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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.
- Biohydrogen fermantative production : energetic valorization of microalgae biomassPublication . Batista, Ana Paula; Ortigueira, Joana; Gouveia, Luisa; Marques, Paula; Alves, Luís; Moura, PatríciaRenewable, sustainable and carbon-neutral energy production is needed to deal with the challenges of growing energy demand and climate change. Hydrogen (H2) is most promising in the succession of fuel evolution, with several technical, socio-economic and environmental benefits to its credit [1]. It is an excellent energy carrier as it has the highest energy content per unit weight/mass of any known fuel (142 kJ/g) and upon oxidation produces only water [1]. H2 is being explored for use in combustion engines and fuel-cell electric vehicles, and it is expected that H2 demand increases significantly in the near and long term [2]. Biological hydrogen production processes are found to be more environmentally friendly and less energy intensive as compared to thermochemical and electrochemical processes [1]. In dark fermentation, carbohydrate-rich substrates can be used to produce bioH2 in a process mediated by hydrogenase enzymes of anaerobic microorganisms. Moreover, residues and byproducts from agricultural and food industries or wastewaters can be used, providing inexpensive energy generation with simultaneous waste treatment [3]. Recently, there has been an increasing interest on using microalgal biomass for biofuels production. Besides oil extraction for biodiesel purposes [4] or sugar extraction for bioethanol production [5-6], microalgal biomass can also be fermented into bioH2. In this work, Scenedesmus obliquus biomass was used as feedstock for biohydrogen production by Enterobacter aerogenes and Clostridium butyricum. The concentration of microalgal biomass used as fermentation substrate was optimized for each microorganism: 2.5 g/L for E. aerogenes and 50 g/L for C. butyricum. The values of hydrogen production by using “wet” (75% moisture) and dried (oven, 80°C) microalgal biomass were compared, as the suppression of an intermediate biomass drying step is economically advantageous. The highest H2 yield (113.1 mL/g algaAFDW) was attained by C. butyricum with dried microalgal biomass. Hydrogen production by E. aerogenes was clearly improved by using S. obliquus wet biomass, generating an H2 yield of 72.3 mL/g algaAFDW
- 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.
- 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.
- KOH for enhanced sugarcane bagasse delignification and further production of sugar-rich hydrolyzates by enzymes applicationPublication . Paixão, Susana M.; Ladeira, S. A.; Arez, B. F.; Martins, M. L. L.; Roseiro, J. Carlos; Alves, LuísLignocellulosic biomass is envisaged as an important raw material for bioethanol production due to its low cost and high availability. Sugarcane bagasse (SCB), a fibrous residue of cane stalks left over after crushing and extraction of the juice from sugarcane; it is one of the largest cellulosic agro-industrial by-products. Tons of SCB are produced in Brazil as a waste of sugar and ethanol industries. This lignocellulosic by-product is a potential renewable source for 2G-bioethanol production. Usually, SCB is pretreated using alkaline and/or acid treatments viewing higher ethanol yields. The main goal of this study was to optimize the delignification of SCB towards the higher availability of glucans and xylans for further enzymatic hydrolysis to obtain sugar-rich syrups that will be more readily fermented to bioethanol. The delignification was carried out by autoclaving the biomass with KOH and the influence of KOH concentration (1-10%) and the autoclave time (10-60 min) were evaluated through a statistical design. Experimental distribution for two factors according to the Doehlert uniform design was used to produce response surfaces. The responses studied in this design were the percentage of hemicellulose, lignin and total polysaccharides. The results showed that from the two factors evaluated, the KOH concentration was the one that most influenced the response observed and that the treatments of SCB with KOH 5-10% for 35 minutes of autoclave at 121ºC and 1 atm led to the highest rates of lignin extraction. Using KOH treatment, a significant reduction of lignin content in SCB was observed, namely from 19% to 5%. Scanning electron micrographs of SCB pre-treated with 10% KOH for 35 minutes demonstrated a change in the structure of the material, with the appearance of broken structures, which can be attributed to the alkaline treatment. To validate the experiments, the SCB pretreated in the optimal conditions (95% of total polysaccharides) was further hydrolyzed with commercial enzymes and the enzymatic hydrolysis performance was evaluated.
- Microalgae as a sustainable raw material for biofuels production and high added value compounds extraction by an integrated biorefinary conceptPublication . Gouveia, Luisa; Matos, Cristina T.; Oliveira, Ana Cristina; Passarinho, Paula; Miranda, J. R.; Paulo, V.; Marques, Paula; Batista, Ana Paula; Moura, Patrícia; Alves, Luís; Ortigueira, Joana; Nobre, B. P.; Palavra, António F.
- Microalgae biomass as fermentation substrate for hydrogen and butyric acid production by clostridium tyrobutyricumPublication . Ortigueira, Joana; Lúcio, M.; Rodrigues, S.; Alves, Luís; Gouveia, Luisa; Moura, PatríciaFossil fuels are a limited type of feedstock, increasingly expensive, and carrying strong polluting properties. The search for alternative sources which can replace fossil fuels without the severe disadvantages that its use conveys is therefore of paramount importance. Microalgae biomass represents an example of such non-food renewable biomass that can be regarded as a valid alternative to fossil fuels. As biomass, microalgae are highly desirable since they are photosynthetic organisms with a very fast growth rate in comparison to higher plants, and their production does not require arable land or potable water. Furthermore, some microalgae are able to store large amounts of oil or sugars, prime materials for the production of biofuels and bulk-chemicals [1]. Scenedesmus obliquus is a microalgae with the referred properties, easily produced at large scale and capable of storing a high amount of sugars under nitrogen shortage. The objective of the present work was to investigate the production of hydrogen and butyrate from S. obliquus hidrolysate by four hydrogen- and butyrate-producing bacterial strains previously isolated by us and identified as Clostridium tyrobutyricum 1T, 2T, 3T and 9P. S. obliquus biomass was produced locally in air-lifts. After harvest, all biomass was submitted to acid pre-treatment [2] resulting in a microalgae hydrolysate with a final concentration of 10.3 g/l of glucose, xylose, arabinose, mannose and galactose. The hydrolysate was used as carbon and energy source for hydrogen and butyrate production by the four C. tyrobutyricum isolates. Hydrogen yields ranged from 0.63, 1.29, 1.36 and 1.24 of mol H2/ mol sugars by strains 1T, 2T, 3T and 9P, respectively. Hydrogen production was accompanied by the production of carbon dioxide and organic acids, mainly butyrate. Butyrate yields were 0.29, 0.49 and 0.48 mol butyric acid/ mol sugars, respectively by C. tyrobutyricum strains 1T, 2T and 3T, and 9P. The best C. tyrobutyricum isolate for combined hydrogen and butyrate production from S. obliquus hydrolysate will be used in further studies of energetic valorisation of spent algal biomass available from both biodiesel and bioethanol processes.
- Optimization of a SSF process using invertase applied to fossil fuels biodesulturizationPublication . Arez, B. F.; Alves, Luís; Roseiro, J. Carlos; Paixão, Susana M.
- Production and characterization of Talf1 yeast invertases and further application to biodesulfurizationPublication . Arez, B. F.; Alves, Luís; Paixão, Susana M.Combustion of fossil fuels generates emissions of numerous toxic gases which in later years have become a major concern internationally. One of the most concerning problems is sulfur and sulfur dioxide, and maximum levels have been established through the years. Biodesulfurization (BDS) could be a complementary technology to the commonly used physico-chemical process. BDS is based on the use of microorganisms for the removal of sulfur even from the most recalcitrant compounds at atmospheric pressure and temperature, making it cheaper and more eco-friendly. However, this bioprocess has a few limitations, such as the high costs of the culture medium, which makes the process very expensive. Thus, in order to reduce its costs, it is important to search for cheaper carbon sources which can contribute to produce the microbial biomass. The goal of this work was the production and characterization of novel Z. bailii strain Talf1 invertases for further application to BDS, in order to expand the usable alternative carbon sources to high sucrose level feedstock, comparing two different approaches: Separated Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF) processes.