Butanol synthesis is catalyzed by six enzymes, namely thiolase, also known as acetyl-CoA acetyltransferase (THL), $\beta$-hydroxybutyryl-CoA dehydrogenase (BHBD), 3-hydroxybutyryl-CoA dehydratase (CRT, also called crotonase), butyryl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase (BAD), and butanol dehydrogenase (BDH). Thiolase has an important role in both acidogenesis and solventogenesis, as it directs the pathway toward forming acetone, butyrate, or butanol. Another important enzyme is NADH-dependent butyraldehyde dehydrogenase, which catalyzes the conversion of butyryl-CoA to butyraldehyde, a key intermediate in butanol formation. The third enzyme, butanol dehydrogenase, catalyzes the reduction of butyraldehyde to butanol using the reducing power of NAD(P)H in the final step of butanol synthesis [21].

CoA-transferase and acetoacetate decarboxylase are the key enzymes involved in acetone synthesis. CoA-transferase catalyzes the formation of the intermediate acetoacetate by transferring CoA from acetoacetyl-CoA to either acetate or butyrate. Acetoacetate decarboxylase (AADC) is the next important enzyme in this pathway, which decarboxylates acetoacetyl-CoA to acetone by releasing a CO₂ molecule [21].

Ethanol synthesis is dependent on two key enzymes, namely, coenzyme A-acylating aldehyde dehydrogenase (CoA-AAD) and NAD(P)H alcohol dehydrogenase [NAD(P)H-ADH]. CoA-AAD is also an important enzyme (enzyme no. 13 in the biosynthetic pathway) that diverts acetyl-CoA towards ethanol synthesis during the fermentation process of C. acetobutylicum American Type Culture Collection (ATCC) 824. NAD(P)H-ADH then converts the acetaldehyde (generated through CoA-AAD action) to ethanol through a reduction process using the reducing power of NAD(P)H [21].

Bioethanol can also be produced through ethanolic fermentation performed by Saccharomyces cerevisiae [24]. Biomethanol, on the other hand, is created through a thermochemical or biochemical pathway. The biochemical path for methanol production involves the anaerobic degradation of biowastes to produce biogas, which involves four stages: hydrolysis, acetogenesis, acidogenesis, and methanogenesis. The microorganisms involved in methanol production are methanotrophic bacteria, including Methylococcus capsulatus and Methylobacterium extorquens [25].

Fermentative biohydrogen production involves the use of anaerobic bacteria, such as Clostridium paraputrificum, C. butyricum, C. beijerinckii [26], and Ruminococcus albus [27], or facultative anaerobic bacteria such as Escherichia coli [28], Enterobacter cloacae [29], and Citrobacter freundii [30]. These bacteria can degrade organic compounds, especially carbohydrates, via an anaerobic fermentative pathway. Fermentation produces hydrogen gas and other byproducts such as acetic acid, butyric acid, and ethanol. As there is an incomplete breakdown of sugar molecules, the H₂ yielded by this process is lower than that produced by photofermentative biohydrogen using phototrophic bacteria, such as the purple bacteria [31]. The overall reaction can be presented as:

(1)$${\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6⟶\mathrm{Y}+{\mathrm{xH}}_2+2\mathrm{C}{\mathrm{O}}_2$$

meanwhile, when Y = acetic acid, the value of x = 4, i.e., 4 moles of H₂ is produced from 1 mole of glucose, whereas when Y = butyric acid or ethanol, then x = 2, i.e., 2 moles of H₂ is produced from 1 mole of glucose [32].

Fermentative biohydrogen production by Clostridium occurs when a proton is the final electron acceptor, forming hydrogen [33]. H₂ production from protons is dependent on the activity of two types of hydrogenases: (i) pyruvate: ferredoxin oxidoreductase, which transfers electrons from reduced ferredoxin to protons and reduces these protons to H₂; (ii) NADH: ferredoxin oxidoreductase, which uses NADH as the electron donor to reduce H⁺ to H₂ [34].

(i) Direct biophotolysis: Photosynthetic biohydrogen production involves using photosynthetic microorganisms such as green algae and cyanobacteria. These microorganisms use light energy to split water into oxygen and hydrogen gas, known as photolysis. The overall reaction can be presented as:

(2)$${\mathrm{H}}_2\mathrm{O}+\text{light}⟶2{\mathrm{H}}_2+{\mathrm{O}}_2$$

in this reaction, water is split into two molecules of hydrogen gas and one molecule of oxygen using light energy. The above reaction scheme (2) can be further subdivided into two steps:

(3)$${\mathrm{H}}_2\mathrm{O}+2\mathrm{F}{\mathrm{d}}_{\mathrm{ox}}⟶2{\mathrm{H}}^{+}+\mathrm{½}{\mathrm{O}}_2+2\mathrm{F}{\mathrm{d}}_{\mathrm{re}}$$

(4)$$2{\mathrm{H}}^{+}+2\mathrm{F}{\mathrm{d}}_{\mathrm{red}}⟷{\mathrm{H}}_2+2\mathrm{F}{\mathrm{d}}_{\mathrm{ox}}$$

the reaction (3) occurs in all oxygenic phototrophs, but the second reaction (4) requires anaerobic or microaerobic conditions. The bidirectional hydrogenases catalyze the reaction (4), i.e., the H₂ production step. There are two types of bidirectional hydrogenases: [Fe–Fe] enzymes, found in algae, and [Ni–Fe]-hydrogenases, found in cyanobacteria [14, 35].

(ii) Indirect biophotolysis: This method uses cyanobacteria and microalgae to produce hydrogen from stored carbohydrates in two steps. In the first step, carbohydrates (such as glycogen and starch) are synthesized in the presence of light. In the second step, these carbohydrates are utilized to produce hydrogen through photofermentation [14].

(5)$${\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6+6{\mathrm{H}}_2\mathrm{O}⟶6\mathrm{C}{\mathrm{O}}_2+12{\mathrm{H}}_2$$

H₂ production is a byproduct of the nitrogen fixation process in the indirect biophotolysis process, and the nitrogenase system represents the enzyme involved [14]. This process is also sensitive to oxygen, as the nitrogenase is deactivated in the presence of O₂. Therefore, H₂ production through this process occurs in specialized cells, such as heterocysts (found in filamentous cyanobacteria such as Nostoc), which lack photosystem II (PS-II) and do not perform the water-splitting reaction that evolves O₂. Alternatively, some phototrophs (such as Cyanothece) perform photosynthesis during the daytime and H₂ production at night [36]. At night, high respiration rates result in a microaerobic intracellular environment that allows N₂ fixation and simultaneous H₂ production by the nitrogenase system [37]. The rate of H₂ production is highest in the absence of nitrogen when nitrogenase exclusively catalyzes the reduction of H⁺ to H₂ [31].

Purple phototrophic bacteria (PPB) mainly produce biohydrogen through anoxygenic photosynthesis. In place of water, these bacteria use hydrogen sulfide as a source of electrons or reducing agents, releasing sulfur as a byproduct rather than oxygen [38]. Purple phototrophic bacteria use light in the near-infrared region ($>$800 nm) for anoxygenic photosynthesis. Hydrogen production by these bacteria is catalyzed by nitrogenases [38]. PPB has demonstrated that it can successfully utilize various organic acids, such as formic, acetic, and butyric acids, to produce H₂ efficiently [39]. Purple bacteria have some other significant useful applications, such as water purification, animal nutrition, biofertilizers, and bioremediation of wastes [31].

(6)$${\mathrm{CH}}_3\mathrm{COOH}+2{\mathrm{H}}_2\mathrm{O}\stackrel{\text{Nitrogenase}}{\to }{\mathrm{H}}_2+{\mathrm{CO}}_2$$

(7)$${\mathrm{C}}_4{\mathrm{H}}_8{\mathrm{O}}_2+6{\mathrm{H}}_2\mathrm{O}\stackrel{\text{Nitrogenase}}{\to }10{\mathrm{H}}_2+4\mathrm{C}{\mathrm{O}}_2$$

Endoglucanases, also called carboxymethyl cellulase (CMCase), cleave internal bonds within the amorphous regions of cellulose, creating nicks that ultimately convert lignocelluloses into simple sugars. These enzymes are valued for their low energy requirements and high specificity, making them cost-effective [64]. Commercial endoglucanase production typically involves fungal strains of Aspergillus and Trichoderma and bacterial strains such as Streptomyces sp. and Bacillus sp. [65]. Endoglucanases operate through two primary mechanisms—retention and inversion—to hydrolyze cellulose [63].

Exoglucanases, also known as cellobiohydrolases (CBHs), work in concert with endoglucanases and $\beta$-glucosidase to break down cellulose. As an exocellulase enzyme, CBH targets the 1,4-$\beta$-D-glycosidic bonds at the ends of cellulose chains. There are two types of cellobiohydrolases: CBH1 and CBH2. CBH1 acts on the reducing end of cellulose, while CBH2 targets the non-reducing end, contributing to cellobiose production [64]. The hydrolysis of cellulose to cellobiose is a key step in limiting the overall cellulose degradation rate. This process relies on the combined action of endoglucanase and cellobiohydrolase (exo–endo synergism), as well as the cooperation between CBH1 and CBH2 (exo–exo synergism), to efficiently remove cellobiosyl residues from both ends of the cellulose chain. Commercially, CBH is produced by various fungal species, including Trichoderma reesei and Penicillium sp. [66], and bacterial strains belonging to Flavobacterium sp. and Paenibacillus sp. CBH enhances the efficiency of cellulose degradation by increasing substrate proximity to glycosyl hydrolases, improving accessibility, and modifying the cellulose surface crystals [66].

The final and crucial step in converting cellulose into simpler sugars is conducted by $\beta$-glucosidases (BGLs). BGL completes the saccharification process by converting cellobiose, generated by exoglucanase, into glucose [67]. Exoglucanase is essential for maintaining the overall rate of cellulase hydrolysis, as cellobiose can inhibit both exoglucanase and endoglucanase. However, BGL can be inhibited by glucose, making its efficiency a critical factor [68]. Commercial production of BGL is often achieved using microorganisms such as Aspergillus sp., Penicillium sp., and certain Bacillus species [63]. Therefore, increasing BGL production is essential to meet the high demands of the biofuel industry. Immobilizing BGL has been shown to enhance the hydrolysis rate [63], which could reduce production costs and streamline the biofuel production process.

Hemicellulose is a key structural polysaccharide found in the cell walls of plants. Hemicellulose is a branched, heteropolymeric carbohydrate composed of a mix of hexose (six-carbon) and pentose (five-carbon) sugars [69]. Hemicellulose is critical in the plant cell wall, providing structural support and contributing to the flexibility and strength of the wall. Hemicellulose also forms a matrix around cellulose fibers and interacts with lignin, another major cell wall component, to stabilize the cell wall structure. This interaction is crucial for maintaining cell wall integrity and overall plant rigidity. The breakdown of hemicellulose is performed by a group of enzymes known as hemicellulases. Xylanases are crucial in enhancing the saccharification of lignocellulosic waste, including hemicelluloses such as xylan. By breaking down these complex polysaccharides, xylanases increase the availability of sugars for fermentation, thereby boosting biofuel production [45]. Both bacterial and fungal species have been employed for xylanase production, including B. subtilis, B. amyloliquefaciens, B. stearothermophilus, Clostridium thermocellum, Streptomyces sp., Aspergillus spp., and Trichoderma spp. [45].

Lignin is a complex organic polymer found in plant cell walls, particularly in wood and bark. It is one of the most abundant natural polymers on Earth, second only to cellulose. Lignin is crucial in providing structural support to plants by reinforcing the cell walls, making them rigid and resistant to decay [70]. This polymer is highly cross-linked, which provides its characteristic hardness and insolubility in water.

Laccase is a multicopper oxidase enzyme that is crucial in lignin degradation. Laccase catalyzes the oxidation of phenolic substrates, leading to the breakdown of lignin into smaller, more manageable components. Laccase is produced by various organisms, including fungi, bacteria, and plants, but it is most commonly associated with white-rot fungi, which are highly efficient lignin degraders [71]. The lignin degradation mechanism employed by laccase involves oxidizing phenolic hydroxyl groups in lignin, generating phenoxy radicals. These radicals can undergo further reactions, including depolymerization, leading to the breakdown of the complex lignin structure into smaller aromatic compounds [46]. Laccase can also act on non-phenolic lignin structures when combined with mediators, which are small molecules that extend the range of substrates that laccase can oxidize. This mediator system enhances the efficiency of lignin degradation, making laccase a powerful tool in biotechnological applications aimed at lignin valorization. In addition to its role in natural lignin degradation, laccase has gained significant attention for its potential in producing biofuels from lignocellulosic biomass [72].

Pectin is a naturally occurring polysaccharide found in the cell walls of plants, particularly in the primary cell walls and middle lamellae. Pectin plays a crucial role in maintaining the structural integrity of plant tissues by acting as a cementing substance that binds cells together. Pectin is composed mainly of galacturonic acid units, which are linked together to form a linear chain, though it can also include various side chains of other sugars [73].

Pectinase plays a crucial role in pectin degradation by cleaving the glycosidic bonds within the pectin molecules. This leads to the depolymerization and solubilization of pectin into simpler sugars such as galacturonic acid. Pectinase is produced by various microorganisms, including bacteria, fungi, and yeasts, as well as by some plants [74]. In biofuel production, pectinases contribute to the breakdown of plant cell walls, facilitating biomass conversion into bioethanol. Specifically, in biomass with lower lignin content, pectinases aid in the degradation of plant materials, enhancing the efficiency of enzymatic conversion processes [47, 75].

Protease enzymes play an important role in the degradation of biomass used to produce biofuels. Indeed, protease enzymes degrade proteins in plant and microbial biomass. While the primary focus in biofuel production often centers on the breakdown of carbohydrates, such as cellulose and hemicellulose, the degradation of proteins is also crucial, particularly in processes involving complex biomass such as algae, which contain significant protein content [76].

In the context of biofuel production, proteases contribute to the overall efficiency of biomass conversion by hydrolyzing proteins into smaller peptides and amino acids. This process not only helps reduce the protein content in the biomass, thereby increasing the relative concentration of fermentable sugars, but also releases nitrogen, which can serve as a nutrient for microbial communities involved in subsequent fermentation processes [76]. The breakdown of proteins by proteases can improve the accessibility of other enzymes, such as cellulases and hemicellulases, to the carbohydrate components in the biomass, thereby enhancing the overall yield of biofuels such as bioethanol and biogas. This leads to a more efficient conversion process, ultimately improving the cost-effectiveness and sustainability of biofuel production.