Dinoflagellates and Toxin Production 227
continuously aerated with a mixture of 2.5% (v/v) CO 2 enriched-air, at a flow rate set at 6.25 L·min
–1
.
The indoor systems were operated under semi-continuous mode, with a photoperiod of 18 h/6 h and an
average light irradiance of 158 µmol m
–2
s
–1
for 230 d, while the outdoors system was operated under
~ 460 µmol m
–2
s
–1
. Average biomass productivities were 0.052 mg L d
–1
and 0.036 g L d
–1
indoors and
outdoors, respectively (Fuentes-Grünewald et al. 2016).
In a previous study, Fuentes et al. (2013) have tested a large-scale method, with two different toxic
dinoflagellates, Alexandium minutum and Karlodinium veneficum; nine bubble column PBRs, with a
total of 350 L, were also used indoors and outdoors. The working volume was established as 315 L
(~ 35 L each column), and agitation was provided by continuously injecting pre-filtered air at a flow rate
of 0.1 vvm, concomitant with a well-mixed supply of nutrients (thus avoiding O 2 accumulation). Light
intensity indoors was 110 µE m
–2
s
–1
, with a light and dark regime of 12 h/12 h, while outdoors it varied
between 200 to 4,000 µE m
–2
s
–1
. Indoor experiments were run in batch mode, with biomass productivities
not surpassing 0.16 g L
−1
d
−1
for both microalgae; the outdoor cultures were run in semi-continuous mode,
and attained biomass productivities of 0.35 g L
−1
d
−1
and 0.22 g L
−1
d
−1
for Alexandium minutum and
Karlodinium veneficum, respectively (Fuentes-Grünewald et al. 2013).
A LED-illuminated bubble column PBR was also designed and built by López-Rosales et al. (2016),
with a working volume of 80 L. The culture was mixed by sparging the vessel with pre-filtered air, at a
superficial aeration velocity below 2.74 x 10
3
m s
–1
(to prevent cell damage) and operated in a sequential
batch. Cell concentrations achieved were ~ 120,0000 cells mL
–1
at the final stage (López-Rosales et al.
2016).
Another approach was attempted by Benstein et al. (2014), namely the use of a biofilm
photobioreactor—a type of bioreactor that relies on immobilization of microalgae in a biofilm on sheetlike surfaces. Symbiodinium voratum was chosen, for implementation in an optimized Twin-Layer PBR
consisting of a thin membrane of polycarbonate with a total area 414 cm
2
placed in transparent poly(methyl
methacrylate) tubes, with inner diameter of 11.4 cm. Several cultivation discs (where microalgae were
inoculated) were produced by punching out discs of 47 mm in diameter, from the outer paper layer of
a thin layer sheet. The cultures were aerated by membrane pumping, at 75 L min
–1
, of filter-sterilized
ambient air. The reactor was placed in a climatic chamber and a rooftop greenhouse, under different
light conditions—the former with light Biolux lamps, intensity of 26 µmol m
–2
s
–1
and photoperiod of
14 h/10 h; and the latter with natural light and external sodium discharge lamp providing ~ 73 µmol m
–2
s
–1
photon irradiance. Biomass concentrations (in dry matter) have reached 1 g m
–2
d
−1
in the former conditions,
but more than doubled in the latter—thus achieving ca. 2.6 g m
–2
d
−1
. An optimized version of this PBR
was tested by employing polycarbonate membranes instead of paper as substrate for immobilization but
using the same conditions, in the greenhouse chamber; 4.3 g m
–2
d
−1
was attained, and higher light intensity
(~ 417 µmol m
–2
s
–1
) led to 11 g m
–2
d
−1
(Benstein et al. 2014).
Several constrains have to be taken into account concerning scalability of cultivation. Large volumes
are commonly associated to decreased productivities when compared to bench-scale experiments. For
that reason, optimization to attain high toxin titers is not easy, and further investigation is needed on the
topic of operation and PBR design—without forgetting safety issues. Furthermore, the understanding of
how a species can turn a higher-producer of this type of value-added products is crucial; special features
required, ecology, behavior, metabolic pathways and genetic factors would all reinforce knowledge on
how to rationally improve toxin production, so as to eventually help expand this niche market.
Concluding remarks
Dinoflagellates are a versatile group of microalgae possessing outstanding features, morphology, and
ecology—exceptionally well adapted to a wide range of environments. They have an impact on the
ecosystem of water columns, and are majorly responsible for occurrence of HABs concomitant with
toxin production. Such events are quite unfavorable for the marine life, and well-established human
activities in coastal areas can be severely affected—with detrimental economic implications. Several
aspects of HABs and toxin production remain to be elucidated: how and why such events take place,
and why and how such toxins are produced. The effective influence of abiotic factors (i.e., nutrition,
continuously aerated with a mixture of 2.5% (v/v) CO 2 enriched-air, at a flow rate set at 6.25 L·min
–1
.
The indoor systems were operated under semi-continuous mode, with a photoperiod of 18 h/6 h and an
average light irradiance of 158 µmol m
–2
s
–1
for 230 d, while the outdoors system was operated under
~ 460 µmol m
–2
s
–1
. Average biomass productivities were 0.052 mg L d
–1
and 0.036 g L d
–1
indoors and
outdoors, respectively (Fuentes-Grünewald et al. 2016).
In a previous study, Fuentes et al. (2013) have tested a large-scale method, with two different toxic
dinoflagellates, Alexandium minutum and Karlodinium veneficum; nine bubble column PBRs, with a
total of 350 L, were also used indoors and outdoors. The working volume was established as 315 L
(~ 35 L each column), and agitation was provided by continuously injecting pre-filtered air at a flow rate
of 0.1 vvm, concomitant with a well-mixed supply of nutrients (thus avoiding O 2 accumulation). Light
intensity indoors was 110 µE m
–2
s
–1
, with a light and dark regime of 12 h/12 h, while outdoors it varied
between 200 to 4,000 µE m
–2
s
–1
. Indoor experiments were run in batch mode, with biomass productivities
not surpassing 0.16 g L
−1
d
−1
for both microalgae; the outdoor cultures were run in semi-continuous mode,
and attained biomass productivities of 0.35 g L
−1
d
−1
and 0.22 g L
−1
d
−1
for Alexandium minutum and
Karlodinium veneficum, respectively (Fuentes-Grünewald et al. 2013).
A LED-illuminated bubble column PBR was also designed and built by López-Rosales et al. (2016),
with a working volume of 80 L. The culture was mixed by sparging the vessel with pre-filtered air, at a
superficial aeration velocity below 2.74 x 10
3
m s
–1
(to prevent cell damage) and operated in a sequential
batch. Cell concentrations achieved were ~ 120,0000 cells mL
–1
at the final stage (López-Rosales et al.
2016).
Another approach was attempted by Benstein et al. (2014), namely the use of a biofilm
photobioreactor—a type of bioreactor that relies on immobilization of microalgae in a biofilm on sheetlike surfaces. Symbiodinium voratum was chosen, for implementation in an optimized Twin-Layer PBR
consisting of a thin membrane of polycarbonate with a total area 414 cm
2
placed in transparent poly(methyl
methacrylate) tubes, with inner diameter of 11.4 cm. Several cultivation discs (where microalgae were
inoculated) were produced by punching out discs of 47 mm in diameter, from the outer paper layer of
a thin layer sheet. The cultures were aerated by membrane pumping, at 75 L min
–1
, of filter-sterilized
ambient air. The reactor was placed in a climatic chamber and a rooftop greenhouse, under different
light conditions—the former with light Biolux lamps, intensity of 26 µmol m
–2
s
–1
and photoperiod of
14 h/10 h; and the latter with natural light and external sodium discharge lamp providing ~ 73 µmol m
–2
s
–1
photon irradiance. Biomass concentrations (in dry matter) have reached 1 g m
–2
d
−1
in the former conditions,
but more than doubled in the latter—thus achieving ca. 2.6 g m
–2
d
−1
. An optimized version of this PBR
was tested by employing polycarbonate membranes instead of paper as substrate for immobilization but
using the same conditions, in the greenhouse chamber; 4.3 g m
–2
d
−1
was attained, and higher light intensity
(~ 417 µmol m
–2
s
–1
) led to 11 g m
–2
d
−1
(Benstein et al. 2014).
Several constrains have to be taken into account concerning scalability of cultivation. Large volumes
are commonly associated to decreased productivities when compared to bench-scale experiments. For
that reason, optimization to attain high toxin titers is not easy, and further investigation is needed on the
topic of operation and PBR design—without forgetting safety issues. Furthermore, the understanding of
how a species can turn a higher-producer of this type of value-added products is crucial; special features
required, ecology, behavior, metabolic pathways and genetic factors would all reinforce knowledge on
how to rationally improve toxin production, so as to eventually help expand this niche market.
Concluding remarks
Dinoflagellates are a versatile group of microalgae possessing outstanding features, morphology, and
ecology—exceptionally well adapted to a wide range of environments. They have an impact on the
ecosystem of water columns, and are majorly responsible for occurrence of HABs concomitant with
toxin production. Such events are quite unfavorable for the marine life, and well-established human
activities in coastal areas can be severely affected—with detrimental economic implications. Several
aspects of HABs and toxin production remain to be elucidated: how and why such events take place,
and why and how such toxins are produced. The effective influence of abiotic factors (i.e., nutrition,