A Review of Liquid and Gaseous Biofuels from Advanced Microbial Fermentation Processes

Abstract

Biofuels are the sustainable counterparts of fossil fuels to meet the increasing energy demands of the current and future generations. Biofuels are produced from waste organic residues with the application of mechanical, thermochemical and biological methods and processes. While mechanical and thermochemical conversion processes involve the use of heat, pressure, catalysts and other physicochemical attributes for the direct conversion of biomass, biological conversion requires microorganisms and their enzymes as biocatalysts to degrade the fermentable substrates into biofuels and biochemicals. This article highlights the advances and opportunities in biological conversion technologies for the development of a closed-loop biorefinery approach. This review highlights the distinction between biological and thermochemical conversion technologies, including a discussion on the pros and cons of the pathways. Different categories of biological conversion processes, such as enzymatic saccharification, submerged fermentation, solid-state fermentation and simultaneous saccharification and fermentation are also discussed in this article. The main essence of this article is the description of different fermentative technologies to produce next-generation biofuels, such as bioethanol, biobutanol, biomethane, biohydrogen and biodiesel. This article provides a state-of-the-art review of the literature and a technical perspective on the bioproduction of bioethanol, acetone–ethanol–butanol fermentation, anaerobic digestion, photo/dark fermentation, and the transesterification of lignocellulosic substrates to produce the above-mentioned biofuels. In addition, recommendations for improving bioprocessing efficiency and biofuel yields are provided in this comprehensive article.

1. Introduction

The increasing energy demand coupled with concerns over climate change and environmental sustainability has fueled the urgent need for renewable fuels [1]. The current worldwide energy matrix today is largely composed of crude oil (31.6%), coal (26.7%), natural gas (23.5%), hydropower (6.7%), nuclear energy (4%), wind energy (3.3%), solar energy (2.1%), biofuels (0.7%) and other renewables (1.4%) (Figure 1) [2]. Fossil fuel combustion releases CO₂ and other greenhouse gases into the atmosphere, which leads to the accumulation of heat-trapping gases and global warming. The fractions of greenhouse gas emissions worldwide vary with CO₂ (76%), CH₄ (16%), N₂O (6%) and fluorinated gases (2%) [3]. Furthermore, the depletion of fossil fuel reserves necessitates the exploration of alternative energy sources. Fossil fuels are finite resources, and as concerns for climate change and global warming intensify, their extraction and use are becoming less endurable. The current atmospheric level of CO₂ is 418 ppm, which is dramatically higher compared to that in 1960 (316 ppm) [4].

Biofuels, on the other hand, can help mitigate these emissions since the carbon released during their combustion is part of a closed carbon cycle, as the biomasses used for their production absorb CO₂ during its growth [5,6]. Biofuels are derived from renewable biomass sources and offer a viable alternative to finite fossil fuels, leading to carbon emissions that are recycled by fresh plants during photosynthesis or are at levels much lower than those that cause environmental deterioration [7]. On the contrary, biofuels are produced from abundant and low-cost bioresources, such as agricultural residues, energy crops, woody biomass and organic fractions of municipal solid waste rich in renewable and replenishable carbon [8].

The conversion of biomass into biofuels can be achieved through mechanical, thermochemical and biological conversion processes. Mechanical processes involve mostly pressing and centrifugation to extract bioactive components, including biofuels from biomass, such as oilseed crops and algae [9]. Thermochemical conversion employs processes such as pyrolysis, liquefaction and gasification to convert biomass into selective solid, liquid and gaseous biofuels utilizing high temperatures, pressures and catalysts. Pyrolysis involves the heating of biomass in the absence of oxygen to produce bio-oil and biochar, which can be further processed into transportation fuels [10]. Gasification converts biomass into synthesis gas, which can be used to produce a variety of biofuels, including hydrogen and synthetic liquid fuels [11]. Liquefaction uses catalysts and solvents to hydrothermally crack organic compounds in biomasses to produce bio-crude oil with fuel properties comparable to liquid transportation fuels [12].

Biological conversion involves the use of enzymes or microorganisms to break down biomass components, such as cellulose and hemicellulose, into monomeric pentose and hexose sugars. These sugars can be fermented to produce value-added biofuels and biochemical precursors, such as bioethanol, biobutanol, biohydrogen, biomethane, acetic acid, butyric acid, lactic acid, etc., through fermentation and anaerobic digestion [13]. Liquid feedstocks, such as industrial organic effluents, landfill leachate and crude glycerol from biodiesel industries can also be pretreated and fermented to biofuels and biochemicals using suitable microorganisms [14]. Although thermochemical conversion offers higher energy efficiency and the potential to convert a broader range of biomass feedstocks in a short time, biological conversion possesses several unique advantages.

Bioprocessing is widely preferred due to its flexibility to utilize biomass containing high moisture, low infrastructure requirement, compatibility for integration with mechanical and thermochemical biorefinery infrastructures, as well as wastewater or sewage treatment facilities for a circular conversion approach and the requirement of moderate processing conditions. This approach can leverage, upgrade, or retrofit existing infrastructures, thus reducing the need for substantial new investments. Biological conversion techniques are more energy-efficient and environmentally friendly compared to thermochemical and mechanical conversion techniques [15]. Lower energy inputs also translate into potentially lower operational costs and a smaller carbon footprint. Biological conversion utilizes biocatalysts, such as enzymes and microorganisms, which can perform catabolic and metabolic reactions under mild conditions [16]. This feature eliminates the need for harsh chemicals and extreme temperatures, reducing the likelihood of unwanted side reactions or the formation of harmful byproducts. The mild reaction conditions also offer greater flexibility in the process design and allow for the utilization of diverse feedstocks.

Biological conversion processes can exhibit high specificity and selectivity in terms of product formation due to the variety of metabolic pathways of microorganisms [17]. Bioprospecting microorganisms and enzymes can be further genetically engineered to demonstrate specific metabolic pathways, leading to exhibiting desired traits, such as maximum substrate utilization, tolerance to substrates, products and inhibitors and the enhanced production of targeted biochemicals and bioproducts [18,19]. This level of control is advantageous for industries requiring pure or specialized compounds, such as nutraceuticals, cosmeceuticals, pharmaceuticals, or platform chemicals. The products generated through bioconversion routes are also biodegradable and have a lower environmental impact compared to thermochemical conversion products, thus minimizing pollution risks and contributing to a more sustainable and circular approach. Besides the main product, certain byproducts generated from bioconversion routes, such as organic acids, proteins, fatty acids, etc. can generate additional revenue streams and enhance the overall economic viability of the process [20].

Despite the many benefits of bioconversion processes, significant knowledge gaps have also been identified that prevent their large-scale commercialization to produce biofuels and biochemicals. The main objective of this article is to highlight the knowledge gaps as well as address measures to counter them. For example, bioconversion processes, such as fermentation and anaerobic digestion, suffer due to the slow biodegradation of substrates, inhibitors and contaminants that limit microbial growth, leading to lower product yields and a high cost of hydrolytic enzymes [21]. The recalcitrance of lignocellulosic biomass also possesses complexities for the removal of lignin and the recovery of hemicellulose and cellulose for fermentation [22]. This requires a variety of pretreatments involving acids, alkalis, solvents, ionic liquids, ozone, liquid ammonia, ultrasonication, enzymes and supercritical fluids, which adds to the overall operating costs of bioprocesses [23].

Biological conversion processes must also be investigated beyond laboratory conditions to demonstrate their potential for on-site application. Microorganisms and enzymes are typically investigated for biorefining processes under controlled laboratory-scale biosystems with selective process variables and regular monitoring by personnel and/or probes. The knowledge generated from these lab-scale biosystems could deviate slightly or substantially upon scaling up these bioprocesses on-site due to a variety of external variables and uncontrolled environmental conditions. Hence, it is essential to acknowledge and evaluate these possible risks before scaling up the bioconversion process for biofuel and biochemical production. This review aims to shed light on the prospects and challenges of different types of bioconversion processes, as well as the next generation of biofuel products that could be produced from waste biomass using advanced microorganisms and enzymes.

2. Classification of Bioconversion Pathways

Bioconversion pathways can be broadly classified into enzymatic hydrolysis, submerged fermentation and solid-state fermentation (Figure 2). Enzymatic saccharification is a key pretreatment step to produce sugars from lignocellulosic biomasses for liquid biofuel production. In enzymatic saccharification, the complex carbohydrates (i.e., polysaccharides) present in the lignocellulosic materials are broken down into monosaccharide sugars for fermentation into biofuels and biochemicals [24]. Enzymatic saccharification is typically performed by using cellulases, hemicellulases (e.g., arabinofuranosidases, esterases, glucuronidases, mannanases, xylanases and xylosidases) and lignin-modifying enzymes to denature cellulose, hemicellulose and lignin frameworks in the biomass [25,26,27]. Compared to other pretreatment methods, one major advantage of enzymatic saccharification is that it can be conducted under relatively mild conditions since enzymes can be deactivated at high temperatures. This reduces energy consumption and the production of unwanted byproducts [28]. The cost and recycling of enzymes remain a major challenge in the commercial-scale production of biofuels through enzymatic saccharification [29]. Efforts are underway to develop more efficient and cost-effective enzyme formulations and to optimize the process parameters to reduce the operating cost and increase efficiency.

Tang et al. [30] found that pretreating rice straw with ethylene glycol and ferric chloride under 1 MPa CO₂ reached 98% cellulose recovery and removed 92% lignin and 90% hemicellulose. Adsul et al. [31] proposed the strategy of developing a cellulolytic enzyme cocktail to improve efficiency and reduce the cost of the conversion of lignocellulosic biomass into biofuels. This study suggests that identifying novel accessory enzymes through metagenomic, proteomic, or microbial bioprospecting approaches can be helpful for scaled-up production and reducing operating costs.

Submerged fermentation is a process that involves the growth of microorganisms in a liquid medium in closed vessels [32]. Submerged fermentation has been widely used as a method for producing biofuels, such as bioethanol and biobutanol from lignocellulosic biomass. During submerged fermentation, microorganisms, such as yeast or bacteria, are inoculated into a liquid medium containing the pentose and hexose sugars extracted from the pretreatment of lignocellulosic biomass [33]. An advantage of the submerged fermentation process is that it can be carried out using a wide range of biomass feedstocks and microorganisms. Additionally, it can be carried out at ambient temperatures and under aerobic or anaerobic conditions [34]. However, the high energy consumption required to heat large volumes of liquid fermentation medium and the need to carefully control the pH and nutrient levels in the medium to optimize the growth of the microorganisms are some key considerations [35,36].

Submerged fermentation is a promising method for producing biofuels and other high-value biochemicals, like phenolics [37], vanillin [38] and pigments [39]. Moreover, research efforts are underway to optimize the process parameters and develop new microorganisms and substrates to increase the fermentation efficiency and reduce operating costs [40]. Additionally, efforts are being made to integrate submerged fermentation with other processes, such as enzymatic saccharification, to further increase the efficiency of biofuel production and maximize substrate utilization and byproduct formation.

Solid-state fermentation is an important methodology of biomolecule manufacturing that involves the cultivation of microorganisms using a solid substrate without the presence or under the near absence of free water [41]. During solid-state fermentation, microorganisms, such as fungi and bacteria, are inoculated onto a solid biomass-based substrate. Subsequently, the microorganisms grow and produce enzymes that break down the complex polysaccharides in the biomass into simple sugars. These simple sugars can then be fermented into biofuels such as bioethanol [42]. Compared to other biomass pretreatments, one of the advantages of solid-state fermentation is that it can be carried out using inexpensive and readily available feedstocks [43]. Additionally, it can produce high yields of biofuels and can be carried out at relatively low temperatures, which requires less energy input. Solid-state fermentation could be integrated into a multistep process to produce biofuels and biochemicals. However, some challenges need to be tackled, including the difficulty of controlling the moisture content and temperature of the substrate, which can affect the growth and activity of the microorganisms [44].

3. Fermentation-Derived Biofuels

3.1. Bioethanol

Bioethanol is a liquid biofuel constituted of a significantly higher oxygen content of 35%, which can decrease vehicular emissions. Bioethanol is often blended with gasoline at flexible propositions for use as a drop-in biofuel in existing motor engines [45]. The use of bioethanol in the replacement of conventional fuels could boost the performance and economy of vehicular engines due to its low carbon footprint. Meanwhile, due to its poor volumetric energy density compared to regular gasoline, the vehicles need more volume of bioethanol, unlike conventional fuels. To overcome this problem, bioethanol can be used as a blending component in conventional liquid fuel. Bioethanol is produced from various lignocellulosic and sugar-based feedstocks like agricultural residues (e.g., straw, bran and stalk), food-processing wastes and different energy crops using pretreatment, enzymatic hydrolysis and fermentation.

Baker’s yeast (Saccharomyces cerevisiae) is a model microorganism that can ferment starch and hexose sugars (i.e., glucose) to produce bioethanol through the simple glycolytic pathway. It should be noted that first-generation biomasses, such as corn, potato and cassava, are rich in starch and/or glucose, which are the preferred feedstocks for biorefineries for fermentation to produce bioethanol because the diversion of these food crops for fuel products instead of consumption led to food-versus-fuel criticism [46]. On the contrary, second-generation or lignocellulosic biomass, such as agricultural and wood-based residues, have emerged as alternative feedstocks for bioethanol and other biofuel production [47]. However, lignocellulosic biomass poses several upstream challenges for fermentation due to the presence of lignin, which hinders the access of enzymes and microorganisms to directly biodegrade holocellulose (cellulose and hemicellulose) sugars. Although the pretreatment of biomass could separate lignin from holocellulose, it may result in the generation of certain degradation products, such as carboxylic acids, phenols and furfurals, which reduce the pH of the hydrolysate and also inhibit the growth and activity of fermenting microorganisms [48]. In addition, S. cerevisiae lacks the natural metabolic pathway to ferment pentose sugars (monomers of hemicellulose), thus impacting the fermentation of lignocellulosic biomass to bioethanol [49]. Nonetheless, continued research and development in biomass pretreatment processes, bioprocess engineering and genetic engineering of microorganisms have led to significant progress in the fermentation of lignocellulosic materials to bioethanol. One such approach is the use of thermophilic bacteria for bioethanol production. Thermophilic bacteria are extremophilic microorganisms that can proliferate under extreme temperatures, pressure, osmosis, salinity and radiation conditions [50]. Thermophilic microorganisms harbor unique enzymes that make them thermally and physiologically robust, with an array of advantages for bioprocessing, such as stability against contamination and inhibitors, enhanced reaction kinetics, improved product yields and accelerated organic matter degradation [51,52,53].

Figure 3 represents the typical glycolytic fermentation pathway to produce ethanol using glucose as a simple sugar. S. cerevisiae deploys the Embden–Meyerhoff–Parnas glycolytic pathway, whereas Zymomonas mobilis uses the Entner–Doudoroff pathway for glucose metabolism [54]. The Entner–Doudoroff pathway is mainly an aerobic route for glucose metabolism and is widely found in Pseudomonas spp. The Entner–Doudoroff pathway theoretically yields 2 moles of adenosine triphosphate (ATP) per mole of glucose fermented to ethanol. On the other hand, the Entner–Doudoroff pathway releases 1 mol/mol of ATP, which also results in low cell mass and allows higher ethanol yields [54].

There have been several studies on the fermentation of sugars to produce bioethanol in the last few decades, a few of which are presented in

Table 1. Summary of significant studies on bioethanol production from waste biomass.

Biomass	Pretreatment	Process Parameters	Ethanol Yield (g/g)	Reference
Barley straw	• Decompression pretreatment • Temperature: 125–175 °C • N2 pressure: 3 MPa	• Hydrolysis (Accellerase 1500, 30 FPU/g cellulase, temperature: 50 °C, agitation: 250 rpm, time: 72 h) • Fermentation: Saccharomyces cerevisiae (1 g per 200 mL, temperature: 22 °C, time: 7 days)	0.43–0.9	Raud et al. [55]
Mesquite stem (Prosopis juliflora)	• Mild acid pretreatment • 3 vol/vol% HNO3 • Temperature: 121 °C • Biomass-to-liquid ratio: 1:10 • Pressure: 0.01 MPa	• Simultaneous saccharification and fermentation (commercial cellulases 12 FPU/g of biomass, Kluyveromyces marxianus MTCC 1389, inoculum concentration: 3 vol/vol%, pH 4.9, temperature: 41 °C, substrate concentration: 5 wt/vol%, time: 72 h)	0.67	Sivarathnakumar et al. [56]
Palm kernel cake	• Steam explosion • Pressure: 0.45 MPa • Reaction time: 15 min	• Hydrolysis (SEB mannanase 17.9 U/g mannan, CTec2 cellulase 10.4 FPU/g glucan, temperature: 50 °C, time: 72 h, agitation: 250 rpm) • Fermentation (Geobacillus thermoglucosidasius TM242, temperature: 60 °C, time: 48 h, agitation: 250 rpm)	0.47	Raita et al. [57]
Rice straw	• Microwave-assisted NaOH pretreatment • Temperature: 100–140 °C • Reaction time: 1–2 min	• Hydrolysis (cellulolytic enzymes produced from Trichoderma reesei NCIM-1052: 20 FPU/g of biomass, time: 64 h), pH 4.8, temperature: 55 °C, fermentation time: 72 h, agitation: 150 rpm) • Fermentation (Pichia stipites NCIM 3499 and Saccharomyces cerevisiae 3186, temperature: 30 °C, time: 74 h, pH 4.5)	0.44	Prasad et al. [58]
Sorghum stalk	• Mild acid pretreatment (H2SO4)	• Fermentation (Thermoanaerobacterium aotearoense SCUT27/ΔargR1864, total sugar: 15 g/L, temperature: 55 °C, agitation: 150 rpm)	0.36	Qu et al. [59]
Soybean straw	• Mild acid pretreatment • 0.1 M H2SO4 • Temperature: 121 °C • Reaction time: 30 min	• Fermentation (Thermoanaerobacterium aotearoense SCUT27/ΔargR1864, total sugar: 15 g/L, temperature: 55 °C, agitation: 150 rpm)	0.34	Qu et al. [59]
Starch-based food waste	• Mild acid pretreatment (H2SO4) • Feedstock concentration: 30 wt/vol%	• Simultaneous saccharification and fermentation (enzyme was produced inside the reactor using Saccharomyces cerevisiae and Fusarium oxysporum, inoculum-to-feedstock: 1:10, pH 6, temperature: 30 °C, time: 94 h, agitation: 80 rpm)	0.1	Prasoulas et al. [60]

Note: Filter paper unit (FPU). The units of bioethanol yields for a few studies have been calculated for presentation in g/g to maintain uniformity.

. In a study by Raud et al. [55], barley straw was used to produce bioethanol using a three-step process, which included liquid hot water pretreatment at 125–175 °C with an external N₂ pressure of 3 MPa, followed by hydrolysis and fermentation. The hydrolysis of pretreated feedstock was performed using a commercial biocatalyst or enzyme (Accellerase 1500) at 50 °C for 72 h. The enzymatic hydrolysis was followed by fermentation using the conventional fungi (S. cerevisiae) at 22 °C for 7 days, which resulted in a bioethanol yield of 0.43–0.9 g/g. The higher pretreatment temperature resulted in enhanced degradation of non-cellulosic moieties, which subsequently increased the availability of cellulose for enzymatic hydrolysis and fermentation. Barley straw required pretreatment for the decomposition of non-cellulosic components and loosening of the strong intermolecular or intramolecular linkages in the lignocellulosic matrix for the enhanced recovery of fermentable sugars. However, food processing wastes comprised of fermentable sugars that could be directly fermented into bioethanol without any intensive pretreatment.

Khoshkho et al. [61] used waste carrot pulp for fermentation using S. cerevisiae at 28 °C in 72 h to produce a bioethanol concentration and yield of 40.6 g/L and 0.57 g/g, respectively. The enhanced production of bioethanol was attributed to the use of beet molasses during the fermentation process, which significantly activated the microbial community due to the presence of pre-fed sugars through molasses. In addition to the two-step fermentation process, consolidated bioprocessing has become more economically feasible for bioethanol fermentation using genetically engineered thermophilic bacteria, such as Thermoanaerobacterium saccharolyticum [62].

In a study by Qu et al. [59], soybean straw and sorghum stalk were used as feedstocks for fermentation after mild acid pretreatment. The fermentation process was performed in an anaerobic environment with an engineered thermophilic bacterium Thermoanaerobacterium aotearoense SCUT27/ΔargR₁₈₆₄ at 55 °C, which provided the highest bioethanol yield of 0.34 and 0.36 g/g from the soybean straw and sorghum stalk, respectively.

Raita et al. [57] used de-oiled palm kernel cake for a three-step conversion into bioethanol. The de-oiled cake was pretreated using a steam explosion technique followed by enzymatic hydrolysis using a bi-enzyme system containing SEB mannanase and CTec2 cellulase at 50 °C for 72 h. The fermentation of hydrolyzed feedstock was performed using a thermophilic bacterium strain Geobacillus thermoglucosidasius TM242, which produced a significantly high yield of bioethanol (0.47 g/g). Unlike mesophilic microbial systems, thermophilic anaerobic bacteria have the advantage of higher bioethanol yield due to the significant reduction of the oxidation reaction and utilization of a wide range of hexose and pentose sugars [63].

Sivarathnakumar et al. [56] utilized mesquite stem as a lignocellulosic feedstock to produce bioethanol, where the biomass was treated with mild nitric acid before saccharification and fermentation processes. The simultaneous saccharification and fermentation process was performed in a single-compartment fermenter in the presence of the commercially available cellulase (activity of 12 FPU/g of biomass) and Kluyveromyces marxianus MTCC 1389 as the fermentative bacteria. The entire saccharification and fermentation were performed at 41 °C and pH 4.9 in 72 h to obtain a bioethanol concentration and yield of 21.5 g/L and 0.67 g/g, respectively.

Tse et al. [64] investigated bioethanol production from various feedstocks and reported that barley straw had a maximum bioethanol yield of 1.14 L/kg compared to that of corn stover, sugarcane bagasse and wheat straw, which revealed a bioethanol yield in the range of 0.4–0.6 L/kg. A bioethanol yield of 1.5 L/kg has also been reported from brown macroalgae [64].

3.2. Biobutanol

Butanol is categorized into four active isomers, 1-butanol, 2-butanol, iso-butanol and tert-butanol. The physicochemical properties of biobutanol suggest it is a superior fuel additive due to its higher calorific value of 29 MJ/L as compared to bioethanol (19.6 MJ/L) [65]. Compared to bioethanol, biobutanol has lower volatility, higher miscibility in gasoline and considerably fewer issues during ignition in vehicle engines [18]. Due to these superior physicochemical properties, biobutanol can be used as an additive in various fossil fuels, like gasoline and diesel, or as a drop-in biofuel without any modification of vehicular engines. In addition to fuel applications, butanol has several applications as a routine laboratory solvent in the chemical, polymer, textiles, paints and cosmetic industries. Biobutanol is biologically produced using one of the oldest fermentation processes, namely acetone–butanol–ethanol (ABE) fermentation using different species of the anaerobic bacteria Clostridium.

The metabolic pathway of ABE fermentation is presented in Figure 4. The production of butanol is regulated by several enzymes, namely acetyl-CoA, acetoacetyl-CoA, 3-hydroxybutyryl-CoA, crotonyl-CoA and butyryl CoA [66]. Clostridium metabolizes glucose to produce pyruvate, which is converted into acetyl-CoA by the tricarboxylic acid cycle. Acetyl-CoA is further converted into acetic acid and butyryl-CoA via acetoacetyl-CoA and 3-hydroxybutyryl-CoA. Butyryl-CoA is metabolized to butyric acid, butyraldehyde and butanol. Acetic acid, butyric acid, acetone, ethanol, H₂ and CO₂ are also obtained as byproducts of ABE fermentation along with butanol. The typical yield ratio of acetone, butanol and ethanol from ABE fermentation orchestrated by Clostridium spp. is 3:6:1 [67]. Recent research efforts have reported developing efficient bioprocesses and microorganisms that can co-utilize the byproducts, such as lactic acid and acetic acid, to produce biobutanol [68]. A few notable studies on ABE fermentation pathways are presented in

Table 2. Summary of significant studies on biobutanol production from waste biomass.

Biomass	Pretreatment	Process Parameters	Biobutanol Concentration, Yield and Productivity	Reference
Cellulose	-	• Clostridium cellulovorans (overexpressing adhE2)	• Concentration: 1.42 g/L, yield: 0.195 g/g, productivity: 5.9 mg/L/h	Yang et al. [69]
Chicken feather and wheat straw	• Alkaline pretreatment • 0.6% NaOH • Temperature: 80 °C • Time: 20 h • Agitation: 130 rpm	• Enzyme: CellicCtec2 enzyme (temperature: 50 °C, pH: 6.3, time: 20 h, agitation: 130 rpm) • Fermentation: Clostridium beijerinckii strain NCIMB 8052 (temperature: 37 °C, time: 48 h)	• Concentration: 4.6 g/L, yield: 0.054 g/g	Branska et al. [70]
Corn cob	• Alkali pretreatment	• Clostridium cellulovorans DSM 743B and Clostridium beijerinckii NCIMB 8052	• Concentration: 11.8 g/L, yield: 0.14 g/g, productivity: 5.9 mg/L/h	Wen et al. [71]
Corn stalk	• Ethanol-assisted alkali pretreatment • 4% NaOH • 60 vol/vol% ethanol • Temperature: 110 °C • Time: 90 min	• Enzymes: Cellulase and xylanase (temperature: 50 °C, agitation: 150 rpm) • Fermentation: Clostridium beijerinckii NCIMB 4110	• Concentration: 12.8 g/L, yield: 0.43 g/g, productivity: 0.18 g/L/h	Tang et al. [72]
Glucose	-	• Fed-batch fermentation (Clostridium acetobutylicum JB200 in 78 h)	• Concentration: 19.1 g/L, yield: 0.21 g/g, productivity: 0.24 g/L/h	Xue et al. [73]
Rice bran and sesame oil cake	• Autoclaved at 121 °C for 20 min	• Clostridium sp. BOH3	• Concentration: 13.5 g/L, yield: 0.1 g/g	Rajagopalan et al. [74]
Rice straw	• H2O2 (0.2 wt/vol%) assisted NaOH (1.5%) pretreatment	• Enzyme (Accellerase 1500) • Fermentation (polyvinyl alcohol-immobilized Clostridium acetobutylicum ATCC 824)	• Concentration: 13.8 g/L, yield: 0.23 g/g, productivity: 0.9 g/L/h	Tsai et al. [75]
Sweet sorghum bagasse	• Alkali pretreatment • 2% NaOH • Feedstock-to-liquid ratio: 1:10 • Temperature: 120 °C • Time: 1 h	• Enzyme: (Cellulase 30 FPU/g, temperature: 50 °C, time 96 h, agitation: 180 rpm) • Fermentation (bioethanol production by Saccharomyces cerevisiae M3013 followed by ABE fermentation by Clostridium acetobutylicum ABE 1201)	• Ethanol yield: 0.144 g/g, butanol yield: 0.02 g/g	Su et al. [76]
Wheat bran, sugarcane bagasse and orange peel	• Microwave-assisted acid and surfactant-based pretreatment	• Enzyme (Aspergillus niger SKN1 and Trametes hirsuta SKH1) • Fermentation (Clostridium acetobutylicum ATCC 824)	• Concentration: 16.5 g/L, yield: 0.24 g/g	Mondal et al. [77]
Yeast extract, rice bran, soybean waste, dried spent yeast, urea, ammonium sulfate	-	• Clostridium beijerinckii TISTR 1461	• Concentration: 11.4 g/L, yield: 0.4 g/g, productivity: 0.32 g/L/h	Narueworanon et al. [78]

Note: The units of bioethanol yields for a few studies have been calculated for presentation in g/g to maintain uniformity.

.

In Table 2, it can be seen that the highest biobutanol production obtained was 19.1 g/L by Clostridium acetobutylicum JB200 [73]. In a study by Yang et al. [69], cellulose fiber was used as the feedstock for ABE fermentation to produce biobutanol using Clostridium cellulovorans through a consolidated bioprocessing pathway. The bacteria overexpressed the adhE2 gene, which is responsible for the conversion of butyryl coenzyme A to butanol. This study resulted in a butanol yield, concentration and productivity of 19.5 wt/wt%, 1.42 g/L and 5.9 mg/L/h, respectively. In another study, Rajagopalan et al. [74] utilized a mixture of two different agro-processing residues, rice bran and sesame oil cake, for biobutanol production by implementing Clostridium sp. BOH3. ABE fermentation was performed using a consolidated bioprocessing route using both feedstocks, resulting in a biobutanol concentration of 13.5 g/L.

Wen et al. [71] used two clostridial strains, Clostridium cellulovorans DSM 743B and Clostridium beijerinckii NCIMB 8052, for the conversion of corn cob into biobutanol. Before the fermentation process, the feedstock was treated with a mild alkali solution for the removal of lignin and loosening of the cellulosic bond structures. Moreover, C. cellulovorans was genetically engineered by removing the acetate and lactic acid-forming genes, acetate kinase and lactate dehydrogenase, respectively. This subsequently overexpressed the butyryl-forming genes to produce butyryl kinase. For C. beijerinckii, the genes responsible for biobutanol formation through organic acid reassimilation and metabolism of pentose sugars, ctfAB, cbei_3833/3834, xylR, cbei_2385, xylT and cbei_0109, were also overexpressed for the enhanced production of biobutanol. The overexpression of these genes diverts the metabolic pathway of the bacteria toward the butyryl formation and subsequently increases the biobutanol yield. In this study, a butanol yield of 14 wt/wt% was obtained with a productivity of 5.9 g/L/h.

Tsai et al. [75] used rice straw as the feedstock to produce biobutanol through a separate hydrolysis and fermentation route. In this preliminary step, the feedstock was pretreated using a mild solution of hydrogen peroxide for better digestion of the feedstock due to the degradation of the lignin and weakening of different cellulosic and hemicellulosic linkages. In this study, the pretreated rice straw was hydrolyzed using the Accellerase 1500 enzyme followed by ABE fermentation using Clostridium acetobutylicum ATCC 824, which produced 23 wt/wt% biobutanol. This study showed a comparatively higher biobutanol yield than the other studies described above, which was attributed to the immobilization of the clostridial stain on polyvinyl alcohol. The immobilization of C. acetobutylicum enhanced cell loading into the fermenter, which decreased the lag phase, increasing the sugar conversion rate and biobutanol yield. Thus, the immobilization of enzymes and microorganisms is considered another technique for the enhancement of biobutanol or other alcohol fermentation processes.

Narueworanon et al. [78] used urea and ammonium sulfate with a mixture of agro-processing waste constituting spent yeast, rice bran and soybean residue to study the effect of nitrogenous material on ABE fermentation. In this study, the fermentation was performed using C. beijerinckii TISTR 1461 strain through a consolidated bioprocessing technique. The study delivered the highest butanol yield of 40 wt/wt%, with a concentration of 11.4 g/L, which was attributed to the external nitrogen source and the genetically engineered C. beijerinckii TISTR 1461.

3.3. Biomethane

Anaerobic digestion is well established as a traditional technique for the bioconversion of organic waste and sludge into biogas or biomethane. This technique is promising for degrading solid residues from sewage treatment plants and fermentation processes to simultaneously produce biomethane while valorizing the wastes [79,80]. As shown in Figure 5, the anaerobic digestion process is constituted of four major steps, including hydrolysis, acidogenesis, acetogenesis and methanogenesis [81]. Biomethane has the potential to replace natural gas for both stationary and mobile applications due to its higher calorific value of around 36 MJ/m³.

In addition to anaerobic digestion, the co-digestion of different organic wastes provides a large corridor for industrial research due to its advantages of the synergistic effects of the substrates to maintain the pH and carbon/nitrogen ratio, which plays a vital role in biomethane production. Anaerobic digestion occurs in the absence of oxygen with the application of common methanogenic bacteria under the genera Methanobrevibacter, Methanococcus, Methanogenium, Methanopyrus, Methanosaeta, Methanosarcina and Methanosphaera.

Syntrophic metabolism mediated through interspecies electron transfer plays a significant role in anaerobic digestion, especially in the oxidation of volatile fatty acids [82]. In this mechanism, the redox mediator generated by the biological oxidation of volatile fatty acids is transferred between methanogenic and non-methanogenic bacteria via interspecies electron transfer. This interspecies hydrogen transfer can decrease the partial pressure of hydrogen to less than 10⁻⁴ atm, facilitating the emergence of acetogenic reactions [83]. Furthermore, anaerobic digestion and co-digestion are also affected by several other experimental factors, such as temperature, inoculum-to-feedstock ratio and incubation time. Some significant research works elaborating on the effects of these process parameters on anaerobic digestion for biomethane production are presented in

Table 3. Summary of significant studies on biomethane production from waste biomass.

Biomass	Pretreatment	Process Parameters	Biomethane or Biogas Yield	Reference
Cow dung (CD) and cotton seed hull (CSH)	Biogas plant slurry	• 500 mL batch reactor • 300 mL of inoculum • CD/CSH ratio: 100:0, 0:100, 50:50, 75:25 and 25:75 • Temperature: 35 °C • pH: 7.5 • Reaction time: 45 days • Stirring: 90 rpm	• CD: 193 mL/g VS • CSH: 33 mL/g VS • CD/CSH (50:50 ratio): 37 mL/g VS • CD/CSH (75:25 ratio): 86 mL/g VS • CD/CSH (25:75 ratio): 23 mL/g VS	Venkateshkumar et al. [84]
Fish waste and activated sludge	Anaerobic digested sludge	• 300 mL batch reactor • Temperature: 37 °C • Reaction time: 50 days • Fish waste loading: 0, 1.5, 3, 6 and 10 wt% • Inoculum/substrate ratio: 1:8	• 1.5 wt% fish waste: 410 mL/g VS • 3 wt% fish waste: 684 mL/g VS	Wu and Song [85]
Fruit and vegetable waste (FVW) and primary sludge (PS)	Activated sludge	• 500 mL batch reactor • FVW/PS ratio: 50:50 • Temperature: 37 °C • Reaction time: 30 days • Inoculum/substrate ratio (50:50 FVW/PS): 2	• Without inoculum: 141 mL/g VS • With inoculum: 295 mL/g VS	Elsayed et al. [86]
Olive pomace (OP) and apple pulp (AP)	Cow slurry (CS)	• 128 L pilot scale reactor • Temperature: 35 °C • pH: 7.5 • Reaction time: 40 days • Stirring: 90 rpm • Feedstock composition: 85 wt% CS, 10 wt% OP and 5 wt% AP	• 216 mL/g VS (CH4 fraction: 52%)	Riggio et al. [87]
Orange peel waste (OPW) and organic fraction of the municipality waste (OFMSW)	Degassed sewage sludge	• 150 mL batch reactor • Temperature: 37 °C and 55 °C • pH: 7.5 • Reaction time: 35 days • OPW/OFMSW ratio: 50:50 • Inoculum/substrate ratio: 1:3	• At 55 °C (thermophilic condition): 432 mL/g VS • At 37 °C (mesophilic condition): 294.6 mL/g VS	Bouaita et al. [88]
Pineapple peel (PP)	Pig slurry (PS)	• 4.8 L continuous stirred reactor • Organic loading rate: 1.46 g VS/L/day • PS/PP ratio: 80:20 • Temperature: 37 °C • pH: 7.5 • Reaction time: 16 days	• 580 mL/g VS	Azevedo et al. [89]
Potato peel (PP) and pig manure (PM)	Anaerobically digested sludge	• 150 mL batch reactor • Temperature: 38 °C • Reaction time: 27 days • Stirring: 150 rpm • Feedstock composition: 50 wt% PP and 50 wt% PM	• 380 mL/g VS	Adeleye et al. [90]
Slaughterhouse waste and sewage sludge	Sludge from the secondary treatment plant (Activated sludge)	• 1 L batch reactor • Total solids: 5% • Inoculum/substrate ratio: 4 • Temperature: 34 °C	• Biogas yield: 631 mL/g VS • CH4 yield: 462 mL/g VS	Latifi et al. [91]

Note: Volatile solids (VS).

.

Latifi et al. [91] utilized the organic waste generated from the slaughterhouse (e.g., blood, meat pieces and feathers) along with sewage sludge for biomethane production through anaerobic co-digestion. The authors optimized the effects of the total solid content of the feedstock (e.g., 5 and 7 wt%) and the inoculum/substrate ratio (e.g., 1, 2 and 4) on biomethane yield. It was observed that at a total solid content of 5 wt% and inoculum/substrate ratio of 4, the co-digestion of the feedstocks produced the highest biogas yield of 631 mL/g volatile solids (VS), with a biomethane fraction of 73% under mesophilic conditions using secondary sludge as the inoculum, which can be considered one of the highest biomethane fraction obtained in biogas. The lower solid-constituted substrate provided a higher biogas yield due to the lower accumulation of volatile fatty acids in the reactor, which lessened the negative effects due to the volatile fatty acid accumulation.

Elsayed et al. [86] studied the effects of inoculum on biomethane generation from the co-digestion of fruit–vegetable waste (FVW) and the primary sludge (PS) using activated sludge (or secondary sludge) as the inoculum. This study delivered the highest biomethane yields of 141 mL/g VS and 295 mL/g VS without and with inoculum, respectively. The optimized parameters, including the temperature, retention time, and the primary sludge-to-FVW and inoculum/substrate ratios were determined as 37 °C, 30 days, 50:50 and 2, respectively. It was observed that feedstock with a higher component of fruits and vegetable waste (PS/FVW ratio of 20:80) delivered the lowest biomethane yield due to the accumulation of volatile fatty acids, which affected the growth and activity of methanogenic bacteria and subsequently decreased the biomethane yield.

The mixing of different substrates in the proper ratio governs the pH and carbon/nitrogen ratio of the medium, which prominently affects the yield of biomethane. In addition to the production of biomethane, anaerobic digestion has become a potential component in wastewater treatment processes. It was employed to remove organic solids from various wastewater and sludge generated from different process industries and municipal solid or liquid wastes [87,90].

3.4. Biohydrogen

Hydrogen is a clean and versatile energy vector and carrier. Hydrogen energy has long been considered energy for the future [92]. The sustainable production of hydrogen from renewable resources plays an important role in the global energy transition. The hydrogen economy is also expected to increase with the implementation of the existing renewable energy strategies [93]. However, the industrial-scale production of hydrogen is still heavily dependent on fossil energy resources through the steam reforming of methane (i.e., natural gas), which is considered not sustainable due to significant carbon emissions [94,95]. Therefore, finding alternatives to produce hydrogen in a renewable manner and with a cleaner process has been a hot research topic for decades.

Currently, there are many different hydrogen production routes mainly categorized into thermochemical (e.g., gasification and pyrolysis), electrochemical (e.g., electrolysis), photocatalytic and biological (e.g., dark fermentation and photo-fermentation) [96]. In the photo and dark fermentation processes, organic matter (e.g., sugars derived from different sources, food waste, agricultural residues, sewage sludge and wastewater) is used as a substrate for biodegradation by microorganisms [97]. The advantages of dark fermentation include (i) the production of biohydrogen without a light source, (ii) a higher bioproduction rate compared to biophotolysis and photo fermentation, (iii) flexibility in using diverse and low-cost feedstocks and (iv) adaptability to perform the fermentation process using existing bioreactor designs [98]. During the photo and dark fermentation processes, several critical factors that significantly impact biohydrogen yield and selectivity include the physicochemical properties and loading of the feedstocks, the type of fermenting microorganism, the process parameters, including the temperature, reaction media, time, agitation, aerobic or anaerobic conditions, bioreactor design and feeding mode (i.e., batch, fed-batch, or continuous) and catabolic enzymes [99,100].

The partial pressure of hydrogen is also a critical factor of dark fermentation. Fermentative biohydrogen production is negatively impacted at a higher partial pressure of hydrogen, which requires the intermittent degassing of the enclosed bioreactors with inert gases, such as N₂, to enhance the liquid-to-gas mass transfer [101]. According to Henry’s Law, the liquid concentration and the partial pressure of hydrogen are theoretically linked at thermodynamic equilibrium [102].

Like ABE fermentation, several Clostridium spp. are also responsible for performing dark fermentation to produce biohydrogen through the metabolism based on NADH-for and pyruvate ferredoxin oxidoreductase (pfor) [103]. The reduced ferredoxin (Fd) and NADH lead to the reduction of H⁺ ions catalyzed by hydrogenase (hyd), thus resulting in biohydrogen production via dark fermentation. As shown in Figure 6, NADH produced from glycolysis is oxidized, resulting in the release of 2 mol of H₂ by the reduction of H⁺ ions. On the other hand, ferredoxin is reduced via the oxidation of pyruvate to acetyl coenzyme A (CoA), leading to biohydrogen production. Two moles of biohydrogen are also produced by the oxidation of the reduced ferredoxin [104]. Hence, totals of 2 moles and 4 moles of H₂ are produced when butyrate and acetate are the final products, respectively. Similarly, a hyperthermophilic anaerobic bacterium, Thermotoga maritima, deploys its iron hydrogenase enzyme to synergistically utilize NADH and ferredoxin to produce biohydrogen [105]. In addition, the anaerobic bacterium Syntrophomonas wolfei hosting a multimeric [FeFe]-hydrogenase enzyme demonstrates biohydrogen production when co-cultured with hydrogen- and/or formate-using methanogen via fatty-acid oxidization [106]. [FeFe]-hydrogenase has been reported to be ferredoxin-independent and NADH-dependent to re-oxidize NADH.

Biohydrogen production from lignocellulosic substrates can be facilitated under thermophilic conditions. Hyperthermophilic bacteria exhibit a theoretical biohydrogen production potential of 4 mol/mol via dark fermentation [107]. Thermophilic bacteria, such as Caldicellulosiruptor saccharolyticus, Thermotoga neapolitana and Thermotoga maritima, demonstrate a wide variety of physiological and metabolic properties to aid dark fermentation and improve biohydrogen production [108,109,110]. Some of these physiological properties distinctive to (hyper)thermophilic bacteria include hydrolytic capability (via glycoside hydrolase), substrate degradation, tolerance to inhibition and stress conditions, thermal stability and enhanced regulation of redox and carbon metabolism pathways [111].

Table 4. Summary of significant studies on biohydrogen production from waste biomass.

Biomass	Microorganism	Bioreactor Type	Biohydrogen Yield or Productivity	Reference
Activated sludge	Actinobacteria, Bacterioidetes, Chloroflexi, Firmicutes and Proteobacteria	Plexiglass bottles	11 mL/g	Li et al. [112]
Algae and glucose	Clostridium pasteurianum	Serum bottles	67 mL/g	Srivastava et al. [113]
Cheese whey	Enterobacter aerogenes 2822	Double-walled cylindrical bioreactor	0.75 mL/g/h	Rao and Basak [114]
Glucose	Clostridium sensu stricto 1	Glass bioreactors	205 mL/g	Zhang et al. [115]
Glucose	Firmicutes, Chloroflexi, Proteobacteria, Synergistetes and Bacteroidetes	Serum vials	250.1 mL/g	Zhang et al. [116]
Potato peel	Clostridium propionicum	Transfusion bottle submerged in a water bath	106.2 mL/g	Wang et al. [117]
Sugar beet pulp	Clostridia and Coriobacteriia	Glass bottles (batch tests), cylindrical glass reactors (semi-continuous tests)	58.9 mL/g	Cieciura-Włoch et al. [118]

Note: The units of biohydrogen yields for a few studies have been calculated for presentation in mL/g to maintain uniformity.

summarizes some recent studies on the fermentative production of biohydrogen from organic wastes.

Chen et al. [97] studied the effects of the temperature of the content of total solids on biohydrogen production via dark fermentation of rice straw operated under thermophilic (55 °C) and mesophilic (37 °C) conditions. The results suggest that the butyric acid fermentation pathway was the primary biohydrogen production route for both the thermophilic and mesophilic dark fermentation processes. The thermophilic dark fermentation process showed a higher biohydrogen yield than that operated under mesophilic conditions. The doubling in total solids content from 6% to 12% resulted in a shift of the conversion pathway, leading to better improvement in the biomethane generation compared to biohydrogen.

Li et al. [112] studied biohydrogen generation from the dark fermentation of activated sludge. The results reveal that the addition of rhamnolipid, an environmentally friendly biosurfactant, improved the biohydrogen yield. Another recent trend to enhance biohydrogen production from dark fermentation is the use of novel nanomaterials, such as Au, Ni, Ag, Cu, Fe, Pd, TiO₂ and activated carbon [119]. In a study by Zhang et al. [115], the addition of cobalt ferrate nanoparticles was found to be effective in increasing biohydrogen yield from the dark fermentation of glucose.

Although there have been many studies on biohydrogen production from biomass via dark fermentation, a few challenges still exist in scaling up this technology. The interaction between different types of nanoparticles and the microbial community in dark fermentation systems should be further studied. It would be beneficial to see more work on techno-economic analyses, sustainability analyses and lifecycle assessments to help guide the industrial-scale designs of bioreactor systems for biohydrogen production.

3.5. Biodiesel

Biodiesel is a fatty acid methyl ester derived from vegetable oils, grease, animal fats, algae, microbial and other lipid sources. The production of biodiesel is a multi-step process that involves the extraction of oil, esterification, transesterification and purification. It has emerged as an alternative drop-in biofuel with enormous potential to reduce greenhouse gas emissions associated with fossil-derived diesel fuel. Biodiesel can also be used in existing diesel engines without requiring any major modifications [120].

Biodiesel production starts with the extraction of oil from the feedstock source via different methods, such as mechanical pressing, solvent extraction, microwave extraction, ultrasonic extraction and supercritical fluid extraction [121]. These oils are utilized as feedstock to produce biodiesel through transesterification in the presence of methanol and different acid catalysts. The most common raw materials used are vegetable oils, such as palm oil, soybean oil, corn oil, grapeseed, cottonseed oil, sunflower oil and canola oil [122]. Recent research advancements have shown promising results from algae as a potential biodiesel feedstock, which not only has the least competition to arable lands but also contributes to carbon fixation and wastewater reclamation [123]. The second step in biodiesel production is transesterification, which involves the reaction of the oil with an alcohol, typically methanol or ethanol, in the presence of a catalyst. The interaction of alcohol and oil produces fatty acid methyl esters, which are the main components of biodiesel. The final step in biodiesel production is the purification of impurities from biodiesel. This process is typically carried out by washing the biodiesel with water or using a dry-washing process [124,125].

Biodiesel production using lipases as biocatalysts has been the subject of numerous studies since it offers several advantages, such as high specificity, mild reaction conditions and environmentally benign process [123]. Lipases function as transesterification catalysts rather than hydrolases, which hydrolyze ester bonds. They facilitate the reaction between triglycerides and alcohol, leading to the production of biodiesel and glycerol. In addition to lipases, other enzymes, such as proteases and cellulases can also be employed in biodiesel production, although they are less commonly used compared to lipases [126]. These enzymes can be used to treat feedstocks that contain impurities like proteins or cellulosic material, which can interfere with the transesterification process. Lipases from microbial sources, such as bacteria, fungi and yeast have been extensively studied [127,128,129]. For example, lipases from Candida antarctica, Rhizopus oryzae and Pseudomonas cepacia have shown promising results in terms of activity and stability in biodiesel production [130].

Table 5. Summary of significant studies on catalytic transesterification for biodiesel production.

Feedstock	Catalyst or Biocatalyst	Biodiesel Yield	Reference
Algae	NaOH	92%	Sivaramakrishnan et al. [131]
Algal oil	Lipase from Candida antarctica	99.1%	Wang et al. [132]
Babassu oil	Lipase from Burkholderia cepacia	>99%	Da Rós et al. [133]
Canola oil	CaO calcined dolomite	99.4%	Korkut and Bayramoglu [134]
Castor oil	La2O3/Na–Y-600	85%	Du et al. [135]
Palm oil	NaAlO2/γ-Al2O3	97.7%	Zhang et al. [136]
Waste cooking oil	CaO/SiO2	91%	Putra et al. [137]
Waste cooking oil	Lipase	>99%	Jayaraman et al. [138]

summarizes a few studies on the production of biodiesel from different feedstocks using catalysts and biocatalytic enzymes.

Wang et al. [132] developed an enzyme-based pathway to convert crude algal oil into fatty acid methyl esters. The researchers used immobilized lipase from C. antarctica for biodiesel production. Furthermore, the authors enlightened the efficacy of different solvents, reaction times and temperatures in biodiesel conversion and recorded a 99.1% efficiency under optimized conditions (i.e., algal oil/tert-butanol ratio of 1:1, temperature of 25 °C and reaction time of 4 h). The authors also reported high stability for the lipase to withstand 41 cycles with minimal energy requirement and reduced wastewater discharge.

Jayaraman et al. [138] reported the efficacy of lipase-based catalysts in the enzymatic production of biodiesel. The authors used waste cooking oil as the source material to achieve 100% conversion to biodiesel with an optimized enzyme concentration of 1.5%, methanol as the solvent and 4 h. Sivaramakrishnan et al. [131] studied the efficacy of two microalgae (i.e., Chlorella and Scenedesmus) for methyl ester production via various solvent systems and cell disruption techniques.

Taher et al. [123] demonstrated biodiesel production via supercritical CO₂ extraction from Nannochloropsis gaditana. The authors reported a 10.5% internal rate of return with a net present value of USD $8.31 million. They found that the transportation of equipment and materials is a significant contributor, with a share of 75% of the total impact. This can be linked to the use of fossil fuels for transportation. The lifecycle profiles are not only oriented with biocatalyst reactions but also importantly impacted with the downstream processes responsible for the purification and obtaining of the desired products.

4. Conclusions and Perspectives

Low-carbon energy sources are stressed and supported under climate change initiatives globally, and with the assumption of carbon neutrality, biomass becomes more favorable. Several factors, such as the type of feedstock and its transportation, feedstock pretreatment and upstream processing, bioprocess optimization, product separation and downstream processing, co-product utilization, product distribution and techno-economic and lifecycle assessments should be considered for the scale-up and commercialization of biomass conversion technologies. First-generation biomasses (e.g., food grains and oilseed crops) have remained the preferred raw materials for fermentation and transesterification to produce biofuels and biochemicals due to the ease of extraction and conversion. However, the criticism surrounding their diversion from the food chain into the biorefinery framework has impacted their utilization as biofuel feedstocks. In contrast, second-generation ‘non-food’ biomasses, including crop and woody residues, are the new drivers for biofuel production. These lignocellulosic biomasses are low-cost and do not compete with arable lands and food supply. Unlike starch-based feedstocks, the processing of lignocellulosic biomasses, such as the removal of lignin and the extraction of monomeric cellulosic and hemicellulosic sugars, is challenging and adds to the cost of the bioconversion process and the final biofuel product. The selection of the upstream process of biomass, including its pretreatment to separate lignin and fractionate cellulose and hemicellulose into monomeric sugar subunits via a variety of mechanical, physicochemical and biological pretreatments, is an important step before fermentation, transesterification and/or anaerobic digestion. The type of pretreatment agent and processing conditions could significantly affect the overall capital cost of the bioconversion process.

Depending on the biofuel production processes, different co-products are generated. Glycerol is a co-product of biodiesel production, while lignin is a byproduct of biomass pretreatment and delignification. Carboxylic acids are also produced from biomass pretreatment and fermentation, along with bioethanol, biobutanol, biohydrogen and biomethane. Solid digestate, dried distiller grains and black liquor are other byproducts of biomass saccharification and fermentation. The environmental benefits, applications and marketability of these products are associated with their treatment, utilization or disposal. Finding suitable and alternative uses for different co-products of a bioprocess can lead to many benefits, such as generating additional revenues and market, subsidized capital expenditures, replacing petrochemicals and synthetic products and closing the waste recycling loop.

The disposal or recycling of biofuels and generated co-products needs to be considered for lifecycle assessment. Lifecycle assessment is a method to evaluate the environmental impacts associated with the entire lifecycle of a product or process, from raw material extraction to the disposal of residual raw material, final products and byproducts. It is frequently used as an industry benchmark for the impact of climate change. Depending on the specific waste management practices, impacts such as emissions, energy use and waste generation can occur, which can impact biodiversity adversely. Water consumption and chemical requirements for carrying out these processes also have a significant impact on the environment and energy requirements. In addition, the transportation of feedstocks to the bioprocessing facility could also contribute to greenhouse gas emissions and air pollution if fossil fuels are used as a primary fuel source for long-hauling heavy vehicles. Biofuels are often upgraded and transported to distribution facilities for different applications or blending with conventional fuels. Transportation involving fossil fuels induces a negative impact on the environment despite the biofuel product being shipped. Furthermore, the infrastructure involved in this stage is also a critical factor of the impact on the environment. The application of biofuels in different sectors results in emissions, which can be linked to negative impacts on air quality and greenhouse gas emissions. However, establishing bioprocessing units in the proximity of farms and biomass storage facilities can minimize carbon emissions.

Techno-economic assessment or cost analysis is a systematic strategy to evaluate the productivity, scale-up potential, adaptability and commercialization of biofuels and biochemicals in a very competitive market dominated by fossil fuels and petrochemicals. However, the cost analysis is fairly limited under several assumptions and considerations. This knowledge gap persists because of a scarcity of large-scale production facilities for many biofuel technologies relying on second- and third-generation ‘non-food’ feedstocks, along with a substantial variation in the expenses relating to the feedstock cost, operating conditions (upstream and downstream), strategies for waste management, byproduct utilization, product recovery and upgrading and other sensitivity parameters that are affected by local geography and legislations internationally. As many countries and international organizations are unifying their efforts to gradually phase out fossil fuels and adopt biofuels, these challenges are expected to be addressed in the near future.