17 Effect of Dilution Rate on Azadinium spinosum and Azaspiracid (AZA). . .
201
Results and Discussion
Cell concentration, mean diameter, cellular volume and toxin content remained
constant at steady states when the dilution rate changed. However, differences were
noticed between reactors in series (R1 and -2). At the different dilution rates studied,
cell concentrations were equal to 190,000 and 215,000 cells mL
1 in R1 and R2
respectively and results for mean diameter and cellular volume were comparable at
all dilution rates but differed between R1 and R2 (Table 17.1). For each steady state
studied, AZAs cell quota increased between bioreactor 1 and -2, showing a positive
effect of bioreactors in series to enhance AZA cellular content. Interestingly,
contrarily to cellular concentration, AZA cell content decreased as dilution rate
increased, ranging from 67 to 24 fg cell
1 for R1 and 98 to 63 fg cell
1 for
R2. Thus, the cell production increased as dilution rate increased whereas AZA
production reached an optimum at 0.25 day
1 of 475 ˙ 17 g day
1 under the
studied conditions.
Batch cultured A. spinosum (strain 3D9 or SM2) produced AZA1 and -2, with
AZA1 as the predominant AZA and with a cell quota ranging from 5 to 40 fg cell
1
(Jauffrais et al. 2010; Salas et al. 2011; Tillmann et al. 2009). In the present study, the
same toxin profiles were found, however, AZAs cell quota of 24–98 fg cell
1 were
obtained depending on the dilution rate. There was a higher toxin concentration at
low growth rate of A. spinosum than higher dilution rate (especially in R1), showing
the necessity of chemostats in series at higher dilution rate to significantly increase
toxin concentration.
As described above, continuous A. spinosum culture was shown to be valuable
for production of AZAs using photobioreactors in series. Subsequently, AZA
extractions were developed to optimise the recovery from bioreactors. Before
filtration, 95 % of the toxin was intracellular, whereas after filtration, 50–70 % of
the toxin was contained in the concentrate and 30–50 % released in the permeate.
The observed variation was time dependent, with longer filtration times leading to
higher proportions of toxin in the permeate.
Intracellular toxin content was recovered as algal paste after centrifugation of the
retentate, however, this procedure caused the loss of some toxin from the supernatant
(˙10 %). To avoid this loss a solid phase adsorption was implemented using
Diaion
® HP20 resin as explained above, this procedure allowed for the recovery
of up to 90 % of the total toxin from the retentate.
Extracellular toxin content was extracted using two procedures, the SPATT bags
and a solid phase extraction procedure. SPATT bags were initially designed as
a monitoring tool to follow and predict micro-algal toxic events around shellfish
production areas (MacKenzie et al. 2004; MacKenzie 2010). The solid phase extraction procedure was implemented for biotoxin extraction from naturally occurring
micro-algal blooms (Rundberget et al. 2007). These two methods allowed good
recovery, however, recovery using SPATT bags showed more variability than the
SPE procedure in the conditions tested. Even though, the procedures for AZAs
extraction from the concentrate and permeate allowed for the recovery of 80 ˙ 5 %
of toxins originally produced by A. spinosum pilot scale culture.
201
Results and Discussion
Cell concentration, mean diameter, cellular volume and toxin content remained
constant at steady states when the dilution rate changed. However, differences were
noticed between reactors in series (R1 and -2). At the different dilution rates studied,
cell concentrations were equal to 190,000 and 215,000 cells mL
1 in R1 and R2
respectively and results for mean diameter and cellular volume were comparable at
all dilution rates but differed between R1 and R2 (Table 17.1). For each steady state
studied, AZAs cell quota increased between bioreactor 1 and -2, showing a positive
effect of bioreactors in series to enhance AZA cellular content. Interestingly,
contrarily to cellular concentration, AZA cell content decreased as dilution rate
increased, ranging from 67 to 24 fg cell
1 for R1 and 98 to 63 fg cell
1 for
R2. Thus, the cell production increased as dilution rate increased whereas AZA
production reached an optimum at 0.25 day
1 of 475 ˙ 17 g day
1 under the
studied conditions.
Batch cultured A. spinosum (strain 3D9 or SM2) produced AZA1 and -2, with
AZA1 as the predominant AZA and with a cell quota ranging from 5 to 40 fg cell
1
(Jauffrais et al. 2010; Salas et al. 2011; Tillmann et al. 2009). In the present study, the
same toxin profiles were found, however, AZAs cell quota of 24–98 fg cell
1 were
obtained depending on the dilution rate. There was a higher toxin concentration at
low growth rate of A. spinosum than higher dilution rate (especially in R1), showing
the necessity of chemostats in series at higher dilution rate to significantly increase
toxin concentration.
As described above, continuous A. spinosum culture was shown to be valuable
for production of AZAs using photobioreactors in series. Subsequently, AZA
extractions were developed to optimise the recovery from bioreactors. Before
filtration, 95 % of the toxin was intracellular, whereas after filtration, 50–70 % of
the toxin was contained in the concentrate and 30–50 % released in the permeate.
The observed variation was time dependent, with longer filtration times leading to
higher proportions of toxin in the permeate.
Intracellular toxin content was recovered as algal paste after centrifugation of the
retentate, however, this procedure caused the loss of some toxin from the supernatant
(˙10 %). To avoid this loss a solid phase adsorption was implemented using
Diaion
® HP20 resin as explained above, this procedure allowed for the recovery
of up to 90 % of the total toxin from the retentate.
Extracellular toxin content was extracted using two procedures, the SPATT bags
and a solid phase extraction procedure. SPATT bags were initially designed as
a monitoring tool to follow and predict micro-algal toxic events around shellfish
production areas (MacKenzie et al. 2004; MacKenzie 2010). The solid phase extraction procedure was implemented for biotoxin extraction from naturally occurring
micro-algal blooms (Rundberget et al. 2007). These two methods allowed good
recovery, however, recovery using SPATT bags showed more variability than the
SPE procedure in the conditions tested. Even though, the procedures for AZAs
extraction from the concentrate and permeate allowed for the recovery of 80 ˙ 5 %
of toxins originally produced by A. spinosum pilot scale culture.