Second-generation Ethanol Production Research Articles

Cassava hydrolysates used as substrates for the production of second-generation ethanol by Saccharomyces cerevisiae ATCC 26602 immobilized in sodium alginate spheres

Cassava has a prominent position in the Brazilian agriculture and economy. During the production of flour, a very large volume of residue is generated. One way to use these residues is through the production of ethanol. In this context, the present research aimed to use cassava residues as raw material for ethanol production. For this, sulfuric acid was used at concentrations of 1.0 to 5.0% during 5, 10 and 15 minutes of heating in an autoclave. The fermentation was carried out by Saccharomyces cerevisiae ATCC 26602, and to achieve a better performance, it was immobilized in sodium alginate spheres. Ethanol production was also estimated using a synthetic medium added with glucose that served as a comparison standard. The results of the experiment showed that the highest concentration of reducing sugars obtained by the hydrolysis of the residue was with the use of 2.0% of H2SO4 with 15 min. heating at 121 ºC, releasing 56.26 g/L of reducing sugars. The parameters used that led to the highest production of ethanol by the yeast, 7.85 g/L of ethanol, were: pH of 6.5, growth temperature of 30 °C, without agitation. The results showed that cassava residues can be used as a potential substrate for ethanol production. Thus, this work presents data on the most suitable conditions for the use of these industrial wastes in order to generate less polluting fuel energy, an increasingly attractive feature in a world where economic and environmental concerns grow every day.

ShF5H1 overexpression increases syringyl lignin and improves saccharification in sugarcane leaves

ABSTRACT The agricultural sugarcane residues, bagasse and straws, can be used for second-generation ethanol (2GE) production by the cellulose conversion into glucose (saccharification). However, the lignin content negatively impacts the saccharification process. This polymer is mainly composed of guaiacyl (G), hydroxyphenyl (H), and syringyl (S) units, the latter formed in the ferulate 5-hydroxylase (F5H) branch of the lignin biosynthesis pathway. We have generated transgenic lines overexpressing ShF5H1 under the control of the C4H (cinnamate 4-hydroxylase) rice promoter, which led to a significant increase of up to 160% in the S/G ratio and 63% in the saccharification efficiency in leaves. Nevertheless, the content of lignin was unchanged in this organ. In culms, neither the S/G ratio nor sucrose accumulation was altered, suggesting that ShF5H1 overexpression would not affect first-generation ethanol production. Interestingly, the bagasse showed a significantly higher fiber content. Our results indicate that the tissue-specific manipulation of the biosynthetic branch leading to S unit formation is industrially advantageous and has established a foundation for further studies aiming at refining lignin modifications. Thus, the ShF5H1 overexpression in sugarcane emerges as an efficient strategy to improve 2GE production from straw.

Sugarcane: A Climate Resilient Devine Crop

Worldwide increasing demands of food, fodder, fuel and other bio-chemicals for erratically increasing population, urbanization and industrialization. Only sugarcane (Saccharum spp.) has great potential as a major feedstock for sugar, biofuel and allied related production. Sugarcane is the lead driver for production of sugar and sweeteners globally. It is considered among the best options and replace the fossil fuels for production of biofuels today due to an exceptional biomass production capacity, high carbohydrate (sugar + fiber) content, and a favourable energy input/output ratio. The conversion of sugarcane biomass into fermentable sugars for second-generation ethanol production is a promising alternative to meet future demands of biofuel production in the world. Biofuels have gained much importance due to the depleting fossil fuel resources and the over-accumulation of CO2 and other greenhouse gases in the environment. Biofuel and paper production from sugarcane can be achieve the targets to replace fossil fuels to scavenge carbon dioxide released into the atmosphere and to attain environmental and economic sustainability. Fermentable sugars (40-60%) of molasses which is diluted with distilled water and by fermentation process (Saccharomyces cerevisae) produce rum (wine). Application of sugarcane by-products, such as press mud, vinasse and bagasse, to improves chemical, physical, and biological properties of soil and enhanced the crop quality and yield. For transfer the live micro-organism from agar slant of laboratory to rhizosphere then sugar press mud play key role in formulating microbial inoculant as suitable carrier. Food yeast Torulopsis utilis, is prepared from molasses used as baker’s and brewer’s yeast in bakery industry. Sugarcane improves the economy of rural population by providing lead source of fodder for livestock management as a sugarcane tops and by-products of sugarcane (molasses treated with urea, mineral mixture treated sugarcane top hays etc.). Scientific interventions have not only helped to improve the cane crop but industrial procedures have also been upgraded resulting in improved production of biofuel, paper, wine etc and other sugarcane by-products used to improve the economy of rural and urban population. However, building a bridge between science and industry requires investments in research, development and transfer of new technologies to the industry as well as specialized personnel to deal with new technological challenges.

Study of acid hydrolysis and submerged fermentation parameters in second generation ethanol production using cassava waste

Biofuels are an alternative to reduce the environmental impacts caused by the use of petroleum-based fuels. Second generation ethanol comes from lignocellulosic materials that are little used or discarded in the environment, including agro-industrial waste. These residues are rich in sugars that can be converted by ethanol-producing microorganisms. In this context, the present work uses cassava waste (Manihot esculenta Crantz) obtained during the processing of starch as a raw material for the production of cellulosic ethanol, as well as evaluating different parameters in acid hydrolysis and during submerged fermentation using the strain Saccharomyces cerevisiae ATCC 26602. Experiments of acid hydrolysis of these residues with sulfuric acid in concentrations of 0.5% to 5.0% (v/v) were carried out. The results demonstrated that 2.0% H2SO4 for 10 min. reaction at 121 ºC/1.1 atm in an autoclave reached the highest release of reducing sugars present in the residue (131.09 g.L-1). The ideal conditions for alcoholic fermentation using yeast were pH 6.5, temperature of 35 ºC, without rotation and initial reducing sugar concentration of 50 g.L-1, resulting in 21.23 g.L-1 of ethanol with productivity of 1.86 g.L-¹.h-¹ and theoretical yield of 96.5% after 12 hours of fermentation. The results indicated that the cassava waste serve as a potential substrate for the production of second generation ethanol. Thus, this study provides data on the most suitable conditions for the use of these industrial wastes aiming at the generation of a renewable fuel, an increasingly attractive feature for the world, where the economic and environmental concern is growing.

Adsorption of cadmium and copper using biochar obtained from the residue of the second-generation ethanol production: kinetics and equilibrium study

The disposal of industrial effluents containing cadmium and copper ions is a significant environmental concern. This study aims to address this issue by evaluating the adsorption capacity of adsorbents derived from residual biomass from second-generation ethanol production. The objective is to effectively remove cadmium and copper ions from aqueous solutions. The biochar utilized in the study was characterized as microporous and exhibited characteristic bands associated with carboxyls and hydroxyls. The pHPZC (point of zero charge) was determined to be 8.0. The kinetic study revealed that the PSO (pseudo-second-order) model provided the best fit, indicating that the adsorption rate depends on the amount of solute adsorbed onto the adsorbent’s surface. In the equilibrium study, it was observed that adsorption occurred in a monolayer, as evidenced by the superior fit of the Langmuir model. Furthermore, the equilibrium experimental data for the bicomponent system was more accurately represented by the empirical models of Extended Freundlich and Modified Redlich-Peterson. These findings demonstrate the potential of biochar derived from the residue of second-generation ethanol production, a novel application, in effectively removing cadmium and copper ions from aqueous solutions.

Production of Lignin-Derived Functional Material for Efficient Electromagnetic Wave Absorption with an Ultralow Filler Ratio.

Industrial lignin, a by-product of pulping for papermaking fibers or of second-generation ethanol production, is primarily served as a low-grade combustible energy source. The fabrication of high-value-added functional materials with industrial lignin is still a challenge. Herein, a three-dimensional hierarchical lignin-derived porous carbon (HLPC) was prepared with lignosulfonate as the carbon source and MgCO3 as the template. The uniform mixing of precursor and template agent resulted in the construction of a three-dimensional hierarchical porous structure. HLPC presented excellent electromagnetic wave (EMW) absorption performance. With a low filler content of 7 wt%, HLPC showed a minimum reflection loss (RL) value of -41.8 dB (1.7 mm, 13.8 GHz), and a maximum effective absorption bandwidth (EAB) of 4.53 GHz (1.6 mm). In addition, the enhancement mechanism of HLPC for EMW absorption was also explored through comparing the morphology and electromagnetic parameters of lignin-derived carbon (LC) and lignin-derived porous carbon (LPC). The three-dimensional hierarchical porous structure endowed the carbon with a high pore volume, resulting in an abundant gas-solid interface between air and carbon for interfacial polarization. This structure also provided conductive networks for conduction loss. This work offers a strategy to synthesize biomass-based carbon for high-performance EMW absorption.

First- and second-generation integrated process for bioethanol production: Fermentation of molasses diluted with hemicellulose hydrolysate by recombinant Saccharomyces cerevisiae.

The integration of first- (1G) and second-generation (2G) ethanol production by adding sugarcane juice or molasses to lignocellulosic hydrolysates offers the possibility to overcome the problem of inhibitors (acetic acid, furfural, hydroxymethylfurfural and phenolic compounds), and add nutrients (such as salts, sugars and nitrogen sources) to the fermentation medium, allowing the production of higher ethanol titers. In this work, an 1G2G production process was developed with hemicellulosic hydrolysate (HH) from a diluted sulfuric acid pretreatment of sugarcane bagasse and sugarcane molasses. The industrial Saccharomyces cerevisiae CAT-1 was genetically modified for xylose consumption and used for co-fermentation of sucrose, fructose, glucose, and xylose. The fed-batch fermentation with high cell density that mimics an industrial fermentation was performed at bench scale fermenter, achieved high volumetric ethanol productivity of 1.59 g L-1 h-1, 0.39 g g-1 of ethanol yield, and 44.5 g L-1 ethanol titer, and shown that the yeast was able to consume all the sugars present in must simultaneously. With the results, it was possible to establish a mass balance for the global process: from pretreatment to the co-fermentation of molasses and HH, and it was possible to establish an effective integrated process (1G2G) with sugarcane molasses and HH co-fermentation employing a recombinant yeast.

Silencing ScGUX2 reduces xylan glucuronidation and improves biomass saccharification in sugarcane.

There is an increasing need for renewable energy sources to replace part of our fossil fuel-based economy and reduce greenhouse gas emission. Sugarcane bagasse is a prominent feedstock to produce cellulosic bioethanol, but strategies are still needed to improve the cost-effective exploitation of this potential energy source. In model plants, it has been shown that GUX genes are involved in cell wall hemicellulose decoration, adding glucuronic acid substitutions on the xylan backbone. Mutation of GUX genes increases enzyme access to cell wall polysaccharides, reducing biomass recalcitrance in Arabidopsis thaliana. Here, we characterized the sugarcane GUX genes and silenced GUX2 in commercial hybrid sugarcane. The transgenic lines had no penalty in development under greenhouse conditions. The sugarcane GUX1 and GUX2 enzymes generated different patterns of xylan glucuronidation, suggesting they may differently influence the molecular interaction of xylan with cellulose and lignin. Studies using biomass without chemical or steam pretreatment showed that the cell wall polysaccharides, particularly xylan, were less recalcitrant in sugarcane with GUX2 silenced than in WT plants. Our findings suggest that manipulation of GUX in sugarcane can reduce the costs of second-generation ethanol production and enhance the contribution of biofuels to lowering the emission of greenhouse gases.

Calcium alginate-immobilized β-glucosidase from Moniliophthora perniciosa: characterization and sugarcane bagasse hydrolysis

The utilization of lignocellulosic materials for second-generation ethanol production via enzymatic catalysts is primarily hindered by enzyme cost. Enzymatic immobilization emerges as a viable solution, enabling enzyme reuse. This study investigated the immobilization of an enzymatic extract obtained from Moniliophthora perniciosa fermentation in calcium alginate spheres using the direct trapping method. Initial tests assessed β-glucosidase activity, showing that a higher concentration of calcium chloride (1 M) alongside larger diameter spheres yielded improved results. The immobilized enzyme was reused for up to 17 cycles without significant loss of activity. The percentage of reducing sugars after 48-h hydrolysis with the supplemented enzymatic extract was 226%, doubling the value achieved with only the free enzymatic extract. The immobilized enzyme retained 50% of its initial activity after 1 h at 80 °C, demonstrating higher activity at pH 6 and 60 °C. These findings suggest that this immobilization technique is simple, economically viable, and effective for the hydrolysis of pretreated sugarcane bagasse.

Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate

Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two β-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both β-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1Δ gre3Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.

Production of second-generation ethanol and carboxymethylcellulose from sugarcane bagasse pretreated with low-cost ionic liquids

Production of second-generation ethanol and carboxymethylcellulose from sugarcane bagasse pretreated with low-cost ionic liquids

Al2(SO4)3 alters the antioxidant mitochondrial metabolism of Botritys cinerea and optimizes the production of cellulose and oxidative degrading enzymes

The enzymes produced by pathogenic fungi, especially Botritys cinerea, deserve specific attention due to the diversity of their applications, mainly in biofuel production, food processing, and the pharmaceutical industry. Thus, this work used Al2(SO4)3 as a stressor in order to evaluate if the stress levels caused by the concentrations of 100, 250, 500, and 1000 ppm were sufficient to increase the production of hydrolytic cellulolytic enzymes (FPase, CMCase, Avicelase, β-glucosidase, xylanase) and oxidative (laccase and manganese peroxidase). The study also evaluated the stress levels in previously treated mycelia of B. cinerea and whether they corresponded to the different states of mitochondrial respiration. Our study indicates that Al2(SO4)3 increased the production of cellulolytic and oxidative enzymes in all concentrations in a dose-dependent manner and that Al2(SO4)3 alters the mitochondrial respiratory rate, with lower ATP productions, indicating that less-coupled mitochondria were obtained and that this may be due to the increase of oxidative stress. Thus, it is plausible to suggest the use of Al2(SO4)3 in the production of cellulolytic enzymes, which could be used in the hydrolysis stage of second-generation ethanol production processes, as it reduces the time required for enzymatic expression applications in industrial processes.

Enzyme technology in bioethanol production from lignocellulosic biomass: Recent trends with a focus on immobilized enzymes

Enzyme immobilization is a useful tool to produce biocatalysts with improved performance, such as high activity, high stability at operational conditions, and easy recovery and reuse. In this context, the production of second-generation ethanol using immobilized enzymes was reviewed. Emphasis was placed on the pre-treatment and/or hydrolysis steps to increase efficiency and reduce the cost of the process. In addition, the process design of bioethanol using immobilized enzymes was critically reviewed. In the enzymatic pre-treatment, laccases, manganese peroxidases, lignin peroxidases, and lytic polysaccharide monooxygenases are the main enzymes involved. When considering processes with heterogeneous biocatalysts, laccases are the most explored enzyme. In the hydrolysis step, cellulases and hemicellulases or a mixture of them are the main enzymes explored in the literature frequently using magnetic nanoparticles as support for enzyme immobilization. Although enzyme immobilization is a mature technology, the use of these biocatalysts is frequently limited to only one step of the bioethanol production process, such as pre-treatment or hydrolysis performed using Benchmark Technology. However, some recent papers have explored innovative design processes using a mixture of immobilized enzymes or co-immobilized enzymes to join some process steps, thereby decreasing the cost of enzymes and equipment.

Exploring the Potential of Coffee Husks as a Raw Material for Second-Generation Ethanol Production

Exploring the Potential of Coffee Husks as a Raw Material for Second-Generation Ethanol Production

Acidogenesis of Pentose Liquor to Produce Biohydrogen and Organic Acids Integrated with 1G–2G Ethanol Production in Sugarcane Biorefineries

Second-generation (2G) ethanol production has been increasingly evaluated, and the use of sugarcane bagasse as feedstock has enabled the integration of this process with first-generation (1G) ethanol production from sugarcane. The pretreatment of bagasse generates pentose liquor as a by-product, which can be anaerobically processed to recover energy and value-added chemicals. The potential to produce biohydrogen and organic acids from pentose liquor was assessed using a mesophilic (25 °C) upflow anaerobic packed-bed bioreactor in this study. An average organic loading rate of 11.1 g COD·L−1·d−1 was applied in the reactor, resulting in a low biohydrogen production rate of 120 mL·L−1 d−1. Meanwhile, high lactate (38.6 g·d−1), acetate (31.4 g·d−1), propionate (50.1 g·d−1), and butyrate (50.3 g·d−1) production rates were concomitantly obtained. Preliminary analyses indicated that the full-scale application of this anaerobic acidogenic technology for hydrogen production in a medium-sized 2G ethanol distillery would have the potential to completely fuel 56 hydrogen-powered vehicles per day. An increase of 24.3% was estimated over the economic potential by means of chemical production, whereas an 8.1% increase was calculated if organic acids were converted into methane for cogeneration (806.73 MWh). In addition, 62.7 and 74.7% of excess organic matter from the 2G ethanol waste stream could be removed with the extraction of organic acid as chemical commodities or their utilization as a substrate for biomethane generation, respectively.

Evaluating the Production of Second-Generation Ethanol by Spathaspora passalidarum Immobilized on Sugarcane Bagasse

Evaluating the Production of Second-Generation Ethanol by Spathaspora passalidarum Immobilized on Sugarcane Bagasse

Improving the Integrated Process of First- and Second-Generation Ethanol Production with Multiperiod Energy Integration

Improving the Integrated Process of First- and Second-Generation Ethanol Production with Multiperiod Energy Integration

Ethanol production from Agave salmiana leaves by semi and simultaneous saccharification and fermentation at high temperature using Kluyveromycesmarxianus

Second-generation ethanol production by separate hydrolysis and fermentation (SHF) currently remains economically unviable, and thus more profitable alternatives are being sought. In the present study, the lignocellulosic biomass (LB) of Agave salmiana leaves was evaluated in the production of bioethanol through the processes of SHF, semi-simultaneous saccharification and fermentation (SSSF), and simultaneous saccharification and fermentation (SSF). The leaves of A. salmiana were subjected to acid pretreatment (1% H2SO4) and then to saccharification at temperatures of 40 and 50 °C. The processes of SHF, SSSF, and SSF were evaluated at 40 °C using the yeast Kluyveromyces marxianus OFF1. The results show that saccharification at 50 °C released up to 103.12 ± 1.07 g/L of reducing sugars, while at a temperature of 40 °C, the value was 78.41 ± 0.80 g/L, and thus the enzyme retained 76% of its activity at the latter temperature. In the fermentation processes, sugar consumption was up to 89% and ethanol yield up to 94%. The maximum ethanol production was recorded with the process of SSF, with concentrations of 51.5 g/L, which were significant and at least 13% higher compared to the other processes. The results show that the thermotolerance of K. marxianus OFF1 allows fermentation at 40 °C and a more efficient SSF process for transforming the sugars from this agro-industrial waste into ethanol.

Evaluating the Potential of Newly Developed Energy Cane Clones for First- and Second-Generation Ethanol Production

The rapid increases in fuel ethanol demand and food security concerns have driven the need for diverse feedstocks in the ethanol production process. Energy cane is an energy crop that is an ideal sustainable biofuel feedstock. The present study evaluated ethanol production of the juice and bagasse of two newly developed energy cane clones, TByEFC08-0035 and TByEFC10-0004. The results of the chemical composition analyses of the juice and bagasse samples revealed that the two energy cane clones contained high contents of both sucrose (15.36–17.95%) and fiber (13.44–24.16%). The maximum ethanol concentrations from the juice on a laboratory scale (87.10 g/L) and on an agronomic scale (1211.76 kg/ha) were recorded for TByEFC10-0004 fermented with a new isolate Kluyveromyces marxianus SJT83, whereas the maximum ethanol concentrations from bagasse on a laboratory scale (9.81 g/L) and on an agronomic scale (790.68 kg/ha) were reached with TByEFC08-0035 fermented with Scheffersomyces shehatae TTC79. The total ethanol yields from the juice and bagasse samples per cultivation area of both energy cane clones were in the range 1294.23–1469.14 kg/ha, being 1.70–1.93 and 1.08–1.23 times higher than the control energy cane Biotec2 variety and the commercial sugar cane Khon Kaen3 variety, respectively. This study revealed the potential of the energy cane clones TByEFC08-0035 and TByEFC10-0004 currently being developed as sugar and lignocellulose substrates for first- and second-generation ethanol industry applications.

Flexible heat integration system in first-/second-generation ethanol production via screening pinch-based method and multiperiod model

In sugarcane biorefineries, energy integration plays a fundamental role regarding by-product destination and final sales profitability. In biorefineries, the by-product of the sugar/ethanol production process – sugarcane bagasse – can be burned in boilers to produce steam to supply the thermal demands of the plant. Normally, the quantity of bagasse from the plant exceeds the quantity necessary for supplying the energy requirements of the plant. This surplus can be used for either (i) additional electricity production and selling to the grid or (ii) second-generation (2G) ethanol production. To maximize profit, the plant should be flexible to produce either electricity or 2G ethanol according to the current market prices of these products. In this work, an approach is presented for designing a flexible heat exchanger network (HEN) based on a multiperiod synthesis. It involves assessing probabilities of occurrence for different bagasse use scenarios. These values are obtained from a pinch-based approach for screening optimal heat integration in a range of ethanol/electricity prices. A multiperiod HEN synthesis problem is then solved with variable flow rates. Results show that maximum bagasse diverted to 2G ethanol production is increased from 66% to 77% with the new HEN compared to the original design considered.