Paul W. Behrens

COMMERCIAL DEVELOPMENTS IN MICROALGAL BIOTECHNOLOGY

A number of important advances have occurred in microalgal biotechnology in recent years that are slowly moving the field into new areas. New products are being developed for use in the mass commercial markets as opposed to the “health food” markets. These include algal‐derived long‐chained polyunsaturated fatty acids, mainly docosahexaenoic acid, for use as supplements in human nutrition and animals. Large‐scale production of algal fatty acids is possible through the use of heterotrophic algae and the adaptation of classical fermentation systems providing consistent biomass under highly controlled conditions that result in a very high quality product. New products have also been developed for use in the development of pharmaceutical and research products. These include stable‐isotope biochemicals produced by algae in closed‐system photobioreactors and extremely bright fluorescent pigments. Cryopreservation has also had a tremendous impact on the ability of strains to be maintained for long periods of time at low cost and maintenance while preserving genetic stability.

The effect of dietary arachidonic acid (20:4n - 6) on growth, survival and resistance to handling stress in gilthead seabream (Sparus aurata) larvae

Abstract The effects of high dietary docosahexaenoic acid (22:6 n −3, DHA) and varying arachidonic acid (20:4 n −6, AA) were tested on growth, survival and resistance to handling stress in 5–35 day old gilthead seabream larvae. Three enrichment treatments differing in their DHA/AA ratios were fed to rotifers ( Brachionus rotundiformis ) and Artemia nauplii. The high DHA (35.9% TFA) enrichment treatment (DHA-PL) contained no AA and included lipid from the heterotrophically grown DHA-rich dinoflagellate Crypthecodinium sp. A second enrichment treatment (AADHA), selected from an earlier screening study, supplemented the high DHA enrichment treatment with an AA-rich lipid (52% TFA) from the heterotrophically grown fungus Mortierella alpina. A third enrichment treatment (ALGA) was the commercial product Algamac 2000, which is devoid of AA, but includes approximately 12.9% of TFA as docosapentaenoic acid (DPA, 22:5 n −6). Rotifers fed the DHA-PL, AADHA and ALGA treatments demonstrated a range of DHA/AA ratios (20.9, 5.6 and 10.1, respectively) as did the Artemia nauplii (25.8, 3.7 and 4.6, respectively). The enriched rotifers were fed to larvae reared in 400 l V-tanks from day 5 to day 19 post-hatching. Following this period, larvae were exposed to controlled handling stress during transfer to 27 l aquaria, where they were then fed the enriched nauplii from day 20 to day 35 post-hatching. Although larval fatty acid profiles reflected the enrichment treatments, there were no marked differences ( P >0.05) in survival and growth in 5–19 day old larvae at the end of rotifer feeding. However, the larvae fed the AA enriched rotifers prior to the handling stress of transfer to the aquaria demonstrated daily and significantly ( P Artemia feeding than larvae fed the AA-deficient (DHA-PL) and ALGA-enriched rotifers. As larvae fed the ALGA, rotifers partially retroconverted DPA to AA in their tissues, the final survival (31.0%) in these larvae was markedly better ( P P Artemia . The results suggest that dietary AA fed prior to handling stress improved survival more effectively than when fed following handling stress. These findings imply, as well, the importance of early larval nutrition on later larval and juvenile survival during crowding, grading and other handling stressors.

Advanced DHA, EPA and ArA enrichment materials for marine aquaculture using single cell heterotrophs

Abstract Heterotrophically grown algae and fungal biomass and their residual materials from an industrial oil extraction process were used as components in marine larval and broodstock diets. Crypthecodinium sp. phospholipid extract and meal, used to enrich rotifers and Artemia nauplii, produced higher levels of DHA and higher DHA/EPA ratios in these zooplankters than Schizochytrium sp. algal whole cell preparation or fish oil-based emulsion. The improved enrichment resulted in enhanced growth of Atlantic halibut larvae, whereas several other marine larvae species (sea bream, European sea bass and striped bass) respond almost equally to all enrichment materials. In addition, a 60% replacement of menhaden oil with algal oil and meal in striped bass broodstock diets resulted in a similar growth increase to that obtained with standard commercial diets. Striped bass broodstock fish diets supplemented with an arachidonic acid (ArA)-rich oil obtained from heterotrophically grown fungi, Mortierella alpina , was shown to have significant benefits on the hatching rate of larvae. These findings demonstrate the potential of single cell heterotrophs as a partial substitute or replacement for fish-based ingredients in aquaculture diets.

Enhanced absorption of docosahexaenoic acid (DHA, 22:6n-3) in Artemia nauplii using a dietary combination of DHA-rich phospholipids and DHA-sodium salts.

The effects of surface-active agents such as phospholipids (PL) and salts of long chain fatty acids in enrichment lipids on emulsification, and hence digestion and absorption of essential fatty acids (EFA) were studied. The advantages obtained by using a mixture of docosahexaenoic acid (DHA, 22:6n-3)-rich phospholipids (DHA-PL) and DHA-salts in the process of live feed enrichment are reported on. Enrichment diets at equal levels of DHA, but with varying proportions of DHA-rich triacylglycerols (DHA-TAG), DHA-PL and DHA-sodium salt (DHA-Na) isolated from heterotrophicaly grown Crypthecodinium sp. algae were fed to instar-II stage Artemia nauplii. Artemia nauplii survival and lipid content after 16 h of enrichment were 74.0±7.8% and 24.3±0.6% (dry weight) respectively, and not significantly affected (P>0.05) with increasing dietary quantities of PL and DHA-salts (up to 40 and 30%, respectively). Artemia lipid class composition was independent of dietary phospholipid level. However, higher proportions of polar lipid fraction was evident in Artemia lipids with the addition of DHA-Na. Dietary inclusion of 20% PL or a mixture of 10% PL and 10% DHA-Na resulted in maximal (P<0.05) absorption of dietary DHA by the Artemia (27.5±2.6 mg/g dry weight) with a DHA to eicosapentaenoic acid (EPA, 20:5n-3) ratio greater than 3. Furthermore, PL was the most efficient dietary fraction to deliver DHA to Artemia nauplii as compared to TAG or unesterified delivery forms.

Microalgae as a source of stable isotopically labeled compounds

Microalgae have the ability to convert inorganic compounds into organic compounds. When they are cultured in the presence of stable (non-radioactive) isotopes (i.e.13CO2,15NO3−,2H2O) their biomass becomes labeled with the stable isotopes, and a variety of stable isotopically-labeled compounds can be extracted and purified from that biomass.Two applications for stable isotopically-labeled compounds are as cell culture nutrients and as breath test diagnostics. Bacteria that are cultured with labeled nutrients will produce bacterial products that are labeled with stable isotopes. The presence of these isotopes in the bacterial products, along with recent developments in NMR technology, greatly reduces the time and effort required to determine the three-dimensional structure of macromolecules and the interaction of proteins with ligands. As breath test diagnostics, compounds labeled with13C are used to measure the metabolism of particular organs and thus diagnose various disease conditions. These tests are based on the principle that a particular compound is metabolized primarily by a single organ, and when that compound is labeled with13C, the appearance of13CO2 in exhaled breath provides information about the metabolic activity of the target organ. Tests of this type are simple to perform, non-invasive, and less expensive than many conventional diagnostic procedures.The commercialization of stable isotopically labeled compounds requires that these compounds be produced in a cost-effective manner. Our approach is to identify microalgal overproducers of the desired compounds, maximize the product content of those organisms, and purify the resulting products.