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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 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.
- 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.
- 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.
- 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.
- Flow cytometric method for cell viability evaluation of Gordonia alkanivorans strain 1B in fossil fuels biodesulfurization processesPublication . Teixeira, A. V.; Silva, Tiago; Silva, Teresa Lopes da; Paixão, Susana M.; Alves, LuísThis work reports the development of a rapid flow cytometric method for the viability assessment of Gordonia alkanivorans strain 1B, a bacterium used in the biodesulfurization process. To demonstrate that it is possible to monitor by flow cytometric analysis changes in this bacterium physiological state, positive controls using the 5(6)-carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) staining mixture were set. The loss of viability of G. alkanivorans resting cells in the presence of different concentrations of 2-hydroxybiphenyl, a very toxic end product of the dibenzotiophene desulfurization process, was assessed over a period of time. The results here reported demonstrate the potential of this technique for the biodesulfurization process monitoring and consequent enhancement.
- Enhancement of dibenzothiophene desulfurization by Gordonia alkanivorans strain 1B using sugar beet molasses as alternative carbon sourcePublication . Alves, Luís; Paixão, Susana M.There are several problems limiting an industrial application of fossil fuel biodesulfurization, and one of them is the cost of culture media used to grow the microorganisms involved in the process. In this context, the utilization of alternative carbon sources resulting from agro-industrial by-products could be a strategy to reduce the investment in the operating expenses of a future industrial application. Recently, Gordonia alkanivorans 1B was described as a fructophilic desulfurizing bacterium, and this characteristic opens a new interest in alternative carbon sources rich in fructose. Thus, the goal of this study was to evaluate the utilization of sugar beet molasses (SBM) in the dibenzothiophene (DBT) desulfurization process using strain 1B. SBM firstly treated with 0.25 % BaCl2 (w/v) was used after sucrose acidic hydrolysis or in a simultaneous saccharification and fermentation process with a Zygosaccharomyces bailii Talf1 invertase (1 %), showing promising results. In optimal conditions, strain 1B presented a ìmax of 0.0795 h.1, and all DBT was converted to 2-hydroxybiphenyl (250 ìM) within 48 h with a maximum production rate of 7.78 ìM h.1. Our results showed the high potential of SBM to be used in a future industrial fossil fuel biodesulfurization process using strain 1B.
- Enhancement of dibenzothiophene biodesulfurization by Gordonia alkanivorans strain 1B using fructose rich culture mediaPublication . Alves, Luís; Silva, Tiago; Arez, B. F.; Paixão, Susana M.The removal of sulfur mediated by microorganisms or biodesulfurization (BDS) is already an extensively studied approach. The first studies were reported in the 50’s and 60’s, but only in the last 20 years have been successful breakthroughs. Through BDS it is possible to remove most of the recalcitrant sulfur compounds to the commonly physico-chemical process at mild operating conditions without molecular hydrogen, resorting to microorganisms. These microorganisms can remove sulfur from dibenzothiphene (DBT), a model compound, and other polycyclic aromatic using them as their sulfur source, making BDS an easy and environmental friendly process. Gordonia alkanivorans strain 1B [1] has been described as a desulphurizing bacterium, able to desulfurize DBT to 2-hydroxybiphenyl (2-HBP), the final product of the 4S pathway, using D-glucose as carbon source. However, both the cell growth and the desulphurization rate can be largely affected by the nutrient composition of the growth medium [2,3,4], due to cofactor requirements of many enzymes involved in BDS biochemical pathway.
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