Biochemical and morphological characterization of freshwater microalga Tetradesmus obliquus (Chlorophyta: Chlorophyceae).

Tetradesmus is a microalgal genus with biotechnological potential due to its rapid production of biomass, which is plenty in proteins, carbohydrates, lipids, and bioactives. However, its morphology and physiology need to be determined to guide better research to optimize the species cultivation and biocompounds processing. Thus, this study describes the biochemistry and morphology of the strain Tetradesmus obliquus BR003, isolated from a sample of freshwater reservoirs in a Brazilian municipality. In the T. obliquus BR003 dry biomass, we identified 61.6% unsaturated fatty acids, and 3.4% saturated fatty acids. Regarding other compounds, 28.50 ± 1.47 g soluble proteins/100 g, 0.14 ± 0.009 g carotenoids/100 g, 0.76 ± 0.013 g chlorophyll a/100 g, and 0.42 ± 0.015 g chlorophyll b/100 g with a chlorophyll a/b ratio of 1.8 were detected. The main chemical elements found were S, Mg, and P. The cells of BR003 were elliptically curved at the ends and without appendages. Histochemical tests showed carbohydrates distributed in the cytoplasm and pyrenoids, some lipid droplets, and proteins. The cytoplasm is rich in vacuoles, rough endoplasmic reticulum, mitochondria, and chloroplasts. The nucleus has a predominance of decondensed chromatin, and the cell wall has three layers. Chloroplasts have many starch granules and may be associated with a spherical central pyrenoid. To the best of our knowledge, this was the first biochemical description combined with ultrastructural morphological characterization of the strain T. obliquus BR003, grown under standard conditions, to demonstrate specific characteristics of the species.

Harvesting of Chlorella sorokiniana BR001 cultivated in a low-nitrogen medium using different techniques / Colheita de Chlorella sorokiniana BR001 cultivada em um meio com baixo teor de nitrogênio utilizando diferentes técnicas

ABSTRACT: The harvesting process is a current challenge for the commercial production of microalgae because the biomass is diluted in the culture medium. Several methods have been proposed to harvest microalgae cells, but there is not a consensus about the optimum method for such application. Herein, the methods based on sedimentation, flocculation, and centrifugation were evaluated on the recovery of Chlorella sorokiniana BR001 cultivated in a low-nitrogen medium. C. sorokiniana BR001 was cultivated using a low-nitrogen medium to trigger the accumulation of neutral lipids and neutral carbohydrates. The biomass of C. sorokiniana BR001 cultivated in a low-nitrogen medium showed a total lipid content of 1.9 times higher (23.8 ± 4.5%) when compared to the biomass produced in a high-nitrogen medium (12.3 ± 1.2%). In addition, the biomass of the BR001 strain cultivated in a low-nitrogen medium showed a high content of neutral carbohydrates (52.1 ± 1.5%). The natural sedimentation-based process was evaluated using a sedimentation column, and it was concluded that C. sorokiniana BR001 is a non-flocculent strain. Therefore, it was evaluated the effect of different concentrations of ferric sulfate (0.005 to 1 g L-1) or aluminum sulfate (0.025 to 0.83 g L-1) on the flocculation process of C. sorokiniana BR001, but high doses of flocculant agents were required for an efficient harvest of biomass. It was evaluated the centrifugation at low speed (300 to 3,000 g) as well, and it was possible to conclude that this process was the most adequate to harvest the non-flocculent strain C. sorokiniana BR001.

RESUMO: O processo de colheita é um desafio atual para a produção comercial de microalgas porque a biomassa é diluída no meio de cultivo. Diversos métodos têm sido propostos para coletar células de microalgas, porém não existe um consenso sobre um método ótimo para tal aplicação. Neste estudo, métodos baseados em sedimentação, floculação e centrifugação foram avaliados na recuperação de Chlorella sorokiniana BR001 cultivada em um meio com baixo teor de nitrogênio. C. sorokiniana BR001 foi cultivada em um meio com baixo teor de nitrogênio para induzir ao acúmulo de lipídeos e carboidratos neutros. A biomassa de C. sorokiniana BR001 cultivada em um meio com baixo teor de nitrogênio apresentou um teor de lipídeos 1,9 vezes superior (23,8 ± 4,5%), quando comparada à biomassa produzida em um meio com alto teor de nitrogênio (12,3 ± 1,2%). Adicionalmente, a biomassa da linhagem BR001 cultivada em um meio com baixo teor de nitrogênio apresentou alto teor de carboidratos neutros (52,1 ± 1,5%). O processo baseado em sedimentação natural foi avaliado utilizando uma coluna de sedimentação e concluiu-se que C. sorokiniana BR001 é uma linhagem não floculante. Portanto, o efeito de diferentes concentrações de sulfato férrico (0,005 a 1 g L-1) ou sulfato de alumínio (0,025 a 0,83 g L-1) foram avaliados no processo de floculação de C. sorokiniana BR001, mas altas doses de floculantes foram necessárias para uma colheita de biomassa eficiente. Também foi avaliada a centrifugação em baixa velocidade (300 a 3.000 g), e foi possível concluir que este processo constituiu o mais adequado para a colheita da linhagem não floculante C. sorokiniana BR001.

Microalgae proteins: production, separation, isolation, quantification, and application in food and feed.

Many countries have been experienced an increase in protein consumption due to the population growth and adoption of protein-rich dietaries. Unfortunately, conventional-based protein agroindustry is associated with environmental impacts that might aggravate as the humankind increase. Thus, it is important to screen for novel protein sources that are environmentally friendly. Microalgae farming is a promising alternative to couple the anthropic emissions with the production of food and feed. Some microalgae show protein contents two times higher than conventional protein sources. The use of whole microalgae biomass as a protein source in food and feed is simple and well-established. Conversely, the production of microalgae protein supplements and isolates requires the development of feasible and robust processes able to fractionate the microalgae biomass in different value-added products. Since most of the proteins are inside the microalgae cells, several techniques of disruption have been proposed to increase the efficiency to extract them. After the disruption of the microalgae cells, the proteins can be extracted, concentrated, isolated or purified allowing the development of different products. This critical review addresses the current state of the production of microalgae proteins for multifarious applications, and possibilities to concatenate the production of proteins and advanced biofuels.

Extraction of proteins from the microalga Scenedesmus obliquus BR003 followed by lipid extraction of the wet deproteinized biomass using hexane and ethyl acetate.

A current problem of the lipid extraction from wet biomass is the formation of emulsions during the mixing of the microalgal biomass and organic solvents. It has been suggested that microalgal proteins play an important role in the formation and stability of such emulsions. Herein, the extraction of proteins of the freshwater microalga Scenedesmus obliquus BR003 was optimized for further extraction of lipids from the wet deproteinized biomass. The optimal (pH 12 at 60 °C for 3 h) and moderate (pH 10.5 at 50 °C for 2 h) conditions of protein extraction resulted in protein yields of 20.6% and 15.4%, respectively. Wet lipid extraction of deproteinized biomass resulted in a less stable emulsion that released twice the solvent than the control biomass. However, the faster separation of phases that occurred during the wet lipid extraction of the deproteinized biomass resulted in a lipid yield twice lower than the control biomass.

Alternative fertilizer-based growth media support high lipid contents without growth impairment in Scenedesmus obliquus BR003.

Nitrogen (N) sources have been target in microalgae cultivation studies, considering their nutritional impact on growth and high costs. Here, we have evaluated the growth of Scenedesmus obliquus BR003, applying alternative low-cost culture media containing ammonium and urea, or combinations of both N sources. The culture media were applied for indoor and outdoor cultivation, followed by growth analyses and metabolic characterization. The alternative culture media B4 and L4 supported higher biomass production (1.4 g L-1) compared to BG11 (nitrate-based medium). In addition, the lipid percentage was higher for B4 (ammonium-based culture medium), reaching up to 25% DW. High contents of carbohydrates (60%) and proteins (40%) were also obtained in media with ammonium and urea, respectively. Considering the lower costs of alternative fertilizer-based media, using ammonium and/or urea as N sources, and the high lipid content observed, we suggest these media as viable for large-scale production of S. obliquus.

Combination of trace elements and salt stress in different cultivation modes improves the lipid productivity of Scenedesmus spp.

The genus Scenedesmus harbors around 120 species, and some strains have been successfully used for mass culture and biotechnological applications. Considering the potential of this genus as a promising feedstock for production of biofuels, mainly biodiesel, it was evaluated the combined effects of trace elements, salinity stress and different cultivation modes (single batch, semi-continuous, and two-stage batch) on lipid productivity of the freshwater strains S. obliquus BR003 and S. bajacalifornicus BR024. Cultivation of BR003 and BR024 applying culture medium supplemented with trace elements and salt stress sustained a higher production of lipids. However, S. obliquus BR003 and S. bajacalifornicus BR024 showed different concentrations of neutral and total lipids when cultivated in batch-based and semi-continuous modes, and the batch-based modes were preferred for the production of lipids and carbohydrates. Consequently, different cultivation strategies coupled with slight salt stress improve the lipid productivity in Scenedesmus strains.

Drying of microalga Scenedesmus obliquus BR003 in a gas dryer at low temperatures / Secagem da microalga Scenedesmus obliquus BR003 em secador a gás em baixas temperaturas

RESUMO: O desenvolvimento de equipamentos eficientes e específicos para a secagem de microalgas é essencial para a exploração comercial destes microrganismos que apresentam alta taxa de crescimento e grande potencial biotecnológico. Os custos de secagem da biomassa de microalgas ainda são elevados e precisam ser reduzidos para a produção de compostos com baixo valor agregado. Portanto, realizou-se o estudo da secagem da microalga Scenedesmus obliquus BR003 utilizando baixas temperaturas. S. obliquus BR003 é uma microalga robusta que apresenta alta produtividade de lipídeos. Em escala laboratorial, observou-se que a biomassa de S. obliquus BR003 foi rapidamente seca em baixas temperaturas entre 50 e 60 ºC. Um secador a gás foi utilizado para avaliar a secagem da biomassa de S. obliquus BR003 em escala piloto. A biomassa foi seca em menos de 24 h utilizando o secador a gás, entretanto, a elevada umidade da biomassa da microalga requereu uma maior renovação de ar na câmara do secador. A análise de fluidodinâmica computacional do secador a gás mostrou dois parâmetros importantes para se obter uma maior efetividade de transferência de calor e massa durante o processo de secagem da biomassa de microalga. Concluiu-se que um secador a gás adequado, para a biomassa de microalgas, deve possuir múltiplos pontos de injeção de ar, e um eficiente sistema de circulação e renovação de ar no interior da câmara de secagem.

ABSTRACT: Development of efficient and specific equipment to dry microalgae is essential for commercial use of these microorganisms that show high growth rates and biotechnological potential. Drying costs of microalgae biomass are still high and they should be reduced for the production of compounds with low added value. Therefore, we evaluated the drying process of the microalga Scenedesmus obliquus BR003 using low temperatures. S. obliquus BR003 is a robust microalga that shows high lipid productivity. At laboratory scale, it was observed that the biomass of S. obliquus BR003 was rapidly dried at low temperatures between 50 and 60 ° C. A gas dryer was used to evaluate the drying of the biomass of S. obliquus BR003 on a pilot-scale. The biomass was dried in less than 24 h using the gas dryer; however, the high moisture of the microalga biomass required a higher air renovation in the drying chamber. Computational fluid dynamics analysis of the gas dryer showed two important parameters to achieve greater effectiveness of heat and mass transfer rates during the drying process of the microalga biomass. It was concluded that a gas dryer suitable for the microalgae biomass should have multiple air injection points, and an efficient circulation and renovation system of air inside the drying chamber.

Fed-batch production of green coconut hydrolysates for high-gravity second-generation bioethanol fermentation with cellulosic yeast.

The residual biomass obtained from the production of Cocos nucifera L. (coconut) is a potential source of feedstock for bioethanol production. Even though coconut hydrolysates for ethanol production have previously been obtained, high-solid loads to obtain high sugar and ethanol levels remain a challenge. We investigated the use of a fed-batch regime in the production of sugar-rich hydrolysates from the green coconut fruit and its mesocarp. Fermentation of the hydrolysates obtained from green coconut or its mesocarp, containing 8.4 and 9.7% (w/v) sugar, resulted in 3.8 and 4.3% (v/v) ethanol, respectively. However, green coconut hydrolysate showed a prolonged fermentation lag phase. The inhibitor profile suggested that fatty acids and acetic acid were the main fermentation inhibitors. Therefore, a fed-batch regime with mild alkaline pretreatment followed by saccharification, is presented as a strategy for fermentation of such challenging biomass hydrolysates, even though further improvement of yeast inhibitor tolerance is also needed.

Green coconut mesocarp pretreated by an alkaline process as raw material for bioethanol production.

Cocos nucifera L., coconut, is a palm of high importance in the food industry, but a considerable part of the biomass is inedible. In this study, the pretreatment and saccharification parameters NaOH solution, pretreatment duration and enzyme load were evaluated for the production of hydrolysates from green coconut mesocarp using 18% (w/v) total solids (TS). Hydrolysates were not detoxified in order to preserve sugars solubilized during the pretreatment. Reduction of enzyme load from 15 to 7.5 filter paper cellulase unit (FPU)/g of biomass has little effect on the final ethanol titer. With optimized pretreatment and saccharification, hydrolysates with more than 7% (w/v) sugars were produced in 48h. Fermentation of the hydrolysate using industrial Saccharomyces cerevisiae strains produced 3.73% (v/v) ethanol. Our results showed a simple pretreatment condition with a high-solid load of biomass followed by saccharification and fermentation of undetoxified coconut mesocarp hydrolysates to produce ethanol with high titer.