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- Identification and characterisation of xylanolytic yeasts isolated from decaying wood and sugarcane bagasse in BrazilPublication . Lara, Carla A.; Santos, Renata O.; Cadete, Raquel, M.; Ferreira, Carla; Marques, SusanaIn this study, yeasts associated with lignocellulosic materials in Brazil, including decaying wood and sugarcane bagasse, were isolated, and their ability to produce xylanolytic enzymes was investigated. A total of 358 yeast isolates were obtained, with 198 strains isolated from decaying wood and 160 strains isolated from decaying sugarcane bagasse samples. Seventy-five isolates possessed xylanase activity in solid medium and were identified as belonging to nine species: Candida intermedia, C. tropicalis, Meyerozyma guilliermondii, Scheffersomyces shehatae, Sugiyamaella smithiae, Cryptococcus diffluens, Cr. heveanensis, Cr. laurentii and Trichosporon mycotoxinivorans. Twenty-one isolates were further screened for total xylanase activity in liquid medium with xylan, and five xylanolytic yeasts were selected for further characterization, which included quantitative analysis of growth in xylan and xylose and xylanase and ß-d-xylosidase activities. The yeasts showing the highest growth rate and cell density in xylan, Cr. laurentii UFMG-HB-48, Su. smithiae UFMG-HM-80.1 and Sc. shehatae UFMG-HM-9.1a, were, simultaneously, those exhibiting higher xylanase activity. Xylan induced the highest level of (extracellular) xylanase activity in Cr. laurentii UFMG-HB-48 and the highest level of (intracellular, extracellular and membrane-associated) ß-d-xylosidase activity in Su. smithiae UFMG-HM-80.1. Also, significant ß-d-xylosidase levels were detected in xylan-induced cultures of Cr. laurentii UFMG-HB-48 and Sc. shehatae UFMG-HM-9.1a, mainly in extracellular and intracellular spaces, respectively. Under xylose induction, Cr. laurentii UFMG-HB-48 showed the highest intracellular ß-d-xylosidase activity among all the yeast tested. C. tropicalis UFMG-HB 93a showed its higher (intracellular) ß-d-xylosidase activity under xylose induction and higher at 30 °C than at 50 °C. This study revealed different xylanolytic abilities and strategies in yeasts to metabolise xylan and/or its hydrolysis products (xylo-oligosaccharides and xylose). Xylanolytic yeasts are able to secrete xylanolytic enzymes mainly when induced by xylan and present different strategies (intra- and/or extracellular hydrolysis) for the metabolism of xylo-oligosaccharides. Some of the unique xylanolytic traits identified here should be further explored for their applicability in specific biotechnological processes.
- Admissibility Grid to Support the Decision for the Preferential Routing of Portuguese Endogenous Waste Biomass for the Production of Biogas, Advanced Biofuels, Electricity and HeatPublication . Crujeira, Teresa; Trancoso, Maria Ascensão; Eusebio, Ana; Oliveira, Ana Cristina; Passarinho, Paula; Abreu, Mariana; Marques, Isabel Paula; Marques, Paula; Marques, Susana; Albergaria, Helena; Pinto, Filomena; Costa, Paula; Andre, Rui N.; Girio, Francisco; Moura, PatríciaABSTRACT: A methodology was developed to assess the allocation of different types of endogenous waste biomass to eight technologies for producing electricity, heat, biogas and advanced biofuels. It was based on the identification of key physicochemical parameters for each conversion process and the definition of limit values for each parameter, applied to two different matrices of waste biomass. This enabled the creation of one Admissibility Grid with target values per type of waste biomass and conversion technology, applicable to a decision process in the routing to energy production. The construction of the grid was based on the evaluation of 24 types of waste biomass, corresponding to 48 sets of samples tested, for which a detailed physicochemical characterization and an admissibility assessment were made. The samples were collected from Municipal Solid Waste treatment facilities, sewage sludges, agro-industrial companies, poultry farms, and pulp and paper industries. The conversion technologies and energy products considered were (trans)esterification to fatty acid methyl esters, anaerobic digestion to methane, fermentation to bioethanol, dark fermentation to biohydrogen, combustion to electricity and heat, gasification to syngas, and pyrolysis and hydrothermal liquefaction to bio-oils. The validation of the Admissibility Grid was based on the determination of conversion rates and product yields over 23 case studies that were selected according to the best combinations of waste biomass type versus technological solution and energy product.
- Correction: Correia et al. Sustainability Assessment of 2G Bioethanol Production from Residual Lignocellulosic Biomass. Processes 2024, 12, 987Publication . Correia, Bárbara; Matos, Henrique A.; Lopes, Tiago; Marques, Susana; Gírio, FranciscoError in Table: In the original publication [1], there was a mistake in Table 8 as published. The presented cost of purchased equipment and Total CAPEX cost shall not differ between the two scenarios. The purchased equipment cost and Total CAPEX cost should be, for both scenarios, 21.35 and 75.66M€, respectively. The corrected Table 8 appears below. The authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated.
- Efficient conversion of agricultural and forest residues into bioethanol: BIOFLEXPOR as flexible technology towards sugar-based biorefineries [Poster]Publication . Marques, Susana; Paixão, Susana M.; Alves, Luís; Gomes, Miguel; Eusebio, Ana; Lopes, Tiago; Coelho, Lucas; Diebold, Eduardo; Gírio, FranciscoABSTRACT: Lignocellulosic ethanol is in the upfront of advanced biofuels to be commercialized worldwide. However, the commercial deployment of 2G ethanol is dependent of high biomass availability and cost-effective supply. In Europe, some agricultural residues are presently underused and constitute attractive renewable resources. In addition, residual forest biomass, non-seasonably available at low cost, might be complementarily used as raw material boosting the economy of biorefineries. In this context, the present work deals with the development of an innovative and sustainable technological strategy to produce advanced bioethanol using agricultural and forestry residual biomass. The bioprocess involves enzymatic hydrolysis of major lignocellulose polysaccharides (cellulose and xylan) with commercial enzymes and fermentation of the resulting sugars. A pre-treatment step should firstly be accomplished to make cellulose more amenable to hydrolytic enzymes, and the prototype is based on a proprietary non-catalysed steam explosion technology, i.e., without the addition of acids and using only high-pressure steam, called FLEXBIO™, which was initially developed in Brazil by the company STEX and since 2019 in partnership with LNEG. The proposed technology has been successfully demonstrated in a relevant environment (TRL 5) for the efficient conversion of corn stover, olive tree pruning and eucalyptus-based forest residual biomass, yielding close to 150 L of ethanol per metric tonne (dry basis) of biomass, corresponding to an overall yield close to 75% of maximal theoretical yield for glucan conversion. Both enzymatic hydrolysis and fermentation steps have achieved yields superior to 85% of the maximal theoretical conversion, and the optimization of process configuration, targeting the best integration with pre-treatment, is now under progress and higher yields will be expected. Given the higher xylan content of corn stover, both cellulose and xylan fractions are pursued. In addition, the upgrading potential of all wastewater streams will also be assessed, by studying the feasibility of its combined use to increase the ethanol yield as alternative to its use for biogas production through anaerobic digestion, with the goal to reach near-zero waste. In conclusion, the present study reveals the industrial potential of this flexible technology that might be applied to implement distinct small-scale sugar-based biorefineries by converting several lignocellulosic raw materials into distinct marketable biofuels/biomaterials, promoting the circular bioeconomy.
- Sustainability Assessment of 2G Bioethanol Production from Residual Lignocellulosic BiomassPublication . Correia, Bárbara; Matos, Henrique A.; Lopes, Tiago; Marques, Susana; Gírio, FranciscoABSTRACT: The development of sustainable biofuels can help to reduce the reliance on fossil fuels and mitigate the impact of climate change. This study analyzes bioethanol production from agro-forestry residual biomass, namely eucalyptus residues and corn stover. The study includes process simulation using Aspen Plus software, followed by economic analysis and life cycle assessment (LCA) with the help of SimaPro software and by applying the environmental footprint (EF) 3.0 method. The economic analysis on the biorefinery’s economic viability, equipment, and production costs reveals a positive decision for bioethanol production from eucalyptus residues due to logistical and transportation costs. The minimum ethanol selling price (MESP) obtained was 2.19 €/L and 2.45 €/L for eucalyptus residues and corn stover, respectively. From the LCA with a functional unit of 1 MJ of ethanol, bioethanol production from eucalyptus residues results in a single score impact of 37.86 µPt, whereas for corn stover, it is 33.47 µPt. In the climate change impact category, the eucalyptus residues scenario has an impact of 0.264 kg CO2 eq/MJ ethanol while corn stover leads to 0.254 kg CO2 eq/MJ ethanol. In-situ enzyme production, heat integration, and the use of renewable energy sources were also analyzed. Combining in situ enzyme production with renewable energy sources lowers CO2 equivalent emissions by 89% for both feedstocks, in comparison to the base-case scenario.
- Conversion of cellulosic materials into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma spp. under SHF and SSF processesPublication . Faria, Nuno Torres; Santos, Marisa V.; Ferreira, Carla; Marques, Susana; Ferreira, Frederico Castelo; Fonseca, CésarBackground: Mannosylerythritol lipids (MEL) are glycolipids with unique biosurfactant properties and are produced by Pseudozyma spp. from different substrates, preferably vegetable oils, but also sugars, glycerol or hydrocarbons. However, solvent intensive downstream processing and the relatively high prices of raw materials currently used for MEL production are drawbacks in its sustainable commercial deployment. The present work aims to demonstrate MEL production from cellulosic materials and investigate the requirements and consequences of combining commercial cellulolytic enzymes and Pseudozyma spp. under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes. Results: MEL was produced from cellulosic substrates, Avicel® as reference (>99% cellulose) and hydrothermally pretreated wheat straw, using commercial cellulolytic enzymes (Celluclast 1.5 L® and Novozyme 188®) and Pseudozyma antarctica PYCC 5048T or Pseudozyma aphidis PYCC 5535T. The strategies included SHF, SSF and fed-batch SSF with pre-hydrolysis. While SSF was isothermal at 28°C, in SHF and fed-batch SSF, yeast fermentation was preceded by an enzymatic (pre-)hydrolysis step at 50°C for 48 h. Pseudozyma antarctica showed the highest MEL yields from both cellulosic substrates, reaching titres of 4.0 and 1.4 g/l by SHF of Avicel® and wheat straw (40 g/l glucan), respectively, using enzymes at low dosage (3.6 and 8.5 FPU/gglucan at 28°C and 50°C, respectively) with prior dialysis. Higher MEL titres were obtained by fed-batch SSF with pre-hydrolysis, reaching 4.5 and 2.5 g/l from Avicel® and wheat straw (80 g/l glucan), respectively. Conclusions: This work reports for the first time MEL production from cellulosic materials. The process was successfully performed through SHF, SSF or Fed-batch SSF, requiring, for maximal performance, dialysed commercial cellulolytic enzymes. The use of inexpensive lignocellulosic substrates associated to straightforward downstream processing from sugary broths is expected to have a great impact in the economy of MEL production for the biosurfactant market, inasmuch as low enzyme dosage is sufficient for good systems performance.
- Biological upgrading of wastes from the pulp and paper industryPublication . Marques, Susana; Alves, Luís; Gírio, Francisco; Santos, J. A. L.; Roseiro, J. CarlosA process for biological upgrading of recycled paper sludge (RPS) was developed based on the enzymatic hydrolysis of major sludge components (cellulose and xylan) with commercial enzymes and fermentation of the resulting sugars into ethanol or lactic acid with adequate microbial strains. The process was implemented performing both steps sequentially (SHF) or simultaneously (SSF). Cellulosic and hemicellulosic fractions of RPS were completely converted by enzymatic hydrolysis (using Celluclast®1.5L with Novozym®188) into the constitutive glucose and xylose. Ethanol was produced from the RPS hydrolysate by the yeast Pichia stipitis CBS 5773. A slightly higher conversion yield was attained on SHF process, corresponding to an ethanol concentration of 19.6 g L-1, but 179 hours were needed. The SSF process was completed after 48 hours of incubation allowing the production of 18.6 g L-1 of ethanol from 178.6 g L-1 of dried RPS, corresponding to an overall conversion yield of 51% of the available carbohydrates on the initial substrate. Maximum production of lactic acid (LA) with Lactobacillus rhamnosus ATCC 7469 was obtained by performing the SSF process: 73 g L-1 of LA was achieved, corresponding to a maximum productivity of 2.9 g L-1 h-1, with 0.97 g LA produced per g of carbohydrates on initial sludge. The present results demonstrate the feasibility of the biological conversion of the ultimate waste obtained in the paper recycling loop into a biofuel (bioethanol) or an important chemical intermediate (LA, precursor of bioplastics), under the concept of a multi-purpose biorefinery.
- Kinetics of the Release Sugars from the Enzymatic and Physico-Chemical Pre-treated Sugarcane Bagasse and Residual Forest BiomassPublication . Codato-Zumpano, Carolina Brito; Gírio, Francisco; Carvalheiro, Florbela; Marques, Susana; Ceccato-Antonini, Sandra Regina; Bastos, ReinaldoABSTRACT: Several pre-treatments are used to release sugars from lignocellulosic materials that are used to produce second-generation ethanol (2G). This study aimed to evaluate the kinetic release of glucose and xylose through the enzymatic and physical treatments of sugarcane bagasse and residual forest biomass, focusing on the ratio between hexose and pentose. Enzymatic hydrolysis after hydrothermal pre-treatment under different conditions, at 170, 170 and 190 degrees C, 170 and 190 degrees C with sulfuric acid, and 170 and 190 degrees C with the Organosolv solvent, all of them for 10 min, were performed with sugarcane bagasse and residual forest biomass, and the kinetic parameters of sugar release were evaluated. The results indicated that compared to hydrothermal and combined hydrothermal and dilute acid hydrolysis, organosolvation process led to higher release of glucose in hydrolysates from both biomasses, with a maximum yield of 14.12 and 33.33 g L-1, respectively. On the other hand, the highest glucose/xylose ratio (about 19), which will facilitate its subsequent use for fermentation, was obtained from sugarcane bagasse after hydrothermal treatment at 170 and 190 degrees C. This ratio was higher for all treatments when compared to untreated biomass, which indicated that temperature and acid affected xylose instead of glucose.
- Biorefining strategy for maximal monosaccharide recovery from three different feedstocks: eucalyptus residues, wheat straw and olive tree pruningPublication . Silva-Fernandes, Talita; Duarte, Luís C.; Carvalheiro, Florbela; Marques, Susana; Loureiro-Dias, M. Conceição; Fonseca, César; Gírio, FranciscoThis work proposes the biorefining of eucalyptus residues (ER), wheat straw (WS) and olive tree pruning (OP) combining hydrothermal pretreatment (autohydrolysis) with acid post-hydrolysis of the liquid fraction and enzymatic hydrolysis of the solid fraction towards maximal recovery of monosaccharides from those lignocellulose materials. Autohydrolysis of ER, WS and OP was performed under non-isothermal conditions (195–230 C) and the non-cellulosic saccharides were recovered in the liquid fraction while cellulose and lignin remained in the solid fraction. The acid post-hydrolysis of the soluble oligosaccharides was studied by optimizing sulfuric acid concentration (1–4% w/w) and reaction time (10–60 min), employing a factorial (22) experimental design. The solids resulting from pretreatment were submitted to enzymatic hydrolysis by applying commercial cellulolytic enzymes Celluclast1.5 L and Novozyme 188 (0.225 and 0.025 g/g solid, respectively). This strategy provides high total monosaccharide recovery or high glucose recovery from lignocellulosic materials, depending on the autohydrolysis conditions applied.
- Monitoring Yeast Cultures Grown on Corn Stover Hydrolysate for Lipid ProductionPublication . Fontes, Afonso; Francisco, Ricardo; Ferreira, Frederico Castelo; Faria, Nuno Torres; Marques, Susana; Reis, Alberto; Moura, Patrícia; Lukasik, Rafal M.; Santos, José A. L.; Silva, Teresa Lopes daABSTRACT: Microbial oils can be used as an alternative sustainable and renewable feedstock to fossil reserves for producing lubricants and polyurethane materials. Two oleaginous yeasts were grown on non-detoxified corn stover hydrolysate supplemented with corn steep liquor and mineral medium in shake flasks. Trichosporon oleaginosus DSM 11815 displayed the highest lipid production. This strain was further cultivated in a bench bioreactor, using the same culture medium, under a batch regime. Flow cytometry was used to monitor the T. oleaginosus culture using the dual staining technique (SYBR Green and PI) for cell membrane integrity detection. Values of 42.28% (w/w) and 0.06 g/Lh lipid content and lipid productivity, respectively, were recorded for T. oleaginosus cultivated in the bench bioreactor operated under a batch regime. During the cultivation, most of the yeast cells maintained their integrity. T. oleaginosus has the potential to be used as an oil microbial source for a wide range of industrial applications. In addition, it is robust in adverse conditions such as lignocellulosic hydrolysate exposure and oxygen-limiting conditions. Flow cytometry is a powerful and useful tool for monitoring yeast cultivations on lignocellulosic hydrolysates for cell count, size, granularity, and membrane integrity detection.
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