated for ABE production (solventogenesis); all these make engineering of this group of microorganisms to selectively produce butanol challenging.

Figure 1.1  Acetone-butanol-ethanol (ABE) metabolic pathway in C. beijerinckii and C. acetobutylicum including nonenzymatic decarboxylation of acetoacetate into acetone and CO2 (Han et al., 2011). With Permission from Springer. Copyright© 20xx.

Some nonsolventogenic Clostridium species such as the Lactobacillus species produces 2-butanol, not 1-butanol, within bacterial microcompartments during anaerobic sugar fermentation via reduction of 2,3-butanediol (2,3-BD) (Russmayer et al., 2019). In this pathway, vitamin B12-dependent glycerol dehydratase first dehydrates meso-2,3-BD into 2-butanone. This processing stage is followed by the reduction of 2-butanone into 2-butanol by an alcohol dehydrogenase (Russmayer et al., 2019). In Fig. 1.2, there is the depiction of the metabolic pathway of 2-butanol formation in microcompartments of Lactobacillus diolivorans during anaerobic fermentation of sugar.

1.3.2. Metabolically modified native butanol-producing microorganisms

A major limitation to large-scale production of butanol by native solventogenic Clostridium species is small tolerance of the cells to biosynthesized solvents, n-butanol being the most toxic. The n-butanol toxicity to solventogenic clostridial cells results in a relatively lesser butanol titer in the bioreactor during ABE fermentation, making the fermentation process less economically feasible. In addition to the lesser butanol titer, other limitations of native solventogenic Clostridium species include production of multiple products (butanol, acetone, ethanol, acetic acid, and butyric acid) during ABE fermentation, sporulation and the small quantity of solvent production, and strain degeneration (Kazemi Shariat Panahi et al., 2019; Lee et al., 2008). Mutagenesis and metabolic engineering can be utilized to address the constraints that were previously described. Chemical or physical mutagens including UV irradiation, nitrosoguanidine, ethyl methanesulfonate, metronidazole, nalidixic acid, and hydrogen peroxide can be used to induce random mutagenesis in solventogenic Clostridium species (Ezeji et al., 2007b). For example, N-methyl-N-nitro-N-nitrosoguanidine was used to induce random mutagenesis in C. beijerinckii NCIMB 8052, which resulted in the production of a stable hyperamylolytic and hyperbutanologenic strain, C. beijerinckii BA101 (Ezeji et al., 2004b). The strain that was developed tolerated 23gL −¹ butanol and produced approximately 33gL −¹ ABE (vs. 15–20gL −¹) in a batch process (Qureshi et al., 2000). Furthermore, the F2-GA strain was developed by a combination of random mutagenesis of C. acetobutylicum CICC 8012 with UV or nitrosoguanidine and genome shuffling by protoplast fusion (Gao et al., 2012). Compared with the wild strain, with F2-GA, there was production of 34.6% more ABE than the wildtype. The fermentation capacity of three carbon sugar-metabolizing solventogenic Clostridium species can also be improved with chemical mutagenesis for butanol production. When glycerol metabolizing C. pasteurianum, which typically ferment crude glycerol into butanol (7.8gL −¹) with there being little productivity (i.e., 0.03gL −¹ h) and yield of 0.36gg −¹ (Taconi et al., 2009), was subjected to chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine, the resulting MBEL_GLY2 strain had a greater butanol production of as much as 17.8gL −¹ with the productivity and yield being 0.43gL −¹ h and 0.30gg −¹, respectively (Malaviya et al., 2012). The genetic modification facilitated the inclusion of crude glycerol, a major by-product of biodiesel industry (Rahimzadeh et al., 2018; Tabatabaei et al., 2019, 2020b), as an inexpensive substrate for biobutanol production (Kazemi Shariat Panahi et al., 2019).

Figure 1.2  Metabolic pathway of 2-butanol formation in microcompartments of Lactobacillus diolivorans during anaerobic fermentation of sugar (Russmayer et al., 2019).

Targeted mutagenesis and gene overexpression can and have been used to improve butanol production in solventogenic Clostridium species. Strategies such as deletion of pathways in competition with butanol production and overexpression of genes in solventogenic Clostridium species to enhance the production of butanol or tolerance to fermentation inhibitors have been implemented successfully. The function of Spo0A, a master regulator of both solventogenesis and sporulation in C. acetobutylicum (Xu et al., 2015), for example, was disrupted by a single base deletion in cac3319, one of the three orphan histidine kinases involved in Spo0A activation. The butanol tolerance and production by the generated C. acetobutylicum JB200 was 67% (21 compared with 12.6gL −¹) and 44.4% (18.2 compared with 12.6gL −¹), respectively, greater than the wildtype, C. acetobutylicum ATCC 55025 (Xu et al., 2015). Similarly, disruption of SMB_G1518 and/or its downstream gene SMB_G1519 can enhance butanol tolerance in C. acetobutylicum (Jia et al., 2012). To improve glycerol utilization by C. beijerinckii NCIMB 8052, two glycerol dehydrogenase (Gldh) genes (dhaD1 and gldA1) from the glycerol hyperutilizing C. pasteurianum (Cp) were overexpressed in C. beijerinckii as a fused protein, thereby increasing glycerol utilization by as much as 43% (Agu et al., 2019). Similarly, Okonkwo et al. increased the tolerance of C. beijerinckii to furfural by two-fold when there was homologous integration and constitutive expression of the Cbei_3974 and Cbei_3904 genes, which encode aldo-keto reductase and previously annotated short chain dehydrogenase/reductase, respectively, in C. beijerinckii NCIMB 8052 (Okonkwo et al., 2019). With the invention of CRISPR-Cas system and adaptation to engineering of solventogenic Clostridium species, more targeted mutagenesis and gene expression to improve butanol production are expected. Utilization of the CRISPR procedures has provided for a very efficient deletion method of genome engineering with a simple cloning method. With this technique, an array of target sequence is transcribed and processed to form CRISPR-RNA (CrRNA) guide Cas nuclease. The target site is subsequently cleaved using protospaceradjacent motif (PAM). Type-II CRISPR analysis is a dual RNA complex guiding system, in which Cas9 is activated only with transactivating CRISPR RNA (TracrRNA) and CrRNA. Using the CRISPR-Cas9n-based genome editing technology, the encoding region of six histidine kinases genes of C. beijerinckii were deleted, with butanol production being increased by 41% when compared with the production by wildtype microbes (Xin et al., 2020). A major limitation of the CRISPR technique, however, is accurate, dependent on homologous recombinant efficiency of the microbe (Bruder et al., 2015; Copeland et al., 2014; Wang et al., 2015b). In another study, the severe autolytic actions (affects cells stability) of C. saccharoperbutylacetonicum was mitigated by identifying the key autolysin gene in the bacteria and deleting it using CRISPR-Cas9 technique; the stability and butanol production characteristics increased significantly (Jiménez-Bonilla et al., 2021).

1.3.3. Metabolically modified nonnative butanol-producing microorganisms

The complex physiology of native solventogenic Clostridium species with associated biphasic fermentation and spore-forming life cycle is a major factor that has contributed to protracted metabolic engineering of this group of microorganisms for optimal butanol production and yield. In many studies, there have been attempts to clone butanol production pathway in nonnative butanol producing and genetically tractable microorganisms such as E. coli (Atsumi et al., 2008a; Dong et al., 2017; Inui et al., 2008), Bacillus subtilis (Nielsen et al., 2009), Pseudomonas putida (Nielsen et al., 2009), Lactobacillus brevis (Berezina et al., 2010), Saccharomyces cerevisiae (Generoso et al., 2015), and Synechococcus elongatus (Lan and Liao, 2011). There is large incentive, therefore, to develop a robust, genetically stabile, and easily utilizable microbial population for butanol production. Substrate cost and sustainability are among other factors affecting the desire to develop nonnative butanol-producing microorganisms as a robust and resourceful biocatalyst for industrial production of butanol. To achieve this goal, several nonnative butanol-producing microorganisms have been metabolically engineered to produce butanol. There is information included in Table 1.1 summarizing some recombinant nonnative butanol-producing strains that have been developed during the past 2decades. Of particular interest is E. coli, a Gram-negative rapid-growing rod-shaped facultative anaerobe, that is non-spore-forming and genetically tractable. It should be noted that E. coli was the first nonnative butanol-producing microorganism in which there was cloning and expression of genes encoding for proteins of the butanol production pathway of C. acetobutylicum. Although the pathway was functional, the butanol titer was less than optimal being about 1gL −¹ (Atsumi et al., 2008a; Inui et al., 2008). The butanol titer was improved by replacing the bcd gene of the original pathway with the ter gene from Treponema denticola, which resulted in a decrease in the negative feedback control of the forward reaction in the butanol production pathway (Bond-Watts et al., 2011; Shen et al., 2011). Furthermore, systematic deletion of 33 native genes and heterologous overexpression of five genes resulted in production of an E. coli strain, EB243, that produced 20gL −¹ butanol with a yield of 34% (Dong et al., 2017). The butanol production pathway has also been successfully transferred into other microorganisms such as C. cellulovorans, C. tyrobutyricum, S. cerevisiae, C. cellulolyticum, C. ljungahlii, C. autoethanogenum, P. furiosus, etc (Table 1.2). The rationale for choosing these microorganisms for metabolic engineering to produce butanol was strategic in terms of growth rate and conditions, substrate utilization, and product formation. For example, C. tyrobutyricum is a nonsolventogenic acidogen, which had advantages compared with solventogenic Clostridium species including minimal autolysis and sporulation rates, greater butanol tolerance, and favorable metabolic pathway functions in conversion of glucose to butyryl-CoA with no acetone production. While overexpression of aldehyde/alcohol dehydrogenase (adhE2) and acetate kinase (ack) gene in C. tyrobutyricum resulted in butanol production (Liu et al., 2006; Yu et al., 2011), when there were relatively greater titers of butyric and acetic acids, there was a lesser butanol yield of about 0.1gg −¹ glucose (Du et al., 2015). Further engineering of these microorganisms resulted in decreased butyrate, acetate, and hydrogen production by 80%–90%, which led to an increased butanol production and yield (Du et al., 2015). The improvements were attributed to improved NADH availability through addition of 500μM methyl viologen because NADH was determined to be the limiting factor for butanol production (Shen et al., 2011). Meanwhile, NADH availability can be improved by manipulation of the electron flow and redirection of carbon flux in C. tyrobutyricum (Liu et al., 2013; Lütke-Eversloh and Bahl,