Growth optimization of marine diatom Amphora sp. by tailoring silica and nitrate concentration

• ¹ Centre of Biological Engineering, Department of Biological Engineering, Portugal

Aquaculture comprises the production of approximately 23 million tonnes of molluscs and crustaceans in 2014 (FAO, 2016). In addition, it is estimated to steadily increase 4.5 % per year (FAO, 2016). Therefore, it is crucial to meet the demand of feedstock in terms of quantity and quality, particularly due to the recognized correlation between malnutrition and illness in crustaceans (Genc et al., 2007; Tareen, 1982). Unsurprisingly, one of the key steps in the microalgae discovery pipeline described by the European Marine Biological Research Infrastructure Cluster (embric) is the growth optimization. Amphora sp. is regularly used as primary food for larvae of highly valued and praised seafood, such as Crassostrea gigas (Pacific oyster), Penaeus semisulcatus (green tiger shrimp), Placopecten megellanicus (sea scallop), Crepidula onyx (limpet), and Halliotis sp. (abalone), among others (Al-Maslamani et al., 2007; Chiu et al., 2007; Daume et al., 2000; Shpigel et al., 1993; Shumway et al., 1987). The standard culture medium for this marine diatom is the f/2+Si culture medium, which contains nitrate as primary nitrogen source for the synthesis of essential biomolecules, such as amino acids and nucleic acids (Dortch, 1982; Khatoon et al., 2009). Moreover, f/2+Si medium possess metasilicate, required for the development of frustules. Using a design of experiments, the concentration of these two compounds was analysed to improve Amphora sp. biomass output. A preliminary screening of nitrate and metasilicate was performed using the concentrations of 0.5-fold, 1-fold, 2-fold, 5-fold and 10-fold the standard concentration to set the experimental design borders. The biomass was estimated through spectrofluorimetry, previously correlated with the diatom cell concentration determined by cytometry. A central composite design was used to establish a quadratic model with the following equation in coded factors (Figure 1): cell ratio = 2.84 + 0.11 x + 0.07 y + 0.16 xy – 0.18 x^2 – 0.24 y^2, where x corresponds to the sodium nitrate concentration and y to sodium metasilicate concentration. The obtained model, besides being statistical significant, is clearly within the designed borders. The optimal conditions determined using the model comprise 1.55 g L-1 of sodium nitrate and 0.15 g L-1 of sodium metasilicate nonahydrate. This corresponds to an increase of 20-fold and 5-fold the quantities of sodium nitrate and sodium metasilicate nonahydrate in the standard medium, respectively. This results in a raise of approximately 0.18 € L-1 of culture medium. Nevertheless, the improvement of Amphora sp. cell ratio is nearly 1.5-fold in just 72 hour of incubation. The use of this optimal formulation in the culture medium of Amphora sp. will result in an important enhancement of the yield of this aquaculture feedstock, implying a reasonable increase of its price. The enhancement of the feedstock availability may denote a considerable improvement of the nutrition efficacy of the larva, crustaceans and marine snails. By reducing the frequency of undernourishment, the occurrence of diseases and mortality may be effectively decreased.