Second-generation Bioethanol Production Research Articles

Agricultural Waste Management by Production of Second-Generation Bioethanol from Sugarcane Bagasse Using Indigenous Yeast Strain.

In the wake of rapid industrialization and burgeoning transportation networks, the escalating demand for fossil fuels has accelerated the depletion of finite energy reservoirs, necessitating urgent exploration of sustainable alternatives. To address this, current research is focusing on renewable fuels like second-generation bioethanol from agricultural waste such as sugarcane bagasse. This approach not only circumvents the contentious issue of food-fuel conflicts associated with biofuels but also tackles agricultural waste management. In the present study indigenous yeast strain, Clavispora lusitaniae QG1 (MN592676), was isolated from rotten grapes to ferment xylose sugars present in the hemicellulose content of sugarcane bagasse. To liberate the xylose sugars, dilute acid pretreatment was performed. The highest reducing sugars yield was 1.2% obtained at a temperature of 121°C for 15min, a solid-to-liquid ratio of 1:25 (% w/v), and an acid concentration of 1% dilute acid H2SO4 that was significantly higher (P < 0.001) yield obtained under similar conditions at 100°C for 1h. The isolated strain was statistically optimized for fermentation process by Plackett-Burman design to achieve the highest ethanol yield. Liberated xylose sugars were completely utilized by Clavispora lusitaniae QG1 (MN592676) and gave 100% ethanol yield. This study optimizes both fermentation process and pretreatment of sugarcane bagasse to maximize bioethanol yield and demonstrates the ability of isolated strain to effectively utilize xylose as a carbon source. The desirable characteristics depicted by strain Clavispora lusitaniae shows its promising utilization in management of industrial waste like sugarcane bagasse by its conversion into renewable biofuels like bioethanol.

Enhancing xylose-fermentation capacity of engineered Saccharomyces cerevisiae by multistep evolutionary engineering in inhibitor-rich lignocellulose hydrolysate.

Major progress in developing Saccharomyces cerevisiae strains that utilize the pentose sugar xylose has been achieved. However, the high inhibitor content of lignocellulose hydrolysates still hinders efficient xylose fermentation, which remains a major obstacle for commercially viable second-generation bioethanol production. Further improvement of xylose utilization in inhibitor-rich lignocellulose hydrolysates remains highly challenging. In this work, we have developed a robust industrial S. cerevisiae strain able to efficiently ferment xylose in concentrated undetoxified lignocellulose hydrolysates. This was accomplished with novel multistep evolutionary engineering. First, a tetraploid strain was generated and evolved in xylose-enriched pretreated spruce biomass. The best evolved strain was sporulated to obtain a genetically diverse diploid population. The diploid strains were then screened in industrially relevant conditions. The best performing strain, MDS130, showed superior fermentation performance in three different lignocellulose hydrolysates. In concentrated corncob hydrolysate, with initial cell density of 1g DW/l, at 35°C, MDS130 completely coconsumed glucose and xylose, producing±7% v/v ethanol with a yield of 91% of the maximum theoretical value and an overall productivity of 1.22g/l/h. MDS130 has been developed from previous industrial yeast strains without applying external mutagenesis, minimizing the risk of negative side-effects on other commercially important properties and maximizing its potential for industrial application.

The S-S bridge mutation between the A2 and A4 loops (T416C-I432C) of Cel7A of Aspergillus fumigatus enhances catalytic activity and thermostability.

Disulfide bonds are important for maintaining the structural conformation and stability of the protein. The introduction of the disulfide bond is a promising strategy to increase the thermostability of the protein. In this report, cysteine residues are introduced to form disulfide bonds in the Glycoside Hydrolase family GH 7 cellobiohydrolase (GH7 CBHs) or Cel7A of Aspergillus fumigatus. Disulfide by Design 2.0 (DbD2), an online tool is used for the detection of the mutation sites. Mutations are created (D276C-G279C; DSB1, D322C-G327C; DSB2, T416C-I432C; DSB3, G460C-S465C; DSB4) inside and outside of the peripheral loops but, not in the catalytic region. The introduction of cysteine in the A2 and A4 loop of DSB3 mutant showed higher thermostability (70% activity at 70°C), higher substrate affinity (Km = 0.081 mM) and higher catalytic activity (Kcat = 9.75 min-1; Kcat/Km = 120.37 mM min-1) compared to wild-type AfCel7A (50% activity at 70°C; Km = 0.128 mM; Kcat = 4.833 min-1; Kcat/Km = 37.75 mM min-1). The other three mutants with high B factor showed loss of thermostability and catalytic activity. Molecular dynamic simulations revealed that the mutation T416C-I432C makes the tunnel wider (DSB3: 13.6 Å; Wt: 5.3 Å) at the product exit site, giving flexibility in the entrance region or mobility of the substrate in the exit region. It may facilitate substrate entry into the catalytic tunnel and release the product faster than the wild type, whereas in other mutants, the tunnel is not prominent (DSB4), the exit is lost (DSB1), and the ligand binding site is absent (DSB2). This is the first report of the gain of function of both thermostability and enzyme activity of cellobiohydrolase Cel7A by disulfide bond engineering in the loop.IMPORTANCEBioethanol is one of the cleanest renewable energy and alternatives to fossil fuels. Cost efficient bioethanol production can be achieved through simultaneous saccharification and co-fermentation that needs active polysaccharide degrading enzymes. Cellulase enzyme complex is a crucial enzyme for second-generation bioethanol production from lignocellulosic biomass. Cellobiohydrolase (Cel7A) is an important member of this complex. In this work, we engineered (disulfide bond engineering) the Cel7A to increase its thermostability and catalytic activity which is required for its industrial application.

Comparative Analysis of Acidic and Alkaline Pretreatment Techniques for Bioethanol Production from Perennial Grasses

This review paper examines acid and alkaline pretreatments on perennial grasses for second-generation (2G) bioethanol production, a relatively unexplored area in this field. It compares the efficiency of these pretreatments in producing fermentable sugar and bioethanol yield. This study finds that alkaline pretreatment is more effective than acidic pretreatment in removing lignin and increasing sugar yield, leading to higher ethanol yields. However, it is costlier and requires longer reaction times than acidic pretreatment, while acidic pretreatment often leads to the formation of inhibitory compounds at higher temperatures, which is undesirable. The economic and environmental impacts of lignocellulosic biomass (LCB) are also assessed. It is revealed that LCB has a lower carbon but higher water footprint and significant costs due to pretreatment compared to first-generation biofuels. This review further explores artificial intelligence (AI) and advanced technologies in optimizing bioethanol production and identified the gap in literature regarding their application to pretreatment of perennial grasses. This review concludes that although perennial grasses hold promise for 2G bioethanol, the high costs and environmental challenges associated with LCB necessitate further research. This research should focus on integrating AI to optimize the pretreatment of LCB, thereby improving efficiency and sustainability in 2G biofuel production.

A Pareto aggregation approach for environmental-economic multi-objective optimization applied on a second-generation bioethanol production model

Multi-objective optimization is an important decision-making tool for energy processes, as multiple targets need to be achieved. These objectives are usually conflicting since a single solution cannot be optimal for all objectives, resulting in a set of Pareto-optimal solutions. Multiple indicators might be available to describe a sustainability objective, such as the environmental impact which is commonly evaluated by performing a life cycle assessment. In this study, Pareto aggregation is proposed as a method which employs a novel multi-objective optimization-based approach as an alternative to the classically used aggregation in life cycle assessment. This method identifies conflicting environmental indicators and performs an aggregation among those that require a trade-off. An environmental-economic optimization of a second-generation bioethanol plant is used to illustrate and evaluate the proposed method. Process parameters from a biochemical conversion pathway flowsheet simulation model are chosen as optimization variables. To reduce the computational time, surrogate models, based on artificial neural networks, are used. Out of the eighteen ReCiPe Midpoint environmental indicators, five were identified as conflicting, resulting in an aggregated environmental objective, which was then traded off with the economic objective function, chosen as the levelized cost of ethanol. Comparison with the widely used single-score EcoIndicator99 showed that the Pareto aggregation method can reduce most of the environmental indicators by up to 6.5%. This research provides an insight on non-redundant objective functions, aiming at reducing the dimensionality of multi-objective optimization problems, while taking into consideration decision-makers’ preferences.

Drivers and Barriers in the Production and Utilization of Second-Generation Bioethanol in India

Second-generation biorefinery refers to the production of different types of biofuels, biomaterials, and biochemicals by using agri-based and other lignocellulosic biomasses as substrates, which do not compete with arable lands, water for irrigation, and food supply. From the perspective of transportation fuels, second-generation bioethanol plays a crucial role in minimizing the dependency on fossil-based fuels, especially gasoline. Significant efforts have been invested in the research and development of second-generation bioethanol for commercialization in both developing and developed countries. However, in different developing countries like India, commercialization of second-generation bioethanol has been obstructed despite the abundance and variety of agricultural feedstocks. This commercial obstruction was majorly attributed to the recalcitrance of the feedstock, by-product management, and marginal subsidies compared to other nations. This article reviews the major roadblocks to the viability and commercialization of second-generation biofuels, especially bioethanol in India and a few other leading developed and developing nations. This article also reviews the biomass availability, technological advancements, investments, policies, and scale-up potential for biorefineries. A thorough discussion is made on the prospects and barriers to research, development, and demonstration as well as strengths, weaknesses, opportunities, and threats for the commercialization of second-generation bioethanol.

Lignin reduction in sugarcane by performing CRISPR/Cas9 site-direct mutation of SoLIM transcription factor

Genetic engineering of plant cell walls is limited for reducing lignocellulose recalcitrance, so mild and/or green-like pretreatment is still required for sequential enzymatic saccharification. Here, we report a method to reduce lignin content in sugarcane stalks using the CRISPR/Cas 9 technique. Three target sequences of SoLIM were designed and fused to pRGEB32. The cassette constructs were introduced into sugarcane calli cv. KK3 through Agrobacterium-mediated transformation. We produced one base substitution and one insertion line for the 1st target site; two insertions, one deletion, and one base substitution for the 2nd target site; and one base substitution and insertion for the 3rd target site. qRT-PCR analysis of SoLIM, SoPAL, SoC4H, and SoCAD showeded that downregulation of SoLIM by single nucleotide insertions or deletions reduced the expression of SoPAL, SoC4H, and SoCAD. Consequently, the edited lines contained 9.74 to 51.46% less lignin content compared to that in the wild-type plants. The syringyl/guaiacyl (S/G) ratio of the edited lines ranged between 0.23 and 0.49, while the wild-type was 0.22. The histochemical evaluation and scanning electron microscopy of the cell walls supported this observation. A low lignin content sugarcane will provide a better feedstock for second-generation bioethanol production.

Production of second generation bioethanol from straw during simultaneous microbial saccharification and fermentation

Production of second generation bioethanol from straw during simultaneous microbial saccharification and fermentation

Adaptive laboratory evolution under acetic acid stress enhances the multistress tolerance and ethanol production efficiency of Pichia kudriavzevii from lignocellulosic biomass

Second-generation bioethanol production using lignocellulosic biomass as feedstock requires a highly efficient multistress-tolerant yeast. This study aimed to develop a robust yeast strain of P. kudriavzevii via the adaptive laboratory evolution (ALE) technique. The parental strain of P. kudriavzevii was subjected to repetitive long-term cultivation in medium supplemented with a gradually increasing concentration of acetic acid, the major weak acid liberated during the lignocellulosic pretreatment process. Three evolved P. kudriavzevii strains, namely, PkAC-7, PkAC-8, and PkAC-9, obtained in this study exhibited significantly higher resistance toward multiple stressors, including heat, ethanol, osmotic stress, acetic acid, formic acid, furfural, 5-(hydroxymethyl) furfural (5-HMF), and vanillin. The fermentation efficiency of the evolved strains was also improved, yielding a higher ethanol concentration, productivity, and yield than the parental strain, using undetoxified sugarcane bagasse hydrolysate as feedstock. These findings provide evidence that ALE is a practical approach for increasing the multistress tolerance of P. kudriavzevii for stable and efficient second-generation bioethanol production from lignocellulosic biomass.

Process optimization for saccharification and fermentation of the Organic Fraction of Municipal Solid Waste (OFMSW) to maximize ethanol production performance

The Organic Fraction of Municipal Solid Waste (OFMSW) contains paper and cardboard, an attractive cellulosic feedstock for bioethanol production. Waste paper/cardboard (WPC) was subjected to mild acid treatment in solids loadings from 20 to 27.5 %. Different doses of commercial enzyme cocktail and various adjuvants facilitating enzymatic saccharification were tested. Enzyme dosing of 3.75 FPU/g fiber with very slow fed-batch saccharification of 27.5 % (w/v) solids from model lignin-free paper pulp resulted in 12.6 % (w/v) total sugar after 74 h. Use of cheap non-catalytic waste protein adjuvants including Soymeal, Protisyr, Feather meal strongly improved enzymatic saccharification and reduced the overall process cost by reducing commercial enzyme dosing. After fermentation with the industrial yeast strain BMD44 (Cellusec® 1.0) an ethanol titer of 5.77 % (v/v) was obtained using a reduced enzyme loading of 2.5 FPU/g of actual industrial WPC fiber. These results show the potential of economically profitable second-generation bioethanol production with WPC fiber.

Geospatial environmental techno-economic assessment of pretreatment technologies for bioethanol production

Second-generation biofuels, starting from lignocellulosic biomass, are considered as a renewable alternative for fossil fuels with lower environmental impact and potentially higher supply and energy security. The economic and environmental performance of second-generation bioethanol production from corn stover in the European Union (EU) is studied, starting in Belgium as base case. A comparative environmental techno-economic assessment has been conducted, with process simulations in Aspen Plus and corn stover availability data in thirteen EU countries to calculate minimum ethanol selling prices (MESP) and Greenhouse gas emissions (GHGe). In this analysis, the emphasis is on the comparison of different pretreatment technologies, namely (i) dilute acid, (ii) alkaline, (iii) steam explosion and (iv) liquid hot water. Dilute acid showed the best economic and environmental performance for the base case scenario. Within the EU, Hungary and Romania presented the lowest MESP for the steam explosion model at 0.39 and 0.43 EUR/L respectively. Poland showed the lowest GHGe, at 0.46 kg CO2eq/L for the alkaline model, mainly due to the avoided product allocation on electricity and its high carbon intensity in the electricity generation sector. The second lowest GHGe were obtained in France for the dilute acid model and are attributed to its low agricultural emissions intensity. This study identifies a location-dependence of the economic and environmental performance of pretreatment technologies, which can be extrapolated from the EU to other large regions around the world and should be taken into consideration by decision-makers.

Long term study of cell wall saccharification and high added value phenolic acids production from Phalaris aquatica L. energy crop

Phalaris aquatica L. is a cool-season perennial grass of the Mediterranean that has been used for forage production, while recently was identified as a novel candidate for sustainable second-generation bioethanol production. This study provides for the first time evidence about this energy crop long-term performance (five years of growth), the cell wall saccharification efficiency and the production of high added value compounds during this period. High biomass production was recorded constantly after the first year of plant growth, with a maximum of 22.4 ± 0.5 t of dry mass (DM) per ha and stable structural polysaccharides concentration 68.8% ± 0.8 w/w DM from the third year on. Independently from crop age, dilute acid pretreatment (120 °C, 1.5% H2SO4, 45 min) promoted the subsequent enzymatic hydrolysis of P. aquatica L. biomass. The dilute acid pre-treatment positive effect is attributed both to the hemicellulose solubilization and the breakage of intermolecular bonds between polysaccharides and phenolic acids, as supported by the present study results These combined acid/enzymatic hydrolysis processes were able to convert more than 80% of cell wall polysaccharides into fermentable sugars, releasing simultaneously a substantial amount of phenolic acids of 2.16% ± 0.48 w/w DM. The ratio of ferulic to p-coumaric acid in the acid hydrolysate can be used for the prediction of the subsequent cellulose enzymatic saccharification efficiency, by the model described here. The protocatechuic acid, which can find many technological applications, was the main bioactive polyphenol in the final hydrolysate with concentration ∼1% w/w DM. These results are supportive of Phalaris aquatica L. suitability as a sustainable biorefinery crop, under the semi-arid Mediterranean climatic conditions contributing to this region biodiversity and prosperity.

Biotechnological Potential of the Stress Response and Plant Cell Death Regulators Proteins in the Biofuel Industry.

Production of biofuel from lignocellulosic biomass is relatively low due to the limited knowledge about natural cell wall loosening and cellulolytic processes in plants. Industrial separation of cellulose fiber mass from lignin, its saccharification and alcoholic fermentation is still cost-ineffective and environmentally unfriendly. Assuming that the green transformation is inevitable and that new sources of raw materials for biofuels are needed, we decided to study cell death-a natural process occurring in plants in the context of reducing the recalcitrance of lignocellulose for the production of second-generation bioethanol. "Members of the enzyme families responsible for lysigenous aerenchyma formation were identified during the root hypoxia stress in Arabidopsis thaliana cell death mutants. The cell death regulatory genes, LESION SIMULATING DISEASE 1 (LSD1), PHYTOALEXIN DEFICIENT 4 (PAD4) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) conditionally regulate the cell wall when suppressed in transgenic aspen. During four years of growth in the field, the following effects were observed: lignin content was reduced, the cellulose fiber polymerization degree increased and the growth itself was unaffected. The wood of transgenic trees was more efficient as a substrate for saccharification, alcoholic fermentation and bioethanol production. The presented results may trigger the development of novel biotechnologies in the biofuel industry.

Construction of Novel Yeast Strains from Candida tropicalis KBKTI 10.5.1 and Saccharomyces cerevisiae DBY1 to Improve the Performance of Ethanol Production Using Lignocellulosic Hydrolysate.

Increased consumption of xylose-glucose and yeast tolerance to lignocellulosic hydrolysate are the keys to the success of second-generation bioethanol production. Candida tropicalis KBKTI 10.5.1 is a new isolated strain that has the ability to ferment xylose. In contrast to Saccharomyces cerevisiae DBY1 which only can produce ethanol from glucose fermentation. The research objective is the application of the genome shuffling method to increase the performance of ethanol production using lignocellulosic hydrolysate. Mutants were selected on xylose and glucose substrates separately and using random amplified polymorphic DNA (RAPD) analysis. The ethanol production using lignocellulosic hydrolysate by parents and mutants was evaluated using a batch fermentation system. Concentrations of ethanol, residual sugars, and by-products such as glycerol, lactate and acetate were measured using HPLC machine equipped with Hiplex H for carbohydrate column and a refraction index detector (RID). Ethanol produced by Fcs1 and Fcs4 mutants on acid hydrolysate increased by 26.58% and 24.17% from parent DBY1, by 14.94% and 21.84% from parent KBKTI 10.5.1. In contrast to the increase in ethanol production on alkaline hydrolysate, Fcs1 and Fcs4 mutants only experienced an increase in ethanol production by 1.35% from the parent KBKTI 10.5.1. Ethanol productivity by Fcs1 and Fcs4 mutants on acid hydrolysate reached 0.042 g/L/h and 0.044 g/L/h. The recombination of the genomes of different yeast species resulted in novel yeast strains that improved resistance performance and ethanol production on lignocellulosic hydrolysates.

Second-Generation Bioethanol Production and Cellulases of Aspergillus niger ITV02 Using Sugarcane Bagasse as Substrate

Second-Generation Bioethanol Production and Cellulases of Aspergillus niger ITV02 Using Sugarcane Bagasse as Substrate

Assessing the future of second-generation bioethanol by 2030 – A techno-economic assessment integrating technology learning curves

Lignocellulosic biomass is the most abundant source of renewable biomass and is seen as a high-potential replacement for petroleum-based resources. The conversion technologies to advanced biofuels are still at a low maturity level, thus allowing for future cost reductions through technological learning. This fact is barely considered in state-of-the-art techno-economic assessments and a structured approach to account for technological learning in techno-economic assessments is needed. In this study, a framework for techno-economic assessments of advanced biofuels, integrating learning curves, is proposed. As a validation of this framework, the economic feasibility of the valorization of corn stover for the production of second-generation bioethanol in Belgium is studied. Process flowsheet simulations in Aspen Plus are developed, with an emphasis on the comparison of four different pretreatment technologies and two plant capacities at 156 dry kt biomass/y and 667 dry kt/y. The dilute acid pretreatment model of the large-scale biorefinery required the lowest minimum learning rate to reach an economically feasible biorefinery by 2030, being 3.9%, almost half as the one calculated for the smaller scale plant. This learning rate seems to be achievable based on learning rates commonly estimated in literature. We conclude that there is a potential for advanced ethanol production in Belgium under the current state of technology for large-scale biorefineries, which require additional biomass imports, when accounting for future cost reductions through learning.

Xylanase enhanced second-generation bioethanol production through simultaneous saccharification and fermentation

This study aimed to correlate hydrolytic enzymes with enhanced bioethanol production during the simultaneous process of saccharification and fermentation for different agricultural wastes. This study screened the activities of hydrolytic enzymes to evaluate the simultaneous process and correlated them with bioethanol production. The results of the simultaneous process showed that cantaloupe peels produced the highest amount of reducing sugars and bioethanol. Cellulase showed maximum activity in the first 24 h, indicating that yeast cells favoured glucose over xylose. The results of the simultaneous saccharification and fermentation experiments revealed a 5-fold decrease in cellulase activity after 72 h. Xylanase activity improved from 79.38 to 95.18 U g−1, and bioethanol production was enhanced from 21.42 to 75.66 g L−1, confirming the capability of the simultaneous process to enhance bioethanol production. Although ethanol production was lower than that obtained with xylose cultivation alone, it was higher than that observed with glucose.

New perspectives for maximizing sustainable bioethanol production from corn stover

In Egypt, the production of second-generation bioethanol from agricultural waste is a thriving method to compensate the excessive usage as a consequence of the outspread of Covid-19. The profusion and renewability of lignocellulosic biomass urge its utilization as a promising feedstock for bioethanol production. However, functional delignification without affecting the cellulose matrices remains the major obstacle to achieving effective enzyme accessibility. This paper highlights a novel physio-chemical combination for corn stover (CS) pretreatment for bioethanol production. The optimum pretreatment condition was achieved using a mixture of 5% maleic acid (MA) and 3% citric acid (CA) for 30 min at an autoclave temperature of 110 °C leading to produce a pretreated CS (MAC) with 99% hemicellulose removal, 90% cellulose recovery, and 80% lignin removal. Characteristics analyses such as; SEM, FTIR, TGA, EDX, elemental, proximate, ultimate, higher heating value (HHV), and functionalization analyses were performed to emphasize the property and structure change of CS before and after the pretreatment. Then, MAC was hydrolyzed by cellulase enzyme and produced 13.5 g/L glucose yield which was fermented by Saccharomyces cerevisiae and produced 10 g/L bioethanol.

Microbial Enzyme Systems in the Production of Second Generation Bioethanol

The primary contributor to global warming has been the careless usage of fossil fuels. Urbanization’s threat to the depletion of these resources has made it necessary to find alternatives due to the rising demand. Four different forms of biofuels are now available and constitute a possible replacement for fossil fuels. The first generation of biofuels is generated from the edible portion of biomass, the second generation is made from the non-edible portion of biomass, the third generation is made from algal biomass, and the fourth generation is made using molecular biology to improve the algal strain. Second-generation biofuels are extremely important because they are derived from non-edible biomass, such as agricultural and agro-industrial wastes rich in cellulose, hemicellulose, pectin, and starch impregnated with lignin, and are hydrolyzed after delignification by physio-chemical or biological pretreatments using ligninases. The enzymes involved in the hydrolysis of feedstocks for the production of second-generation bioethanol, a highly acceptable biofuel, are discussed in this article. Furthermore, the article discusses various fermentation technologies as well as significant developments in second-generation biofuel production by combining various microbial enzyme systems.

Evolving Robust and Interpretable Enzymes for the Bioethanol Industry.

Obtaining a robust and applicable enzyme for bioethanol production is a dream for biorefinery engineers. Herein, we describe a general method to evolve an all-round and interpretable enzyme that can be directly employed in the bioethanol industry. By integrating the transferable protein evolution strategy InSiReP 2.0 (In Silico guided Recombination Process), enzymatic characterization for actual production, and computational molecular understanding, the model cellulase PvCel5A (endoglucanase II Cel5A from Penicillium verruculosum) was successfully evolved to overcome the remaining challenges of low ethanol and temperature tolerance, which primarily limited biomass transformation and bioethanol yield. Remarkably, application of the PvCel5A variants in both first- and second-generation bioethanol production processes (i. Conventional corn ethanol fermentation combined with the in situ pretreatment process; ii. cellulosic ethanol fermentation process) resulted in a 5.7-10.1 % increase in the ethanol yield, which was unlikely to be achieved by other optimization techniques.