Dinoflagellates and Toxin Production 225
concerning resistance to shear stress exists. For instance, Protoceratium reticulatum is a thecate organism
highly sensitive to shear, whereas Crypthecodinium cohnii—bearing a similar structure, is more robust.
The latter is able to support values of energy dissipation rates (EDRs) of 5.8×10
5
cm
2
s
–3
(Hu et al. 2007),
without visible damage—while the former cannot exceed the marginal value of 0.8 cm
2
s
–3
(GarcíaCamacho et al. 2007). Therefore, shear sensitivity of such type of microalgae is apparently speciesdependent (Berdalet et al. 2007; Gallardo-Rodríguez et al. 2015). Several studies, encompassing various
orders of dinoflagellates, have shown that EDRs values in the range [0.011, 10] cm
2
s
−3
generally inhibit
dinoflagellate growth (Gallardo-Rodríguez et al. 2012a). Only EDR under 1 cm
2
s
–3
(~ 0.1W m
–3
) seems
not to trigger detrimental effects upon dinoflagellate cells (Berdalet et al. 2007).
The levels of turbulence stood by dinoflagellates are generally one to two orders of magnitude smaller
than those that produce damage in most suspended plant and animal cells; the latter is among the most
sensitive to shear in bioreactors (Gallardo-Rodríguez et al. 2012a). Previous strategies proven successful
in commercial large-scale cultivation of these types of cells (Varley and Birch 1999; Juhl et al. 2001)
cannot be extrapolated to dinoflagellates—due to complex circadian cycles and unique metabolism.
Biotechnological cultivation of dinoflagellates—some advances
For many decades, studies related to dinoflagellates remained focused on systematic of bloom dynamics and
HABs. Conversely, scare attempts were devoted to development of controlled cultures of dinoflagellates
in bioreactors (Gallardo-Rodríguez et al. 2012a). Scientists have recently invested a lot of effort into this
latter issue—motivated by the outstanding potential of dinoflagellate-derived compounds (e.g., GallardoRodríguez et al. 2010; García-Camacho et al. 2011; Beuzenberg et al. 2012; López-Rosales et al. 2015;
Fuentes-Grünewald et al. 2016; López-Rosales et al. 2016).
In this regard, controlled cultivation is a must, and safety issues must be taken into account for
culture scale up. Cultivation the dinoflagellates in open systems (i.e., raceways, natural lagoons)—
despite being easily operated and having low input requirements, does not seem a feasible option for
safety reasons and/or environmental contamination. Only dinoflagellates producing interesting non-toxic
or relatively benign compounds might be candidates for such reactor configurations (Gallardo-Rodríguez
et al. 2012a). For instance, Karenia brevis—a producer of brevetoxins is known to lyse when cultures
are aerated. Releasing such type of toxins in the environment can be extremely dangerous for humans
or other organisms, as it works like an aerosol (Abraham et al. 2005; Flewelling et al. 2005; Fleming
et al. 2007). Moreover, production of toxins as final product—with potential application as medicines,
would be improbable under this system; contamination of metabolites would raise several risks for drug
development and/or human treatment. On the other hand, the direct effect of weather would probably
constrain dinoflagellate growth, as many species are deeply sensitive to physico-chemical alterations and
possess a complex metabolism (Carvalho et al. 2006).
Taking such drawbacks into consideration, the logical alternative is employing enclosed systems,
as is the case of photobioreactors (PBRs). PBRs tend to be more complex and expensive than open
systems, but permit better control of culture environment (Chisti 2007). Many studies on dinoflagellate
cultivation have resorted to just flask or bottle cultures at bench scale; a few studies have, however,
surfaced dealing with larger scale (Zittelli et al. 2013). Depending on the bioreactor technologies
employed, volumes ranging from 4 L to 700 L and distinct cultivation strategies have been attempted,
with the goal of achieving high biomass productivities, as well as higher biotoxin concentrations. The
culture systems described for those purposes range from carboys (a type of container made of glass
or plastic), chemostats or stirred-tanks, to classical airlift, bubble column, tubular reactor or flat-plate
PBR—chiefly the designs used for conventional microalga mass culture. For instance, Pan et al. (1999)
produced a toxic Prorocentrum lima in 36 L-glass carboys with 18 L-working volume, at about 90 µmol
photons m
−2
s
−1
, 14 h/10 h light/dark cycle and gentle aeration, and obtained an increase from ca. 2,000
to 11,000 cells mL
−1
, and from ca. 20 to 220 nMol DSP toxins (okadaic acid and dinophysis toxins)
for 40 d (Pan et al. 1999). Also, Loader and coworkers (2007) have tested, in polycarbonate carboys
of 14 L (in a total of 226 L), growth of Protoceratium reticulatum and production of yessotoxin and
furanoyessotoxin with the light/dark cycles applied in batch culture for 43 d. Between 200 to 15,000
concerning resistance to shear stress exists. For instance, Protoceratium reticulatum is a thecate organism
highly sensitive to shear, whereas Crypthecodinium cohnii—bearing a similar structure, is more robust.
The latter is able to support values of energy dissipation rates (EDRs) of 5.8×10
5
cm
2
s
–3
(Hu et al. 2007),
without visible damage—while the former cannot exceed the marginal value of 0.8 cm
2
s
–3
(GarcíaCamacho et al. 2007). Therefore, shear sensitivity of such type of microalgae is apparently speciesdependent (Berdalet et al. 2007; Gallardo-Rodríguez et al. 2015). Several studies, encompassing various
orders of dinoflagellates, have shown that EDRs values in the range [0.011, 10] cm
2
s
−3
generally inhibit
dinoflagellate growth (Gallardo-Rodríguez et al. 2012a). Only EDR under 1 cm
2
s
–3
(~ 0.1W m
–3
) seems
not to trigger detrimental effects upon dinoflagellate cells (Berdalet et al. 2007).
The levels of turbulence stood by dinoflagellates are generally one to two orders of magnitude smaller
than those that produce damage in most suspended plant and animal cells; the latter is among the most
sensitive to shear in bioreactors (Gallardo-Rodríguez et al. 2012a). Previous strategies proven successful
in commercial large-scale cultivation of these types of cells (Varley and Birch 1999; Juhl et al. 2001)
cannot be extrapolated to dinoflagellates—due to complex circadian cycles and unique metabolism.
Biotechnological cultivation of dinoflagellates—some advances
For many decades, studies related to dinoflagellates remained focused on systematic of bloom dynamics and
HABs. Conversely, scare attempts were devoted to development of controlled cultures of dinoflagellates
in bioreactors (Gallardo-Rodríguez et al. 2012a). Scientists have recently invested a lot of effort into this
latter issue—motivated by the outstanding potential of dinoflagellate-derived compounds (e.g., GallardoRodríguez et al. 2010; García-Camacho et al. 2011; Beuzenberg et al. 2012; López-Rosales et al. 2015;
Fuentes-Grünewald et al. 2016; López-Rosales et al. 2016).
In this regard, controlled cultivation is a must, and safety issues must be taken into account for
culture scale up. Cultivation the dinoflagellates in open systems (i.e., raceways, natural lagoons)—
despite being easily operated and having low input requirements, does not seem a feasible option for
safety reasons and/or environmental contamination. Only dinoflagellates producing interesting non-toxic
or relatively benign compounds might be candidates for such reactor configurations (Gallardo-Rodríguez
et al. 2012a). For instance, Karenia brevis—a producer of brevetoxins is known to lyse when cultures
are aerated. Releasing such type of toxins in the environment can be extremely dangerous for humans
or other organisms, as it works like an aerosol (Abraham et al. 2005; Flewelling et al. 2005; Fleming
et al. 2007). Moreover, production of toxins as final product—with potential application as medicines,
would be improbable under this system; contamination of metabolites would raise several risks for drug
development and/or human treatment. On the other hand, the direct effect of weather would probably
constrain dinoflagellate growth, as many species are deeply sensitive to physico-chemical alterations and
possess a complex metabolism (Carvalho et al. 2006).
Taking such drawbacks into consideration, the logical alternative is employing enclosed systems,
as is the case of photobioreactors (PBRs). PBRs tend to be more complex and expensive than open
systems, but permit better control of culture environment (Chisti 2007). Many studies on dinoflagellate
cultivation have resorted to just flask or bottle cultures at bench scale; a few studies have, however,
surfaced dealing with larger scale (Zittelli et al. 2013). Depending on the bioreactor technologies
employed, volumes ranging from 4 L to 700 L and distinct cultivation strategies have been attempted,
with the goal of achieving high biomass productivities, as well as higher biotoxin concentrations. The
culture systems described for those purposes range from carboys (a type of container made of glass
or plastic), chemostats or stirred-tanks, to classical airlift, bubble column, tubular reactor or flat-plate
PBR—chiefly the designs used for conventional microalga mass culture. For instance, Pan et al. (1999)
produced a toxic Prorocentrum lima in 36 L-glass carboys with 18 L-working volume, at about 90 µmol
photons m
−2
s
−1
, 14 h/10 h light/dark cycle and gentle aeration, and obtained an increase from ca. 2,000
to 11,000 cells mL
−1
, and from ca. 20 to 220 nMol DSP toxins (okadaic acid and dinophysis toxins)
for 40 d (Pan et al. 1999). Also, Loader and coworkers (2007) have tested, in polycarbonate carboys
of 14 L (in a total of 226 L), growth of Protoceratium reticulatum and production of yessotoxin and
furanoyessotoxin with the light/dark cycles applied in batch culture for 43 d. Between 200 to 15,000