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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.
- 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).
- 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.
- Carbon footprint assessment of microalgal biomass production, hydrothermal liquefaction and refining to sustainable aviation fuel (SAF) in mainland PortugalPublication . Pires, Renata; Silva, Tiago; Ribeiro, Cláudia; Costa, Luis; Matos, Cristina T.; Costa, Paula; Lopes, Tiago; Gírio, Francisco; Silva, CarlaABSTRACT: Industrial liquid effluents (e.g., from fertilizer industry) and flue gas streams (e.g., CO2-rich, from cement industry) arise as an opportunity for waste valorization. Microalgae are suitable biomass for assimilating both effluents at the cultivation stage. Under a biorefinery concept, given the urge for energy transition in the aviation sector, this research explores the transformation of a microalgae consortium grown at an industrial site in Portugal and its subsequent harvesting, hydrothermal liquefaction (HTL), and bio-oil refining. A life cycle assessment (LCA) approach is undertaken with two functional units (FU): 1 kg of microalgae dry-cell weight (dw) and 1 MJ of bio-jet fuel. The latter follows an attributional approach with energy allocation for comparison with the Carbon Offsetting and Reduction Scheme for International Aviation (CORSIA) guidelines. HTL is based on data from bench-scale experiments and literature, whereby the Petroleum Refinery Life Cycle Inventory Model (PRELIM) is used to mimic bio-oil refining. Following this approach, achieving Sustainable Aviation Fuel (SAF) compliance requires net-zero electricity (0 gCO2eq/kWh), with an HTL bio-oil yield of 55.6 % dw (the maximum observed), a minimum refining bio-jet fuel yield of at least 16 %. Alternatively, an HTL bio-oil yield of 36.9 % dw (the median observed) with a refining efficiency of at least 24.3 %.
- 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.
- Biodesulfurization biorefinery using Gordonia alkanivorans strain 1B: life cycle inventory of the integrated processPublication . Silva, Tiago; Silva, Carla; Paixão, Susana M.; Alves, LuísABSTRACT: High sulfur concentrations are a problem common to fossil fuels and derivatives (such as oil and coal), as well as many new generation fuels and biofuels (such as pyrolysis oils, syngas, biogas or even biodiesel). If the sulfur present in these fuels is released into the atmosphere it can result in SO2/SOx emissions, leading to environmental damage, and health issues. Transportation fuels have sulfur limits that go below 5000 ppm in ships, 3000 ppm in airplanes and 10 ppm in cars, and without treatment fuels can have several thousand ppm of sulfur. As such, they must be submitted to desulfurization, typically through a thermochemical process known as hydrodesulfurization, in which H2 is combined with the fuel at high temperatures and pressures, in the presence of metal catalysts. However, this process has significant environmental impacts. Usually, it depends on hydrogen and heat/steam produced from natural gas, totalizing 4.17 kg natural gas per 2.89 kg sulfur removed. It also involves high electricity and water consumption (approximately 2.9 kWh and 86.9 kg, respectively, per 2.89 kg sulfur removed). Furthermore, these impacts are greater for lower sulfur demands (Burgess & Brennan, 2001). Thus, there has been a search for alternative/complementary processes, one of which is biodesulfurization (BDS). It consists of the use of microorganism that consume the sulfur present in the fuels, at ambient temperature and pressure, without the need for metal catalysts. BDS still presents several bottlenecks, common to many microbial processes, such as low conversion rates and high production costs for the microbial biocatalyst. To surpass these limitations researchers have pursued different strategies: minimization/optimization of culture medium and culture conditions; employment of cheaper alternative nutrient sources; exploitation of added value products. Gordonia alkanivorans strain 1B is a bacterium known for its biodesulfurization properties. It has demonstrated several characteristics which make it interesting: it can perform BDS of different compounds, several of which extremely recalcitrant for the thermochemical process; it has very low nutritional needs; it can be cultivated on several alternative carbon sources; it has been shown to produce two different types of added value products: carotenoids and biosurfactants (Alves et al., 2015; Silva et al., 2020, 2022). Therefore, G. alkanivorans strain 1B is the ideal candidate for a biodesulfurization biorefinery, that simultaneously removes sulfur from fuels and produces carotenoids and biosurfactants.
- Development of a bench-scale photobioreactor with a novel recirculation system for continuous cultivation of microalgaePublication . Tavares, João; Silva, Tiago; Paixão, Susana M.; Alves, LuísABSTRACT: Microalgae cultivation can be used to increase the sustainability of carbon emitting processes, converting the CO2 from exhaust gases into fuels, food and chemicals. Many of the carbon emitting industries operate in a continuous manner, for periods that can span days or months, resulting in a continuous stream of gas emissions. Biogenic CO2 from industrial microbiological processes is one example, since in many cases it becomes unsustainable to stop these processes on a daily or weekly basis. To correctly sequester these emissions, microalgae systems must be operated under continuous constant conditions, requiring photobioreactors (PBRs) that can act as chemostats for long periods of time. However, in order to optimize culture parameters or study metabolic responses, bench-scale setups are necessary. Currently there is a lack of studies and design alternatives using chemostat, since most works focus on batch assays or semi-continuous cultures. Therefore, this work focused on the development of a continuous bench-scale PBR, which combines a retention vessel, a photocollector and a degasser, with an innovative recirculation system, that allows it to operate as an autotrophic chemostat, to study carbon sequestration from a biogenic CO2-rich constant air stream. To assess its applicability, the PBR was used to cultivate the green microalga Haematococcus pluvialis using as sole carbon source the CO2 produced by a coupled heterotrophic bacterial chemostat. An air stream containing ≈0.35 vol% of CO2, was fed to the system, and it was evaluated in terms of stability, carbon fixation and biomass productivity, for dilution rates ranging from 0.1 to 0.5 d−1. The PBR was able to operate under chemostat conditions for more than 100 days, producing a stable culture that generated proportional responses to the stimuli it was subjected to, attaining a maximum biomass productivity of 183 mg/L/d with a carbon fixation efficiency of ≈39% at 0.3 d−1. These results reinforce the effectiveness of the developed PBR system, making it suitable for laboratory-scale studies of continuous photoautotrophic microalgae cultivation.
- 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.
- Ionic liquids toward enhanced carotenoid extraction from bacterial biomassPublication . Silva, Tiago; Alves, Luís; Salgado, Francisco; Roseiro, J. Carlos; Lukasik, Rafal M.; Paixão, Susana M.ABSTRACT: Carotenoids are high added-value products primarily known for their intense coloration and high antioxidant activity. They can be extracted from a variety of natural sources, such as plants, animals, microalgae, yeasts, and bacteria. Gordonia alkanivorans strain 1B is a bacterium recognized as a hyper-pigment producer. However, due to its adaptations to its natural habitat, hydrocarbon-contaminated soils, strain 1B is resistant to different organic solvents, making carotenoid extraction through conventional methods more laborious and inefficient. Ionic liquids (ILs) have been abundantly shown to increase carotenoid extraction in plants, microalgae, and yeast; however, there is limited information regarding bacterial carotenoid extraction, especially for the Gordonia genus. Therefore, the main goal of this study was to evaluate the potential of ILs to mediate bacterial carotenoid extraction and develop a method to achieve higher yields with fewer pre-processing steps. In this context, an initial screening was performed with biomass of strain 1B and nineteen different ILs in various conditions, revealing that tributyl(ethyl)phosphonium diethyl phosphate (IL#18), combined with ethyl acetate (EAc) as a co-solvent, presented the highest level of carotenoid extraction. Afterward, to better understand the process and optimize the extraction results, two experimental designs were performed, varying the amounts of IL#18 and EAc used. These allowed the establishment of 50 µL of IL#18 with 1125 µL of EAc, for 400 µL of biomass (cell suspension with about 36 g/L), as the ideal conditions to achieve maximal carotenoid extraction. Compared to the conventional extraction method using DMSO, this novel procedure eliminates the need for biomass drying, reduces extraction temperatures from 50 °C to 22 ± 2 °C, and increases carotenoid extraction by 264%, allowing a near-complete recovery of carotenoids contained in the biomass. These results highlight the great potential of ILs for bacterial carotenoid extraction, increasing the process efficiency, while potentially reducing energy consumption, related costs, and emissions.