Enhanced ethanol production using hydrophobic resin detoxified Pine forest litter hydrolysate and integrated fermentation process development supplementing molasses.

Globally escalating ethanol demand necessitates the use of hybrid technologies integrating first- and second-generation biofuel feedstocks for achieving the futuristic targets of gasoline replacement with bioethanol. In present study, an optimized two-step sequential pre-treatment (first dilute alkali, then dilute acid) of Pine forest litter (PFL) was developed. Furthermore, the saccharification of pre-treated PFL was optimized through Response Surface Methodology using Box-Behnken Design, wherein 0.558 g/g of reducing sugar was released under the optimized conditions (12.5% w/v of biomass loading, 10 FPU/g of PFL enzyme loading, 0.15% v/v Tween-80 and 48 h incubation time). Moreover, during hydrolysate fermentation using Saccharomyces cerevisiae NCIM 3288 strain, 22.51 ± 1.02 g/L ethanol was produced. Remarkably, hydrophobic resin (XAD-4) treatment of PFL hydrolysate, significantly removed inhibitors (Furfural, 5-hydroxymethylfurfural and phenolics) and increased ethanol production to 27.38 ± 1.18 g/L. Furthermore, during fermentation of molasses supplemented PFL hydrolysate (total initial sugar: 100 ± 3.27 g/L), a maximum of 46.02 ± 2.08 g/L ethanol was produced with 0.482 g/g yield and 1.92 g/l/h productivity. These findings indicated that the integration of molasses to lignocellulosic hydrolysate, would be a promising hybrid technology for industrial ethanol production within existing bio-refinery infrastructure.

Effects of corn drying and storage conditions on flour, starch, feed, and ethanol production: a review.

The objective was to review the effects of the drying and storage conditions of corn on the physical-chemical quality in the processing of starch and flour, in the production of animal feed, and in the industrialization of ethanol. Initially, the review presented an overview of the post-harvest stages of corn grains, highlighting drying and storage. The main drying and storage methods used for corn grains were presented. Among the drying conditions, the air temperature was the main factor that affected the properties of starch, flour, feed, and ethanol produced from corn. It was verified that the corn grains submitted to drying at temperatures below 60 °C obtained better results in the industry. In storage, in addition to the storage time, factors such as temperature and moisture content of the grains affected the physical-chemical quality of the processed products. In this stage, the moisture content below 14% and the storage temperature below 25 °C conserved the physical-chemical quality of the grains and obtained better processing results. Further studies are needed to assess the effects of the drying and storage conditions of corn on the properties of flour, starch, animal feed, and, mainly, ethanol production.

Evasion of Cas9 toxicity to develop an efficient genome editing system and its application to increase ethanol yield in Fusarium venenatum TB01.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) system is a powerful genome editing tool that has been successfully established in some filamentous fungi due to its high flexibility and efficiency. However, the potential toxicity of Cas9 restricts the further popularization and application of this system to some degree. The AMA1 element is a self-replicator derived from Aspergillus nidulans, and its derived vectors can be readily lost without selection. In this study, we eliminated Cas9 toxicity to Fusarium venenatum TB01 based on 100% AMA1-based Cas9 expression vector loss. Meanwhile, two available endogenous Pol III promoters (FvU6374 and Fv5SrRNA) used for sgRNA expression of the CRISPR/Cas9 system were excavated. Compared to FvU6374 (40-50%), Fv5SrRNA exhibited higher single-gene editing efficiency (> 85%), and the efficiency of simultaneous editing of the two genes using Fv5SrRNA was over 75%. Based on this system, a butanediol dehydrogenase encoding gene FvBDH was deleted, and the ethanol yield in variants increased by 52% compared with that of the wild-type. The highly efficient CRISPR/Cas9 system developed here lays the technical foundation for advancing the development of F. venenatum TB01 through metabolic engineering, and the obtained FvBDH gene-edited variants have the potential to simultaneously produce mycoprotein and ethanol by further gene modification and fermentation process optimization in the future.Key points• Cas9 toxicity disappeared and DNA-free gene-edited strains obtained after vector loss• Promoter Fv5SrRNA conferred TB01 higher gene editing efficiency than FvU6374•Deletion of the FvBDH gene resulted in a 52% increase in ethanol yield.

In Situ Corn Fiber Conversion for Ethanol Improvement by the Addition of a Novel Lignocellulolytic Enzyme Cocktail.

Corn mashes have high-viscosity and high-sugar characteristics, which hinders yeast-fermentation efficiency and the ethanol yield increase. The excessive viscosity of corn mash is caused by the unutilized cellulose in corn kernel fiber. A novel lignocellulolytic enzymes cocktail with strong substrate specificity was prepared for high-viscosity, high-sugar corn mash. The in situ conversion of corn mashes with novel lignocellulolytic enzymes at the optimum cellulase dosage of 50 FPU/L resulted in about 12% increased ethanol concentration compared with the reference mash at different batch-fermentation scales. Adding the lignocellulolytic enzymes caused the greatest decrease in viscosity of corn mash and residual sugars by 40.9% and 56.3%, respectively. Simultaneously, the application of lignocellulolytic enzymes increased the value of the dried distiller's grain with solubles (DDGS) by increasing the protein content by 5.51%. The in situ conversion of cellulose can decrease the fermentation broth viscosity and improve the rheological property, thereby improving the ethanol yield. With the same amount of material, the application of the novel enzymes cocktail can enhance the ethanol yield by more than 12%. A quarter of the ethanol yield increase was due to the further hydrolysis of starch, while three quarters to cellulose. Thus, this technology will increase the net revenue of bioethanol industrialization.

Ethanol yield calculations in biorefineries.

The ethanol yield on sugar during alcoholic fermentation allows for diverse interpretation in academia and industry. There are several different ways to calculate this parameter, which is the most important one in this industrial bioprocess and the one that should be maximized, as reported by Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91). On the one hand, the various methods currently employed in industry provide dissimilar results, and recent evidence shows that yield has been consistently overestimated in Brazilian sugarcane biorefineries. On the other hand, in academia, researchers often lack information on all the intricate aspects involved in calculating the ethanol yield in industry. Here, we comment on these two aspects, using fuel ethanol production from sugarcane in Brazilian biorefineries as an example, and taking the work of Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91.) as a starting point. Our work is an attempt to demystify some common beliefs and to foster closer interaction between academic and industrial professionals from the fermentation sector. Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91).

Novel ethanol production using biomass preprocessing to increase ethanol yield and reduce overall costs.

BACKGROUND: Ethanol biorefineries need to lower their overall production costs to become economically feasible. Two strategies to achieve this are to reduce costs using cheaper feedstocks or to increase the ethanol production yield. Low-cost feedstocks usually have high non-structural components (NSC) content; therefore, a new process is necessary to accommodate these feedstocks and overcome the negative effects of NSC. This study developed a novel ethanol biorefinery process including a biomass preprocessing step that enabled the use of lower-cost feedstocks while improving ethanol production without detoxification (overliming). Two types of poplar feedstocks were used, low-quality whole-tree chips (WTC) and high-quality clean pulp chips (CPC), to determine if the proposed process is effective while using feedstocks with different NSC contents. RESULTS: Technical assessment showed that acidic preprocessing increased the monomeric sugar recovery of WTC from 73.2% (untreated) to 87.5% due to reduced buffering capacity of poplar, improved sugar solubilization during pretreatment, and better enzymatic hydrolysis conversion. Preprocessing alone significantly improved the fermentability of the liquid fraction from 1-2% to 49-56% for both feedstocks while overliming improved it to 45%. Consequently, it was proposed that preprocessing can substitute for the detoxification step. The economic assessment revealed that using poplar WTC via the new process increased annual ethanol production of 10.5 million liters when compared to using CPC via overliming (base case scenario). Also, savings in total operating costs were about $10 million per year when using cheaper poplar WTC instead of CPC, and using recycled water for preprocessing lowered its total operating costs by 45-fold. CONCLUSIONS: The novel process developed in this study was successful in increasing ethanol production while decreasing overall costs, thus facilitating the feasibility of lignocellulosic ethanol biorefineries. Key factors to achieving this outcome included substituting overliming by preprocessing, enabling the use of lower-quality feedstock, increasing monomeric sugar recovery and ethanol fermentation yield, and using recycled water for preprocessing. In addition, preprocessing enabled the implementation of an evaporator-combustor downstream design, resulting in a low-loading waste stream that can be treated in a wastewater treatment plant with a simple configuration.

Co-culture of Saccharomyces cerevisiae (VS3) and Pichia stipitis (NCIM 3498) enhances bioethanol yield from concentrated Prosopis juliflora hydrolysate.

Biphasic acid hydrolysates and enzymatic hydrolysates from carbohydrate-rich Prosopis juliflora, an invasive perennial deciduous shrub of semi-arid regions, were used for bioethanol production. Saccharomyces cerevisiae and Pichia stipitis were used for fermentation of hexoses and pentoses. P. juliflora acid hydrolysate with an initial sugar concentration of 18.70 ± 0.16 g/L was concentrated to 33.59 ± 0.52 g/L by vacuum distillation. The concentrated hydrolysate was pretreated and fermented by mono- and co-culture methods either singly or in combination with enzyme hydrolysate and ethanol yields were compared. Monoculture with S. cerevisiae (VS3) and S. cerevisiae (NCIM3455) yielded maximum ethanol of 36.6 ± 1.83 g/L and 37.1 ± 1.86 g/L with a fermentation efficiency of 83.94 ± 4.20% and 84.20 ± 4.21%, respectively, after 36 h of fermentation. The ethanol yield obtained was 0.428 ± 0.02 g/g substrate and 0.429 ± 0.02 g/g substrate with a productivity of 1.017 ± 0.051 g/L/hand 1.031 ± 0.052 g/L/h, respectively. P. stipitis (NCIM3498) yielded maximum ethanol of 24 g/L with ethanol yield of 0.455 ± 0.02 g/g substrate and a productivity of 1.004 ± 0.050 g/L/h after 24 h of fermentation. With concentrated acid hydrolysate as substrate, S. cerevisiae (VS3) produced ethanol of 8.52 ± 0.43 g/L, whereas S. cerevisiae (NCIM3455) produced 5.96 ± 0.30 g/L of ethanol. P.stipitis (NCIM3498) produced 4.52 ± 0.23 g/L of ethanol by utilizing 14.66 ± 0.87 g/L of sugars. Co-culture with S. cerevisiae (VS3) addition after 18 h of addition of P. stipitis (NCIM3498) to the mixture of concentrated acid hydrolysate and enzyme hydrolysate produced 13.86 ± 0.47 g/L of ethanol with fermentation efficiency, ethanol yield and productivity of 87.54 ± 0.54%, 0.446 ± 2.36 g/g substrate and 0.385 ± 0.014 g/L/h, respectively. Hence, it is concluded that co-culture with S. cerevisiae and P. stipitis is feasible, further scaling up of fermentation of P. juliflora substrate for bioethanol production.

High-Throughput Phenotyping of Bioethanol Potential in Cereals Using UAV-Based Multi-Spectral Imagery.

Bioethanol production obtained from cereal straw has aroused great interest in recent years, which has led to the development of breeding programs to improve the quality of lignocellulosic material in terms of the biomass and sugar content. This process requires the analysis of genotype-phenotype relationships, and although genotyping tools are very advanced, phenotypic tools are not usually capable of satisfying the massive evaluation that is required to identify potential characters for bioethanol production in field trials. However, unmanned aerial vehicle (UAV) platforms have demonstrated their capacity for efficient and non-destructive acquisition of crop data with an application in high-throughput phenotyping. This work shows the first evaluation of UAV-based multi-spectral images for estimating bioethanol-related variables (total biomass dry weight, sugar release, and theoretical ethanol yield) of several accessions of wheat, barley, and triticale (234 cereal plots). The full procedure involved several stages: (1) the acquisition of multi-temporal UAV images by a six-band camera along different crop phenology stages (94, 104, 119, 130, 143, 161, and 175 days after sowing), (2) the generation of ortho-mosaicked images of the full field experiment, (3) the image analysis with an object-based (OBIA) algorithm and the calculation of vegetation indices (VIs), (4) the statistical analysis of spectral data and bioethanol-related variables to predict a UAV-based ranking of cereal accessions in terms of theoretical ethanol yield. The UAV-based system captured the high variability observed in the field trials over time. Three VIs created with visible wavebands and four VIs that incorporated the near-infrared (NIR) waveband were studied, obtaining that the NIR-based VIs were the best at estimating the crop biomass, while the visible-based VIs were suitable for estimating crop sugar release. The temporal factor was very helpful in achieving better estimations. The results that were obtained from single dates [i.e., temporal scenario 1 (TS-1)] were always less accurate for estimating the sugar release than those obtained in TS-2 (i.e., averaging the values of each VI obtained during plant anthesis) and less accurate for estimating the crop biomass and theoretical ethanol yield than those obtained in TS-3 (i.e., averaging the values of each VI obtained during full crop development). The highest correlation to theoretical ethanol yield was obtained with the normalized difference vegetation index (R 2 = 0.66), which allowed to rank the cereal accessions in terms of potential for bioethanol production.

Simultaneous saccharification and fermentation of native rye, wheat and triticale starch.

BACKGROUND: The increasing global demand for starchy raw material requires new methods for obtaining ethanol from a range of plants using environmentally friendly methods. Granular starch-hydrolyzing enzymes (GSHE) can effectively support the development of the distillery industry. RESULTS: The aim of this study was to evaluate the effectiveness of simultaneous saccharification and fermentation of native rye, wheat or triticale starch. Mashes were prepared using methods that limit water and energy consumption (pre-hydrolysis at 35 °C for 30 min). The results show that the degree of starch saccharification depended on the raw material. However, the highest yields of ethanol were obtained with 100 kg of triticale mashes (38.9 ± 1.4 L absolute alcohol) as compared to rye and wheat mashes. The concentration of dry matter (between 250 and 280 g L-1 ) in the mashes was not associated with a decrease in ethanol yield and improved efficiency in the case of wheat and triticale. CONCLUSION: Simultaneous saccharification and fermentation offers a low-cost and environmentally friendly alternative to existing procedures for industrial ethanol production, which may be of particular interest to raw-spirit producers, as well as to the food and fermentation industry at large. © 2019 Society of Chemical Industry.

Ethanol production potential from AFEX™ and steam-exploded sugarcane residues for sugarcane biorefineries.

BACKGROUND: Expanding biofuel markets are challenged by the need to meet future biofuel demands and mitigate greenhouse gas emissions, while using domestically available feedstock sustainably. In the context of the sugar industry, exploiting under-utilized cane leaf matter (CLM) in addition to surplus sugarcane bagasse as supplementary feedstock for second-generation ethanol production has the potential to improve bioenergy yields per unit land. In this study, the ethanol yields and processing bottlenecks of ammonia fibre expansion (AFEX™) and steam explosion (StEx) as adopted technologies for pretreating sugarcane bagasse and CLM were experimentally measured and compared for the first time. RESULTS: Ethanol yields between 249 and 256 kg Mg-1 raw dry biomass (RDM) were obtained with AFEX™-pretreated sugarcane bagasse and CLM after high solids loading enzymatic hydrolysis and fermentation. In contrast, StEx-pretreated sugarcane bagasse and CLM resulted in substantially lower ethanol yields that ranged between 162 and 203 kg Mg-1 RDM. The ethanol yields from StEx-treated sugarcane residues were limited by the aggregated effect of sugar degradation during pretreatment, enzyme inhibition during enzymatic hydrolysis and microbial inhibition of S. cerevisiae 424A (LNH-ST) during fermentation. However, relatively high enzyme dosages (> 20 mg g-1 glucan) were required irrespective of pretreatment method to reach 75% carbohydrate conversion, even when optimal combinations of Cellic® CTec3, Cellic® HTec3 and Pectinex Ultra-SP were used. Ethanol yields per hectare sugarcane cultivation area were estimated at 4496 and 3416 L ha-1 for biorefineries using AFEX™- or StEx-treated sugarcane residues, respectively. CONCLUSIONS: AFEX™ proved to be a more effective pretreatment method for sugarcane residues relative to StEx due to the higher fermentable sugar recovery and enzymatic hydrolysate fermentability after high solids loading enzymatic hydrolysis and fermentation by S. cerevisiae 424A (LNH-ST). The identification of auxiliary enzyme activities, adequate process integration and the use of robust xylose-fermenting ethanologens were identified as opportunities to further improve ethanol yields from AFEX™- and StEx-treated sugarcane residues.

Bioethanol Potential of Energy Sorghum Grown on Marginal and Arable Lands.

Field experiments were conducted in marginal lands, i.e., sub-humid climate and saline-land (SHS) and semi-arid climate and wasteland (SAW), to evaluate ethanol potential based on the biomass yield and chemical composition of biomass type (var. GN-2, GN-4, and GN-10) and sweet type (var. GT-3 and GT-7) hybrids of energy sorghum [Sorghum bicolor (L.) Moench] in comparison with sub-humid climate and cropland (SHC) in northern China. Results showed that environment significantly (p < 0.05) influenced plant growth, biomass yield and components, and subsequently the ethanol potential of energy sorghum. Biomass and theoretical ethanol yield of the crop grown at SHS (12.2 t ha-1 and 3,425 L ha-1, respectively) and SAW (8.6 t ha-1 and 2,091 L ha-1, respectively) were both statistically (p < 0.001) lower than values at the SHC site (32.6 t ha-1 and 11,853 L ha-1, respectively). Higher desirable contents of soluble sugar, cellulose, and hemicellulose were observed at SHS and SHC sites, while sorghum grown at SAW possessed higher lignin and ash contents. Biomass type sorghum was superior to sweet type as non-food ethanol feedstock. In particular, biomass type hybrid GN-10 achieved the highest biomass (17.4 t ha-1) and theoretical ethanol yields (5,423 L ha-1) after averaging data for all environmental sites. The most productive hybrid, biomass type GN-4, exhibited biomass and theoretical ethanol yields >42.1 t ha-1 and 14,913 L ha-1, respectively, at the cropland SHC site. In conclusion, energy sorghum grown on marginal lands showed a very lower ethanol potential, indicating a considerable lower possibility for being used as commercial feedstock supply when compared with that grown on regular croplands. Moreover, screening suitable varieties may improve energy sorghum growth and chemical properties for ethanol production on marginal lands.

Bioethanol from sugarcane bagasse: Focused on optimum of lignin content and reduction of enzyme addition.

To investigate the effect of delignification on enzymatic saccharification and ethanol fermentation of sugarcane bagasse (SCB), NaClO, NaOH, and Na2CO3 were used to prepare SCB with different lignin contents. We found that a lignin content of approximately 11% was sufficient for enzymatic saccharification and fermentation. Based on this result, an economical delignification pretreatment method using a combination of acid and alkali (CAA) was applied. Lignin content of 11.7% was obtained after CAA pretreatment with 0.5% w/v H2SO4 at 140 °C for 10 min and 1.0% w/v NaOH at 90 °C for 60 min. Presaccharification-simultaneous saccharification and fermentation (P-SSF) of the CAA-pretreated SCB resulted in an ethanol concentration of 43.8 g/L and an ethanol yield of 81.7%, with an enzyme loading of 15 FPU/g-CAA-pretreated SCB. Enzyme activities (filter paper, carboxymethyl cellulase, and ß-glucosidase activities) were determined in liquid phase during P-SSF, indicating that the residual cellulase activity could be further used. Thus, fed-batch P-SSF was carried out, and an ethanol concentration of 43.1 g/L and an ethanol yield of 80.4% were obtained with an enzyme loading of 10 FPU/g-CAA-pretreated SCB. Fed-batch P-SSF was found to be effective to reduce enzyme loading.

Effect of ultraviolet radiation on physiological and biochemical properties of yeast Saccharomyces cerevisiae during fermentation of ultradispersed starch raw material

Background: Study of correlation between pretreatment of yeast with ultraviolet radiation and efficiency of further fermentation of wort made of ultrafine grain particles to ethanol. Results: We investigated three races of industrial yeast Saccharomyces cerevisiae (native and irradiated by ultraviolet). Physiological properties during fermentation of starchy wort were tested in all variants. It was shown that activation of the yeast by ultraviolet radiation allows to further increase the ethanol yield by 25% on average compared with the native yeast races when using thin (up to micro- and nano-sized particles) or standard grain grinding. Conclusions: Using mechanical two-stage grinding of starchy raw materials and ultraviolet pretreatment of yeast, the efficiency of saccharification of starch and fermentation of wort to ethanol was increased.

Low Phenotypic Penetrance and Technological Impact of Yeast [GAR +] Prion-Like Elements on Winemaking.

[GAR +] prion-like elements partially relieve carbon catabolite repression in Saccharomyces cerevisiae. They have been hypothesized to contribute to wine yeast survival and alcohol level reduction, as well as communication with bacteria and stuck fermentation. In this work, we selected [GAR +] derivatives from several genetic backgrounds. They were characterized for phenotypic penetrance, heritability and confirmed as prion-like through curing by desiccation. In terms of fermentation kinetics, the impact of the prion on anaerobic wine fermentation (natural grape juice) was either neutral or negative, depending on the genetic background. Likewise, residual sugars were higher or similar for [GAR +] as compared to the cognate [gar -] strains. The prions had little or no impact on glycerol and ethanol yields; while acetic acid yields experienced the highest variations between [GAR +] and [gar -] strains. Strains analyzed under aerobic conditions followed the same pattern, with either little or no impact on fermentation kinetics, ethanol or glycerol yield; and a clearer influence on volatile acidity. Although no clear winemaking advantages were found for [GAR +] strains in this work, they might eventually show interest for some combinations of genetic background or winemaking conditions, e.g., for reducing acetic acid yield under aerated fermentation.

High-throughput analysis of chemical components and theoretical ethanol yield of dedicated bioenergy sorghum using dual-optimized partial least squares calibration models.

BACKGROUND: Due to its chemical composition and abundance, lignocellulosic biomass is an attractive feedstock source for global bioenergy production. However, chemical composition variations interfere with the success of any single methodology for efficient bioenergy extraction from diverse lignocellulosic biomass sources. Although chemical component distributions could guide process design, they are difficult to obtain and vary widely among lignocellulosic biomass types. Therefore, expensive and laborious "one-size-fits-all" processes are still widely used. Here, a non-destructive and rapid analytical technology, near-infrared spectroscopy (NIRS) coupled with multivariate calibration, shows promise for addressing these challenges. Recent advances in molecular spectroscopy analysis have led to methodologies for dual-optimized NIRS using sample subset partitioning and variable selection, which could significantly enhance the robustness and accuracy of partial least squares (PLS) calibration models. Using this methodology, chemical components and theoretical ethanol yield (TEY) values were determined for 70 sweet and 77 biomass sorghum samples from six sweet and six biomass sorghum varieties grown in 2013 and 2014 at two study sites in northern China. RESULTS: Chemical components and TEY of the 147 bioenergy sorghum samples were initially analyzed and compared using wet chemistry methods. Based on linear discriminant analysis, a correct classification assignment rate (either sweet or biomass type) of 99.3% was obtained using 20 principal components. Next, detailed statistical analysis demonstrated that partial optimization using sample set partitioning based on joint X-Y distances (SPXY) for sample subset partitioning enhanced the robustness and accuracy of PLS calibration models. Finally, comparisons between five dual-optimized strategies indicated that competitive adaptive reweighted sampling coupled with the SPXY (CARS-SPXY) was the most efficient and effective method for improving predictive performance of PLS multivariate calibrations. CONCLUSIONS: As a dual-optimized methodology, sample subset partitioning combined with variable selection is an efficient and straightforward strategy to enhance the accuracy and robustness of NIRS models. This knowledge should facilitate generation of improved lignocellulosic biomass feedstocks for bioethanol production. Moreover, methods described here should have wider applicability for use with feedstocks incorporating multispecies biomass resource streams.

Adaptation to low pH and lignocellulosic inhibitors resulting in ethanolic fermentation and growth of Saccharomyces cerevisiae.

Lignocellulosic bioethanol from renewable feedstocks using Saccharomyces cerevisiae is a promising alternative to fossil fuels owing to environmental challenges. S. cerevisiae is frequently challenged by bacterial contamination and a combination of lignocellulosic inhibitors formed during the pre-treatment, in terms of growth, ethanol yield and productivity. We investigated the phenotypic robustness of a brewing yeast strain TMB3500 and its ability to adapt to low pH thereby preventing bacterial contamination along with lignocellulosic inhibitors by short-term adaptation and adaptive lab evolution (ALE). The short-term adaptation strategy was used to investigate the inherent ability of strain TMB3500 to activate a robust phenotype involving pre-culturing yeast cells in defined medium with lignocellulosic inhibitors at pH 5.0 until late exponential phase prior to inoculating them in defined media with the same inhibitor cocktail at pH 3.7. Adapted cells were able to grow aerobically, ferment anaerobically (glucose exhaustion by 19 ± 5 h to yield 0.45 ± 0.01 g ethanol g glucose(-1)) and portray significant detoxification of inhibitors at pH 3.7, when compared to non-adapted cells. ALE was performed to investigate whether a stable strain could be developed to grow and ferment at low pH with lignocellulosic inhibitors in a continuous suspension culture. Though a robust population was obtained after 3600 h with an ability to grow and ferment at pH 3.7 with inhibitors, inhibitor robustness was not stable as indicated by the characterisation of the evolved culture possibly due to phenotypic plasticity. With further research, this short-term adaptation and low pH strategy could be successfully applied in lignocellulosic ethanol plants to prevent bacterial contamination.

Non-conventional Yeast Species for Lowering Ethanol Content of Wines.

Rising sugar content in grape must, and the concomitant increase in alcohol levels in wine, are some of the main challenges affecting the winemaking industry nowadays. Among the several alternative solutions currently under study, the use of non-conventional yeasts during fermentation holds good promise for contributing to relieve this problem. Non-Saccharomyces wine yeast species comprise a high number or species, so encompassing a wider physiological diversity than Saccharomyces cerevisiae. Indeed, the current oenological interest of these microorganisms was initially triggered by their potential positive contribution to the sensorial complexity of quality wines, through the production of aroma and other sensory-active compounds. This diversity also involves ethanol yield on sugar, one of the most invariant metabolic traits of S. cerevisiae. This review gathers recent research on non-Saccharomyces yeasts, aiming to produce wines with lower alcohol content than those from pure Saccharomyces starters. Critical aspects discussed include the selection of suitable yeast strains (considering there is a noticeable intra-species diversity for ethanol yield, as shown for other fermentation traits), identification of key environmental parameters influencing ethanol yields (including the use of controlled oxygenation conditions), and managing mixed fermentations, by either the sequential or simultaneous inoculation of S. cerevisiae and non-Saccharomyces starter cultures. The feasibility, at the industrial level, of using non-Saccharomyces yeasts for reducing alcohol levels in wine will require an improved understanding of the metabolism of these alternative yeast species, as well as of the interactions between different yeast starters during the fermentation of grape must.

Hemicellulosic ethanol production by immobilized cells of Scheffersomyces stipitis: effect of cell concentration and stirring.

Bioconversion of hemicellulosic hydrolysate into ethanol plays a pivotal role in the overall success of biorefineries. For the efficient fermentative conversion of hemicellulosic hydrolysates into ethanol, the use of immobilized cells system could provide the enhanced ethanol productivities with significant time savings. Here, we investigated the effect of 2 important factors (e.g., cell concentration and stirring) on ethanol production from sugarcane bagasse hydrolysate using the yeast Scheffersomyces stipitis immobilized in calcium alginate matrix. A 2(2) full factorial design of experiment was performed considering the process variables- immobilized cell concentration (3.0, 6.5 and 10.0 g/L) and stirring (100, 200 and 300 rpm). Statistical analysis showed that stirring has the major influence on ethanol production. Maximum ethanol production (8.90 g/l) with ethanol yield (Yp/s) of 0.33 g/g and ethanol productivity (Qp) of 0.185 g/l/h was obtained under the optimized process conditions (10.0 g/L of cells and 100 rpm).

Effect of environment and variety on the relationships of wheat grain physical and chemical characteristics with ethanol yield.

BACKGROUND: Following the Renewable Transport Fuel Obligation (RTFO), there is an increasing demand for wheat grain for liquid biofuel in the UK. In order to enhance productivity of the bioethanol industry, good quality wheat must be used. RESULTS: A total of 84 grain samples comprising 14 varieties collected from 11 sites in two harvest years were analysed for a range of grain quality parameters and ethanol yield (EY). The grain quality parameters studied were starch and protein concentration, specific weight, grain density, packing efficiency, thousand-grain weight (TGW), grain length, width, length/width ratio and hardness index. Regression analysis was used to establish the relationships between grain quality parameters and EY. Apart from grain length and density, all grain parameters had significant relationships with EY. In the order of importance, protein concentration, TGW, packing efficiency and specific weight showed good relationships with EY. All other parameters, including starch concentration, showed a poor correlation with EY. EY and the relationship with the grain parameters were affected more by environment than by variety. Some sites gave consistently higher EY than others. When site and variety were considered with TGW and protein, a good prediction of EY could be made (variance accounted for = 87%). CONCLUSION: Combining TGW and protein concentration could be a better indicator of EY than the current practice of specific weight and protein.

Strategy for adapting wine yeasts for bioethanol production.

The Saccharomyces cerevisiae wine yeast strains 71B-1122 and K1-V1116 were used to derive strains that could tolerate and produce higher ethanol yields. Respiratory-deficient mutants resistant to 500 microg/mL lycorine were isolated. Two mutants, 71B-1122 YEBr L3 and K1-V1116 YEBr L4, were shown to achieve about 10% and 18% improvement in their glucose-to-ethanol conversion efficiency compared to their respective parent strains. The K1-V1116 YEBr L4 in particular can tolerate an ethanol yield of 18.8 +/- 0.8% at 3.5 weeks of fermentation and continued to consume most of the sugar until less than 1% glucose was left.