Selenium-enriched Nannochloropsis oceanica for aquaculture feed

This thesis aimed at investigating the accumulation and speciation of Se in N. oceanica, and its applicability as an organic dietary Se supplement in aquafeed at both laboratory and pilot-scale. Strategies were explored to improve Se accumulation and uptake efficiency. Finally, the Se-enriched N. oceanica was fed to gilthead seabream (Sparus aurata) and its bioavailability assessed. Marine microalgal species are cultivated in artificial or natural seawater which is rich in salts such as sodium (Na), calcium (Ca), and magnesium (Mg). In Chapter 2 an artificial seawater medium was formulated. The effect of washing the microalgal biomass prior to elemental composition determination was investigated. It was found that washing the biomass twice with ammonium formate enabled the removal of seawater components such as Na, K, and Ca. Furthermore, it was found that unwashed biomass resulted in the oversaturation of the ICP-OES detector. Washing the microalgal biomass enabled for the removal of cell debris, the excess Na, K, Ca present in the leftover medium, and a more accurate elemental analysis. The concentration of the other elements was determined in the culture medium and biomass samples. The elements present in the biomass were present in the following order: P > S > K > Mg > Ca> Fe > Na > Zn > Mn > Cu > Co ˜ Mo. Se enrichment in N. oceanica was investigated by identifying the optimal inorganic Se form and concentration for Se-enriched N. oceanica biomass production at laboratory and pilot-scale (Chapter 3). Strategies such as sulphur (S) and phosphorus (P) limitation were explored to improve Se uptake efficiency (Chapter 4, 5). In Chapter 3 the two inorganic forms (sodium selenate and selenite) were assessed as Se sources for the production of Se-enriched N. oceanica. Initially, the toxicity of these two inorganic Se forms was assessed (EC50 selenite = 163.82 µM, EC50 selenate = 32.93 µM). Subsequently, the accumulation of Se was evaluated in N. oceanica. It was concluded that sodium selenite was more efficiently accumulated than sodium selenate (up to 2.16 gSe/kgbiomass). In addition, the daily uptake of Se was studied in a tubular reactor in AlgaePARC (Wageningen, The Netherlands) over several batch processes in a greenhouse supplemented with artificial light. Se accumulation was positively correlated with cell growth. Throughout the sequential batch runs the Se accumulation remained relatively stable (0.10-0.16 gSe/kgbiomass). The Se inclusion in the biomass at pilot-scale (1300 L) was similar to that achieved in laboratory scale experiments (0.15 L). The biomass produced at AlgaePARC was later used for the gilthead seabream trials in Chapter 6. In Chapter 4, the effect of S on Se accumulation and speciation was investigated by using S deplete and replete conditions. N. oceanica was cultivated under different S concentrations, with and without Se. Se accumulation increased up to 8-fold under Se deplete conditions. Se speciation demonstrated that N. oceanica is capable of transforming inorganic Se into organic. The average relative abundance of organic Se species was 98.2% selenomethionine (SeMet), 1.4% selenocystine (SeCys2), and 0.4% selenomethyl selenocysteine (SeMeSeCys). Total fatty acid content was not significantly affected by S limitation or the presence of Se, but a decrease in EPA was observed with S limitation. In Chapter 5, the Se uptake efficiency in N. oceanica was investigated by altering P and Se concentrations in the media. Overall, P limitation (= 200 µM) increased Se accumulation and uptake efficiency. The highest Se accumulation was observed for the culture treated with 10 µM of P and 30 µM of Se, which was 16-fold higher than the control condition (P 2470 µM, Se 30 µM). The highest Se uptake efficiency was observed for P 100 µM and Se 5 µM (82% Se uptake efficiency), which led to an increase of 3.6-fold compared to the control condition (P 2470, Se 5). N. oceanica was cultivated in a flat-panel photobioreactor (PBR) in batch operation with either 250 µM or 750 µM of P in the medium and Se uptake was monitored daily. Se uptake increased with P limitation and was correlated with cell growth. In Chapter 6, fish trials with gilthead seabream were performed to assess the uptake and bioavailability of different dietary Se sources in aquafeed (SPAROS, Ltd). No negative effects on fish growth were observed from the different dietary Se sources. Se-enriched yeast and Se-enriched microalgae resulted in an increase in Se accumulation in fish muscle compared to inorganic Se. It was concluded that Se-enriched N. oceanica is a promising organic source of Se and that further work should focus on the Se balance and its effect on the environment. Finally, in Chapter 7 the previous chapters were discussed and the current status of Se-enriched microalgae evaluated. The different strategies used in this thesis to improve Se accumulation and uptake efficiency were evaluated and compared to the literature. The optimum method for Se-enriched production to maximise Se uptake efficiency and minimise Se in the wastewater was implementing phosphorus limitation in a repeated batch production process. A perspective was given on the potential of incorporation of Se-enriched microalgae in aquafeed as an organic supplement. Se-enriched N. oceanica can be considered an organic Se supplement due to its high organic Se content (62.6% to 87.6%). The possible demand for Se supplementation was explored and the current cost of microalgae production was discussed. The discussion focused on a price range for Se-microalgae production for a 250 m2 to 1 hectare microalgae farm with an estimated cost of €123 and €64.2 per kilo, respectively. This would allow for modular microalgae farm productions which would aim to meet the demand for Se supplementation which could range from 146,000 tonnes to 1.46 million tonnes of dry Se-enriched microalgae biomass per year by 2025. With further innovations in the microalgal biotechnology field, Se-enriched microalgae could become a commercial reality and contribute towards sustainable aquaculture intensification.