1. Introduction

Increased utilization of fossil fuels leads to rapid depletion of its sources which in turn increases interest to produce biofuels from acetone-butanol-ethanol (ABE) fermentation. Use of natural sources to produce biobutanol and bioethanol from ABE fermentation makes the production process more sustainable, renewable and environmental benign leading to decreased dependency on fossil fuel reservoirs (Mahalingamet al., 2022). Alcoholic biofuels bioethanol and biobutanol produced through ABE fermentation can be blended with gasoline.However, bioethanol has a lower heat of combustion than gasoline makes it unsuitable and limits its blending of 5–10% with gasoline for present IC engines. While biobutanol with similar physicochemical properties as that of gasoline serves as a potential transportation fuel compatible with present engine (Moholkar et al. 2019). Apart from this biobutanol offers several advantages such as, high boiling point, non-corrosive nature, lower moistureabsorption, lower volatility and reduced Reid vapor pressure, which decreases its tendency to evaporate quickly and lowers the risk of explosiveness

During World War I, a microbial approach for generating butanol using ABE fermentation was pioneered and established as a largest fermentation process during 20^th century. However, increased emergence of petrochemical industry, microbial route of ABE fermentation was declined (Gottumukkala et al., 2019). Biobutanol is an exciting liquid transportation fuel that has a net-zero carbon footprint, butitsproduction for purposes has been hindered by the conflict between using crops for food or fuel which limits the utilization of conventional materials such as sugar and starch which leads to intrinsic challenges of recalcitrant nature of lignocellulosic feedstock. Feedstock, a prime component in the ABE fermentation, it defines the process economy and productivity of biodiesel (Kumar et al., 2012). Chaim Weizmann, called as a father of industrial fermentation after discovery of Clostridum acetobutylicum for butanol production through ABE fermentation. Over the years tremendous development has been done in ABE fermentation in terms of identification of new substrate and its pretreatment, isolation of new competitive strains, optimization of down strea m process to increase yield and productivity (Gottumukkala et al., 2019). Researchers reported limitations of biobutanol production i.e. self-inhibition due to its toxicity, also nutrient depletion ceases the fermentation and resulted in lower productivity and yield (Nalawade et al., 2023). Biobutanol is generated via ABE fermentation. Nonetheless, the extraction of butanol from the ABE solvent remains a costly and intricate process. As a result, the ABE solvent is utilized directly to reduce purification expenses. (Mahalingam et al., 2022). However, the information about ABE as a biofuel, its progress in process development and technoeconomical analysis are dispersed and not well-coordinated. Hence, the present review paper explicitly deals with the potential of ABE fermentation as a biofuel and limitation and challenges during ABE fermentation.

2. ABE Fermentation

Commonly used industrial strains for ABE production are dedicated to genera Clostridium, they are well-known for their capacity to digest both simple and complex carbohydrates, including as glucose, cellulose, and sucrose (Li et al., 2019). Generally, several clostridia species exhibits almost similar metabolic pathways, illustrated in Figure 1. These pathways can be broken down into three distinct stages: 1. acidogenesis, 2. solventogenesis, and 3. sporogenesis, which collectively result in the generation of three primary products: (1) solvents (including ABE), (2) organic acids (such as acetic and butyric acid), and (3) gases (carbon dioxide and hydrogen) (Li et al., 2020).

The process begins with the uptake of glucose or other fermentable carbohydrates by C. acetobutylicum. Glucose is metabolized through glycolysis, resulting in the production of pyruvate. Further pyruvatе is thеn dеcarboxylatеd by thе еnzymе pyruvatе: fеrrеdoxin oxidorеductasе, producing acеtyl-CoA and rеducing fеrrеdoxin . Then acetyl-CoA can be converted into acetate through the acetate kinase and phosphate acetyl transferase enzymes. A significant portion of acetyl-CoA is further converted into acetone, butanol, and ethanol through the ABE pathway. This pathway includes the following steps: initially acetyl-CoA gets converted into acetoacetyl-CoA through the enzyme acetoacetyl-CoA: acetate/butyrate:CoA-transferase, followed by reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA by the NADPH-dependent 3-hydroxybutyryl-CoA dehydrogenase. Theen 3-hydroxybutyryl-CoA gets converted into butyryl-CoA by the 3-hydroxybutyryl-CoA dehydratase enzyme and ultimately converted to butanol through a series of enzymatic reactions, including butyraldehyde formation and reduction to butanol. Furthermore, acetone is produced by the decarboxylation of acetoacetate. Moreover, acetobutylicum can also produce ethanol through a separate pathway that involves the conversion of acetyl-CoA to acetaldehyde by the enzyme acetaldehyde dehydrogenase and then the reduction of acetaldehyde to ethanol by alcohol dehydrogenase. Acetyl-CoA can also be converted into butyrate through a series of enzymatic reactions. These reactions involve enzymes like butyryl-CoA dehydrogenase and butyrate kinase. ABE fermentation pathway is tightly regulated and can be influenced by environmental factors, such as nutrient availability and pH. The production of acetone, butanol, and ethanol is often associated with the metabolic shift from acidogenesis (production of organic acids like acetate and butyrate) to solventogenesis (production of ABE) (Li et al., 2019).

C. acetobutylicum and C. beijerinckii are most popular industrial strains, however, C. pasteurianum, C. saccharoperbutylacetonicum andC. sporogenes, wild type strains exhibits more industrial potential in ABE production under strictly anaerobic condition (Gottumukkala et al., 2019). These wild type strains suffers from several drawback such as sluggish growth, reduced cell density during solventogenic phase and weak solvent tolerance (Qureshi and Blaschek, 2001).

               Fig. 1 Intracellular metabolic pathway in C. acetobutylicum dedicated to ABE production (Li et al., 2019)

Tsai et al. (2020) have used PVA-immobilized C. acetobutylicum for biobutanol production using lignocellulosic feedstock such as rice straw, sugarcane bagasse and microalgal hydrolysate. A higher butanol productivity of  0.90 g/L/h, yield of0.23 g biobutanol/g glucose and titer of 13.80 g/L was achieved using rice straw as feedstock (Tsai et al., 2020). During the pre-genomic era, high butanol-producing strains were obtained by mutagenesis of clostridia through physical or chemical methods. Qureshi and Blaschek, (2001) used mutant strainC. beijerinckii BA101 which hydrolyzes starch effectively and produces 27–29 g/ l of solvents. Moreover, utilization of sodium acetate enhances solvent production to 33 g/l (Qureshi and Blaschek, 2001). Further, to boost butanol yield and productivity, researchers focused on manipulating genes involved in solvent synthesis and modifying the metabolic regulatory system. Schwarz et al. (2017), reported in-frame deletion mutants of pivotal genes particularlyhydA (hydrogenase), rex (Redox response regulator) and dhaBCE (glycerol dehydratase) linked to solvent production in C. pasteurianum. hydA mutant exhibits elevated levels of ethanol production (64.3 ± 3.2 mM after 24 h) over rex mutant. However, rexmutant shows highest butanol titre 133.3 ± 1.8 mM compared to hydA mutant. Furthermore, increased n-butanol titres result from the inactivation of both rex and hydA, which are the initial steps towards using C. pasteurianum as a possible strain for the industrial production of ABE (Schwarz et al., 2017). With the byproduct acetone declining by 31.2%, the hydA disrupted strain of C. acetobutylicum ATCC 5502 was able to produce 18.3% more butanol, suggesting that the suppression of hydrogenase regulated redox balance for the selective suppression of acetone formation. Furthermore, they employed methyl viologen which shifted the carbon flux and produces elevated butanol yield of 0.28 g/g with reduced acetone formation (Du et al. 2021).Using Targe Tron technology, the acetoacetate decarboxylase gene (adc) in hyperbutanol producing C. acetobutylicum EA 2018 was disrupted. In the adc-disrupted mutant, acetone synthesis decreased to about 0.21 g/L while the butanol ratio increased from 70% to 80.05% (Jiang et al., 2009). Jang et al. (2012) obtained increased butanol yield by strengthening C. acetobutylicum's direct butanol-forming flow. Through batch fermentation, 18.9 g/liter of butanol was generated, yielding 0.71 mol butanol/mol glucose—levels 160% and 245% greater than those achieved with the wild type. A fed-batch culture of this modified strain with in situ recovery yielded 585.3 g of butanol from 1,861.9 g of glucose, with a productivity of 1.32 g/liter/h and a yield of 0.76 mol butanol/mol glucose.(Jang et al., 2012).

Consolidated Bioprocessing (CBP) is a bioengineering approach that aims to streamline the production of biofuels or biochemicals by combining multiple processes into a single step. When it comes to butanol production, CBP approaches

seek to integrate the fermentation of biomass feedstocks and the conversion of sugars into ABE within a single microorganism or bioprocess. Wen et al. (2019) discusses three strategies for CBP. These includes modifying solventogenic Clostridia to secrete/cell display cellulases or cellulosomes, butanol pathway engineering of cellulolytic Clostridia, and mixed-culture of butanol-producing and cellulolytic Clostridia, as shown in Fig. 2. CBP is a potential method for the near future cellulosic butanol manufacturing on a huge scale (Wen et al., 2017).

Researchers started focusing on improving ABE fermentation through enzyme modification as an alternative strategy, however significant progress has not been achieved. Thiolases are essential enzymes for metabolic pathways that produce carbon–carbon bonds as they catalyze the condensation of two acetyl–CoA molecules into acetoacetyl–CoA. C. acetobutylicum's thiolase was specially altered by substituting three amino acids (R133G, H156N, G222V) to decrease sensitivity towards coenzyme A (CoA‐SH) and considerably increases butanol titers by 46% and 18%, respectively (Li et al. 2020). Clostridium acetobutylicum produces acetone from acetoacetyl-CoA by the action of the enzymes acetoacetate decarboxylase and coenzyme A transferase. The genes ctf and adhE, which together encode a likely polyfunctional aldehyde/alcohol dehydrogenase, create a shared transcription unit, whereas the adc gene, which codes for the former enzyme, is arranged in a monocistronic operon. This genetic configuration may represent physiological needs during solventogenesis (Durre et al., 1995).

3. Feedstock for ABE fermentation

The main feedstock for acetone-butanol-ethanol fermentation is typically starchy or sugary substrates, such as corn or molasses, which contain a high concentration of carbohydrates can be turned into fermentable sugars( Li et al., 2019).Other potential feedstock options for ABE fermentation include microalgal biomass, lignocellulosic biomass (Fathima et al., 2016; Paniagua-García et al., 2018). These feedstocks offer several advantages, such as their abundance and low cost.

Furthermore, their utilization helps in reducing the dependence on food-based sources like corn and sugarcane, making the process more sustainable (Tsai et al., 2020). Table 1 represents the different feedstock and theit corresponding ABE titre.

Qureshi et al. (2008a), produced ABE in batch reactors employing C. beijerinckiiand alkaline peroxide treated and enzymatically hydrolized wheat straw Acid and enzyme hydrolyzed corn fibre as a substrate in ABE production using C. Beijerinckii (Qureshi et al., 2008b). Enzymatic hydrolysis and detoxification are common pre-treatment steps used in the biofuel conversion of lignocellulosic biomass. These processes are critical in reducing the complexity of lignocellulosic biomass and making it more accessible for subsequent conversion into biofuels. Enzymatic hydrolysis is the process of breaking down cellulose and hemicellulose into simpler sugars such as glucose and xylose using enzymes such as cellulases and xylanases. However, one major challenge in enzymatic hydrolysis is the presence of inhibitors in the biomass, which can hinder the activity of the enzymes and reduce the efficiency of the process (Gottumukkala et al., 2019). To overcome this challenges in enzymatic hydrolysis, Paniagua-García et al. (2018), have used hydrolysate obtained from switchgrass for ABE fermentation without prior enzymatic hydrolysis or detoxification steps using C. beijerinckii CECT 508 (Paniagua-García et al., 2018). To maximize the utilization of fresh corn stalks, the juice, which contains sugars, served as the exclusive source for ABE production, without the addition of any extra nutrients. Additionally, the bagasse, remaining after juice extraction, was employed as the immobilization matrix. In this setup, a total of 21.34 grams per liter (g/L) of ABE was generated in the immobilized batch cells. Continuous multiple stages ABE fermentation process demonstrated excellent stability and tremendous promise for use in upcoming industrial applications (Chang et al., 2016). Baral and Shah, (2016) used corn stover feedstock treated with dilute sulfuric acid for ABE fermentation. The approach of vacuum fermentation, simultaneous saccharification, and recovery holds promise for raising ABE yield.

The process of producing ABE from lignocellulosic feedstock offers the potential to generate its own energy needs from the raw materials, making it self-sustaining in terms of bio-energy. This means that the net energy value (NEV) matches the gross energy value (GEV) of the feedstock (Haigh et al., 2018). In contrast, the molasses-ABE process would necessitate either importing biomass from a nearby industrial source or relying on fossil fuels for energy during the production process, as described by Haigh et al. (2018). Although the lignocellulosic-based approach may not be as energy-efficient as the molasses-butanol method, probably it exhibit superior environmental performance owed to its ability to generate its own energy from bio-resources.

4. Techno-economical analysis

In the biological production process, the production of ABE suffers from several disadvantages. Therefore, techno-economic analysis (TEA) of ABE fermentation involves evaluating the economic feasibility and viability of the process. The majority of the total production cost, around 85%, is influenced by the fixed capital investment and the cost of the feedstock. To make the process commercially viable, there is a need to achieve a significant reduction in production cost, aiming for approximately a 55% reduction. A substantial portion of the fixed capital investment, around 50%, is allocated to the pretreatment and hydrolysis units, highlighting the critical necessity for enhancing the preliminary phases of the butanol fermentation (Gottumukkala et al., 2019). The effectiveness of this approach can be improved by choosing a highly efficient strain, using more cost-effective sugar sources, and implementing advanced recovery techniques. These process optimizations have a potential to result in a more economically efficient production of ABE .

Fig.2 CBP approaches for butanol production (Wen et al. 2019)