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Review

Fermentative Butanol Production—Perspectives and Scale-Up Challenges

by
Seedhabadee Ganeshan
* and
Mehmet Çağlar Tülbek
Saskatchewan Food Industry Development Centre, Saskatoon, SK S7M 5V1, Canada
*
Author to whom correspondence should be addressed.
Encyclopedia 2025, 5(2), 50; https://doi.org/10.3390/encyclopedia5020050
Submission received: 26 February 2025 / Revised: 26 March 2025 / Accepted: 5 April 2025 / Published: 9 April 2025
(This article belongs to the Section Chemistry)
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Abstract

:
Sustainable solutions to the use of petrochemical products have been increasingly sought after in recent years. While alternatives such as biofuels have been extensively explored and commercialized, major challenges remain in using heterogeneous feedstocks and scaling-up processes. Among biofuels, higher alcohols have recently gained renewed interest, especially in the context of upcycling agri-food residues and other industrial organic wastes. One of the higher alcohols produced via fermentation is butanol, which was developed over a century ago. However, the commercial production of butanol is still not widespread, although diverse feedstocks are readily available. Hydrolysis of the feedstocks and scale-up challenges in the fermentation and purification of butanol are recurring bottlenecks. This review addresses the current state of fermentative butanol production and opportunities to address scale-up challenges, including purification. With the significant interest and promise of precision fermentation, this review also addresses some of the recent advances and potential for enhanced fermentative butanol production.

1. Introduction

Our dependence on fossil fuels is immeasurable, and despite our best efforts to become less dependent, the availability of alternatives is still yet to supplant existing petrochemical-derived products. The staggering increase in primary energy consumption over many decades reinforces our astounding dependence on fossil fuels (Figure 1), which was only made more significant with the dawn of the Industrial Revolution [1,2,3]. Interestingly, in 2019, there was a decrease in primary energy consumption due to the COVID-19 pandemic caused by the SARS-CoV-2 coronavirus, which led to a global lockdown and a reduction in industrial activity and, indeed, human commute in general. However, the pandemic has also offered a glimpse into what a reduction in energy consumption might look like on the overall climate change outlook and humanity’s existence per se. One of the debatable questions was whether COVID-19 could lead to extinction, not because of the COVID-19 disease, but rather due to reduced industrial activity that could lead to temperature increases due to the lack of the dimming effects of aerosols generally associated with industrial activity protecting against such rapid increases in temperatures [4]. While this debate regarding the extinction of life is unlikely to be resolved in the near future, the increasing trend in primary energy consumption derived from fossil fuels since the dip observed in 2019 certainly calls for renewed perspectives into renewable sources of energy for environmental sustainability. Butanol, one of the most promising and widely studied alternatives, is worth revisiting in this context.
While butanol can be chemically synthesized from petrochemical fossil fuels via the hydrolysis of haloalkanes or hydration of alkenes [5], sustainable or renewable sources for energy production have been a long-standing area of focus, particularly with respect to feedstocks and thermochemical or biological approaches. Biofuels, for example, have been routinely used to produce products such as biodiesel and ethanol from feedstocks such as lignocellulosic biomass [6]. The term “biofuels” has been applied to the use of biomass for the production of fuels, be it gas, liquid, or solid, including products such as biomethanol, bioethanol, biohydrogen, biodiesel, biochar, bio-synthetic gas, etc. [7]. Since biomass is a renewable alternative, it was perceived as an ideal substrate for the production of biofuels. Subsequently, the strategy of biofuel production shifted from chemical conversions to biological conversions using microorganisms. The use of microorganisms for the production of value-derived products using biomass is not new and has been in existence for millennia. Beer, for example, has been documented to have been produced and consumed as far back as 7000 years ago in China [8]. Archaeological excavations have revealed that a clay tablet dated back to 6000 BC with likely one of the oldest beer recipes inscribed on it in Mesopotamia [9]. Similarly, the earliest chemical evidence for wine from a Neolithic village’s pottery jar was dated to have been produced around 5400–5000 BC [10]. Whether this is intentional production is still debatable. However, archaeological evidence for an intentionally fermented beverage that was not produced by the well-established Vitis vinifera or its ancestors was found and dated over 7000 BC in a Neolithic area near the Yellow River in China [11]. Since then, the use and exploitation of microorganisms for the production of other value-derived products has slowly gained prominence to the extent that any organic residue can become a source of feedstock for microbial activity.
In our current context, the selected feedstocks for the production of the various biofuels have been categorized into generations based on their chronological usage, including the production of biobutanol, which is the focus of this review. Butanol has been seen as ideal due to its higher energy content, lower volatility, lower absorption of water, easier blending with gasoline and, importantly, its compatibility with conventional combustion engines compared to ethanol [12,13] (Table 1). Besides its potential application as an alternative to fossil fuels, butanol has had applications in the chemical industry for the production of methacrylate esters, butyl acrylate, butyl glycol ether, butyl acetate, and plasticizers. Globally, over 50% of the produced butanol is converted to acrylate and methacrylate esters [12,14]. According to Market Research Future, the estimated size of the biobutanol market in 2024 was USD 1.4 billion and is expected to increase from USD 1.18 billion in 2025 to USD 3.5 billion by 2034, with an expected compound annual growth rate (CAGR) of about 13.18% between the forecast period of 2025 to 2034 [15]. The optimism around biobutanol is due to the fact that technological advancements have allowed for reduced cost of production and higher efficiency at scale [15]. Nonetheless, there are still a limited number of industrial biobutanol manufacturing facilities globally [16]. Thus, the objective of this review is to offer some perspectives on biobutanol production, scale-up challenges, and the promise of precision fermentation. While fermentative aspects of biobutanol production have been exhaustively addressed, some of the recurring bottlenecks include inhibitory accumulation of the biobutanol, which precludes higher titers of butanol. Furthermore, many of the studies have focused on laboratory scale process optimization, which is unfortunately not amenable to scale-up. Similarly, metabolic engineering approaches to enhance biobutanol production have been conducted [17], including the use of synthetic biology [18]. Biobutanol production is also likely to receive further impetus due to extensive research efforts toward utilizing feedstocks from agri-food waste streams, which offer new perspectives into upcycling and sustainability approaches in a circular economy context. The development of engineered strains capable of more efficient acetone–butanol–ethanol (ABE) fermentation will certainly aid in the use of agri-food waste streams. However, engineered strains are not available for broader use and have not been reported to be commercially used. Therefore, general ABE fermentation is still dependent on available wild-type strains, which, as mentioned above, are prone to inhibition due to increasing concentrations of butanol during fermentation or other potential inhibitors arising from the side streams.

2. Chemical and Biological Synthesis of Butanol

The production of butanol from fossil fuels is certainly feasible [22]. However, such a pathway would not contribute to reduced dependence on fossil fuels, although it has been seen as more cost-effective with significantly higher efficiencies in terms of material and energy [23]. Notwithstanding this current economic discrepancy in chemical synthesis versus biological synthesis, there are other considerations that can be taken as to which particular approach would introduce more long-term sustainable and economical solutions (Figure 2). From a sustainability perspective, the abundance of lignocellulosic biomass augurs well for further investments into enhancing biobutanol production efficiency and lowering the cost of production.
Among the three chemical synthesis processes, viz., crotonaldehyde synthesis, Reppe synthesis, and oxo synthesis (also known as hydroformylation), established for butanol production [21,24] (Figure 3), crotonaldehyde synthesis was one of the earliest approaches up to the 1950s, only to be superseded by oxo synthesis [23]. Crotonaldehyde synthesis occurs via aldol condensation of two acetaldehyde molecules at room temperature and atmospheric pressure to produce 3-hydroxybutyraldehyde, the reaction being catalyzed by an alkali [23]. Subsequently, acidification with acetic acid or phosphoric acid results in a dehydration reaction, leading to the production of crotonaldehyde. Hydrogenation of the latter in the presence of a copper catalyst gives rise to butanol [21,23,24]. In Reppe synthesis, carbonylation of propylene occurs in the presence of carbon monoxide, hydrogen, and a catalyst, resulting in n-butanol and iso-butanol synthesis [24]. The catalyst is generally a tertiary ammonium salt or polynuclear iron carbonyl hydrides, and the reaction proceeds at a temperature of 100 °C and a pressure of 0.5–2 × 106 Pa [24]. Oxo or hydroformylation synthesis is a fundamental step in the production of higher aldehydes and alcohols and has seen major improvements in the use of rhodium-based catalysts [25]. The efficiency and lower cost of production using the Oxo process led to crotonaldehyde synthesis becoming obsolete, in spite of the former being performed at elevated temperatures (80–200 °C) and pressures (20–30 MPa), yielding 75% n-butanol and 25% iso-butanol [21]. However, further improvements have allowed the process to be conducted at lower pressures (1–5 × 106 Pa), yielding 95% n-butanol and 5% iso-butanol [24]. While chemical synthesis is a viable option, it still involves some fossil oil-derived materials such as ethylene, propylene, and triethylaluminium or carbon monoxide and hydrogen [26,27]. Furthermore, most of the chemical synthetic approaches involve the use of catalysts—for example, for the selective synthesis of 1-butanol from ethanol over strontium phosphate hydroxyapatite catalysts [28].
The biosynthetic pathway for butanol production in microbial systems is generally well understood, with a solventogenic pathway and an acidogenic pathway [30,31] (Figure 4). During exponential growth, mostly organic acids such as acetic acid and butyric acid are produced as a result of sugar metabolism. Due to the production of the acids, the pH decreases, which leads to the triggering of the solventogenic phase, wherein the acids are metabolized to produce ethanol and butanol [32,33]. Essentially, the metabolic pathway for ABE is the glycolytic (Embden–Meyerhof–Parnas) pathway, with the starting fermentable sugar being glucose [34], converting one glucose molecule to two pyruvates, resulting in the production of two molecules of ATP and two molecules of NADH (Figure 4). It should also be noted that besides hexoses, pentoses can also be metabolized, resulting in the synthesis of pentose 5-phosphate, fructose 6-phosphate, and glyceraldehyde 3-phosphate catalyzed by transaldolases, transketolases, pyruvate, and then, in turn, CO2 and Acetyl-CoA [34,35,36]. The latter is then converted to acetone, butanol, butyrate, ethanol, and other intermediates.

3. Feedstocks and Agri-Food Industry Residues’ Valorization

The fermentative production of butanol has existed for over a century and is commonly referred to as acetone–butanol–ethanol (ABE) fermentation via anaerobic Clostridium species [38]. First described by Weizmann at the beginning of the 20th century [39], it was subsequently referred to as the Weizmann process [40]. Although the microorganism identified by Weizmann was named “BY” and capable of fermenting a variety of starchy substrates to produce butanol and acetone, it was later named Clostridium acetobutylicum [41]. The latter is nowadays one of the most commonly associated microorganisms for fermentative butanol production using various feedstock sources.
With regard to feedstocks, from the initial simple types termed first generation, additional generations have been recognized based on complexity and the source of feedstocks or microorganisms used [42] (Figure 5).
Simple substrates such as molasses from sugarcane or starches from grain crops were considered very accessible and abundant and were, therefore, very popular in the early revolution of the biofuel industries. However, it became apparent that in the urgency to reduce dependence on fossil fuels, there was a significant encroachment into the food chain by siphoning away food for fuel. This valid concern led to a shift toward other sources of feedstocks, such as lignocellulosic materials, which became known as second-generation biofuel, and algae, which became known as third-generation biofuels. Genetically modified microorganisms have been considered fourth-generation biofuels (Table 2). Although the titers and yields are variable and certainly indicate that more improvements need to be made, the availability of genome editing tools is likely to contribute to further improvements.
Irrespective of the feedstock or microorganisms, this “siloed” approach to considering the feedstocks is obsolete in the current context. When attempting to use feedstocks from various sources, there is certainly overlap in complexity, and these generational categorizations are no longer necessary. For example, food wastes and agri-food wastes range in complexity from being a mix of simple sugars, starches, proteinaceous, oily, or cellulosic consistencies (Figure 6). The focus, therefore, has to be designing approaches to efficiently process and/or ferment heterogeneous feedstocks.
For example, the demand for plant-based proteins has increased significantly in recent years due to considerations for sustainable production, health, and ethics [53]. Among the options, pulse-derived proteins have emerged as ingredients of choice in the vegetarian/vegan lifestyle diet [54], and the side stream starches (which are of low value) can typically be harnessed to derive additional value by way of fermentative butanol production. Thus, in this scenario, the side stream starch from the grain is not necessarily part of the food chain and is neither first-generation nor any other subsequent generations. This type of side stream residue from the agri-food industry is likely to become a significant source of feedstocks in the near future in addition to other feedstocks. In Canada in 2021, the combined production of dry peas, lentils, and chickpeas was about 4 million tons [55]. One of the challenges for the pulse industry is to continue to add value to side streams like starches. Considering the pulse starch concentration of peas, lentils, and faba bean on a dry weight basis ranging from 40 to 50% [56], it has recently been touted as a feedstock for biobutanol production for value addition.
While pulse starch side streams for biobutanol production are still in the research stage, wheat starch wastewater has been used [57]. Corn stover employing a simultaneous saccharification and fermentation (SSF) approach has been used for fermentative butanol production [58]. The most common approach to ABE fermentation using biomass or starches is through SSF. However, SSF is not without its limitations, such as the presence of inhibitors. However, the inhibitory compounds are more prevalent in complex feedstocks such as lignocellulosic or algal biomass [59]. The choice of any specific approach will depend on the initial experimentation and results. Furthermore, the energy required to pre-treat some of the more complex feedstocks is significant and can add to the cost of processing, although their sugar contents can be as high as 50% [42,60].

4. Scale-Up Challenges and Purification

One of the most commonly overlooked aspects of fermentation is scale-up. All too often, process development and optimization are conducted at small scales with limited foresight into process translation to large scale [61]. Such translation includes an outlook on logistics, finances, timelines, and ultimate objectives [62]. In essence, a techno-economic analysis (TEA) would need to be conducted prior to embarking on a large-scale venture besides the typical process optimization. While there are different approaches to address scale-up challenges, a Process Hazards Analysis (PHA) approach is considered very intuitive for risk management and mitigating risks [61]. Commonly used in the industrial engineering/manufacturing space for risk management and risk mitigation for safety and process operation, it has been sparingly used in the fermentation industry. The food processing and beverage industries employ Good Manufacturing Practices (GMPs), Good Hygiene Practices (GHPs), Quality Management Systems (QMS), and Hazard Analysis and Critical Control Points (HACCPs) to ascertain the safety of the food and beverage for human consumption (for reviews, see [63,64]). However, in industrial fermentation, such practices are limited to ensuring consistent productivity, yield and titers, and, of course, returns. Although biobutanol production at an industrial scale has been established, many of the research and development aspects still neglect to envisage the practicality, feasibility, and economic aspects of the scaled-up process. Thus, a PHA approach would be very useful to guarantee successful scale-up in the identification and assessment of risks and providing mitigating options.
Generally, PHA is defined as the systematic, comprehensive, and analytical review of a process based on a number of standards for the identification and assessment of processes and associated operational hazards and their potential effects [65]. PHA strategies emerged as a result of repeating serious incidents due to operators, personnel, machinery, oversight, etc., with the result that productivity, revenue, public trust, and overall dependability on meeting demands were obvious. Due to this, many of the large manufacturing entities have established policies for PHAs [66] in some form with ranging complexities. In addition to commonly practiced safety procedures and following Standard Operating Procedures (SOPs), PHA incorporates operational safety of processes while also addressing non-safety related potential failures, which lead to failed processes and economic losses. It is important to note that basic operational and general safety procedures would have been implemented prior to proceeding with PHA approaches for desired projects [67]. PHA, in the case of biobutanol production, would start with feedstock selection, pre-treatment conditions and complexity, the fermentation process itself, ABE purification, economic feasibility, financial investments, and finally, available platforms for scaling up production.
For example, one of the challenges in ABE production is the selection of feedstocks and process flow improvement that can be translated to large-scale production. After judiciously optimizing the performance and productivity of the selected strain at a small scale using defined media components, not enough resources are allocated to achieve equal or superseding productivity with other feedstocks, especially complex ones. Improving process flow is also important to minimize the duration of fermentation while maximizing yield. Considering the first-generation feedstocks, in the past, saccharification for biofuel production was conducted separately from fermentation in a two-step process. This approach was inefficient and cost-intensive. Improvements have led to the establishment of a single-step process wherein simultaneous saccharification and fermentation (SSF) are conducted in the bioreactor [68]. Other improvements included the use of microbial strains capable of saccharification, followed by fermentative butanol production [69] or conducting both simultaneously as co-cultures [70].
The more complex feedstocks would require more energy to break down and will therefore add to the higher cost of production. Thus, a first-generation feedstock such as starch will require less energy to release the sugars compared to cellulosic feedstocks. While both feedstocks undergo a saccharification step, the cellulosic feedstock has to be broken down into finer particles and hydrolyzed by acid treatment prior to the saccharification step [71,72]. Therefore, in the context of scaling up, these pre-treatments and the process flow to fermentation significantly influence the efficiency of biobutanol production.
Scale-up challenges during fermentation per se have been widely addressed in a number of reviews (e.g., [61,62,73,74]) and for biobutanol production (e.g., [57,69,75]. Suffice it to state that the fermentation processes and designs are well established, with the bottlenecks being in the inhibitory accumulation of butanol and the removal and purification of butanol from fermentation broth. Even in this regard, numerous studies have been conducted, but mostly limited to a small scale or pilot scale and not demonstrated at production or manufacturing scales. Some of the strategies used to address inhibitory butanol concentrations have included the removal of broth and replenishing with fresh media or in-line distillation and removal of accumulated butanol, etc. Commonly, however, batch, fed-batch, continuous, or combination thereof have been used [76]. For example, simply adding butyric acid at a very low concentration was found to promote ABE production by C. beijerinckii strain NCIMB 8052, resulting in a 31% increase in a final butanol titer and a 133% increase in butanol productivity [77]. While this research was performed at a laboratory scale in a benchtop bioreactor, the economic feasibility of butyric acid supplementation at a large scale needs to be further investigated. Besides the inhibitory accumulation of butanol during fermentation, inhibitors derived from lignocellulosic hydrolysis can reduce butanol titers. In a fed-batch strategy using Clostridium beijenrinckii NRRL B-598 and hydrolyzed wheat straw, such inhibitors were effectively eliminated by timing the feed to coincide with the late acidogenesis and early solventogenesis phases, leading to a titer of 7.0 g/L butanol [78]. Employing a detoxification strategy using strong acid cation exchange with wheat straw hydrolysates for fermentation with Clostridium acetobutylicum CICC 8012, a titer of 7.42 g/L butanol was obtained [79]. Toxicity from hydrolysates has also been observed in other biomasses, such as sweet sorghum bagasse, with primary hydrolysis resulting in poor butanol titers, but secondary hydrolysis resulted in 8.9 g/L butanol [80]. In a subsequent study, it was demonstrated that a primary fermentation with the secondary hydrolysate allowed for vigorous cell growth, and when followed by a slow feed of the primary hydrolysate, butanol titers could be increased to 12 g/L [81].
One of the most adaptable strategies for large-scale ABE production is undoubtedly continuous fermentation. This approach allows for the removal of broth during fermentation and replenishment with new media. Thus, the inhibitory effects of butanol can be obviated. The removed broth can be adapted for in-line distillation after the removal of cells and/or cell cycling. The latter approach has been gaining attention due to its higher productivity [82], but scalability is still not clearly defined. In another small-scale study, continuous fermentation in combination with gas stripping was shown to increase productivity by 320% over a 21-day period and was intentionally terminated after this period [83]. Cell cycling, on the other hand, relies on soundly selected ultrafiltration systems, with productivity ranging from 4.1 to 7.55 g/L/h [84,85]. The combination of continuous fermentation and cell recycling via ultrafiltration has steadily improved and has led to a biobutanol productivity of 10.7 g/L/h [86]. However, as with other studies, scale-up feasibility is yet to be demonstrated. Attempts at in situ separation have also been undertaken, including liquid–liquid extraction [87], gas stripping [88], adsorption [89], and pervaporation [90]. It is thus obvious that there are numerous options for increasing the productivity, yield, and titer of biobutanol, but a successful outcome will depend on the adaptability of the process to large scale cost-effectively.
With Clostridial species being predominant in fermentative butanol production [91], it has been observed that these species themselves are inherently affected by metabolic oscillations during fermentation [92,93]. Oscillations are common features of biological systems, the most studied being those associated with circadian rhythms and day and night cycles. The occurrence of metabolic oscillations in Clostridium species can have major implications for scaling up the production of fermentative butanol (reviewed in [94]). For example, during glycerol fermentation by Clostridium pasteurianum, it was observed that there was self-synchronized oscillatory metabolism coinciding with higher glycerol feed unrelated to butanol accumulation [92]. This was deemed a potential problem for scaling up production if glycerol feed concentrations were to be increased for high butanol productivity. The study also reported that lower temperatures stopped the self-synchronized oscillations and could be restored by increasing the temperature [92].

5. The Potential of Precision Fermentation for Improved Biobutanol Production

There have been significant advances in fermentation technology with regard to biobutanol production, including engineered microorganisms. As mentioned earlier, the latter has been considered the fourth generation of biofuels, but as such, it should be considered a system for use with any type of feedstock. Maximizing biobutanol production using any type of feedstock should be the focus if any commercial success is to be attained. With available technologies in genome engineering combined with the availability of heterogeneous feedstocks in the context of upcycling and circular economy, the need for more efficient strains is imperative.
More recently, the advent of precision fermentation has opened new opportunities for metabolic engineering to be more targeted [73]. While mainly focused on specific target molecules, it has the potential to be applied more broadly, including in biobutanol production. It encompasses synthetic biology and a whole repertoire of other tools, such as omics, artificial intelligence, bioinformatics, systems biology, computational biology, and functional characterization. Relying extensively on an understanding of microbial genomes and metabolic functions, it is a perfect approach to enhance biobutanol production in the context of heterogeneous biomass use and to enhance microbial efficiency [95]. Essentially considered an advanced fermentation system, it has the ability to precisely produce specific molecules with very tightly controlled manufacturing practices to enhance the yield of desired products at reduced cost [96]. In the case of biobutanol production, the metabolic engineering of species such as Clostridium would be directed to reduce wasteful pathways and direct synthesis toward butanol [97]. Further advances in technology by way of Internet of Things (IoT)-based sensors have enabled the operation of a low-cost prototype bioreactor for biobutanol production and sample collection and monitoring [98], resulting in improved biobutanol production.
While precision fermentation is yet to have a significant impact on biobutanol production efficiency enhancement, several conventional strain improvement strategies, such as mutagenesis, directed evolution, genome engineering, and genome editing, have been employed (reviewed in [99]). Interestingly, while the concept of using microorganisms as cell factories in line with precision fermentation for the production of single molecules is a significant topic of discussion, especially in the food industry, elements of it have already been practiced in fermentative butanol pathway modification, albeit unconsciously. For example, while it has been well-established that increased production of butanol inhibits cell growth, thereby limiting high productivity, it became conceivable that the biosynthetic pathway could be modified to produce butyric acid, which could further be processed downstream for conversion to butanol [100]. Indeed, the approach taken in this study included the knockout of genes encoding phosphotransacetylase and acetate kinase in an attempt to shut down the acetate-producing pathway. However, the acetate production was not significantly reduced, although butyric acid production was increased with no inhibitory effects on cell growth [100]. In another study in Clostridium acetobutylicum JB200 using the ClosTron group II intron-based system, a histidine kinase gene, cac3319, was inactivated and led to an increase in butanol production in addition to enhanced tolerance to the increased butanol concentration [101].
Intricately associated with precision fermentation and synthetic biology approaches for engineering microorganisms is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology, commonly abbreviated CRISPR/Cas9. CRISPRs [102], or Short Regularly Spaced Repeats, as they were originally termed [103], are unique to prokaryotes (archaea and bacteria), featuring direct repeats of 21–37 bp. Associated with the CRISPR loci were Cas genes [102]. It was subsequently shown that the CRISPR/Cas9 system, as a bacterial immune system mechanism against infecting bacteriophages, could be harnessed for editing genes [104]. With the availability of this new gene editing tool and recent advances, attempts to edit the butanol pathway have been made. With Clostridium species being naturally able to produce butanol, editing the butanol biosynthetic pathway for increased efficiency would have been the target of choice. However, due to the complex physiological nature of Clostridium species and limited genetic manipulation information, other systems were explored for butanol pathway engineering. Thus, using CRISPR/Cas9 editing in Escherichia coli, butanol production was achieved in a micro-aerobic environment [105]. Further knowledge and understanding of the underlying mechanisms of native CRISPR/Cas systems in Clostridium have enabled tailoring the endogenous systems to edit the butanol biosynthetic pathway. Using Clostridium tyrobutyricum, a hyper-butyrate producer, the butyrate:acetate CoA transferase (cat1) gene was replaced with aldehyde/alcohol dehydrogenase genes (adhE1 or adhE2), leading to the near elimination of butyrate production converting the C. tyrobutyricum strain into a hyper-butanol producer [51]. It is likely that with further research into precision fermentation, synthetic biology, and gene editing using the CRISPR/Cas9 system, there will be enhanced biobutanol production for commercially viable manufacturing.

6. Conclusions

The production of biobutanol is certainly not new, having been in existence for over a century. However, the challenges have been in scale-up production and commercial viability. Although the value of biobutanol as a green alternative is well recognized, there has not been enough focus on the establishment of large-scale production research and deep investigation into the inhibitory aspects of butanol accumulation. The opportunities for refinement of the process are currently readily available by way of precision fermentation and advanced manufacturing systems, including AI. Furthermore, the abundance of diverse feedstocks in the context of upcycling and circular economy opens up new opportunities for metabolic engineering of microbial systems to either direct the metabolic pathway for the production of butanol or for metabolizing complex agri-food residues for the release of the fermentable sugars. Of the currently operational commercial biobutanol production facilities, Celtic Renewables (https://www.celtic-renewables.com/, accessed 7 April 2025) in Scotland is producing biobutanol from wastes that include food, drink, and agriculture. Gevo (https://gevo.com/product/isobutanol/, accessed 7 April 2025) in the USA produces iso-butanol from agriculture residues in a side-by-side operation, including ethanol production, thus sharing various streams and minimizing CAPEX and OPEX. However, for further proliferation of commercial biobutanol production facilities globally, the outlined challenges need to be addressed.

Author Contributions

Conceptualization, S.G. and M.Ç.T.; writing—original draft preparation, S.G. and M.Ç.T.; writing—review and editing, S.G. and M.Ç.T.; project administration, S.G.; funding acquisition, S.G. and M.Ç.T. All authors have read and agreed to the published version of the manuscript.

Funding

Funding for biobutanol research in our laboratory from the Saskatchewan Ministry of Agriculture-Agriculture Development Fund (ADF Project # 20220241) is gratefully acknowledged.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflict of interest and declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Primary energy consumption, including natural gas, oil, and coal, over two centuries and into the 21st century (data sources: [2,3]).
Figure 1. Primary energy consumption, including natural gas, oil, and coal, over two centuries and into the 21st century (data sources: [2,3]).
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Figure 2. Some advantages and disadvantages of chemical versus biological synthesis for the production of butanol.
Figure 2. Some advantages and disadvantages of chemical versus biological synthesis for the production of butanol.
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Figure 3. Chemical synthesis of butanol. (a) Crotonaldehyde synthesis; (b) Reppe synthesis; (c) oxo synthesis. Adapted from [21,23,29].
Figure 3. Chemical synthesis of butanol. (a) Crotonaldehyde synthesis; (b) Reppe synthesis; (c) oxo synthesis. Adapted from [21,23,29].
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Figure 4. Schematic of biosynthetic pathway for fermentative butanol. Adapted from [14,22,34,37].
Figure 4. Schematic of biosynthetic pathway for fermentative butanol. Adapted from [14,22,34,37].
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Figure 5. The four generations of feedstocks used for biobutanol production.
Figure 5. The four generations of feedstocks used for biobutanol production.
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Figure 6. Some examples of abundant unconventional feedstocks from agri-food industries and other sources.
Figure 6. Some examples of abundant unconventional feedstocks from agri-food industries and other sources.
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Table 1. Isomers of butanol and other alcohols, as well as some of their characteristics (sources: [19,20,21]).
Table 1. Isomers of butanol and other alcohols, as well as some of their characteristics (sources: [19,20,21]).
Encyclopedia 05 00050 i001n-ButanolEncyclopedia 05 00050 i002sec-ButanolEncyclopedia 05 00050 i003iso-ButanolEncyclopedia 05 00050 i004tert-ButanolEncyclopedia 05 00050 i005MethanolEncyclopedia 05 00050 i006EthanolEncyclopedia 05 00050 i007Propanol
Boiling point (°C)11899.51088364.778.197
Melting point (°C)−90−115−10825.7−97.6−114.1−126
Density (Kg/L)0.810.8060.8020.7910.7920.7891.049
Flash point (°C)35312811121415
Motor octane number7832948997–104100–106-
Table 2. Butanol production from the selected feedstocks from the four generations. “-“ indicates data not available.
Table 2. Butanol production from the selected feedstocks from the four generations. “-“ indicates data not available.
FeedstockMicroorganismTiter (g/L)Yield (g/g)References
1st Generation
Corn starchClostridium acetobutylicum ATCC 82411.2-[43]
Cassava starchClostridium acetobutylicum SE3616.120.32[44]
2nd Generation
SwitchgrassClostridium saccharoperbutylacetonicum N1–48.60.16[45]
Wheat strawClostridium beijenrinckii P26012.00.2[46]
3rd Generation
Green alga (Ulva lactuca)Clostridium beijerinckii NCIMB 80523.00.35[47]
Microalga (Chlorella sorokinianaCY1)Clostridium acetobutylicum ATCC 8243.860.13[48]
Macroalga (Rhizoclonium spp.)Clostridium beijerinckii TISTR 1461 2.55-[49]
4th Generation
Genome shufflingClostridium acetobutylicum GX0120.1-[50]
CRISPR-CasClostridium tyrobutyricum ATCC 2575515.0–26.20.23–0.35[51]
CRISPR-CasClostridium acetobutylicum DSM 7921.3–13.3-[52]
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Ganeshan, S.; Tülbek, M.Ç. Fermentative Butanol Production—Perspectives and Scale-Up Challenges. Encyclopedia 2025, 5, 50. https://doi.org/10.3390/encyclopedia5020050

AMA Style

Ganeshan S, Tülbek MÇ. Fermentative Butanol Production—Perspectives and Scale-Up Challenges. Encyclopedia. 2025; 5(2):50. https://doi.org/10.3390/encyclopedia5020050

Chicago/Turabian Style

Ganeshan, Seedhabadee, and Mehmet Çağlar Tülbek. 2025. "Fermentative Butanol Production—Perspectives and Scale-Up Challenges" Encyclopedia 5, no. 2: 50. https://doi.org/10.3390/encyclopedia5020050

APA Style

Ganeshan, S., & Tülbek, M. Ç. (2025). Fermentative Butanol Production—Perspectives and Scale-Up Challenges. Encyclopedia, 5(2), 50. https://doi.org/10.3390/encyclopedia5020050

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