Use of whole-slurry from autohydrolyzed Eucalyptus wood for bioethanol production

The development of a cost-effective process on large-scale is one of the most important targets in the second generation bioethanol. The use of pretreated whole-slurry allows savings in washing-steps and water consumption. In this work the whole-slurry from pretreated Eucalyptus wood (EW) was used for the bioethanol production by saccharification and fermentation process. Firstly, EW was submitted to autohydrolysis treatment and the slurry obtained was employed for the optimization of enzymatic saccharification using an experimental design. The optimized conditions were employed for bioethanol production using a robust industrial Saccharomyces cerevisiae strain. The highest ethanol concentration obtained was 50 g/L corresponding to an ethanol conversion of 95 %

Genetic engineering approaches for enhanced lignocellulosic-based bioprocesses

Book of Abstracts of CEB Annual Meeting 2017Lignocellulosic biomass is the most abundant, low-cost, bio-renewable resource. It has a recognised potential as a sustainable platform for the production of biofuels and other bio-chemicals. To improve the accessibility of the cellulose component from complex lignocellulosic structures to the enzymes, a pretreatment step is necessary. Enzymatic saccharification of resulting whole slurry is highly desirable as it avoids the solid-liquid separation step, the need for detoxification and related waste disposal problem, and increases final sugar concentration. However, lignin residues and other inhibitory compounds resulting from pretreatment negatively affect the digestibility of the whole slurry and compromise fermentation efficiency. To tackle these pitfalls, genetic engineering strategies have been developed and integrated in the process to improve both stages. For improving the fermentation efficiency, our approach has been to intensify the process by using high solid loadings and both pentose and hexoses fractions, enriching sugar concentration available for fermentation. To work under such demanding conditions robust yeast strains are crucial. We have selected natural robust yeast isolates and identified key genes necessary for yeast growth and maximal fermentation rate in hydrolysates. Selected robust yeast chassis have been metabolic engineered for cofermentation of glucose and xylose from hemicellulose fraction using a novel metabolic assembly tool and key tolerance genes expression has been simultaneously evaluated for the valorization of biomass of different origins. Results obtained pointed to the importance of designing from the very beginning a tailor-made yeast considering the specific raw material and process [1]. The flexibility of the metabolic assembly tool developed and the selected robust yeast backgrounds envisioned the developing of effective yeast platforms for biomass processing into different products. For improving the saccharification of whole slurry, our strategy has been to use the efficient recombinant protein production system from Escherichia coli to produce hydrolysis enhancers, namely a family 3 carbohydrate-binding module (CBM3). The purified CBM3 was used as an additive in the enzymatic hydrolysis of the whole slurry from hydrothermally-pretreated Eucalyptus globulus wood among other biotechnological applications [2]. The results obtained show an increase in glucose yield when CBM3 was added, compensating the negative effect of inhibitors on the enzymatic efficiency of whole slurry saccharification. Thus, CBM3 is a valid additive for enhanced lignocellulosics saccharification and a valuable alternative to costly additives (e.g. BSA) as it can be affordably obtained from heterologous bacterium or integrated in the developed yeast platforms, thus contributing to more cost-efficient and environmental-friendly biomass conversion bioprocesses.info:eu-repo/semantics/publishedVersio

Effects of mechanically separated dairy cow slurry on grazing performance

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Integrated approach for effective bioethanol production using whole slurry from autohydrolyzed Eucalyptus globulus wood at high-solid loadings

One of the most important targets and challenges in the second generation bioethanol is the development of a cost-effective process on large-scale. In this context, the high solid loading on saccharification and fermentation and the use of whole-slurry from pretreatment could be promising alternatives to obtain high ethanol concentrations and to decrease operational costs and wastewater. In this work, Eucalyptus globulus wood was submitted to non-isothermal autohydrolysis treatment (Tmax = 210 °C) and the whole-slurry obtained was assayed for the optimization of enzymatic saccharification at different solid and enzymes (CTec2 and HTec2) loadings using a Box–Behnken experimental design. Under the optimized conditions (liquid solid ratio 6.4 g/g, cellulase to substrate ratio 22.5 FPU/g and hemicellulase to substrate ratio 500 UI/g), two strategies were evaluated for ethanol production (Simultaneous Saccharification and Fermentation, SSF and Presaccharification and Simultaneous Saccharification and Fermentation, PSSF), using an industrial and robust Saccharomyces cerevisiae strain. High concentrations of ethanol (50.2 and 48.8 g/L) and productivities (0.63 and 0.55 g/L h) were obtained by SSF and PSSF, respectively. The SSF process proved to be an advantageous strategy to obtain concentrations >6% (v/v) of ethanol with elevated conversion (95%) even employing high solid loading and non-detoxified hydrolysate. Following an integrated optimization process, cost-effective bioethanol production conditions from whole-slurry E. globulus wood were determined and validated experimentally, representing a step-forward towards its industrial implementation.The authors A. Roman and F.B. Pereira thank to the "Fundacao para a Ciencia e a Techologia" (FCT, Portugal) for their fellowships (grant number, SFRH/BPD/77995/2011 and SFRH/BD/64776/2009, respectively) and Gil Garrote (University of Vigo, Spain) for assistance in the pre-treatment of EGW. Research described in this article was financially supported by FEDER and FCT: Strategic Project PEst-OE/EQB/LA0023/2013, Project "BioInd-Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028'' Co-funded by the Programa Operacional Regional do Norte (ON. 2 - O Novo Norte) QREN, FEDER

Comparative performance of precommercial cellulases hydrolyzing pretreated corn stover

<p>Abstract</p> <p>Background</p> <p>Cellulases and related hydrolytic enzymes represent a key cost factor for biochemical conversion of cellulosic biomass feedstocks to sugars for biofuels and chemicals production. The US Department of Energy (DOE) is cost sharing projects to decrease the cost of enzymes for biomass saccharification. The performance of benchmark cellulase preparations produced by Danisco, DSM, Novozymes and Verenium to convert pretreated corn stover (PCS) cellulose to glucose was evaluated under common experimental conditions and is reported here in a non-attributed manner.</p> <p>Results</p> <p>Two hydrolysis modes were examined, enzymatic hydrolysis (EH) of PCS whole slurry or washed PCS solids at pH 5 and 50°C, and simultaneous saccharification and fermentation (SSF) of washed PCS solids at pH 5 and 38°C. Enzymes were dosed on a total protein mass basis, with protein quantified using both the bicinchoninic acid (BCA) assay and the Bradford assay. Substantial differences were observed in absolute cellulose to glucose conversion performance levels under the conditions tested. Higher cellulose conversion yields were obtained using washed solids compared to whole slurry, and estimated enzyme protein dosages required to achieve a particular cellulose conversion to glucose yield were extremely dependent on the protein assay used. All four enzyme systems achieved glucose yields of 90% of theoretical or higher in SSF mode. Glucose yields were reduced in EH mode, with all enzymes achieving glucose yields of at least 85% of theoretical on washed PCS solids and 75% in PCS whole slurry. One of the enzyme systems ('enzyme B') exhibited the best overall performance. However in attaining high conversion yields at lower total enzyme protein loadings, the relative and rank ordered performance of the enzyme systems varied significantly depending upon which hydrolysis mode and protein assay were used as the basis for comparison.</p> <p>Conclusions</p> <p>This study provides extensive information about the performance of four precommercial cellulase preparations. Though test conditions were not necessarily optimal for some of the enzymes, all were able to effectively saccharify PCS cellulose. Large differences in the estimated enzyme dosage requirements depending on the assay used to measure protein concentration highlight the need for better consensus methods to quantify enzyme protein.</p

Bioethanol production from whole slurry of hydrothermally treated brewer´s spent grain at high solid loadings

Portuguese Foundation for Science and Technology (FCT) under the scope of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Authors acknowledge Fermentum-Engenharia das Fermentações Lda. for providing the raw materials usedinfo:eu-repo/semantics/publishedVersio

Effect of hemicellulose liquid phase on the enzymatic hydrolysis of autohydrolyzed Eucalyptus globulus wood

In this work, Eucalyptus globulus wood was pretreated under non-isothermal autohydrolysis process at 210, 220, and 230 °C, obtaining a pretreated solid with high cellulose content and a hemicellulosic liquid phase (HLP) containing mainly xylose, acetic acid, furfural, xylooligosaccharides, and phenolic compounds. The maximum concentration of xylooligosaccharides (8.97 g/L) and phenolic compounds (2.66 g/L) was obtained at 210 and 230 °C, respectively. To evaluate the effect of HLP addition on the enzymatic hydrolysis using unwashed pretreated solid as substrate, different proportions of HLP were studied. Also, in order to use the whole slurry on enzymatic hydrolysis, the supplementation of xylanases was evaluated. Glucose concentration of 107.49 g/L (corresponding to 74.65 % of conversion) was obtained using pretreated solid at 220 °C liquid/solid ratio (LSR) of 4 g/g and enzyme solid ratio (ESR) of 25 FPU/gwithout the addition of HLP. Thus, it was shown that the unwashed pretreated solids are susceptible to enzymatic hydrolysis contributing to reduce operational cost (water consumption). Additionally, the influence of the inhibitory compounds in the HLP was shown to affect the enzymatic hydrolysis. Results indicated that 82.52 g/L of glucose (59.37 % of conversion) was obtained, using 100 % of HLP at LSR of 4 g/g and ESR of 16 FPU/g at 210 °C of pretreated solid. However, a positive effect was shown on the enzymatic hydrolysis when the xylanases were added using 100 % of HLP, increasing to 35 and 27 % in the glucose production with respect to whole slurry without addition of xylanases.The authors A. Romani and F. B. Pereira thank to the Portuguese Foundation for Science and Technology (FCT, Portugal) for their fellowships (grant number, SFRH/BPD/77995/2011 and SFRH/BD/64776/2009, respectively)

Quantification of priming and CO2 emission sources following the application of different slurry particle size fractions to a grassland soil

The highest emissions of CO2 from soils and most pronounced priming effect (PE) from soils generally occur immediately after slurry application. However, the influence of different particle size slurry fractions on net soil C respiration dynamics and PE has not been studied. Therefore, a slurry separation technique based on particle sizes was used in the present study. Six distinct fractions (>2000, 425–2000, 250–425, 150–250, 45–150, 250 μm fractions. The overall contribution of slurry C to total CO2 emissions was higher in smaller slurry particle size treatments in the first days after application. The addition of the various slurry fractions to soil caused both significant positive and negative PEs on the soil organic matter mineralization. The timing and type (positive or negative) of PE depended on the slurry particle size. Clearly, farm based separation pre-treatment leading to two or more fractions with different particle sizes has also the potential to reduce or modify short-term CO2 emissions immediately after slurry application to soil

Recombinant family 3 carbohydrate-binding module as a new additive for enhanced enzymatic saccharification of whole slurry from autohydrolyzed eucalyptus globulus wood

By-products resulting from lignocellulosics pretreatment affect the digestibility of resulting whole slurries, but this can be minimized by additives supplementation. In this work, a family 3 carbohydrate-binding module (CBM3), recombinantly produced from Escherichia coli, was used as additive in the enzymatic hydrolysis of the whole slurry from autohydrolyzed Eucalyptus globulus wood (EGW). At the higher dosage used (30 mg/gsolids), CBM3 led to an increase in glucose yield from 75 to 89%. A similar result was obtained for bovine serum albumin (BSA) (11% increase), which has a well-documented additive effect. CBM3 had no effect on the non-productive binding of enzymes, since it could not bind to EGW lignin, while it rapidly bound to cellulose, as shown by fluorescence microscopy. CBM3 is a valid additive for enhanced lignocellulosic saccharification and a valuable alternative to costly additives (e.g. polyethylene glycol) as it can be affordably produced from heterologous bacterium, thus contributing to more cost-efficient biomass valorization bioprocesses.This work was developed under the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. The research leading to the reported results has received funding from Fundação para a Ciência e a Tecnologia (FCT) through the project MultiBiorefinery (POCI-01–0145-FEDER-016403) and through grants to C. Oliveira (SFRH/BPD/110640/2015) and D. Gomes (SFRH/BD/88623/2012).info:eu-repo/semantics/publishedVersio

Steam Explosion Pretreatment of Beechwood. Part 1: Comparison of the Enzymatic Hydrolysis of Washed Solids and Whole Pretreatment Slurry at Different Solid Loadings

Steam explosion is a well-known process to pretreat lignocellulosic biomass in order to enhance sugar yields in enzymatic hydrolysis, but pretreatment conditions have to be optimized individually for each material. In this study, we investigated how the results of a pretreatment optimization procedure are influenced by the chosen reaction conditions in the enzymatic hydrolysis. Beechwood was pretreated by steam explosion and the resulting biomass was subjected to enzymatic hydrolysis at glucan loadings of 1% and 5% employing either washed solids or the whole pretreatment slurry. For enzymatic hydrolysis in both reaction modes at a glucan loading of 1%, the glucose yields markedly increased with increasing severity and with increasing pretreatment temperature at identical severities and maximal values were reached at a pretreatment temperature of 230 °C. However, the optimal severity was 5.0 for washed solids enzymatic hydrolysis, but only 4.75 for whole slurry enzymatic hydrolysis. When the glucan loading was increased to 5%, glucose yields hardly increased for pretreatment temperatures between 210 and 230 °C at a given severity, and a pretreatment temperature of 220 °C was sufficient under these conditions. Consequently, it is important to precisely choose the desired conditions of the enzymatic hydrolysis reaction, when aiming to optimize the pretreatment conditions for a certain biomass