Current Trends of Cellulosic Ethanol Technology from the Perspective of Industrial Development

Abstract

Driven by the energy transition within the framework of the United Nations Framework Convention on Climate Change, second-generation (2G) ethanol stands out as a technical and sustainable alternative to fossil fuels. Although first-generation ethanol, produced from saccharine and starchy feedstocks, represents an advance in mitigating emissions, its expansion is limited by competition with areas destined for food production. In this context, 2G ethanol, obtained from residual lignocellulosic biomass, emerges as a strategic route for diversifying and expanding the renewable energy matrix. Thus, this work discusses the current state of 2G ethanol technology based on the gradual growth in production and the consolidation of this route over the last few years. Industrial second-generation ethanol plants operating around the world demonstrate the high potential of agricultural waste as a raw material, particularly corn straw in the United States, which offers a lower cost and significant yield in the production of this biofuel. Similarly, in Brazil, sugarcane by-products, especially bagasse and straw, are consolidating as the main sources for 2G ethanol, integrated into the biorefinery concept and the valorization of by-products obtained during the 2G ethanol production process. However, despite the wide availability of lignocellulosic biomass and its high productive potential, the consolidation of 2G ethanol is still conditioned by technical and economic challenges, especially the high costs associated with pretreatment stages and enzymatic cocktails, as well as the formation of inhibitory compounds that compromise the efficiency of the process. Genetic engineering plays a particularly important role in the development of microorganisms to produce more efficient enzymatic cocktails and to ferment hexoses and pentoses (C₆ and C₅ sugars) into ethanol. In this scenario, not only are technological limitations important but also public policies and tax incentives, combined with the integration of the biorefinery concept and the valorization of (by)products, which prove fundamental to reducing costs, increasing process efficiency, and ensuring the economic viability and sustainability of second-generation ethanol.

1. Introduction

The growing demand for renewable energy sources has gained prominence in global debates. The most recent Conference of the Parties (COP) of the United Nations Framework Convention on Climate Change, held in November 2025 in Brazil, was focused on climate finance and the energy transition. The event brought together global leaders to discuss mechanisms in support of these objectives, encouraging mitigation and adaptation actions, and biofuels have their place in this transaction. Furthermore, there is a growing movement by industries toward a circular economy, prioritizing the reuse of waste that would otherwise be discarded, which is converted into new bioproducts with added value [1].

In this context, ethanol emerges as a key topic when discussing renewable energy. Generated from sugars found in several biomass sources, such as sugarcane and corn, 1G or first-generation ethanol offers advantages, since according to the idea of an environmental balance, its production emits less carbon dioxide (CO₂) compared with gasoline [2,3]. Currently, the world’s largest producers of 1G ethanol are the United States and Brazil, which have been seeking alternatives to conventional raw materials, as these directly compete with areas used for food cultivation, a critical issue given the population growth worldwide [2,3].

Alternatively, cellulosic or second-generation (2G) ethanol can be produced using industrial and agricultural waste rich in fermentable sugars structured into lignocellulose and can help the balance of food vs. fuel shown by the biomasses applied in 1G technology. Lignocellulosic biomass, such as wood and crop residues, is composed primarily of cellulose and hemicelluloses acting as sources of sugars, and lignin, a complex and resistant polyphenolic structure, which is not converted by fermentation to ethanol. Lignocellulosic biomass represents a promising raw material for ethanol production because it is readily available in various regions around the world and does not necessitate the allocation of fertile land areas, such as land used for food crops. In addition, considering the context of biorefinery, this biomass type can generate products beyond ethanol, such as renewable electricity through the burning of residues not converted into ethanol, or obtain value-added products (for example, phenols and sugar derivatives) generated during the processing steps, increasing economic viability and aligning with the idea of a circular economy [4]. While cellulosic ethanol gained from the conversion of lignocellulosic biomass enables the valorization of municipal and forestry waste, promotes a circular economy, and contributes to the reduction in environmental pollution, as a main drawback, the conversion of lignocellulosic biomass into ethanol necessitates challenging and costly technological steps, such as pretreatment and hydrolysis, which are necessary to break down the lignocellulosic structure and release sugars, followed by fermentation and distillation stages [5,6].

Reviews on cellulosic ethanol available in the literature are focused on pretreatment steps [7,8,9,10], aspects related to hydrolysis and fermentation efficiencies [8,9,10,11,12,13,14], and optimization of treatment stages [15,16]. Meanwhile, information about current policies and trends [17,18] as well as knowledge about successful industrial applications is not widespread [3,19]. Accordingly, this review aims to provide a more integrated picture of the progress of cellulosic ethanol production on the global stage, addressing processing stages, main costs, incentive policies, as well as future prospects.

2. Bioethanol Production

Initiatives to mitigate ozone layer pollution have become increasingly relevant topics, driven by technological and population growth. In this context, the adoption of renewable fuels (biofuels) has gained prominence as a global concern, since reducing CO₂ emissions indirectly contributes to the recovery of the ozone layer by helping with temperature control and reducing the probability of undesirable chemical reactions that degrade the ozone layer. First-generation ethanol (1G) stands out as a viable alternative due to its low greenhouse gas emissions, both during combustion and through the capture of CO₂ resulting from the cultivation of biomass used as a feedstock [20]. According to Figure 1, world bioethanol production is already showing a recovery following the COVID-19 pandemic, with the United States and Brazil accounting for over 80% of global production, relying on corn and sugarcane as the primary biomass source, respectively.

In comparison with first-generation (1G) crops, the application of lignocellulosic biomasses (second generation—2G) is still modest, but increases continuously. Annual global production of residues containing lignocellulosic biomass is estimated at approx. 1.3 billion tons, with the main share consisting of agricultural and forestry residues [2,3,23]. In the USA, approximately 215 million tons of lignocellulosic biomass residues are produced annually, mainly corn and wheat straw [24]. Comparatively, in Brazil, approximately 196.2 million tons of residues are produced, with sugarcane bagasse and straw accounting for the major share [25]. In Europe, the main residue is wheat stalk, with an annual production of approx. 15 million tons [24]. In the case of Indonesia, studies indicate that the country has significant potential for cellulosic ethanol production due to the abundant availability of palm oil residues, which could offer economic benefits, such as job creation and reduced oil imports. As an illustration, the growth of cellulosic ethanol production in Indonesia in 2024 and its prospects from 2026 to 2032 are presented in Figure 2.

In 2024, the US accounted for 52% of yearly global ethanol production, with 16.1 billion gallons, followed by Brazil with 8.78 billion gallons (28% of global production). India (1.63 billion gallons) and the European Union (1.44 billion gallons) together contributed 5%, while China produced 2% (1.2 billion gallons) [27].

In the US, the main feedstock for bioethanol production is corn, whose starch is converted into sugar, from which ethanol fuel is obtained [28]. Kumar and Sinha [20] estimate that the production of 1 L of ethanol may consume, on average, 2.54 kg of corn. In 2024, the value of bioethanol production per bushel of corn was USD 4.71, with a yield of 10.6 L per bushel (2.8 gallons per bushel). The cost of corn is estimated at USD 61/ton, and that of corn stover is estimated at USD 58.50/ton. In view of these cost estimates, US companies have invested in the production of cellulosic ethanol, focusing on technologies for converting corn residues, such as straw, into ethanol [29]. The largest 2G ethanol producers in the US are POET LCC and Archer Daniels Midland. In 2018, POET increased its production capacity to 27 ethanol plants [3]. In 2022, the USA produced around 1.4 million gallons of cellulosic ethanol [30,31].

In Brazil, sugarcane remains the highest-yielding raw material for bioethanol production. It is estimated that 10–14 kg of sugarcane or 4–5 kg of sugarcane molasse are required to produce 1 L of ethanol, with the yield ranging between 6000 and 8000 L/ha, costing on average BRL 4.13 per liter [20,32]. According to RPA News magazine [33], the average cost of sugarcane cultivation in 2024 was BRL 18,014 per hectare. Furthermore, corn has been gaining prominence in Brazilian bioethanol production, reaching a 32.8% increase compared to the previous year [34].

In order to reduce costs and follow a circular economy approach, companies have been investing in agricultural waste as a source of sugar. According to the national energy plan developed by the Ministry of Mines and Energy of Brazil [35], the cost of producing sugarcane is BRL 35/ton, while the cost of producing bagasse is BRL 19.6/ton. In 2024, Raízen S/A opened the world’s largest cellulosic ethanol plant, with a production capacity of 82,000 m³/year. Located in the Bonfim Bioenergy Park, in Guariba (São Paulo), the unit uses sugarcane straw and bagasse as raw materials [36]. The costs of sugarcane and sugarcane bagasse production are estimated at USD 186/ton (approx. BRL 1.031/ton) and USD 36.38/ton (approx. BRL 201/ton), respectively [37].

In Europe, 1G ethanol is produced primarily from sugar beets, which account for about 30% of ethanol production in the European Union [38]. In France, the region’s leading producer achieves a productivity of up to 9000 L of ethanol per hectare of sugar beets, which costs approx. EUR 2.5 per liter. In 2024, the production cost for this crop amounted to EUR 2967/ha [39,40]. To account for high crop cultivation costs, Europe has also been investing in the production of cellulosic ethanol, which now accounts for about 25% of the global market and is obtained primarily from forestry residues [41]. The main biomasses used in the production of lignocellulosic ethanol and their productivity in L/ton of biomass and L/ha of cultivated area are presented in

Table 1. Ethanol yield from different biomass sources.

Biomass	Ethanol (L/ton of Biomass)	Ethanol (L/ha)	Reference
1st-generation biomasses—saccharine and starch-based
Sugarcane	483	5930	[42,43]
Cassava	137–180	2210	[44]
Sweet potato	171	1470	[43,45]
Sugar beet	110	6650	[43,46]
Sorgo	390	2500	[47,48]
Corn	390	5685	[48]
2nd-generation biomasses—cellulose-based
Rice straw	517	5014.9 *	[42]
Barley straw	490	1107.4 *	[42]
Wheat straw	460	1163.8 *	[42]
Corn stover	362–456	2606.4–3283.2 *	[13]
Sugarcane bagasse	318–500	2544–4000 * or 6360–10,000 *	[13]

* Values obtained by multiplying the ethanol yield (L) per biomass (ton) by the crop yield in ton/ha for each biomass [49]. The following values were considered: for rice straw, the yield from Spain of 9.7 ton/ha [50]; for barley straw and corn stover, the crop yields from the United Kingdom of 2.26 and 2.53, respectively [51]; for corn stover, the yield of 7.2 ton/ha from the USA [52]; and a range of 8–20 ton/ha was applied for sugarcane bagasse in Brazil [53].

.

Therefore, the growth in cellulosic ethanol production worldwide is notable, driven by both economic factors as well as measures aimed at mitigating environmental pollution. On the other hand, the use of these residues faces some obstacles with regards to their lignocellulosic composition, resulting in high costs for biomass pretreatment and the acquisition of enzymes necessary for the conversion of sugars into ethanol, which significantly impacts the final cost of the process [17].

3. Lignocellulose, Pretreatment, Hydrolysis, and Fermentation

Lignocellulosic biomass can be divided into four main categories: agricultural residues, industrial residues, municipal solid waste, and woody forest residues. Agricultural residues include sugarcane bagasse, rice straw, corn cob, and straw, as well as wheat straw. These residues have been widely studied as sustainable, highly available alternatives that contribute to the circular economy. However, these materials require costly pretreatment in order to access sugars contained in their structure. Therefore, process optimization is essential for overcoming these challenges and advancing cellulosic ethanol production [5,6]. The use of industrial and municipal waste provides an environmentally friendly alternative, in addition to being generated in large quantities. However, its composition is highly variable and, in some cases, may not only require pretreatment stages to release sugars from the lignocellulosic biomass matrix but also additional pretreatment steps to remove contaminants. Forest woody residue can be classified in soft woods (pies, cedar, cypress) and hard woods (poplar, cotton wood, willow), which are very abundant and low-cost. It is estimated that 65% of the biomass energy potential of woody forest residues originates from wood chips, sawdust, and bark [2,7]. Due to the broad range of variations in chemical composition of lignocellulosic biomass, its detailed characterization is required prior to application on an industrial scale.

3.1. Chemical Composition of Lignocellulosic Biomass

Lignocellulosic materials are composed primarily of three polymers: hemicellulose, cellulose, and lignin, respectively, accounting for 20–40%, 30–60%, and 15–25% of the dry biomass. Hemicellulose and cellulose contain sugars, which can be converted to ethanol, while lignin acts as a polyphenolic protective layer, which is highly resistant to biological and chemical degradation and is insoluble in water, making cellulose removal a challenge in the industrial application of biomass [2,37]. Another challenge in cellulosic ethanol production is the variation in chemical composition between different types of biomasses, as presented in Table 2.

The use of machine learning (ML) in biorefineries has gained prominence as a promising tool for process optimization. Based on information such as the chemical composition of biomass and growth rate of yeast, ML algorithms can identify patterns and correlations for the selection of the most suitable biomasses along with optimal process parameters for bioethanol production. The computing methods of Decision Trees, Artificial Neural Networks (ANNs), and Gradient Boosting are frequently applied [54]. In addition to understanding the chemical and structural characteristics of biomass to improve process efficiency, it is also of fundamental importance to evaluate the yield of raw material per hectare, aiming at ensuring the viability of the system.

Cellulose is the most interesting polymer for 2G ethanol production since it is composed of glucose linked by β-1,4-glycosidic bonds, the most suitable sugar for alcoholic fermentation. Despite this, cellulose possesses hydrogen bonds that result in a rigid structure, insoluble in water and resistant to hydrolysis, although it has hydrolyzable amorphous regions [2,37,55]. In turn, hemicellulose is a heteropolymer with a branched structure, composed of hexoses and pentoses (glucose and xylose, for example) and acidic sugars. It is linked to cellulose by hydrogen bonds and ether and ester linkages and is of interest for ethanol production since its monomers can also be converted into ethanol by fermentation [2,55]. Lignin represents the greatest challenge in cellulosic ethanol production because it is a complex and hydrophobic aromatic polymer that coats cellulose and hemicellulose, hindering access to sugars. It is composed of carbon–carbon and ether bonds, requiring the application of efficient and economically viable pretreatment methods for its removal or at least de-structuration [37,55]. A simplified structure of lignocellulose in plant cells is presented in Figure 3.

Based on the chemical composition of lignocellulosic biomass, biorefineries perform a series of pretreatment stages to convert carbohydrates into ethanol efficiently, although technical and economic aspects are challenging and must be considered in each process and biomass type. Several studies have focused on optimizing this step, with lignin separation considered the most complex and costly phase of the process.

Table 2. Lignocellulosic composition of different biomasses.

Composition of Lignocellulosic Biomasses (% in Dry Basis)
Biomass	Cellulose	Hemicellulose	Lignin	Reference
Sugarcane bagasse	45.00	32.00	17.00	[56]
Sugarcane straw	36.90	19.70	13.70	[57]
Corn husk	54.69 ± 0.15	27.13 ± 1.80	8.780 ± 1.10	[58]
Rice husk	49.63 ± 0.98	10.44 ± 0.28	21.76 ± 0.91	[58]
Corn stalk	36.89	20.42	17.38	[59]
Corn stover	37.50	22.40	17.6	[60]
Rice straw	33.42 ± 0.03	28.52 ± 0.81	4.54 ± 0.20	[61]
Coconut bran	20.95 ± 2.14	7.19 ± 0.99	41.29 ± 4.1	[61]
Rape straw	35.00 ± 0.02	28.62 ± 0.12	3.44 ± 0.56	[61]
Pine bark	25.20 ± 0.86	6.50 ± 0.12	36.91 ± 1.50	[61]
Sawdust	54.40 ± 0.13	13.43 ± 0.51	18.00 ± 0.44	[61]
Banana leaves	43.34	34.34	15.00	[62]
Coconut husk	38.86	30.00	34.96	[63]
Pineapple leaf	70.42	23.13	4.330	[64]
Cocoa shell	21.20	20.12	51.93	[64]
Olive tree residue	32.30	28.50	32.31	[65]
Miscanthus ginantus	41.08	24.52	27.00	[66]
Miscanthus sinensis	44.12	29.79	19.52	[67]
Miscanthus sacchariflorus	44.57	29.11	20.34	[67]
Elephant grass	22.00	24.00	24.00	[68]
Belulang grass (Eleusine indica)	34.16	31.15	10.92	[69]

Table 2. Lignocellulosic composition of different biomasses.

Composition of Lignocellulosic Biomasses (% in Dry Basis)
Biomass	Cellulose	Hemicellulose	Lignin	Reference
Sugarcane bagasse	45.00	32.00	17.00	[56]
Sugarcane straw	36.90	19.70	13.70	[57]
Corn husk	54.69 ± 0.15	27.13 ± 1.80	8.780 ± 1.10	[58]
Rice husk	49.63 ± 0.98	10.44 ± 0.28	21.76 ± 0.91	[58]
Corn stalk	36.89	20.42	17.38	[59]
Corn stover	37.50	22.40	17.6	[60]
Rice straw	33.42 ± 0.03	28.52 ± 0.81	4.54 ± 0.20	[61]
Coconut bran	20.95 ± 2.14	7.19 ± 0.99	41.29 ± 4.1	[61]
Rape straw	35.00 ± 0.02	28.62 ± 0.12	3.44 ± 0.56	[61]
Pine bark	25.20 ± 0.86	6.50 ± 0.12	36.91 ± 1.50	[61]
Sawdust	54.40 ± 0.13	13.43 ± 0.51	18.00 ± 0.44	[61]
Banana leaves	43.34	34.34	15.00	[62]
Coconut husk	38.86	30.00	34.96	[63]
Pineapple leaf	70.42	23.13	4.330	[64]
Cocoa shell	21.20	20.12	51.93	[64]
Olive tree residue	32.30	28.50	32.31	[65]
Miscanthus ginantus	41.08	24.52	27.00	[66]
Miscanthus sinensis	44.12	29.79	19.52	[67]
Miscanthus sacchariflorus	44.57	29.11	20.34	[67]
Elephant grass	22.00	24.00	24.00	[68]
Belulang grass (Eleusine indica)	34.16	31.15	10.92	[69]

3.2. Stages of Cellulosic Ethanol Production

The conversion of biomass into 2G ethanol represents a significant challenge due to compositional variability, high lignin content, and the highly crystalline structure of the cellulosic matrix. Therefore, in the upstream stage, biomass pretreatment is required to break down natural barriers, mainly consisting of lignin and hemicellulose, exposing the cellulosic fibers. Additionally, once cellulose and hemicellulose become accessible to enzymes, enzymatic hydrolysis can be applied to degrade the polymeric sugar matrix into sugar oligomers and monomers. In the subsequent step, the fermentation stage, microorganisms are inoculated to ferment oligomeric and monomeric sugars, converting them into ethanol. The next step involves downstream processes, such as distillation, where ethanol is separated and purified by distilling the fermented broth, and stillage separation and reuse [3,7,70].

Co-products can be obtained from the 2G ethanol process, and their valorization can support the economic and energetic balance of the system. In particular, the lignin fraction can be used either in its raw form, to produce energy by combustion or, following further treatment stages, in an integrated biorefinery, to generate other products such as aromatic chemicals, phenolic compounds, adhesives, resins, etc. Stillage can be used to generate methane and biofertilizer by anaerobic digestion, and CO₂ can be used as an industrial component or as a stimulant for plant photosynthesis in a greenhouse.

A simplified flowchart of the processing steps performed in a conventional 2G ethanol plant is presented in Figure 4. Additional valorization pathways may involve the chemical compounds resulting from the applied biomass degradation processes with potential market value, such as furfural, hydroxymethylfurfural (HMF), and acetic acid. The valorization of a broad range of by-products can be considered in the context of a biorefinery, which emphasizes the need for fractioning and extraction of a broad range of bioproducts from lignocellulosic biomass.

3.2.1. Biomass Pretreatment

As mentioned, the pretreatment step aims to disrupt/reduce the lignin and hemicellulose matrix to extract polysaccharides, making the cellulose and hemicellulose substrate more accessible for subsequent processing stages. These methods are classified as physical, chemical, physicochemical, and biological, each with their own advantages and disadvantages, as summarized in

Table 3. Positive and negative aspects in biomass pretreatment methods [7,15,71].

Pretreatment Method	Advantages	Disadvantages
Physical
Mechanical crushing	Decreasing cellulose crystallinity, and altering the fiber morphology of the treated material	High energy consumption, leading to increased processing costs
Ultrasonic	Rapid operation, particle size reduction, enhanced cellulose accessibility, and no requirement for additional reagents	Costly equipment, high energy consumption with poor efficiency, expensive for large-scale production
Microwave	Simple and fast operation, short processing time, energy efficiency, rapid heating, degradation of cellulose structure, and no additional reagents required	Expensive, low delignification, production of inhibitors, required combination with other pretreatment technologies, scaling-up issues
Chemical
Alkaline	Reduces cellulose absorption due to efficient lignin removal and low cost; when combined with acid, produces pure cellulose with minimal by-product formation	Salt formation necessitates neutralization, chemical recovery processes, extended residence time
Acid	Low costs, favorable results when combined with alkaline treatment, effectively hydrolyzes hemicellulose	Hazardous, toxic, and corrosive chemicals, requiring neutralization, detoxification, chemical recovery processes, and use of anti-corrosive materials, yield losses due to sugar degradation
Ionic liquid	Breaks down oxygen-containing structures, operates without chemical reagents, easy to use, low energy consumption	The solvent is volatile and costly, with recovery relying on ion exchange chromatography (an expensive method)
Organosolv	High solid loading, obtaining high-purity lignin for further applications	High cost, requirement for separation, recovery, and reuse of solvent
Physicochemical
Liquid hot water	Does not require washing, chemical recovery, or detoxification steps	Longer residence time, use of large volumes of water, costly equipment, high energy consumption for heating and water evaporation
Steam explosion	Uses of chemicals not required, low water consumption, low environmental impact, efficiently combined with other pretreatment methods	Yield losses due to sugar degradation, high energy input required for steam heating
Ammonia	Low amounts of co-products, reduces enzyme requirement in the hydrolysis stage	Separation, recovery, and recycling of ammonia incur high costs
Oxidative	Reduced treatment severity through combined addition of oxygen and alkali to the wet oxidation process, hence reduced formation of inhibitors	Due to high costs, solvents must be separated, recovered, and reused
Biological
Bacteria and fungi or enzymes	Selective, chemical-free, and energy-efficient	Yield losses due to bioconversion, long incubation times, low productivity, high sensitivity to inhibition

. Furthermore, pretreatment methods can be combined with each other to achieve greater process efficiency [7]. The choice of the appropriate pretreatment method may depend mainly on the biomass considered and its chemical composition.

Physical pretreatment methods (milling, microwave, ultrasound, pyrolysis, and pulsed electric field) degrade lignin by applying pressure, temperature (60–210 °C), vaporization, irradiation, grinding, and milling. The latter two processes focus more on reducing the size of the raw material, aiming to increase the contact surface area. Physical pretreatment methods are ineffective alone, as they do not reduce cellulose crystallinity, nor do they break down only the lignin fraction. They are more commonly used for biomass preprocessing and increase the surface area for an additional pretreatment step. One of the most applicable methods in physical pretreatment are milling and irradiation [7,72].

Chemical pretreatment (alkali, acid, organosolv, ionic liquids, solvents) uses products such as acids, organic solvents, and alkali, among others, to release cellulose. Sulfuric and hydrochloric acids are commonly used in these treatments, acting as catalysts for breaking polymer bonds and dissolving hemicellulose, while cellulose remains broadly unchanged. Alkaline treatment uses sodium hydroxide, lime, and Na₂CO₃ to dissolve lignin and break inter- and intra-unit bonds, leaving the cellulose intact. Ammonia can also be used in this type of pretreatment; however, its use increases the cost of the process, as it requires an additional recovery step [7,72].

Physicochemical treatment processes include steam explosion, liquid hot water, wet oxidation, ammonia, CO₂ explosion, and oxidative treatment. Physical–chemical treatments are the most effective, especially thermochemical treatments, with the ability to reduce crystallinity and delignify and depolymerize the biomass. Steam explosion is one of the most efficient methods, combining high pressure and temperature, causing a thermal and mechanical shock that ruptures lignin. As additional advantages, steam explosion can be combined with other pretreatment methods and does not require chemicals.

Biological pretreatment methods rely on microorganisms such as fungi, brown fungi, hard rot fungi, soft rot fungi, bacteria, and archaea for lignin removal. Although low-cost and environmentally friendly, the processes are still very slow compared to other methods, which hinders their industrial application [7,72]. Efficiencies of different pretreatments applied to different biomasses are displayed in

Table 4. Process conditions in pretreatment methods of lignocellulosic biomasses.

Pretreatment: Type, Technique, Biomass and Reference	Biomass Composition (%) and Process Conditions	Pretreatment Results (%)
Physical Ultrasound—Sugarcane bagasse [73]	Untreated biomass — Lignin: 27.0 ± 0.1, Hemicellulose: 32.0 ± 0.2, and Cellulose: 38.0 ± 0.4. Pretreated biomass — Lignin: 20.7 ± 0.5, Hemicellulose: 29.3 ± 0.8, and Cellulose: 46.9 ± 0.6.	95.60 (Cellulose recovery) 26.70 (Hemicellulose removal) 58.14 (Lignin removal)
Physical Ultrasound, pulsed electric fields, and high voltage—Walnut shells [74]	Untreated biomass — Lignin: 34.92 ± 0.66. Temperature of water: 70 °C, Solid–liquid ratio: 1:10 w/v, Potential ultrasound: 400 W, Ultrasound frequency: 12 kHz, and Maximum voltage of PEF: 40 kV PEF.	33.40 (Cellulose recovery) 17.03 (Lignin removal)
Physical Milling— Sugarcane bagasse 5% w/v [75]	Untreated biomass — Lignin: 22.48 ± 0.05, Hemicellulose: 27.73 ± 0.09, and Cellulose: 44.13 ± 0.12. 90 stainless steel balls with diameter 2 cm, Agitation rate: 250 rpm, Time: 4 h and pauses between 10 min.	Significant particle size reduction (not specified)
Chemical Alkaline—Sugarcane bagasse 10% w/v [76]	Untreated biomass — Lignin: 22.10 ± 0.8, Hemicellulose: 25.80 ± 0.6, and Cellulose: 42.50 ± 0.6. Pretreated biomass — Lignin: 12.3 ± 0.5, Hemicellulose: 22.8 ± 0.9, and Cellulose: 47.4 ± 0.7. NaOH concentration: 1% (w/v), Temperature: 50 °C, and Time: 2 h.	21.40 (Cellulose recovery) 38.10 (Hemicellulose removal) 38.10 (Lignin removal)
Chemical Alkaline + organosolv—Sugarcane bagasse 10% w/v [76]	Untreated biomass — Lignin: 22.1 ± 0.8, Hemicellulose: 25.8 ± 0.6, and Cellulose: 42.5 ± 0.6. Pretreated biomass — Lignin: 8.1 ± 0.3, Hemicellulose: 8.0 ± 0.4, and Cellulose: 66.1 ± 0.7. Phase 1: NaOH: 1% (w/v), T: 50 °C, and Time: 2 h. Phase 2: H3PO4: 85% v/v with chilled acetone, T: 50 °C, and Time: 1 h.	28.40 (Cellulose recovery) 85.80 (Hemicellulose removal) 83.20 (Lignin removal)
Chemical Organosolv— Sugarcane bagasse 10% w/v [76]	Untreated biomass — Lignin: 22.10 ± 0.8, Hemicellulose: 25.80 ± 0.6, and Cellulose: 42.50 ± 0.6. Pretreated biomass — Lignin: 15.0 ± 0.6, Hemicellulose: 15.0 ± 0.8 and Cellulose: 54.5 ± 0.4. Phosphoric acid concentration 85%, v/v with chilled acetone, Temperature: 50 °C, and Time: 1 h.	19.30 (Cellulose recovery) 63.40 (Hemicellulose removal) 56.00 (Lignin removal)
Chemical Ammonia— Sugarcane bagasse [73]	Untreated biomass — Lignin: 27.0± 0.1, Hemicellulose: 32.0 ± 0.2, and Cellulose: 38.0 ± 0.4. Pretreated biomass — Lignin: 50.4 ± 0.1, Hemicellulose: 26.8 ± 0.0 and Cellulose: 50.4 ± 0.1.	95.40 (Cellulose recovery) 39.70 (Hemicellulose removal) 47.20 (Lignin removal)
Chemical Ionic liquid (IL)— Wheat straw [77]	Untreated biomass — Lignin: 18.80 ± 0.58, Cellulose: 35.69 ± 0.33, and Hemicellulose: 26.14 ± 0.28 xylan and 3.54 ± 0.11 arabinan. Pretreated biomass — Lignin: 3.73 ± 0.24, Cellulose: 53.52 ± 0.48, and Hemicellulose: 10.55 ± 0.14 of xylan and 0 arabinan. Triethyl ammonium hydrogen sulfate, Water: 20% wt, Solid solvent ratio: 1:5 g/g, Time: 3 h, and T: 130 °C.	64.00 (Hemicellulose removal) 80.16 (Lignin removal)
Biological Biologic degradation—Cornstalk [78]	Microorganism: Irpex lacteus, and Time: 42 days.	62.50 (Glucan preservation) 40.30 (Xylan preservation) 37.60 (Lignin removal)
Biological Biologic degradation— Bamboo culms [79]	Untreated biomass — 49.6 ± 1.5% of total sugars. Pretreated biomass — 42.9 ± 10.9% glucose, 0.4 ± 0.1% galactose, 1.0 ± 0.3% arabinose and 16.0 ± 4.8% xylose. Microorganism: Punctualaria sp. TUFC20056, and Time: 3 months.	53.30 ± 4.4 (Lignin removal)
Physicochemical Microwave—arginine based deep eutectic solvents— Bamboo 5% w/v [80]	Pretreated biomass — glucan: 50, xylan: 20, and Lignin: 30. Arginine:lactic acid ratio: 1:7, and Temperature: 120 °C.	~80.00 (Glucan recovery) 76.90 (Xylan removal) 84.80 (Lignin removal)
Physicochemical Microwave assisted green solvents — Corn stover [81]	Untreated biomass — Lignin: 22.21 ± 0.11, Hemicellulose: 22.13 ± 0.39, and Cellulose: 36.59 ± 0.45. Pretreated biomass — Lignin: 5.55 ± 0.29, Hemicellulose: 0 and Cellulose: 79.00 ± 5.84. Microwave irradiation: 800 W, T: 160 °C, Time: 30 min, DES: 20 g, and ChCl:LA ratio: 1:2.	88.64 retention of cellulose 100.0 (Hemicellulose removal) 89.75 (Lignin removal)
Physicochemical Microwave—alkali —Corncob [82]	Untreated biomass — Lignin: 20.68, Hemicellulose: 32.65, and Cellulose: 45.35. Pretreated biomass — Lignin: 5.51, Hemicellulose: 22.14 and Cellulose: 71.69. Microwave power: 691.73 W, NaOH: 3.4%, and Irradiation time: 15.5.	32.19 (Hemicellulose removal) 73.36 (Lignin removal)
Physicochemical Microwave assisted sodium chlorite method— Rice straw [83]	Untreated biomass — Lignin: 14.69 ± 1.4, Hemicellulose: 31.70 ± 0.76, and Cellulose: 34.19 ± 0.32. Microwave power: 671 W, Irradiation time: 8.66 min, Bleaching solution: 2.670%, and Bleaching time: 1 h.	Mass yield of 75.90% (Holocellulose + lignin (13.6%)) 93.51 (Lignin removal)
Physicochemical Steam explosion and biologic degradation— Corn straw 1.6 g/L [84]	Untreated biomass — Lignin: 16.37 ± 3.69, Hemicellulose: 24.91 ± 4.80, and Cellulose: 39.54 ± 5.43. Immersed in water for 10 min, 1.6 Mpa, and Time steam explosion: 180 s. Microbial consortium Saccharomycetales (98.92%) and the bacteria of J-6 were mainly Shinella sp. (47.38%), Cupriavidus sp. (29.84%), and Bosea sp. (7.96%). Time for biotreatment: 7 days.	44.24 (Lignin removal)
Physicochemical Induced eletric field-aided dilute acid—Wheat straw 25 g/L [85]	Pretreated biomass — glucan: 33.33 ± 0.19, xylan: 22.74 ± 0.56 and arabinan: 4.31 ± 0.08. Acid concentration: 1%, Time: 5 h, and Temperature: 90 °C.	73.6 (Hemicellulose removal)
Physicochemical Organic solvent and steam explosion— Spruce biomass 6.2% w/w [86]	Untreated biomass — Lignin: 32.60, Hemicellulose: 17.60, and Cellulose: 37.60. Pretreated biomass — Lignin: 15.4, Hemicellulose: 4.0, and Cellulose: 72.0. T: 200 °C, Time: 30 min, and Sulfuric acid concentration: 1% (w/w).	16.30 (Cellulose recovery) 90.20 (Hemicellulose removal) 79.40 (Lignin removal)
Physicochemical Ultrasound assisted ammonia Sugarcane bagasse [73]	Untreated biomass — Lignin: 27.0 ± 0.1, Hemicellulose: 32.0 ± 0.2, and Cellulose: 38.0 ± 0.4. Particle size: 0.2740 mm, Sonication time: 45 min, Ammonia concentration: 10%, LSR 10 mL/g, and Temperature: 80 °C.	95.78 (Cellulose recovery) 58.14 (Lignin removal)

.

Furthermore, during this stage, inhibitory compounds capable of interfering with subsequent hydrolysis and fermentation processes can be generated. The main inhibitory compounds are acetic acid and levulinic acid, value-added by-products, as well as phenolic compounds, furfural and hydroxymethylfurfural (HMF), with the latter two compounds capable of inhibiting the growth of yeast and bacteria. The inhibitory concentration of acetic acid hydrolysates in lignocellulosic ethanol ranges from 0.10–1.10% w/v and may amount to 0.32% w/v for levulinic acid and 0.01–0.59% w/v for HMF (hydroxymethylfurfural) [2,87]. For example, washing of pretreated biomass may be applied as an essential biomass detoxification method for the removal of inhibitory compounds generated during pretreatment of lignocellulosic biomass, such as organic acids, phenols, and furans. Washing of pretreated biomass reduces liquor toxicity and improves the efficiency of the subsequent stages of enzymatic hydrolysis and fermentation. Conversely, detoxification of the liquor containing pretreated biomass, which contains fermentable sugars and inhibitors, can be achieved by physical methods, such as vacuum evaporation, adsorption, and membrane filtration; chemical methods, such as neutralization, precipitation, and ionization; or biological methods, which rely on enzymes or microorganisms to enhance the fermentation [12,88,89].

Currently, combined or multi-stage pretreatment methods are gaining ground. However, there is still a need to develop more economical methodologies with a lower energy demand, low inhibitor formation, and lower pollution potential. Physical pretreatments are essential for biomass preprocessing and important for increasing the contact surface area, favoring subsequent combination with physicochemical and chemical treatments.

In this context, physicochemical treatments have played a fundamental role, and a highly regarded technology is the hydrothermal pretreatment, a low-corrosion method achieving high-energy conversion without the use of catalysts. This technique consists of denaturing the cell wall and degrading hemicellulose through contact with water at critical temperatures. The efficiency of the process increases as contact with the biomass surface increases. If combined with a chemical treatment, its efficiency can be further increased. Future research should focus on optimizing current pretreatment methods, such as their biomass loading, residence time, and operational costs, as well as considering the real scale and aligning with environmental sustainability [7,8,9,10].

Regarding chemical processes, they are considered limited when using low catalyst concentrations such as acid or base concentrations up to 1%, and combining them with other techniques could be more efficient, however they greatly increase the severity of the process what can potentially compromising the degradation of sugars and the possibility of forming inhibitors, degradation compounds, and salts, as well as increasing the corrosiveness of the equipment.

In addition, pretreatments that use chemical compounds and/or solvents, such as ammonia and organosolv, are only applicable if chemical compounds can be recovered in a separation unit and recycled within the process. Finally, biological processes, due to their high cost related to the use of enzymes and microbial propagation or resulting from slow microorganism growth, are not yet competitive.

3.2.2. Hydrolysis of Pretreated Biomass

The hydrolysis stage aims to convert polysaccharides present in cellulose (mainly) and hemicellulose fractions into monosaccharide sugars and can be performed through acidic or enzymatic processes. Acid hydrolysis is generally performed with sulfuric acid (H₂SO₄) or hydrochloric acid (HCl). Concentrated acid treatment can be performed at around 50 °C and at concentrations of 70–90%, resulting in high biomass conversion yields, but causing corrosion in equipment and requiring an additional neutralization step [2,90], hindering large-scale application. In turn, dilute acid treatment requires higher temperatures up to 140–150 °C with low acid concentrations (2–5%), but these processing conditions may result in yield losses due to sugar degradation and enhanced production of inhibitory compounds. In order to circumvent this problem, hydrolysis is generally initiated at moderate temperatures of 170–190 °C to solubilize amorphous regions of hemicellulose, and temperature is subsequently increased to 200–230 °C in order to disrupt the crystalline structure of cellulose [2,90]. Considering the hydrolysis step for ethanol production, the acid hydrolysis is not applied at an industrial scale.

Considering enzymatic hydrolysis, it is preferable due the brand condition for action and specificity in the attack of biomass components and consists of a mixture of enzymes used to cleave cellulose and hemicellulose polymers into monomers, primarily glucose. A broad range of hydrophilic enzymes, including cellulases, hemicellulases, and auxiliary enzymes, are applied. Nevertheless, natural barriers such as the high lignin content and crystallinity of cellulose prevent these enzymes from contacting the substrate, causing low process efficiency, so the choice of biomass pretreatment is essential to secure the efficiency of enzymatic action [91,92].

Cellulases form a family of hydrolytic enzymes exhibiting several types of degradative activity, including endoglucanase, exoglucanase, and β-glucosidase (cellobiohydrolase). The process begins with endoglucanase breaking the glycosidic bonds in the cellulose chain, releasing reducing and non-reducing ends. Exoglucanase subsequently acts on these ends to remove cellulose dimers (cellobiose). Finally, cellobiose molecules are hydrolyzed by beta-glucosidase, yielding glucose (Figure 5a). Furthermore, cellulose hydrolysis can be further facilitated by lytic polysaccharide monooxygenases (LPMOs or LMPOs) as non-hydrolytic auxiliary enzymes, which can break β-1,4-glycosidic bonds of polysaccharide chains through oxidation of C₁ or C₄ carbons of glucose monomers mediated by O₂ or H₂O₂ as an oxidant, facilitating access to the rigid structure of cellulose and increasing the hydrolytic efficiency of cellulases [90,91,92].

Cellulase used in the hydrolysis process for ethanol production is generally obtained from microorganisms, and filamentous fungi and some bacteria stand out due to their high biological capacity for secreting extracellular enzymes. Endoglucanases are produced by the following bacterial genera: Thermomonospora, specifically Thermomospora fusca, and Clostridium. Exoglucanases and β-glucanases are produced by the bacterial genus Streptomyces and from the fungal genera Trichoderma, Aspergillus, Penicillium, and Rhizopus. LPMO enzymes have been isolated from archaea, bacteria, and filamentous fungi [91].

Endo-1,4-β-D-xylanase, an enzyme of the hemicellulase class, breaks down hemicellulose into xylose oligomers, which are in turn converted to xylose by exo-β-xylosidase. Auxiliary enzymes such as β-D-glucuronidase, β-L-arabinase, and β-mannanase break down polymers from other sugar types present in hemicellulose [93,94,95,96]. This mechanism is summarized in Figure 5b. Hemicellulase enzymes have been obtained from the bacteria Bacillus, Streptomyces, Actinomyces, and Xyllella, as well as from the filamentous fungi Aspergillus and Thermophilus [93].

3.2.3. Fermentation of the Hydrolyzed Liquor

Fermentation converts sugars extracted from biomass (glucose, xylose, and others) into ethanol through the action of microorganisms. It can occur separately from the hydrolysis step (Separated Hydrolysis and Fermentation, SHF), simultaneously (Simultaneous Saccharification and Fermentation, SSF), simultaneously but with co-fermentation of the pretreatment liquor (SSF-co), or with all steps, including enzyme production, occurring in one reactor (Consolidated Bioprocessing, CBP), the latter still being at the conceptual stage or applicable with efficiency at the laboratory scale. In SHF, sugars released during hydrolysis are transported to the reactor containing fermenting microorganisms. Disadvantages of this method include the high costs and extended duration of the process, decreasing productivity. Furthermore, very high concentrations of sugar from hydrolysis at the beginning of the fermentation proocess may inhibit the inoculum [90].

Simultaneous saccharification and fermentation (SSF) is carried out in a single reactor containing enzymes capable of releasing carbohydrates, along with fermenting microorganisms, so that hydrolysis and fermentation take place concurrently, thus preventing the issue related to the high inhibitory sugar concentration and higher contamination possibility exhibited by the SHF process. Nevertheless, a major challenge of SSF is that process optimization should encompass optimal conditions for both hydrolysis and fermentation stages, hence rendering the process more complex and sensitive, i.e., both processes should respect the production rates of enzymes and microorganisms, acting in a synergistic way to achieve higher ethanol productivity.

SSF-co is based on the same principle as SSF, but with fermenting microorganisms being able to ferment both hexoses such as glucose, resulting from cellulose hydrolysis, as well as pentoses, resulting from hemicellulose hydrolysis (generally from the pretreatment liquor), and requires the inclusion of microbes in symbiotic relationships, since the yeast Saccharomyces cerevisiae is the most commonly applied strain for industrial bioethanol production and lacks the ability to ferment pentose sugars obtained from the hydrolysis of hemicellulose. Consolidated bioprocessing (CBP) is based on the same configuration as SSF-co, but with enzyme production occurring in the same reactor, so additional enzyme-producing microorganisms are present in the reactor together with fermentative microorganisms [90]. Figure 6 summarizes the different configurations mentioned before, which are also exemplified by published studies compiled in Table 5.

The yeast S. cerevisiae is the main strain used in fermentation to produce bioethanol from glucose, fructose, and sucrose. However, fermentation requires cells to exhibit thermotolerance and resistance to inhibitors such as phenolic compounds, furans, and weak acids. Therefore, these capacities have been improved through genetic engineering. Additionally, studies show that the genes HSP82, SSA4, and HSP104p encode heat shock proteins and can improve yeast temperature resistance. Also, the proteins ASR1 and FPS1 are essential for yeast activity in the presence of alcohol inhibitors.

On the other hand, studies show that a diversity of yeasts is still to be explored for bioethanol production [97]. Considering that S. cerevisiae is unable to efficiently metabolize xylose, genetic recombination studies are currently implemented to improve xylose conversion and increase the ethanol yield. Another approach is the use of other yeasts, such as Scheffersomyces stipitis, Candida shehatae, and Kluyveromyces marxianus, which are capable of metabolizing pentoses [98].

Examples of these strategies are applied in the literature. For example, in the study reported by Jawad et al. [99], S. cerevisiae and Clostridium beijerinckii were co-cultivated in non-detoxified corn straw hydrolysate, after acid pretreatment and enzymatic hydrolysis with Cellic CTec2, obtaining a production of 20.8 g/L of ethanol. In addition, Li et al. [100] applied S. cerevisiae LF1 and S. cerevisiae 6M-15, strains genetically modified to co-ferment C₆ and C₅ sugars, from corn fiber pretreated with acid and hydrolyzed with cellulases produced by the filamentous fungi Penicillium oxalicum and Trichoderma reesei, under controlled fermentation conditions, and they obtained 0.48 g ethanol/g sugar.

Table 5. Examples of process conditions and results of lignocellulosic biomass hydrolysis and fermentation.

Biomass Composition and Pretreatment	Hydrolysis Condition and Efficiency	Fermentation Condition and Efficiency	Reference
Old fibers—palm trees Cellulose: 62.31 ± 0.98%, Hemicellulose: 5.80 ± 0.44%, and Lignin: 21.12 ± 1.13%. Mechanical and thermochemical pretreatment: — Temperature: 55 °C and Time: 12 h; Chemical pretreatment — Temperature: 220 °C, and Concentration: H2SO4 0.025M. Treated biomass: Lignin: 28.45%, and Holocellulose (Cellulose + Hemicellulose): 49.61%.	Enzymes Celluclast 1.5 L and Novozyme® 188 (36.6 FPU of cellulases and 356.4 IU of β-glucosidase by pretreated biomass). Temperature: 50 °C, Rotating speed: 450 rpm, Time: 72 h, and TRS: 34.60 g/L, with 0.46–0.49 g TRS per g pretreated biomass (0.31–0.35 g glucose/g pretreated biomass).	SHF—Phoenix dactylifera L. Temperature: 30 °C, pH: 4.5, and Rotating speed: 450 rpm. Hydrolyzed fibers: glucose: 33.61 g/L, arabinose: 0.41 g/L, xylose: 0.61 g/L, andgalactose: 0.13 g/L. Hydrolysis liquor with 34.60 TRS g/L. Results: 10.88 g ethanol/100 g pretreated biomass and 0.38 g ethanol/g TRS.	[101]
Rice straw Cellulose: 31.90 ± 0.20%, Hemicellulose: 28.10 ± 0.27%, and Lignin: 3.80 ± 0.40%. Biological pretreatment.	Pecoramyces sp. F1. 8.33 g/L rice straw.	CBP—Zymomonas mobilis ATCC 31821. Time of treatment: 4 days. Results: 0.32 g ethanol/g glucose, 0.98 g/L, and ethanol fermentation efficiency of 92%.	[102]
Palm wood Cellulose: 19.80%, Hemicellulose: 10.90%, and Lignin: 11.00%. Chemical pretreatment (3% sulfuric, nitric, and phosphoric acids), and biomass concentration 10% (w/v). Treated biomass: Lignin: 8.00–10.20%, Hemicellulose: 7.30–9.10%, and Cellulose: 23.30–20.50%.	Cellulase produced by T reesei MTCC 4876 during growth in pretreated biomass. pH: 4.5, and Agitation: 150 rpm. Reducing sugar: 33.15–52.65 g/L, Time: 96 h, and Total Sugars: 70 g/L.	SHF—K. marxianus MTCC 1389. Temperature: 45 °C, Agitation rate: 156 rpm, pH: 5, Substrate concentration: 8.0% (v/v), Inoculum size: 3.2% (v/v), and Ethanol yield of 22.90 g/L.	[103]
Sugarcane bagasse Chemical pretreatment applying Temperature: 145 °C, Pressure: 1 atm, Time: 12 min, and Concentration acid: 0.5% H2SO4 v/v) and 10% w/v of bagasse. Lignin: 34.73 ± 3.58, Cellulose: 44.80 ± 0.04, and Hemicellulose: 2.02 ± 0.79.	Cellulolytic Cocktail Cellic® CTec2 (12.5 FPU/g pretreated bagasse). Biomass concentration: 16.5% of pretreated bagasse.	SSF—S. cerevisiae AGY001 (CRISPR/Cas9 modified). Treatment duration: 48 h. First stage: Aerobic regime, Temperature: 30 °C; Rotating speed: 200 rpm agitation overnight. Second stage: semi-anaerobiosis, Temperature: 40 °C. OD600: 1.0. Results: Ethanol yield of 6.88 ± 0.84 g/L and 89% fermentation efficiency.	[104]
Sugarcane bagasse Cellulose: 44%, Hemicellulose: 24%, and Lignin: 19%. Hydrothermal pretreatment applying Temperature: 195 °C, Time: 10 min, Agitation: 300 rpm, and Ratio: 1:10 w/v. Pretreated solid fraction: Cellulose: 59%, Hemicellulose: 12%, and Lignin: 26%.	Enzymes secreted by S. cerevisiae AC14 (Cellobiohydrolase I, Cellobiohydrolase II, Endoglucanase, β-Glucosidase, Xylanase, β-Xylosidase, Acetylxylan esterase). Cell concentration: 80 g/L.	CBP—S. cerevisiae. 10 g/L of pretreated bagasse. OD600: 100, pH: 5.5, Temperature: 35 °C, Time: 7 h, and Hydrothermal liquor (g/L): xylose: 4.22, glucose: 0.38, cellobiose: 1.15, and Total xylooligomers: 14.68. Results: 1.86 g/L/h, 13 g/L, and 89% of ethanol productivity, concentration, and fermentation efficiency, respectively.	[105]
Corncob residue Chemical pretreatment with 0.1 M citric acid and trisodium citrate, and pH 6.2. Treated biomass: Lignin: 35.0%, Hemicellulose: 5.4%, and Cellulose: 62.7%.	Cellic® CTec3 8.8 FPU/g of solids. Time of enzymatic hydrolysis: 72 h. 25% dry matter. Temperature: 35 °C, pH: 5.0, Agitation: 150 rpm, and Inoculation ratio: 1% w/w. Cellulose conversion: 85%.	SHF—S. cerevisiae, Zymomonas mobilis, and genetically related engineered strains. Glucose initial: 140 g/L. Temperature: 35 °C, pH: 5.0, Agitation: 200 rpm, and Time: 144 h. Results: 62.41 g/L of ethanol and 6.89 g ethanol/100 g biomass.	[106]
Corn stover 12.60 ± 0.6% Lignin. Chemical pretreatment applying Steam explosion; Temperature: 210 °C, Residence time: 5 min, and Initial moisture content: 30%. Treated biomass: Lignin: 23.10 ± 1.5%.	Novozyme® 188 (60 FPU/g glucan). Solid loading: 20% (12% glucan loading).	SSF—S. cerevisiae 80000012 a. Temperature: 39 °C, pH: 4.8, 50% nutrients added; 12% glucan loading. OD600: 4. Initial sugar concentration: glucan: 31.10 ± 1.1% and xylan: 17.10 ± 0.7%. Results: 59.8 g/L, 77.2%, and 78.6% of ethanol yield, sugars, and glucan fermentation efficiency.	[107]
Rice straw Chemical pretreatment with Dilute NaOH 10% w/v, Temperature: 121 °C, Pressure: 15 psi, and Time: 45 min.	15 FPU of cellulase blend (Sigma-Aldrich®, St. Louis, MO, USA). 5% of pretreated biomass. pH 4.8 with 50 mM citrate buffer.	SSF—K. marxianus MSS6.3, 5% w/v dried rice straw, Time of treatment: 48 h, Temperature: 45 °C, Rotating speed: 150 rpm, and OD600: 1. Initial sugars in g/L: glucose: 24.20, xylose: 7.400, and arabinose: 1.30 g/L. Results: 10.9 g/L of ethanol, 85% fermentation efficiency, productivity of 0.22 g L−1 h−1, and overall efficiency of 46%.	[108]
Corn stover Lignin: 11.00%, Glucan: 34.10%, and Xylan: 20.40%. Chemical pretreatment, AFEX treatment of ammonia to biomass loading 1.0 g/g dry biomass, Water loading 0.6 g/g dry biomass, Temperature: 140 °C, and Residence time: 15 min.	Enzymes produced during cultivation of Clostridium phytofermentans ATCC 700394.	CBP—Clostridium phytofermentans ATCC 700394. 0.5% (w/w) glucan loading AFEX-Corn Stover. Agitation rate: 200 rpm, Temperature: 30 °C, Inoculation: 5% v/v, pH: 7, and Time: 10 days. Results: 76% glucan conversion, 88% xylan conversion, 2.8 g/L of ethanol yield, and ethanol/acetate ratio: 1.08.	[109]
Corncob Prepared based on 70% (w/w) concentrated xylose crystallization mother. Liquid supplemented with 364.0 g/L glucose and volume ratio of 1:1.	Pre-cultured cells of Escherichia B0013-2021HPA (Phosphoenolpyruvate:glucose phosphotransferase (PTase), pyruvate decarboxylase (PDC), and alcohol dehydrogenase II (ADH II).	SHF—E. coli B0013-2021HPA3 l. Volume containing 300 mL of hydrolysate for growth + 300 mL of hydrolysate for ethanol production. pH: 7. Initial sugar concentration (g/L): glucose: 266.0, xylose: 200.0, galactose: 27.00, and arabinose: 114.5. Results: 127.7 total g ethanol produced and 2.85 g/L/h of productivity.	[110]
Rice husk — Cellulose: 37.74%, Hemicellulose: 33.85%, and Lignin: 9.67 + Corn cob — Cellulose: 36.84%, Hemicellulose: 24.61%, and Lignin 13.73%.	Cellulase and xylanase. Cellulolytic activities: 219.9 ± 18.64 FPU/mL. Xylanolytic activities: 333.4 ± 22.74 U/mL.	SSF-co—S. cerevisiae and K. marxianus. Ratio: 50:50 v/v; Time of treatment: 72 h; and 124.6 g of TRS/L of hydrolysate. Results: 55.56 ± 0.19 g/L of ethanol, 88.32% fermentation efficiency, and overall efficiency of 46.70%.	[111]
Brewing spent grain, barley straw, and oak shavings Biological pretreatment with Time: 7–18 days at Temperature: 25 °C.	Trichoderma viride cellulolytic enzymes. Temperature: 25 °C, Time: 21 days, and 3.4 L solution final.	SHF—S. cerevisiae. pH: 5, Time: 14 days, and Temperature: 30 °C. Ethanol fermentation efficiency of 65%.	[112]
Sugarcane bagasse Lignin: 25.21%, xylan: 21.04%, and glucan: 39.60%. Chemical pretreatment applying Concentration: 20% NH4OH, Liquid ratio: 1:10, Temperature: 50 °C, and Time: 48 h. Treated biomass: Lignin: 19.49%, xylan: 5.20%, and glucan: 45.96%.	Cellic® CTec2 (10 FPU/gdry biomass). 40% solid loading, Temperature: 50 °C, Agitation rate: 150 rpm, Laccase mediator (200 U/g dry biomass), and Time: 96 h.	SHF—Candida tropicalis Y-27290. Inoculum concentration of 10.00%, Temperature: 28 °C, Agitation rate: 200 rpm. Initial sugars: glucose: 157.6 g/L and xylose: 57.12 g/L. Results: 72.40 ± 1.9 g/L ethanol produced, 0.46 ± 0.03 g/g glucose ethanol productivity, 1.21 ± 0.02 g/L/h volumetric ethanol productivity, and 89% ethanol yield.	[113]
Wheat straw Lignin: 22%, xylan: 22%, and glucan: 36.7%. Dry matter: 94.40%. Chemical pretreatment applying Wheat straw loading of 7.5% w/w, Temperature: 140 °C, Time: 20.5 min, and H2SO4 concentration: 1% w/w. Treated biomass: xylose: 22 g/L and glucan: 29 g/L.	Enzymes: Spezyme CP cellulase and Novozyme® 188. Enzyme loading: 15 FPU gglucan−1 and 30 CBU gglucan−1.	CBP—T. reesei Rut C30, S. cerevisiae, and Scheffersomyces stipites. Agitation rate: 150 rpm, and Temperature: 28 °C. Initial sugars: glucose: 29 g/L and xylose: 22 g/L. Result: 67% ethanol fermentation efficiency.	[114]
Sugarcane bagasse Cellulose: 39.72 ± 0.33%, Hemicelluloses: 26.96 ± 2.11%, and Lignin: 40.98 ± 0.01%. Chemical pretreatment with Solution of sodium hydroxide 15 g/L, and hydrogen peroxide: 2%, 50 g of bagasse, and pH 7. Treated biomass: Cellulose: 72.1%, Hemicellulose: 24.4%, and Lignin and ash: 3.5%.	Enzymes obtained during microorganisms’ growth. Cellulase concentration: 100 FPU/mL.	SSF-co—Zymomonas mobilis (PVA immobilized cells) and Pichia stipitis (suspended cells). 20 g/L pretreated bagasse. Cell loading: 40% w/v and OD600: 20. Initial sugars: glucose 28.2 g/L, xylose 6.9 g/L, cellobiose 7.5 g/L, and Cellulose 20 g/L. Results: 0.414 g ethanol/g cellulose, 81.20% of theoretical yield, and 0.705 g/L/h ethanol productivity.	[115]

SHF: separated hydrolysis and fermentation; SSF: simultaneous saccharification and fermentation; SSF-co: co-fermentation; CBP: consolidated bioprocess; TRS: total reducing sugars; FPU: filter paper unit; OD₆₀₀: optical density at 600 nm.

Table 5. Examples of process conditions and results of lignocellulosic biomass hydrolysis and fermentation.

Biomass Composition and Pretreatment	Hydrolysis Condition and Efficiency	Fermentation Condition and Efficiency	Reference
Old fibers—palm trees Cellulose: 62.31 ± 0.98%, Hemicellulose: 5.80 ± 0.44%, and Lignin: 21.12 ± 1.13%. Mechanical and thermochemical pretreatment: — Temperature: 55 °C and Time: 12 h; Chemical pretreatment — Temperature: 220 °C, and Concentration: H2SO4 0.025M. Treated biomass: Lignin: 28.45%, and Holocellulose (Cellulose + Hemicellulose): 49.61%.	Enzymes Celluclast 1.5 L and Novozyme® 188 (36.6 FPU of cellulases and 356.4 IU of β-glucosidase by pretreated biomass). Temperature: 50 °C, Rotating speed: 450 rpm, Time: 72 h, and TRS: 34.60 g/L, with 0.46–0.49 g TRS per g pretreated biomass (0.31–0.35 g glucose/g pretreated biomass).	SHF—Phoenix dactylifera L. Temperature: 30 °C, pH: 4.5, and Rotating speed: 450 rpm. Hydrolyzed fibers: glucose: 33.61 g/L, arabinose: 0.41 g/L, xylose: 0.61 g/L, andgalactose: 0.13 g/L. Hydrolysis liquor with 34.60 TRS g/L. Results: 10.88 g ethanol/100 g pretreated biomass and 0.38 g ethanol/g TRS.	[101]
Rice straw Cellulose: 31.90 ± 0.20%, Hemicellulose: 28.10 ± 0.27%, and Lignin: 3.80 ± 0.40%. Biological pretreatment.	Pecoramyces sp. F1. 8.33 g/L rice straw.	CBP—Zymomonas mobilis ATCC 31821. Time of treatment: 4 days. Results: 0.32 g ethanol/g glucose, 0.98 g/L, and ethanol fermentation efficiency of 92%.	[102]
Palm wood Cellulose: 19.80%, Hemicellulose: 10.90%, and Lignin: 11.00%. Chemical pretreatment (3% sulfuric, nitric, and phosphoric acids), and biomass concentration 10% (w/v). Treated biomass: Lignin: 8.00–10.20%, Hemicellulose: 7.30–9.10%, and Cellulose: 23.30–20.50%.	Cellulase produced by T reesei MTCC 4876 during growth in pretreated biomass. pH: 4.5, and Agitation: 150 rpm. Reducing sugar: 33.15–52.65 g/L, Time: 96 h, and Total Sugars: 70 g/L.	SHF—K. marxianus MTCC 1389. Temperature: 45 °C, Agitation rate: 156 rpm, pH: 5, Substrate concentration: 8.0% (v/v), Inoculum size: 3.2% (v/v), and Ethanol yield of 22.90 g/L.	[103]
Sugarcane bagasse Chemical pretreatment applying Temperature: 145 °C, Pressure: 1 atm, Time: 12 min, and Concentration acid: 0.5% H2SO4 v/v) and 10% w/v of bagasse. Lignin: 34.73 ± 3.58, Cellulose: 44.80 ± 0.04, and Hemicellulose: 2.02 ± 0.79.	Cellulolytic Cocktail Cellic® CTec2 (12.5 FPU/g pretreated bagasse). Biomass concentration: 16.5% of pretreated bagasse.	SSF—S. cerevisiae AGY001 (CRISPR/Cas9 modified). Treatment duration: 48 h. First stage: Aerobic regime, Temperature: 30 °C; Rotating speed: 200 rpm agitation overnight. Second stage: semi-anaerobiosis, Temperature: 40 °C. OD600: 1.0. Results: Ethanol yield of 6.88 ± 0.84 g/L and 89% fermentation efficiency.	[104]
Sugarcane bagasse Cellulose: 44%, Hemicellulose: 24%, and Lignin: 19%. Hydrothermal pretreatment applying Temperature: 195 °C, Time: 10 min, Agitation: 300 rpm, and Ratio: 1:10 w/v. Pretreated solid fraction: Cellulose: 59%, Hemicellulose: 12%, and Lignin: 26%.	Enzymes secreted by S. cerevisiae AC14 (Cellobiohydrolase I, Cellobiohydrolase II, Endoglucanase, β-Glucosidase, Xylanase, β-Xylosidase, Acetylxylan esterase). Cell concentration: 80 g/L.	CBP—S. cerevisiae. 10 g/L of pretreated bagasse. OD600: 100, pH: 5.5, Temperature: 35 °C, Time: 7 h, and Hydrothermal liquor (g/L): xylose: 4.22, glucose: 0.38, cellobiose: 1.15, and Total xylooligomers: 14.68. Results: 1.86 g/L/h, 13 g/L, and 89% of ethanol productivity, concentration, and fermentation efficiency, respectively.	[105]
Corncob residue Chemical pretreatment with 0.1 M citric acid and trisodium citrate, and pH 6.2. Treated biomass: Lignin: 35.0%, Hemicellulose: 5.4%, and Cellulose: 62.7%.	Cellic® CTec3 8.8 FPU/g of solids. Time of enzymatic hydrolysis: 72 h. 25% dry matter. Temperature: 35 °C, pH: 5.0, Agitation: 150 rpm, and Inoculation ratio: 1% w/w. Cellulose conversion: 85%.	SHF—S. cerevisiae, Zymomonas mobilis, and genetically related engineered strains. Glucose initial: 140 g/L. Temperature: 35 °C, pH: 5.0, Agitation: 200 rpm, and Time: 144 h. Results: 62.41 g/L of ethanol and 6.89 g ethanol/100 g biomass.	[106]
Corn stover 12.60 ± 0.6% Lignin. Chemical pretreatment applying Steam explosion; Temperature: 210 °C, Residence time: 5 min, and Initial moisture content: 30%. Treated biomass: Lignin: 23.10 ± 1.5%.	Novozyme® 188 (60 FPU/g glucan). Solid loading: 20% (12% glucan loading).	SSF—S. cerevisiae 80000012 a. Temperature: 39 °C, pH: 4.8, 50% nutrients added; 12% glucan loading. OD600: 4. Initial sugar concentration: glucan: 31.10 ± 1.1% and xylan: 17.10 ± 0.7%. Results: 59.8 g/L, 77.2%, and 78.6% of ethanol yield, sugars, and glucan fermentation efficiency.	[107]
Rice straw Chemical pretreatment with Dilute NaOH 10% w/v, Temperature: 121 °C, Pressure: 15 psi, and Time: 45 min.	15 FPU of cellulase blend (Sigma-Aldrich®, St. Louis, MO, USA). 5% of pretreated biomass. pH 4.8 with 50 mM citrate buffer.	SSF—K. marxianus MSS6.3, 5% w/v dried rice straw, Time of treatment: 48 h, Temperature: 45 °C, Rotating speed: 150 rpm, and OD600: 1. Initial sugars in g/L: glucose: 24.20, xylose: 7.400, and arabinose: 1.30 g/L. Results: 10.9 g/L of ethanol, 85% fermentation efficiency, productivity of 0.22 g L−1 h−1, and overall efficiency of 46%.	[108]
Corn stover Lignin: 11.00%, Glucan: 34.10%, and Xylan: 20.40%. Chemical pretreatment, AFEX treatment of ammonia to biomass loading 1.0 g/g dry biomass, Water loading 0.6 g/g dry biomass, Temperature: 140 °C, and Residence time: 15 min.	Enzymes produced during cultivation of Clostridium phytofermentans ATCC 700394.	CBP—Clostridium phytofermentans ATCC 700394. 0.5% (w/w) glucan loading AFEX-Corn Stover. Agitation rate: 200 rpm, Temperature: 30 °C, Inoculation: 5% v/v, pH: 7, and Time: 10 days. Results: 76% glucan conversion, 88% xylan conversion, 2.8 g/L of ethanol yield, and ethanol/acetate ratio: 1.08.	[109]
Corncob Prepared based on 70% (w/w) concentrated xylose crystallization mother. Liquid supplemented with 364.0 g/L glucose and volume ratio of 1:1.	Pre-cultured cells of Escherichia B0013-2021HPA (Phosphoenolpyruvate:glucose phosphotransferase (PTase), pyruvate decarboxylase (PDC), and alcohol dehydrogenase II (ADH II).	SHF—E. coli B0013-2021HPA3 l. Volume containing 300 mL of hydrolysate for growth + 300 mL of hydrolysate for ethanol production. pH: 7. Initial sugar concentration (g/L): glucose: 266.0, xylose: 200.0, galactose: 27.00, and arabinose: 114.5. Results: 127.7 total g ethanol produced and 2.85 g/L/h of productivity.	[110]
Rice husk — Cellulose: 37.74%, Hemicellulose: 33.85%, and Lignin: 9.67 + Corn cob — Cellulose: 36.84%, Hemicellulose: 24.61%, and Lignin 13.73%.	Cellulase and xylanase. Cellulolytic activities: 219.9 ± 18.64 FPU/mL. Xylanolytic activities: 333.4 ± 22.74 U/mL.	SSF-co—S. cerevisiae and K. marxianus. Ratio: 50:50 v/v; Time of treatment: 72 h; and 124.6 g of TRS/L of hydrolysate. Results: 55.56 ± 0.19 g/L of ethanol, 88.32% fermentation efficiency, and overall efficiency of 46.70%.	[111]
Brewing spent grain, barley straw, and oak shavings Biological pretreatment with Time: 7–18 days at Temperature: 25 °C.	Trichoderma viride cellulolytic enzymes. Temperature: 25 °C, Time: 21 days, and 3.4 L solution final.	SHF—S. cerevisiae. pH: 5, Time: 14 days, and Temperature: 30 °C. Ethanol fermentation efficiency of 65%.	[112]
Sugarcane bagasse Lignin: 25.21%, xylan: 21.04%, and glucan: 39.60%. Chemical pretreatment applying Concentration: 20% NH4OH, Liquid ratio: 1:10, Temperature: 50 °C, and Time: 48 h. Treated biomass: Lignin: 19.49%, xylan: 5.20%, and glucan: 45.96%.	Cellic® CTec2 (10 FPU/gdry biomass). 40% solid loading, Temperature: 50 °C, Agitation rate: 150 rpm, Laccase mediator (200 U/g dry biomass), and Time: 96 h.	SHF—Candida tropicalis Y-27290. Inoculum concentration of 10.00%, Temperature: 28 °C, Agitation rate: 200 rpm. Initial sugars: glucose: 157.6 g/L and xylose: 57.12 g/L. Results: 72.40 ± 1.9 g/L ethanol produced, 0.46 ± 0.03 g/g glucose ethanol productivity, 1.21 ± 0.02 g/L/h volumetric ethanol productivity, and 89% ethanol yield.	[113]
Wheat straw Lignin: 22%, xylan: 22%, and glucan: 36.7%. Dry matter: 94.40%. Chemical pretreatment applying Wheat straw loading of 7.5% w/w, Temperature: 140 °C, Time: 20.5 min, and H2SO4 concentration: 1% w/w. Treated biomass: xylose: 22 g/L and glucan: 29 g/L.	Enzymes: Spezyme CP cellulase and Novozyme® 188. Enzyme loading: 15 FPU gglucan−1 and 30 CBU gglucan−1.	CBP—T. reesei Rut C30, S. cerevisiae, and Scheffersomyces stipites. Agitation rate: 150 rpm, and Temperature: 28 °C. Initial sugars: glucose: 29 g/L and xylose: 22 g/L. Result: 67% ethanol fermentation efficiency.	[114]
Sugarcane bagasse Cellulose: 39.72 ± 0.33%, Hemicelluloses: 26.96 ± 2.11%, and Lignin: 40.98 ± 0.01%. Chemical pretreatment with Solution of sodium hydroxide 15 g/L, and hydrogen peroxide: 2%, 50 g of bagasse, and pH 7. Treated biomass: Cellulose: 72.1%, Hemicellulose: 24.4%, and Lignin and ash: 3.5%.	Enzymes obtained during microorganisms’ growth. Cellulase concentration: 100 FPU/mL.	SSF-co—Zymomonas mobilis (PVA immobilized cells) and Pichia stipitis (suspended cells). 20 g/L pretreated bagasse. Cell loading: 40% w/v and OD600: 20. Initial sugars: glucose 28.2 g/L, xylose 6.9 g/L, cellobiose 7.5 g/L, and Cellulose 20 g/L. Results: 0.414 g ethanol/g cellulose, 81.20% of theoretical yield, and 0.705 g/L/h ethanol productivity.	[115]

SHF: separated hydrolysis and fermentation; SSF: simultaneous saccharification and fermentation; SSF-co: co-fermentation; CBP: consolidated bioprocess; TRS: total reducing sugars; FPU: filter paper unit; OD₆₀₀: optical density at 600 nm.

4. Characteristics of 2G Ethanol Plants Worldwide

Increasing demand for alternative and renewable energy sources, allied with the need to mitigate environmental impacts associated with fossil fuels, is driving the bioethanol industry to invest in research and development of new technological innovations, encompassing the study of various biomass types as feedstocks to produce second-generation ethanol, especially biomass residues and biowastes. These efforts are resulting in a transition from the laboratory scale to large industrial units (biorefineries), capable of operating with high efficiency and sustainability [116,117].

In the United States, most components of the maize crop (cobs, ears, leaves, husks, and stems) will be used as material to produce 2G ethanol by the LIBERTY Project biorefinery operated by POET-DSM Advanced Biofuels (Emmetsburg, Iowa), achieving biofuel generation on a scale of 25 million gallons per year. Based on an existing 1G corn ethanol plant, more easily degradable above-ground parts (upper stalks) will be applied as feedstock in the new bioethanol plant, while the lower parts (corn stover/stubble), more difficult to valorize, will be returned to the soil [118].

In Brazil, the production of second-generation ethanol goes through a process of intense development and transformation. An example is the Costa Pinto sugarcane plant launched in 2015, belonging to Raízen Energia S/A (Piracicaba, Brazil) and located in the state of São Paulo. Initially, this unit used a technology based on genetically modified yeasts, proposed by Iogen Energy, and transformed the waste from the production of sugar and 1G ethanol from sugarcane bagasse into additional 2G ethanol, reaching a production of 40,000 ton ethanol between the years 2019 and 2020. Then, in 2025, it was announced that the bioethanol plant would change its focus to a research and development unit for the biofuel sector. Recently, in 2024, a new 2G ethanol production plant was inaugurated by the company, also located in the state of São Paulo, which promises to be the largest 2G ethanol plant in the world, with a production capacity of 82,000 m³/year. In Alagoas, the BioFlex plant, installed in 2017 by GranBio Investimentos SA, will continue to produce lignocellulosic ethanol through sugarcane straw conversion and, starting in 2025, the plant will also valorize sugarcane bagasse from the vertix energy variety, a species specially developed by the company for bioenergy production [36,119]. According to Malik [120], the ethanol production capacity of this plant will reach a mark of 60,000 L by 2024–2025.

In developing countries such as India, public policies are being implemented to encourage second-generation ethanol production, leading to the installation of demonstration plants dedicated to developing 2G ethanol production lines to commercialize the technology [121]. One of the initial milestones of this movement was the installation of an experimental plant in Kashipur, in the state of Uttarakhand, in 2016. The project, led by the Mumbai Institute of Chemical Technology in partnership with the Department of Biotechnology, converted a variety of waste materials, such as sugarcane bagasse, rice and wheat straw, cotton stalks, bamboo, and chopped wood, into approximately 300 L of ethanol for each ton of feedstock used. Similarly, the Indian Oil Corp. refinery in the city of Panipat implemented a rice straw bioethanol production plant, with a production of 100 m³/d. Furthermore, in partnership with Chempolis, Assam Bio-Refinery implemented a bioethanol production plant with a capacity of 60,000 m³/year, in addition to 11,000 tons of acetic acid, 19,000 tons of furfural, and 144 GWh of green energy [120,121].

In Austria, the 2G bioethanol plant of AustroCel Hallein GmbH (Hallein, Austria) generates 30,000 m³/year of bioethanol from pulp mill waste using a continuous production process with Valmet DNA-controlled automation. In Norway, the biorefinery of Borregaard AS (Sarpsborg, Norway) produces 20,000 m³/year of high-purity (99.9%) ethanol annually, free of genetically modified organisms and antibiotics, from Norway Spruce, an inherent by-product of paper pulp processing [122].

In Romania, with significant co-funding from the European Union in association with major companies in the framework of the BBI (Bio-Based Industries) joint undertaking, Clariant AG (Podari, Romania) built a large-scale commercial cellulosic bioethanol plant, using agricultural waste as an input. Full production was reached in 2020, processing 250,000 t of feedstock and generating 50,000 t ethanol annually, using sunliquid^® technology [123]. Nevertheless, following economic losses, the unit was subsequently taken over by Cordem Biochem GmbH (Frankfurt, Germany), with plans to repurpose the unit for 1G ethanol production.

Given the strategic importance of biorefineries for the use of renewable resources, several facilities have already been implemented in different regions of the world (

Table 6. Industrial plants operating around the world for bioethanol production.

Company Name/ Facility, City/Country, and Feedstock	Process Characteristics	Capacity	References
Praj Industries (Pune, India) Sugarcane bagasse, rice straw, agricultural wastes	ENFINITY® pretreatment technology, pretreated only by steam; customized cellulolytic enzyme, optimized yeasts, enzymatic hydrolysis (efficiently cleave C5 and C6 sugars), and Separated Hydrolysis and Fermentation (SHF).	4400 (ton/year)/TRL	[14,125]
Clariant (Straubing, Germany) Wheat straw, corn stover, miscanthus, sugarcane bagasse	SUNLIQUID® pretreatment technology,; mechanical and thermal pretreatment, integrated process including optimized enzyme production and simultaneous conversion of C5 and C6 into ethanol, and Separated Hydrolysis and Fermentation + Simultaneous Saccharification and Co-Fermentation (SHF + SSF-co).	4500/TRL, to be repurposed for 1G ethanol production	[14,126]
INBICON (Fredericia, Denmark) Straw, corn	Three-stage continuous process: mechanical conditioning of the biomass, hydrothermal pretreatment conducted at 180–200 °C, and enzymatic hydrolysis performed at high solid loading. The hydrothermal pretreatment based on autohydrolysis (compressed hot water without the addition of chemical catalysts), and Separated Hydrolysis and Fermentation (SHF).	4300 ton/year	[126]
ABENGOA (Sevilha, Spain) Corn	The process employs steam explosion technology for biomass pretreatment, sulfuric acid-catalyzed steam explosion followed by enzymatic hydrolysis using proprietary, in-house-produced enzymes. ST1 Gothenburg.	75,000 ton/year	[126]
Borregaard AS (Sarpsborg, Norway) Sugarcane bagasse, straw, wood, energy crops, and others	BALI™ sulfite pretreatment for biomass conversion into sugars or fermentation, cellulase enzymes, and Separated Hydrolysis and Fermentation (SHF).	365/TRL	[14,122]
GranBio (São Miguel dos Campos, Brazil) Lignocellulosic biomass	PROESA® pretreatment technology, uncatalyzed steam explosion pretreatment followed by simultaneous saccharification and fermentation, Novozymes enzymes, DSM yeasts, and Simultaneous Saccharification and Co-Fermentation (SSF-co).	30,000 m3/year, to be repurposed as integrated biorefinery	[127,128]
AGRANA Biorefinery (Pischelsdorf, Austria) Wheat, maize	-	250,000 m3/year	[129]
Zeitz Commercial (Zeitz, Germany) Sugar beet, grain	-	-	[129]
ST1 Gothenburg, Ethanol (Etanolix) Plant (NEOT) Commercial (Gothenburg, Sweden) Residues: biowaste and process residue from local bakeries and bread from shops	Etanolix® plan, alcohol fuels from cellulosic sugars, process units: feedstock reception and pretreatment, enzymatic hydrolysis, fermentation, and ethanol distillation, and Separated Hydrolysis and Fermentation (SHF).	5000 m3 of advanced bioethanol	[129,130]
AustroCel Biorefinery—Hallein. Commercial (Hallein, Austria) Cellulose waste	No pretreatment, Spent Sulfite Liquor (SSL), different yeast strains, hydrolysis not separately reported, sugars are already dissolved in brown liquor, and Separated Hydrolysis and Fermentation (SHF).	160,000 ton/year	[129,131]
Biochemtex-Crescentino (Crescentino, Italy) Lignocellulosic biomass	PROESA® pretreatment technology (hydrolysis is proprietary), enzymatic hydrolysis, and Simultaneous Saccharification and Co-Fermentation (SSF-co).	-	[129,132]
Domsjö Fabriker Commercial (Örnsköldsvik, Sweden) Forestry raw material:wood	No pretreatment, sulfite pulping process acts as a pretreatment, Spent Sulfite Liquor (SSL), hydrolysis not separately reported, sugars are already dissolved in liquor, and Separated Hydrolysis and Fermentation (SHF).	20,000 tons	[129,133]
Abengoa Bioenergy (St. Louis, MO, USA) Wheat straw, corn stover, and various perennial grasses	Dilute acid and steam explosion pretreatment, cellulase enzymes, proprietary in-house enzyme production for the enzymatic hydrolysis stage, and Separated Hydrolysis and Fermentation (SHF).	25,000 ton/year	[134,135]
LanzaTech (Skokie, IL, USA) Woody biomass	No pretreatment, gaseous fermentation; syngas/CO/H2, and acetogenic microbes use CO and H2 to produce ethanol.	15,000 ton/year	[136,137]

), strengthening 2G ethanol production, as well as the economic and energy balance. This reflects not only technical advancements but also the search for sustainable alternatives to fossil fuels. However, the implementation and operation of biorefineries does not depend exclusively on biochemical fundamentals or the viability of the technologies involved. It is also necessary to consider logistical factors, existing infrastructure, biomass supply chains, industrial and environmental risks, market penetration, socioeconomic aspects, and the political context of each region [124].

5. Enzymatic Cocktails Applied for 2G Ethanol Production

As mentioned in the previous sections, several stages are involved in the process of producing ethanol from lignocellulosic biomass, including hydrolysis, the process responsible for converting polysaccharides present in the biomass into fermentable monosaccharides. Hydrolysis of lignocellulosic biomass requires the association of a range of different steps acting on the degradation of cellulose from both its internal structures and ends, such as endo-1,4-glucanases and exo-1,4-glucanases exposing cellobiose, along with β-glucosidase converting cellobiose into glucose [116,138].

The optmization of the enzymatic hydrolysis steps still faces many challenges: (i) achieving high and stable hydrolysis rates through the development of enzymatic cocktails with applicability in several biomass types; (ii) using a low enzyme load during the process; (iii) decreasing production costs, including techniques for fermentation and concentration/purification of the enzymes; (iv) increasing the stability of enzymatic cocktails in conservation and in the process; and (v) improving the synergistic effect of the several enzymes present in the cocktail. Also, in each point, genetic engineering can be used to develop new and improved enzymatic cocktails.

The industrial market of enzyme production is already providing well-established enzyme cocktails, such as Accellerase^® 1500 and Accellerase^® TRIO, formed by a range of different enzymes that include exoglucanase, endoglucanase, hemicellulase, and betaglucosidase, both produced by DuPont Industrial Biosciences LLC (Wilmington, DE, USA) [139]. The first cocktail has a similar composition to Viscozyme^® L from Novonesis A/S (formerly/previously Novozymes), displaying exoglucanase, endoglucanase, hemicellulase, and betaglucosidase activities, produced by Aspergillus sp. and applicable to the degradation of pectin-rich biomasses such as sugar pulp, while the latter product is obtained from a genetically modified strain of the fungus T. reesei, allowing its application in different lignocellulosic biomasses, such as straw, bagasse, wood chips, etc.

Further enzyme cocktails were previously produced by DuPont (previously Genencor), such as Multifect displaying xylanase and pectinase activities, containing β-glucosidase, xylanase, α-arabinofuranosidase, β-xylosidase, α-galactosidase, p-coumaryl esterase, and feruloyl esterase [116,140,141]. Novonesis still produces the Xylio^® Pre xylanase, which is commonly used for bleaching in the paper industry, and also provides cellulase-based enzyme cocktails such as Cellic^® Ctec, first generation and improved cost-competitiveness, which was developed into Cellic^® Ctec2 and Cellic^® CTec3 with higher activity, especially β-glucosidase, and now Cellic^® CTec3 HS, encompassing both cellulase and hemicellulase, which is applied for a high solid load as in 2G ethanol production plants. It is important to mention that the previous hemicellulase-containing cocktails directed to ethanol production were Cellic^® HTec and Cellic^® HTec2 (generally used in combination with Cellic^® Ctec cocktails). Lee and Yu [142] produced bioethanol from acacia wood and applied pretreatment with sulfuric acid followed by hydrolysis with the cellulose and hemicellulase enzyme cocktail Cellic^® CTec3, achieving a glucose conversion of 73.8% and a maximum ethanol yield of 94.9%. Although the enzyme market and bioprocesses have evolved over the recent years, Novonesis (previously Novozymes) provides enzyme preparations with high β-glucosidase, xylanase, α-arabinofuranosidase, β-xylosidase, α-galactosidase, and ferulic esterase activities from Aspergillus sp. as Viscozyme^® L, previously Novozyme^® 188, applied in the fruit juice industry [116,140,142].

In addition, companies such as DSM-Firmenich (Leeuwarden, Holland), based in the Netherlands, Dyadic International Inc. (Jupiter, FL, USA), located in the United States, and Metgen (Kaarina, Finland), in Finland, also produce and/or supply enzyme cocktails for use in second-generation bioethanol production processes in biorefineries, though branding and ownership of the product lines may be evolving due to ongoing consolidation of companies in the biotechnology sector. The enzyme cocktail formulated by DSM-Firmenich was obtained through the action of the fungus Rasamsonia emersonii and characterized by its ability to act at temperatures up to 65 °C. It contained LPMO, thus reducing the enzyme concentration required for biomass degradation by up to 40%. Furthermore, Dyadic, through its AlternaFuel CMAX™ formulation, offered highly efficient enzyme preparations derived from the C1 platform, based on the fungal strain Myceliophthora thermophila. The CMAX3 version reduced the required enzyme dose by up to five times, and the more recent CMAX5 further improved efficiency at a lower cost. These enzymes operated across a wide range of temperatures and pH levels, with proven industrial-scale applications, optimizing the hydrolysis of lignocellulosic biomass [143,144].

These companies are not only focused on developing highly efficient enzyme cocktails covering a wide range of biomasses with different characteristics but also on reducing the manufacturing costs of these compounds, making the technology more economically viable. In recent years, research and development may have achieved a cost reduction from USD 2 to approximately USD 0.3 per gallon of ethanol [143,144,145,146].

Several studies have focused on enzyme cocktails to find the best synergistic relationship between them, based on parameters such as the proportion of each enzyme in the mixture, as well as their individual characteristics. Raheja et al. [121] developed a customized cocktail for 2G ethanol, called Remzyme, composed of accessory and core enzymes, such as recombinant lytic polysaccharide monooxygenase, cellobiohydrolase, and xylanase, using a simplex lattice mixture design, achieving a saccharification efficiency of up to 98.59% on unwashed rice straw paste pretreated with steam/acid. The results were compared with the commercially available Cellic^® CTec3 enzyme cocktail, which showed a slightly lower efficiency of max. 97.47% compared with the cocktail proposed by the authors.

Similarly, Aishwarya and Goyal [138] developed a new enzyme cocktail with high potential for use in biorefineries using lignocellulosic material as the feedstock. For this, they studied the synergy between chimeric enzymes (endoglucanase and β-glucosidase), cellobiohydrolase, endo-1,4-β-xylanase, β-xylosidase, and arabinofuranosidase in a ratio of 9:4:2:2:3, respectively, along with the influence of the addition of Triton X-100 on the saccharification capacity of this cocktail, achieving 76% efficiency in the hydrolysis of elephant grass pretreated with alkaline hydrogen peroxide at 4.2% (v/v), 101.8 °C for 147.6 min.

Additionally, Maibam and Goyal [147] developed a new crude recombinant bacterial enzyme cocktail to be used in the pretreatment of delignified rice straw pretreated with a choline chloride: acetic acid mixture at molar ratio of 1:3.59, temperature of 126 °C, and treatment duration of 2.5 h, performing the SSF. The authors achieved a total ethanol yield of 182 L/ton raw biomass while applying an enzyme dosage of 83.7 FPU/g with a duration of fermentation of 12.2 h at 33.7 °C.

Finally, Prajapati et al. [148] evaluated an enzyme cocktail of cellulase and hemicellulase obtained from the fungus Aspergillus tubingensis NKBP-55 for the saccharification of sugarcane bagasse (SCB). The results showed that under parametrically optimized conditions with an SCB load of 7%, and an enzyme load of 1 U/mg biomass, glucose, xylose, and arabinose were released at a total concentration of 20 mg/L, enabling fermentation by the yeast C. shehatae NCIM 3501 and generation of ethanol at a maximum concentration of 15.54 g/L, achieving a fermentation efficiency of 77.9%.

6. Fermentative Microorganisms and Strategies for Process Optimizing

During the fermentation step, the main points addressed by the recent studies are the following: (i) co-cultivation between S. cerevisiae and pentose-fermenting strains, (ii) genetic engineering of S. cerevisiae focused on co-fermentation (glucose and xylose mainly) and resistance to cultivation stresses such as the presence of inhibitors (e.g., degradation compounds and salts), and (iii) genetic engineering or co-cultivation of strains that can secrete cellulase and hemicellulase enzymes to perform better configurations such as SSF, SSF-co, and CBP.

The process of transforming sugars present in pretreated and hydrolyzed lignocellulosic biomass into alcohol is generally carried out by yeasts. The main strain is S. cerevisiae, widely used in industrial 1G ethanol production. This yeast is highly efficient in glucose fermentation but cannot ferment pentoses such as xylose. On the other hand, strains such as Scheffersomyces stipitis, Pachysolen tannophilus, K. marxianus, and C. shehatae can ferment xylose and can be applied in co-cultivation with S. cerevisiae as a consortium [149].

In addition, the broad variety of compounds obtained from the hydrolysis of lignocellulosic biomass, including hexoses, pentoses, methyl pentoses, and uronic acids, has motivated the investigation of yeast types that can act efficiently on a wide variety of sugars, especially with genetically modified/manipulated microorganisms. For example, Zhao et al. [150] developed a recombinant strain of S. cerevisiae, named BLH510, displaying high efficiency in the fermentation of sugars present in lignocellulosic hydrolysates, even in a medium containing inhibitory compounds. The strain was obtained through protoplast fusion between parental strains LF1, efficient in the co-fermentation of glucose and xylose, and BLN26, resistant to inhibitory compounds, combined with metabolic engineering. As a result, BLH510 was able to completely consume glucose, xylose, cellobiose, and xylooligosaccharides in up to 84 h, producing 33.96 g/L of ethanol and achieving up to 84.3% of the theoretical ethanol yield. The increased stress resistance (mixed inhibitors and xylose, for example) of the strain was attributed to intracellular accumulation of trehalose at a concentration of 239.3 mg.g⁻¹ of biomass. These results highlight the potential of BLH510 for industrial applications in the production of 2G bioethanol and demonstrate the effectiveness of combining protoplast fusion with metabolic engineering for the development of robust strains.

In another study, Yan et al. [151] obtained three S. cerevisiae strains with a high xylose utilization capacity by inserting genes coding for artificial ancestral isomerases into the diploid yeast genome, followed by laboratory evolution and screening of colonies with the best fermentation performance. The strains obtained were able to consume 40 g/L of xylose within 14 h as well as co-ferment 80 g/L of glucose together with 40 g/L of xylose in just 18 h. Furthermore, the authors proposed a simpler process for bioethanol production from corn stover, which included biomass pretreatment by sulfuric acid densification (DLC(sa)), use of a low cellulase dosage of 14.81 FPU.g⁻¹, and elimination of the stages of washing, detoxification, and supplementation with external nitrogen sources. This strategy, using unwashed corn stover, yielded up to 54.8 g/L of ethanol. The yeast also took advantage of nitrogen sources already present in the hydrolysate, thus eliminating the need for nitrogen addition in the medium.

Depending on the type of pretreatment used in the biomass preparation stage, secondary compounds may be generated, which inhibit commonly used yeast strains, such as S. cerevisiae. Therefore, research is underway to produce more resistant and adaptable strains in view of increasing process efficiency. Klanrit et al. [152] addressed the challenges posed by inhibitory compounds present in lignocellulosic hydrolysates, proposing the use of the unconventional yeast Saccharomycodes ludwigii APRE2 for efficient production of bioethanol from non-detoxified sugarcane bagasse hydrolysate (SBH) at 37 °C. This strategy eliminated the requirement for costly detoxification steps, resulting in an improved economic viability of the process. Furthermore, the authors applied central composite design (CCD) to optimize fermentation conditions. Even in the presence of inhibitors such as organic acids and furfurals, S. ludwigii APRE2 achieved an ethanol yield of 38.11 g/L, with a productivity of 1.59 g·L⁻¹·h⁻¹, corresponding to 0.45 g·g⁻¹ of biomass. The superior performance of the strain compared with other yeasts under similar conditions highlights its industrial potential for sustainable bioethanol production without the need for additional pretreatments.

Similarly, Zhang et al. [84] carried out a study focused on developing a yeast with greater tolerance to the inhibitory compounds of sodium salts produced during the dilute acid pretreatment of corn straw and bagasse, aiming to ensure the efficient and complete conversion of hydrolyzed sugars, including xylose. To this end, the authors used laboratory adaptive evolution and metabolic engineering mechanisms to promote the evolution of S. cerevisiae, creating the new strain eGXI-1.0, which may meet industrial expectations for ethanol production from lignocellulosic biomass regarding resistance and the ability to metabolize both glucose and xylose.

Finally, Procópio et al. [153] modified S. cerevisiae strain SR8A6S3 to express Neurospora crassa enzymes, including β-xylosidases GH43-2 and GH43-7 and CDT-2 transporter, replacing GRE3 and SOR1 genes, linked to xylitol production. The new strain, SR8A6S3-CDT-2-GH34-2/7, was able to simultaneously consume xylooligosaccharides (XOS), xylose, and acetate, which are common components of lignocellulosic hydrolysates besides glucose. When cultivated in hemicellulosic hydrolysate, the strain produced 60% more ethanol and 12% less xylitol than the control strain and obtained an 84% higher yield of ethanol from xylan. As highlighted by these results, the economic profitability of 2G ethanol may be improved through yeast improvement for inhibitor resistance and enhanced conversion of hemicellulosic hydrolysate, combined with process simplification, particularly the removal of washing/detoxification stages of biomass hydrolysate gained from enhanced yeast resistance.

7. Government Incentives Worldwide for Cellulosic Ethanol

To make 2G bioethanol from lignocellulosic biomass an attractive option for investors, many countries combine environmental policies, such as low emission requirements, with economic incentives and supply chain support. Effective policies should combine performance incentives with research and infrastructural support. Apart from technical and economic challenges, the successful commercialization of lignocellulosic bioethanol also depends on political structures for positive social support and engagement. Government incentives, such as subsidies, fiscal credits, mixed mandates, and renewable fuel standards, can significantly shape market competitiveness, mitigate investment risks, and ensure long-term industry viability.

The United States stands out as a world leader in promoting cellulosic ethanol, supported by a robust set of public policies that combine regulatory mandates, tax incentives, and investments in research, such as the federal Volumetric Ethanol Tax Credit and renewable energy identification numbers as part of the Renewable Fuel Standard [154,155].

Since the 2000s, the United States has implemented the Renewable Fuel Standard (RFS), a policy that mandates the inclusion of advanced biofuels, such as second-generation ethanol, within the national energy mix. The second version of the program (RFS2) established ambitious targets, including a total volume of 36 billion gallons of biofuels by 2022 and the requirement of 5.6 billion liters of cellulosic biofuels by 2025, with a projected supplementary volume of 1.38 billion gallons for 2025 [156,157].

In addition, tax incentives have played a crucial role in making these technologies economically viable. Notable among these is the 45Q credit, aimed at carbon capture in biorefineries, offering up to USD 1.01 per gallon produced, depending on the carbon intensity of the fuel [88,158]. At the state level, several states offer additional incentives, such as tax exemptions and direct financing; the state of Minnesota, for example, grants up to USD 120,000 per quarter to biorefineries that demonstrate the production of cellulosic ethanol [159]. This institutional support has been fundamental in enabling commercial plants such as the POET-DSM plant in Iowa, which uses corn stover as a feedstock.

Additionally, the Bioenergy Technologies Office, affiliated with the U.S. Department of Energy (DOE), announced an investment of USD 12 million to boost the development of integrated biorefinery technologies, with the goal of reducing emissions in the transportation and industrial sectors [160]. Complementing this, Aui and Wang [161] indicated that the continuation and strengthening of these regulatory and financial instruments could significantly increase the production of cellulosic ethanol and estimated bioethanol production to grow from 20.78 million up to 65.94 million liters by 2050, provided that generous subsidies and mandates are adopted.

In this sense, Brazil also stands out as one of the leading countries in promoting 2G ethanol with robust regulatory, fiscal, and financial policies [25,162]. For this purpose, the RenovaBio program was established in 2017, setting annual decarbonization targets for the transportation sector and issuing Decarbonization Credits [163,164,165]. This program has been fundamental in providing economic support for fuels with a lower carbon footprint. Complementing the RenovaBio program, the Future Fuels Law (Law 14.993/2024) was sanctioned with the objective of accelerating decarbonization in the transport sector [159]. This legislation provides, among other measures, for an increase in ethanol content in gasoline of up to 30% (E30) by 2025, which should boost the demand for 2G bioethanol [166,167,168]. Financially, the BNDES (Brazilian Development Bank) has played a central role in scaling up production, approving the provision of BRL 1 billion for the construction of a new Raízen plant in Andradina (São Paulo) in 2025, with a capacity of 82 million liters/year [169,170].

The European Union (EU) has also adopted a strategic and comprehensive approach to fostering the production and use of lignocellulosic ethanol. One of the main regulatory milestones in this context is the Renewable Energy Directive (RED II), which requires that, by 2030, at least 3.5% of fuels used in the transport sector should be produced from advanced renewable sources, including 2G ethanol [171]. RED II also establishes rigorous sustainability criteria, ensuring that only technologies with a low environmental impact are encouraged [172]. In addition, the policy provides for so-called “double counting” for advanced low-carbon biofuels, such as 2G ethanol, which represents an additional bonus to EU countries for reaching national targets [173,174]. As for RED III, the main change involves an increase in the share of renewable fuels in the transport sector, along with the phasing out of first-generation fuels, which may significantly alter the EU fuel market by 2030 [175].

Additionally, several European countries have implemented specific incentive policies. Sweden, Denmark, and Finland, for example, offer tax exemptions for second-generation biofuels [17,176]. Germany, on the other hand, has distinguished itself by financing technological demonstration projects, such as the Clariant plant in Straubing, producing ethanol from wheat straw [177,178], which may still be repurposed for 1G ethanol production due to economic difficulties. In Denmark, the EUDP (Energy Technology Development and Demonstration Program) has invested in projects such as the Inbicon biomass refinery for the development and commercialization of cellulosic ethanol [179].

The European Union has invested heavily in research and innovation on cellulosic ethanol, especially through Horizon 2020 and Horizon Europe programs. Between 2014 and 2020, approximately 370 M EUR were allocated to research into biofuels from waste, in addition to 55 M EUR from the European Regional Development Fund. The EU also financed, with 24.7 M EUR, a pioneering plant in Romania for bioethanol production from straw. These investments, which include more than 200 M EUR for technologies such as enzymatic hydrolysis and pyrolysis, aim at making second-generation ethanol economically viable and meeting renewable energy targets for 2020 and 2030 [180,181,182].

Cellulosic ethanol production in Asia has received significant incentives from several countries, particularly India, China, and Thailand, which are implementing specific policies aimed at promoting second-generation biofuels from agricultural waste. In India, the revised National Biofuel Policy strengthened the PM JI-VAN Yojana program, offering subsidies and financing for 2G plants, while the state of Odisha, in 2025, launched the Interest Subvention Scheme to support 29 local projects [183,184,185]. Furthermore, India has also established an ambitious plan to blend 20% ethanol by 2025–2026, prioritizing raw materials such as straw and bagasse, and instituted the Viability Gap Funding (VGF), with a ceiling of INR 1.5 billion (approximately USD 20 million) per project for the commissioning of up to 12 commercial plants. Estimates indicate that this support can reduce cellulosic ethanol production costs by up to 13% for smaller units and 8% for larger units [26,186]. In China, the 14th Five-Year Plan (2021–2025) and guidelines from the National Energy Administration reinforce the priority given to non-food biofuels [187]. Additionally, by the end of 2025, China aims to transition to commercial-scale production of 2G ethanol, which is manufactured by converting vegetation unsuitable for human consumption [128]. In Thailand, incentives include tax reductions for ethanol derived from agricultural waste, with the long-term promotion of bioethanol supported by the Alternative Energy Development Plan (AEDP 2015) aiming to produce four million liters by 2036 [188,189,190]. These efforts reveal a dynamic scenario in Asia, where integrated policies of subsidies, targeted financing, and tax incentives have been instrumental in driving the economic development of 2G ethanol.

In other Latin American countries such as Colombia, Mexico, Argentina, Chile, and Peru, incentives are still more modest, but more favorable incentives may still be under development. In Colombia, federal legislation provides for VAT exemptions, tax deductions, and investment incentives in bioenergy, especially in rural areas, favoring the environment for the adoption of advanced biofuels [191,192].

In Mexico, the new Biofuels Law was approved in 2025, establishing guidelines to encourage the production, use, and commercialization of biofuels, focusing on the use of organic waste and biomass from marginal lands in view of promoting the circular economy as well as energy transition. The legislation prioritizes bioethanol from non-food crops, such as sugarcane, and excludes from its scope commercial disputes over biomass transactions [193]. Aligned with national climate goals, the country aims to achieve 35% renewable energy by 2024 and reduce greenhouse gas emissions by 22% by 2030 [194].

In Argentina, although in the absence of specific federal legislation for 2G, the country maintains a mandatory 12% ethanol blend in gasoline and has promoted regional and sectoral initiatives that favor its adoption [195]. The government, through the Ministry of Energy, has supported the modernization of refineries and distillery infrastructure aimed at ethanol production, including partnerships with companies such as Raízen S/A, which maintains investments in 2G plants in the country, focusing on the production of sustainable biofuels and integration with the Brazilian market [196]. On the other hand, Chile, Peru, and other Latin American nations already have policies aimed at renewable energies in general but still lack specific mandates or regulatory and financial incentives directed at cellulosic ethanol, which inhibits investments in this technological route.

8. Lignocellulosic Biomass Biorefinery

A biorefinery is a sustainable facility that enables the complete conversion of biomass into fuels, energy, chemicals, and valuable materials through conversion technologies such as thermochemical and biochemical processes. Its concept is based on the valorization of biomass as a raw material, adopting an integrated management approach for by-products that would otherwise be considered waste, turning each fraction of the material into useful inputs, thereby promoting efficiency, sustainability, and waste reduction [197,198]. The biorefining of residual biomass is an integrated process that comprises the stages of pretreatment, hydrolysis, and fermentation, along with subsequent processing steps [199].

Modern biorefinery technologies applied to lignocellulosic biomass should integrate two main stages: (i) the fractionation of lignin, cellulose, and hemicellulose using sustainable and low-cost methods; and (ii) the integrated biorefining of these fractions through processes such as thermochemical, biochemical, chemical, and physical/mechanical conversion, depending on the raw material and the desired product. In recent decades, research and development efforts have focused specifically on the production of fuels, with a focus on 2G ethanol [200,201,202]. In general, two different processing pathways may be applied to convert lignocellulosic biomass into biofuels: thermochemical and biochemical processing, as shown in Figure 7.

Both pathways have specificities in terms of technologies employed, types of products obtained, and processing time. Biochemical processes, such as fermentation, anaerobic digestion, and enzymatic hydrolysis, use biological catalysts, such as enzymes and microorganisms, and require a relatively long processing time, ranging from hours to weeks, to convert lignocellulosic biomass into products such as biogas, bioethanol, biobutanol, biohydrogen, and biomethane. On the other hand, thermochemical processes are characterized by significantly faster conversion rates, ranging from seconds to minutes, and use heat and chemical reactions to transform the biomass into bio-oils, charcoal, synthesis gas, and other intermediate products. The main thermochemical technologies include pyrolysis, gasification, hydrothermal liquefaction, and torrefaction [2,203,204].

Prior to conversion, biomass typically requires pre-processing through mechanical, chemical, biological, or combined treatment routes. These steps are essential for deconstructing the biomass structure, making its components accessible for subsequent processes and increasing the overall efficiency of the system. Mechanical processes, such as pressing, fractionation, and size reduction, can promote the separation of components and the generation of fractions with different chemical compositions. Chemical methods, such as acid hydrolysis, oxidation, and esterification, involve reactions to alcohols or acids aimed at producing compounds of interest. In addition, extraction techniques may be applied prior to biomass pretreatment in order to recover valuable compounds from biomass and prevent their degradation during subsequent processing steps [201,205,206].

The biochemical route typically requires a sequence of steps, starting with the pretreatment of the biomass, followed by enzymatic hydrolysis of the polysaccharides, microbial fermentation of the released sugars, and subsequent concentration/purification of the obtained bioproduct. Depending on the microorganism used, bioalcohols (such as bioethanol and biobutanol) may be obtained, in addition to biohydrogen. In anaerobic digestion, the synergy and combination of biological acidogenesis, acetogenesis, and methanogenesis reactions performed by microorganisms result in the production of biogas, which can subsequently be purified as biomethane [204,205,207].

On the other hand, thermochemical processing does not require biological catalysts and can be defined as the application of heat or controlled oxidation for the generation of biomaterials, generally resulting in the production of solid, liquid and gaseous biofuels. During these steps, intermediates such as synthesis gas, bio-oil, and char are formed in specific reactors, which can subsequently be converted into liquid and gaseous fuels, such as methanol, ethanol, hydrogen, and synthetic biodiesel [203,208,209].

Biorefineries, by definition, have sustainable characteristics. However, for biorefineries to effectively generate positive socioeconomic impacts, it is essential that they operate with high technical and economic performance, associated with minimal environmental impacts. In this context, several challenges must be considered in the planning and operation of these units, including (i) minimizing the generated waste streams, (ii) diversifying the energy matrix, (iii) using different types of biomasses as raw material, and (iv) developing new products with industrial value. Nevertheless, these aspects are still often addressed in a fragmented way, while an integrated and systemic perspective is lacking, which compromises the full utilization of the sustainable potential of biorefineries [127,210,211].

An effective strategy to increase the sustainability of these systems is the application of criteria promoting efficient optimization and integration of mass and energy streams, considering biorefineries as integrated biomass processing centers. This approach combines the complete utilization of raw materials along with the optimization of processes, resulting in increased economic performance and energy efficiency while significantly reducing environmental impacts of the processing plant [211,212].

In the Brazilian context, companies like Raízen S/A demonstrate the possibility to maximize the use of biomass derived from sugarcane. Following this approach, 2G ethanol produced from lignocellulosic residues may be directed both for export and for industrial and pharmaceutical applications, while conventional ethanol (1G), obtained from sugarcane juice, may be used further as biofuel. By-products such as vinasse may also be valued, either as an agricultural input (fertigation) or as a raw material for biogas production, which reinforces the concept of full resource utilization [211,213,214].

Modern biorefineries have evolved to function as integrated processing centers for lignocellulosic biomass. In addition to ethanol, various valuable co-products such as electricity, lignin, organic acids, and biogas may be produced, increasing revenue streams and improving economic viability. While technological routes for converting carbohydrates into fermentable sugars are well established, the utilization of lignin remains a technological challenge. Traditionally treated as energy waste and burned for heat generation, lignin is, in fact, the largest natural reservoir of aromatic compounds and may constitute a promising renewable raw material for the synthesis of high value-added products [205,215].

In this sense, the development of effiient pathways for lignin valorization emerges as a crucial strategy to increase the economic viability of biorefineries. It is estimated that more than 100 million tons of lignin are generated annually by the pulp, paper, and cellulosic ethanol industries [215,216]. Several studies have focused on the development of technologies capable of fractionating, depolymerizing, and processing lignin into aromatic and phenolic compounds, which have potential applications in the pharmaceutical, cosmetic, chemical, and biomaterials industries. Although lignin valorization is still mostly at the research and development stage, advances in this field are fundamental to ensuring greater competitiveness for biorefineries, promoting increased revenue and better utilization of biomass in a future “atomic economy”, and providing direct support for the bioeconomy sector [215,216].

Additionally, by-products of the bioethanol process, such as vinasse and CO₂ from fermentation, have been widely recognized for their commercial value and applicability across different industrial sectors. Vinasse, in addition to being used as a fertilizer, may also be converted into bioenergy through anaerobic digestion, while CO₂ may be captured and purified for various industrial uses [142,217,218].

It is worth noting that selective pretreatment techniques can be used to hydrolyze hemicellulose into fermentable sugars, optimizing fermentation and allowing the recovery of high-value compounds such as xylitol and furfural. However, the conversion of cellulose into sugars is slower and requires high temperatures, increasing energy consumption and the formation of inhibitory by-products such as formic acid, acetic acid, furfural, 5-hydroxymethylfurfural, and phenols [17,219,220]. Some by-products marketed by 2G ethanol refineries (companies) are presented in

Table 7. Biorefineries operating around the world for bioethanol production.

Company, City/Country and Feedstock	Process	Co-Products	References
Lenzing AG—wood-based fibers. Commercial (Lenzing, Austria) Lignocellulose form agricultural or forest residues	Lignocellulosic biorefinery, employs pulping, Modal, and Lyocell processes; platform distribution comprises 40% pulp production, 50% bioenergy generation, and 10% bio-based materials.	Pulp, Bioenergy, Acetic acid, Furfural, Magnesium Lignosulfonate, Soda ash, Sodium sulfate, Xylose	[129]
Versalis Biorefinery (Crescentino, Italy) Hardwood (poplar), Agricultural residuals	A three-platform biorefinery (C5/C6 sugars, lignin, and power/heat) designed for the production of bioethanol from lignocellulosic feedstocks (operated at 13 MWe) utilizes saturated steam.	Lignin, Green electricity, Disinfectants	[129]
AGRANA Biorefinery (Pischelsdorf, Austria) Wheat, maize	Two-platform system (starch and C5/C6 sugars) that integrates wheat starch processing with an existing bioethanol plant. Agricultural feedstocks are first processed to produce wheat starch and gluten, while residual streams and additional grains are directed to the bioethanol unit. The process yields high-quality bioethanol, recovered CO2 for the food and beverage industry, and non-GMO animal feed.	Protein-rich animal feed, Gluten, Biogenic CO2	[129,221]
GranBio (São Miguel dos Campos, Brazil) Sugarcane bagasse	Steam explosion, with or without acid catalysis. High recovery of C5 sugars in the liquid fraction, while retaining C6 sugars in the solid fiber, although inhibitory compounds are generated, mainly acetic acid, formic acid, and phenolics derived from lignin. The process includes continuous biomass feeding, precise control of steam pressure and residence time in the pretreatment reactor, and minimization of steam losses.	Acetic acid, Furfural, Lignin	[222,223]
Raízen (Guariba, Brazil) Lignocellulosic biomass		Lignin, Vinasse, Filter cake	[223,224]
Godavari Biorefineries Limited (Sakharwadi, India) Sugarcane molasses	Utilizes sugarcane juice and syrup in ethanol production. Alternates between sugar and ethanol production. Features vertical integration in ethanol production from molasses, supplies inputs to the ethyl acetate chain, planned to expand cellulose and cellulose derivative production from bagasse, and is a world leader in the manufacture of high-purity crude palm oil (MPO), reinforcing its profile as an integrated biorefinery.	Acetic acid, Butanol, Ethyl acetate	[225]
CropEnergies Bioethanol GmbH (Zeitz, Germany) Lignocellulosic biomass	C5 and C6 sugars can be converted, through fermentation or catalysis, into various products, with bioethanol being the main industrial application. Cellulose can be used for the production of microcrystalline or nanocrystalline cellulose (MCC and NCC), while lignin can be used to obtain aromatic products and other chemicals. The process requires thermochemical pretreatment of the biomass, followed by cellulose hydrolysis and sugar fermentation.	Neutral alcohol, Protein animal feed, Liquefied CO2	[226]
Bioamber (Sarnia, ON, Canada) Grain-based	Conversion of glucose into succinic acid by metabolically enhanced/proprietary microorganisms. Steps for separating succinic acid and fermentation using membranes.	Succinic acid	[227]
AVA Biochem Ltd. (Muttenz, Switzerland) Lignocellulosic biomass	COBRIS™ (Conversion of Biomass to Renewable Industrial Substances) is a patented aqueous hydrothermal technology that promotes the conversion of renewable sugars from biomass into high-value-added chemicals. It is a sustainable, non-toxic, and scalable process developed for industrial applications.	5-Hydroxymethyl- furfural (HMF)	[228]

.

9. Bottlenecks for the Consolidation of Technology and Increased Production

Despite recognized environmental advantages associated with 2G ethanol produced from lignocellulosic residues, the commercial viability of the process still faces several technical, economic, and logistical bottlenecks. The complex structure of lignocellulosic biomass makes the conversion process into ethanol significantly more challenging than when using raw materials rich in sucrose or starch. The process involves multiple interdependent steps, such as biomass pretreatment, hydrolysis of polysaccharides into fermentable sugars, fermentation of monosaccharides, comprising both hexoses and pentoses, and isolation of ethanol from the fermented mixture [229,230].

Biomass conversion into sugars represents one of the main technological obstacles, where a significant challenge lies in the requirement for selective lignin removal, since lignin acts as a physical barrier preventing access to polysaccharides for their further valorization. The lignin removal process should be effective, while being performed under mild pretreatment conditions in order to preserve the integrity of polysaccharides while maintaining economic viability. Efficient lignin extraction is fundamental to increasing enzyme access to cellulose and, consequently, improving the efficiency of enzymatic hydrolysis [229].

The introduction of innovative pretreatment technologies, such as those assisted by hydrodynamic cavitation, emerges as an alternative to improve economic parameters and reduce the energy intensity of the process [229]. Furthermore, the costs of enzymes are still high, and their production, use, and environmental impact are not always transparently accounted for in Life Cycle Assessments (LCAs), mainly due to the scarcity of reliable public data, resulting from commercial barriers and confidentiality policies on the part of manufacturers [231].

Additionally, the high capital cost and complexity of the industrial processes involved limit the competitiveness of 2G ethanol compared to fossil fuels and first-generation (1G) ethanol. The need for a continuous and diversified supply of biomass throughout the year, efficient energy integration between processing stages, development of fermentation technologies suitable for a high biomass load, and genetic modification of microorganisms to produce enzymes, fermenting pentoses, and/or support inhibitors such as degradation compounds and salts formed during pretreatment steps and with a tolerance to a high ethanol concentration are critical factors for enabling large-scale 2G ethanol production [232].

From an economic point of view, challenges are further amplified by the difficulty of obtaining an immediate financial return from the production of ethanol as a single product. One strategy is to direct the 2G ethanol process towards the generation of more valuable products such as those present in chemical, pharmaceutical, and hygiene/cleaning industries while 1G ethanol is linked to the biofuels market (2G (0.8–1.5 USD/L ethanol) > 1G (0.45–0.65 USD/L ethanol) [233,234]). Accordingly, product diversification and valorization of by-products have proven to be promising strategies to increase the financial sustainability of biorefineries. Studies show that secondary streams from the process can be valorized to obtain high-value-added bioproducts, such as biopigments, biopolymers, biosurfactants, and xylitol, among others. The exploration of new valorization routes, such as decarbonization credits and the expansion of the product portfolio, may strengthen the economic feasibility of 2G ethanol and increase its attractiveness in the global market [235,236,237,238].

Even so, 2G or hybrid (1G/2G) biorefineries continue to face structural challenges related to economic sustainability and public acceptance. Despite the difficulties faced in the production and commercialization of cellulosic ethanol, the production of 2G ethanol presents promising prospects in the context of the global energy transition. With the biorefinery market projected to grow to USD 52.68 billion by 2027 [239], technological advancements, sustainability, and the strengthening of the bioeconomy are the main drivers.

Key prospective areas to improve the viability of 2G ethanol include reducing raw material costs, improving pretreatments, developing more efficient enzymatic cocktails, optimizing fermentation strategies, and obtaining more robust microorganisms to produce enzymes or ethanol more efficiently. Strategies such as the co-production of bioproducts, integration with renewable sources, and sustainable use of land and water are essential to increase the efficiency and competitiveness of the process. The success of 2G ethanol will depend on integrated actions between technological innovation, logistics, and public policies, consolidating it as a sustainable alternative for the global energy matrix. The main challenges for the production of cellulosic ethanol are presented in Figure 8.

10. Conclusions and Future Perspectives

2G ethanol production is in constant growth and represents one of the most promising alternatives for the energy transition, leveraging lignocellulosic residues and reducing the dependence on fossil fuels. This technology reinforces the principles of the circular economy by transforming agro-industrial waste into value-added biofuels, contributing to climate change mitigation and meeting global decarbonization goals. However, biomass conversion pathways still face technical and economic barriers, primarily related to pretreatment, enzymatic efficiency, and costs associated with industrial-scale production.

Under these circumstances, scientific and technological advances are still essential in making the process more competitive and sustainable. In particular, the development of optimized enzyme cocktails, genetically enhanced microorganisms, and innovative pretreatments, such as cavitation-assisted or hybrid catalysts, hold the potential to increase hydrolysis and fermentation efficiency, reducing costs and improving productivity. Furthermore, the valorization of lignin and other by-products in integrated biorefinery processes may increase economic profitability, diversify the product portfolio, and strengthen the bioeconomy sector.

However, the full feasibility of 2G ethanol depends not only on technological advancements but also on the creation of favorable institutional and regulatory environments. International experience shows that tax incentives, decarbonization credits, subsidies, and mixed mandates are crucial for reducing investment risks and stimulating large-scale expansion.

Therefore, the integration of science, industry, and government is essential to strengthen supply chains, ensure an adequate logistics infrastructure, and consolidate the competitiveness of lignocellulosic 2G ethanol in relation to fossil fuels and first-generation (1G) ethanol. Accordingly, the future of 2G ethanol is directly linked to the ability to reconcile environmental sustainability, economic efficiency, and political support. Through the combination of technological innovation, robust public policies, and strategies for the full use of biomass, lignocellulosic ethanol is poised to establish itself as a pillar of the global renewable energy matrix. More than just fuel, it represents a driver of socioeconomic transformation, capable of strengthening the bioeconomy sector, generating skilled jobs, and promoting sustainable development in different regions of the world.