Increase in larval shellfish survival and expansion of shellfish seed availability through novel feed sources

Shellfish seed availability is limited in New England in the spring when farmers need it most, and this is due to several interrelated factors. There are only a handful of hatcheries in New England, and they are hampered by high costs of startup and production, which can limit expansion and increases in indoor area to produce microalgae feed and larval organisms. This in turn leads to a cap on shellfish seed produced per unit area, which subsequently leads to a lack of available shellfish seed, and higher costs for seed to aquaculture producers in New England. Paramount to this issue is the lack of viable space to produce microalgae for feed, which is the backbone of any shellfish hatchery.

The limiting factor in growing enough microalgae is the square footage inside the hatchery needed to grow the algae, with the primary issue being the low density of microalgae cells to media volume within each culture unit. As an example, one of the most common species of algae grown in shellfish hatcheries is Tetraselmis suecica, which is a high lipid dinoflagellete, and is great food for conditioning adults prior to spawn, and for feeding shellfish set after metamorphosis. At the highest density, this organism can reach up to 3 million cells per milliliter, which only results in 0.5 mg of biomass per liter, or over 99.9% water in culture. This low density is a function of how photoautotrophic microalgae grows. Typical autotrophic microalgae grows just like any other plant, which requires nitrogen, phosphorus, and minerals and nutrients, which the plant uses in conjunction with light absorbed in their chloroplasts to produce sugars, which the cells use to grow and divide. Therefore, the limiting factor for photoautotrophs, given sufficient nutrients and culture media conditions, is light availability. This is limited in microalgae culture, because as the culture volumes increase, and as the density of cells increases, there is naturally shading as the cells move further from the light source. There is a known maximum cell density per culture volume, and it is a function of the species, light penetration and volume of culture vessel or distance from light source, all other culture factors being equal.

However, certain species, such as Tetraselmis suecica, can take in nutrients and create energy to growth and divide through a completely different method, known as heterotrophic growth, or fermentation. In this method, the same microalgae species is grown in the dark, in the complete absence of light, and is given nitrogen and a carbon source, such as glucose of peptone. Due to the fact that light is no longer a factor, the cultures can reach far higher densities, and the same species of Tetraselmis suecica can reach 50 grams per liter as opposed to 0.5 grams per liter when grown as an autotroph. This would also allow for greater efficiency and lower cost per unit to produce shellfish seed, which would allow for lower prices for farmers to purchase shellfish seed.

In this project two different species of dinoflagellate microalgae will be grown through heterotrophic methods, and feed the microalgae to larval and post-set shellfish to investigate clearance rates and subsequent growth and survival. The two species to be cultured are Crypthecodinium cohnii and Tetraselmis suecica.

Crypthecodinium cohnii is a nonphotosynthetic, heterotrophic marine dinoflagellate in which nearly 30–50% of its constituent fatty acids is DHA, other polyunsaturated fatty acids are present in trace amounts. It is a chloroplast-lacking heterotrophic marine species which means that it naturally uses other sources of energy different to light (Jiang and Chen, 2000). This species is ideal for shellfish hatchery work in that it is high in DHA, which is a critical component of egg production and larval development. This species has been studied extensively and is currently represents the major commercially grown source for DHA (Sijtsma et al., 2010). This species has been studied as an enrichment feed to rotifers, as a feed for copepods and as a substitute for fish-derived oils for fish feeds (Ganuza et al., 2008; Eryalçın  et al., 2015; Jakobsen et al., 2018).

Tetraselmis suecica is a standard microalgae species as part of most commercial shellfish hatcheries suite of feed components to broodstock and post-set animals for decades (Helm, 1977; Robert et al., 2001). This species has a high lipid content, consisting of both EPA and DHA, both commonly accepted as crucial for developing gamates and juvenile shellfish. This species is commonly grown photosynthetically, with lower biomass per unit volume, and therefore in this work will be grown in a manner which will provide the same high quality shellfish feed, while being produced in a manner which is over three times greater yield.

The major macronutrients to be provided for microalgal growth are carbon, nitrogen, phosphorus and potassium, with trace elements and vitamins provided in the culture medium, with silica which  is required for the cultivation of diatoms. Heterotrophic cultivation eliminates the requirement of light because organic carbon sources are provided. Carbon is the most important nutrient to build biomass which varies between 17 and 65% of total biomass depending upon the microalgal species. Various monosaccharides like glucose, galactose, mannose, sugar alcohols or carboxylic acids like acetate or disaccharides like sucrose and lactose can be applied as the carbon source. Nitrogen, which accounts for about 5–10% of the biomass content, is often supplied as peptone or urea, and the preference for nitrogen source varies with each microalgal strain. Heterotrophy in microalgae is an aerobic process, and in high cell density, the supply of oxygen concentration for cell growth is critical, and agitation is a way of evenly distributing the supplied oxygen. Heterotrophic cultivation is particularly well suited for lipid accumulation, because of the high carbon content supplied in the media, and the C/N ratio plays a key role in lipid accumulation of microalgal cultures which will benefit successful shellfish growth and development (Anderson, 2005).