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- D-lactic acid production from hydrothermally pretreated, alkali delignified and enzymatically saccharified rockrose with the metabolic engineered Escherichia coli strain JU15Publication . Fernandes, M. C.; Alves Ferreira Caturra, Júnia Aparecida; Duarte, Luís C.; Pereira, Helena; Carvalheiro, Florbela; Martinez, AlfredoABSTRACT: Rockrose lignocellulosic residues (RR) were selectively fractionated for hemicellulose separation using autohydrolysis, followed by an alkaline treatment to solubilize the lignin. The cellulose-enriched solids were used to study the effect of solid loading (SL: 2-10%) and enzyme dosage (ED: 6.34-23.66 FPU/g dry biomass) on saccharification using a Doehlert experimental design, followed by fermentation with the metabolic engineered Escherichia coli strain JU15 to produce D-lactic acid (DLA). Pretreatment increased glucan content and enzymatic digestibility up to 84%. A significant positive effect of SL and ED was found for glucose production, but SL negatively impacted glucose yield. DLA concentrations and productivity varied from 8.85 to 32.98 g/L and 1.11 to 2.17 g/(Lh), respectively. Overall process efficiency strongly depended on saccharification yield and varied from 33 to 71%. These results indicate that sequential autohydrolysis, delignification, and fermentation of RR may be a potential relevant strategy for D-lactic production in the biorefinery framework.
- Production of Oligosaccharides from Pine Nut Shells by AutohydrolysisPublication . Torrado, Ivone; Dionísio, Ana; Fernandes, M. C.; Roseiro, Luisa B.; Carvalheiro, Florbela; Pereira, Helena; Duarte, Luís C.ABSTRACT: Pinus pinea nuts are commercial relevant Mediterranean edible forest nuts, with an increasing production and market value, whose industrial processing yields a lignocellulosic by-product, the pine nut shells, currently only used for combustion. Little research has been done on pine nut shells that could support a value-added application for this residue. This work studies for the first time the production of oligosaccharides by autohydrosis, and aims at an integrated upgrade within the biorefinery framework. Autohydrolysis was explored in the temperature range between 150 and 230 degrees C (corresponding to severity factors 2.13-4.63). Oligosaccharides, mainly xylo-oligosaccharides (95% of the total), were the key soluble products, reaching 28.7 g/100 g of xylan of the feedstock at the optimal conditions (log R-0 4.01). Other products were monosaccharides and phenolic compounds that reached 7.8 and 4.7 g/L, respectively, under the most severe conditions. The stability of the oligosaccharides at different temperatures (room, 37 degrees C and 100 degrees C) and pH (between 1 and 11) grant them significant market potential in the food and pharma sectors. The pre-treated pine nut shells by autohydrolysis presented an improved, although low, enzymatic digestibility (14%), and an improved high-heating value, therefore advising their further valorization by thermochemical pathways.
- Cistus ladanifer as a Potential Feedstock for Biorefineries: A ReviewPublication . Alves Ferreira Caturra, Júnia Aparecida; Duarte, Luís C.; Fernandes, M. C.; Pereira, Helena; Carvalheiro, FlorbelaABSTRACT: Cistus ladanifer (rockrose) is a widespread shrub species in the Mediterranean region well known due to its production of labdanum gum, especially in the hot season. Its leaves and branches can be subjected to different extraction and distillation processes to produce various types of extracts. The natural extracts of C. ladanifer have several applications, especially in the perfumery and cosmetics sector. C. ladanifer extracts, in addition to presenting interesting odoriferous properties, are also known for their bioactive properties, such as antioxidant and antimicrobial. Use of this species in animal feed or phytostabilisation of mining areas has also been successfully applied. On the other hand, the lignin and polysaccharides that are the major fractions from Cistus residues can be relevant sources of high-value products in a biorefinery framework. Recently, it has been reported that the residues obtained from the essential oil industry can sustain production of significant amounts of other marketable products, namely phenolic compounds, oligomeric and monomeric sugars, lignin, and lactic acid. All these applications show the potential of C. ladanifer as a raw material to be fully valued in a biorefinery context, contributing to important revenues and generating an associated marketable biobased product portfolio.
- D-Lactic acid production from Cistus ladanifer residues: Co-fermentation of pentoses and hexoses by Escherichia coli JU15Publication . Alves Ferreira Caturra, Júnia Aparecida; Carvalheiro, Florbela; Duarte, Luís C.; Ferreira, Ana R. P.; Martinez, Alfredo; Pereira, Helena; Fernandes, M. C.ABSTRACT: 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.
- Delignification of Cistus ladanifer Biomass by Organosolv and Alkali ProcessesPublication . Alves Ferreira Caturra, Júnia Aparecida; Lourenço, Ana; Morgado, Francisca; Duarte, Luís C.; Roseiro, Luisa B.; Fernandes, M. C.; Pereira, Helena; Carvalheiro, FlorbelaABSTRACT: Residues of Cistus ladanifer obtained after commercial steam distillation for essential oil production were evaluated to produce cellulose enriched solids and added-value lignin-derived compounds. The delignification of extracted (CLRext) and extracted and hydrothermally pretreated biomass (CLRtreat) was studied using two organosolv processes, ethanol/water mixtures (EO), and alkali-catalyzed glycerol (AGO), and by an alkali (sodium hydroxide) process (ASP) under different reaction conditions. The phenolic composition of soluble lignin was determined by capillary zone electrophoresis and by Py-GC/MS, which was also used to establish the monomeric composition of both the delignified solids and isolated lignin. The enzymatic saccharification of the delignified solids was also evaluated. The ASP (4% NaOH, 2 h) lead to both the highest delignification and enzymatic saccharification (87% and 79%, respectively). A delignification of 76% and enzymatic hydrolysis yields of 72% were obtained for AGO (4% NaOH) while EO processes led to lower delignification (maximum lignin removal 29%). The residual lignin in the delignified solids were enriched in G- and H-units, with S-units being preferentially removed. The main phenolics present in the ASP and AGO liquors were vanillic acid and epicatechin, while gallic acid was the main phenolic in the EO liquors. The results showed that C. ladanifer residues can be a biomass source for the production of lignin-derivatives and glucan-rich solids to be further used in bioconversion processes.
- Evaluation of different fractionation methods for the simultaneous protein and carbohydrate extraction from microalgaePublication . Martins, Pedro L.; Duarte, Luís C.; Pereira, Helena; Reis, Alberto; Carvalheiro, FlorbelaABSTRACT: The production of high-value products from microalgae, one of the preferred emerging biorefineries' feedstocks, relies on the crucial step of biomass fractionation. In this work, the fractionation of Chlorella vulgaris and Scenedesmus obliquus biomass was tested for protein extraction using a wide range of physical, chemical, and enzymatic treatment combinations, including ultrasound, cell homogenizer, cellulase, and alcalase combinations in aqueous and alkali extraction conditions. The impact of these processes on biomass carbohydrates was also evaluated. Alkaline-assisted ultrasound treatments using alcalase presented the highest protein extraction yield, reaching 90 g/100 g protein on C. vulgaris, closely followed by the same treatment in aqueous conditions (85 g/100 g protein). The same aqueous treatment achieved the best performance on S. obliquus, reaching 82 g/100 g protein. All treatments on both microalgae partially solubilized the polysaccharide fraction with all alkaline treatments solubilizing over 50 g/100 g sugars for all conditions. Overall, all the treatments applied were effective methods for biomass fractionation, although they showed low selectivity regarding the individual extraction of protein or carbohydrates.
- Microwave-assisted hydrothermal processing of pine nut shells for oligosaccharide productionPublication . Torrado, Ivone; Neves, Beatriz Guapo; Fernandes, M. C.; Carvalheiro, Florbela; Pereira, Helena; Duarte, Luís C.ABSTRACT: Pine nut shells, a biomass residue from the Mediterranean Pinus pinea pine nut industrial processing, were treated by microwave-assisted autohydrolysis to produce xylo-oligosaccharides. Microwave-assisted processes provide alternative heating that may reduce energy input and increase overall process efficiency. The autohydrolysis treatments were performed under isothermal and non-isothermal operations within a wide range of operational conditions (temperature/reaction times) covering several severity regimes (as measured by the log R-0 severity factor). The composition of the autohydrolysis liquors was determined in terms of oligo- and monosaccharides, aliphatic acids and degradation compounds. The process was highly selective towards hemicelluloses hydrolysis and liquid streams containing a mixture of oligomeric compounds (mainly xylo-oligosaccharides) could be obtained under relatively mild operation conditions (190 degrees C, 30 min) with a maximal oligosaccharides' concentration of 18.48 g/L. The average polymerization degree of the obtained oligosaccharides was characterised by HPLC, showing that for the optimal conditions a mixture of oligomers with DPs from 2 to 6.