Intensive production of the harpacticoid copepod Tigriopus californicus in a zero-effluent ‘green water’ bioreactor

Aquaculture is looking for substitutes for fishmeal and fish oil to maintain its continued growth. Zooplankton is the most nutritious option, but its controlled mass production has not yet been achieved. In this context, we have developed a monoalgal ‘green water’ closed-loop bioreactor with the microalgae Tetraselmis chui that continuously produced the harpacticoid copepod Tigriopus californicus. During 145 days of operation, the 2.2 m3 bioreactor produced 3.9 kg (wet weight) of Tigriopus with (dry weight) 0.79 ± 0.29% eicosapentaenoic acid (EPA), 0.82 ± 0.26% docosahexaenoic acid (DHA), 1.89 ± 0,60% 3S,3’S-astaxanthin and an essential amino acid index (EAAI) of 97% for juvenile Atlantic salmon. The reactor kept the pH stable over the operation time (pH 8.81 ± 0.40 in the algae phase and pH 8.22 ± 2.96 in the zooplankton phase), while constantly removed nitrate (322.6 mg L−1) and phosphate (20.4 mg L−1) from the water. As a result of the stable pH and nutrient removal, the bioreactor achieved zero effluent discharges. The upscaling of monoalgal, closed-loop ‘green water’ bioreactors could help standardize zooplankton mass production to supply the aquafeeds industry.

Protein Enrichment of Wheat Bread with Microalgae: Microchloropsis gaditana, Tetraselmis chui and Chlorella vulgaris

Cell wall disrupted and dried Microchloropsis gaditana (Mg), Tetraselmis chui (Tc) and Chlorella vulgaris (Cv) microalgae biomasses, with or without ethanol pre-treatment, were added to wheat bread at a wheat flour substitution level of 12%, to enrich bread protein by 30%. Baking performance, protein quality and basic sensory properties were assessed. Compared to wheat, Mg, Tc and Cv contain higher amounts of essential amino acids and their incorporation markedly improved protein quality in the bread (DIAAS 57–66 vs 46%). The incorporation of microalgae reduced dough strength and bread volume and increased crumb firmness. This was most pronounced for Cv and Tc but could be improved by ethanol treatment. Mg gave adequate dough strength, bread volume and crumb structure without ethanol treatment. To obtain bread of acceptable smell, appearance, and colour, ethanol treatment was necessary also for Mg as it markedly reduced the unpleasant smell and intense colour of all algae breads. Ethanol treatment reduced the relative content of lysine, but no other essential amino acids. However, it also had a negative impact on in vitro protein digestibility. Our results show that Mg had the largest potential for protein fortification of bread, but further work is needed to optimize pre-processing and assess consumer acceptance.

Development of High-Protein Vegetable Creams by Using Single-Cell Ingredients from Some Microalgae Species

The aim of this paper was to develop high-protein vegetable creams through the incorporation of microalgae. Single-cell ingredients from Arthrospiraplatensis (spirulina), Chlorella vulgaris, Tetraselmis chui, and Nannochloropsis oceanica were incorporated at two levels of addition (1.5% and 3.0%) to a standard vegetable cream (STD). Effects of incorporation were assessed in terms of physicochemical and rheological attributes as well as nutritional labeling facts. Creams formulated with 3% A. platensis, N. oceanica, or T. chui showed strong color differences (6 < ΔE < 12) compared to STD; creams formulated with 1.5% A. platensis, T. chui, or N. oceanica showed perceptible differences (3 < ΔE < 6); and those made with C. vulgaris at 1.5 and 3% exhibited small differences (ΔE < 2). Moisture content, water activity, pH, syneresis, and °Brix did not show significant changes. Adding microalgae increased Bostwick consistency and decreased the consistency coefficient (K) except in creams made with A. platensis, which showed comparable values to STD. Principal component analysis indicated that creams made with 1.5% C. vulgaris were the most similar to STD considering all evaluated parameters. Estimation of the nutritional labeling facts showed that the four formulations could be labeled as having “high protein content” following the present EU legislation.

Selection and validation of reference genes for quantitative real-time PCR in the green microalgae Tetraselmis chui

Quantitative real-time reverse transcription PCR (RT-qPCR) is a highly sensitive technique that can be applied to analyze how genes are modulated by culture conditions, but identification of appropriate reference genes for normalization is a critical factor to be considered. For this reason, the expression stability of 18 candidate reference genes was evaluated for the green microalgae Tetraselmis chui using the widely employed algorithms geNorm, NormFinder, BestKeeper, the comparative ΔCT method, and RefFinder. Microalgae samples were collected from large scale outdoor photobioreactors during the growing phase (OUT_GP), and during the semi-continuous phase at different times of the day (OUT_DC). Samples from standard indoor cultures under highly controlled conditions (IND) were also collected to complement the other data. Different rankings for the candidate reference genes were obtained depending on the culture conditions and the algorithm employed. After comparison of the achieved ranks with the different methods, the references genes selected for samples from specific culture conditions were ALD and EFL in OUT_GP, RPL32 and UBCE in OUT_DC, and cdkA and UBCE in IND. Moreover, the genes EFL and cdkA or EFL and UBCE appeared as appropriate combinations for pools generated from all samples (ALL). Examination in the OUT_DC cultures of genes encoding the large and small subunits of ADP-glucose pyrophosphorylase (AGPL and AGPS, respectively) confirmed the reliability of the identified reference genes, RPL32 and UBCE. The present study represents a useful contribution for studies of gene expression in T. chui, and also represents the first step to set-up an RT-qPCR platform for quality control of T. chui biomass production in industrial facilities.

Evaluación de dietas microalgales para pre-semillas de la ostra de mangle Crassostrea rhizophorae (Güilding, 1828) y su crecimiento en condiciones exteriores

Con el fin de garantizar un nivel adecuado de las macromoleculas energéticas, que permitan su transferencia exitosa al ambiente natural, se estudió la supervivencia, el crecimiento y la composición bioquímica (carbohidratos, lípidos y proteínas) de pre-semillas (“spat”) (talla inicial 3.77 ± 0.64 mm) de ostra de mangle Crassostrea rhizophorae, alimentada con tres microalgas tropicales: Chaetoceros sp. Cepa Araya (Ch-A), Isochrysis galbana (Ig) y Tetraselmis chui (Tc), ofertadas como dietas unialgales o mezcladas en dietas bialgales y trialgales. La composición bioquímica de las diferentes cepas de microalgas fue también determinada. En el bioensayo bajo condiciones de laboratorio (hatchery), se utilizaron tres acuarios (réplicas con 21 semillas) para cada una de las dietas. Adicionalmente tres réplicas se confinaron en el medio ambiente (control). Después de 36 días, se analizó sus contenidos bioquímicos y registraron las variables biométricas, y los juveniles se transfirieron al ambiente exterior (tres réplicas) y se cultivaron en suspensión durante 30 días. Al final del cultivo en el medioambiente se obtuvieron de nuevo sus variables bioquímicas y biométricas. Sin embargo, debido a una alta mortalidad ocurrida en las réplicas de control (≈80%), no fue posible obtener más datos de estas últimas, después de 36 días. Durante el cultivo en el medio ambiente, se tomaron muestras semanales de las variables ambientales. En general, en el cultivo en el laboratorio, los mayores valores biométricos y bioquímicos se obtuvieron en los organismos alimentados con la dieta monoalgal (Ch-A), dietas bialgales (Ch-A + Tc y Ch-A + Ig) y la trialgal (Ch-A+Ig+Tc), una tendencia que se mantuvo en el cultivo en el medio ambiente. Lo anterior se atribuyó a la contribución equilibrada de las biomoléculas ofrecidas anteriormente por estas dietas. Estos resultados evidenciaron el uso de la energía contenida en carbohidratos y lípidos, por parte de las ostras juveniles, una vez que fueron transferidas al medio ambiente, donde estas fuentes de energía probablemente fueron catabolizadas para compensar la escasa disponibilidad de alimentos (baja Clorofila a y materia orgánica) observada en el sitio de cultivo en el medio ambiente.