The potential of native and engineered Clostridia for biomass biorefining

Since their first industrial application in the acetone-butanol-ethanol (ABE) fermentation in the early 1900s, Clostridia have found large application in biomass biorefining. Overall, their fermentation products include organic acids (e.g., acetate, butyrate, lactate), short chain alcohols (e.g., ethanol, n-butanol, isobutanol), diols (e.g., 1,2-propanediol, 1,3-propanediol) and H2 which have several applications such as fuels, building block chemicals, solvents, food and cosmetic additives. Advantageously, several clostridial strains are able to use cheap feedstocks such as lignocellulosic biomass, food waste, glycerol or C1-gases (CO2, CO) which confer them additional potential as key players for the development of processes less dependent from fossil fuels and with reduced greenhouse gas emissions. The present review aims to provide a survey of research progress aimed at developing Clostridium-mediated biomass fermentation processes, especially as regards strain improvement by metabolic engineering.

The very next sections will provide a survey of the main raw materials for Clostridium fermentation (Section 2) and the metabolic pathways involved (Section 3).The following sections will summarize research progress in production of some of the most valuable fuels and chemicals by Clostridia (Table 1), with special focus on strain improvement by metabolic engineering.

Feedstocks for Clostridium fermentation
Overall, Clostridia can ferment a wide range of substrates comprising soluble sugars (e.g., glucose, xylose, fructose, lactose, cellobiose), polysaccharides (e.g., starch, cellulose), glycerol and gaseous carbon compounds (CO, CO 2 ) (Wang and Yin, 2021;Fernández-Blanco et al., 2023).Since feedstock expenditure may account for more than 70% of the total fermentation cost, the use of waste biomass is preferable to more expensive substrates such as pure sugars or edible crops, especially for producing bulk compounds such as fuels and platform chemicals (Gu et al., 2011;Abo et al., 2019;Rawoof et al., 2021).As a comparison, sugar costs about 460 US$/ton, pulp grade wood (a lignocellulosic biomass) can be calculated at 43-54 US$/ton of fermentable sugars and no cost can be referred to food waste (Nuss and Gardner, 2013;Gharehkhani et al., 2015;International Sugar Organization, 2019;Qureshi et al., 2020).
Carbohydrate-rich feedstocks include: i) lignocellulosic biomass; ii) (micro) algae biomass; iii) food waste; iv) municipal waste; v) agro-industrial effluents.Large availability and low cost of lignocellulosic feedstocks, such as wastes from agriculture (e.g., straws, stalks, wood fibers) and food processing (e.g., bagasse, mushroom compost) make it an ideal raw material for biorefining processes (Wang and Yin, 2021).However, this is offset by its complex composition (mainly consisting of cellulose, hemicellulose and lignin) and innate recalcitrance to biodegradation (Lynd, 2017).(Micro) algae biomass has gained considerable attention owing to its fast growth, no requirement of farmland, low demand for growth conditions and no or reduced lignin content which makes its hydrolysis and fermentation easier than plant biomass (Liu et al., 2012a;Ortigueira et al., 2015a;Ortigueira et al., 2015b;Fonseca et al., 2020).Globally, food waste (a very abundant biomass with high starch content) represents almost one-third of food produced for human consumption, and its disposal significantly contributes to greenhouse gas emission (Parthiba Karthikeyan et al., 2018;Qin et al., 2018;Zhang et al., 2020a;Su et al., 2022).Carbohydrate-rich raw materials also include some municipal wastes such as sewage sludge (Yin and Wang, 2016;Yin and Wang, 2019), paper waste (Liu et al., 2006) and garden wastes (leaves, branches) (Yang et al., 2019a).However, most of them have a complex composition (e.g., organics are mostly encapsulated in microbial cells in sewage sludge, paper and garden wastes are rich in lignocellulose) and generally need pretreatment prior to fermentation (Wang and Yin, 2018).Carbohydrate-rich agro-industrial effluents comprise sugarcane juice, molasses, cassava wastewater and cheese whey (Wang and Yin, 2021).
Glycerol is the main by-product (10% w/w) obtained by transesterification or saponification reactions aimed at producing oleochemicals such as biodiesel (Yang et al., 2012).The substantial increase in the biodiesel industry in the recent years has led to massive production of crude glycerol and drop in prices, hence, turned glycerol into a waste stream rather than a by-product (Ciriminna et al., 2014;Russmayer et al., 2019).
C1 gases (CO, CO 2 ) are part of the greenhouse gases (mainly CO 2 ) contributing to global warming and climate change (Canatoy et al., 2022).The use of these compounds as fermentation feedstocks can decrease their emission into the atmosphere by human activities.Among the industries that use fossil fuels for generating power and heat, steelmaking process emits about 50% of the carbon used as CO (Bengelsdorf et al., 2018).CO, CO 2 (and H 2 ) are also the major components of syn (thesis) gas which can be generated from natural gas, by gasification of coal, oil, biomass (e.g., agricultural and municipal waste) and by recycling used plastics (Köpke et al., 2010;Arslan et al., 2019;Zhang et al., 2020b).Syngas has extensively been used as a feedstock in the chemical industry, but this requires precise CO/H 2 ratio and expensive gas purification from interfering contaminants (Köpke et al., 2010).Chemoautotrophic Clostridia are far more tolerant to such contaminants and already industrially used for ethanol production from these feedstocks (by companies such as Coskata, INEOS Bio, LanzaTech) (Köpke et al., 2010).However, low gas-liquid mass transfer rate (due to poor solubility of these gases in water) results in low cell densities and fermentation efficiency and is the main limit of this technology (Fernández-Blanco et al., 2023).

Heterotrophic and autotrophic fermentative pathways of Clostridia
Clostridia include bacteria with heterotrophic and autotrophic metabolism.In saccharolytic strains, glucose is generally converted to pyruvate through the Embden Meyerhof Parnas (EMP) pathway, since most Clostridia lack the oxidative part of the pentose phosphate pathway (Crown et al., 2011;Koendjbiharie et al., 2020;Foulquier et al., 2022).It is worth noting that clostridial EMP pathway may contain a number of atypical reactions with respect to the traditional glycolysis which affect electron distribution among the different redox cofactors [pyridine cofactors, ferredoxin (Fd)], cellular pools of energy carriers (e.g., adenine/guanine nucleotides, pyrophosphate) and pathway thermodynamics (Iddar et al., 2002;Zhou et al., 2013;Scott et al., 2019;Jacobson et al., 2020).Some heterotrophic Clostridia can also ferment more reduced substrates than carbohydrates such as glycerol which is converted to dihydroxyacetone phosphate (DHAP) and enters the EMP pathway (Yoo et al., 2016;Agu et al., 2019;Sarma et al., 2019).

Hydrogen
Hydrogen gas (H 2 ) is an optimal energy carrier featuring high energy content (122 kJ/g) and clean combustion product (Table 1) (Akhlaghi and Najafpour-Darzi, 2020).Biological production of H 2 has gained attention over traditional technologies (e.g., steam reforming of CH 4 , coal gasification) (Valle et al., 2019;Akhlaghi and Najafpour-Darzi, 2020) because it does not rely upon usage of fossil fuels and has reduced CO 2 emissions (Lepage et al., 2021).However, current technologies for biological production of H 2 need to improve their yield and cost competitiveness (Lepage et al., 2021;Nirmala et al., 2023).
Bio-H 2 production by the so called dark processes, that is anaerobic fermentation of organic compounds by a number of heterotrophic microbes (mainly bacteria), is generally considered more effective than light-driven processes (i.e., direct and indirect biophotolysis, photofermentation by means of photosynthetic microorganisms) (Das and Veziroglu, 2008;Mudhoo et al., 2011;Arizzi et al., 2021;Cao et al., 2022).Among microorganisms catalyzing dark fermentation, obligate anaerobes such as Clostridium spp.feature 2-fold higher maximum theoretical H 2
So far, most of these investigations achieved limited H 2 yield enhancement (generally comprised between 15% and 80%) with H 2 yield still lower than 2 mol/mol hexose in most engineered strains (Mazzoli et al., 2024).One likely reason is that only one gene has generally been down-or upregulated in each engineered strain.For instance, in Clostridia multiple hydrogenase-encoding genes are present, with different roles and expression levels, and the balance of their activity can be a key to improved performances (Land et al., 2020;Arizzi et al., 2021;Morra, 2022;Fasano et al., 2024).Combination of multiple advantageous metabolic modifications (e.g., overexpression of evolving H 2 ases, elimination of alternative pathways, optimization of sugar metabolism) in one strain seems among the most obvious implementation.More extensive application of adaptive laboratory evolution could be an additional tool that avoid or implement complex rational genetic Fermentative pathways in Clostridia, production of C4-compounds.2-ketoisovalerate decarboxylase (KivD) was not found in Clostridia but a Lactococcus lactis gene coding for it was engineered in C. thermocellum (Lin et al., 2015).Abbreviations: Acr, acetoin reductase; Adh, alcohol dehydrogenase; Ald, α-acetolactate decarboxylase; Aldh, aldehyde dehydrogenase; Als, α-acetolactate synthase; Dhad, dihydroxy acid dehydratase; DHAP, dyhydroxyacetone phosphate; Fd, ferredoxin; Kari, keto acid reductoisomerase; KivD, L. lactis 2-ketoisovalerate decarboxylase; Kor, ketoisovalerate ferrodoxin-dependent reductase; Pfl, pyruvate-formate lyase; Pfor, pyruvate:ferredoxin oxidoreductase.
engineering to tackle all these issues and enhance H 2 production of microorganisms (Tunca et al., 2023).Along with strain optimization, the use of improved fermentation conditions (e.g., pH, temperature, substrate concentration, H 2 partial pressure), mode (e.g., continuous fermentation) and bioreactor configuration is pivotal (Cai et al., 2013;Wang et al., 2014;Łukajtis et al., 2018;Son et al., 2021;Julkipli et al., 2023).From this standpoint, the improvement of systems for reducing H 2 partial pressure in the bioreactor (e.g., stirring the growth medium, sparging the growth medium with inert gas, removing gas by a vacuum pump, selectively removing H 2 by active membranes) is essential for overcoming thermodynamic barriers of biological H 2 production.The Δ r G' m (i.e., the change in Gibbs free energy associated with a metabolic reaction/pathway when all the reactants have a concentration of 1 mM) for the production of H 2 by oxidation of glucose to acetate (through the EMP pathway) can vary from −205.1 to 0.2 kJ/mol for dissolved H 2 concentration ranging from 10 -9 M-1 M (Flamholz et al., 2012).Promising results have also been reported through the development of processes based on syntrophic microbial chains, such as two-step fermentation (e.g., dark fermentation + photofermentation) (Ramprakash et al., 2022) or co-cultures (e.g., H 2 producing bacteria + bacteria able to perform anaerobic respiration) (Zhang et al., 2023b) providing complete oxidation of Clostridium fermentation by-products.Improvement of H 2 yield obtained by combining dark-and photo-fermentation was significantly higher (≈100-200%) (Ramprakash et al., 2022) than those reported for co-cultures (≈30-45%) (Zhang et al., 2023b).The efficiency of syntrophic co-cultures could be enhanced within the framework of microbial electrolysis cells in which additional electric voltage is used to increase H 2 yield from organic compound oxidation (Bora et al., 2022).

Ethanol
Ethanol is the most broadly produced biofuel today, with over 16 billion gallons produced in the United States alone in 2019 (Table 1) (Ethanolrfa, 2019).It is typically blended with gasoline for use in spark-ignited engines (10% in the United States, 27% in Brazil), but can also be catalytically upgraded to longer-chain fuel molecules, such as jet fuel and gasoline (Hannon et al., 2020;Mazzoli and Olson, 2020).3.1 EJ/year of ethanol is currently obtained from sugar cane (30%) and cereals (50%) (Lynd et al., 2017).However, projected future global demand of bioethanol and the need for more massive reduction of greenhouse gas emission will require a significant contribution by bioethanol derived from other feedstocks (e.g., lignocellulose, CO 2 ) (Wang et al., 2015;Lynd et al., 2017).Today, bioethanol is primarily produced by fermentation of mono-or disaccharides using Saccharomyces cerevisiae or Zymomonas mobilis.Nonetheless, these organisms cannot directly grow on cheap feedstocks such as lignocellulose or syngas (Himmel et al., 2007).Substantial research has been dedicated to develop recombinant cellulolytic yeast or Z. mobilis strains, yet the maximum cellulosic ethanol titer obtained through direct biomass fermentation by these strains (≤10 g/L) is far lower than what is generally considered as necessary for commercial application (titer = 40 g/L, yield = 1.8 mol/mol hexose) (Dien et al., 2003;Kojima et al., 2013;Todhanakasem et al., 2019;Anandharaj et al., 2020).Clostridium thermocellum and Thermoanaerobacterium saccharolyticum have been important alternative microbial paradigms for one-step production of ethanol from cellulose or hemicellulose, respectively (Figure 1) (Mazzoli and Olson, 2020).On the other hand, acetogenic Clostridia such as C. ljungdahlii (Phillips et al., 1993) and C. ragsdalei (Sun et al., 2018) have been investigated as promising ethanol producers through C1-gas fermentation.
T. saccharolyticum is a thermophilic anaerobic bacterium which can ferment xylan (the main polymer in hemicellulose) and all the most abundant monosaccharides of plant biomass (e.g., glucose, mannose, xylose, galactose, and arabinose) although it cannot use cellulose (Herring et al., 2016).The wild-type T. saccharolyticum accumulates ethanol as its main fermentation product (yield = 1 mol/mol xylose) but also significant amounts of acetate and lactate (Shaw et al., 2008).Disruption of genes involved in production of acetate (phosphotransacetylase, pta, and acetate kinase, ack) and adaptation to high xylose concentration resulted in homoethanologenic phenotype with a maximum ethanol titer = 37 g/L (Shaw et al., 2008).In a following study, higher performing T. saccharolyticum strains were obtained by introducing additional genetic modifications such as the expression of a urease and the disruption of an operon involved in exopolysaccharide biosynthesis (Herring et al., 2016).These strains were able to produce up to 70 g/L of ethanol from a mixture of cellobiose and maltodextrin.However, much lower ethanol titer (26 g/L) was obtained by fermentation of a hemicellulose extract (Herring et al., 2016).Interestingly, co-culture of C. thermocellum and T. saccharolyticum (or other hemicellulolytic microbes such as (Thermoanaerobacterium thermosaccharolyticum and Herbinix spp.) has been performed which enabled one-pot fermentation of both the cellulose and hemicellulose components of plant biomass (He et al., 2011;Jiang et al., 2013;Froese et al., 2019;Beri et al., 2020).
As mentioned above, acetogenic Clostridia have been studied for ethanol production via C1-gas fermentation.This generally occurs in two steps, that is acetogenesis precedes ethanol formation (solventogenesis) (Fernández-Blanco et al., 2023).Gas fermenting Clostridia can produce ethanol through two pathways that is: i) reduction of acetyl-CoA by aldehyde/alcohol dehydrogenase or; ii) reduction of acetate to acetaldehyde by acetaldehyde ferredoxin oxidoreductase (Aor) and then reduction of acetaldehyde by alcohol dehydrogenase (Figure 1) (Zhang et al., 2020b).Ethanol production is triggered by stress conditions limiting cell growth such as acidic pH and/or lack of nutrients (Daniell et al., 2012;Fernández-Naveira et al., 2016;Al-Shorgani et al., 2018).The highest ethanol titer reported through C1-gas fermentation (48 g/L) was achieved in 1993 by Philips and co-workers by using a C. ljungdahlii strain growing on syngas (Phillips et al., 1993).This result was obtained after 560 h fermentation in a stirred tank bioreactor with cell recirculation, using an optimized growth medium and high gasliquid mass transfer.This study indicated that a medium with pH range of 4.0-4.5 promoted ethanol accumulation.However, far lower ethanol titers were obtained in more recent investigations (Fernández-Blanco et al., 2023).As far as we know, the highest ethanol titer (16.25 g/L) reported by the latest studies was obtained through syngas fermentation by C. ragsdalei in a medium supplemented with poultry litter biochar (Sun et al., 2018).As regards strain improvement by metabolic engineering, significant increase of ethanol production (50%-180%) by C. autoethanogenum or C. carboxidivorans was obtained by either inactivation (Liew et al., 2017) or overexpression (Lu et al., 2019) of adhE genes (encoding bifunctional alcohol/aldehyde dehydrogenase).However, the maximum ethanol titers obtained through autotrophic growth of these strains were ≤ 3 g/L (Liew et al., 2017;Cheng et al., 2019).
In conclusion, Clostridia provide promising paradigms for production of ethanol from low-cost feedstocks, such as lignocellulose and C1-gas.Research progress is at a more advanced stage as regards lignocellulose fermentation (also in reason of the higher number of studies), while investigation of C1-gas fermentation still needs substantial efforts.
One of the main hurdles in engineering LA hyper-production in anaerobic (hemi) cellulolytic microbes is their limited acid tolerance (Yang et al., 2013;Mazzoli et al., 2022;Svetlitchnyi et al., 2022).Growth at regulated pH through base addition was essential for major increase of the LA production of a Thermoanaerobacterium aotearoense strain deficient in acetate production (Yang et al., 2013).This growth condition resulted in nearly homolactic fermentation (LA yield = 0.93 g/g glucose) with a final LA titer up to 47 g/L (Yang et al., 2013).As base addition is complicated and expensive at the industrial scale, use of strains with high acid tolerance is recommended (Singhvi et al., 2018).Ideally, a microbial host should tolerate pH conditions around the pK a (s) of the produced acid (typically in the range 3-5 for organic acids) (Skoog et al., 2018).So far, C. thermocellum and Caldicellulosyruptor sp.strains have been obtained by adaptive laboratory evolution which have increased tolerance to LA (Mazzoli et al., 2022;Svetlitchnyi et al., 2022).An evolved Caldicellulosyruptor sp.strain showed more than 10-fold increase in LA titer and was able to produce up to 70 g/L LA through batch fermentation of microcrystalline cellulose (Svetlitchnyi et al., 2022).This titer is comparable to that obtained through fermentation of cellulosic biomass hydrolysate by lactic acid bacteria (Rawoof et al., 2021).However, it should be noted that pH regulation of Caldicellulosyruptor sp.cultures was still necessary (Svetlitchnyi et al., 2022).In fact, acid stress depends on both the decrease of pH and the nature of the acid, i.e., more hydrophobic carboxylic acids generally are more toxic (Jarboe et al., 2013;Wilbanks and Trinh, 2017).Irrespective of the strategy used (random mutagenesis, strain evolution, rational engineering), no research could lower the acidic pH limit allowing a microorganism to grow beyond 0.5 pH unit such as in the case of the anaerobic cellulolytic bacteria Clostridium cellulovorans and Fibrobacter succinogenes (Wu et al., 2017;Wen et al., 2020a;Mazzoli, 2021).More intense effort in this direction is needed as regards LA hyperproducing cellulolytic Clostridia.

Overview of C3-compound production by Clostridia
Among the C3 compounds considered here, production of 1,3-PDO by C. butyricum is the most established (Table 2), although the lack of efficient genetic tools hampers strain improvement (e.g., increase product tolerance, reduce by-products formation) and increase in process efficiency which has stimulated research on other clostridial strains (e.g., C. acetobutylicum).A few engineered clostridial strains have shown promising potential for direct fermentation of lignocellulose to lactic acid although their limited acid tolerance constitutes a major issue.As reported also for other microorganisms (Table 2), 1,2-PDO production by Clostridia is intrinsically hindered by the high cost of substrates or by accumulation of toxic intermediates which need to be addressed (e.g., by metabolic engineering).

Improving butanol production in natural solventogenic Clostridia
Butanol has high potential as a drop-in fuel (namely, it can be fed to spark ignited engines without any modification) which adds to its applications as paint, polymer and plastic precursor (Table 1) (Gu et al., 2011;Campos-Fernández et al., 2012;Jiang et al., 2015).ABE fermentation is still the most economically viable route for biobutanol production, yet, it is affected by several drawbacks especially under industrial conditions: i) high cost of feedstock and substrate inhibition; ii) low butanol titer (≤20 g/L), yield (≈0.33 g/g), and productivity (<0.5 g/L/h); iii) important formation of by-products which increase butanol purification costs; iv) poor understanding of Clostridium physiology (Green, 2011;Gu et al., 2011;Abo et al., 2019;Li et al., 2020c).Current research aimed to improve butanol production by native solventogenic Clostridia includes: i) enhancing butanol yield, titer and tolerance; ii) expanding fermentation substrates to low-cost feedstocks (e.g., food waste, lignocellulosic biomass and C1-gases) (Zhang et al., 2020a;Fernández-Blanco et al., 2023;Re and Mazzoli, 2023).
Four Clostridium species, C. acetobutylicum, C. beijerinckii, C. saccaroperbutylacetonicum and C. saccharoacetobutylicum, can biosynthesize significant butanol amounts (Figure 4) (Keis et al., 2001;Huang et al., 2010).The growth of these strains develops through three phases that is acidogenesis, solventogenesis and sporogenesis which is subjected to complex metabolic regulation whose molecular details are still poorly understood (Li et al., 2020c).The exponential phase is characterized by accumulation of acids (mainly acetic and butyric acid), ATP and reduced pyridine cofactors.Growth medium acidification and accumulation of ATP, NAD(P)H and possibly other metabolites (e.g., butyrylphosphate, formic acid) contribute to shift the metabolism towards solvent (acetone, butanol, ethanol) production and is accompanied by acid re-assimilation (Zhao et al., 2005;Wang et al., 2011b;Li et al., 2020c).Biomass production, nonassimilation of acids, and accumulation of other carbohydrates contribute to lower the actual butanol yield with respect to the theoretical maximum (1 mol/mol of glucose, that is 0.41 g/g) (Li et al., 2020c).
Significant enhancement of butanol production by natural solventogenic Clostridia has been obtained by: i) strain improvement (by mutagenesis, metabolic engineering, adaptive laboratory evolution); ii) optimization of the growth medium; iii) optimization of the fermentation process (e.g., high cell density fermentation, use of in situ product recovery techniques) [extensively reviewed by Li et al. (2020c)].Further improvement of bacterial strains through mutagenesis is hampered by the complex and unknown metabolic/phenotypic changes generated by random gene mutations (Palsson and Zengler, 2010).Rational metabolic engineering has targeted genes involved in: i) butanol biosynthetic pathway; ii) pathways competing for carbon and electrons; iii) butanol tolerance (illustrated in Section 4.4.1.4);iv) redox homeostasis; v) energy homeostasis; vi) regulation of acidogenesis-solventogenesis shift and solvent production (Wietzke and Bahl, 2012;Nguyen et al., 2018;Li et al., 2020c;Dai et al., 2021;Re and Mazzoli, 2023).
The impairment of biosynthetic pathways for alternative fermentation products (e.g., acetate, acetone, butyrate, ethanol, lactate) (Figures 1-4) has generally decreased by-product accumulation and enhanced butanol yield (Lee et al., 2012;Nguyen et al., 2018) but sometimes generated undesired phenotypes (e.g., defective acid assimilation or growth rate) (Li et al., 2020c).Elimination of acetone production (a non-fuel solvent with high corrosivity) (dos Santos Vieira et al., 2019) has been reported to increase acetate (and ethanol) generation and decrease butanol accumulation (Jiang et al., 2009;Lehmann et al., 2012).Instead, overexpression of secondary-alcohol dehydrogenases (sAdh) (coupled with upregulation of acetone pathway and disruption of butyrate production) in C. acetobutylicum resulted in efficient conversion of acetone to isopropanol (up to 8 g/L) (Dusséaux et al., 2013;dos Santos Vieira et al., 2019;Zhang et al., 2023a).This strategy generated isopropanol, butanol, ethanol (IBE) mixture that, differently from the traditional ABE fermentation, can be directly applied to spark-ignition engines without the need to remove by-products (Peralta-Yahya and Keasling, 2010;Zhang et al., 2023a).Engineering of C. acetobytylicum aldehyde/alcohol dehydrogenases has been used to increase substrate specificity for butyryl-CoA and increase butanol over ethanol production (Cho et al., 2019).
So far, the largest increases in butanol titer and/or productivity have been obtained by engineering the fermentation process, that is by using fed-batch or continuous configuration, and/or using immobilized cells and/or using in situ product recovery (e.g., pervaporation, adsorption, liquid-liquid extraction, gas stripping, vacuum fermentation) (Nguyen et al., 2018;Li et al., 2020c), leading to alleviation of substrate and/or product inhibition.So far, the highest butanol titer (550 g/L) was obtained by an engineered C. acetobutylicum strain in a continuous high cell density bioreactor with in situ alcohol extractive distillation (Table 2) (Nguyen et al., 2018).However, these improved fermentation systems are generally associated with higher technical complexity and cost (Nguyen et al., 2018;Li et al., 2020c).

Engineering butanol production in non-native Clostridium hosts
Engineering butanol production in non-native hosts has at least two possible advantages: i) no production of other solvents which allows simplified downstream processing for product purification; ii) selection of hosts that can naturally ferment low-cost substrates (e.g., lignocellulose, CO 2 ).Much interest has been attracted by the hyper-butyrate producer C. tyrobutyricum (Bao et al., 2020).In fact, this microorganism has high metabolic flux toward butyryl-CoA and a high butanol tolerance (>15 g/L) (Yu et al., 2011).In addition, C. tyrobutycum has rarely been reported to be subjected to bacteriophage infection, which is a common issue of industrial ABE fermentation (Bao et al., 2020).A butanol hyper-producing C. tyrobutyricum was developed by overexpressing the C. acetobutylicum bifunctional acetaldehyde-alcohol dehydrogenase AdhE2 and disrupting the gene encoding butyrate:acetate CoA transferase (Cat1, which catalyzes butyrate production) which can generate 26.2 g/L butanol through glucose fermentation (Figure 4) (Lee et al., 2016;Zhang et al., 2018).This butanol titer is actually higher than those reported for native butanol producers.Hence, C. tyrobutyricum appears to be a microbial platform with high potential for butanol production, at a level similar or higher than other non-native butanol producers such as E. coli (Shen et al., 2011) or the yeast Arxula adeninivorans (Kunze and Haehnel, 2011).
Butanol production has mainly been engineered in three cellulolytic Clostridia, C. cellulolyticum (Gaida et al., 2016) C. cellulovorans (Yang et al., 2015) and C. thermocellum (Tian et al., 2019b).Reduced or imbalanced biosynthesis of butanol pathway enzymes, enzyme instability, insufficient availability of co-factors and unfavorable reaction thermodynamics have probably contributed to a variable extent to modest butanol titer (<0.5 g/ L) in C. cellulolyticum and C. thermocellum (Gaida et al., 2016;Tian et al., 2019b).Fewer genetic modifications enabled butanol production in C. cellulovorans (it is naturally equipped with a butyryl-CoA biosynthetic pathway) (Yang et al., 2015;Wen et al., 2019) which likely contributed to its higher butanol titer (4.96 g/L) from lignocellulosic biomass (Wen et al., 2020b).Similar metabolic engineering strategies used for ABE fermenting strains [e.g., impairing alternative fermentative pathways, enhancing NAD(P) H availability, dysregulating redox homeostasis] are likely to succeed also in further improving butanol formation in C. cellulovorans (and other cellulolytic Clostridia) (Bao et al., 2021;Re and Mazzoli, 2023).However, the efficiency of genetic tools for manipulating C. cellulovorans is still limited (Wen et al., 2017).Apart from the native ability to produce butanol of C. carboxidivorans, the butanol pathway has been engineered in another C1-gas fermenting Clostridium, namely, C. ljungdahlii (Köpke et al., 2010;Lauer et al., 2022b).However, butanol titer < 0.2 g/L was obtained by this engineered strain through autotrophic growth.An alternative approach to bioconvert C1-gases into alcohols with longer chain than C2 consists in co-culturing gas fermenting strains with chainelongating strains (Fernández-Blanco et al., 2023).Different gas fermenting Clostridia (e.g., C. aceticum, C. autoethanogenum, C. carboxidivorans, C. ljungdahlii) have been co-cultured with the chain-elongating model strain Clostridium kluyvery resulting in production of C4-C8 alcohol mixtures (maximum alcohol titer generally ≤ 1 g/L).(Diender et al., 2016;Richter et al., 2016;Bäumler et al., 2022;Fernández-Blanco et al., 2022).In fact, C. kluyvery is able to elongate the chain of acetate and ethanol (produced by acetogenic Clostridia) to butyrate, hexanoate and octanoate by using reverse β-oxidation.On the other side, acetogenic strains can reduce these fatty acids to their corresponding alcohols (Figure 4).One of the main limitations of this approach is that acetogenic bacteria usually have an optimum pH for growth close to 6, while that of C. kluyveri is close to neutrality (Fernández-Blanco et al., 2023).

Production of isobutanol
Isobutanol is an attractive vehicle fuel with energy density similar to butanol and higher octane number, which is advantageous for blending into gasoline (Chen and Liao, 2016).Moreover, isobutanol can be dehydrated to isobutene, which can then be converted to C8-C12 alkenes to be used as jet fuel (Lin et al., 2015).Additional uses of isobutanol and its derivatives are as solvents, additives in paints, ink ingredients, and extractants for organic compounds (Table 1) (Nawab et al., 2024).A few microorganisms such as Lactococcus lactis, S. cerevisiae, Pichia pastoris and Candida sp. can naturally produce very little isobutanol amounts (≤0.44 g/L) (Nawab et al., 2024).In S. cerevisiae and lactic acid bacteria, isobutanol is biosynthesized through the diversion of 2-ketoisovalerate (an intermediate of valine and isoleucine biosynthesis) which is decarboxylated to isobutyraldehyde by 2-ketoisovalerate decarboxylase and finally reduced (Figure 3) (Hazelwood et al., 2008).Higher levels of isobutanol have been produced by engineered microbial platforms (Nawab et al., 2024) with studies targeting Corynebacterium glutamicum (Yamamoto et al., 2013;Hasegawa et al., 2020) and E. coli (Atsumi et al., 2008) reporting the highest titers (≥20 g/L) through glucose fermentation.However, much lower isobutanol titer (1.88 g/L) was obtained through fermentation of cheaper feedstocks such as pretreated corn stover (Minty et al., 2013).
Advantageously, native production of higher isobutanol levels (1.6 g/L) has been observed in the cellulolytic bacterium C. thermocellum (Holwerda et al., 2014).Isobutanol biosynthetic pathway in C. thermocellum differs from that found in lactic acid bacteria or S. cerevisiae in that 2-ketoisovalerate is converted to isobutyraldehyde in two steps (Figure 3) (Lin et al., 2015).First, ferredoxin-dependent ketoisovalerate reductase (KOR) catalyzes oxidative decarboxylation of 2-ketoisovalerate to isobutyryl-CoA by which is then reduced to isobutyraldehyde (Figure 3).Isobutanol production of C. thermocellum was enhanced by overexpressing autologous acetohydroxy acid synthase (Als), keto acid reductoisomerase (Kari) and dihydroxy acid dehydratase (Dhad) and introducing L. lactis 2-ketoisovalerate decarboxylase (KivD) (Figure 3) (Lin et al., 2015).In optimized growth conditions, the most efficient engineered C. thermocellum strain produced 5.4 g/L of isobutanol from cellulose, which corresponds to 41% of the theoretical yield (Lin et al., 2015).Since this concentration is close to the isobutanol tolerance of the wild type C. thermocellum (Tian et al., 2019a), it is likely that isobutanol production in this microorganism is limited by tolerance.Less successful engineering of isobutanol production (titer ≤ 0.66 g/L) was reported in other cellulolytic Clostridia, i.e., C. cellulolyticum (Higashide et al., 2011) and C. cellulovorans (Wen et al., 2022).Recently, an original approach was reported which consisted in producing a mixture of butanol and isobutanol through direct fermentation of alkali extracted deshelled corn cobs (Wen et al., 2022).To this aim, artificial consortia were developed that included engineered strains of C. cellulovorans and C. beijerinckii which were able to generate up to 1.05 g/L isobutanol and 6.22 g/L butanol in the same fermentation (Wen et al., 2022).Efforts to engineer the isobutanol pathway into autotrophic Clostridia have also been reported (Weitz et al., 2021).However, fermentation of a syngas mixture (50% CO, 45% H 2 , 5% CO 2 ) by an engineered C. ljungdahlii strain could only produce ≈ 70 mg/L isobutanol (Weitz et al., 2021).

Improving butanol/isobutanol tolerance
Butanol (and isobutanol) toxicity is among the main issues of biological production of this chemical(s).These compounds are inherently more noxious than other established biofuels, such as ethanol, owing to their higher hydrophobicity (Heipieper et al., 2007;Wilbanks and Trinh, 2017).The toxicity of butanol is mainly due to impairment of structure and functions of biological membranes, dissipation of proton motive force and ATP pools and protein denaturation (Bowles and Ellefson, 1985;Tomas et al., 2004;Alsaker et al., 2010;Venkataramanan et al., 2015).Even native butanol-producing strains, e.g., C. acetobutylicum, can typically tolerate up to 1%-2% v/v butanol (Huang et al., 2010;Nicolaou et al., 2010).
Increasing microbial tolerance to butanol/isobutanol is a key aspect for enhancing their biological production especially as regards final titer.To this aim, responses to butanol stress have been studied in several microbial species that showed the implication of a very complex network of mechanisms only partially understood (Re and Mazzoli, 2023).Hence, increasing butanol tolerance by targeted gene manipulation (e.g., overexpression of protein chaperones) has so far attained only limited results.Random approaches (e.g., random mutagenesis, genome shuffling, adaptive evolutionary engineering) proved to be more suitable strategies to select for multiple-gene trait combinations conferring higher butanol resistance such as for mutant C. acetobutylicum strains able to tolerate up to 3%-4% (v/v) butanol (Liu et al., 2012b;Liu et al., 2013).Interestingly, the adaptive evolution strategy used to develop a butanol-hypertolerant C. thermocellum also enhanced tolerance to isobutanol to the same extent (i.e., 15 g/L), suggesting that similar physiological mechanisms allow cells to cope with both compounds (Tian et al., 2019a).Interestingly, most of the strains characterized by increased tolerance (and equipped with butanol pathway) also showed higher butanol production which highlights the importance of this line of research in improving biobutanol production (Re and Mazzoli, 2023).
Metabolic engineering strategies targeting global gene regulators involved in stress response could advantageously contribute to future development of butanol/isobutanol hypertolerant Clostridia (Jones et al., 2016;Mazzoli, 2021;Xu et al., 2021).Recent studies have indicated that small non-coding RNAs (sRNAs) and RNA chaperones (e.g., Hfq) have important roles in the ability of microorganisms to tolerate a variety of stresses such as butanol exposure (Venkataramanan et al., 2013;Jones et al., 2016;Sun et al., 2017;Costa et al., 2021).

Overview of C4-compound production by Clostridia
Extensive research has been dedicated to improving butanol production by ABE fermenting strains and non-native butanol producers.Although native solventogenic Clostridia are still the most established microorganisms for industrial production of butanol, other engineered strains are promising alternative candidates on traditional substrates (e.g., C. tyrobutyricum) or lignocellulosic feedstocks (e.g., C. cellulovorans).More limited progress has been reported as regards the generation of isobutanol through direct fermentation of lignocellulose or C1gases.Yet, a major challenge in the biological production of butanol/isobutanol is the development of hyper-tolerant strains which still requires substantial efforts.Despite the high commercial interest in 2,3-BDO, the levels so far produced by Clostridia are modest both from gaseous substrates and sugars.
Recently, hexanol production was engineered in another gas fermenting Clostridium (C.ljungdhalii) (Lauer et al., 2022b).Two sets of genes encoding the acetyl-CoA-to-hexanoyl-CoA pathway of C. kluyvery, and the gene coding for the aldehyde-alcohol dehydrogenase AdhE2 of C. acetobutylicum (for the reduction of hexanoyl-CoA to hexanol) were integrated into the C. ljungdahlii chromosome (Figure 4).The engineered C. ljungdahlii showed improvement of both butanol and hexanol production (Lauer et al., 2022b).Fermentation in 2 L bioreactor, with continuous CO 2 -H 2 supplementation and pH regulation (pH = 6) resulted in the production of 0.122 g/L hexanol.Limited hexanol titer was attributed to inefficient biosynthesis of some enzymes of the acetyl-CoA-to-hexanoyl-CoA pathway.Therefore, additional genes from C. carboxidivorans (encoding thiolase, crotonase, 3hydroxybutyryl-CoA dehydrogenase and butyryl-CoA dehydrogenase complex) (Figure 4) were introduced in the genome of the engineered C. ljungdahlii.The final strain produced 0.251 g/L hexanol through fermentation of 20% CO 2 , 80% H 2 gas mixture (Oh et al., 2022).
As for other products obtained through gas fermentation, significant research towards higher hexanol production has been focused on optimizing the fermentation process such as growth conditions, media composition, gas composition or supply (Oh et al., 2022).Substantial improvement was obtained by using in situ extraction of hexanol, which reduces hexanol accumulation in the fermentation medium (Kottenhahn et al., 2021;Oh et al., 2023).It is worth remembering that hexanol is even more toxic than butanol because of its longer carbon chain (Heipieper et al., 2007).Supplementation of 1 g/L hexanol is enough for reducing C. ljungdahlii growth and 5 g/L caused total growth cessation (Oh et al., 2023).Supplementation of small quantities of a biocompatible extractant (i.e., oleyl alcohol) and ethanol (as a precursor) to C. carboxidivorans P7 cultures growing on CO enabled production of 8.45 g/L hexanol, which is the highest titer reported so far (Oh et al., 2023).

Medium (C4-C8) chain esters
Short-and medium-chain (C2-C12) esters such as ethyl acetate, butyl acetate, isobutyl acetate and butyl butyrate, have a broad range of application as flavors, fragrances, pharmaceuticals, green solvents and advanced biofuels (Table 1) (Lee and Trinh, 2020;Wang et al., 2021a).The traditional methods for synthesizing short-and medium-chain fatty acid esters are mainly based on concentrated sulfuric acid-mediated esterification of acids and alcohols and are affected by serious health and environmental issues (Cull et al., 2000;Jermy and Pandurangan, 2005).Alternative chemical strategies based on ionic liquid catalysis can reduce these problems to some extent, but they are expensive and not stable (Tankov et al., 2017).In nature, microbes and plants can biosynthesize several esters which encounters the increasing consumer preference for natural and sustainable products (Lee and Trinh, 2020;Seo et al., 2021).Some yeasts and lactic acid bacteria can naturally form esters but with limited efficiency (Abeijón Mukdsi et al., 2009;Kruis et al., 2018).This has stimulated research aimed to engineer more efficient producers (Rodriguez et al., 2014;Kruis et al., 2017).
One of the key aspects of engineering ester production in Clostridia relies on overproduction of stable AATs with desired substrate specificity (Seo et al., 2021).Most characterized AATs derive from plants or yeasts which results in poor biosynthesis, solubility and/or thermostability in prokaryotes (Horton and Bennett, 2006;Noh et al., 2018;Li et al., 2020a;Fang et al., 2020).Studies have been dedicated to optimize AATs structure and/or biosynthesis (Seo et al., 2021).In addition, limited knowledge is available on substrate specificities of AATs (Noh et al., 2018;Kruis et al., 2019).Both these aspects were tackled to develop an ester overproducing C. saccharoperbutylaceticum (Feng et al., 2021).In addition, systems metabolic engineering was used to increase availability of NADH and ester precursor(s) (namely, acetyl-CoA).The most efficient strain was able to produce up to 20.3 g/L butyl acetate (and 0.9 g/L butyl butyrate) through glucose fermentation and up to 17.8 g/L butyl acetate by fermentation of corn stover hydrolysate (Feng et al., 2021).Few metabolic modifications were able to convert C. tyrobutyricum (an efficient butyrate producer) into high performing butyl butyrate producing strain, namely, enhancing butyryl-CoA pathway (by upregulating CtfAB encoding CoA transferase, Figure 4), engineering butyryl-CoA reduction to butanol (by expressing C. acetobutylicum AdhE2), and introducing the wild strawberry alcohol acyltransferase (VAAT) (Guo et al., 2023).Additional reducing power for ester production was supplied by using a reduced growth substrate, that is mannitol, instead of glucose.Fed batch fermentation of mannitol at regulated pH and supplementation of hexadecane for in situ extraction of esters led to ≈ 63 g/L butyl butyrate (yield = 0.21 mol/mol, theoretical maximum is 0.5 mol/mol) (Table 2) (Guo et al., 2023).This is by far the highest medium chain ester titer obtained by microbial fermentation.Additional introduction of heterologous α-amylase and α-glucosidase enabled the engineered C. tyrobutyricum to produce high amounts of butyl butyrate (26.8 g/L) through batch fermentation of starch from a nonfood crop (i.e., cassava) (Guo et al., 2024).As mentioned above for other hydrophobic products (e.g., butanol, hexanol), in situ extraction of esters significantly contributed to increase fermentation efficiencies of the engineered strains by limiting toxicity linked to product accumulation in the growth medium (Fang et al., 2020;Feng et al., 2021;Guo et al., 2023;Guo et al., 2024).
In conclusion, the variety of acyl-CoA, acids and alcohols produced by Clostridia confers them the ability to produce a panel of commercially interesting medium-chain esters in high amounts.Yet, challenges of this research area reside in efficient expression of AATs with desired substrate specificity and improving the availability of carbon precursors and reducing power.

Conclusion
The studies summarized in the present review illustrate the key role of Clostridia in the development of sustainable biomass biorefining processes.Clostridia are already the reference cell factories for fuels such as butanol and among the most promising microbial platforms for production of valuable chemicals (e.g., ethanol, lactate, H 2 , hexanol, medium chain esters) through direct fermentation of low cost biomasses (e.g., lignocellulose, C1-gases) (Table 2).Significant progress has also been reported as regards clostridial production of 1,2-PDO and 1,3-PDO, whose titers are not far from those obtained by the highest performing microorganisms (e.g., engineered E. coli) (Table 2).However, for the full deployment of Clostridium potential in commercial applications further advances at multiple levels are necessary.
Extensive investigations on the physiology and metabolism of Clostridia over the last decades have revealed peculiar characteristics (e.g., atypical pathway reactions, sophisticated regulation of enzyme activity) and subtle differences between the different strains which affect carbon and electron flux and pathway thermodynamics.We believe that further advances in detailed understanding the metabolic network of Clostridia will be major sources of inspiration for improving current metabolic engineering strategies.To this aim, further development of genetic tools for manipulating Clostridia is also necessary, especially for strains which are recalcitrant to the currently available methods (e.g., Clostridium diolis) or which shows low genetic tractability (e.g., C. cellulovorans).
Apart from strain improvement by metabolic engineering (enhancement of product yield, titer, productivity; increase of tolerance to toxic products), research aimed to optimize the fermentation process (e.g., growth media and conditions, fermentation mode, bioreactor configuration, use of syntrophic co-cultures) including extraction and purification of products will be essential to achieve the efficiency required by industrial application.Coping with the different feedstocks (e.g., insoluble solids such as lignocellulose versus gaseous substrates) and products (e.g., acids versus hydrophobic solvents versus gaseous products) described in the present review will certainly require far different process optimization.The efficiency of gas fermentation in inherently limited by the low solubility of CO/CO 2 in water.Developing suitable systems for improving gas-liquid mass transfer is among the priority of this research area.Accumulation of acids (e.g., lactic acid) or solvents (e.g., butanol, hexanol, medium chain esters) reduces Clostridium fermentation efficiency due to limited tolerance of cells these chemicals.Both strain improvement by metabolic engineering and process engineering (e.g., pH regulation, in situ recovery of solvents) should synergistically provide a cost sustainable solution.As regards biological production of H 2 , the improvement of systems for reducing H 2 partial pressure in the bioreactor (e.g., stirring the growth medium, sparging the growth medium with inert gas, removing gas by a vacuum pump, selectively removing H 2 by active membranes) is essential for overcoming thermodynamic barriers of biological H 2 production.We are confident that thanks to the contribution of these different approaches a rapid improvement of Clostridium-mediated biorefinery processes suitable for their industrial application will be possible.

TABLE 1
Most recent estimation of economic parameters and applications of some top value chemicals produced by Clostridia.

TABLE 2
Comparison between production of top value chemicals by Clostridia (bold) and other high performing microorganisms.
(Continued on following page)Frontiers in Bioengineering and Biotechnology frontiersin.org

TABLE 2 (
Continued) Comparison between production of top value chemicals by Clostridia (bold) and other high performing microorganisms.Frontiers in Bioengineering and Biotechnology frontiersin.orgcosmetic, pharmaceutical industry and the synthesis of biodegradable solvents and plastics (e.g., polylactide) (Table 1) (Abdel-Rahman et al., 2011; Abdel-Rahman et al., 2013; Alves de Oliveira et al., 2018).About 90% of global LA production is obtained by microbial fermentation producing pure L-or D-LA enantiomer (chemical synthesis generates a racemic mixture) (Abdel-Rahman et al., 2013; Alves de Oliveira et al., 2018) which is required for plastic polymer synthesis (Abdel-Rahman and Sonomoto, 2016), and food and pharmaceutical applications