Hemicellulose biorefineries: a review on biomass pretreatments

Biomass pretreatment (BP) plays a crucial role in a lignocellulose feedstock-based biorefinery (LCFBR) for processing of three major output streams (cellulose, hemicelluloses and lignin) into chemicals and biofuels. BP includes processing of lignocellulosic material (LCM) under aqueous, dilute acid or alkaline media to obtain a cellulosic fraction, which is then fermented to produce bioethanol. Hemicellulose is usually treated as a secondary stream due to lack of efficient fermentation of hemicellulosic sugars to ethanol. This review provides BPs assuming that hemicellulose stream should be integrated in LCFBR as a primary fraction for converting into value-added compounds other than bioethanol. Different LCM treatments are analyzed foreseeing bio-based products possible to obtain from hemicellulose path

Hydrothermal processing of corn residues:process optimisation and products characterisation

Hydrothermal processing was used as pre-treatment method for the selective solubilisation of hemicellulose from corn residues (leaves and stalks). The raw material was treated at a liquidto- solid ratio of 10 g/g, under non-isothermal conditions (150-240ºC) and the effect of treatment on the composition of both liquid and solid phases was evaluated. The yields of solid residue and soluble products, e.g., oligosaccharides, monosaccharides, acetic acid and degradation compounds, such as furfural, hydroxymethylfurfural are presented and interpreted using the severity factor (log R0). The operational conditions leading to the maximum recovery of XOS (53% of initial (arabino)xylan) and for highest glucan content of the solid residue (64%) were established for log R0 of 3.75 and 4.21, respectively. Under the severest condition 95% of xylan was selectively solubilised and 90% of initial glucan was recovered on the solid residue, making it very attractive for further processing in a biorefinery framework

Fermentation of biomass-derived syngas to ethanol and acetate by clostridium ljungdahlii

In the biochemical pathway of lignocellulosics conversion into fuels, a significant portion of biomass cannot be hydrolysed to fermentable sugars and remains as waste substrate that, due to its recalcitrance, is not converted to ethanol by microorganisms. In terms of product yield, this residual biomass represents renewable feedstock that is being wasted, which contradicts the target of 100% feedstock utilisation. The gasification of this biomass constitutes an alternative to circumvent this problem, as the produced synthesis gas (syngas) can be used as substrate for microorganisms that are able to convert CO, CO2 and H2 into important bulk chemicals and biofuels, such as ethanol, acetate and butanol [1,2]. Thus, syngas fermentation to ethanol and acetate can be regarded as a possible process to increase the overall product yield from lignocellulosic feedstock. Some advantages of fuels and chemicals production through syngas fermentation over metal catalyst conversion are the possibility of utilisation of the whole biomass regardless its quality, the independence of a fixed H2:CO ratio for the bioconversion process, a higher specificity of the microbial biocatalyst over chemical catalysts, and the bioreactor operation at ambient conditions [3]. However, syngas fermentation also presents several limitations, such as low yields and poor solubility of the gaseous substrate in the liquid phase. The objective of the present study was to evaluate C. ljungdahlii as microbial catalyst capable of fermenting syngas produced by gasification of spent solids obtained after lignocellulosic biomass saccharification and fermentation into ethanol. The heterotrophic and autotrophic growth of C. ljungdahlii were compared. Parameters such as bacterial growth, acetate and ethanol production, substrate consumption, and bioconversion yields were evaluated. In order to overcome the problem of gas diffusion in the liquid phase, fermentations were conducted at different total pressure

Simplex optimization and mathematical modeling of wheat straw dilute acid hydrolysisand

Wheat straw is an interesting biorefinery raw material, due to its abundance, chemical composition, and cost. Among the different pretreatments suitable for its processing, dilute acid hydrolysis still presents some benefits due to its simplicity. Nevertheless, it requires a careful optimization to avoid excessive by-products formation and catalyst spending. An attractive and simple optimization approach is the Sequential Simplex Method, an iterative procedure that enables to rapidly screen a large area ofoperational conditions and effectively encircle the optimal. In this work, dilute acid hydrolysis of wheat straw was optimized to selectively hydrolyze the hemicellulose fraction and obtain a pentose-rich fermentable hydrolyzate. The influence oftime (up to 180 min), and sulfuric acid concentration (up to 4%, w/w) were studied. The hydrolyzates obtained in the optimized conditions mainly contain free sugars (total content higher than 46 giL). The main potential microbial inhibitors found were acetic acid, furfural, and HMF, in concentrations lower than 4.8,1.7 and 0.3 giL, respectively. Empirical models describing the influence ofthe studied variables on sugars and by-products formation were validated for the entire domain. Sulfuric acid concentration was found to be the most influential variable, although both variables are statistically significant for xylose recovery. Interaction effects play a significant (negative) role. Data was also modeled based on the combined severity parameter (CS) and the results of these two approaches are compared and discussed. These hydrolyzates were easily utilized by Debaryomyces hansenii, a natural pentose assimilating yeast

Prebiotic xylo-oligosaccharides as high-value co-products on an integrated biorefinery approach from lignocellulosic feedstock

The present work proposes the production of prebiotic xylo-oligosaccharides (XOS) as high-value co-products of the Lignocellulose Feedstock Biorefinery concept, foreseeing potential applications on food, feed and nutraceutical industries. Autohydrolysis was used to selectively solubilise the hemicellulosic fraction of several xylan-rich, widely available, agricultural, agro-industrial and forestry by-products: corn cobs, brewery’s spent grain and Eucalyptus wood chips. The soluble hemicellulose-rich and the solid cellulose- and lignin-rich fractions were separated, and the crude XOS-rich hydrolysates were further purified by gel filtration chromatography. Selected fractions of purified XOS within the desired ranges of polymerization degree were characterised and their prebiotic potential was investigated in in vitro fermentations by bifidobacteria, lactobacilli and intestinal inocula. Parameters such as bacterial growth and XOS consumption were evaluated and compared with commercially available xylo-oligosaccharides. The differences observed were considered of relevance for the formulation of symbiotic preparations and the future design of targeted, tailor-made prebiotic xylo-oligosaccharides

The pros and cons of the dedicated upgrade of the hemicellulosic sugar stream in a biorefinery framework

The challenge of the future integrated biorefineries is the full economically utilization of all biomass components with the simultaneously production of fuels and chemicals, preferably of added-value. This can only be achieved by the selective fractionation of the lignocellulosic biomass into its polymeric components, thus increasing their individual upgradeability to enhance the process economics. To reach this goal, the fractionation methods used are of crucial importance. Yet, many of the most widely accepted biochemical biorefineries potential lay-outs, are mainly concerned with cellulose hydrolysis and fermentation and the hemicellulosic fractions are, at best, clamped with cellulose, averting its differential upgrade. Therefore, a change in perspective by which the fractionation processes, as well as the overall biorefinery lay-out, are thought and evaluated is needed. The objective of this work is to review, compare and discuss the main advantages and bottlenecks of the currently available biomass pre-treatment technologies, particularly those leading to the selective fractionation of hemicelluloses. The advantages and disadvantages of the methods will be analysed foreseeing the added-value products possible to obtain from the hemicellulose path, and the most relevant factors which influence both product yield and consistency. Actually, the chemical composition and structural diversity of hemicelluloses constitutes an opportunity for the production of many chemicals, which has not yet been fully exploited. The integration of potential added-value products, e.g. oligosaccharides, polyols, and enzymes in a biorefinery framework will also be presented and discussed based on data for the upgrade of agro-food industrial residues and by-products. Examples will compare the use of mild processes for the selective recovery of hemicelluloses such autohydrolysis and dilute acid hydrolysis of brewery's spent grain, wheat straw, and eucalypt wood and the biotechnological processing of the hydrolysates. It is foreseen that hemicellulose-derived chemicals will become an ever more relevant category of products, as they hold a promise of economic benefit for the biorefineries

Caracterização do investimento em TIC nos hospitais públicos portugueses: análise dos anos 2015 e 2016

Mestrado em GestãoA transformação digital e a disseminação das tecnologias de informação e comunicação (TIC) ganham especial importância face à atual conjuntura económica, desempenhando um papel chave na transformação da estratégia, estrutura, cultura e processos de uma organização. Consequentemente, no setor da saúde, este processo reveste-se de um caráter ainda mais importante, isto porque falamos de instituições de saúde, cuja natureza, estrutura e complexidade diferem de qualquer outro tipo de organização. As TIC permitem que uma instituição de saúde responda de forma mais rápida e eficaz, ajustando-se às necessidades e reinventando os processos na prestação dos serviços de saúde. Esta temática tem sido estudada, porém o interesse tem-se acentuado recentemente, havendo ainda algumas dúvidas sobre o processo e a aceitação das TIC. As TIC desempenham um papel de destaque nas organizações de saúde, sendo expectável que este papel se intensifique ainda mais. A medicina moderna, centrada nos pacientes, assente em estratégias inovadoras de prestação de cuidados de saúde, afigura o caminho que todos sabemos estar a percorrer. Neste sentido, torna-se pertinente conhecer e perceber como se caracteriza e como é feito o investimento em TIC pelos hospitais públicos portugueses, bem como as principais barreiras e facilitadores encontrados na implementação destas tecnologias.Digital transformation and the dissemination of information and communication technologies (ICT) gain special importance in the current economic environment, playing a key role in transforming organization's strategy, structure, culture and processes. Consequently, in the health sector, this process is even more important, because we are referring to health institutions, whose nature, structure and complexity differ from any other type of organization. ICT enables a health institution to respond more quickly and effectively, adjusting to needs and reinventing processes in the provision of health services. This subject has been studied, but the interest has recently been accentuated, with some doubts about the process and the acceptance of ICT. ICT plays an important role in health organizations, and it is expected that this role will intensify even more. Modern and patient-centered medicine, based on innovative health care strategies, is the path we all know we are going through. In this sense, it becomes pertinent to know and understand how ICT investment by portuguese public hospitals is characterized, as well as the main barriers and facilitators found in the implementation of these technologies

The role of ABC proteins in the mechanism of action of promising ruthenium anticancer agents

Tese de mestrado em Química, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017O cancro é um termo genérico para um vasto grupo de doenças que podem afetar qualquer parte do nosso corpo. Esta doença é definida pela proliferação anormal de células. Estas células anómalas podem invadir outros tecidos e órgãos formando assim metástases. O cancro, considerado uma doença mundial e que afeta diversas faixas etárias, continua a ser uma preocupação para a população e, nomeadamente, para os cientistas. A investigação nesta área já é longa e felizmente conta já com importantes avanços. No entanto, apesar de todos os progressos, continuam a existir obstáculos para o tratamento cem por cento eficaz. Um desses obstáculos é a resistência das células cancerígenas aos fármacos, o que limita consideravelmente a eficácia dos mesmos. Esta resistência deve-se a vários fatores sendo, um deles, a existência de um tipo de proteínas transportadoras, denominadas transportadores ABC, que se encontram sobre expressas nas células cancerígenas e que atuam sobre os fármacos levando ao seu rápido efluxo para fora da célula limitando, assim, a sua capacidade de ação sobre as células cancerígenas. A resistência a fármacos refere-se à capacidade das células cancerígenas para resistirem a uma variedade estrutural de fármacos anticancerígenos, levando a um dos maiores problemas da quimioterapia. Na realidade, este tipo de resistência é responsável pelo fracasso de mais de 90 % dos tratamentos em cancro. A família ABC (ATP binding cassette) é constituída por várias proteínas, sendo que atualmente as mais conhecidas, e aqui estudadas são: P-gp ou ABCB1, MRP1 ou ABCC1, MRP2 ou ABCC2 e ABCG2 ou BCRP. Apesar de existirem várias teorias que procuram explicar os seus mecanismos de ação, a certeza é que estas proteínas transportadoras permitem a expulsão dos fármacos, aumentando, em consequência, a resistências das células cancerígenas a estes fármacos. Os estudos de elucidação dos mecanismos bioquímicos que permitem combater esta resistência aos fármacos têm-se centrado principalmente na identificação de inibidores seletivos destas proteínas que bloqueiem a passagem dos fármacos para o exterior da célula cancerígena. A maior limitação até agora tem sido encontrar inibidores específicos para cada transportador, que ao mesmo tempo apresentem baixa citotoxicidade para células saudáveis e de alta eficiência. Por isso, a investigação nesta área continua a ser uma prioridade. Foi neste âmbito que o Laboratorio de Química Organometálica da Faculdade de Ciências da Universidade de Lisboa, Portugal, juntamente com o “Drug Resistance and Membrane Proteins team” em Lyon, França, avaliou, durante a realização desta tese de Mestrado, o papel que diversos transportadores ABC têm no mecanismo de ação de uma família de complexos organometálicos de ruténio ciclopentadienilo, “RuCp” (Cp = η5-C5H5). Foram estudados sete compostos, todos contendo o fragmento ‘Ru(η5-CpR)(PPh3)(bipiridina-R)’, com potencial atividade anticancerígena e anteriormente desenvolvidos pelo Laboratório de Química Organometálica. Entre eles, encontram-se os compostos de ruténio-polímero PMC78 e PMC85 que foram escolhidos devido ao seu elevado peso molecular que permite uma maior facilidade de acumulação destes compostos no interior das células pelo efeito de EPR (“enhanced permeation and retention effect”). Para além disso, estes compostos revelaram melhores citotoxicidades que a cisplatina para as linhas celulares do ovário A2780 e mama MCF7 e MDA-MB-231, e parecem ser capazes de ultrapassar os mecanismos de resistência de células cancerígenas (resultados obtidos por comparação entre a linha celular A2780 sensível e A2780CisR, resistente à cisplatina) . Para além destes compostos, foi também escolhido o composto PMC79, composto parental dos anteriores, com a mesma estrutura, mas sem as cadeias de polímero na sua estrutura. O composto PMC79 apresenta uma boa citotoxicidade relativamente à cisplatina para as mesmas linhas celulares. No entanto, para este composto o nível de acumulação nas células A2780 sensíveis foi muito superior que nas resistentes. Devido a estes resultados, o PMC79 foi também escolhido para este trabalho para se tentar perceber em maior detalhe qual o(s) transportadores ABC responsáveis por este efeito. O composto LCR134, [Ru(η5-Cp)(PPh3)(bipiridina-biotina)][CF3SO3], foi também escolhido uma vez que é baseado no PMC79, mas onde foram adicionadas duas moléculas de biotina (vitamina H ou B7) à bipiridina. A inclusão desta biomolécula poderá ser vantajosa devido à capacidade de se ligar a recetores da membrana celular das células cancerígenas. A biotina é essencial para o nosso organismo e tem sido frequentemente utilizada em diversos estudos reportando a sua facilidade de transporte para dentro das células cancerígenas. Os três compostos restantes, pertencem à subfamília de ruténio η5-metilciclopentadienilo e foram escolhidos com o objetivo de se conseguir obter uma correlação entre a sua atividade biológica e os substituintes na bipiridina. Desta forma, para se estudar o papel dos transportadores ABC no mecanismo de ação destes compostos, utilizaram-se diversas técnicas, tais como o teste de viabilidade celular para avaliar a citotoxicidade de cada composto através do cálculo do IC50, citometria de fluxo para verificar a percentagem de inibição de cada composto para os transportadores ABC, citometria de massa para quantificar a percentagem de acumulação do ruténio nas células, e docking molecular para a caracterizar a ligação de compostos ao sitio ativo da proteína P-gp. Todos os compostos obtiveram bons resultados ao nível da citotoxicidade para a linha celular cancerígena 2008C (1.1 - 4.5 μM), assim como bons níveis de internalização celular de ruténio. Os resultados obtidos permitiram concluir que compostos mesmo estruturalmente muito similares, possuem atividades biológicas distintas. Verificou-se que os compostos de ruténio-polímero, PMC78 e PMC85, são mais citotóxicos para células sobre expressas com transportadores (P-gp e MRP1, respetivamente) do que sem transportadores. O PMC78 demonstrou também que seria um bom inibidor para a P-gp. Todos estes fatores levaram a indicar que o uso do polilactídeo poderá potenciar a ação anticancerígena de compostos não poliméricos. Observou-se também que o uso do fragmento da bipiridina funcionalizada com duas moléculas de biotina poderá potenciar a capacidade anticancerígena dos compostos, visto que o complexo LCR134 revelou ser muito bom inibidor da P-gp. Cálculos de docking molecular mostram que é possível que haja competição entre o LCR134 e o conhecido substrato Rodamina 123 pelo centro ativo da P-gp . Os compostos LCR136 e RT11, pertencentes à família η5-MeCp, foram os compostos que revelaram os melhores resultados ao nível das suas atividades inibidoras e a melhor internalização para as linhas com os transportadores ABC estudados, sugerindo uma correlação entre as suas atividades e a sua internalização celular. Para além disso, revelaram melhor citotoxicidade para células sobre expressas. Os compostos PMC79 e RT12, são os compostos estruturalmente mais parecidos, onde a única diferença é a existência do grupo metil no ciclopentadienilo para o RT12. Os resultados mostraram que estes dois compostos têm atividades biológicas muito parecidas. Ambos são mais citotóxicos para as células sem sobre expressão de transportadores do que para as células sobre expressas e parecem não terem qualquer efeito inibitório para este tipo de células resistentes, contrariamente aos outros compostos estudados. Concluindo, pode-se afirmar que o grupo -CH2OH, comum aos dois compostos e que os distingue dos restantes, terá um papel importante no efluxo dos mesmos, tornando-os substratos dos transportadores ABC. Decorrente da avaliação dos estudos biológicos realizados, foi sintetizado com sucesso um novo complexo de ruténio, [Ru(η5-(Me-C5H4)(PPh3)(bipiridina-biotina)][CF3SO3] (Ru2). Este composto foi analisado por técnicas espetroscópicas como o RMN (1H, 31P, 13C e técnicas bidimensionais), UV-Vis e FT-IR, e a sua pureza foi determinada por análises elementares. O complexo revelou também adequada estabilidade em meio celular (variação menor que 5 % às 24 h) e caráter lipofílico (logPo/w= 1,6), o que nos assegurou continuação para os estudos biológicos neste novo composto. Foi então avaliado, para Ru2, a viabilidade celular nas linhas celulares utilizadas anteriormente. Contrariamente aos resultados previamente obtidos, este novo complexo de ruténio é muito menos citotóxico para NIH3T3 WT, NIH3T3-P-gp e 2008C, sendo que não é citotóxico para as outras linhas celulares estudadas. Percebe-se também que este composto é um substrato para a P-gp e não tem qualquer efeito inibitório para esta ou outra proteína transportadora. Concluindo, pode-se afirmar que a coordenação da biotina e do grupo η5-MeCp na mesma estrutura parece modificar a capacidade inibitória para P-gp e MRP2 como tinham os compostos LCR134, RT11 e LCR136. Este resultado revelou ser muito interessante, e como tal deve ser explorado em trabalhos futuros. Deste modo este trabalho apresenta pela primeira vez o estudo de novos compostos de ruténio com fragmento ‘Ru(η5-CpR)(PPh3)(bipiridina-R)’ em células sobre expressas por transportadores ABC. A descoberta de que estes complexos de ruténio são inibidores para proteínas transportadoras abre novas possibilidades relativamente aos seus mecanismos de ação. Para além disso, tal como observado para outros compostos da literatura, verificou-se que pequenas alterações estruturais desencadeiam respostas biológicas muito diferentes mostrando a importância deste tipo de estudos que relaciona a estrutura com a atividade. O objetivo deste trabalho foi então concluído com sucesso revelando que compostos de ‘Ru(η5-CpR)(PPh3)(bipiridina-R)’ poderão constituir uma ferramenta importante para o combate ao cancro, especialmente em cancros resistentes.Cancer is a global disease that affects most of the age ranges and is still one of the biggest concerns for the scientists worldwide. The research in this area is exhaustive and, fortunately, important developments are done year after year. However, there are some obstacles for the successful treatment such as multidrug resistance (MDR) that limits the drug efficacy. The main reason for this resistance lies in one type of proteins called ABC transporters. These proteins are overexpressed in cancer cell lines and allow the efflux of the drug out from the cell. P-gp or ABCB1, MRP1 or ABCC1, MRP2 or ABCC2 and ABCG2 or BCRP are the most studied proteins belonging to the ABC family. Although the transport mechanism of each pump is still missing, one thing that the scientists are sure is that these proteins are responsible for the efflux of molecules out of the cells. To try to avoid this efflux, the identification of selective inhibitors that block the drugs efflux is being explored. The main challenge of this research is to find compounds that can act as high effective inhibitors while presenting low toxicity for healthy cells. Within this frame, the Organometallic Chemistry Laboratory from Faculdade de Ciências da Universidade de Lisboa, Portugal, and the Drug Resistance and Membrane Proteins in Lyon, France, studied the role of several ABC transporters on the mechanism of action of new ruthenium cyclopentadienyl compounds “Ru(η5-Cp)”. All the complexes were cytotoxic for the cell lines overexpressed and not overexpressed with ABC transporters and also for one cancer cell line, 2008C. Four compounds (PMC78, LCR134, RT11, LCR136) exhibited specific inhibitory activity for some of the ABC transporters studied. The amount of ruthenium internalization on the cell lines was also quantified by mass cytometry (CyTOF), indicating that, in all cases, the compounds are internalized. A molecular docking study was also carried out for one of the structures (LCR134) in P-gp protein revealing that a competition between LCR134 and the P-gp substrate might happen. With the aim of optimizing the inhibitory activity of this family of compounds, a new ruthenium complex was synthesized, [Ru(η5-MeCp)(PPh3)(bipy-biot)][CF3SO3] Ru2, bearing the structural features inducing the best inhibition effects: a biotin molecule and a η5-MeCp ligand. This compound was characterized by the usual techniques (NMR, UV-Vis and IR spectroscopies) and its purity was assessed by elemental analyses. Ru2 was found to be very stable in cell medium (less than 5% variation over 24 h) and it has an hydrophobic character (logPo/w= 1.6), allowing us to carry on with the biological evaluation. The new compound was evaluated in the same cell lines as the previous compounds. Interestingly, this compound is much less cytotoxic for NIH3T3 WT, NIH3T3-P-gp and 2008C cell lines than the previously compounds studied, and is non-cytotoxic for all the other cell lines. Moreover, it seems that this compound is a substrate for P-gp pumps and does not have any inhibitory effect. To conclude, we can say that the biotin and η5-MeCp motifs in the same complex do not improve the inhibitory potential, resulting, in contrast, in the loss of the inhibitory capacity. Altogether, the proposed aims for this work were successfully achieved and allowed us to unravel an unprecedented mechanism of action for ruthenium cyclopentadienyl complexes that can be used as tool to fight the multidrug resistance in cancer

Engineered Ashbya gossypii for single-cell oil production from non-detoxified Eucalyptus bark hydrolysate

[Excerpt] Ashbya gossypii is a filamentous fungus industrially used for riboflavin production, a bioprocess in which downstream product recovery is facilitated by the ability of this fungus to undergo autolysis during the late stationary phase of growth or at low temperature [1]. In addition to riboflavin, engineered A. gossypii strains are capable of producing other compounds of interest for the food and feed industry, among which Single-Cell Oils (SCOs) from media containing mixed formulations of detoxified corn-cob hydrolysate, sugarcane molasses or crude glycerol [2]. [...]This work was supported by Compete 2020, Portugal 2020 and Lisboa 2020 through MoveToLowC (POCI-01-0247-FEDER-046117) and by FCT through the strategic funding of UIDB/04469/2020 and project ESSEntial (PTDC/BII-BTI/1858/2021).info:eu-repo/semantics/publishedVersio

Single-cell oil production by engineered Ashbya gossypii from non-detoxified lignocellulosic biomass hydrolysate

In this work, microbial lipid production from non-detoxified Eucalyptus bark hydrolysate (EBH) with oleaginous xylose-utilizing Ashbya gossypii strains was explored. The best producing strain from a set of engineered strains was identified in synthetic media mimicking the composition of the non-detoxified EBH (SM), the lipid profile was characterized, and yeast extract and corn steep liquor (CSL) were pinpointed as supplements enabling a good balance between lipid accumulation, biomass production, and autolysis by A. gossypii. The potential of the engineered A. gossypii A877 strain to produce lipids was further validated and optimized with minimally processed inhibitor-containing hydrolysate and high sugar concentration, and scaled up in a 2 L bioreactor. Lipid production from non-detoxified EBH supplemented with CSL reached a lipid titer of 1.42 g/L, paving the way for sustainable single-cell oil production within the concept of circular economy and placing lipids as an alternative by-product within microbial biorefineries.This study was supported by Compete 2020, Portugal 2020, and Lisboa 2020 through MoveToLowC (POCI-01-0247-FEDER-046117) and by the Portuguese Foundation for Science and Technology (FCT) through the strategic funding of UIDB/04469/2020 and project ESSEntial (PTDC/BII-BTI/1858/2021). The authors gratefully acknowledge RAIZ (Forest and Paper Research Institute) for providing the Eucalyptus bark material, and Novozymes A/S for supplying Cellic® CTec3 HS. The technical assistance of STEX company (Aveiro, Portugal) on the operation of pilot-scale infrastructure of steam explosion and enzymatic hydrolysis is also acknowledged.info:eu-repo/semantics/publishedVersio