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

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

Orientation of doubly excited states in N2

We have measured the total fluorescent intensity and circular polarization of light emitted in 3p 4Po → 3s4P transitions of excited neutral nitrogen atoms created by the photofragmentation of the N2 molecule with circularly polarized light having energies between 21 and 26 eV. The intensity measurements show the effect of predissociation of the N2 Rydberg series R(C) 1Σ+u states by non-Rydberg doubly excited resonances (NRDERs), while nonzero values of circular polarization allow us to unambiguously identify the presence of a directly excited NRDER with 1Πu symmetry in this energy range

Orientation of doubly excited states in N-2

We have measured the total fluorescent intensity and circular polarization of light emitted in 3p4Po→3s4P transitions of excited neutral nitrogen atoms created by the photofragmentation of the N2 molecule with circularly polarized light having energies between 21 and 26 eV. The intensity measurements show the effect of predissociation of the N2 Rydberg series R(C) 1Σ+u states by non-Rydberg doubly excited resonances (NRDERs), while nonzero values of circular polarization allow us to unambiguously identify the presence of a directly excited NRDER with 1Πu symmetry in this energy range

Quantitative analysis of azaspiracids in Azadinium spinosum cultures, with a focus on azaspiracid methyl-esters as extraction artefacts

Azaspiracids (AZAs) are secondary metabolites of Azadinium spinosum that can accumulate in shellfish and cause food poisoning when consumed. We describe here an analytical procedure for the determination of AZAs in cultures of A. spinosum with a focus on the formation of AZA methyl esters as artefacts during extraction and sample pretreatment. A. spinosum cells were collected from bioreactor cultures using centrifugation or filtration. Different extraction procedures were evaluated for formation of methyl ester artefacts, yield, and matrix effects. Filtration of cultures using glass-fibre filters led to increased formation of methyl esters, and centrifugation is recommended for recovery of cells. The extraction solvent (methanol (MeOH), acetone, and acetonitrile (MeCN)) did not significantly affect the yield of AZAs as long as the organic content was 80% or higher. However, the use of MeOH as extraction solvent led to increased formation of methyl esters. AZA1 recovery over two successive extractions was 100% at the 95% confidence level for acetone and MeOH. In standardaddition experiments, no significant matrix effects were observed in extracts of A. spinosum or Azadinium obesum up to a sample size of 4.5×109 μm3. Moreover, experiments carried out to clarify the formation and structure of methylated AZA analogues led to the description of two AZA methyl esters and to the correction of the chemical structures of AZAs29–32