Production and Isolation of Azaspiracid-1 and -2 from Azadinium spinosum Culture in Pilot Scale Photobioreactors

Abstract

Azaspiracid (AZA) poisoning has been reported following consumption of contaminated shellfish, and is of human health concern. Hence, it is important to have sustainable amounts of the causative toxins available for toxicological studies and for instrument calibration in monitoring programs, without having to rely on natural toxin events. Continuous pilot scale culturing was carried out to evaluate the feasibility of AZA production using Azadinium spinosum cultures. Algae were harvested using tangential flow filtration or continuous centrifugation. AZAs were extracted using solid phase extraction (SPE) procedures, and subsequently purified. When coupling two stirred photobioreactors in series, cell concentrations reached 190,000 and 210,000 cell·mL−1 at steady state in bioreactors 1 and 2, respectively. The AZA cell quota decreased as the dilution rate increased from 0.15 to 0.3 day−1, with optimum toxin production at 0.25 day−1. After optimization, SPE procedures allowed for the recovery of 79 ± 9% of AZAs. The preparative isolation procedure previously developed for shellfish was optimized for algal extracts, such that only four steps were necessary to obtain purified AZA1 and -2. A purification efficiency of more than 70% was achieved, and isolation from 1200 L of culture yielded 9.3 mg of AZA1 and 2.2 mg of AZA2 of >95% purity. This work demonstrated the feasibility of sustainably producing AZA1 and -2 from A. spinosum cultures.

Dissolved azaspiracids are absorbed and metabolized by blue mussels (Mytilus edulis)

“…spinosum strain 3D9 was used to study accumulation of AZA1 and -2 in mussels. The alga was grown in a stirred photobioreactor (100 L) operated in chemostat mode at a dilution rate of 0.2 day -1 under the environmental conditions of Jauffrais et al (2012b).…”

“…Biomass (200 L) was collected by tangential flow filtration, followed by adsorption of AZAs onto activated HP-20 Diaion resin (25 g) from the sonicated algal concentrate (± 1 L). The washed resin was placed in a glass column and eluted with acetone (3 × 50 mL), and the eluate evaporated and reconstituted in 10 mL of methanol (Jauffrais et al, 2012b). This crude algal extract contained 110 ± 6 µg.mL -1 of AZA1 plus AZA2 (ratio 79:21).…”

“…Since its discovery, the North Sea strain of A. spinosum has been cultured for toxin production in pilot-scale photobioreactors (Jauffrais et al, 2012b). However, while the organism produces AZA1 and -2, it does not produce the metabolites formed in shellfish (AZA3 to -23, reviewed in Rehmann et al (2008)) McCarron et al, 2009).…”

Cardiovascular Toxicity from Marine Envenomation

“…Production of azaspiracid-1 and azaspiracid-2-the two most common forms of the toxin-has only very recently been achieved in the marine algae Azadinium spinosum. 22 Isolation of the toxin and its analogs in shellfish and its presence in Protoperidinium crassipes blooms has been well documented in the coastal waters of northwestern Africa, western Europe, and eastern Canada; it is most prevelant from midsummer to mid-winter. 23 A vast number of azaspiracid analogs have been detected in shellfish tissue though azaspiracid-1 or azaspiracid-2 are typically predominant with azaspiracid-3 and other analogs forming a minority of total azaspiracids; a possible role of metabolic bioconversion in shellfish has been suggested given that only azaspiracid-1, azaspiracid-2, and azaspiracid-3 have been detected outside of these animals.…”

Gambierol: Synthetic Aspects

“…As a final approach, a pH controlled and stirred bioreactor (R1) in continuous culture mode was connected in series to a second one (R2) to induce a phase of maturation (growth stagnation but continuing toxin production) in the second bioreactor 54 .…”