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- New dual-stage pH control fed-batch cultivation strategy for the improvement of lipids and carotenoids production by the red yeast Rhodosporidium toruloides NCYC 921Publication . Dias, Carla; Sousa, Sofia; Caldeira, João; Reis, Alberto; Silva, Teresa Lopes daThe optimal medium pH to produce biomass and fatty acids by the red yeast Rhodosporidium toruloides NCYC 921 is 4.0, and to produce carotenoids is 5.0. Based on this difference, a dual-stage pH control fed-batch cultivation strategy for the enhancement of lipids and carotenoids production by this yeast was studied. The results showed that when the yeast growth phase was conducted at pH 4.0, and the products accumulation phase was conducted at pH 5.0, biomass, total fatty acid and total carotenoid productivities were significantly improved comparing with the yeast fed batch cultivations carried out at fixed medium pH (4 or 5). Under dual-stage pH control conditions, the biomass, carotenoids and lipids productivities attained 2.35 g/L h, 0.29 g/L h and 0.40 g/L h, respectively. It was also observed that the oxygen played a major role in the yeast carotenoid production.
- The Use of Oleaginous Yeasts and Microalgae Grown in Brewery Wastewater for Lipid Production and Nutrient Removal: A ReviewPublication . Dias, Carla; Santos, José A. L.; Reis, Alberto; Silva, Teresa Lopes daABSTRACT: Brewery wastewater has been proposed as an attractive low-cost substrate for microbial lipid production for oleaginous yeast and microalga with promising results. For each liter of beer produced, from 3 to 10 L of wastewater are generated which can be used as culture medium for autotrophic or heterotrophic metabolism. This strategy allows reducing the culture medium cost, as well as obtaining high lipid contents and other high value compounds which can make the process profitable. Additionally, the use of industrial effluents/wastes as substrates for microbial growth can be a strategy to treat them based on the circular economy rules. This review presents the different brewery wastewater treatment strategies using oleaginous yeast and microalga pure and mixed cultures for the concomitant wastewater treatment and lipids/carotenoids production so far reported, highlighting the benefits/disadvantages of such strategies and comparing their performance in terms of wastewater treatment, lipids and carotenoids production between pure and mixed cultures performance.
- Lipid and carotenoid production by a Rhodosporidium toruloides and Tetradesmus obliquus mixed culture using primary brewery wastewater supplemented with sugarcane molasses and ureaPublication . Dias, Carla; Nobre, B. P.; Santos, J. A. L.; Reis, Alberto; Silva, Teresa Lopes daABSTRACT: In this study, Rhodosporidium toruloides and Tetradesmus obliquus were used for lipid and carotenoid production in mixed cultures using primary brewery wastewater (PBWW) as a culture medium, supplemented with sugarcane molasses (SCM) as a carbon source and urea as a nitrogen source. To improve biomass, lipid, and carotenoid production by R. toruloides and T obliquus mixed cultures, initial SCM concentrations ranging from 10 to 280 g L-1 were tested. The medium that allowed higher lipid content (26.2% w/w dry cell weight (DCW)) and higher carotenoid productivity (10.47 mu g L-1 h(-1)) was the PBWW medium supplemented with 100 g L-1 of SCM and 2 g L-1 of urea, which was further used in the fed-batch mixed cultivation performed in a 7-L bioreactor. A maximum biomass concentration of 58.6 g L-1 and maximum lipid content of 31.2% w/w DCW were obtained in the fed-batch cultivation. PBWW supplemented with SCM was successfully used as a low-cost medium to produce lipids and carotenoids in a R. toruloides and T obliquus mixed culture, with higher productivities than in pure cultures, which can significantly reduce the cost of the biofuels obtained.
- Concomitant wastewater treatment with lipid and carotenoid production by the oleaginous yeast Rhodosporidium toruloides grown on brewery effluent enriched with sugarcane molasses and ureaPublication . Dias, Carla; Reis, Alberto; Santos, J. A. L.; Silva, Teresa Lopes daABSTRACT: In this study, secondary brewery wastewater (SBWW) supplemented with sugarcane molasses (SCM) was used for SBWW treatment with concomitant lipid and carotenoid production by the yeast Rhodosporidium toruloides NCYC 921. In order to improve the biomass production, ammonium sulfate, yeast extract and urea were tested as nitrogen sources. Urea was chosen as the best low-cost nitrogen source. A fed-batch cultivation was carried out with SBWW supplemented with 10 g L−1 of sugarcane molasses as carbon source, and 2 g L−1 of urea as nitrogen source. A maximum biomass concentration of 42.5 g L−1 was obtained at t=126.5 h and the maximum biomass productivity was 0.55 g L−1 h−1 at t=48.25 h. The maximum lipid content was 29.9 % w/w (DCW) at t=94 h of cultivation and the maximum carotenoid content was 0.23 mg g−1 at 120 h of cultivation. Relatively to the SBWW treatment, after the batch phase, 45.8 % of total Kjeldahl nitrogen removal, 81.7 % of COD removal and 100 % of sugar consumption were observed. Flow cytometry analysis revealed that 27.27 % of the cells had injured membrane after the inoculation. This proportion was reduced to 10.37 % at the end of the cultivation, indicating that cells adapted to the growth conditions.
- Sequential carotenoids extraction and biodiesel production from Rhodosporidium toruloides NCYC 921 biomassPublication . Passarinho, Paula; Oliveira, Bruno; Dias, Carla; Teles, Marta; Reis, Alberto; Silva, Teresa Lopes daABSTRACT: A new process for co-extraction and separation of fatty acids and carotenoids from Rhodosporidium toruloides NCYC 921 biomass in order to achieve full exploitation of the yeast lipidic fraction is described. A saponification of the wet yeast biomass was performed using a potassium hydroxide solution (1.1 M) in ethanol 96%, at 65 °C for 180 min. In the carotenoid extraction step, a biphasic system with an organic: aqueous phases ratio of 0.49 mL/mL and a water content of 18.9% (w/w) was used. In the presence of an acid catalyst, the fatty acid fraction was esterified into fatty acids ethyl esters. The yeast biomass downstream processing allowed reaching a fatty acid and total carotenoids recovery yields of 91.0% and 85.2%, respectively. The process reported here takes advantage of various components of the yeast biomass, therefore maximizing the value derived from the biomass feedstock, with a minimal environmental impact within the frame of circular bioeconomy.
- Effect of brewery effluent inhibitors on Rhodotorula toruloides NCYC 921 cells grown in pure and mixed cultures at pH 4 and 6Publication . Dias, Carla; Santos, J. A. L.; Reis, Alberto; Silva, Teresa Lopes daABSTRACT: The presence of inhibitor compounds in the culture medium can cause severe effects on the microorganisms cells. Brewery wastewaters present organic acids (acetic, propionic and butyric acids) which can severely affect yeast cells metabolism, when grown in pure cultures, although in mixed cultures they are able to develop. To understand the physiological changes on Rhodotorula toruloides (formerly Rhodosporidium toruloides) cells when fermenting in the presence of the organic acids present in brewery wastewater, pure and mixed cultures with the microalga Tetradesmus obliquus were performed in a synthetic medium containing the same organic acids concentrations that are present in brewery wastewater at pH 4 and 6. It was concluded that, at pH 4, the organic acids effects in the yeast cells were much more toxic than at pH 6. Moreover, mixed cultures can be an advantage over heterotrophic pure cultures as the microalga is able to contribute for the consumption of potential inhibitors for the yeast.
- Rhodosporidium toruloides and Tetradesmus obliquus Populations Dynamics in Symbiotic Cultures, Developed in Brewery Wastewater, for Lipid ProductionPublication . Dias, Carla; Gouveia, Luisa; Santos, J. A. L.; Reis, Alberto; Silva, Teresa Lopes daABSTRACT: In this work, primary brewery wastewater (PBWW) and secondary brewery wastewater (SBWW) separately, or mixed at the ratios of 1:1 (PBWW:SBWW) and 1:7 (PBWW:SBWW), with or without supplementation with sugarcane molasses (SCM), were used as culture media for lipid production by a mixed culture of the oleaginous yeast Rhodosporidium toruloides NCYC 921 and the microalgae Tetradesmus obliquus (ACOI 204/07). Flow cytometry was used to understand the dynamics of the two micro-organisms during the mixed cultures evolution, as well as to evaluate the physiological states of each microorganism, in order to assess the impact of the different brewery effluent media composition on the microbial consortium performance. Both brewery wastewaters (primary and secondary) without supplementation did not allow R. toruloides heterotrophic growth. Nevertheless, all brewery wastewater media, with and without SCM supplementation, allowed the microalgae growth, although the yeast was the dominant population. The maximum total biomass concentration of 2.17 g L-1 was achieved in the PBWW mixed cultivation with 10 g L-1 of SCM. The maximum lipid content (14.86% (w/w DCW)) was obtained for the mixed culture developed on SBWW supplemented with 10 g L-1 of SCM. This work demonstrated the potential of using brewery wastewater supplemented with SCM as a low-cost culture medium to grow R. toruloides and T. obliquus in a mixed culture for brewery wastewater treatment with concomitant lipid production.
- Carob pulp syrup : a potential Mediterranean carbon source for carotenoids production by Rhodosporidium toruloides NCYC 921Publication . Martins, Vasco; Dias, Carla; Caldeira, João; Duarte, Luís C.; Reis, Alberto; Silva, Teresa Lopes daABSTRACT: Carob pulp syrup (CPS) was used as carbon source to produce carotenoids from Rhodosporidium toruloides. To increase the carbon concentration in the growth medium aiming at the carotenoid production improvement, the CPS was concentrated and two different total sugar (TS) concentrations (195.6 g/L and 548.7 g/L) were studied. CPS 195.6 g/L TS contained 4.1 g/L and 0.7 g/L of hydroxymethyl furfural (HMF) and furfural, respectively. CPS 548.7 g/L TS contained 17.7 g/L of HMF and 1.2 g/L of furfural, respectively. >42% of metabolically active cells (with intact membrane and enzymatic activity) were detected throughout the course of the yeast cultivation on CPS 195.6 g/L TS. On the contrary, the proportion of metabolically active cells was always below 28% during the yeast cultivation on CPS 548.7 g/L TS. Nevertheless, the maximum carotenoid content and productivity (0.42 mg/g and 0.43 mg/Lh, respectively) were obtained when using CPS 548.7 g/L TS.
- Biofuels and high value added products from the yeast Rhodosporidium toruloides NCYC 921: Strategies towards a true cost-effective and environmentally sustainable integrated multiproduct driven biorefinery [Poster]Publication . Reis, Alberto; Dias, Carla; Caldeira, João; Teles, Marta; Oliveira, Bruno; Passarinho, Paula; Silva, Teresa Lopes daSingle-cell oils (SCO) have been considered a promising source of 3rd generation biofuels mainly in the final form of biodiesel. However, its high production costs have been a barrier towards the commercialization of this commodity. The fast growing yeast Rhodosporidium toruloides NCYC 921 has been widely reported as a potential SCO producing yeast. In addition to its well-known high lipid content (that can be converted into biodiesel), is rich in high value added products such as carotenoids with commercial interest. The process design and integration may contribute to reduce the overall cost of biofuels and carotenoid production and is a mandatory step towards their commercialization. The present work addresses the biomass disruption, extraction, fractionation and recovery of products with special emphasis on high added valued carotenoids (beta-carotene, torulene, torularhodin) and fatty acids directed to biodiesel. The chemical structure of torularhodin with a terminal carboxylic group imposes an additional extra challenge in what concern its separation from fatty acids. The proposed feedstock is fresh biomass pellet obtained directly by centrifugation from a 5L fed-batch fermentation culture broth. The use of a wet instead of lyophilised biomass feedstock is a way to decrease processing energy costs and reduce downstream processing time. These results will contribute for a detailed process design. Gathered data will be of crucial importance for a further study on Life-Cycle Assessment (LCA).
- Primary brewery wastewater as feedstock for the yeast Rhodosporidium toruloides and the microalga Tetradesmus obliquus mixed cultures with lipid productionPublication . Dias, Carla; Reis, Alberto; Santos, J. A. L.; Gouveia, Luisa; Silva, Teresa Lopes daABSTRACT: Rhodosporidium toruloides and Tetradesmus obliquus pure and mixed cultures were grown on primary brewery wastewater (PBWW), with and without supplementation of sugarcane molasses (SCM) and urea. R. toruloides developed in pure cultures was not able to grow on PBWW, with or without supplementation, but grew in mixed cultures with the micmalga T. obliquus in all media. In contrast, all T. obliquus pure cultures developed on PBWW, with and without supplementation, were able to develop. Higher biomass productivity (149.3 mg L-1 h(-1)) was obtained for the mixed culture using PBWW supplemented with 10 g L-1 of SCM and 2 g L-1 of urea. Therefore, a strategy to stimulate lipid production by R. toruloides and T. obliquus grown in mixed cultures with higher SCM concentrations (20, 40 and 100 g L-1 of SCM) was developed. The maximum lipid content (26.3 % (w/w DCW)) was obtained for the mixed culture developed on PBWW, supplemented with 100 g L-1 of SCM plus 2 g L-1 of urea, at t = 121 h. This work demonstrated the feasibility of using PBWW supplemented with SCM and urea as culture medium to grow R. toruloides and T. obliquus and to produce microbial lipids.