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• Jauffrais, T
• Kilcoyne, J
• Séchet, V
• Herrenknecht, C
• Truquet, P
• Hervé, F
• Bérard, J
• Nulty, C
• Taylor, S
• Tillmann, U
• Miles, C
• Hess, P
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• Jauffrais, T
• Kilcoyne, J
• Séchet, V
• Herrenknecht, C
• Truquet, P
• Hervé, F
• Bérard, J
• Nulty, C
• Taylor, S
• Tillmann, U
• Miles, C
• Hess, P
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• Jauffrais, T
• Kilcoyne, J
• Séchet, V
• Herrenknecht, C
• Truquet, P
• Hervé, F
• Bérard, J
• Nulty, C
• Taylor, S
• Tillmann, U
• Miles, C
• Hess, P

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Mar. Drugs 2012, 10(6), 1360-1382; doi:10.3390/md10061360

Article

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

Thierry Jauffrais ^1,* , Jane Kilcoyne ² , Véronique Séchet ¹ , Christine Herrenknecht ³ , Philippe Truquet ¹ , Fabienne Hervé ¹ , Jean Baptiste Bérard ⁴ , Cíara Nulty ² , Sarah Taylor ¹ , Urban Tillmann ⁵ , Christopher O. Miles ⁶  and Philipp Hess ^1,*

¹ Ifremer, EMP/PHYC Laboratory, Rue de l'Ile d'Yeu, 44311 Nantes, France ² Marine Institute, Rinville, Oranmore, Co., Galway, Ireland ³ Nantes Atlantic University, MMS EA2160, 9 rue Bias, 44035 Nantes, France ⁴ Ifremer, BRM/PBA Laboratory, Rue de l'Ile d'Yeu, 44311 Nantes, France ⁵ Alfred Wegener Institute, Am Handelshafen 12, D-27570 Bremerhaven, Germany ⁶ Norwegian Veterinary Institute, P. O. Box 750 Sentrum, 0106 Oslo, Norway

* Authors to whom correspondence should be addressed.

Received: 15 May 2012; in revised form: 1 June 2012 / Accepted: 4 June 2012 / Published: 13 June 2012

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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.

Keywords: solid phase extraction; photobioreactor; chemostat; dinoflagellate; micro-algae; LC-MS/MS; tangential flow filtration; azaspiracid; HP-20

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