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- A new combined approach to improved lipid production using a strictly aerobic and oleaginous yeastPublication . Guerreiro, Fábio; Constantino, Ana; Emília Lima-Costa, Maria; Raposo, SaraMicrobial lipids have potential applications in energy, and food industry, because most of those lipids are triacylglycerol with long-chain fatty-acids that are comparable to conventional vegetable oils and can be obtained without arable land requirement. Rhodosporidium toruloides is a strictly aerobic strain, where oxygen plays a crucial role in growth, maintenance, and metabolite production, such as lipids and carotenoids. Dissolved oxygen concentration is one of the major factors affecting yeast physiological and biochemical characteristics. In this context, different approaches have been developed to increase available oxygen by the increasing the aeration and the addition of an oxygen-vector. The growth of R. toruloides in 2-L mechanical stirred tank reactor equipped with 1 or 2 porous spargers and a 70 C/N ratio, revealed a lipid content of 0.47 and 0.52 g/g and a lipidic productivity of 0.16 and 0.17 g/L day, respectively. The oxygen-vector addition, increased the lipidic productivity for 0.20 g/L day and a lipid contend of 0.51 g of lipids/g of biomass. The combined approach, combining high aeration (AA), and 1% of n-dodecane addition (DA), produced a significant improvement in the lipid accumulation (62%, w/w), when compared with the DA (51%, w/w) and the AA (52%, w/w) approaches. The increasing of lipids accumulation and smaller culture time are key factors for the success of scale-up and profitability of a bioprocess.
- Nitrogen Sources Screening for Ethanol Production Using Carob Industrial WastesPublication . Raposo, Sara; Constantino, Ana; Rodrigues, F.; Rodrigues, Brígida; Lima-Costa, Maria EmíliaNowadays, bioethanol production is one of the most important technologies by the necessity to identify alternative energy resources, principally when based on inexpensive renewable resources. However, the costs of 2nd-generation bioethanol production using current biotechnologies are still high compared to fossil fuels. The feasibility of bioethanol production, by obtaining high yields and concentrations of ethanol, using low-cost medium, is the primary goal, leading the research done today. Batch Saccharomyces cerevisiae fermentation of high-density sugar from carob residues with different organic (yeast extract, peptone, urea) and inorganic nitrogen sources (ammonium sulfate, ammonium nitrate) was performed for evaluating a cost-effective ethanol production, with high ethanol yield and productivity. In STR batch fermentation, urea has proved to be a very promising nitrogen source in large-scale production of bioethanol, reaching an ethanol yield of 44 % (w/w), close to theoretical maximum yield value and an ethanol production of 115 g/l. Urea at 3 g/l as nitrogen source could be an economical alternative with a great advantage in the sustainability of ethanol production from carbohydrates extracted from carob. Simulation studies, with experimental data using SuperPro Design software, have shown that the bioethanol production biorefinery from carob wastes could be a very promising way to the valorization of an endogenous resource, with a competitive cost.
- Chemo-enzymatic saccharification strategy of microalgae chlorella sorokinianaPublication . Constantino, Ana; Rodrigues, Brígida; Raposo, SaraBiofuel production using microalgae attracted much attention because it can be cultured using CO2 and sunlight. With high carbohydrate content, microalgae have the potential to be used as a fermentation feedstock for bioethanol production. In present work, chemo-enzymatic saccharification of Chlorella sorokiniana microalgae were investigated. Chemical hydrolysis of the biomass followed by enzymatic hydrolysis and was also evaluated the effect of combining the two enzymes and the sequential addition. The effect of α-amylase concentrations was analyzed in ranged between 50 and 8000 U/g of biomass and for amyloglucosidase between 90 and 600 U/g of biomass. The higher concentrations showed the highest conversion of reducing sugars. The α-amylase concentration 8000 U/g of biomass presented a conversion of 43.06 ± 2.92% (w/w), while amyloglucosidase with 600 U/g of biomass obtained 76.57 ± 6.42% (w/w). The combination of two enzymes simultaneously was more efficient than the sequential addition for low enzyme concentrations (α-amylase 50 U/g and amyloglucosidase 90 U/g) with a total reducing sugar of 22.78 ± 3.06 and 16.92 ± 2.06% (w/w), respectively. On the other hand, using the higher enzymes concentrations, no difference was observed between the two addition strategies, 58.9 ± 3.55 and 57.05 ± 2.33% (w/w) for the sequential and simultaneous, respectively. Both strategies didn’t present advantage, since the amyloglucosidase enzyme alone produced slightly higher results. Even thought, the obtained results showed successfully performed saccharification of microalgal biomass and clearly point to microalgae use for saccharification and subsequent bioethanol production.
- Kinetic and energetic parameters of carob wastes fermentation by Saccharomyces cerevisiae: crabtree effect, ethanol toxicity, and invertase repressionPublication . Rodrigues, Brígida; Peinado, J. M.; Raposo, Sara; Constantino, Ana; Quintas, Célia; Emília Lima-Costa, MariaCarob waste is a useful raw material for the second-generation ethanol because 50% of its dry weight is sucrose, glucose, and fructose. To optimize the process, we have studied the influence of the initial concentration of sugars on the fermentation performance of Saccharomyces cerevisiae. With initial sugar concentrations (S0) of 20 g/l, the yeasts were derepressed and the ethanol produced during the exponential phase was consumed in a diauxic phase. The rate of ethanol consumption decreased with increasing S0 and disappeared at 250 g/l when the Crabtree effect was complete and almost all the sugar consumed was transformed into ethanol with a yield factor of 0.42 g/g. Sucrose hydrolysis was delayed at high S0 because of glucose repression of invertase synthesis, which was triggered at concentrations above 40 g/l. At S0 higher than 250 g/l, even when glucose had been exhausted, sucrose was hydrolyzed very slowly, probably due to an inhibition at this low water activity. Although with lower metabolic rates and longer times of fermentation, 250 g/l is considered the optimal initial concentration because it avoids the diauxic consumption of ethanol and maintains enough invertase activity to consume all the sucrose, and also avoids the inhibitions due to lower water activities at higher S0.
- Alternative chemo-enzymatic hydrolysis strategy applied to different microalgae species for bioethanol productionPublication . Constantino, Ana; Rodrigues, Brígida; Leon, R.; Barros, Raúl; Raposo, SaraMicroalgae have been considered third generation feedstock for biofuel production based on the expectation that large amounts of algal biomass can be cultivated at an acceptable cost. Transformation of biomass into ethanol requires a saccharification step, where complex carbohydrates are broken down by hydrolysis into sugars that can be fermented to bioethanol. Carbohydrate mobilization is hampered by the recalcitrance of the cell envelope of microalgal cells, because complex structural polysaccharides are difficult to depolymerize and make internal carbohydrate reserves inaccessible to hydrolysis. Saccharification can be accomplished by either acidic hydrolysis, enzymatic treatment or a combination of both. The present work focused on the chemo-enzymatic hydrolysis of lyophilized biomass of different microalgae and subsequent fermentation of hydrolysates with higher reducing sugar content. A chemo-enzymatic hydrolysis strategy was defined, consisting of an acid pretreatment carried out at high pressure and temperature, followed by incubation with Amyloglucosidase and finally by incubation with alpha-Amylase, the opposite order of the conventional use of these enzymes. An increase of reducing sugar yield of about one third was observed, and this strategy was successfully applied to a broad group of microalgae, resulting in maximum release yields of at least 34.0 +/- 1.0 g total reducing sugar/100 g dry biomass. For bioethanol production studies, the microalgae hydrolysates of Chlorella sorokiniana, Tetraselmis sp. (Necton) and Skeletonema sp. were selected according to their high reducing sugar content. High ethanol production was achieved with all hydrolysates, with ethanol yields close to the theoretical maximum and the highest ethanol concentrations so far reported under comparable conditions. Chlorella sorokiniana stood out as the best hydrolysate for ethanol production, with an ethanol yield of 0.464 +/- 0.013 g/g reducing sugar and ethanol productivity of 0.344 +/- 0.020 g/L.h.
- Chemical and physical pretreatments of microalgal biomassPublication . Rodrigues, Brígida; Borges, Rodrigo; Castro, Maria; Constantino, Ana; Raposo, SaraNon-axenic microalga Chlorella sorokiniana was cultivated in batch cultures, and its total sugar composition was determined. The microalga under study showed a total sugar concentration of 21.44 ± 0.46% (w/w). The effects of freeze-drying, oven-drying, freezing and thawing, chemical and the combination of hydrothermal and chemical pretreatments were evaluated. In the combined pretreatment different concentrations of H2SO4 and reaction times were also optimized. It was possible to determine that the sugar extraction yields more significant were 59.5% for the lyophilization, 6.2% with 6 cycles of freeze and thawing and around 100% for 2 and 4% (v/v) of H2SO4 at 121 °C for 30 min. Some of the methods that were described in this study are interesting to facilitate cost-efficient conversion of microalgal biomass into biofuels.
- Microalgae growth in different trophic conditionsPublication . Constantino, Ana; Jardim, Beatriz; Leon, Antonio; Leon, Rosa; Rodrigues, Brígida; Raposo, SaraMicroalgae growth in different trophic conditions ( Poster Presentation) in Abstract Book 5th Algaeurope Conference
- Biofuels production by chlorella sorokiniana in a biorefinery perspectivePublication . Constantino, Ana; Glória, Patrícia; Rodrigues, Brígida; Leon, Rosa; Barros, Raúl; Raposo, SaraBiofuels Production By Chlorella Sorokiniana In A Biorefinery Perspective (Poster Presentation) in Abstract Book 5th Algaeurope Conference