Search

Trees constitute promising renewable feedstocks for biorefinery using biochemical conversion, but their recalcitrance restricts their attractiveness for the industry. To obtain trees with reduced recalcitrance, large-scale genetic engineering experiments were performed in hybrid aspen blindly targeting genes expressed during wood formation and 32 lines representing seven constructs were selected for characterization in the field. Here we report phenotypes of five-year old trees considering 49 traits related to growth and wood properties. The best performing construct considering growth and glucose yield in saccharification with acid pretreatment had suppressed expression of the gene encoding an uncharacterized 2-oxoglutarate-dependent dioxygenase (2OGD). It showed minor changes in wood chemistry but increased nanoporosity and glucose conversion. Suppressed levels of SUCROSE SYNTHASE, (SuSy), CINNAMATE 4-HYDROXYLASE (C4H) and increased levels of GTPase activating protein for ADP-ribosylation factor ZAC led to significant growth reductions and anatomical abnormalities. However, C4H and SuSy constructs greatly improved glucose yields in saccharification without and with pretreatment, respectively. Traits associated with high glucose yields were different for saccharification with and without pretreatment. While carbohydrates, phenolics and tension wood contents positively impacted the yields without pretreatment and growth, lignin content and S/G ratio were negative factors, the yields with pretreatment positively correlated with S lignin and negatively with carbohydrate contents. The genotypes with high glucose yields had increased nanoporosity and mGlcA/Xyl ratio, and some had shorter polymers extractable with subcritical water compared to wild-type. The pilot-scale industrial-like pretreatment of best-performing 2OGD construct confirmed its superior sugar yields, supporting our strategy.

The extraction of bioactive compounds and cellulose saccharification are potential directions for the valorization of spent mushroom substrate (SMS). Therefore, investigating the suitability of different extraction methods for recovering bioactive compounds from SMS and how the extraction affects the enzymatic saccharification is of uppermost relevance. In this work, bioactive compounds were extracted from Pleurotus spp. SMS using four extraction methods. For Soxhlet extraction (SoE), a 40:60 ethanol/water mixture gave the highest extraction efficiency (EE) (69.9–71.1%) among the seven solvent systems assayed. Reflux extraction with 40:60 ethanol/water increased the extraction yield and EE compared to SoE. A shorter reflux time yielded a higher extraction of carbohydrates than SoE, while a longer time was more effective for extracting phenolics. The extracts from 240 min of reflux had comparable antioxidant activity (0.3–0.5 mM GAE) with that achieved for SoE. Ultrasound-assisted extraction (UAE) at 65 °C for 60 min allowed an EE (~82%) higher than that achieved by either reflux for up to 150 min or SoE. Subcritical water extraction (SWE) at 150 °C resulted in the best extraction parameters among all the tested methods. Vanillic acid and chlorogenic acid were the primary phenolic acids identified in the extracts. A good correlation between the concentration of caffeic acid and the antioxidant activity of the extracts was found. Saccharification tests revealed an enhancement of the enzymatic digestibility of SMS cellulose after the extraction of bioactive compounds. The findings of this initial study provide indications on new research directions for maximizing the recovery of bioactive compounds and fermentable sugars from SMS.

This study aimed to explore the viability of converting potato peel waste into bioethanol using a custom-produced multi-enzyme preparation. Various pretreatment approaches were employed on the potato peels, including thermal, chemical, and thermo-chemical methods. These methods involved boiling for 30 and 60 min, steaming at different pressures and durations, and applying different concentrations of chemicals, including H2SO4, HNO3, CH3COOH, HCl, NaOH, Ca(OH)2, KOH, NH3, and H2O2, either individually or in combination with steam treatment. The pretreated potato peels were subsequently subjected to enzymatic hydrolysis using a crude multi-enzyme cocktail obtained from solid-state fermentation of wheat bran by a naturally occurring strain of Aspergillus niger P-19. This enzyme cocktail consisted of cellulases, hemicellulases, pectinase, and amylases. The most effective pretreatment combination involved the use of 3% H2SO4 followed by steam treatment under pressure, and enzymatic hydrolysis utilizing the crude multi-enzyme preparation. This combination resulted in the highest concentration of reducing sugars (141.04 ± 12.31 g/L), with a carbohydrate conversion rate of 98.49% when a substrate loading of 20% was used. As a result, an ethanol yield of 43.2 ± 3.82 g/L, representing 21.6% based on dry matter, was achieved. Furthermore, supplementing the medium with peptone, (NH4)(H2PO4), and ZnSO4 at a concentration of 0.1% w/v each, along with solid loadings of 22% and 24%, respectively, achieved yield improvements of 51.67 g/L and 54.75 g/L. However, the maximum productivity of 23.4% was observed with a 22% loading, compared to a yield of 22.8% with a 24% solid loading, based on dry matter.

The synthesis of ideal, well-defined, functionalized lignins is of great significance for realizing the full valorization potential of lignocellulosic biomass. Here, the application of acidified triethylammonium triflate is reported, allowing for the selective benzylic alkoxylation of different lignins at room temperature. Full benzylic alkoxylation of typically hard-to-process, high-molecular-weight lignin was achieved in this medium by simple mixing with different alcohols. Limited lignin degradation was observed, and lignin modification was also achieved within the lignocellulosic matrix. This allows the method to be used as a biomass pretreatment, which was shown to also enhance enzymatic saccharification. The reactivity, stability, and hydrophobicity of the lignin can be tuned based on the alcohol used. Effective recovery of the ionic liquid and recycling of the ionic liquid was achieved. Thus, a straightforward mild method is demonstrated to introduce new functionality in lignin, allowing it to be tuned towards novel applications.

The role of lignin in enzymatic saccharification of cellulose involving lytic polysaccharide monooxygenase (LPMO) was investigated in experiments with the solid and liquid fractions of pretreated Norway spruce from a biorefinery demonstration plant using hydrothermal pretreatment and impregnation with sulfur dioxide. Pretreated biomass before and after enzymatic saccharification was characterized using HPAEC, HPLC, Py-GC/MS, 2D-HSQC NMR, FTIR, and SEM. Chemical characterization indicated that relatively harsh pretreatment conditions resulted in that the solid phase contained no or very little hemicellulose but considerable amounts of pseudo-lignin, and that the liquid phase contained a relatively high concentration (∼5 g/L) of lignin-derived phenolics. As judged from reactions continuously supplied with either air or nitrogen gas, lignin and lignin fragments from both the solid and the liquid phases efficiently served as reductants in LPMO-supported saccharification. When air was used to promote LPMO activity, the enzymatic conversion of cellulose after 72 h was 25% higher in reactions with pretreated solids and buffer, and 14% higher in reactions with pretreatment liquid and microcrystalline cellulose. Research in this area is useful for designing efficient saccharification steps in biochemical conversion of lignocellulosic biomass.

Dans un contexte de transition énergétique, l’utilisation de la biomasse lignocellulosique comme source d’énergie renouvelable pour la production de biocarburants a le potentiel de diminuer notre dépendance vis-à-vis des énergies fossiles polluantes et de réduire de façon radicale notre empreinte carbone. Autre que les résidus forestiers et agricoles; la biomasse lignocellulosique peut provenir des cultures énergétiques dédiées à base de plantes à croissance rapide comme les saules (Salix spp.). En effet, ces plantes ligneuses sont faciles à mettre en place et produisent de hauts rendements pendant plusieurs années. L’eau et les nutriments (essentiellement l’azote) étant cependant les principaux facteurs limitants à leur développement. L'utilisation des effluents municipaux constituent une alternative intéressante à l'irrigation conventionnelle et à la fertilisation par des engrais chimiques dont la synthèse même contribue à l’augmentation des gaz à effet de serre. En effet, les eaux usées qu’elles soient partiellement et /ou totalement traitées contiennent encore de grandes quantités d’azote et de phosphore qui peuvent être captées par les saules améliorant d’autant leur croissance et leur productivité. La phytofiltration; c.-à-d. l’utilisation de filtre végétal est donc un concept séduisant; permettant d’une part de solutionner des problématiques sociétales et environnementales telles que la gestion des grands volumes d’effluents organiques et la préservation des milieux naturels et d’autre part de valoriser les contaminants qui sont en solution dans les effluents pour promouvoir le développement des plantes et en augmenter les rendements de la biomasse. Cette biomasse lignocellulosique pourrait aussi servir de matière première pour la production de biocarburant, néanmoins, il est important que l’emphase soit mise non seulement sur les quantités des bois produits, mais aussi sur leur qualité. En effet, lors de la production de biocarburant lignocellulosique, l’efficacité de la conversion biochimique d’une biomasse donnée est étroitement liée à la composition chimique de cette dernière. L’objectif de cette thèse est de déterminer l’effet de l’irrigation par des effluents organiques sur la structure anatomique et la composition chimique du bois de divers génotypes de saules et de comprendre les répercussions d’une altération du bois, si elles existent, sur la récalcitrance du bois à l’hydrolyse enzymatique. Une première étude a été conduite sur une plantation de saules Salix miyabeana ‘SX67’. Le but de cette étude était de déterminer l’effet à moyen terme de l’irrigation par des eaux usées avec traitement primaire (et donc ayant gardé une forte portion de sa charge organique et azotée) en premier lieu sur la physiologie et ensuite sur les caractéristiques intrinsèques du bois telles que sa composition chimique ou encore la structure hydraulique et mécanique. Dans un premier temps, les résultats ont montré que la fertigation a altéré la morphologie des feuilles ainsi que les stratégies d’utilisation du carbone ce qui a permis de maximiser la surface photosynthétique et d’améliorer le rendement quantique par unité de surface foliaire. Aussi, des analyses histologiques ont permis de déceler une élongation de la longueur des stomates chez les plantes fertiguées, engendrant une augmentation de la conductance stomatique et inévitablement celle du taux de carbone assimilé (la photosynthèse) permettant ainsi de promouvoir la croissance et le développement des plantes irriguées par les eaux usées et d’en améliorer la productivité. Dans un second temps, les analyses chimiques du bois du cultivar Salix miyabeana ‘SX67’ ont montré que pour les plantes qui ont été irriguées par des eaux usées, il y a eu une diminution de la fraction des extractibles ainsi qu’une augmentation de la fraction cellulose sans toutefois que cela soit accompagné d’une diminution de la lignine. Les analyses histologiques ont aussi dévoilé une différence de la densité des vaisseaux et de leur diamètre entre les plantes fertiguées et les témoins, toutefois, il ne peut être exclu que cette différence soit en partie occasionnée et/ ou accentuée par la méthode d’échantillonnage qui cible des régions différentes de la plante. Dans une deuxième étude, la biomasse issue de trois expériences distinctes, réalisées au Canada (Beaverlodge et Whitecourt) et en Grande-Bretagne (Hillsborough) et dans lesquelles différents cultivars de saules ont été irrigués durant trois années de croissance par différents effluents organiques (des eaux usées avec traitement secondaire et des effluents de laiterie non traités), a été analysée afin d’étudier les conséquences de la fertigation sur la structure anatomique et la composition chimique du bois ainsi que sur sa récalcitrance à la saccharification enzymatique. Pour tous les sites, les résultats n’ont pas montré de différence pour les variables anatomiques et la densité du bois entre les plantes témoins et celles fertiguées , suggérant que ces cultivars étaient résilients à l’application d’effluents organiques. Cependant la fertigation a eu un effet sur la composition chimique et la récalcitrance du bois. La réponse des plantes a varié en fonction du site avec pas d’effets pour les cultivars de Beaverlodge, une diminution du taux d’extractibles et une augmentation des rendements en sucre pour certains cultivars de Whitecourt et une augmentation du contenu en xylose et une diminution des sucres libérées pour la plupart des génotypes de Hillsborough. Ces différences dans la réponse peuvent suggérer que des facteurs spécifiques aux sites tels que les conditions environnementales et la composition des eaux usées pourraient avoir influencé la composition chimique du bois ainsi que sa récalcitrance à l’hydrolyse enzymatique. Suite à l’hydrolyse enzymatique, nous avons constaté que la récalcitrance d’une biomasse donnée n’est pas exclusivement liée à son contenu en cellulose étant donné que pour plus d’un cultivar, le rendement en glucose a été plus élevé que pour d’autres génotypes qui avaient une plus grande teneur en cellulose. Les analyses chimiques des différents cultivars ont permis de déceler une grande variabilité dans la teneur en cellulose (glucose) ainsi que dans l’indice de la récalcitrance entre les génotypes d’un même site, toutefois, la différence était plus prononcée entre les génotypes de sites différents. Aucun génotype n’étant commun aux trois sites nous ne pouvons cependant conclure clairement sur ce point qui mériterait donc des études subséquentes afin d’affiner plus encore la sélection des variétés au niveau local. Cette thèse a permis de démontrer le potentiel des saules pour filtrer de grandes quantités d’effluents tout en apportant des informations nouvelles quant aux impacts sur la composition chimique et la récalcitrance de la biomasse produite dans ces circonstances. Les travaux ici présentés pouvant être l’assise nécessaire pour une optimisation efficace de cette phytotechnologie, tant du point de vue du choix variétal que de la modulation de la fertigation en vue de la double utilisation de la plantation productrice de biomasse pour les biocarburants et en tant que filtre végétal., In the current context of transition to more sustainable energies, the use of lignocellulosic biomass as a renewable energy for biofuels production has the potential to decrease our dependence on polluting fossil fuels and drastically reduce our carbon footprint. Other than forestry and agricultural residues; lignocellulosic biomass can come from dedicated energy crops based on fast growing plants such as willows (Salix spp.). Indeed, these woody plants are easy to establish and produce high yields for several years, water and nutrients (mainly nitrogen) yet being the main plant development limiting factors. However, the use of municipal effluents is an interesting alternative to traditional irrigation and fertilization with chemical fertilizers, the very synthesis of which contributes to the increase in greenhouse gases. Indeed, wastewater, whether partially and /or fully treated, still contains large amounts of nitrogen and phosphorus which can be captured by willows, further improving their growth and productivity. Phytofiltration; i.e. the use of plant-based filters is therefore an attractive concept; allowing on one hand to solve societal and environmental problems such as the management of large volumes of organic effluents and the preservation of natural environments; on the other hand to recover contaminants to promote plant development and increase biomass yields. Then, this lignocellulosic biomass could be used for the production of biofuel. But it is now important to focus research work on wood quality rather than on quantities produced. Indeed, during the production of lignocellulosic biofuel, biochemical conversion efficiency of a given biomass is closely linked to its chemical composition. Therefore, the objective of this thesis is to determine the effect of irrigation by organic effluents on the anatomical structure and chemical composition of wood of various genotypes of willows and to understand the latter consequences on its recalcitrance to enzymatic hydrolysis. A first study was carried out on a plantation of Salix miyabeana ‘SX67’ willows. The aim of this study was to determine the medium-term effect of irrigation by wastewater with primary treatment (with still a large portion of its organic and nitrogen load) first on plant physiology and then on intrinsic characteristics of wood such as its chemical composition or its hydraulic and mechanical structure. First, the results showed that fertigation altered leaf morphology as well as carbon utilization strategies with an increased photosynthetic area and improved quantum yield per unit leaf area. Also, histological analyzes have made it possible to detect in fertigated plants stomata length elongation and therefore an increase in stomatal conductance and of the rate of assimilated carbon (photosynthesis) that promoted plant growth and development. In a second step, the chemical analyzes of the wood of the same Salix miyabeana ‘SX67’ plants showed that wastewater irrigation decreased the fraction of extractables as well as increased cellulose content, no change was observed for lignin fraction. Histological analyzes also revealed a difference in the vessel density and diameter between fertigated plants and controls, however it cannot be excluded that this difference is partly caused and /or accentuated by the sampling method chosen (region of the plant where the samples were taken). In a second study, the biomass resulting from three separate experiments carried out in Canada (Beaverlodge and Whitecourt) and in Great Britain (Hillsborough) where different cultivars of willows were irrigated for three years by different organic effluents (wastewater with secondary treatment and primary dairy farm wastewater) was analyzed in order to study the consequences of fertigation on the anatomical structure and the chemical composition of wood as well as its recalcitrance to enzymatic saccharification. For all sites, the results showed no difference between control and fertigated irrigated plants neither for anatomical variables nor for wood density, suggesting that these cultivars would have been resilient to the application of organic effluent. Regarding the effect of fertigation on wood chemical composition and recalcitrance, the plants response varied between sites. No effects was observed for Beaverlodge cultivars but we measured a decrease in extractables content and an increase of sugar yields for some cultivars of Whitecourt and finally, an increase in xylose content and a decrease of the rate of sugar yields for most of Hillsborough genotypes. This difference in response between the three sites may suggest that site-specific factors such as wastewater composition but also distinct environmental conditions may have influenced the chemical composition of the wood as well as its recalcitrance to enzymatic deconstruction. Following enzymatic hydrolysis, we found that the recalcitrance of a given biomass is not exclusively related to its cellulose content since for more than one cultivar glucose yield was higher than for others which had higher cellulose content. Finally, chemical analyzes of the different cultivars made it possible to detect a great variability in the content of cellulose (glucose) as well as in the index of recalcitrance between genotypes planted on the same site, but the differences were much more pronounced between different sites. However, no genotype being common to the three sites, we cannot clearly conclude on this point, which would therefore merit subsequent studies in order to further refine the selection of varieties at the local level. This thesis allows to demonstrate the potential of willows to filter large quantities of effluents while providing new information on the impacts on the chemical composition and the recalcitrance of the biomass produced under these circumstances. The work presented here may be the necessary basis for an effective optimization of this phytotechnology, both from the point of view of varietal choice and of the modulation of fertigation in the perspective of both the plantation producing biomass for biofuels and as a cost-effective planted filter.

The Sustainable Development Goals along with national policies pave the way to a sustainable, circular, and resource efficient development model. The environmental scenario could change with the promotion of biofuels such as bioethanol. Recent research on bioethanol aspires to reduce the costs production, via the optimization of process variables and the increase in ethanol yields. This study presented a stepwise upscaling of bioethanol production from dried source-separated municipal biowaste. Three different scales (250 mL, 4 L, 100 L) were examined applying advanced ethanol production via simultaneous saccharification and fermentation. The bioprocess runs at each of the three scales and produced very similar ethanol yields, indicating excellent scalability. The validated optimum conditions at the pilot scale were 25% solids loading, Spirizyme 40 μL/g starch, NS87014 175 μL/g cellulose, and 2% S. cerevisiae. The results from the pilot trials were very successful and repeatable. Τhe mean ethanol yield was 86.60 ± 4.91%, while the structural component such as starch and cellulose were efficiently hydrolysed. The produced ethanol was recovered and purified meeting the standards of absolute ethanol, rendering it suitable for industrial uses and for biofuel use as well. Energy consumption aspects were discussed as well. Conclusively, all the stages of the value chain for source-separated biowaste valorisation (collection, treatment, added value product recovery) were successfully showcased.

Rice straw is an important feedstock for second-generation biorefineries, the majority being conventional white rice. This research investigated the potential of the straw of black rice Chakhao Poireiton, a rice variety of increasing importance, for biorefining. Pre-treatment of black rice straw was carried out in a pressurised microwave reactor and free sugar release was measured following digestion with Cellic® CTec3. This was compared to white rice straw. Pre-treatment (100 − 200° C for 5 min) did not drastically impact the sugar composition but brought about an enhanced release of glucose after enzymatic hydrolysis from 25 to 50% (black rice) and from 26 to 55% (white rice). For xylose digestibility, the increase was around seven-fold for black rice (8 to 57%) and five-fold for white rice (13 to 64%). This improvement in digestibility of the straw samples could have come from modification in the lignocellulose structural features making the polysaccharides more susceptible to hydrolysis. Black rice could be a suitable feedstock for a combined biorefinery–food and nutraceuticals from grain (much higher than white rice) and the straw for fermentable sugars comparable to white rice straw for biofuels.

Erianthus arundinaceus (ER) is greatly appreciated among domestic energy crops in Japan for the production of fermentable sugars from lignocellulosic polysaccharides. In this study, we developed an efficient Ca(OH)2-based pretreatment of both stems and leaves of ER at ambient temperature with the addition of a washing step for enzymatic saccharification. The recoveries of glucans and xylans in the pretreated ER after four countercurrent washing cycles were 91 and 76 %, respectively, the former being considerably higher than that of rice straw (RS) (72 %). Their saccharification ratios in the washed sample under the pressure of 1 atm CO2 were 80 and 92.5 %, respectively. The application of this simple sugar production process from ER would further support the domestic bioprocess development. ER is also foreseen to provide the additional feedstock favorable for harvesting from winter to spring in Japan, preventing a risk for feedstock shortage generated by single harvesting such as RS.

Many countries have their biofuel policy programs in place as part of their overall strategy to achieve sustainable development. Among biofuels, bioethanol as a promising alternative to gasoline is of substantial interest. However, there is limited availability of a sufficient quantity of bioethanol to meet demands due to bottlenecks in the present technologies to convert non-edible feedstocks, including lignocelluloses. This review article presents and critically discusses the recent advances in the pretreatment of lignocellulosic biomass, with a focus on the use of green solvents, including ionic liquids and deep eutectic solvents, followed by enzymatic saccharification using auxiliary proteins for the efficient saccharification of pretreated biomass. Different techniques used in strain improvement strategies to develop hyper-producing deregulated lignocellulolytic strains are also compared and discussed. The advanced techniques employed for fermentation of mixed sugars contained in lignocellulosic hydrolysates for maximizing bioethanol production are summarized with an emphasis on pathway and transporters engineering for xylose assimilation. Further, the integration of different steps is suggested and discussed for efficient biomass utilization and improved ethanol yields and productivity.

n this study, glucan-rich solids, and xylose-rich hydrolysates obtained from Cistus ladanifer distillery residues (CLR) were used for D-lactic acid (D-LA) production by the D-lactogenic Escherichia coli strain JU15. Firstly, hemicellulosic hydrolysates obtained by the autohydrolysis process were submitted to dilute sulfuric acid catalysed post-hydrolysis. The influence of operational conditions on oligosaccharides hydrolysis was assessed by the combined severity parameter (CS) in the range of 1.1-2.3. The optimum post-hydrolysis conditions were found for CS of 1.6 (300 mM H2SO4, 15 min, 121 degrees C). Subsequent detoxification procedures on post hydrolysed liquors were carried out, where 9.1% (w/v) powdered activated charcoal enabled a full removal of furfural, 5-hydroxymethylfurfural (HMF), and phenolic compounds together with a reduction of acetic acid (37%), and formic acid (27%). Diverse fermentation modes using detoxified and non-detoxified hydrolysates, as well using previously NaOH delignified glucan-rich solids alone (SHF or SSF) or together with pentoses liquors (SSCF) (5% loading) were performed. For all the tested conditions, both hemicellulose-and cellulose-derived sugars can be efficiently used as the carbon source to produce D-lactic acid by E. coli JU15 with a D-LA yield always surpassing 92 g(D-LA)/100 g sugars. info:eu-repo/semantics/publishedVersion

Upscaling of biomass pretreatment from laboratory scale to a bench-scale reactor is one of the important steps in the application of the pretreatment for pilot or commercial scale. This study reports the optimization of pretreatment conditions, namely reaction temperature and time, by one factor at a time (OFAT) method for the enhancement of enzymatic saccharification of oil palm empty fruit bunch (OPEFB). OPEFB was pretreated using high temperature-pressure steam reactor with different reaction temperatures (160, 170, 180, 190, 200 °C) and times (10, 20, 30, 40, 50 min). The effectiveness of the pretreatment was determined based on chemical compositions of raw OPEFB and OPEFB pulp and sugar production from enzymatic saccharification of the OPEFB pulp . Solubilized components from OPEFB, such as glucose, xylose, formic acid, acetic acid, 5-hydroxymethyl furfural (HMF), and furfural in the hydrolysate that generated during steam pretreatment were also determined. Pretreatment at 180 °C for 20 min provide s the highest sugar yields (97.30% of glucose yield per initial cellulose and 88.86% of xylose yield per initial hemicellulose). At the optimum condition, 34.9% of lignin and 30.75% of hemicellulose are successfully removed from the OPEFB and resulted in 3.43 delignification selectivity. The relationship between severity factor and by-products generated and the sugars obtained after enzymatic saccharification are discussed. The pulp of OPEFB at the optimum condition was also characterized for its morphological characteristic by scanning electron microscopy (SEM) and crystallinity by X-ray diffractometry (XRD) . These pulp characteristics are then compared with those of the raw OPEFB . The steam pretreatment cause s some fiber disruptions with more defined and opened structures and increase s the crystallinity index (CrI) by 2.9% compared to the raw OPEFB.

Photosynthetic microorganisms are considered excellent feedstock for biofuel production in developing biomass production technologies. A study was conducted to evaluate ethanol production with the sequential enzymatic saccharification and fermentation of Arthrospira platensis (Spirulina) biomass with the metabolically engineered Escherichia coli strain MS04. A. platensis was cultivated semicontinuously in an open raceway pond, and the carbohydrate content was determined to be as high as 40%. The enzymatic saccharification was designed to release the maximum amount of glucose. After 40 h of enzymatic saccharification, 27 g L−1 of monosaccharides was obtained. These slurries were fermented with ethanologenic bacteria, achieving 12.7 g L−1 ethanol after 9 h of fermentation, which corresponds to 92% conversion yield of the glucose content in the hydrolysate, 0.13 g of ethanol per 1 g of Spirulina biomass and a volumetric productivity of 1.4 g of ethanol L−1 h−1. Therefore, we conclude that it is possible, in a short time, to obtain a high ethanol yield corresponding to 160 L per ton of dry biomass with a high productivity.

One of the major concerns for utilizing ionic liquid on an industrial scale is the cost involved in the production. Despite its proven pretreatment efficiency, expenses involved in its usage hinder its utilization. A better way to tackle this limitation could be overcome by studying the recyclability of ionic liquid. The current study has applied the Box–Behnken design (BBD) to optimize the pretreatment condition of rice straw through the usage of 1-ethyl-3-methylimidazolium acetate (EMIM-Ac) as an ionic liquid. The model predicted the operation condition with 5% solid loading at 128.4 °C for 71.83 min as an optimum pretreatment condition. Under the optimized pretreatment condition, the necessity of the best anti-solvent was evaluated among water, acetone methanol, and their combinations. The study revealed that pure methanol is the suitable choice of anti-solvent, enhancing the highest sugar yield. Recyclability of EMIM-Ac coupled with anti-solvent was conducted up to five recycles following the predicted pretreatment condition. Fermentation studies evaluated the efficacy of recycled EMIM-Ac for ethanol production with 89% more ethanol production than the untreated rice straw even after five recycles. This study demonstrates the potential of recycled ionic liquid in ethanol production, thereby reducing the production cost at the industrial level.

Biochemical conversion of lignocellulosic feedstocks to advanced biofuels and other bio-based commodities typically includes physical diminution, hydrothermal pretreatment, enzymatic saccharification, and valorization of sugars and hydrolysis lignin. This approach is also known as a sugar-platform process. The goal of the pretreatment is to facilitate the ensuing enzymatic saccharification of cellulose, which is otherwise impractical due to the recalcitrance of lignocellulosic feedstocks. This review focuses on hydrothermal pretreatment in comparison to alternative pretreatment methods, biomass properties and recalcitrance, reaction conditions and chemistry of hydrothermal pretreatment, methodology for characterization of pretreatment processes and pretreated materials, and how pretreatment affects subsequent process steps, such as enzymatic saccharification and microbial fermentation. Biochemical conversion based on hydrothermal pretreatment of lignocellulosic feedstocks has emerged as a technology of high industrial relevance and as an area where advances in modern industrial biotechnology become useful for reducing environmental problems and the dependence on fossil resources.

Hydrolysis lignin, i.e., the hydrolysis residue of cellulosic ethanol plants, was extracted with the green solvent γ-valerolactone (GVL). Treatments at 170–210 ◦C were performed with either non-acidified GVL/water mixtures (NA-GVL) or with mixtures containing sulfuric acid (SA-GVL). SA-GVL treatment at 210 ◦C resulted in the highest lignin solubilization (64% (w/w) of initial content), and 76% of the solubilized mass was regenerated by water induced precipitation. Regenerated lignins were characterized through compositional analysis with sulfuric acid, as well as using pyrolysis–gas chromatography/mass spectrometry (Py-GC/MS), high-performance size-exclusion chromatography (HPSEC), solid-state cross-polarization/magic angle spinning 13C nuclear magnetic resonance (CP/MAS 13C NMR) spectroscopy, 1 H–13C heteronuclear single-quantum coherence NMR (HSQC NMR), and Fourier-transform infrared (FTIR) spectroscopy. The characterization revealed that the main difference between regenerated lignins was their molecular weight. Molecular weight averages increased with treatment temperature, and they were higher and had broader distribution for SA-GVL lignins than for NA-GVL lignins.

Brown rice is nutritionally superior to white rice, yet oil rancidity can be problematic during processing and storage regarding sensory attributes. Germinating brown rice is known to generally increase some health-promoting compounds. In response to increasing the consumption of plant-based beverages, we sprouted unstabilized brown rice, using green technologies and saccharification enzymes for value-added beverages. ‘Rondo’ paddy rice was dehulled, sorted and germinated, and beverages were produced and compared against non-germinated brown and white brewers rice beverages. The preliminary germinated brown rice beverage contained significantly higher concentrations of total lipids, diacylglycerols, triacylglycerols, free sterols, phytosterol esters and oryzanols than both non-germinated brown and white rice beverages. White rice beverages had significantly higher free fatty acids. Significant lipid losses occurred during sieving, yet novel germinated brown rice beverages contained appreciable levels of valuable health-beneficial lipids, which appeared to form natural emulsions. Further pilot plant investigations should be scaled-up for pasteurization and adjusted through emulsification to ameliorate sieving losses.

Hydrolysis lignin, i.e., the hydrolysis residue of cellulosic ethanol plants, was extracted with the green solvent γ-valerolactone (GVL). Treatments at 170–210 °C were performed with either non-acidified GVL/water mixtures (NA-GVL) or with mixtures containing sulfuric acid (SA-GVL). SA-GVL treatment at 210 °C resulted in the highest lignin solubilization (64% (w/w) of initial content), and 76% of the solubilized mass was regenerated by water-induced precipitation. Regenerated lignins were characterized through compositional analysis with sulfuric acid, as well as using pyrolysis–gas chromatography/mass spectrometry (Py-GC/MS), high-performance size-exclusion chromatography (HPSEC), solid-state cross-polarization/magic angle spinning 13C nuclear magnetic resonance (CP/MAS 13C NMR) spectroscopy, 1H–13C heteronuclear single-quantum coherence NMR (HSQC NMR), and Fourier-transform infrared (FTIR) spectroscopy. The characterization revealed that the main difference between regenerated lignins was their molecular weight. Molecular weight averages increased with treatment temperature, and they were higher and had broader distribution for SA-GVL lignins than for NA-GVL lignins.

Inhibitors formed during pretreatment impair lignocellulose bioconversion by making enzymatic saccharification and microbial fermentation less efficient, but conditioning of slurries and hydrolysates can improve fermentability and sometimes also enzymatic digestibility. Conditioning of pretreated softwood using four industrial reducing agents (sodium sulfite, sodium dithionite, sodium borohydride, and hydrogen) was compared with standard methods, such as overliming and treatment with activated charcoal. A dosage of approx. 1 mM sulfur oxyanion (sulfite or dithionite) per percent water-insoluble solids (WIS) in the slurry was found to result in good fermentability. Treatment of 10–20% WIS slurries with 15 mM sulfur oxyanion under mild reaction conditions (23°C, pH 5.5) resulted in sulfonation of the solid phase and saccharification improvements of 18–24% for dithionite and 13–16% for sulfite. Among the different conditioning methods studied, treatment of slurries with sodium sulfite was superior with respect to cost-efficient improvement of fermentability. Treatments of slurry or pretreatment liquid with 15 mM sulfite or dithionite resulted in 58–76% reduction of the content of formaldehyde. The comparison indicates that conditioning of pretreated biomass using sulfur oxyanions warrants further attention.

Auto-catalyzed hydrothermal pretreatment (A-HTP) and sulfuric-acid-catalyzed hydrothermal pretreatment (SA-HTP) were applied to quinoa stalks in order to reduce their recalcitrance towards enzymatic saccharification. Prior to pretreatment, quinoa stalks were extracted with either water or a 50:50 (v/v) ethanol–water mixture for removing saponins. Extraction with water or aqueous ethanol, respectively, led to removal of 52 and 75% (w/w) of the saponins contained in the raw material. Preliminary extraction of quinoa stalks allowed for a lower overall severity during pretreatment, and it led to an increase of glucan recovery in the pretreated solids (above 90%) compared with that of non-extracted quinoa stalks (73–74%). Furthermore, preliminary extraction resulted in enhanced hydrolysis of hemicelluloses and lower by-product formation during pretreatment. The enhancement of hemicelluloses hydrolysis by pre-extraction was more noticeable for SA-HTP than for A-HTP. As a result of the pretreatment, glucan susceptibility towards enzymatic hydrolysis was remarkably improved, and the overall conversion values were higher for the pre-extracted materials (up to 83%) than for the non-extracted ones (64–69%). Higher overall conversion was achieved for the aqueous ethanol-extracted quinoa stalks (72–83%) than for the water-extracted material (65–74%).

Banana is a major fruit crop throughout the world with abundant lignocellulose in the pseudostem and rachis residues for biofuel production. In this study, we collected a total of 11 pseudostems and rachis samples that were originally derived from different genetic types and ecological locations of banana crops and then examined largely varied edible carbohydrates (soluble sugars, starch) and lignocellulose compositions. By performing chemical (H2SO4, NaOH) and liquid hot water (LHW) pretreatments, we also found a remarkable variation in biomass enzymatic saccharification and bioethanol production among all banana samples examined. Consequently, this study identified a desirable banana (Refen1, subgroup Pisang Awak) crop containing large amounts of edible carbohydrates and completely digestible lignocellulose, which could be combined to achieve the highest bioethanol yields of 31–38% (% dry matter), compared with previously reported ones in other bioenergy crops. Chemical analysis further indicated that the cellulose CrI and lignin G-monomer should be two major recalcitrant factors affecting biomass enzymatic saccharification in banana pseudostems and rachis. Therefore, this study not only examined rich edible carbohydrates for food in the banana pseudostems but also detected digestible lignocellulose for bioethanol production in rachis tissue, providing a strategy applicable for genetic breeding and biomass processing in banana crops.

Background Salix (willow) species represent an important source of bioenergy and offer great potential for producing biofuels. Salix spp. like many hardwoods, produce tension wood (TW) characterized by special fibres (G-fibres) that produce a cellulose-rich lignin-free gelatinous (G) layer on the inner fibre cell wall. Presence of increased amounts of TW and G-fibres represents an increased source of cellulose. In the present study, the presence of TW in whole stems of different Salix varieties was characterized (i.e., physical measurements, histochemistry, image analysis, and microscopy) as a possible marker for the availability of freely available cellulose and potential for releasing d-glucose. Stem cross sections from different Salix varieties (Tora, Björn) were characterized for TW, and subjected to cellulase hydrolysis with the free d-glucose produced determined using a glucose oxidase/peroxidase (GOPOD) assay. Effect of cellulase on the cross sections and progressive hydrolysis of the G-layer was followed using light microscopy after staining and scanning electron microscopy (SEM). Results Tension wood fibres with G-layers were developed multilaterally in all stems studied. Salix TW from varieties Tora and Björn showed fibre G-layers were non-lignified with variable thickness. Results showed: (i) Differences in total % TW at different stem heights; (ii) that using a 3-day incubation period at 50 °C, the G-layers could be hydrolyzed with no apparent ultrastructural effects on lignified secondary cell wall layers and middle lamellae of other cell elements; and (iii) that by correlating the amount of d-glucose produced from cross sections at different stem heights together with total % TW and density, an estimate of the total free d-glucose in stems can be derived and compared between varieties. These values were used together with a literature value (45%) for estimating the contribution played by G-layer cellulose to the total cellulose content. Conclusions The stem section-enzyme method developed provides a viable approach to compare different Salix varieties ability to produce TW and thus freely available d-glucose for fermentation and biofuel production. The use of Salix stem cross sections rather than comminuted biomass allows direct correlation between tissue- and cell types with d-glucose release. Results allowed correlation between % TW in cross sections and entire Salix stems with d-glucose production from digested G-layers. Results further emphasize the importance of TW and G-fibre cellulose as an important marker for enhanced d-glucose release in Salix varieties.

Background Pretreatment is a critical step required for efficient conversion of woody biomass into biofuels and platform chemicals. Fungal pretreatment is regarded as one of the most promising technology for woody biomass conversion but remains challenging for industrial application. The exploration of potential fungus strain with high efficient delignification and less processing time for woody biomass pretreatment will be valuable for development of biorefinery industry. Here, a newly isolated white-rot basidiomycete Peniophora incarnate T-7 was employed for poplar wood pretreatment. Results The chemical component analysis showed that cellulose, hemicellulose and lignin from poplar wood declined by 16%, 48% and 70%, respectively, after 7 days submerged fermentation by P. incarnate T-7. Enzymatic saccharification analysis revealed that the maximum yields of glucose and xylose from 7 days of P. incarnate T-7 treated poplar wood reached 33.4% and 27.6%, respectively, both of which were enhanced by sevenfold relative to the untreated group. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), X-ray diffraction (XRD) and pyrolysis gas chromatography–mass spectrometry (Py-GC/MS) characterization confirmed that lignocellulosic structure of poplar wood was largely broken by P. incarnate T-7, including delignification and de-crystalline of cellulose. Meanwhile, lignin component of poplar wood was selectively degraded by P. incarnate T-7, and G-type unit of lignin was preferentially attacked by the strain. Furthermore, quantitative proteomic analysis revealed that a considerable amount of lignocellulolytic enzymes were detected in the secretory proteins of P. incarnate T-7, especially with high abundance of lignin-degrading enzymes and hemicellulases. Combination of quantitative proteomic with transcriptomic analysis results showed that most of those lignocellulolytic enzymes were highly upregulated on poplar wood substrate compared to glucose substrate. Conclusions This study showed that P. incarnate T-7 could selectively delignify poplar wood by submerged fermentation with short time of 7 days, which greatly improved its enzymatic saccharification efficiency. Our results suggested that P. incarnate T-7 might be a promising candidate for industrial woody biomass pretreatment.

Generally, Ca(OH)2 pretreatment of lignocellulosics for fermentable sugar recovery requires a subsequent washing step for calcium removal and pH control for optimized saccharification. However, washing Ca(OH)2-pretreated feedstock with water is considered problematic because of the low solubility of Ca(OH)2 and its adsorption to biomass. In this study, we estimated the availability of carbonated water for calcium removal from the slurry of Ca(OH)2-pretreated rice straw (RS). We tested two kinds of countercurrent washing sequences, four washings exclusively with water (W4) and two washings with water and subsequent two washings with carbonated water (W2C2). The ratios of calcium removal from pretreatment slurry after washing were 64.2 % for the W4 process and 92.1 % for the W2C2 process. In the W2C2 process, 49 % of the initially added calcium was recovered as CaO by calcination. In enzymatic saccharification tests under a CO2 atmosphere at 1.5 atm, in terms of recovery of both glucose and xylose, pretreated, feedstock washed through the W2C2 process surpassed that washed through the W4 process, which could be attributed to the pH difference during saccharification: 5.6 in the W2C2 process versus 6.3 in the W4 process. Additionally, under an unpressurized CO2 atmosphere at 1 atm, the feedstock washed through the W2C2 process released 78.5 % of total glucose residues and 90.0 % of total xylose residues. Thus, efficient removal of calcium from pretreatment slurry would lead to not only the recovery of added calcium but also the proposal of a new, simple saccharification system to be used under an unpressurized CO2 atmosphere condition.

Commonly, the agricultural waste, i.e. lignocellulosic biomass is disposed through combustion causing air pollution with production of PM2.5 and PM10 particles. However, it has been found that these biomasses can be used as source for the production of biofuels and other valuable biochemicals. Though deconstruction of lignocellulosic biomass is challenging due to its complex structure. In this study, rice straw (RS) was pretreated using potassium permanganate (KMnO4) to enhance the enzymatic saccharification efficiency. The study was carried out by varying the operational factors in pretreatment, including temperature (30-90°C), time (30-360 min) and concentration of KMnO4 (0.5-3.0, % w/v), respectively, based on Box-Behnken design (BBD). Through multi-regression analysis of the experimental data obtained after pretreatment, the optimum conditions were determined. The optimum conditions for temperature, time and potassium permanganate concentration were 48.09°C, 360 min, and 1.36% w/v, respectively. The saccharifications of pretreatment and untreated rice straw were carried out using Cellic Ctec2. The reducing sugar was determined by using DNS method and the yields of the untreated and pretreated RS were 32.38 and 49.011 mg/mL, respectively. The results showed that the sugar for pretreated RS were 1.51 fold times higher compared to untreated RS. Therefore, this work illustrates the pretreatment efficiency for KMnO4 to enhance the reducing sugar yield during saccharification, which can be used for biofuel and value-added product productions.

A cachaça é a bebida fermentada e destilada obtida a partir do caldo de cana, apresentando características sensoriais peculiares. Estima-se que, no Brasil, em 2015, foram produzidos 8 milhões de litros e a cachaça foi exportada para mais de 60 países, demonstrando a sua importância econômica. Apesar disto, o seu processo de produção apresenta baixo rendimento e ainda gera um grande volume de resíduos (bagaço e vinhaça). Assim, diversas estratégias devem ser avaliadas visando não apenas a otimização das fermentações, mas também a obtenção de cachaças com perfil químico distinto contribuindo para a incrementação de atributos sensoriais e agregando maior valor de mercado. Dentre as estratégias se destacam: o pré tratamento do caldo de cana; a utilização de microrganismos não convencionais e a incorporação de compostos bioativos. Com isso, este trabalho teve por objetivo a produção de enzimas lignolíticas por Streptomyces thermocerradoensis I3 em farelo de trigo e em farinhas de rizomas de dois vegetais extraídos do solo do Cerrado: Cochlospermum regium e Jatropha elliptica, visando obter extrato enzimático para posterior aplicação no pré-tratamento do caldo de cana para obtenção de cachaças. Observou-se que dos meios testados o farelo de trigo se mostrou o melhor, e que os extratos produzidos foram capazes de promover a hidrólise das farinhas, o que promoveu o aumento da concentração de açúcares redutores e compostos fenólicos totais, sugerindo o uso desse extrato para sacarificação em farinhas, promovendo maior acesso a estes compostos. Na segunda etapa do estudo, o objetivo foi a utilização do enzimático obtido do Streptomyces sp. I7 no pré tratamento do caldo de cana, para posterior fermentação deste caldo utilizando três microrganismos diferentes: Saccharomyces cerevisiae; levedura para produção de cerveja Belgian Ale (M27) e por último, um microrganismo isolado de caldo de cana a partir da fermentação espontânea. Verificou-se que independente do microrganismo utilizado na etapa de fermentação, o pré tratamento com o extrato enzimático promoveu aumento da produção de etanol. Além disto, o pré tratamento enzimático gerou cachaças com perfis químicos distintos comprovados por análises de ressonância magnética nuclear. Por fim, foi avaliada a utilização do extrato enzimático associada ao ultrassom de baixa frequência para incorporação de compostos fenólicos extraídos do Zingiber officinale Roscoe em cachaça adquirida em mercado local. Foi constatado que o método empregado favoreceu a extração de açúcares redutores e compostos fenólicos, com destaque para o ácido gálico e caféico. O consumo de bebidas alcóolicas com maior teor de compostos fenólicos quando comparada ao consumo de bebidas sem estes componentes poderia trazer benefícios à saúde dos consumidores. Cachaça is the fermented and distilled beverage obtained from sugarcane juice, with peculiar sensory characteristics. It is estimated that in Brazil, in 2015, 8 million liters were produced, and cachaça was exported to more than 60 countries, showing its economic importance. Despite this, its production process has a low yield and still generates a large volume of waste (bagasse and stillage). Thus, several strategies must be evaluated aiming not only to optimize fermentation but also to obtain cachaças with a different chemical profile, contributing to the increase of sensory attributes and adding greater market value. Among the strategies, the following stand out: pre-treatment of sugarcane juice; the use of non-conventional microorganisms, and the incorporation of bioactive compounds. Thus, this work aimed to produce ligninolytic enzymes by Streptomyces thermocerradoensis I3 in wheat bran and rhizome flours from two vegetables extracted from the Cerrado soil: Cochlospermum regium and Jatropha elliptica, to obtain enzymatic extract for later application in the pre-treatment of sugarcane juice to obtain cachaça. It was observed that the wheat bran proved to be the best of the tested medium and that the extracts produced were able to promote the hydrolysis of the flours, which promoted an increase in the concentration of reducing sugars and total phenolic compounds, suggesting the use of this extract for saccharification in flours, promoting greater access to these compounds. In the second stage of the study, the objective was to use the enzyme obtained from Streptomyces sp. I7 in the pre-treatment of the sugarcane juice, for subsequent fermentation of this juice using three different microorganisms: Saccharomyces cerevisiae; yeast to produce Belgian Ale beer (M27), and finally a microorganism isolated from sugarcane juice from spontaneous fermentation. It was found that regardless of the microorganism used in the fermentation step, the pre-treatment with the enzymatic extract promoted an increase in ethanol production. Also, the enzymatic pretreatment generated cachaças with distinct chemical profiles proven by nuclear magnetic resonance analysis. Finally, the use of enzymatic extract associated with low-frequency ultrasound for the incorporation of phenolic compounds extracted from Zingiber officinale Roscoe in cachaça acquired in the local market was evaluated. It was found that the method employed favored the extraction of reducing sugars and phenolic compounds, with emphasis on gallic and caffeic acids. The consumption of alcoholic beverages with a higher content of phenolic compounds, when compared to the consumption of beverages without these components, could bring benefits to the health of consumers.

We estimated the effect of residual lignin and pulp swelling on the rate of enzymatic saccharification to increase production of ethanol from acid sulfite pulp (SP) by means of enzymatic treatment. The resolution ratio of hardwood (Acacia mearnsii) SP after the enzymatic treatment was lower compared to softwood (Larix leptolepis) SP even though lignin content of hardwood SP was lower. The pyrolysis–gas chromatography/mass spectrometry (Py-GC/MS) analysis revealed that the residual lignin in hardwood SP could more easily adsorb enzyme compared to softwood SP, and the residual lignin in hardwood SP should interfere with the binding of the enzyme to cellulose. The beating treatment of pulp increased the swelling of pulp. The enzymatic saccharification rate was increased by the beating treatment. On the other hand, the delignification treatment was more effective than the beating treatment at enhancing enzymatic saccharification of both hardwood and softwood SPs. We found that the delignification process should be considered a high-priority technique for enhancing enzymatic saccharification of SP.

Fast-growing broad-leaf tree species can serve as feedstocks for production of bio-based chemicals and fuels through biochemical conversion of wood to monosaccharides. This conversion is hampered by the xylan acetylation pattern. To reduce xylan acetylation in the wood, the Hypocrea jecorina acetyl xylan esterase (HjAXE) from carbohydrate esterase (CE) family 5 was expressed in hybrid aspen under the control of the wood-specific PtGT43B promoter and targeted to the secretory pathway. The enzyme was predicted to deacetylate polymeric xylan in the vicinity of cellulose due to the presence of a cellulose-binding module. Cell-wall-bound protein fractions from developing wood of transgenic plants were capable of releasing acetyl from finely ground wood powder, indicative of active AXE present in cell walls of these plants, whereas no such activity was detected in wild-type plants. The transgenic lines grew in height and diameter as well as wild-type trees, whereas their internodes were slightly shorter, indicating higher leaf production. The average acetyl content in the wood of these lines was reduced by 13%, mainly due to reductions in di-acetylated xylose units, and in C-2 and C-3 mono-acetylated xylose units. Analysis of soluble cell wall polysaccharides revealed a 4% reduction in the fraction of xylose units and an 18% increase in the fraction of glucose units, whereas the contents of cellulose and lignin were not affected. Enzymatic saccharification of wood from transgenic plants resulted in 27% higher glucose yield than for wild-type plants. Brunauer-Emmett-Teller (BET) analysis and Simons' staining pointed toward larger surface area and improved cellulose accessibility for wood from transgenic plants compared to wood from wild-type plants, which could be achieved by HjAXE deacetylating xylan bound to cellulose. The results show that CE5 family can serve as a source of enzymes for in planta reduction of recalcitrance to saccharification.

Polyhydroxybutyrates (PHBs) are a class of biopolymers produced by different microbial species and are biodegradable and biocompatible in nature as opposed to petrochemically derived plastics. PHBs have advanced applications in medical sector, packaging industries, nanotechnology and agriculture, among others. PHB is produced using various feedstocks such as glycerol, dairy wastes, agro-industrial wastes, food industry waste and sugars. Current focus on PHB research has been primarily on reducing the cost of production and, on downstream processing to isolate PHB from cells. Recent advancements to improve the productivity and quality of PHB include genetic modification of producer strain and modification of PHB by blending to develop desirable properties suited to diversified applications. Selection of feedstock plays a critical role in determining the economic feasibility and sustainability of the process. This review provides a bird's eye view of the suitability of different waste resources for producing polyhydroxybutyrate; providing state-of the art information and analysis.

The improvement of the saccharification of rice straw is one of the strategies to reduce the sophisticated pretreatment that results in high cost and is unfriendly to the environment. We explored the cell wall features in tetraploid rice and highlighted the enhanced saccharification of tetraploid with large biomass. Results showed that lignin content and S/G ratio reduced to 17.09% and 0.37, respectively, in tetraploid straw by the determination of the pyGC-MS method. After the pretreatment, the cellulose crystallinity index decreased from 63.22% to 57.65% in tetraploid straw, which is lower than that of pretreated diploid straw. Surface topological analysis of SEM images indicated that tetraploid straw was more susceptible to the pretreatment. Tetraploid straw showed a strong advantage in the process of enzymatic hydrolysis. The enzyme efficiency reached the highest value of 77.60%, and the rate of enzyme reaction was improved to make the reaction saturated earlier than conventional rice. We concluded that the high saccharification has resulted from the alteration of lignin and cellulose in tetraploid rice. Our research provides an improved green feedstock for bioenergy, and the tetraploid rice straw shows the potential utilization value in bioethanol production.

Enzymatic saccharification of Laminaria japonica seaweed biomass was optimized by four independent factors (enzyme dose, hydrolysis time, pH, and temperature) using response surface methodology (RSM). To confirm the significance of the quadratic model, an analysis of variance (ANOVA) was performed, and the F-value of 8.76 showed that the regression model was highly significant (&le, 0.1%). In the accuracy study, average recoveries were in the range of 97.00% to 98.32%. The optimum experimental conditions were an enzyme dose of 8.2%, a hydrolysis time of 26 h, a pH of 4.1, and a temperature of 43 . Temperature was the most important factor in the enzymatic saccharification. A relatively low temperature and short hydrolysis time were shown to improve the yield of reducing sugars.

In a time of great dependence on fossil fuels and rising need for sustainable, renewable resources, seaweeds can be a valuable asset to the new and emerging bioeconomy. Norway is in a unique position because its particularly long coastline can provide an alternative to the use of terrestrial biomass, as brown seaweeds are more environmentally friendly to exploit. The polysaccharides in brown seaweed can be processed enzymatically to release fermentable sugars, which can be used further as a carbon source in biorefineries. Biorefineries process biomass in order to produce fuel, energy, chemicals and value-added products. To utilize as much of the carbohydrates in brown seaweed as possible, successful enzymatic degradation of these components is a key step in the process. In this study, the aim was to combine an enzyme cocktail for the degradation of alginate, laminarin and cellulose in Saccharina latissima (L.), a brown seaweed harvested from the Trondheim fjord. Endolytic and exolytic alginate lyases AMOR_PL7A and AMOR_PL17A, laminarinases (or β-1,3- glucanases) GH16 and GH5, and cellulases GH6.4 and therm3 were combined on ground S. latissima for the release of sugars, and compared to the effect of commercial cellulase cocktail Cellic® CTec2. The abovementioned enzymes were added to 15% DM (w/v) ground seaweed, and degradation yield measured both as reducing sugars with the 3,5-Dinitrosalicylic acid (DNS) assay and as glucose with high-performance liquid chromatography (HPLC). Reaction products were measured after 48 hours incubation in a Thermomixer at 50°C, and the enzyme cocktail successfully released oligosaccharides and monomeric sugars from the main carbohydrates in the seaweed. Reaction product analysis from degradation of sodium alginate with AMOR_PL7A and AMOR_PL17A was conducted with MALDI-TOF mass spectrometry and 1H NMR, which showed that these enzymes combined releases monomeric DEHU from alginate. The results from this thesis indicate that an enzyme cocktail can be applied to S. latissima for saccharification of its polysaccharides, which is a very essential step towards more efficient processing of macroalgae in a biorefinery in the future. I ei tid der avhengnaden av fossile ressursar er stor og høvet for berekraftige, fornybare ressursar veks, kan tare bli verdifulle i den nye veksande bioøkonomien. Norge er i ein unik posisjon grunna den store kystlinja som omgjev landet, fordi utnytting av tare kan gi eit meir miljøvennleg alternativ til bruken av landbasert biomasse. Polysakkarida i bruntare kan bli prosessert med enzym for å sleppe ut fermenterbare sukker, som kan brukas vidare som karbonkjelde i bioraffineri. Bioraffineri prosesserer biomass for produksjon av brensel, energi, kjemikaliar og høgverdi-produktar. For å nytta så stor del som mogleg av karbohydrata i bruntare er det naudsynt med suksessfull enzymatisk nedbryting av desse. I denne studien vart målet å kombinere ein enzymblanding for nedbryting av alginat, laminarin og cellulose i Saccharina latissima (L.), ein bruntare hausta frå Trondheimsfjorden. Dei endolytiske og eksolytiske alginat lyasane AMOR_PL7A og AMOR_PL17A, laminarinasane (eller β-1,3-glucanasar) GH16 og GH5, og cellulasane GH6.4 og therm3 vart kominert på malt S. latissima for å sjå på sukkerutslepp, og samanlikna med den kommersielle cellulaseblandinga Cellic® CTec2. Dei nemnde enzyma blei tilført til 15% tørrstoff (i vekt/volum) malt tare, og nedbrytingsutbyttet målt både som reduserande sukker med 3,5-Dinitrosalicylsyre (DNS) analyse og som glukose med HPLC. Reaksjonsprodukta vart målt etter 48 timars inkubering i ein Thermomixer på 50°C, og enzymblandinga resulterte i utsleppet av oligosakkarid og monomerisk sukker frå dei største karbohydratkomponenta i taren. Produktanalyse etter nedbryting av sodium alginat med AMOR_PL7A og AMOR_PL17A vart gjennomført med MALDI-TOF massespektrometri og 1H NMR, og dette viste at desse enzyma kombinert braut ned alginat til monomerisk DEHU. Resultata frå denne oppgåva indikerer at ei enzymblanding kan bli tilsett for sakkarifisering av polysakkarida i S. latissima, noko som er eit svært essensielt steg mot ei meir effektiv prosessering av makroalgar i eit bioraffineri i framtida. Foods of Norway M-KJEMI

Made available in DSpace on 2020-12-12T02:26:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Pycnoporus sanguineus MCA 16 was cultivated by solid state fermentation on lignocellulosic residues, at 40 °C. The highest endoglucanase, exoglucanase, β-glucosidase, xylanase, β-xylosidase and laccase activities (200, 29.1, 52.1, 44.2, 1.81 and 22.04 U g−1, respectively) were obtained when using the mixture of wheat bran and soybean meal (1:1 w/w) as substrates, at 96 h of cultivation. Peroxidases highest activities were observed at 48 h (14.61 and 10.02 U g−1 for manganese and lignin peroxidase, respectively). Enzymatic saccharification of sugarcane bagasse submitted to hydrothermal alkaline pretreatment provided glucose at 7.32 g L−1 when using solid load of 13.5% (dry basis), 288.3 total units of endoglucanase/g of cellulose, 130 h and 57 °C. Ligninases present in the enzymatic solution provided a reduction of 82.3% in the concentration of total phenolic compounds during saccharification. The hydrolysate was used for ethanol production by Saccharomyces cerevisiae CAT-1 and a yield of 73.5% was obtained. São Paulo State University (UNESP) Institute of Biosciences Letters and Exact Sciences São Paulo State University (UNESP) Institute of Chemistry São Paulo State University (UNESP) Faculty of Pharmaceutical Sciences São Paulo State University (UNESP) Institute of Biosciences Letters and Exact Sciences São Paulo State University (UNESP) Institute of Chemistry São Paulo State University (UNESP) Faculty of Pharmaceutical Sciences

The susceptibility of untreated and sulfuric-acid-pretreated aspen, birch, and spruce to analytical enzymatic saccharification was studied in relation to their chemical composition and physical-structural features. The analytical data collected covered the mass fractions of lignin, carbohydrates, and extractives, the release of acetic acid, formic acid, and uronic acids by acid and alkaline hydrolysis, crystallinity and crystallite size, syringyl: guaiacyl (S:G) ratio of lignin, cellulose accessibility, FTIR spectra, images from SEM and fluorescence microscopy, and susceptibility to enzymatic saccharification using enzyme mixtures with and without supplementary xylanase.In the absence of pretreatment the mass fraction yield of Glc on the original dry wood in the analytical enzymatic saccharification increased in the order birch (16 g kg−1) < spruce (35 g kg−1) < aspen (150 g kg−1). After acid pretreatment, the order changed to spruce (170 g kg−1)

Wood industry based on sengon (Falcataria moluccana) material has developed rapidly and cause the huge wastes. Lignocellulosic materials, such as sengon sawdust, offer a great potential as cheap and abundant feedstock for biofuels production. Ozone oxidation pretreatment was carried out on sengon wastes to improve fermentable sugar production by enzymatic saccharification. Sengon wood (SW), sengon media (SM), and sengon spent media (SMM), that was waste of media after mushroom cultivation, treated with ozone for different treatment time were enzymatically saccharified. Then the decrease of hydrolysis weight, reducing sugar yield, and monosaccharide yields were determined. Ozone treatment resulted in Klason lignin degradation in the all samples, resulting in the improvement of subsequent enzymatic saccharification. Ozone treatment with the duration more than 30 min was not suitable for SMM sample. Glucose yield after saccharification from the ozone-treated sample for 60 min was the highest among all samples in SW and SM treated, but for SMM it was 30 min. Based on these results, we concluded that mushroom cultivation can reduce the ozone time treatment and it is an effective treatment to improve sugar yield by enzymatic saccharification of sengon wastes. Gabungan Pra-perlakuan Ozonolisis dan Budidaya Jamur Sebelum Sakarifikasi Enzimatis Serbuk Kayu Sengon (Falcataria moluccana)IntisariIndustri kayu berbahan baku kayu sengon (Falcataria moluccana) saat ini tengah berkembang dengan sangat pesat dan hal ini menyebabkan timbulnya banyak limbah kayu sengon. Materi lignosellulosa, seperti limbah serbuk kayu sengon, merupakan potensi yang sangat baik sebagai bahan baku bio-fuel karena murah dan banyak tersedia. Praperlakuan dengan oksidasi ozon pada limbah kayu sengon dilakukan untuk meningkatkan produksi gula reduksi melalui proses sakarifikasi enzimatis. Serbuk sengon (SW), media sengon (SM), dan limbah media sengon (SMM), yang merupakan sisa dari media budidaya jamur, diberi perlakuan dengan ozon pada berbagai lama waktu perlakuan. Sampel yang sudah diberi perlakuan ozon tersebut kemudian dianalisis kandungan kimianya dan di-sakarifikasi secara enzimatis. Selanjutnya diukur laju hidrolisis, kadar gula pereduksi, dan kadar monosakarida. Perlakuan dengan ozon menyebabkan degradasi kadar Klason lignin di semua sampel, sehingga mengakibatkan peningkatan kemampuan sakarifikasi dari enzim. Perlakuan ozon dengan lama waktu lebih dari 30 menit tidak sesuai untuk sampel SMM. Kadar glukosa hasil sakarifikasi yang tinggi diperoleh dari sampel SW dan SM yang diberi perlakuan ozon selama 60 menit, serta SMM yang diberi perlakuan 30 menit. Berdasarkan hasil ini, dapat disimpulkan bahwa perlakuan jamur dapat mengurangi lama waktu perlakuan ozon dan praperlakuan efektif untuk meningkatkan kadar gula reduksi yang dihasilkan dari sakarifikasi enzimatis limbah kayu sengon.

This work describes the feasibility of using rice milling by-products as feedstock for bioethanol. Starch-rich residues (rice bran, broken, unripe and discolored rice) were individually fermented (20%w/v) through Consolidated Bioprocessing by two industrial engineered yeast secreting fungal amylases. Rice husk (20%w/v), mainly composed by lignocellulose, was pre-treated at 55°C with alkaline peroxide, saccharified through optimized dosages of commercial enzymes (Cellic® CTec2) and fermented by the recombinant strains. Finally, a blend of all the rice by-products, formulated as a mixture (20%w/v) according to their proportions at milling plants, were co-processed to ethanol by optimized pre-treatment, saccharification and fermentation by amylolytic strains. Fermenting efficiency for each by-product was high (above 88% of the theoretical) and further confirmed on the blend of residues (nearly 52g/L ethanol). These results demonstrated for the first time that the co-conversion of multiple waste streams is a promising option for second generation ethanol production.

Softwood bark contains a large amounts of extractives—i.e., soluble lipophilic (such as resin acids) and hydrophilic components (phenolic compounds, stilbenes). The effects of the partial removal of water-soluble extractives before acid-catalyzed steam pretreatment on enzymatic digestibility were assessed for two softwood barks—Norway spruce and Scots pine. A simple hot water extraction step removed more than half of the water-soluble extractives from the barks, which improved the enzymatic digestibility of both steam-pretreated materials. This effect was more pronounced for the spruce than the pine bark, as evidenced by the 30 and 11% glucose yield improvement, respectively, in the enzymatic digestibility. Furthermore, analysis of the chemical composition showed that the acid-insoluble lignin content of the pretreated materials decreased when water-soluble extractives were removed prior to steam pretreatment. This can be explained by a decreased formation of water-insoluble “pseudo-lignin” from water-soluble bark phenolics during the acid-catalyzed pretreatment, which otherwise results in distorted lignin analysis and may also contribute to the impaired enzymatic digestibility of the barks. Thus, this study advocates the removal of extractives as the first step in the processing of bark or bark-rich materials in a sugar platform biorefinery. Electronic supplementary material The online version of this article (doi:10.1007/s12010-017-2577-2) contains supplementary material, which is available to authorized users.

Amphipathic lignin derivatives (A-LDs) prepared from the black liquor of soda pulping of Japanese cedar are strong accelerators for bioethanol production under a fed-batch simultaneous enzymatic saccharification and fermentation (SSF) process. To improve the bioethanol production concentration, conditions such as reaction temperature, stirring program, and A-LDs loadings were optimized in both small scale and large scale fed-batch SSF. The fed-batch SSF in the presence of 3.0 g/L A-LDs at 38 degrees C gave the maximum ethanol production and a high enzyme recovery rate. Furthermore, a jar-fermenter equipped with a powerful mechanical stirrer was designed for 1.5 L-scale fed-batch SSF to achieve rigorous mixing during high substrate loading. Finally, the 1.5 L fed-batch SSF with a substrate loading of 30% (w/v) produced a high ethanol concentration of 87.9 g/L in the presence of A-LDs under optimized conditions.

Amphipathic lignin derivatives (ALDs), prepared from hardwood acetic acid lignin and softwood soda lignin via coupling with a mono-epoxylated polyethylene glycol, have been reported to improve the enzymatic saccharification efficiency of lignocellulose while maintaining significant residual cellulase activity after saccharification. We previously demonstrated that the effect of ALDs was caused by a direct interaction between ALDs and Cel6A (or CBH II). In this study, a different ALD was prepared from softwood kraft lignin in addition to aforementioned ALDs. The interactions between all the ALDs and the enzymes other than Cel6A, such as Cel7A and Cel7B, in a cellulase cocktail were investigated using surface plasmon resonance. The kraft lignin-based ALD showed the highest residual cellulase activity among all ALDs and an improved cellulolytic enzyme efficiency similar to those of the other ALDs. All ALDs were found to directly associate with major enzymes in the cellulase cocktail, Cel6A and Cel7A (or CBH I), but not with Cel7B (or EG I). In addition, the ALDs showed a much higher affinity to amino groups than to hydroxy and carboxy groups. In contrast, polyethylene glycol (molecular mass 4000 Da), one part of the ALD and a previously reported enzymatic saccharification enhancer, did not adsorb onto any enzymes in the cellulase cocktail or the amino group. Size exclusion chromatography demonstrated that the ALDs formed self-aggregates in both water and chloroform; the formation process in the latter was especially unique. Therefore, we conclude that the high residual cellulase activity is attributed to the direct association of ALD aggregates with the CBH group.

Miscanthus is a promising fiber crop with high potential for sustainable biomass production for a biobased economy. The effect of biomass composition on the processing efficiency of miscanthus biomass for different biorefinery value chains was evaluated, including combustion, anaerobic digestion and enzymatic saccharification for the production of bioethanol. Biomass quality and composition was analyzed in detail using stem and leaf fractions of summer (July) and winter (March) harvested biomass of eight compositionally diverse Miscanthus sinensis genotypes. Genotype performance in tests for enzymatic saccharification, anaerobic digestion and combustion differed extensively. The variation between the best and the worst performing genotype was 18% for biogas yield (ml g -1 dm) and 42% for saccharification efficiency (glucose release as %dm). The ash content of the best performing genotype was 62% lower than that of the genotype with the highest ash content and showed a considerably high ash melting temperature during combustion. Variation between genotypes in biomass quality for the different thermochemical bioconversion processes was shown to be strongly correlated to differences in biomass composition. The most important traits that contributed favorably to biogas yields and saccharification efficiency were a high content of trans-ferulic acid, a high ratio of para-coumaric acid to lignin and a low lignin content. Additionally, a high content of hemicellulosic polysaccharides positively affected saccharification efficiency. Low contents of ash and inorganic elements positively affect biomass quality for combustion and low potassium and chloride contents contributed to a higher ash melting temperature. These results demonstrate the potential for optimizing and exploiting M. sinensis as a multipurpose lignocellulosic feedstock, particularly for bioenergy applications.

The NREL developed, atmospheric pressure, low severity Deacetylation and Mechanical Refining (DMR) process under atmospheric pressure, generates highly fermentable, low toxicity sugar syrups and highly reactive lignin streams. The dilute alkali deacetylation step saponifies acetyl groups from the hemicellulosic fraction of biomass into a black liquor waste stream that also contains solubilized acetic, ferulic, p-coumaric acids and lignin-carbohydrate complexes (LCCs), oligosaccharides, and solubilized lignin. Valorization of the soluble components in the deacetylation black liquor waste stream requires characterization of the black liquor. WeOur analyses show that glucan, xylan and lignin were three main components in black liquor and the oligosaccharides in the black liquor were mainly xylan dimers and trimers with arabinofuranose branch groups mainly on the xylooligomers. GPC chromatograms showed that the black liquor also contained oligomeric lignin moieties with molecular weights ranging between 1800 to ~10,000 Da. Enzymatic saccharification of the black liquor was conducted based on the compositional and structural information. Incorporation of Novozymes Cellic® CTec2 and HTec2 commercial cellulase and hemicellulase enzyme preparations with various accessary enzymes showed up to 57% hydrolysis of the xylooligosaccharides in the black liquor to monomeric sugars, which could be used to improve biorefinery carbon utilization in biological fermentations to improve end product fuel or bioproduct volumes per metric tonne of biomass processed. This is so far the first study about characterizing DMR black liquor and exploring breaking down the oligosaccharides in black liquor for potential use.

Background Salix spp. are high-productivity crops potentially used for lignocellulosic biofuels such as bioethanol. In general, pretreatment is needed to facilitate the enzymatic depolymerization process. Biomass resistance to degradation, i.e., biomass recalcitrance, is a trait which can be assessed by measuring the sugar released after combined pretreatment and enzymatic hydrolysis. We have examined genetic parameters of enzymatic sugar release and other traits related to biorefinery use in a population of 286 natural Salix viminalis clones. Furthermore, we have evaluated phenotypic and genetic correlations between these traits and performed a genomewide association mapping analysis using a set of 19,411 markers. Results Sugar release (glucose and xylose) after pretreatment and enzymatic saccharification proved highly variable with large genetic and phenotypic variations, and chip heritability estimates (h2) of 0.23–0.29. Lignin syringyl/guaiacyl (S/G) ratio and wood density were the most heritable traits (h2 = 0.42 and 0.59, respectively). Sugar release traits were positively correlated, phenotypically and genetically, with biomass yield and lignin S/G ratio. Association mapping revealed seven marker–trait associations below a suggestive significance threshold, including one marker associated with glucose release. Conclusions We identified lignin S/G ratio and shoot diameter as heritable traits that could be relatively easily evaluated by breeders, making them suitable proxy traits for developing low-recalcitrance varieties. One marker below the suggestive threshold for marker associations was identified for sugar release, meriting further investigation while also highlighting the difficulties in employing genomewide association mapping for complex traits. Electronic supplementary material The online version of this article (10.1186/s13068-019-1479-7) contains supplementary material, which is available to authorized users.

The aromatic composition of lignins is considered an important trait that affects the physico-chemical properties of lignocellulosic biomass. However, our knowledge of the relationship between lignin structure and biomass utilization properties remains limited, especially in monocotyledonous grass species, despite their potential as biomass feedstocks. In this study, we used recently produced rice transgenic lines with distinct lignin monomer compositions, i.e., guaiacyl (G)/syringyl (S)/p-hydroxyphenyl (H) aromatic unit ratios, to study the impact of lignin composition on the chemical reactivity, enzymatic saccharification efficiency and calorific value of rice lignocellulose. The H-lignin-enriched rice transgenic line showed significantly enhanced biomass saccharification efficiency after alkali and acid pretreatments and even without any pretreatment, whereas the S-lignin-enriched rice transgenic line displayed enhanced saccharification efficiency after liquid hot water pretreatment. While we detected no significant differences in biomass heating values between the transgenic rice materials tested, analysis of synthetic lignins comprising only G, S or H units suggested that increased ratios of G or H units could increase the heating value of lignin-based solid biofuels.

Developing economical and sustainable fractionation technology of lignocellulose cell walls is the key to reaping the full benefits of lignocellulosic biomass. This study evaluated the potential of metal chloride-assisted p-toluenesulfonic acid (p-TsOH) hydrolysis at low temperatures and under acid concentration for the co-production of sugars and lignocellulosic nanofibrils (LCNF). The results indicated that three metal chlorides obviously facilitated lignin solubilization, thereby enhancing the enzymatic hydrolysis efficiency and subsequent cellulose nanofibrillation. The CuCl2-assisted hydrotropic pretreatment was most suitable for delignification, resulting in a relatively higher enzymatic hydrolysis efficiency of 53.2%. It was observed that the higher residual lignin absorbed on the fiber surface, which exerted inhibitory effects on the enzymatic hydrolysis, while the lower lignin content substrates resulted in less entangled LCNF with thinner diameters. The metal chloride-assisted rapid and low-temperature fractionation process has a significant potential in achieving the energy-efficient and cost-effective valorization of lignocellulosic biomass.

Resumo: Os ruminantes são fontes de microrganismos produtores de enzimas lignocelulolíticas, as quais podem ser utilizadas para diferentes aplicações biotecnológicas, como a degradação da fibra do bagaço de cana-de-açúcar (BCA) para uso alimentar ou para produção de bioetanol. Enzimas mais específicas são obtidas quando o próprio substrato de interesse é utilizado na produção dos microrganismos. Nesse sentido, o presente trabalho direcionou a microbiota ruminal de ovinos Morada Nova para a degradação do BCA objetivando extrair as proteínas aderidas à fibra (PAFs) e caracterizá-las quanto ao potencial de degradação do BCA in natura e pré-tratado com 10, 20, 30, 40 ou 50% de NaOH. O conteúdo ruminal foi obtido via fístula, filtrado, lavado com Solução Salina Tamponada (PBS, pH 7,2 sem Ca2+ e Mg2+) e as PAFs extraídas com ureia 8 mmol.L-1 durante 15 minutos. Após serem filtradas e dialisadas contra água (cut off de 12 kDa) as PAFs foram liofilizadas. A atividade total de celulase foi determinada pelo ensaio do ácido 3,5-dinitrosalicílico (DNS) em pH 3,99 e papel de filtro. As PAFs mostraram maior atividade enzimática em pH 3,99. O pH 11,71 foi identificado como o de máxima extração de proteína. A temperatura de 50 ºC por 60 minutos manteve a atividade específica de celulase total das PAFs e essas também resistiram ao aquecimento a 100 °C por 60 minutos. Em relação aos substratos, as PAFs apresentaram maior atividade específica para xilanase (7,01 ± 0,19 U.mg-1) seguida por endoglucanase (4,71 ± 1,65 U.mg-1), exoglucanase (2,68 ± 0,42 U.mg-1) e celulase total (0,62 ± 0,05 U.mg-1). Quanto aos substratos BCA in natura e pré-tratado, a atividade enzimática das PAFs aumentou de forma proporcional à concentração de NaOH utilizado no pré-tratamento, ao mesmo tempo em que se observou na fibra melhor solubilização da hemicelulose e lignina, e aumento da cristalinidade. As fibras de BCA in natura e pré-tratada foram utilizadas como matriz cromatográfica e observou-se interação das PAFs com as matrizes, com destaque para as matrizes de BCA pré-tratadas com 20% e 40% NaOH. Em conclusão, as enzimas ruminais de ovinos Morada Nova foram capazes de degradar o BCA, sendo sua eficiência elevada ajustando-se o pH e a temperatura reacional, assim como realizando o pré-tratamento alcalino do BCA. Ainda, a própria fibra de BCA pré-tratada pode ser utilizada para isolar e caracterizar enzimas ruminais lignocelulolíticas. Abstract: Ruminants are sources of microorganisms that produce lignocellulolytic enzymes which in turn can be used for different biotechnological applications, such as in degrading sugarcane bagasse (SCB) fibers for food use or for producing bioethanol. More specific enzymes are obtained when the substrate of interest itself is used in producing the microorganisms. In this sense, the present work directed Morada Nova sheep ruminal microbiota to degrade SCB, aiming at extracting proteins adhered to the fibers (PAFs), characterizing them as to the potential degradation of the in natura SCB and those pretreated with 10, 20, 30, 40 or 50% NaOH. The ruminal content was obtained via fistula, filtered, washed with Phosphate Buffer Saline (pH 7.2 without Ca2+ and Mg2+) and the PAFs were extracted with 8 mmol.L-1 urea for 15 minutes. The PAFs were then lyophilized after filtration and dialysis against water (cut-off of 12 kDa). Total cellulase activity was determined by the 3,5-dinitrosalicylic acid (DNS) assay at pH 3.99 and filter paper. The PAFs showed higher enzymatic activity at pH 3.99. The pH of 11.71 was identified as the maximum protein extraction. A temperature of 50 ºC for 60 minutes maintained the specific activity of PAFs total cellulase and they also resisted heating at 100 °C for 60 minutes. Regarding substrates, PAFs showed higher specific activity for xylanase (7.01 ± 0.19 U.mg-1), followed by endoglucanase (4.71 ± 1.65 U.mg-1), exoglucanase (2.68 ± 0.42 U.mg-1) and total cellulase (0.62 ± 0.05 U.mg-1). For the SCB substrates in natura and pretreated, the enzymatic activity of PAFs increased in proportion to the increased concentration of NaOH used in the pretreatment, while at the same time better hemicellulose and lignin solubilization were observed in the fiber, as well as increased crystallinity. In natura and pretreated SCB fibers were used as a chromatographic matrix and interaction of the PAFs with the matrices was observed, especially the pretreated SCB matrices with 20% and 40% NaOH. In conclusion, Morada Nova sheep ruminal enzymes were able to degrade SCB fibers, with higher efficiency by adjusting the pH and reaction temperature, as well as performing alkaline pretreatment of the SCB. In addition, the pretreated SCB fiber itself can be used to isolate and characterize lignocellulolytic ruminal enzymes. Made available in DSpace on 2019-09-14T00:45:39Z (GMT). No. of bitstreams: 1 CNPC2019UPA2.pdf: 2470990 bytes, checksum: 085f8bef67701f73830fb19ff0ccc57d (MD5) Previous issue date: 2019 Tese (Doutorado em Biotecnologia) - Universidade Estadual do Ceará, Fortaleza. Orientador: Antônio Sílvio do Egito (Embrapa Caprinos e Ovinos); Coorientadora: Hévila Oliveira Salles (Embrapa Caprinos e Ovinos).

Most of the agricultural wastes in developing countries are disposed of by on-site combustion leading to unmanaged environmental pollutions. Conversion of agricultural wastes to value-added products, such as bioethanol and biogas, is a promising method to reduce agro-waste after harvesting seasons. In this study, Citrus maxima peels (Pomelo peels) was selected to be converted to reducing sugars, which could be a raw material to produce other value-added products. To promote enzymatic hydrolysis reactions, pomelo peels were pretreated with sodium hydroxide by variations of three pretreatment parameters, including temperature (50-100 ºC), time (0.5-6 h), and concentration of NaOH (0.5-3.0 M). Box-Behnken design (BBD) was applied in Response Surface Methodology (RSM) to determine the optimized pretreatment condition and to find the relationship between pretreatment factors and reducing sugar yields. The predicted optimal pretreatment condition was determined to be at 94.28 ºC, 4.5h, 2.17M with reducing sugar yield of 98.9 mg/g of dried pomelo peels. The results clearly showed that reducing sugar yields obtained from pretreated pomelo peels were 1.87 folds higher than untreated biomass (52.81 mg/g of pomelo peels). Therefore, this study demonstrated the potential of pomelo peels to be used as an alternative raw material for value-added products rather than being a landfill or causal agent of pollution.

WOS: 000391185500014 This study examined the suitability of boron derivatives as pretreatment chemicals with regard to their efficiency in the enzymatic saccharification of sunflower stalks. It was hypothesized that the utilization of boron derivatives might improve enzymatic digestibility. The aim of this study was to examine the effect of NaOH and the boron derivatives sodium borohydride [NaBH4], calcined tincal [Na2B4O7(1-5)H2O], colemanite [CaB3O4(OH)(3)H2O], boron oxide [B2O3], borax decahydrate [Na(2)B(4)O(7)10H(2)O], disodium octaborate tetrahydrate [Na(2)B(8)O(13)4H(2)O] and boric acid [H3BO3] on enzymatic saccharification. Results showed that the boron derivatives prevented peeling reactions and preserved more glucan; however, the NaOH removed the highest amounts of xylan and lignin from the structure. Significant xylan removal was observed with H3BO3, while B2O3 was as effective as NaBH4 and NaOH for selective lignin removal. The highest enzymatic saccharification yield (59,5%) resulted with the 2% NaOH pretreatment, whereas slightly lower yields were achieved with some boron derivatives (1% NaBH4-45%; 2% B2O3-40,2%; and 2% H3BO3-36,7%).

Lignocellulosic biomass is a candidate for future renewable energy resources. Choice of optimum biomass types and biological conversion techniques requires well-founded assessment of the digestibility determining the conversion efficiency. The aim of this study was to investigate and evaluate the digestibility of miscanthus samples that were tested using three methods: 3,5-dinitrosalicylic acid assay (DNS), anaerobic batch digestion test, and high-throughput pretreatment and hydrolysis method, including a grinding and hydrothermal pretreatment prior to the analysis (HTPH). The miscanthus samples were expected to have different digestibilities due to maturity stage, dry matter content and the implementation of extrusion as a mechanical pretreatment. The results of the DNS and the biogas batch test methods were highly correlated (R2 between 0.75 and 0.92), but not with the results of the HTPH method. The DNS and biogas batch test showed that digestibility differed between samples, probably due to the degree of lignification and content of soluble sugars. For the HTPH method, the digestibility for biorefining was the same irrespective of the variation in the other analyses. The HTPH method had higher biomass use efficiency, closely followed by the biogas batch test running for 91 days on the mechanically pretreated biomass. The HTPH method provided information on the overall quantity of carbohydrates that can be made available from a given biomass. Additionally, DNS and biogas batch test visualize the variation in digestibility between biomass types caused by lignification and particle. The study concludes that the choice of evaluation method for miscanthus will depend on the bioenergy conversion method used and that important information on the interaction between physio-chemical pretreatment and biological accessibility of the biomass can be obtained by comparing the methods. This information will enable sound decisions on the future choice of bioenergy conversion technologies.