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- Selecting low-cost carbon sources for carotenoid and lipid production by the pink yeast Rhodosporidium toruloides NCYC 921 using flow cytometryPublication . Freitas, Claúdia; Parreira, Teresa M.; Roseiro, J. Carlos; Reis, Alberto; Silva, Teresa Lopes daThe present work studied low-cost carbon sources for carotenoid and lipid production using the yeast Rhodosporidum toruloides NCYC 921. Carob pulp syrup and sugarcane molasses at different concentrations were used as low-cost carbon sources in R. toruloides batch cultivations. Carob pulp syrup containing a total sugar concentration of 75 g L1 induced the highest total fatty acid productivity (1.90 g L1 h1) and the highest carotenoid productivity (9.79 lg L1 h1). Flow cytometric analysis revealed that most of the yeast cells (>60%) grown on carob pulp syrup displayed intact polarised membranes, conversely to the cells grown on sugarcane molasses, wherein a large proportion (>45%) displayed permeabilised cytoplasmic membranes.
- Citometria de fluxo: funcionalidade celular on-line em bioprocessosPublication . Silva, Teresa Lopes da; Reis, Alberto; Hewitt, Christopher; Roseiro, J. Carlos
- Biological upgrading of wastes from the pulp and paper industryPublication . Marques, Susana; Alves, Luís; Gírio, Francisco; Santos, J. A. L.; Roseiro, J. CarlosA process for biological upgrading of recycled paper sludge (RPS) was developed based on the enzymatic hydrolysis of major sludge components (cellulose and xylan) with commercial enzymes and fermentation of the resulting sugars into ethanol or lactic acid with adequate microbial strains. The process was implemented performing both steps sequentially (SHF) or simultaneously (SSF). Cellulosic and hemicellulosic fractions of RPS were completely converted by enzymatic hydrolysis (using Celluclast®1.5L with Novozym®188) into the constitutive glucose and xylose. Ethanol was produced from the RPS hydrolysate by the yeast Pichia stipitis CBS 5773. A slightly higher conversion yield was attained on SHF process, corresponding to an ethanol concentration of 19.6 g L-1, but 179 hours were needed. The SSF process was completed after 48 hours of incubation allowing the production of 18.6 g L-1 of ethanol from 178.6 g L-1 of dried RPS, corresponding to an overall conversion yield of 51% of the available carbohydrates on the initial substrate. Maximum production of lactic acid (LA) with Lactobacillus rhamnosus ATCC 7469 was obtained by performing the SSF process: 73 g L-1 of LA was achieved, corresponding to a maximum productivity of 2.9 g L-1 h-1, with 0.97 g LA produced per g of carbohydrates on initial sludge. The present results demonstrate the feasibility of the biological conversion of the ultimate waste obtained in the paper recycling loop into a biofuel (bioethanol) or an important chemical intermediate (LA, precursor of bioplastics), under the concept of a multi-purpose biorefinery.
- New at-line flow cytometric protocols for determining carotenoidcontent and cell viability during Rhodosporidium toruloides NCYC 921batch growthPublication . Freitas, Claúdia; Nobre, B. P.; Gouveia, Luisa; Roseiro, J. Carlos; Reis, Alberto; Silva, Teresa Lopes daRhodosporidium toruloides NCYC 921 batch growth was monitored as a means to evaluate the yeastbiomass potential as a source for the production of carotenoids and other lipids.Carotenoid content, cell viability and size were assessed by multiparameter flow cytometry. Thesaponifiable lipid fraction was assayed by gas–liquid chromatography.The carotenoid production increased during the stationary phase, reaching 78 g/g while the total fattyacid content attained 32% (w/w) at the end of the fermentation. The fatty acid profile was suitable forbiodiesel purposes.As the yeast cells entered the stationary phase, the proportion of cells with depolarised mitochon-drial membrane and cells with permeabilised cytoplasmic membrane increased, attaining 65% and 14%,respectively. Nevertheless, a high proportion of cells (82%) showed esterase activity.These results demonstrated that flow cytometry can be a powerful at-line technique to monitor thetotal carotenoids and cell viability during the yeast growth, being useful for the yeast process optimisationat lab and pilot scales.
- 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.
- Isolation and identification of Magnusiomyces capitatus as a lipase-producing yeast from olive mill wastewaterPublication . Salgado, Vera; Fonseca, César; Silva, Teresa Lopes da; Roseiro, J. Carlos; Eusebio, AnaABSTRACT: Olive mill wastewaters (OMW) are effluents originated from olive oil extraction. As an oil-rich residue, OMW is a potential source of lipase-producing microorganisms and a complex medium potentially suitable for lipase production. The aim of the present study was to isolate yeasts with the ability to produce extracellular lipases from OMW. Thirty-two yeast isolates were obtained and screening for esterase/lipase activity using rapid plate detection methods allowed the selection of five isolates. Subsequently, extracellular lipolytic activity was determined in shake-flasks, and the best activity was found in the isolate JT5 (0.85 U/mL). This isolate was identified as Magnusiomyces capitatus by DNA sequencing. Growth and lypolytic activities by M. capitatus JT5 were assessed in undiluted OMW, and optimization of lipase production was achieved by a positive interaction of two factors (oxygen availability and nitrogen concentration). The highest lipase activity (1.4 U/mL) was obtained at NH4Cl concentration of 2.8 g/L and kLa of 0.65 min−1. The growth of M. capitatus JT5 in a stirred tank bioreactor, using undiluted OMW, allowed the improvement of lipase production (up to 3.96 U/mL) by increasing olive oil concentration in the medium, under the selected conditions of nitrogen concentration and oxygen availability. This study highlighted the isolate M. capitatus JT5 as a lipase-producing microorganism that is able to grow in undiluted OMW under controlled conditions. Results obtained in shake-flasks have been reproduced satisfactorily in the stirred tank bioreactor.
- 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.
- Rede de laboratórios para a energia e geologiaPublication . Roseiro, J. Carlos; Trancoso, Maria Ascensão; Cunha Diamantino, Teresa; Carvalho, Maria João; Machado Leite, MárioRESUMO: O Laboratório Nacional de Energia e Geologia (LNEG), nas suas áreas de competências reuniu as competências de anteriores instituições com um histórico de trabalho em Laboratórios com acreditação desde os anos 80. No LNEG, estes Laboratórios focaram-se em atividades da área de energia e geologia, mantendo as suas acreditações mas sentiram a necessidade de trabalhar em rede.
- 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.
- Optimization of a biphasic biodesulfurization systemPublication . Silva, Tiago; Paixão, Susana M.; Roseiro, J. Carlos; Alves, LuísABSTRACT: Many of the new generation fuels, although more sustainable, share some of the problems inherent to fossil fuels. Depending on the biomass/material that originated them, they can present different contaminants that can lead to environmental problems. Sulfur is one of the most common and problematic contaminants in fuels. It is released into the atmosphere in the form of SOx, leading to the formation of acid rains, which cause drastic environmental and infrastructural problems, as well as several types of health issues. High sulfur concentrations in fuels also result in a loss of efficiency of motors and energy generation systems, mostly due to corrosion and catalyst poisoning. The current thermochemical desulfurization process, hydrodesulfurization (HDS), is energy demanding, pollutant and has low efficiency against more complex organosulfur molecules. This led researchers to look for new alternatives. Biodesulfurization (BDS), is, as the name implies, the biological removal of sulfur from fuels using microorganisms as living biocatalysts. If correctly employed this process could be more efficient and less pollutant, since microorganisms directly target the sulfur atoms, even those present in complex molecular structures, such as dibenzothiophene (DBT). Moreover, microbial activity occurs at much lower temperatures and pressures, without the need for metal catalysts, resulting in a lower energy demand. While BDS is a promising technology, it is still at a low development stage, mostly due to some bottlenecks, which have been hindering its large-scale application. Similarly, to other biotechnological processes, it presents lower reaction rates, when compared to HDS, since it depends on the use of living organisms as catalysts. Furthermore, it must be performed under conditions that allow the microorganisms to maintain biological activity, limiting the range of applications. These conditions vary greatly depending on the microorganism selected, and their optimization can significantly increase the biodesulfurization activity of a biocatalyst.
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