Lignin Extraction by Using Two-Step Fractionation: A Review.

Lignocellulosic biomass represents the most abundant renewable carbon source on earth and is already used for energy and biofuel production. The pivotal step in the conversion process involving lignocellulosic biomass is pretreatment, which aims to disrupt the lignocellulose matrix. For effective pretreatment, a comprehensive understanding of the intricate structure of lignocellulose and its compositional properties during component disintegration and subsequent conversion is essential. The presence of lignin-carbohydrate complexes and covalent interactions between them within the lignocellulosic matrix confers a distinctively labile nature to hemicellulose. Meanwhile, the recalcitrant characteristics of lignin pose challenges in the fractionation process, particularly during delignification. Delignification is a critical step that directly impacts the purity of lignin and facilitates the breakdown of bonds involving lignin and lignin-carbohydrate complexes surrounding cellulose. This article discusses a two-step fractionation approach for efficient lignin extraction, providing viable paths for lignin-based valorization described in the literature. This approach allows for the creation of individual process streams for each component, tailored to extract their corresponding compounds.

Severity factor kinetic model as a strategic parameter of hydrothermal processing (steam explosion and liquid hot water) for biomass fractionation under biorefinery concept.

Hydrothermal processes are an attractive clean technology and cost-effective engineering platform for biorefineries based in the conversion of biomass to biofuels and high-value bioproducts under the basis of sustainability and circular bioeconomy. The deep and detailed knowledge of the structural changes by the severity of biomasses hydrothermal fractionation is scientifically and technological needed in order to improve processes effectiveness, reactors designs, and industrial application of the multi-scale target compounds obtained by steam explosion and liquid hot water systems. The concept of the severity factor [log10 (Ro)] established>30 years ago, continues to be a useful index that can provide a simple descriptor of the relationship between the operational conditions for biomass fractionation in second generation of biorefineries. This review develops a deep explanation of the hydrothermal severity factor based in lignocellulosic biomass fractionation with emphasis in research advances, pretreatment operations and the applications of severity factor kinetic model.

Novel sustainable alternatives for the fashion industry: A method of chemically recycling waste textiles via acid hydrolysis.

The fashion industry has a considerable environmental impact, especially due to the increased generation of waste textiles as a result of fast fashion business models. Although fiber-to-fiber recycling processes are being developed, such a process is in reality a downcycling process, in which the mechanical properties of the textile fibers are impoverished with each cycle. Thus, new alternatives are required to completely close the fashion loop through chemically recycling textile fibers unfit for other types of recycling or resale due to their poor quality. We have evaluated the possibility of using acid hydrolysis to directly depolymerize the cotton fibers in waste textiles to produce a glucose solution, which could subsequently be used for the production of chemicals or fuels. Although a one-step procedure with sulfuric acid was unable to deliver high glucose production, it was possible to achieve a glucose yield over 90% through a two-step procedure, in which concentrated and dilute sulfuric acid were combined to exploit the benefits of both concentrations. Glucose concentrations around 40 g/L were achieved by increasing the solids loading in the two-step process, which might be sufficiently high for the fermentation of the solution into high-value products. Thus, this study demonstrates that it would be possible to chemically recycle (cellulose-based) waste textiles via acid hydrolysis, which, if correctly designed, could avoid the need to use enzymes to achieve high conversion efficiencies.

Towards circular fashion - transforming pulp mills into hubs for textile recycling.

Most waste textiles are currently incinerated or landfilled, which is becoming an increasing environmental problem due to the ever-increasing consumption of textiles in the world. New recycling processes are required to address this problem and, although textile-to-textile recycling would be preferable, many researchers have suggested implementing processes based on the depolymerization of the textile fibers. We suggest integrating textile recycling with pulp mills, which would reduce the cost of depolymerizing the textile fibers and, at the same time, would diversify the product portfolio of the pulp mill, transforming the facility into a true biorefinery. This integration would be based on using green liquor as the pretreatment agent in the textile recycling process, as well as energy integration between the two processes. Na2CO3 was used to identify the conditions under which this pretreatment should be performed. Temperature and residence time proved to be critical in the efficacy of the pretreatment, as suitable values were required to ensure partial solubilization of the waste textiles. The conditioning of the pretreated material also had an important effect on the process, as it ensured a suitable environment for the enzymatic depolymerization while maintaining the changes in the material caused by pretreatment. Pretreatment was then performed with industrial green liquor, showing that the efficiency of textile recycling was about 70% when integrated in a pulp mill.

A strategy for synergistic ethanol yield and improved production predictability through blending feedstocks.

BACKGROUND: The integration of first- and second-generation bioethanol processes has the potential to accelerate the establishment of second-generation bioethanol on the market. Cofermenting pretreated wheat straw with a glucose-rich process stream, such as wheat grain hydrolysate, in a simultaneous saccharification and fermentation process could address the technical issues faced during the biological conversion of lignocellulose to ethanol. For example, doing so can increase the final ethanol concentration in the broth and mitigate the effects of inhibitors formed during the pretreatment. Previous research has indicated that blends of first- and second-generation substrates during simultaneous saccharification and fermentation have synergistic effects on the final ethanol yield, an important parameter in the process economy. In this study, enzymatic hydrolysis and simultaneous saccharification and fermentation were examined using blends of pretreated wheat straw and saccharified wheat grain at various ratios. The aim of this study was to determine the underlying mechanisms of the synergy of blending with regard to the yield and volumetric productivity of ethanol. RESULTS: Replacing 25% of the pretreated wheat straw with wheat grain hydrolysate during simultaneous saccharification and fermentation was sufficient to decrease the residence time needed to deplete soluble glucose from 96 to 24 h and shift the rate-limiting step from ethanol production to the rate of enzymatic hydrolysis. Further, a synergistic effect on ethanol yield was observed with blended substrates, coinciding with lower glycerol production. Also, blending substrates had no effect on the yield of enzymatic hydrolysis. CONCLUSIONS: The effects of substrate blending on the volumetric productivity of ethanol were attributed to changes in the relative rates of cell growth and cell death due to alterations in the concentrations of substrate and pretreatment-derived inhibitors. The synergistic effect of substrate blending on ethanol yield was attributed in part to the decreased production of cell mass and glycerol. Thus, it is preferable to perform simultaneous saccharification and fermentation with substrate blends rather than pure substrates with regard to yield, productivity, and the robustness of the process.

Hemicellulose Recovery from Spent-Sulfite-Liquor: Lignin Removal by Adsorption to Resins for Improvement of the Ultrafiltration Process.

In this work, three polymeric resins were examined as alternatives for the separation of hemicellulose and lignin. The aim was to remove the lignin from spent-sulfite-liquor (SSL) prior to ultrafiltration, producing a hemicellulose-rich retentate with high purity, and increase the capacity of the membrane filtration. The lignin in the SSL was sulfonated; thus, two of the resins were anion exchangers and 1 was hydrophobic. The data from the equilibrium studies and adsorption kinetics were fitted to established models, and the results were interpreted based on these observations. The strongly basic anion exchanger performed best with regard to lignin removal. The adsorption followed the Sips isotherm, indicating that the process was cooperative with chemisorption as the main reaction between the adsorbate and adsorbent based on the kinetics. Regeneration of the adsorbent was also possible, wherein 100 g/L NaCl was sufficient to recover 98% of the lignin. The lignin removal had a positive effect on the ultrafiltration process, in which the flux increased by 38% and the extent of separation between the hemicellulose and lignin rose from 17% to 59%.

Elucidation of Changes in Cellulose Ultrastructure and Accessibility in Hardwood Fractionation Processes with Carbohydrate Binding Modules.

We have recently presented a sequential treatment method, in which steam explosion (STEX) was followed by hydrotropic extraction (HEX), to selectively fractionate cellulose, hemicellulose, and lignin in hardwood into separate process streams. However, above a treatment severity threshold, the structural alterations in the cellulose-enriched fraction appeared to restrict the enzymatic hydrolyzability and delignification efficiency. To better understand the ultrastructural changes in the cellulose, hardwood chips were treated by single (STEX or HEX) and combined treatments (STEX and HEX), and the cellulose accessibility quantified with carbohydrate-binding modules (CBMs) that bind preferentially to crystalline (CBM2a) and paracrystalline cellulose (CBM17). Fluorescent-tagged versions of the CBMs were used to map the spatial distribution of cellulose substructures with confocal laser scanning microscopy. With increasing severities, STEX increased the apparent crystallinity (CBM2a/CBM17-ratio) and overall accessibility (CBM2aH6 + CBM17) of the cellulose, whereas HEX demonstrated the opposite trend. The respective effects could also be discerned in the combined treatments where increasing severities further resulted in higher hemicellulose dissolution and, although initially beneficial, in stagnating accessibility and hydrolyzability. This study suggests that balancing the severities in the two treatments is required to maximize the fractionation and simultaneously achieve a reactive and accessible cellulose that is readily hydrolyzable.

Introducing low-quality feedstocks in bioethanol production: efficient conversion of the lignocellulose fraction of animal bedding through steam pretreatment.

BACKGROUND: Animal bedding remains an underutilized source of raw material for bioethanol production, despite the economic and environmental benefits of its use. Further research concerning the optimization of the production process is needed, as previously tested pretreatment methods have not increased the conversion efficiency to the levels necessary for commercialization of the process. RESULTS: We propose steam pretreatment of animal bedding, consisting of a mixture of straw and cow manure, to deliver higher ethanol yields. The temperature, residence time and pH were optimized through response-surface modeling, where pretreatment was evaluated based on the ethanol yield obtained through simultaneous saccharification and fermentation of the whole pretreated slurry. The results show that the best conditions for steam pretreatment are 200 °C, for 5 min at pH 2, at which an ethanol yield of about 70% was obtained. Moreover, the model also showed that the pH had the greatest influence on the ethanol yield, followed by the temperature and then the residence time. CONCLUSIONS: Based on these results, it appears that steam pretreatment could unlock the potential of animal bedding, as the same conversion efficiencies were achieved as for higher-quality feedstocks such as wheat straw.

Strain-dependent variance in short-term adaptation effects of two xylose-fermenting strains of Saccharomyces cerevisiae.

The limited tolerance of Saccharomyces cerevisiae to the inhibitors present in lignocellulosic hydrolysates is a major challenge in second-generation bioethanol production. Short-term adaptation of the yeast to lignocellulosic hydrolysates during cell propagation has been shown to improve its tolerance, and thus its performance in lignocellulose fermentation. The aim of this study was to investigate the short-term adaptation effects in yeast strains with different genetic backgrounds. Fed-batch propagation cultures were supplemented with 40% wheat straw hydrolysate during the feed phase to adapt two different pentose-fermenting strains, CR01 and KE6-12. The harvested cells were used to inoculate fermentation media containing 80% or 90% wheat straw hydrolysate. The specific ethanol productivity during fermentation was up to 3.6 times higher for CR01 and 1.6 times higher for KE6-12 following adaptation. The influence of physiological parameters such as viability, storage carbohydrate content, and metabolite yields following short-term adaptation demonstrated that short-term adaptation was strain dependent.

Galactoglucomannan Recovery with Hydrophilic and Hydrophobic Membranes: Process Performance and Cost Estimations.

In this study, we compared the GR51PP (hydrophobic/polysulfone) membrane with a series of hydrophilic (regenerated cellulose) membranes with the aim of increasing the retention of products and decreasing membrane fouling. The raw material used was a sodium-based spent sulfite liquor from the sulfite pulping process of spruce and pine. The results show that the hydrophilic membranes were superior to the hydrophobic membranes in terms of higher fluxes (up to twice the magnitude), higher product retentions and less fouling (up to five times lower fouling). The fouling was probably caused by pore blocking as observed in earlier studies. However, the hydrophilic membranes had a lower affinity for lignin, which was indicated by the lower retention and fouling. This also resulted in a separation degree, which was higher compared with the hydrophobic membrane, thus yielding a higher galactoglucomannan (GGM) purity. 2D HSQC NMR results show that no major structural differences were present in the hydrophilic and hydrophobic retentates. A techno-economical evaluation resulted in the RC70PP being chosen as the most cost-efficient membrane in terms of flux and product recovery.

Sequential fractionation of the lignocellulosic components in hardwood based on steam explosion and hydrotropic extraction.

BACKGROUND: The forest biorefinery plays an important part in the evolving circular bioeconomy due to its capacity to produce a portfolio of bio-based and sustainable fuels, chemicals, and materials. To tap into its true potential, more efficient and environmentally benign methods are needed to fractionate woody biomass into its main components (cellulose, hemicellulose, and lignin) without reducing their potential for valorization. This work presents a sequential fractionation method for hardwood based on steam pretreatment (STEX) and hydrotropic extraction (HEX) with sodium xylene sulfonate. By prehydrolyzing the hemicellulose (STEX) and subsequently extract the lignin from the cellulose fraction (HEX), the major wood components can be recovered in separate process streams and be further valorized. RESULTS: Using autocatalyzed STEX and HEX, hemicellulose (> 70%) and lignin (~ 50%) were successfully fractionated and recovered in separate liquid streams and cellulose preserved (99%) and enriched (~ twofold) in the retained solids. Investigation of pretreatment conditions during HEX showed only incremental effects of temperature (150-190 °C) and hold-up time (2-8 h) variations on the fractionation efficiency. The hydrolyzability of the cellulose-rich solids was analyzed and showed higher cellulose conversion when treated with the combined process (47%) than with HEX alone (29%), but was inferior to STEX alone (75%). Protein adsorption and surface structure analysis suggested decreased accessibility due to the collapse of the fibrillose cellulose structure and an increasingly hydrophobic lignin as potential reasons. CONCLUSION: This work shows the potential of sequential STEX and HEX to fractionate and isolate cellulose, hemicellulose, and a sulfur-free lignin in separate product streams, in an efficient, sustainable, and scalable process.

Evaluation of Sequential Processing for the Extraction of Starch, Lipids, and Proteins From Wheat Bran.

In line with the need to better utilize agricultural resources, and valorize underutilized fractions, we have developed protocols to increase the use of wheat bran, to improve utilization of this resource to additional products. Here, we report sequential processing for extraction of starch, lipids, and proteins from wheat brans with two different particle sizes leaving a rest-material enriched in dietary fiber. Mild water-based extraction of starch resulted in maximum 81.7 ± 0.67% yield. Supercritical fluid extraction of lipids by CO2 resulted in 55.2 ± 2.4% yield. This was lower than the corresponding yield using Soxhlet extraction, which was used as a reference method, but allowed a continued extraction sequence without denaturation of the proteins remaining in the raw-material. Alkaline extraction of non-degraded proteins resulted in a yield corresponding to one third of the total protein in the material, which was improved to reach 62 ± 8% by a combination of wheat bran enzymes activation followed by Osborne fractionation. The remaining proteins were extracted in degraded form, resulting in maximum 91.6 ± 1.6% yield of the total proteins content. The remaining material in both fine and coarse bran had a fiber content that on average corresponded to 73 ± 3%. The current work allows separation of several compounds, which is enabling valorization of the bran raw-material into several products.

Pretreatment for biorefineries: a review of common methods for efficient utilisation of lignocellulosic materials.

The implementation of biorefineries based on lignocellulosic materials as an alternative to fossil-based refineries calls for efficient methods for fractionation and recovery of the products. The focus for the biorefinery concept for utilisation of biomass has shifted, from design of more or less energy-driven biorefineries, to much more versatile facilities where chemicals and energy carriers can be produced. The sugar-based biorefinery platform requires pretreatment of lignocellulosic materials, which can be very recalcitrant, to improve further processing through enzymatic hydrolysis, and for other downstream unit operations. This review summarises the development in the field of pretreatment (and to some extent, of fractionation) of various lignocellulosic materials. The number of publications indicates that biomass pretreatment plays a very important role for the biorefinery concept to be realised in full scale. The traditional pretreatment methods, for example, steam pretreatment (explosion), organosolv and hydrothermal treatment are covered in the review. In addition, the rapidly increasing interest for chemical treatment employing ionic liquids and deep-eutectic solvents are discussed and reviewed. It can be concluded that the huge variation of lignocellulosic materials makes it difficult to find a general process design for a biorefinery. Therefore, it is difficult to define "the best pretreatment" method. In the end, this depends on the proposed application, and any recommendation of a suitable pretreatment method must be based on a thorough techno-economic evaluation.

Removal of Water-Soluble Extractives Improves the Enzymatic Digestibility of Steam-Pretreated Softwood Barks.

Softwood bark contains a large amounts of extractives-i.e., soluble lipophilic (such as resin acids) and hydrophilic components (phenolic compounds, stilbenes). The effects of the partial removal of water-soluble extractives before acid-catalyzed steam pretreatment on enzymatic digestibility were assessed for two softwood barks-Norway spruce and Scots pine. A simple hot water extraction step removed more than half of the water-soluble extractives from the barks, which improved the enzymatic digestibility of both steam-pretreated materials. This effect was more pronounced for the spruce than the pine bark, as evidenced by the 30 and 11% glucose yield improvement, respectively, in the enzymatic digestibility. Furthermore, analysis of the chemical composition showed that the acid-insoluble lignin content of the pretreated materials decreased when water-soluble extractives were removed prior to steam pretreatment. This can be explained by a decreased formation of water-insoluble "pseudo-lignin" from water-soluble bark phenolics during the acid-catalyzed pretreatment, which otherwise results in distorted lignin analysis and may also contribute to the impaired enzymatic digestibility of the barks. Thus, this study advocates the removal of extractives as the first step in the processing of bark or bark-rich materials in a sugar platform biorefinery.

Impact of Lignin Content on the Properties of Hemicellulose Hydrogels.

Hemicellulose is a promising renewable raw material for the production of hydrogels. This polysaccharide exists in large amounts in various waste streams, in which they are usually impure and heavily diluted. Several downstream processing methods can be combined to concentrate and purify the hemicellulose. However, such an approach can be costly; hence, the effect of impurities on the formation and properties of hydrogels must be determined. Lignin usually exists in these waste streams as a major impurity that is also difficult to separate. This compound can darken hydrogels and decrease their swellability and reactivity, as shown in many studies. Other properties and effects of lignin impurities are equally important for the end application of hydrogels and the overall process economy. In this work, we examined the feasibility of producing hydrogels from hemicelluloses that originated from sodium-based spent sulfite liquor. A combination of membrane filtration and anti-solvent precipitation was used to extract and purify various components. The influence of the purity of hemicellulose and the addition of lignosulfonates (emulated impurities in the downstream processing) to the crosslinking reaction mixture on the mechanical, thermal, and chemical properties of hydrogels was determined.

Process design of SSCF for ethanol production from steam-pretreated, acetic-acid-impregnated wheat straw.

BACKGROUND: Pretreatment is an important step in the production of ethanol from lignocellulosic material. Using acetic acid together with steam pretreatment allows the positive effects of an acid catalyst to be retained, while avoiding the negative environmental effects associated with sulphuric acid. Acetic acid is also formed during the pretreatment and hydrolysis of hemicellulose, and is a known inhibitor that may impair fermentation at high concentrations. The purpose of this study was to improve ethanol production from glucose and xylose in steam-pretreated, acetic-acid-impregnated wheat straw by process design of simultaneous saccharification and co-fermentation (SSCF), using a genetically modified pentose fermenting yeast strain Saccharomyces cerevisiae. RESULTS: Ethanol was produced from glucose and xylose using both the liquid fraction and the whole slurry from pretreated materials. The highest ethanol concentration achieved was 37.5 g/L, corresponding to an overall ethanol yield of 0.32 g/g based on the glucose and xylose available in the pretreated material. To obtain this concentration, a slurry with a water-insoluble solids (WIS) content of 11.7 % was used, using a fed-batch SSCF strategy. A higher overall ethanol yield (0.36 g/g) was obtained at 10 % WIS. CONCLUSIONS: Ethanol production from steam-pretreated, acetic-acid-impregnated wheat straw through SSCF with a pentose fermenting S. cerevisiae strain was successfully demonstrated. However, the ethanol concentration was too low and the residence time too long to be suitable for large-scale applications. It is hoped that further process design focusing on the enzymatic conversion of cellulose to glucose will allow the combination of acetic acid pretreatment and co-fermentation of glucose and xylose.

Techno-economic evaluation of integrated first- and second-generation ethanol production from grain and straw.

BACKGROUND: Integration of first- and second-generation ethanol production can facilitate the introduction of second-generation lignocellulosic ethanol production. Consolidation of the second-generation with the first-generation process can potentially reduce the downstream processing cost for the second-generation process as well as providing the first-generation process with energy. This study presents novel experimental results from integrated first- and second-generation ethanol production from grain and wheat straw in a process development unit. The results were used in techno-economic evaluations to investigate the feasibility of the plant, in which the main co-products were distiller's dried grains with solubles and biogas. RESULTS: An overall glucose to ethanol yield, of 81 % of the theoretical, based on glucose available in the raw material, was achieved in the experiments. A positive net present value was found for all the base case scenarios and the minimal ethanol selling price varied between 0.45 and 0.53 EUR/L ethanol. The revenue increased with combined xylose and glucose fermentation and biogas upgrading to vehicle fuel quality. A decrease in the biogas yield from 80 to 60 % also largely affects the net present value. The energy efficiency for the energy content in products available for sale compared with the incoming energy content varied from 74 to 80 %. CONCLUSIONS: One of the two main configurations can be chosen when designing an integrated first- and second-generation ethanol production plant from grain and straw: that producing biogas or that producing distiller's dried grains with solubles from the xylose sugars. The choice depends mainly on the local market and prices for distiller's dried grains with solubles and biogas, since the prices for both co-products have fluctuated a great deal in recent years. In the current study, however, distiller's dried grains with solubles were found to be a more promising co-product than biogas, if the biogas was not upgraded to vehicle fuel quality. It was also concluded that additional experimental data from biogas production using first- and second-generation substrates are required to obtain improved economic evaluations.

Influence of bark on fuel ethanol production from steam-pretreated spruce.

BACKGROUND: Bark and bark-containing forest residues have the potential for utilization as raw material for lignocellulosic ethanol production due to their abundance and low cost. However, the different physical properties and chemical composition of bark compared to the conventionally used wood chips may influence the spruce-to-ethanol bioconversion process. This study assesses the impact of bark on the overall bioconversion in two process configurations, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF), utilizing steam-pretreated spruce bark and wood mixtures. RESULTS: Mixtures of different proportions of spruce bark and wood chips were subjected to SO2-catalyzed steam pretreatment at 210°C for five minutes, which has been shown to be effective for the pretreatment of spruce wood chips. The final ethanol concentration was the highest without bark and decreased significantly with increasing proportions of bark in both process configurations. However, this decrease cannot be attributed solely to the lower availability of the carbohydrates in mixtures containing bark, as the ethanol yield also decreased, from 85 to 59% in SSF and from 84 to 51% in SHF, as the mass fraction of bark was increased from 0 to 100%. CONCLUSIONS: The results show that it was significantly more difficult to hydrolyse spruce bark to monomeric sugars than wood chips. Bark had an adverse effect on the whole bioconversion process due to its lower enzymatic hydrolyzability. On the other hand, bark inclusion had no detrimental effect on the fermentability of steam-pretreated spruce wood and bark mixtures. It was also observed that lower amounts of inhibitory degradation products were formed during the steam pretreatment of spruce bark than during the steam pretreatment of wood chips.

Optimizing ethanol and methane production from steam-pretreated, phosphoric acid-impregnated corn stover.

Pretreatment is of vital importance in the production of ethanol and methane from agricultural residues. In this study, the effects of steam pretreatment with phosphoric acid on enzymatic hydrolysis (EH), simultaneous saccharification and fermentation (SSF), anaerobic digestion (AD) and the total energy output at three different temperatures were investigated. The effect of separating the solids for SSF and the liquid for AD was also studied and compared with using the whole slurry first in SSF and then in AD. Furthermore, the phosphoric acid was compared to previous studies using sulphuric acid or no catalyst. Using phosphoric acid resulted in higher yields than when no catalyst was used. However, compared with sulphuric acid, an improved yield was only seen with phosphoric acid in the case of EH. The higher pretreatment temperatures (200 and 210 °C) resulted in the highest yields after EH and SSF, while the highest methane yield was obtained with the lower pretreatment temperature (190 °C). The highest yield in terms of total energy recovery (78 %) was obtained after pretreatment at 190 °C, but a pretreatment temperature of 200 °C is, however, the best alternative since fewer steps are required (whole slurry in SSF and then in AD) and high product yields were obtained (76 %).

Effects of production and market factors on ethanol profitability for an integrated first and second generation ethanol plant using the whole sugarcane as feedstock.

BACKGROUND: Sugarcane is an attractive feedstock for ethanol production, especially if the lignocellulosic fraction can also be treated in second generation (2G) ethanol plants. However, the profitability of 2G ethanol is affected by the processing conditions, operating costs and market prices. This study focuses on the minimum ethanol selling price (MESP) and maximum profitability of ethanol production in an integrated first and second generation (1G + 2G) sugarcane-to-ethanol plant. The feedstock used was sugarcane juice, bagasse and leaves. The lignocellulosic fraction was hydrolysed with enzymes. Yields were assumed to be 95% of the theoretical for each of the critical steps in the process (steam pretreatment, enzymatic hydrolysis (EH), fermentation, solid/liquid separation, anaerobic digestion) in order to obtain the best conditions possible for ethanol production, to assess the lowest production costs. Techno-economic analysis was performed for various combinations of process options (for example use of pentoses, addition of leaves), EH conditions (water-insoluble solids (WIS) and residence time), operating cost (enzymes) and market factors (wholesale prices of electricity and ethanol, cost of the feedstock). RESULTS: The greatest reduction in 2G MESP was achieved when using the pentoses for the production of ethanol rather than biogas. This was followed, in decreasing order, by higher enzymatic hydrolysis efficiency (EHE), by increasing the WIS to 30% and by a short residence time (48 hours) in the EH. The addition of leaves was found to have a slightly negative impact on 1G + 2G MESP, but the effect on 2G MESP was negligible. Sugarcane price significantly affected 1G + 2G MESP, while the price of leaves had a much lower impact. Net present value (NPV) analysis of the most interesting case showed that integrated 1G + 2G ethanol production including leaves could be more profitable than 1G ethanol, despite the fact that the MESP was higher than in 1G ethanol production. CONCLUSIONS: A combined 1G + 2G ethanol plant could potentially outperform a 1G plant in terms of NPV, depending on market wholesale prices of ethanol and electricity. Therefore, although it is more expensive than 1G ethanol production, 2G ethanol production can make the integrated 1G + 2G process more profitable.