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- Hydrothermal pretreatment of several lignocellulosic mixtures containing wheat straw and two hardwood residues available in Southern EuropePublication . Silva-Fernandes, Talita; Duarte, Luís C.; Carvalheiro, Florbela; Loureiro-Dias, M. Conceição; Fonseca, César; Gírio, FranciscoThis work studied the processing of biomass mixtures containing three lignocellulosic materials largely available in Southern Europe, eucalyptus residues (ER), wheat straw (WS) and olive tree pruning (OP). The mixtures were chemically characterized, and their pretreatment, by autohydrolysis, evaluated within a severity factor (log R0) ranging from 1.73 up to 4.24. A simple modeling strategy was used to optimize the autohydrolysis conditions based on the chemical characterization of the liquid fraction. The solid fraction was characterized to quantify the polysaccharide and lignin content. The pretreatment conditions for maximal saccharides recovery in the liquid fraction were at a severity range (log R0) of 3.65–3.72, independently of the mixture tested, which suggests that autohydrolysis can effectively process mixtures of lignocellulosic materials for further biochemical conversion processes.
- 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.
- 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.
- Production of glycolipid biosurfactants, mannosylerythritol lipids, from pentoses and D-glucose/D-xylose mixtures by Pseudozyma yeast strainsPublication . Faria, Nuno Torres; Santos, Marisa V.; Fernandes, Pedro; Fonseca, Luís Lopes; Fonseca, César; Ferreira, Frederico CasteloThe pentose-assimilating capacity of yeasts from the genus Pseudozyma, P. antarctica PYCC 5048T, P.aphidis PYCC 5535Tand P. rugulosa PYCC 5537T, was exploited towards the production of mannosylery-thritol lipids (MEL), a glycolipid with biosurfactant properties. The three strains tested were able togrow on d-xylose and l-arabinose with similar maximum specific growth rates to those estimated on d-glucose (around 0.2 h-1). The highest MEL titres (4.8–5.4 g/l) and yields (0.11–0.14 g/g) from d-xylosewere found in P. antarctica PYCC 5048T, which presented similar values to those estimated on D-glucose and on D-xylose/D-glucose mixtures. P. rugulosa PYCC 5537T showed a pattern of sugar conversion into MEL similar to P. antarctica, but at 40% lower titres. P. aphidis PYCC 5535T presented lower MEL titres from D-xylose (1.2 g/l) than from D-glucose (3.4 g/l). Nitrate supply increased sugar consumption rate and, when accompanied by D-glucose or D-xylose feeding, additional biomass production. In this case, sugar was completely consumed before sugar feeding at day 7, but not when feeding is performed at day 4. Higher MEL titres where obtained for the later condition reaching 7.3 g/l and 5.8 g/l, in fed-atchcultures with glucose and xylose, respectively.
- Isolation and identification of Magnusiomyces capitatus as a lipase-producing yeast from olive mill wastewaterPublication . Salgado, Vera; Fonseca, César; Silva, Teresa Lopes da; Roseiro, J. Carlos; Eusebio, AnaABSTRACT: Olive mill wastewaters (OMW) are effluents originated from olive oil extraction. As an oil-rich residue, OMW is a potential source of lipase-producing microorganisms and a complex medium potentially suitable for lipase production. The aim of the present study was to isolate yeasts with the ability to produce extracellular lipases from OMW. Thirty-two yeast isolates were obtained and screening for esterase/lipase activity using rapid plate detection methods allowed the selection of five isolates. Subsequently, extracellular lipolytic activity was determined in shake-flasks, and the best activity was found in the isolate JT5 (0.85 U/mL). This isolate was identified as Magnusiomyces capitatus by DNA sequencing. Growth and lypolytic activities by M. capitatus JT5 were assessed in undiluted OMW, and optimization of lipase production was achieved by a positive interaction of two factors (oxygen availability and nitrogen concentration). The highest lipase activity (1.4 U/mL) was obtained at NH4Cl concentration of 2.8 g/L and kLa of 0.65 min−1. The growth of M. capitatus JT5 in a stirred tank bioreactor, using undiluted OMW, allowed the improvement of lipase production (up to 3.96 U/mL) by increasing olive oil concentration in the medium, under the selected conditions of nitrogen concentration and oxygen availability. This study highlighted the isolate M. capitatus JT5 as a lipase-producing microorganism that is able to grow in undiluted OMW under controlled conditions. Results obtained in shake-flasks have been reproduced satisfactorily in the stirred tank bioreactor.
- Screening and Engineering Yeast Transporters to Improve Cellobiose Fermentation by Recombinant Saccharomyces cerevisiaePublication . Kretzer, Leonardo; Knychala, Marília; Silva, Lucca C.; Fontoura, Isadora C.C.; Leandro, Maria José; Fonseca, César; Verstrepen, Kevin J.; Stambuk, BorisABSTRACT: Developing recombinant Saccharomyces cerevisiae strains capable of transporting and fermenting cellobiose directly is a promising strategy for second-generation ethanol production from lignocellulosic biomass. In this study, we cloned and expressed in the S. cerevisiae CEN.PK2-1C strain an intracellular beta-glucosidase (SpBGL7) from Spathaspora passalidarum and co-expressed the cellobiose transporter SiHXT2.4 from Scheffersomyces illinoinensis, and two putative transporters, one from Candida tropicalis (CtCBT1 gene), and one from Meyerozyma guilliermondii (MgCBT2 gene). While all three transporters allowed cell growth on cellobiose, only the MgCBT2 permease allowed cellobiose fermentation, although cellobiose consumption was incomplete. The analysis of the beta-glucosidase and transport activities revealed that the cells stopped consuming cellobiose due to a drop in the transport activity. Since ubiquitinylation of lysine residues at the N- or C-terminal domains of the permease are involved in the endocytosis and degradation of sugar transporters, we constructed truncated versions of the permease lacking lysine residues at the C-terminal domain (MgCBT2 Delta C), and at both the C- and N-terminal domain (MgCBT2 Delta N Delta C) and co-expressed these permeases with the SpBGL7 beta-glucosidase in an industrial strain. While the strain harboring the MgCBT2 Delta C transporter continued to produce incomplete cellobiose fermentations as the wild-type MgCBT2 permease, the strain with the MgCBT2 Delta N Delta C permease was able to consume and ferment all the cellobiose present in the medium. Thus, our results highlight the importance of expressing cellobiose transporters lacking lysine at the N- and C-terminal domains for efficient cellobiose fermentation by recombinant S. cerevisiae.
- Life cycle assessment of advanced bioethanol production from pulp and paper sludgePublication . Sebastião, Diogo; Gonçalves, Margarida S.; Marques, Susana; Fonseca, César; Gírio, Francisco; Oliveira, Ana Cristina; Matos, Cristina T.This work evaluates the environmental performance of using pulp and paper sludge as feedstock for the production of second generation ethanol. An ethanol plant for converting 5400 tons of dry sludge/year was modelled and evaluated using a cradle-to-gate life cycle assessment approach. The sludge is a burden for pulp and paper mills that is mainly disposed in landfilling. The studied system allows for the valorisation of the waste, which due to its high polysaccharide content is a valuable feedstock for bioethanol production. Eleven impact categories were analysed and the results showed that enzymatic hydrolysis and neutralisation of the CaCO3 are the environmental hotspots of the system contributing up to 85% to the overall impacts. Two optimisation scenarios were evaluated: (1) using a reduced HCl amount in the neutralisation stage and (2) co-fermentation of xylose and glucose, for maximal ethanol yield. Both scenarios displayed significant environmental impact improvements.
- High cellulase-free xylanases production by Moesziomyces aphidis using low-cost carbon and nitrogen sourcesPublication . Faria, Nuno Torres; Marques, Susana; Cerejo, Joana; Vorobieva, Ekaterina; Fonseca, CésarABSTRACT: Background Enzymes involved in xylan hydrolysis have several industrial applications. Selection of efficient microbial hosts and scalable bioreaction operations can lower enzyme production costs and contribute to their commercial deployment. This work aims at investigating the Moesziomyces aphidis yeast cultivation conditions that deliver maximal xylanase titres, yields and productivities using low-cost nitrogen (N) and carbon (C) sources. Results NaNO3 and KNO3 supplementation improved xylanase production 2.9- and 2.7-fold (against 67.2 U mL(-1)), respectively, using xylan as C source. Interestingly, the use of KNO3, instead of NaNO3, results in 2- to 3-fold higher specific activity, highlighting the potassium ion role. In addition, this study investigates synergetic effects on using ionic and organic N sources. A 4.9-fold increase in xylanase production, with high specific activity, is attained combining KNO3 and corn steep liquor (CSL). Exploring the previous findings, this study reports one of the highest extracellular xylanase production titres (864.7 U mL(-1)) by yeasts, using a media formulation containing dilute-acid pre-treated brewery spent grains (BSG), as C source and inducer, supplemented with KNO3 and CSL. Replacement of dilute-acid pre-treatmed BSG by untreated BSG had low impact on xylanase production, of only 6%. Conclusion Efficient production of M. aphidis xylanolytic enzymes, using low-cost N and C sources, is attractive for deployment of on-site enzyme production targeting different biotechnological applications under circular economy and biorefinery concepts. Potential xylanases end-users include industries such as brewing (using BSG as substrate for enzyme production), pulp and paper (benefiting from the cellulase-free xylanase activity) or lignocellulosic ethanol (for cellulase supplementation).
- Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiaePublication . Knychala, Marília; Santos, Angela A. dos; Kretzer, Leonardo; Gelsleichter, Fernanda; Leandro, Maria José; Fonseca, César; Stambuk, BorisABSTRACT: In previous work, we developed a Saccharomyces cerevisiae strain (DLG-K1) lacking the main monosaccharide transporters (hxt-null) and displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities. This strain proved to be a useful chassis strain to study new glucose/xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Proteins with high amino acid sequence similarity (78-80%) to SsXUT1 were identified from Spathaspora passalidarum and Spathaspora arborariae genomes. The characterization of these putative transporter genes (SpXUT1 and SaXUT1, respectively) was performed in the same chassis strain. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested (including glucose and xylose), but after being grown in maltose, the uptake of C-14-glucose and C-14-xylose was detected. While SsXUT1 lacks lysine residues with high ubiquitinylation potential in its N-terminal domain and displays only one in its C-terminal domain, both SpXUT1 and SaXUT1 transporters have several such residues in their C-terminal domains. A truncated version of SpXUT1 gene, deprived of the respective 3 '-end, was cloned in DLG-K1 and allowed growth and fermentation in glucose or xylose. In another approach, two arrestins known to be involved in the ubiquitinylation and endocytosis of sugar transporters (ROD1 and ROG3) were knocked out, but only the rog3 mutant allowed a significant improvement of growth and fermentation in glucose when either of the XUT permeases were expressed. Therefore, for the efficient heterologous expression of monosaccharide (e.g., glucose/xylose) transporters in S. cerevisiae, we propose either the removal of lysines involved in ubiquitinylation and endocytosis or the use of chassis strains hampered in the specific mechanism of membrane protein turnover.
- Comparative life cycle assessment of first- and second-generation ethanol from sugarcane in BrazilPublication . Maga, Daniel; Thonemann, Nils; Hiebel, Markus; Sebastião, Diogo; Lopes, Tiago; Fonseca, César; Gírio, FranciscoABSTRACT: Purpose: The use of bagasse and trash from sugarcane fields in ethanol production is supposed to increase the ethanol yield per hectare, to reduce the energy demand, greenhouse gas emissions, and other environmental impacts. In this article, different technological options of ethanol production are investigated and quantified looking at potential environmental impacts. The first generation ethanol from sugarcane is compared to stand-alone second-generation ethanol as well as an integrated first- and second-generation ethanol production. Methods: The method applied for this life cycle assessment follows the ISO standards 14040/44. The data used in this life cycle assessment is mainly derived from process simulation, literature, and primary data collection. Background data was taken from databases such as GaBi and ecoinvent. The life cycle impact assessment follows the default methods at midpoint level recommended by the International Reference Life Cycle Data System. The calculations were performed using the GaBi 7 life cycle assessment software. It is assumed that 50% of sugarcane trash is recovered and used for second-generation ethanol production, whereas the other 50% remain in the field to maintain soil fertility and to prevent soil erosion. In the case of first-generation ethanol, the same amount of trash is used for energy generation. Results and discussion: The results of the life cycle impact assessment show that, compared to first-generation ethanol, secondgeneration ethanol from sugarcane in Brazil allows significant reductions in all investigated impact categories except resource depletion. Resource depletion, however, is strongly influenced by the demand for ammonium phosphate which is needed for inoculum preparation. Integrated first- and second-generation ethanol production also allows reductions in most of the environmental impacts except for global warming, photochemical ozone depletion, and resource depletion. The yield of ethanol per hectare increases since bagasse and trash are used for the production of second-generation ethanol. Consequently, the results show that agricultural land occupation is reduced for integrated first- and second-generation ethanol by approximately 11%, whereas second-generation ethanol allows reduction of land use by approximately a factor of 30. Conclusions: The use of bagasse and trash for ethanol production allows both the reduction of several environmental impacts and land use, in particular, because impacts caused by sugarcane cultivation are avoided. For the integrated first- and second generation ethanol scenario, it is important to further reduce the total energy demand in order to achieve self-sufficiency for the plant energy and to avoid additional emissions from burning fossil fuels.