198
T. Jauffrais et al.
(Salas et al. 2011), France (Nezan et Siano, personal communication), Mexico
(Hernandez-Becerril et al. 2010), Argentina (Akselman and Negri 2012), and AZA
occurrences are now recognized as a worldwide phenomenon. Until now, AZAs
were purified and isolated from contaminated bivalves, as it has been originally
carried out with other marine biotoxins: okadaic acid group toxins, brevetoxins,
saxitoxins, yessotoxins, domoic acid, cyclic imines and pectenotoxins (Rundberget
et al. 2007). However, severe toxic events are required to obtain pure standards
from contaminated bivalves, even though recovery has been improved recently and
the number of purification steps required to purify AZAs from complex matrices
reduced (Kilcoyne et al. 2012; Perez et al. 2010).
The primary AZA producer is now identified and adapted to culture, furthermore,
natural occurring blooms are hard to predict and/or to find; preventing in situ direct
extraction of AZA as developed by Rundberget et al. (2007). Thus, to avoid AZA1
and -2 scarcities it is important to have a sustainable production of toxins from
A. spinosum culture for toxicological studies, and for instrument calibration in
continuous monitoring programs.
The aim of this study was to evaluate the feasibility of AZA production from
A. spinosum produced in a continuous series of pilot scale photobioreactors. We
describe here how dilution rate influences cell concentration as well as toxin
production in pilot scale chemostat bioreactors in series and the use of different solid
phase extraction procedures to recover AZAs from large volumes of A. spinosum
culture (200 L) after tangential flow filtrations.
Materials and Methods
Culture Conditions and Measurement
The strain (3D9) of Azadinium spinosum was the source of AZA1 and -2 for the
experiment. The algae were produced in two chemostats of 100 L each, operated
in series at different dilution rates (0.15, 0.2, 0.25 and 0.3 day
1 ). Culture medium
was a K modified medium (Keller et al. 1987), without NH 4 Cl, tris buffer and with
Na 2 SeO 3 (10
8 M).
The photobioreactors were operating using the following conditions : the pH
was maintained at 7.9 using CO 2 addition, T D 18
ı C, a photon flux density of
200 mol m
2
s
1 on one side of the reactor, and a photoperiod of 16 h of light
and 8 h of dark (Jauffrais et al. 2010). A Rushton turbine was homogenizing the
algae at 40 rpm. Algae were collected in a harvesting tank (300 L), aerated and
maintained at 18
ı C (Fig. 17.1).
A particle counter (Beckman, Multisizer 3 Coulter counter) was used daily to
assess cell concentrations (cells mL
1 ), average size (m) and cellular volume
(m
3
mL
1 ). The bioreactors were considered at steady state after a minimum of
5 days at the same micro-algal concentration (˙5 %).
T. Jauffrais et al.
(Salas et al. 2011), France (Nezan et Siano, personal communication), Mexico
(Hernandez-Becerril et al. 2010), Argentina (Akselman and Negri 2012), and AZA
occurrences are now recognized as a worldwide phenomenon. Until now, AZAs
were purified and isolated from contaminated bivalves, as it has been originally
carried out with other marine biotoxins: okadaic acid group toxins, brevetoxins,
saxitoxins, yessotoxins, domoic acid, cyclic imines and pectenotoxins (Rundberget
et al. 2007). However, severe toxic events are required to obtain pure standards
from contaminated bivalves, even though recovery has been improved recently and
the number of purification steps required to purify AZAs from complex matrices
reduced (Kilcoyne et al. 2012; Perez et al. 2010).
The primary AZA producer is now identified and adapted to culture, furthermore,
natural occurring blooms are hard to predict and/or to find; preventing in situ direct
extraction of AZA as developed by Rundberget et al. (2007). Thus, to avoid AZA1
and -2 scarcities it is important to have a sustainable production of toxins from
A. spinosum culture for toxicological studies, and for instrument calibration in
continuous monitoring programs.
The aim of this study was to evaluate the feasibility of AZA production from
A. spinosum produced in a continuous series of pilot scale photobioreactors. We
describe here how dilution rate influences cell concentration as well as toxin
production in pilot scale chemostat bioreactors in series and the use of different solid
phase extraction procedures to recover AZAs from large volumes of A. spinosum
culture (200 L) after tangential flow filtrations.
Materials and Methods
Culture Conditions and Measurement
The strain (3D9) of Azadinium spinosum was the source of AZA1 and -2 for the
experiment. The algae were produced in two chemostats of 100 L each, operated
in series at different dilution rates (0.15, 0.2, 0.25 and 0.3 day
1 ). Culture medium
was a K modified medium (Keller et al. 1987), without NH 4 Cl, tris buffer and with
Na 2 SeO 3 (10
8 M).
The photobioreactors were operating using the following conditions : the pH
was maintained at 7.9 using CO 2 addition, T D 18
ı C, a photon flux density of
200 mol m
2
s
1 on one side of the reactor, and a photoperiod of 16 h of light
and 8 h of dark (Jauffrais et al. 2010). A Rushton turbine was homogenizing the
algae at 40 rpm. Algae were collected in a harvesting tank (300 L), aerated and
maintained at 18
ı C (Fig. 17.1).
A particle counter (Beckman, Multisizer 3 Coulter counter) was used daily to
assess cell concentrations (cells mL
1 ), average size (m) and cellular volume
(m
3
mL
1 ). The bioreactors were considered at steady state after a minimum of
5 days at the same micro-algal concentration (˙5 %).