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- 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.
- Optimization of low sulfur jerusalem artichoke juice for fossil fuels biodesulfurization processPublication . Silva, Tiago; Paixão, Susana M.; Roseiro, J. Carlos; Alves, LuísMost of the world’s energy is generated from the burning of fossil fuels such as oil and its derivatives. When burnt, these fuels release into the atmosphere volatile organic compounds, sulfur as sulfur dioxide (SO2) and the fine particulate matter of metal sulfates. These are pollutants which can be responsible for bronchial irritation, asthma attacks, cardio-pulmonary diseases and lung cancer mortality, and they also contribute for the occurrence of acid rains and the increase of the hole in the ozone layer. For these reasons countries around the world imposed legal maxima to sulfur concentration on fuels. Forcing companies to develop methods of removing the sulfur contained in the oil. The most common is hydrodesulfurization which employs high pressures and temperatures associated with complex metal catalysts making it extremely expensive. So, it becomes important to explore alternatives such as biodesulfurization (BDS). This process is based on the use of microorganisms for the removal of sulfur form even from the most recalcitrant compounds at atmospheric pressure and temperature, making it cheaper and more eco-friendly. However it still presents some drawbacks, such as being easily inhibited in the presence of sulfates, which have been shown to have great inhibitory effect even in amounts as low as 6 mg/l [1]. In order to further reduce the costs associated with BDS it is possible to explore alternative carbon sources, as previously shown with carob pulp syrup and recycled paper sludge [1, 2]. The main objective of this work is the optimization of sulfate removal, from Jerusalem artichoke juice, in order to use it as an alternative carbon source for BDS.
- 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.
- 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, LuísThe most commonly method used for sulfur removal from fossil fuels is hydrodesulfurization, a physico-chemical process at very high temperatures and pressures. An alternative to this process is biodedesulfurization (BDS), a microbiological process that works at atmospheric pressure and temperature making it easier to work with and less expensive. It also as the advantage of easily desulfurizing recalcitrant sulfur compounds which are hard to remove by hydrodesulfurization [1]. Several bacteria species, such as Gordonia alkanivorans strain1B [2], are able to desulfurize dibenzothiophene, a model compound used commonly in BDS studies, to 2-hydroxybiphenyl (2-HBP) via the 4S pathway without destroying the carbon structure [3], therefore maintaining the fuel potential energy. BDS limitations are related with process parameters and with the cost of maintaining bacterial cultures so to enhance the BDS process, it is necessary to monitor how changes in the experimental system affect the microbial cells viability and consequently the process efficiency. An alternative method to conventional microbial techniques to determine cell viability is flow cytometry. This method provides a fast and accurate quantitative method for measurement of thousands of individual cells, based on scattered light and fluorescence emitted by specific dyes. The goal of this study was to develop a rapid method for viability assessment of G. alkanivorans cells using flow cytometry for further application to monitor and optimize BDS processes.
- Fructose rich alternative carbon sources for enhanced fossil fuels biodesulfurizationPublication . Silva, Tiago; Paixão, Susana M.; Alves, LuísBiodesulfurization allows the removal of recalcitrant sulfur from fossil fuels at mild operating conditions with the aid of microorganisms. However the production of biocatalysts still has elevated costs which hinder its industrial application. So the use of agro-industrial by-products and wastes, as alternative carbon sources could present an opportunity to cheapen the process. In previous works we showed that Gordonia alkanivorans strain 1B has the ability to use materials such as recycled paper sludge hydrolysate and carob pulp liquor to grow and desulfurize after some optimization. Since this is a fructophilic bacterium it is important to compare the use of carbon sources progressively richer in fructose.
- Zygosaccharomyces bailii strain talf1 inulinases: a versatile tool for bioprocessesPublication . Paixão, Susana M.; Teixeira, Pedro D.; Silva, Tiago; Teixeira, A. V.; Alves, LuísFructans are one of the most abundant non-structural polysaccharides found in a wide range of plants. Inulin is a polydisperse fructan polymer composed by linear chains of b-2, 1-linked D-fructofuranose molecules terminated by a glucose residue through a sucrose-type linkage at the reducing end. Inulin or inulin-rich materials can be actively hydrolyzed into fermentable sugars (glucose and fructose) using inulinases and then further used within bioprocesses.
- On the road to cost-effective fossil fuel desulfurization by Gordonia alkanivorans strain 1BPublication . Pacheco, Marta; Paixão, Susana M.; Silva, Tiago; Alves, LuísABSTRACT: Biodesulfurization (BDS) is an ecofriendly process that uses microorganisms to efficiently remove sulfur from fossil fuels. To make the BDS process economically competitive with the deep hydrodesulfurization process, which is currently used in the oil industry, it is necessary to improve several factors. One crucial limitation to be overcome, common within many other biotechnological processes, is the cost of the culture medium. Therefore, an important line of work to make BDS scale-up less costly is the optimization of the culture medium composition aiming to reduce operating expenses and maximize biocatalyst production. In this context, the main goal of this study was on the minimization of inorganic key components of sulfur-free mineral (SFM) medium in order to get the maximal production of efficient desulfurizing biocatalysts. Hence, a set of assays was carried out to develop an optimal culture medium containing minimal amounts of nitrogen (N) and magnesium (Mg) sources and trace elements solution (TES). These assays allowed the design of a SFMM (SFM minimum) medium containing 85% N-source, 25% Mg-source and 25% TES. Further validation consisted of testing this minimized medium using two carbon sources: the commercial C-source (glucose + fructose) versus Jerusalem artichoke juice (JAJ) as a cheaper alternative. SFMM medium allowed microbial cells to almost duplicate their specific desulfurization rate (q(2-HBP)) for both tested C-sources, namely from 2.15 to 3.39 mu moL g(-1) (DCW) h(-1) for Fru + Glu and from 1.91 to 3.58 mu moL g(-1) (DCW) h(-1) for JAJ, achieving a similar net 2-hydroxybiphenyl produced per g of consumed sugar (similar to 17 mu moL g(-1)). These results point out the great advantage of using cheaper culture medium that in addition enhances the bioprocess effectiveness, paving the way to a sustainable scale-up for fossil fuel BDS.
- Influence of the carbon source on Gordonia alkanivorans strain 1B resistance to 2-hydroxybiphenyl toxicityPublication . Teixeira, A. V.; Paixão, Susana M.; Silva, Teresa Lopes da; Alves, LuísThe viability of bacteria plays a critical role in the enhancement of fossil fuels biodesulfurization efficiency since cells are exposed to toxic compounds such as 2-hydroxybiphenyl (2-HBP), the end product of dibenzothiophene (DBT) biodesulfurization. The goal of this work was to study the influence of the carbon source on the resistance of Gordonia alkanivorans strain 1B to 2-HBP. The physiological response of this bacterium, pregrown in glucose or fructose, to 2-HBP was evaluated using two approaches: a growth inhibition toxicity test and flow cytometry. The results obtained from the growth inhibition bioassays showed that the carbon source has an influence on the sensitivity of strain 1B growing cells to 2-HBP. The highest IC50 value was obtained for the assay using fructose as carbon source in both inoculum growth and test medium (IC50-48 h=0.464 mM). Relatively to the evaluation of 2-HBP effect on the physiological state of resting cells by flow cytometry, the results showed that concentrations of 2-HBP >1 mM generated significant loss of cell viability. The higher the 2-HBP concentration, the higher the toxicity effect on cells and the faster the loss of cell viability. In overall, the flow cytometry results highlighted that strain 1B resting cells grown in glucose-SO4 or glucose-DBT are physiologically less resistant to 2-HBP than resting cells grown in fructose-SO4 or fructose-DBT, respectively.