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- 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.
- 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.
- 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.
- Energetic and environmental evaluation of microalgae biomass fermentation for biohydrogen productionPublication . Ferreira, Ana F.; Ortigueira, Joana; Alves, Luís; Gouveia, Luisa; Moura, Patrícia; Silva, Carla M.This paper presents an energetic and environmental evaluation of the fermentative hydrogen production from the sugars of Scenedesmus obliquus biomass hydrolysate by Clostridium butyricum. The main purpose of this work was to evaluate the potential of H2 production and respective energy consumptions and CO2 emissions in the global fermentation process: hydrolysis of S. obliquus biomass, preparation of the fermentation medium, degasification and incubation. The scale-up to industrial production was not envisaged. Energy consumption and CO2 emissions estimations were based on SimaPro 7.1 software for the preparation of the fermentation medium and the use of degasification gas, nitrogen. The functional unit of energy consumption and CO2 emissions was defined as MJ and grams per 1 MJ of H2 produced, respectively. The electricity consumed in all hydrogen processes was assumed to be generated from the Portuguese electricity production mix. 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 consumed 281-405 MJ/MJH2 of energy and emitted 24-29 kgCO2/ MJH2. The fermentation stages with the highest values of energy consumption and CO2 emissions were identified for future energetic and environmental process optimisation.
- Properties of Anoxybacillus sp. 3M Xylanases and further application towards sugar rich hydrolysatesPublication . Alves, Luís; Ladeira, S. A.; Arez, B. F.; Paixão, Susana M.This study aimed to optimize the production of xylanases by Anoxybacillus sp. strain 3M, a thermophilic bacterium isolated from terrestrial hot springs (temperature of 90°C) samples collected on S. Miguel, Azores, Portugal, in batch fermentation testing several agroindustrial byproducts as inducer substrates (BSG - Brewer's spent grain, wheat straw, sugarcane bagasse, and corn cobs). In addition, the xylanases produced by this bacterium with the best inducer substrate were characterized for their optimal pH, temperature and stability. The results for xylanase production showed that the higher levels of xylanases were obtained in growth medium containing 1% (w / v) BSG (1.35 U/mL), but the xylanolytic activity was also observed when wheat straw (1.32 U/mL), sugarcane bagasse (0.80 U/mL), corn cobs (0.30 U/mL) and commercial xylan (0.21 U/mL) were used as substrates. The extracellular crude enzymatic extract from Anoxybacillus sp. 3M was then characterized for its optimal temperature and pH and stability. The best enzyme activity was observed at a temperature of 60 ºC and pH 5.3, and the enzyme retained 100% of its original activity after 96 h at 60 °C and pH 7.0. Zymogram of native gel analysis of the different culture supernatants revealed the presence of an enzyme complex with a molecular weight of 420 kDa. This xylanase may be considered as a biocatalyst thermotolerant and it is interesting for biotechnological applications. Further application of Anoxybacillus 3M crude enzymatic extract to BSG and commercial xylan revealed the presence of xylose and xylooligosaccharides, mainly X2 and X3, in the hydrolizates produced.
- Influence of culture conditions towards optimal carotenoid production by Gordonia alkanivorans strain 1BPublication . Fernandes, Ana S.; Paixão, Susana M.; Silva, Tiago; Roseiro, J. Carlos; Alves, LuísABSTRACT: With the increasing awareness on the toxicity of several synthetic dyes, demand for pigments from natural sources, such as microbial carotenoids, has gained interest as a promising safe alternative colour additive. In this study, a surface response methodology based on the Doehlert distribution for two factors [% of glucose in a mixture of glucose + fructose (10 g/L total sugars), and sulfate concentration] was used towards the optimal carotenoids production by Gordonia alkanivorans strain 1B in the presence of light (400 lx). Time influence on pigment production by this bacterium was also evaluated, as well as the cell viability profile during longer incubation periods at optimal conditions. Indeed, the highest carotenoid production (2596-3100 mu g/g(DCW)) was obtained when strain 1B was cultivated in the optimal conditions: glucose 10 g/L and sulfate >= 22 mg/L, in the presence of light for 19 days at 30 degrees C, 150 rpm. Flow cytometry showed that the highest production was somehow related with the cellular stress. These results highlight the great potential of strain 1B as a new hyperpigment producer to be exploited towards several applications.