Second-generation Bioethanol Production Research Articles

Valorization of Bombax ceiba Waste into Bioethanol Production through Separate Hydrolysis and Fermentation and Simultaneous Saccharification and Fermentation

In this study, Seed pods of B. ceiba were used as a novel, cheap, and sustainable feedstock for second-generation bioethanol production. B. ceiba waste was pretreated with NaOH under different conditions using a Box–Behnken design (BBD) with three factors and three levels. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and scanning electron microscopy (SEM) were used to investigate the chemical, structural, and morphological modifications made by pretreatment. NaOH pretreatment followed by steam was more effective as it offered 60% cellulose and 9% lignin at 10% substrate loading, 5% NaOH conc., and 4 h residence time. Samples with maximum cellulose were employed for ethanol production by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) using indigenously produced cellulase as well as commercial cellulase. HPLC analysis revealed the best saccharification (50.9%) at 24 h and the best ethanol yield (54.51 g/L) at 96 h of fermentation in SSF using commercial cellulose by Saccharomyces cerevisiae. SSF offered a better production of bioethanol from seed pods than SHF. The implications of the work support the notion that B. ceiba waste could be utilized for large-scale bioethanol production.

Improvement of cell-tethered cellulase activity in recombinant strains of Saccharomyces cerevisiae.

Consolidated bioprocessing (CBP) remains an attractive option for the production of commodity products from pretreated lignocellulose if a process-suitable organism can be engineered. The yeast Saccharomyces cerevisiae requires engineered cellulolytic activity to enable its use in CBP production of second-generation (2G) bioethanol. A promising strategy for heterologous cellulase production in yeast entails displaying enzymes on the cell surface by means of glycosylphosphatidylinositol (GPI) anchors. While strains producing a core set of cell-adhered cellulases that enabled crystalline cellulose hydrolysis have been created, secreted levels of enzyme were insufficient for complete cellulose hydrolysis. In fact, all reported recombinant yeast CBP candidates must overcome the drawback of generally low secretion titers. Rational strain engineering can be applied to enhance the secretion phenotype. This study aimed to improve the amount of cell-adhered cellulase activities of recombinant S. cerevisiae strains expressing a core set of four cellulases, through overexpression of genes that were previously shown to enhance cellulase secretion. Results showed significant increases in cellulolytic activity for all cell-adhered cellulase enzyme types. Cell-adhered cellobiohydrolase activity was improved by up to 101%, β-glucosidase activity by up to 99%, and endoglucanase activity by up to 231%. Improved hydrolysis of crystalline cellulose of up to 186% and improved ethanol yields from this substrate of 40-50% in different strain backgrounds were also observed. In addition, improvement in resistance to fermentation stressors was noted in some strains. These strains represent a step towards more efficient organisms for use in 2G biofuel production. KEY POINTS: • Cell-surface-adhered cellulase activity was improved in strains engineered for CBP. • Levels of improvement of activity were strain and enzyme dependent. • Crystalline cellulose conversion to ethanol could be improved up to 50%.

Predicting the techno-economic performance of a large-scale second-generation bioethanol production plant: a case study for Kenya

Predicting the techno-economic performance of a large-scale second-generation bioethanol production plant: a case study for Kenya

Exploring Natural Fermented Foods as a Source for New Efficient Thermotolerant Yeasts for the Production of Second-Generation Bioethanol

Considering the cost-effectiveness of bioethanol production at high temperatures, there is an enduring need to find new thermotolerant ethanologenic yeasts. In this study, a total of eighteen thermotolerant yeasts were isolated from various natural fermented products in Morocco. Ethanol production using 50 g/L glucose or 50 g/L xylose as the sole carbon source revealed potential yeasts with high productivities and volumetric ethanol productivities at high temperatures. Based on molecular identification, the selected thermotolerant fermentative isolates were affiliated with Pichia kudriavzevii, Kluyveromyces marxianus, and Kluyveromyces sp. During the simultaneous saccharification and fermentation of lignocellulosic biomass at a high temperature (42 °C), the designated yeast P. kudriavzevii YSR7 produced an ethanol concentration of 22.36 g/L, 18.2 g/L and 6.34 g/L from 100 g/L barley straw (BS), chickpea straw (CS), and olive tree pruning (OTP), respectively. It also exhibited multi-stress tolerance, such as ethanol, acetic acid, and osmotic tolerance. Therefore, the yeast P. kudriavzevii YSR7 showed promising attributes for biorefinery-scale ethanol production in the future.

Synergistic effects of inhibitors and osmotic stress during high gravity bioethanol production from steam-exploded lignocellulosic feedstocks

High gravity second-generation bioethanol production (solid loading ≥30% w w−1) suffers from prolonged fermentation times and low ethanol productivities due to high concentrations of toxic by-products (inhibitors) and high monosaccharide concentrations. To better understand inhibition during high gravity bioethanol production, this study focused on ethanol production from a variety of different steam-exploded feedstocks with different inhibitor concentrations. The effects of inhibitors on enzymatic hydrolysis and fermentation were investigated by selective removal of inhibitors by means of biological detoxification. The degree of lignocellulose depolymerization and inhibitor formation during steam explosion varied significantly between different feedstock types. Subsequent enzymatic hydrolysis and fermentation indicated that lignocellulosic inhibitors had no significant effect on cellulolytic enzymes, but showed strong inhibition on fermentation performance. Biological detoxification of furan aldehydes and organic acids had no impact on monosaccharide yield during enzymatic hydrolysis, but reduced the fermentative lag phase by up to 48 h. While the detoxification significantly improved fermentation performance, the maximal ethanol yield of 52.6 g L−1 with wheat straw or miscanthus could not be increased. A key finding of this study was that the effect of lignocellulosic inhibitors is greatly increased in environments with high osmotic stress. This synergistic effect is hypothesized to be caused by an inhibitor-induced redox imbalance, impeding the cells’ ability to counteract osmotic stress.

Second-generation bioethanol production from phytomass after phytoremediation using recombinant bacteria-yeast co-culture

Sustainable management of harvested hyperaccumulating plants used in phytoremediation of toxic metals is considered quite challenging. The biomass of hyperaccumulators can be an attractive feedstock for bioethanol production, while removal of toxic metals and efficient ethanol conversion systems are necessary to ensure safe and optimum bioethanol production. This study aimed to develop a compatible and efficient consolidated bioprocessing system (CBP) to produce bioethanol from phytomass of arsenic (As)-hyperaccumulatorPteris vittata. A recombinant kefir yeastKluyveromyces marxianus(KR7 strain) contained six cellulase genes and two recombinantBacillus subtilis(Type 1 and Type 2 strains) carrying cellulosomal genes in different arrangements were used, and the saccharification and fermentation of P. vittata phytomass were investigated through one-step (KR7 strain only) and co-culture (B. subtilis + KR7 strain) methods. The results of reducing sugar showed that KR7 strain possesses higher saccharification ability than bothB. subtilisstrains. While the co-culture of B. subtilisType 2 strain with KR7 strain gave the highest ethanol yield, which achieved 48.5% ethanol yield from P. vittataphytomass after 3 d cultivation. In addition, simple pretreatment by shaking P. vittata with 1% of HNO3 efficiently removed more than 80% of As from the phytomass, suggesting its applicability and feasibility for efficient As extraction to ensure efficient and non-toxic ethanol production. Our results confirm that the developed recombinant bacteria-yeast co-culture possesses a great potential for optimum bioethanol production from As-hyperaccumulators-derived phytomass via CBP.

Integrated production of furfural and second-generation bioethanol from Eucalyptus wood residues: experimental results and process simulation

Integrated production of furfural and second-generation bioethanol from Eucalyptus wood residues: experimental results and process simulation

Producción de celulasas de Aspergillus niger-ITV-02 a partir de residuos lignocelulósicos de maíz

Cellulases are enzymes used in the hydrolysis of lignocellulosic residues for second-generation (2G) bioethanol production, the low availability in the market, and the high-cost impacts in the 40% of the total cost of 2G bioethanol production. Therefore, it is necessary to look for sustainable alternatives for its production. Cellulases are mainly produced by Aspergillus niger, and their activity can be affected by nitrogen concentration, the use of surfactants, and the carbon source, among others. The objective of this work was to identify enzymes with CMCase and β-glucosidase activity of A. niger ITV-02 from low-cost lignocellulosic residues such as corn and cob stubble in two culture media (M1 and M2). The results showed that using corn stover and medium M2 increased the volumetric activity 1.4 and 1.7 times, respectively compared to corn cob and medium M2. Likewise, enzymes with cellulase activity were identified using carboxymethyl cellulose (CMC) and 4- methyl-lumberyl-β-D-glucopyranoside (MUG). The enzymes identified in MUG correspond to a glucoamylase, two β-glucosidases, and an exo β-xylosidase. In CMC the following were identified: exo-β-1,3-glucanase, endo -β-1,4 xylanase arabinosidase, and endoglucanase.

Second Generation Bioethanol Production: Green Alternative Energy for Future Energy Crisis

Emerging environmental pollution and depleting fossil fuel resources issues are creating a requirement for more environmentally friendly alternative renewable fuels. In that case bioethanol has gathered much attention as a fuel additive and fuel enhancer. In the current study, corncob and paper waste were used as lignocellulosic biomass and followed the simultaneous saccharification and fermentation via bacterial pathways to produce second generation bioethanol. Both biomasses were pretreated initially using mechanical and chemical pretreatments and they were hydrolysed by two bacterial strains; Bacillus sp. and Norcadiopsis sp. and fermented by Achromobacter sp. which were previously isolated. The bioethanol production was followed by hydrolysing of biomass by Bacillussp. and Norcadiopsis sp. for 72 hours at 37° C and then the fermentation was done using the bacterium Achromobacter sp. for 72 hours at 37° C. Detecting gas trapped in Durham tubes and Solid Phase Micro Extraction coupled with GCMS were used to screen and quantify the ethanol production respectively. The highest ethanol percentage (v/v) was obtained from alkaline pretreated paper waste hydrolysed by the Bacillus sp. (0.734±0.154%) and the lowest ethanol percentage (v/v) was obtained from alkaline pretreated corncob hydrolysed by the Norcadiopsis sp. (0.155±0.154%). Acid pretreated corncobs, alkaline pretreated corncobs, and acid pretreated paper waste which was hydrolysed by Bacillus sp. and fermented by Achromobacter sp. produced ethanol percentages (v/v) of 0.726±0.154%, 0.564±0.154%, and 0.657±0.154% respectively. Acid pretreated corncobs, acid pretreated paper waste, and alkaline pretreated paper waste which was hydrolysed by Norcadiopsissp. and fermented by Achromobacter sp. produced ethanol percentages (v/v) of 0.587±0.154%, 0.599±0.154%, and 0.627±0.154% respectively. Thus, the results of the study revealed that both corncobs and paper waste have high potential for bioethanol production, and paper waste is the best feedstock for second-generation bioethanol production among used biomasses. 
 Keywords: Bioethanol production, Lignocellulosic biomass, Corncobs, Paper waste

Qualitative assessment of bioethanol production sustainability applying the GBEP methodology: a comparative case between coconut husks and sugarcane bagasse

The conventional production of bioethanol takes place through sugar cane processing. Given that Brazil generates abundant lignocellulosic residues, other bioethanol production routes from residual biomasses have been increasingly considered. In this context, this study aimed to assess the sustainability of two second-generation bioethanol (E2G) production routes, one from sugarcane bagasse and the other from coconut husks. To this end, nine indicators proposed by the GBEP (Global Bioenergy Partnership) methodology, namely GHG emissions, non-GHG emissions, water use and efficiency, income changes, bioenergy sector jobs, incidence of occupational injuries, illnesses and deaths, productivity, net energy balance and increased gross value from bioenergy production, were applied. Following the two bioethanol production route assessments through indicator application, a conclusive graphic outlook was constructed to identify the most sustainable route. The sugarcane bagasse production route derives from the Iogen technology and is employed at Raízen's Costa Pinto Plant, which produces this biofuel on an industrial scale, while the coconut husk production route is currently being improved on a bench scale. The indicator analysis demonstrates that, despite the coconut husk route exhibiting greater social sustainability, with better employee remuneration and less frequent injuries, illnesses and occupational deaths, the sugarcane bagasse route shows greater environmental and economic sustainability, due to lower GHG emissions and water extraction, besides higher productivity, and higher net production income. Therefore, the comparative analysis produced by applying the GBEP methodology demonstrates that the sugarcane bagasse route is the most sustainable regarding the production of second-generation bioethanol.

Identification of a New Endo-β-1,4-xylanase Prospected from the Microbiota of the Termite Heterotermes tenuis.

Xylanases are hemicellulases that break down xylan to soluble pentoses. They are used for industrial purposes, such as paper whitening, beverage clarification, and biofuel production. The second-generation bioethanol production is hindered by the enzymatic hydrolysis step of the lignocellulosic biomass, due to the complex arrangement established among its constituents. Xylanases can potentially increase the production yield by improving the action of the cellulolytic enzyme complex. We prospected endo-β-1,4-xylanases from meta-transcriptomes of the termite Heterotermes tenuis. In silico structural characterization and functional analysis of an endo-β-1,4-xylanase from a symbiotic protist of H. tenuis indicate two active sites and a substrate-binding groove needed for the catalytic activity. No N-glycosylation sites were found. This endo-β-1,4-xylanase was recombinantly expressed in Pichia pastoris and Escherichia coli cells, presenting a molecular mass of approximately 20 kDa. Enzymatic activity assay using recombinant endo-β-1,4-xylanase was also performed on 1% xylan agar stained with Congo red at 30 °C and 40 °C. The enzyme expressed in both systems was able to hydrolyze the substrate xylan, becoming a promising candidate for further analysis aiming to determine its potential for application in industrial xylan degradation processes.

Selection of sustainable lignocellulose biomass for second-generation bioethanol production for automobile vehicles using lifecycle indicators through fuzzy hybrid PyMCDM approach

Petroleum prices are increasing day by day due to the essential requirement of fuel to run automobiles, agricultural engines and standby power plants. More utilization of fossil fuels leads to global warming, environmental pollution, ozone depletion, and climate change. The second-generation ethanol is produced from various feedstock, including agricultural wastes and lignocellulose biomass sources doesn’t affect the food security in many countries and the blending of bio-ethanol in gasoline reduced the greenhouse gas emissions. As large quantity of Lignocellulose biomass is available in India; the present study aims to select sustainable Lignocellulose biomass for the production of second-generation ethanol in Indian context. Five Lignocellulose biomass (Sugarcane Bagasse, Rice straw, wheat straw, Moringa, and Vetiver) were identified and prioritized using Multi-Criteria Decision Making (MCDM) methodology. This study followed the Hybrid MCDM approach to prioritize the Lignocellulose biomass. Python-based Evaluation Based on Distance from Average Solution (PyEDAS) MCDM algorithm was developed, and the final ranking is compared with COmplex PRoportional ASsessment (COPRAS), Technique for Order of Preference by Similarity to Ideal Solution (TOPSIS) & Preference Ranking Organization Method for Enrichment Evaluations (PROMETHEE). From the results, Vetiver grass ranked as the best alternative, and Sugarcane bagasse ranked as the worst alternative among five Lignocellulose biomass sources.

Enzymatic Biocatalysis of Biomass from Aquatic Plant Phragmite Karka for Second-Generation Bioethanol Production

In the context of energy transition and the reduction of greenhouse gas emissions, the production of second-generation bioethanol is also recognized as a promising way to reduce our dependence on fossil fuels. Then, the present studies aim to evaluate the enzymatic biocatalysis of biomass from aquatic plant Phragmite karka in the second-generation bioethanol production. Results obtained revealed a rapid decrease of °Brix during the fermentation of musts and underlined the efficacy of enzyme hydrolysis. The rate of sugar consumption by yeasts is between 32.43 and 70.27%. The yield of ethanol production of yeasts indicated that Angel Brand Thermal-tolerant alcohol active dry yeast was the best yeast strain for this fermentation. These findings underline the potential of Phragmite karka plant materials in the perspective of intensive production of second-generation bioethanol.

Modelling of the Simultaneous Saccharification and Fermentation for a Pine Sawdust Biorefinery

The decline in world oil reserves evidences the need to diversify the sources of equivalent raw materials. The use of biomass is one of the most explored alternatives. This work evaluates the second-generation bioethanol (2G) production from a pine sawdust soda- ethanol pulp by simultaneous hydrolysis and fermentation (SSF) to obtain the best conditions for scaling up the process. Experimental designs have been used to find mathematical models that define the complex situation jointly varying time with other variables (enzyme load and temperature). Time periods in the full model varied from 0 h to 72 h. Given the results (curve shape differences), it was decided to split the design in two, covering periods from 0 h to 24 h (Model I) and from 24 to 72 h (Model II). The pulp chemical composition was 80.2% glucans, 7.2% xylans, 0.3% galactans, 8.4% mannans, 3.7% lignin. Cellic® Ctec2 cellulolytic enzymes were used for saccharification and Saccharomyces cerevisiae IMR 1181 (SC 1181) yeast for fermentation. The best conditions found in the two designs were 30 FPU g−1 glucans, 39 °C, 24 h for Model I, and 30 FPU g−1 glucans, 35 °C, 72 h for Model II. Fermentation optimal values were 63.23 and 81.93 for Models I and II, respectively.

One-pot bioethanol production from brewery spent grain using the ethanologenic Escherichia coli MS04

Lignocellulosic biomass residues constitute an alternative for energy generation. In this work, brewery spent grain (BSG) were used as feedstock to produce ethanol using a recombinant Escherichia coli MS04 strain. A sequential process involving pretreatment, saccharification and fermentation steps in one-pot bioreactors, without solids removal, was proposed. Different diluted acid pretreatment times (1 and 1.5 h) using a 15% (w/w) solids load to obtain high monosaccharides yields were evaluated. After the pretreatment and saccharification, the proposed strategy converted the polysaccharides contained in BSG into fermentable pentoses and hexoses, obtaining a syrup composed by monosaccharides (60 g/L), furfural (0.39 g/L), hydroxymethylfurfural (0.07 g/L) and acetic acid (2.56 g/L), which was afterwards fermented for the bioethanol generation. After 30 h, ethanol production resulted in 29.5 ± 0.41 g/L, which means a volumetric productivity of 0.98 g EtOH/Lh based on the consumption of the glucose, xylose and arabinose. These results mean a global yield of 251 L EtOH/ton BSG of this feedstock and were accomplished without the inhibition caused by acetic acid formation (6.87 g/L). Furthermore, the proposed process in this work was developed in a sequential pathway, which comprises an operational advantage to exploit the BSG in the production of second-generation bioethanol.

Cardoon Hydrolysate Detoxification by Activated Carbon or Membranes System for Bioethanol Production

Advanced biofuels incorporation into the transportation sector, particularly cellulosic bioethanol, is crucial for attaining carbon neutrality by 2050, contributing to climate changes mitigation and wastes minimization. The world needs biofuel to be commercially available to tackle the socioeconomic challenges coming from the continued use of fossil fuels. Cynara cardunculus (cardoon) is a cheap lignocellulosic raw biomass that easily grows in Mediterraneous soils and is a potential renewable resource for a biorefinery. This work aimed to study the bioethanol production from cardoon hemicellulosic hydrolysates, which originated from dilute sulfuric acid hydrolysis pretreatment. A detoxification step to remove released microbial fermentative inhibitors was evaluated by using both activated carbon adsorption and a nanofiltration membrane system. The Scheffersomyces stipitis CBS5773 yeast and the modified Escherichia coli MS04 fermentation performances at different experimental conditions were compared. The promising results with E. coli, using detoxified cardoon by membrane nanofiltration, led to a bioethanol volumetric productivity of 0.30 g·L−1·h−1, with a conversion efficiency of 94.5%. Regarding the S. stipitis, in similar fermentation conditions, volumetric productivity of 0.091 g·L−1·h−1 with a conversion efficiency of 64.9% was obtained. Concluding, the production of bioethanol through detoxification of hemicellulosic cardoon hydrolysate presents a suitable alternative for the production of second-generation bioethanol, especially using the modified E. coli.

Increase in Fermentable Sugars of Olive Tree Pruning Biomass for Bioethanol Production: Application of an Experimental Design for Optimization of Alkaline Pretreatment

Olive Tree Pruning (OTP) biomass can be considered a suitable source of fermentable sugars for the production of second-generation bioethanol. The present study proposes a remarkable alternative for the valorization of olive tree pruning residues. OTP biomass was processed using a sequential calcium hydroxide pretreatment/enzymatic hydrolysis. A 24–1 half fractional factorial design was adopted for the screening of process variables and a central composite design was used for the optimization stage. Temperature and lime loading resulted statistically significant. The following optimal conditions were obtained: 0.01 g of Ca(OH)2/g of dry material, 20 g of H2O/g of dry material at 160 °C for 2 h. The mathematical model that governs this alkaline pretreatment was obtained with a 76% adjusted determination coefficient, which means that it is a good representation of the process. Under optimal operating conditions, 13% of the cellulose and 88% of the hemicellulose was solubilized. Moreover, the fermentable sugar content increased 1800% compared with the initial conditions, obtaining 240 g of glucose per kg of OTP residue. The fermentable sugars obtained after the calcium hydroxide pretreatment and enzymatic hydrolysis of OTP biomass yielded 2.8 g of ethanol/100 g of raw material.

Lignocellulosic Biomass Valorization for Bioethanol Production: a Circular Bioeconomy Approach.

Lignocellulosic biomass generated from different sectors (agriculture, forestry, industrial) act as biorefinery precursor for production of second-generation (2G) bioethanol and other biochemicals. The integration of various conversion techniques on a single platform under biorefinery approach for production of biofuel and industrially important chemicals from LCB is gaining interest worldwide. The waste generated on utilization of bio-resources is almost negligible or zero in a biorefinery along with reduced greenhouse gas emissions, which supports the circular bioeconomy concept. The economic viability of a lignocellulosic biorefinery depends upon the efficient utilization of three major components of LCB—cellulose, hemicellulose and lignin. The heterogeneous structure and recalcitrant nature of LCB is main obstacle in its valorization into bioethanol and other value-added products. The success of bioconversion process depends upon methods used during pre-treatment, hydrolysis and fermentation processes. The cost involved in each step of the bioconversion process affects the viability of cellulosic ethanol. The lignocellulose biorefinery has ample scope, but much-focused research is required to fully utilize major parts of lignocellulosic biomass with zero wastage. The present review entails lignocellulosic biomass valorization for ethanol production, along with different steps involved in its production. Various value-added products produced from LCB components were also discussed. Recent technological advances and significant challenges in bioethanol production are also highlighted in addition to future perspectives.Graphical abstract

Waste-Based Second-Generation Bioethanol: A Solution for Future Energy Crisis

The demand for more environmentally friendly alternative renewable fuels is growing as fossil fuel resources are depleting significantly. Consequently, bioethanol has attracted interest as a potentially viable fuel. The key steps in second-generation bioethanol production include pretreatment, saccharification, and fermentation. The present study employed simultaneous saccharification and fermentation (SSF) of cellulose through bacterial pathways to generate second-generation bioethanol utilizing corncobs and paper waste as lignocellulosic biomass. Mechanical and chemical pretreatments were applied to both biomasses. Then, two bacterial strains, Bacillus sp. and Norcadiopsis sp., hydrolysed the pretreated biomass and fermented it along with Achromobacter sp., which was isolated and characterized from a previous study. Bioethanol production followed by 72 h of biomass hydrolysis employing Bacillus sp. and Norcadiopsis sp., and then 72 h of fermentation using Achromobacter sp. Using solid phase micro extraction combined with GCMS the ethanol content was quantified. SSF of alkaline pretreated paper waste hydrolysed by Bacillus sp. following the fermentation by Achromobacter sp. showed the maximum ethanol percentage of 0.734±0.154. Alkaline pretreated corncobs hydrolyzed by Norcadiopsis sp. yielded the lowest ethanol percentage of 0.155±0.154. The results of the study revealed that paper waste is the preferred feedstock for generating second-generation bioethanol. To study the possible use of ethanol-diesel blends as an alternative biofuel E2, E5, E7, and E10 blend emulsions were prepared mixing commercially available diesel with ethanol. The evaluated physico-chemical characteristics of the ethanol-diesel emulsions fulfilled the Ceypetco requirements except for the flashpoint revealing that the lower ethanol-diesel blends are a promising alternative to transport fuels. As a result, the current study suggests that second generation bioethanol could be used as a renewable energy source to help alleviate the energy crisis..

Integral approach for second-generation bio-ethanol production and wastewater treatment using peanut shell waste: yield, removal, and ANN studies

Integral approach for second-generation bio-ethanol production and wastewater treatment using peanut shell waste: yield, removal, and ANN studies