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Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient CBP yeast. Despite showing a close relatedness to commercial wine yeast and the benchmark Ethanol Red, a unique profile of gene copy number variations (CNVs) was found in L20, mainly encoding membrane transporters and secretion pathway proteins but also the fermentative metabolism. Moreover, the genome annotation disclosed seven open reading frames (ORFs) in L20 that are absent in the reference S288C genome. Genome engineering was successfully implemented for amylase production. However, with equal amylase gene copies, L20 proved its proficiency as a good enzyme secretor by exhibiting a markedly higher amylolytic activity than Ethanol Red, in compliance to the findings of the genomic exploration. The recombinant L20 dT8 exhibited the highest amylolytic activity and produced more than 4 g/L of ethanol from 2% starch in a CBP setting without the addition of supplementary enzymes. Based on the performance of this strain, an amylase/glucoamylase ratio of 1:2.5 was suggested as baseline for further improvement of the CBP ability. Overall, L20 showed important traits for the future construction of a proficient CBP yeast. As such, this work shows that natural S. cerevisiae strains can be used for the expression of foreign secreted enzymes, paving the way to strain improvement for the starch-to-bioethanol route. [ABSTRACT FROM AUTHOR]

The production of lignocellulosic ethanol calls for a robust fermentative yeast able to tolerate a wide range of toxic molecules that occur in the pre-treated lignocellulose. The concentration of inhibitors varies according to the composition of the lignocellulosic material and the harshness of the pre-treatment used. It follows that the versatility of the yeast should be considered when selecting a robust strain. This work aimed at the validation of seven natural Saccharomyces cerevisiae strains, previously selected for their industrial fitness, for their application in the production of lignocellulosic bioethanol. Their inhibitor resistance and fermentative performances were compared to those of the benchmark industrial yeast S. cerevisiae Ethanol Red, currently utilized in the second-generation ethanol plants. The yeast strains were characterized for their tolerance using a synthetic inhibitor mixture formulated with increasing concentrations of weak acids and furans, as well as steam-exploded lignocellulosic pre-hydrolysates, generally containing the same inhibitors. The eight non-diluted liquors have been adopted to assess yeast ability to withstand bioethanol industrial conditions. The most tolerant S. cerevisiae Fm17 strain, together with the reference Ethanol Red, was evaluated for fermentative performances in two pre-hydrolysates obtained from cardoon and common reed, chosen for their large inhibitor concentrations. S. cerevisiae Fm17 outperformed the industrial strain Ethanol Red, producing up to 18 and 39 g/L ethanol from cardoon and common reed, respectively, with ethanol yields always higher than those of the benchmark strain. This natural strain exhibits great potential to be used as superior yeast in the lignocellulosic ethanol plants. [ABSTRACT FROM AUTHOR]

The development of a yeast that converts raw starch to ethanol in one step (called consolidated bioprocessing) could yield large cost reductions in the bioethanol industry. The aim of this study was to develop an efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production. A native and codon-optimized variant of the Aspergillus awamori glucoamylase gene were expressed in the S. cerevisiae Y294 laboratory strain. Codon optimization resulted to be effective and the synthetic sequence sGAI was then δ-integrated into a S. cerevisiae strain with promising industrial fermentative traits. The mitotically stable recombinant strains showed high enzymatic capabilities both on soluble and raw starch (2425 and 1140 nkat/g dry cell weight, respectively). On raw corn starch, the engineered yeasts exhibited improved fermentative performance with an ethanol yield of 0.42 (g/g), corresponding to 75 % of the theoretical maximum yield. [ABSTRACT FROM AUTHOR]

Bioethanol obtained from agro-food wastes could contribute to decrease the dependency on fossil resources, reduce the impact of fossil fuels on the environment, and mitigate the food versus fuel debate. This study is aimed to investigate the availability of residual inexpensive agro-food biomasses that could feed a second-generation bioethanol plant located in a specific area of North Eastern Italy. After the identification of all crops in the area, more than 40 agro-food residues were analyzed for their availability and compositions in terms of water, polysaccharides, and sugars potentially convertible into bioethanol. 574,166 Mg of residual wet lignocellulosic biomass corresponding to 297,325 Mg of dry material were found available for bioethanol conversion. The most promising substrates were wheat straw and vine shoots. Based on the chemical composition of residues, the potential attainable ethanol was determined. Theoretical potential ethanol production was estimated at nearly 72,000 Mg per year. This quantity extensively exceeds the minimum yearly capacity of a sustainable bioethanol plant previously identified as around 50,000 Mg of ethanol. Taken together, these results demonstrate that, in the analyzed area, agro-food residues are available in an amount that could sustain bioethanol production in a specific and restricted district. Techno-economical evaluations are in progress to assess the actual feasibility of installing a second generation bioethanol production plant in the area of interest. [ABSTRACT FROM AUTHOR]

An engineered yeast producing all the cellulases needed for cellulose saccharification could produce ethanol from lignocellulose at a lower cost. This study aimed to express fungal β-glucosidases in Saccharomyces cerevisiae to convert cellobiose into ethanol. Furthermore, two engineering platforms (laboratory vs industrial strain) have been considered towards the successful deployment of the engineered yeast under simulated industrial conditions. The industrial S. cerevisiae M2n strain was engineered through the δ-integration of the β-glucosidase Pccbgl1 of Phanerochaete chrysosporium. The most efficient recombinant, M2n[pBKD2- Pccbgl1 ]-C1, was compared to the laboratory S. cerevisiae Y294[ Pccbgl1 ] strain, expressing Pccbgl1 from episomal plasmids, in terms of cellobiose fermentation in a steam exploded sugarcane bagasse pre-hydrolysate. Saccharomyces cerevisiae Y294[ Pccbgl1 ] was severely hampered by the pre-hydrolysate. The industrial M2n[pBKD2- Pccbgl1 ]-C1 could tolerate high inhibitors-loading in pre-hydrolysate under aerobic conditions. However, in oxygen limited environment, the engineered industrial strain displayed ethanol yield higher than the laboratory Y294[ Pccbgl1 ] only when supplemented with supernatant containing further recombinant β-glucosidase. This study showed that the choice of the host strain is crucial to ensure bioethanol production from lignocellulose. A novel cellobiose-to-ethanol route has been developed and the recombinant industrial yeast could be a promising platform towards the future consolidated bioprocessing of lignocellulose into ethanol. [ABSTRACT FROM AUTHOR]

The use of solid starchy waste streams to produce value-added products, such as fuel ethanol, is a priority for the global bio-based economy. Despite technological advances, bioethanol production from starch is still not economically competitive. Large cost-savings can be achieved through process integration (consolidated bioprocessing, CBP) and new amylolytic microbes that are able to directly convert starchy biomass into fuel in a single bioreactor. Firstly, CBP technology requires efficient fermenting yeast strains to be engineered for amylase(s) production. This study addressed the selection of superior yeast strains with high fermentative performances to be used as recipient for future CBP engineering of fungal amylases. Twenty-one newly isolated wild-type Saccharomyces cerevisiae strains were screened at 30 °C in a simultaneous saccharification and fermentation (SSF) set up using starchy substrates at high loading (20% w/v) and the commercial amylases cocktail STARGEN™ 002. The industrial yeast Ethanol Red™ was used as benchmark. A cluster of strains produced ethanol levels (up to 118 g/L) significantly higher than those of Ethanol Red™ (about 109 g/L). In particular, S. cerevisiae L20, selected for a scale-up process into a 1-L bioreactor, confirmed the outstanding performance over the industrial benchmark, producing nearly 101 g/L ethanol instead of 94 g/L. As a result, this strain can be a promising CBP host for heterologous expression of fungal amylases towards the design of novel and efficient starch-to-ethanol routes. [ABSTRACT FROM AUTHOR]

Abstract: Wheat bran, low-cost residue of the milling industry, is an interesting substrate for ethanol production. This study reports on the pre-treatment of wheat bran for high hexose and pentose recovery in the following enzymatic hydrolysis step using optimised dosages of commercial enzymes. Acid addition, milling and heat treatment were compared in terms of total sugar yield and inhibitory by-product release. The maximum total sugar amount was obtained when limited concentrations of acid were added to milled bran at the pre-treatment step. The whole unfiltered hydrolysates were fermented by highly fermentative wild type yeasts. A glucose-to-ethanol conversion of 95% was attained by Saccharomyces cerevisiae s1, while Saccharomyces diastaticus ATCC 13007 proved effective for the secretion of extracellular glucoamylase resulting in additional ethanol production. [Copyright &y& Elsevier]

In view of the potential application of pyrolysis-based biotechnologies, it is crucial to look for novel microorganisms able to convert pyrolysis-derived products, in particular bio-oil water-soluble constituent, into valuable compounds. For the first time, this paper proposed a survey on a collection of bacterial, yeast, and fungal strains with well-known industrial properties as well as new bacterial isolates in order to select microbes able to both tolerate bio-oil inhibitors and convert bio-oil into valuable products. This survey found that bio-oil aqueous phase (BOAP) obtained from intermediate pyrolysis could be metabolized as it is by fungal strains whereas several dilutions are needed to do not hamper cell viability of many tested yeast and bacterial isolates. To process BOAP into valuable products, the yeast Saccharomyces cerevisiae L13, selected as the most industrially relevant tested strain, was adopted to convert bio-oil aqueous fraction hydrolysate into ethanol without any detoxification step. The fermenting performances were much greater than those of the benchmark yeast strain and S. cerevisiae L13 proved to be a strong candidate for bioethanol production from BOAP hydrolysates. This study demonstrated that the search for microorganisms is a promising approach to the future development of pyrolysis oil-based biorefinery platforms. Image 1 • Bio-oil was obtained from intermediate pyrolysis of wood. • 204 microbes with industrial traits was tested for bio-oil tolerance. • 17 newly isolated bacteria able to use bio-oil as carbon source were obtained. • The most tolerant yeast produced high ethanol yields without detoxification step. [ABSTRACT FROM AUTHOR]

Wheat bran, generated from the milling of wheat, represents a promising feedstock for the production of bioethanol. This substrate consists of three main components: starch, hemicellulose and cellulose. The optimal conditions for wheat bran hydrolysis have been determined using a recombinant cellulase cocktail (RCC), which contains two cellobiohydrolases, an endoglucanase and a β-glucosidase. The 10% (w/v, expressed in terms of dry matter) substrate loading yielded the most glucose, while the 2% loading gave the best hydrolysis efficiency (degree of saccharification) using unmilled wheat bran. The ethanol production of two industrial amylolytic Saccharomyces cerevisiae strains, MEL2[TLG1-SFA1] and M2n[TLG1-SFA1], were compared in a simultaneous saccharification and fermentation (SSF) for 10% wheat bran loading with or without the supplementation of optimised RCC. The recombinant yeast S . cerevisiae MEL2[TLG1-SFA1] and M2n[TLG1-SFA1] completely hydrolysed wheat bran’s starch producing similar amounts of ethanol (5.3 ± 0.14 g/L and 5.0 ± 0.09 g/L, respectively). Supplementing SSF with RCC resulted in additional ethanol production of about 2.0 g/L. Scanning electron microscopy confirmed the effectiveness of both RCC and engineered amylolytic strains in terms of cellulose and starch depolymerisation. This study demonstrated that untreated wheat bran could be a promising ready-to-use substrate for ethanol production. The addition of crude recombinant cellulases improved ethanol yields in the SSF process and S . cerevisiae MEL2[TLG1-SFA1] and M2n[TLG1-SFA1] strains can efficiently convert wheat bran’s starch to ethanol. [ABSTRACT FROM AUTHOR]

Abstract: Wheat bran, an abundant residue of the flour milling industry, is a promising feedstock for bioethanol production. The first objective of the present study was to define the optimised hydrolysis conditions for wheat bran that gave both high sugars concentration and little or no inhibitors formation. This low-cost material was subjected to acid treatment and then hydrolysed using increasing dosages of commercial enzymes. With the aim of selecting a robust yeast suitable for industrial ethanol production, new Saccharomyces cerevisiae yeasts were isolated. Among the strains having remarkable ethanol yields from glucose in a minimal broth with high sugars concentrations, S. cerevisiae MEL2 was selected to be used for wheat bran fermentation. The strain produced ethanol levels higher than the benchmark industrial yeast S. cerevisiae DSM70449, exhibiting an outstanding ethanol yield of 96% of the theoretical and also showing promising fermentative activity on the whole unfiltered hydrolysates. This study demonstrated that dilute acid pre-treatment and the use of a properly selected yeast had a great impact on the overall ethanol yield from wheat bran. [Copyright &y& Elsevier]