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- A new biosurfactant/bioemulsifier from Gordonia alkanivorans Strain 1B: production and characterizationPublication . Silva, Tiago; Paixão, Susana M.; Tavares, João; Gil, Catia V.; Torres, Cristiana A. V.; Freitas, Filomena; Alves, LuísABSTRACT: Biosurfactants and bioemulsifiers (BS/BE) are naturally synthesized molecules, which can be used as alternatives to traditional detergents. These molecules are commonly produced by microorganisms isolated from hydrocarbon-rich environments. Gordonia alkanivorans strain 1B was originally found in such an environment, however little was known about its abilities as a BS/BE producer. The goal of this work was to access the potential of strain 1B as a BS/BE producer and perform the initial characterization of the produced compounds. It was demonstrated that strain 1B was able to synthesize lipoglycoprotein compounds with BS/BE properties, both extracellularly and adhered to the cells, without the need for a hydrophobic inducer, producing emulsion in several different hydrophobic phases. Using a crude BS/BE powder, the critical micelle concentration was determined (CMC = 16.94 mg/L), and its capacity to reduce the surface tension to a minimum of 35.63 mN/m was demonstrated, surpassing many commercial surfactants. Moreover, after dialysis, emulsification assays revealed an activity similar to that of Triton X-100 in almond and sunflower oils. In benzene, the E-24 value attained was 83.45%, which is 30% greater than that of the commercial alternative. The results obtained highlight for the presence of promising novel BS/BE produced by strain 1B.
- Energy requirement and CO2 emissions of bioH2 production from microalgal biomassPublication . Ferreira, Ana F.; Ortigueira, Joana; Alves, Luís; Gouveia, Luisa; Moura, Patrícia; Silva, Carla M.This paper presents the life cycle inventory (LCI) of hydrogen production by Clostridium butyricum fermentation of Scenedesmus obliquus hydrolysate. The main purpose of this work was to evaluate the potential of H2 production from microalgal biomass and the respective energy consumption and CO2 emissions in the bioconversion process considering the microalga production, acid hydrolysis of S. obliquus biomass, preparation of the inoculum and culture media, and fermentation. The scale-up to industrial production was not envisaged. The hydrogen yield obtained in this work was 2.9 ± 0.3 mol H2/mol sugars in S. obliquus hydrolysate. Results show that this process of biological production of hydrogen can achieve 7270 MJ/MJH2 of energy consumption and 670Kg CO2/MJH2. The microalgal culture is the stage responsible for 98% of these total final values due to the use of artificial lighting. All stages and processes with the highest values of energy consumption and CO2 emissions were identified for future energetic and environmental optimisation.
- Production and characterization of a novel yeast extracellular invertase activity towards improved dibenzothiophene biodesulfurizationPublication . Arez, B. F.; Alves, Luís; Paixão, Susana M.The main goal of this work was the production and characterization of a novel invertase activity from Zygosaccharomyces bailii strain Talf1 for further application to biodesulfurization (BDS) in order to expand the exploitable alternative carbon sources to renewable sucrose-rich feedstock. The maximum invertase activity (163 U ml.1) was achieved after 7 days of Z. bailii strain Talf1 cultivation at pH 5.5–6.0, 25 °C, and 150 rpm in Yeast Malt Broth with 25 % Jerusalem artichoke pulp as inducer substrate. The optimum pH and temperature for the crude enzyme activity were 5.5 and 50 °C, respectively, and moreover, high stability was observed at 30 °C for pH 5.5–6.5. The application of Talf1 crude invertase extract (1 %) to a BDS process by Gordonia alkanivorans strain 1B at 30 °C and pH 7.5 was carried out through a simultaneous saccharification and fermentation (SSF) approach in which 10 g l.1 sucrose and 250 ìM dibenzothiophene were used as sole carbon and sulfur sources, respectively. Growth and desulfurization profiles were evaluated and compared with those of BDS without invertase addition. Despite its lower stability at pH 7.5 (loss of activity within 24 h), Talf1 invertase was able to catalyze the full hydrolysis of 10 g l.1 sucrose in culture medium into invert sugar, contributing to a faster uptake of the monosaccharides by strain 1B during BDS. In SSF approach, the desulfurizing bacterium increased its ìmax from 0.035 to 0.070 h.1 and attained a 2-hydroxybiphenyl productivity of 5.80 ìM/h in about 3 days instead of 7 days, corresponding to an improvement of 2.6-fold in relation to the productivity obtained in BDS process without invertase addition.
- 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.
- Production of carotenoids and biosurfactants by Gordonia Alkanivorans Strain 1B using food residues and derivatives [Poster]Publication . Silva, Tiago; Paixão, Susana M.; Alves, LuísABSTRACT: Through different bioprocesses, microorganisms, such as yeasts and bacteria, ferment and transform residue streams into high added value products, such as carotenoids and biosurfactants. Gordonia alkanivorans strain 1B is one of such bacteria, capable of consuming and transforming many types of residues. It is mostly known for its biodesulfurizing ability and it was recently described as a producer of both carotenoids and biosurfactants. In previous works, strain 1B has been cultivated on different sugar rich alternative carbon sources. However, it was shown, that in order to promote surfactant production, the microorganisms should be exposed to inducing factors, such as lipids and alcohols. This work focusses on valorisation of residues from the restaurant and food industry, and derivatives from their processing, by using them as carbon sources to grow the bacterium and produce carotenoids and surfactants.
- Fermentation of xylose-rich substrates by the haloarchaeon halorhabdus utahensis towards high value-added bioproducts [Poster]Publication . Alves, Luís; Paixão, Susana M.; Silva, Tiago; Squillaci, G.; Serino, I.; Morana, A.ABSTRACT: Research that focuses on the use of high value-added bioproducts for industrial applications is essential for the implementation of sustainable approaches forecasting a bio-based economy. The effective use of biomass feedstocks, particularly lignocellulosic materials, in large-scale applications will evolve from innovative research aimed at the development and implementation of biorefineries established for specific feedstocks. In this context, an important step is the concept of fractionating biomass into its core constituents (cellulose, hemicellulose and lignin) for further enhanced valorization. Contrary to the valorization of cellulose fraction, which has been extensively studied, there is a gap in the valorization of the hemicellulose fraction (xylose- rich substrate) towards bioproducts. In this context, the present work aims to explore the ability of the haloarchaeon Halorhabdus utahensis (DSM-12940) to ferment xylose (or xylose-rich substrates) to high added-value bioproducts, such as pigments, exopolysaccharides (EPS) and polyhydroxyalkanoates (PHAs).
- A novel β-xylosidase from Anoxybacillus sp. 3M towards an improved agro-industrial residues saccharificationPublication . Marcolongo, L.; La Cara, F.; Del Monaco, G.; Paixão, Susana M.; Alves, Luís; Ionata, E.; Marques, Isabel PaulaABSTRACT: An intracellular β-xylosidase (AbXyl), fromthe thermoalkaline Anoxybacillus sp. 3M,was purified and characterized. The homodimeric enzyme (140 kDa) was optimally active at 65 °C and pH 5.5, exhibited half life of 10 h at 60 °C, 78 and 88% residual activity after 24 h, at pH 4.5 and 8.0, respectively. Fe2+, Cu2+, Al3+, Ag+ and Hg2+inhibited the enzyme; the activity was moderately stimulated by SDS and not influenced by β-mercaptoethanol. In the presence of p-nitrophenyl-β-D-xylopyranoside, AbXyl exhibited Km of 0.19 mM, Kcat of 453.29 s−1, KcatKm−1 of 2322 s−1mMandwas moderately influenced by xylose (Ki 21.25mM). The enzyme hydrolyzed xylo-oligomers into xylose and catalyzed transxylosilation reactions also in presence of alcohols as acceptors, producing xylo-oligosaccharides and alkyl-xylosides. Finally AbXyl was applied towards a statistically optimized process of brewery's spent grain bioconversion, highlighting the important role of this biocatalyst in reaching high yields of fermentable sugars.
- Ability of Gordonia alkanivorans strain 1B for enhanced desulfurization of dibenzothiophene and its derivatives using fructose as carbon sourcePublication . Alves, Luís; Silva, Tiago; Fernandes, A. S.; Paixão, Susana M.In order to keep up the strict sulfur limits on fossil fuels and their derivatives, refineries commonly use a desulfurization method, which combines high temperatures and pressures with molecular hydrogen known as hydrodesulfurization (HDS). However, the effectiveness of HDS to desulfurize recalcitrant organic aromatic compounds such as dibenzothiophene (DBT) or its derivatives is low. Biodesulfurization (BDS) has been described as a promising complementary technique to HDS. Using microorganisms, BDS is able of desulfurize several recalcitrant compounds usually present in fossil fuels at mild temperatures and pressures without hydrogen, making it a simple and eco-friendly process. In this context and based in the fructophilic behavior of the desulfurizing bacterium, Gordonia alkanivorans strain 1B, several recalcitrant sulfur sources were tested in BDS assays using fructose as carbon source. So, strain 1B was used in desulfurization assays testing 4-mDBT, 4,6-dmDBT and 4,6-deDBT, as sulfur source, in comparison with DBT. Growth and desulfurization kinetics using the different sulfur sources were evaluated and the desulfurization rates were determined by GC analysis of x-DBT consumed. The results showed that the strain 1B using fructose as carbon source was able to fully desulfurize all the sulfur compounds tested in less than 121 hours. For 4-mDBT, 4,6-dmDBT and 4,6-deDBT the maximal bacterial growth rates obtained were 0.072 h-1, 0.069 h-1 and 0.095 h-1 with maximum desulfurization rates of 1.58, 4.84 and 4.30 umol g(DCW)-1 h-1, respectively. In comparison with previous results obtained for max of strain 1B in glucose as carbon source and DBT as sulfur source (0.025 h-1), all the m_ max obtained in this study highlight once more the importance of use fructose as carbon source, independently of sulfur source. In addition, contrary to what has been described for other strains, the desulfurization rates obtained for the compounds with two alkyl groups were higher than for DBT (2.12 umol g(DCW)-1 h-1). In fructose, the desulfurization of 4,6-dmDBT and 4,6-deDBT by strain 1B were more than 2-fold in comparison with that for DBT. These promising results indicate the high potential of use this bacterium towards fossil fuels BDS.
- 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.
- Advances in the reduction of the costs inherent to fossil fuels’ biodesulfurization towards its potential industrial applicationPublication . Paixão, Susana M.; Arez, B. F.; Silva, Tiago; Alves, LuísBiodesulfurization (BDS) process consists on the use of microorganisms for the removal of sulfur from fossil fuels. Through BDS it is possible to treat most of the organosulfur compounds recalcitrant to the conventional hydrodesulfurization (HDS), the petroleum industry’s solution, at mild operating conditions, without the need for molecular hydrogen or metal catalysts. This technique results in lower emissions, smaller residue production and less energy consumption, which makes BDS an eco-friendly process that can complement HDS making it more efficient. BDS has been extensively studied and much is already known about the process. Clearly, BDS presents advantages as a complementary technique to HDS; however its commercial use has been delayed by several limitations both upstream and downstream the process. This study will comprehensively review and discuss key issues, like reduction of the BDS costs, advances and/or challenges for a competitive BDS towards its potential industrial application aiming ultra low sulfur fuels.