Second-generation Bioethanol Production Research Articles

Potentials, Challenges, and Genetic and Genomic Resources for Sugarcane Biomass Improvement.

Lignocellulosic biomass has become an emerging feedstock for second-generation bioethanol production. Sugarcane (Saccharum spp. hybrids), a very efficient perennial C4 plant with a high polyploid level and complex genome, is considered a top-notch candidate for biomass production due to its salient features viz. fast growth rate and abilities for high tillering, ratooning, and photosynthesis. Energy cane, an ideal type of sugarcane, has been bred specifically as a biomass crop. In this review, we described (1) biomass potentials of sugarcane and its underlying genetics, (2) challenges associated with biomass improvement such as large and complex genome, narrow gene pool in existing commercial cultivars, long breeding cycle, and non-synchronous flowering, (3) available genetic resources such as germplasm resources, and genomic and cell wall-related databases that facilitate biomass improvement, and (4) mining candidate genes controlling biomass in genomic databases. We extensively reviewed databases for biomass-related genes and their usefulness in biofuel generation. This review provides valuable resources for sugarcane breeders, geneticists, and broad scientific communities involved in bioenergy production.

Exergetic Analysis between a First-Generation Bioethanol Production Plant and a Second-Generation Plant Coupled to Conventional Process

Large consumption of non-renewable fuels in the world has strongly encouraged the search for biofuels, due to inevitable shortage of fossil energy sources and catastrophic problems generated by their use. Under these circumstances the so called second generation biofuel production has been highlighted, where lignocellulosic materials such as sugarcane bagasse are used to produce bioethanol, unlike the traditional first-generation process, whose feedstock is sugarcane. Therefore, this work aimed to perform a comparative exergetic analysis between first and second-generation bioethanol production processes, in order to evaluate from the exergetic point of view, which of these processes is the most feasible. It was found an overall exergetic efficiency of 60.89 % for the exclusively first-generation plant, whereas for the integrated plant, it was obtained 41.58 %. In this way, insertion of new stages in production process, required by the second generation, has caused consequently an increase of irreversibilities and losses.

A novel closed-circuit circulation system about integrated ethanol-methane fermentation process based on the subcritical water pretreatment of corn stover

Abstract For the purpose to fully reutilize wastewater originated in second-generation bioethanol production from corn stover, a novel closed-circuit circulation system of integrated ethanol-methane fermentation process was successfully established based on the subcritical water pretreatment, in which the wastewater was fully recycled for 20 batches. It was indicated that the pH of recycling liquid decreased with the increasing of cycle numbers, which led to the constant enhancement of the pretreatment efficiency as approximately 60% hemicellulose and 50% lignin degradation as well as more than 80% cellulose reserved. Meanwhile, the performance of both ethanol (2.76–6.94 g/L) and methane (382.56–2631.24 mL/L) fermentation reached stability and trended to be self-improved during the 20 batches, which indicated that the designed integrated process was stable and applicable. Furthermore, the variety of microbial community and other processes showed intimate correlation with the physiochemical properties of the recycling liquid. It was promising to apply the system in bioenergy industries to fully reutilize of the wastewater in the future.

Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield

BackgroundReduction or elimination of by-product formation is of immediate economic relevance in fermentation processes for industrial bioethanol production with the yeast Saccharomyces cerevisiae. Anaerobic cultures of wild-type S. cerevisiae require formation of glycerol to maintain the intracellular NADH/NAD+ balance. Previously, functional expression of the Calvin-cycle enzymes ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK) in S. cerevisiae was shown to enable reoxidation of NADH with CO2 as electron acceptor. In slow-growing cultures, this engineering strategy strongly decreased the glycerol yield, while increasing the ethanol yield on sugar. The present study explores engineering strategies to improve rates of growth and alcoholic fermentation in yeast strains that functionally express RuBisCO and PRK, while maximizing the positive impact on the ethanol yield.ResultsMulti-copy integration of a bacterial-RuBisCO expression cassette was combined with expression of the Escherichia coli GroEL/GroES chaperones and expression of PRK from the anaerobically inducible DAN1 promoter. In anaerobic, glucose-grown bioreactor batch cultures, the resulting S. cerevisiae strain showed a 31% lower glycerol yield and a 31% lower specific growth rate than a non-engineered reference strain. Growth of the engineered strain in anaerobic, glucose-limited chemostat cultures revealed a negative correlation between its specific growth rate and the contribution of the Calvin-cycle enzymes to redox homeostasis. Additional deletion of GPD2, which encodes an isoenzyme of NAD+-dependent glycerol-3-phosphate dehydrogenase, combined with overexpression of the structural genes for enzymes of the non-oxidative pentose-phosphate pathway, yielded a CO2-reducing strain that grew at the same rate as a non-engineered reference strain in anaerobic bioreactor batch cultures, while exhibiting a 86% lower glycerol yield and a 15% higher ethanol yield.ConclusionsThe metabolic engineering strategy presented here enables an almost complete elimination of glycerol production in anaerobic, glucose-grown batch cultures of S. cerevisiae, with an associated increase in ethanol yield, while retaining near wild-type growth rates and a capacity for glycerol formation under osmotic stress. Using current genome-editing techniques, the required genetic modifications can be introduced in one or a few transformations. Evaluation of this concept in industrial strains and conditions is therefore a realistic next step towards its implementation for improving the efficiency of first- and second-generation bioethanol production.

Review of Second Generation Bioethanol Production from Residual Biomass.

In the context of climate change and the depletion of fossil fuels, there is a great need for alternatives to petroleum in the transport sector. This review provides an overview of the production of second generation bioethanol, which is distinguished from the first generation and subsequent generations of biofuels by its use of lignocellulosic biomass as raw material. The structural components of the lignocellulosic biomass such as cellulose, hemicellulose and lignin, are presented along with technological unit steps including pretreatment, enzymatic hydrolysis, fermentation, distillation and dehydration. The purpose of the pretreatment step is to increase the surface area of carbohydrate available for enzymatic saccharification, while minimizing the content of inhibitors. Performing the enzymatic hydrolysis releases fermentable sugars, which are converted by microbial catalysts into ethanol. The hydrolysates obtained after the pretreatment and enzymatic hydrolysis contain a wide spectrum of sugars, predominantly glucose and xylose. Genetically engineered microorganisms are therefore needed to carry out co-fermentation. The excess of harmful inhibitors in the hydrolysate, such as weak organic acids, furan derivatives and phenol components, can be removed by detoxification before fermentation. Effective saccharification further requires using exogenous hemicellulases and cellulolytic enzymes. Conventional species of distiller's yeast are unable to ferment pentoses into ethanol, and only a very few natural microorganisms, including yeast species like Candida shehatae, Pichia (Scheffersomyces) stipitis, and Pachysolen tannophilus, metabolize xylose to ethanol. Enzymatic hydrolysis and fermentation can be performed in a number of ways: by separate saccharification and fermentation, simultaneous saccharification and fermentation or consolidated bioprocessing. Pentose-fermenting microorganisms can be obtained through genetic engineering, by introducing xylose-encoding genes into metabolism of a selected microorganism to optimize its use of xylose accumulated in the hydrolysate.

Physical and Chemical Characterization of Agave tequilana Bagasse Pretreated with the Ionic Liquid 1-Ethyl-3-Methylimidazolium Acetate

Pretreatment of lignocellulosic biomass remains a bottleneck for the production of second-generation bioethanol. Conventional pretreatments have had moderated success in meeting the criteria to be considered cost and energy efficient; however, ionic liquids have shown promising results. 1-ethyl-3-methylimidazolium acetate is one of the most successful ionic liquids used for biomass dissolution. The physical and chemical changes of ionic liquid pretreated lignocellulosic materials must be thoroughly studied to maximize the benefits of this technology. Agave tequilana bagasse (a major source of residual lignocellulosic biomass in Mexico) was pretreated using ionic liquid at four different temperatures: 40, 80, 120 and 160 °C. Untreated and pretreated samples were chemically characterized, and their physical properties were studied using X-ray diffraction, thermogravimetric analysis, attenuated total reflection Fourier-transform infrared spectroscopy, scanning electron microscopy, and surface area analysis using a BET model. Positive effects in the physical characteristics of the bagasse were obtained: an increase in surface area, decrease in cellulose crystallinity, and lignin content were observed at temperatures up to 120 °C. Higher temperatures were detrimental for these characteristics.

Bioethanol Production From Turkish Hazelnut Husk Process Design and Economic Evaluation

The main objective of the current study is to develop and assess the preliminary synthetic design steps of an innovative and unprecedented bioprocess plant converting Turkish hazelnut husk into lignocellulosic ethanol with an emphasis on economic evaluation. Valorization of this agricultural waste would provide a promising economic potential and long-term sustainability with acceptable environmental impact. Preliminary economic evaluations are performed on several scenarios where the effects of changing various process design and operational inputs such as designed plant capacity, evaporation unit operation efficiency, and biocatalyst and nitrogen source prices are simulated. The total capital investment for the base case scenario with an annual throughput of 180,000 metric tons (MT) hazelnut husk is just above USD 111 million. The annual operational costs for this case amount to USD 61 million. Assuming a sales price of USD 1.50 per kg of bioethanol (achieved via governmental subsidy and tax incentives) the return on investment of the project comes to 12.61% with a 8 year payback period. An uncertainty analysis performed on the annual hazelnut husk availability and biocatalyst and nitrogen source price fluctuations establishes a basis for further design of the process taking into account the risk factors associated with the project. The case studies and the uncertainty analysis confirm the fact that production of second generation bioethanol from hazelnut husk in Turkey is a worthwhile endeavor with an economic potential especially with additional social and environmental advantages.

Investigation of different pretreatment methods of Mediterranean-type ecosystem agricultural residues: characterisation of pretreatment products, high-solids enzymatic hydrolysis and bioethanol production

ABSTRACTAgricultural and agro-industrial lignocellulosic residues represent an important renewable resource for the production of fuels and chemicals towards a bio-based economy. Olive pruning, vineyard pruning and almond shells are important residues from agricultural activities in Mediterranean-type ecosystems. In the current work, bioethanol production from the above three types of agro-residues was studied, focusing on the effect of different pretreatment methods on enzymatic saccharrification efficiency of cellulose and production of second-generation bioethanol. Dilute acid, hydrothermal and steam explosion pretreatments were compared in order to remove hemicellulose and facilitate the subsequent enzymatic hydrolysis of the hemicellulose-deficient biomass to glucose. Enzymatic hydrolysis was performed in a free-fall mixing reactor enabling high solids loading of 23% w/w. This allowed hydrolysis of up to 67% of available cellulose in almond shells and close to 50% in olive pruning samples, and facilitated high ethanol production in the subsequent fermentation step; the highest ethanol concentrations achieved were 47.8 g/L for almond shells after steam explosion and 42 g/L for hydrothermally pretreated olive pruning residue.

Strain Breeding Enhanced Heterologous Cellobiohydrolase Secretion by Saccharomyces cerevisiae in a Protein Specific Manner.

The yeast Saccharomyces cerevisiae has a long association with alcoholic fermentation industries and has received renewed interest as a biocatalyst for second-generation bioethanol production. Rational engineering strategies are used to create yeast strains for consolidated bioprocessing of lignocellulosic biomass. Although significant progress is made in this regard with the expression of different cellulolytic activities in yeast, cellobiohydrolase (CBH) titers remain well below ideal levels. Through classical breeding, S. cerevisiae strains with up to twofold increased CBH secretion titers is obtained in strains expressing a single gene copy. An increase of up to 3.5-fold in secreted cellobiohydrolase activity is subsequently shown for strains expressing the heterologous gene on a high copy episomal vector. To our knowledge, this is the first report of classical breeding being used to enhance heterologous protein secretion and also the most significant enhancement of CBH secretion in yeast yet reported. This enhanced secretion phenotype is specific for cellobiohydrolase I secretion, indicating that reporter protein properties might be a major determining factor for efficient protein secretion in yeast. By exploring the latent potential of different S. cerevisiae strains, the authors show that the allele pool of various strains is a valuable engineering resource to enhance secretion in yeast.

Growth and expression of relevant metabolic genes of Clostridium thermocellum cultured on lignocellulosic residues.

The plant cell wall is a source of fermentable sugars in second-generation bioethanol production. However, cellulosic biomass hydrolysis remains an obstacle to bioethanol production in an efficient and low-cost process. Clostridium thermocellum has been studied as a model organism able to produce enzymatic blends that efficiently degrade lignocellulosic biomass, and also as a fermentative microorganism in a consolidated process for the conversion of lignocellulose to bioethanol. In this study, a C. thermocellum strain (designated B8) isolated from goat rumen was characterized for its ability to grow on sugarcane straw and cotton waste, and to produce cellulosomes. We also evaluated C. thermocellum gene expression control in the presence of complex lignocellulosic biomasses. This isolate is capable of growing in the presence of microcrystalline cellulose, sugarcane straw and cotton waste as carbon sources, producing free enzymes and residual substrate-bound proteins (RSBP). The highest growth rate and cellulase/xylanase production were detected at pH 7.0 and 60°C, after 48h. Moreover, this strain showed different expression levels of transcripts encoding cellulosomal proteins and proteins with a role in fermentation and catabolic repression.

English

Beetles of the Passalidae family live and feed on decaying wood and their guts are richly colonized by yeasts. The goal of this research was to prospect xylolytic yeasts with potential for the production of second-generation bioethanol. Therefore, 83 specimens of beetles belonging to the Passalidae and the Scarabaeidae families were collected in the Amazonian rainforest in Itacoatiara - AM, Brazil. Morphological differences of the beetles were identified and 25 chosen specimens were dissected. Yeasts from galleries inhabited by beetles and from insect guts were isolated. Isolates were previously selected through tolerance tests for temperature, ethanol and xylose assimilation capacity. Those isolates were then submitted to a panel of conditions related to ethanol production. The ethanol production reached 24.70 g.L-1 and the xylitol production reached 21.66 g.L-1. One of the isolates with a promising profile was identified as Spathaspora roraimanensis and six as Spathaspora passalidarum. Three isolates showed to be more promising and, curiously, all came from the gut of the species Popilius marginatus (Percheron, 1835). In plate testing, however, the isolates obtained from galleries showed a greater capacity to assimilate xylose. As reported in this field of study, no isolate tolerated all conditions tested. Wild isolates with this profile may be used for testing larger-scale ethanol production, genetic engineering, or evolutionary techniques. Key words: Beetles, bioethanol, Popilius marginatus, xylose.

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae.

Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes d-xylose in the presence of high d-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8, or SSN6, which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10, HXT13, HXT15, and HXT16 Mutant cells showed increased uptake rates of d-xylose in the presence of d-glucose, as well as elevated maximum rates of metabolism (Vmax) for both d-glucose and d-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders d-xylose metabolism less sensitive to d-glucose inhibition due to an elevated transport rate of d-xylose into the cell.IMPORTANCE The yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast.

De novo Transcriptome Analysis of Miscanthus lutarioriparius Identifies Candidate Genes in Rhizome Development.

HIGHLIGHT De novo transcriptome profiling of five tissues reveals candidate genes putatively involved in rhizome development in M. lutarioriparius.Miscanthus lutarioriparius is a promising lignocellulosic feedstock for second-generation bioethanol production. However, the genomic resource for this species is relatively limited thus hampers our understanding of the molecular mechanisms underlying many important biological processes. In this study, we performed the first de novo transcriptome analysis of five tissues (leaf, stem, root, lateral bud and rhizome bud) of M. lutarioriparius with an emphasis to identify putative genes involved in rhizome development. Approximately 66 gigabase (GB) paired-end clean reads were obtained and assembled into 169,064 unigenes with an average length of 759 bp. Among these unigenes, 103,899 (61.5%) were annotated in seven public protein databases. Differential gene expression profiling analysis revealed that 4,609, 3,188, 1,679, 1,218, and 1,077 genes were predominantly expressed in root, leaf, stem, lateral bud, and rhizome bud, respectively. Their expression patterns were further classified into 12 distinct clusters. Pathway enrichment analysis revealed that genes predominantly expressed in rhizome bud were mainly involved in primary metabolism and hormone signaling and transduction pathways. Noteworthy, 19 transcription factors (TFs) and 16 hormone signaling pathway-related genes were identified to be predominantly expressed in rhizome bud compared with the other tissues, suggesting putative roles in rhizome formation and development. In addition, a predictive regulatory network was constructed between four TFs and six auxin and abscisic acid (ABA) -related genes. Furthermore, the expression of 24 rhizome-specific genes was further validated by quantitative real-time RT-PCR (qRT-PCR) analysis. Taken together, this study provide a global portrait of gene expression across five different tissues and reveal preliminary insights into rhizome growth and development. The data presented will contribute to our understanding of the molecular mechanisms underlying rhizome development in M. lutarioriparius and remarkably enrich the genomic resources of Miscanthus.

Integrating starchy substrate into cellulosic ethanol production to boost ethanol titers and yields

Starchy grains as current major feedstocks for bioethanol production are competing with food supply; therefore, lignocellulosic biomass was pursued as an alternative feedstock for bioethanol production due to its high availability and low price. Commercial production of second-generation bioethanol from lignocellulosic biomass is still under development as significant challenges of low fermentation efficiency, low ethanol titers, high enzyme cost, and high water usage remain to be addressed. In this research, sugar-rich substrates such as starchy grains were integrated into cellulosic ethanol production, which could boost ethanol titers and ethanol yields. The substrates with various ratios of corn flour and hydrothermal treated corn stover (4:12; 8:8; 12:4; 12:12) were evaluated on the ethanol concentration and ethanol yield via simultaneous saccharification and fermentation. Ethanol concentration and ethanol yield decreased with increasing amounts of treated corn stover in the mixtures. The maximum ethanol concentration (68.7g/L) was achieved at the corn flour and corn stover ratio of 12:12 using raw starch granular enzyme with the ethanol yield of 86.0%, whereas the maximum ethanol yield was obtained at the corn flour and corn stover ratio of 12:4 as it contained higher amounts of corn flour. All the ethanol concentrations from various mixtures of corn flour and corn stover were higher than 37.9g/L from the control with 100% of treated corn stover (16%, w/v). Saccharification and fermentation processes were optimized to reduce energy cost and the optimized process was able to complete ethanol fermentation within 48h.

Valorization of carob waste: Definition of a second-generation bioethanol production process

The aim of this work was to develop a strategy for second-generation ethanol production from carob solid waste issued from Lebanese food industry. The pros and cons of submerged (SF) and solid-state fermentations (SSF) using S. cerevisiae on ethanol yield and productivity were compared, including the respective roles of upstream and downstream processes, such as the size reduction, or sugar and ethanol recovery processes. The design of experiments methodology was applied. Experimental results demonstrated that SSF applied to cut carob waste from carob syrup preparation was simpler to operate and more cost-effective, maintained yield and productivity (0.458g ethanol/g consumed sugar and 4.3g/(kg waste)/h) in comparison to SF (0.450g ethanol/g consumed sugar and 5.7g/(kg waste)/h), and was able to achieve ethanol production up to 155g/(kg waste) at low water demand, while SF reached only 78g/(kg waste) due to the limitations of the sugar extraction pretreatment.

Saccharification of cellulose in the ionic liquids and glucose recovery

High-efficiency hydrolysis of lignocellulose is critical for the production of second-generation bioethanol. In the present work, the acid hydrolysis of cellulose in ionic liquids (ILs) 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), 1-allyl-3-methylimidazolium chloride ([Amim]Cl), and 1-ethyl-3-methylimidazolium chloride ([Emim]Cl), respectively, was investigated and aqueous two-phase systems were constructed by adding salt solutions to ILs to recover glucose from cellulose hydrolyzates in the ILs. The effects of reaction temperature, reaction time and acid consumption on the cellulose hydrolysis efficiencies in the ILs were determined. The optimal cellulose hydrolysis conditions in ILs were found to be [Bmim]Cl, a reaction time of 0.5 h, an acid consumption of 0.25 mL/g (cellulose) and a reaction temperature of 100 °C. The yield of glucose under the optimal hydrolysis conditions reached 92.88%. IL-based aqueous two-phase systems were formed by adding NaOH, K3PO4, or K2HPO4 into the ILs containing glucose or cellulose hydrolyzates, which were able to partition the glucose into the bottom salt-rich phase. Under the optimum condition, 4 vol 50% NaOH or K3PO4 solution was able to partition 90% glucose, resulting in 16 g/L glucose in the salt-rich phase that could be further used for ethanol fermentation.

Marine Enzymes and Microorganisms for Bioethanol Production.

Bioethanol is a potential alternative fuel to fossil fuels. Bioethanol as a fuel has several economic and environmental benefits. Though bioethanol is produced using starch and sugarcane juice, these materials are in conflict with food availability. To avoid food-fuel conflict, the second-generation bioethanol production by utilizing nonfood lignocellulosic materials has been extensively investigated. However, due to the complexity of lignocellulose architecture, the process is complicated and not economically competitive. The cultivation of lignocellulosic energy crops indirectly affects the food supplies by extensive land use. Marine algae have attracted attention to replace the lignocellulosic feedstock for bioethanol production, since the algae grow fast, do not use land, avoid food-fuel conflict and have several varieties to suit the cultivation environment. The composition of algae is not as complex as lignocellulose due to the absence of lignin, which renders easy hydrolysis of polysaccharides to fermentable sugars. Marine organisms also produce cold-active enzymes for hydrolysis of starch, cellulose, and algal polysaccharides, which can be employed in bioethanol process. Marine microoorganisms are also capable of fermenting sugars under high salt environment. Therefore, marine biocatalysts are promising for development of efficient processes for bioethanol production.

Combustion of lignin-rich residues with coal in a pilot-scale bubbling fluidized bed reactor

The production of second-generation bioethanol generates a waste stream consisting of lignin-rich residues whose valorization has to be found. Fluidized bed combustion technology can be considered as a promising and viable option to recover thermal power from lignin-rich residues of a second-generation bioethanol production plant. This work aims at investigating the combustion of lignin-rich residues with coal in a pilot-scale bubbling fluidized bed combustor (FBC). An experimental campaign was carried out to study gaseous and particulate emissions and thermal regimes during the combustion of mixtures of coal with 30%w of lignin-rich residues varying bed temperature, excess air and fluidization velocity. A few experimental tests were carried out for comparison using only coal, a mixture of coal with 40%w of lignin-rich residues and a mixture of coal with 20%w of wood chips. The analysis of the experimental results mainly highlights that: 1) the gaseous emissions did not significantly change with respect to coal or to reference biomass-coal mixture at least until the content of lignin-rich residues in the mixture was 30–40%w; 2) the particulate emissions increased with the percentage of residues content, but, at the same, the particulate carbon concentration was significantly reduced. Bottom bed particles were analyzed at the end of each experiments, highlighting the absence of agglomerates. However, a significant enrichment of metals like Fe, Mg, Na, Ca and K, most of them probably coming from the ash of lignin-rich residues, was observed when the FBC was operated under conditions which emphasize ash accumulation inside the bed.

Engineering a wild-type diploid Saccharomyces cerevisiae strain for second-generation bioethanol production.

BackgroundThe cost-effective production of second-generation bioethanol, which is made from lignocellulosic materials, has to face the following two problems: co-fermenting xylose with glucose and enhancing the strain’s tolerance to lignocellulosic inhibitors. Based on our previous study, the wild-type diploid Saccharomyces cerevisiae strain BSIF with robustness and good xylose metabolism genetic background was used as a chassis for constructing efficient xylose-fermenting industrial strains. The performance of the resulting strains in the fermentation of media with sugars and hydrolysates was investigated.ResultsThe following two novel heterologous genes were integrated into the genome of the chassis cell: the mutant MGT05196 N360F, which encodes a xylose-specific, glucose-insensitive transporter and is derived from the Meyerozyma guilliermondii transporter gene MGT05196, and Ru-xylA (where Ru represents the rumen), which encodes a xylose isomerase (XI) with higher activity in S. cerevisiae. Additionally, endogenous modifications were also performed, including the overproduction of the xylulokinase Xks1p and the non-oxidative PPP (pentose phosphate pathway), and the inactivation of the aldose reductase Gre3p and the alkaline phosphatase Pho13p. These rationally designed genetic modifications, combined with alternating adaptive evolutions in xylose and SECS liquor (the leach liquor of steam-exploding corn stover), resulted in a final strain, LF1, with excellent xylose fermentation and enhanced inhibitor resistance. The specific xylose consumption rate of LF1 reached as high as 1.089 g g−1 h−1 with xylose as the sole carbon source. Moreover, its highly synchronized utilization of xylose and glucose was particularly significant; 77.6% of xylose was consumed along with glucose within 12 h, and the ethanol yield was 0.475 g g−1, which is more than 93% of the theoretical yield. Additionally, LF1 performed well in fermentations with two different lignocellulosic hydrolysates.ConclusionThe strain LF1 co-ferments glucose and xylose efficiently and synchronously. This result highlights the great potential of LF1 for the practical production of second-generation bioethanol.Electronic supplementary materialThe online version of this article (doi:10.1186/s40643-016-0126-4) contains supplementary material, which is available to authorized users.

Bioprospecting thermotolerant ethanologenic yeasts for simultaneous saccharification and fermentation from diverse environments

Lignocellulosic biomass, a promising renewable energy source, can be used for the production of second generation bioethanol. Simultaneous saccharification and fermentation (SSF), the process which alleviates the problem of separate hydrolysis and fermentation (SHF), requires thermotolerant ethanologenic yeast for bioethanol production. Therefore, ten yeast strains isolated from diverse sources, belonging to various genera like Saccharomyces, Candida, Pichia and Wickerhamomyces were evaluated for their thermotolerance, sugar utilization pattern, inhibitor tolerance and ethanol production potential with glucose, xylose and alkali pretreated paddy straw. All the tested strains were found to be thermotolerant, capable of significant growth at 40°C. Candida tropicalis Y6 was capable of utilizing a wide range of sugars as compared with other yeast isolates. Strains of Candida showed better inhibitor tolerance as compared to Saccharomyces and Pichia strains and exhibited only 5.1-18.8% and 4.7-7.9% reduction in growth with furfural and 5-hydroxymethyl furfural, respectively. Saccharomyces cerevisiae JRC6, isolated from distillery waste, produced ethanol with 88.3% and 89.1% theoretical efficiency at 40°C and 42°C, respectively, from glucose. This strain also produced significantly higher amount of ethanol (3.8g/L) with better fermentation efficiency (87.9%) from alkali pretreated paddy straw at 40°C, as compared with the other yeast strains. Therefore, S.cerevisiae JRC6, based on its ability to ferment sugars at a higher temperature, can be a promising candidate for production of ethanol from lignocellulosic biomass via SSF process.