PRODUCTION OF OKADAIC ACID IN
PROROCENTRUM LIMA THROUGH MIXOTROPHIC
CULTURES WITH
MACROALGAL POLYSACCHARIDES
PRODUCCIÓN DE ÁCIDO OKADAICO EN
PROROCENTRUM LIMA MEDIANTE CULTIVOS
MIXOTRÓFICOS CON POLISACÁRIDOS MACROALGALES
Roberto Pino Selles
Universidad de Panamá, Panamá
Gonzalo Álvarez Vergara
Universidad Católica del Norte, Chile
Eduardo Uribe
Universidad Católica del Norte, Chile
Geovanna Parra Riofrío
Universidad de Guayaquil, Ecuador
Michael Araya
Centro de Investigación y Desarrollo Tecnológico en Algas, Chile
Francisco Álvarez Segovia
Centro de Investigación y Desarrollo Tecnológico en Algas, Chile
pág. 3053
DOI: https://doi.org/10.37811/cl_rcm.v9i3.17924
Production of Okadaic Acid in Prorocentrum Lima Through Mixotrophic
Cultures with Macroalgal Polysaccharides
Roberto Pino Selles1
roberto.pino@up.ac.pa
https://orcid.org/0000-0002-0782-2459
Universidad de Panamá
Panamá
Gonzalo Álvarez Vergara
gmalvarez@ucn.cl
https://orcid.org/0000-0001-5812-1559
Universidad Católica del Norte Sede Coquimbo
Chile
Eduardo Uribe
euribe@ucn.cl
https://orcid.org/0000-0003-4913-8038
Universidad Católica del Norte Sede Coquimbo
Chile
Geovanna Parra Riofrío
geovannaparrar@ug.edu.ec
https://orcid.org/0000-0001-5400-2181
Universidad de Guayaquil
Ecuador
Michael Araya
mmaraya@ucn.cl
https://orcid.org/0000-0001-6142-7823
Centro de Investigación y Desarrollo
Tecnológico en Algas (CIDTA)
Chile
Francisco Álvarez Segovia
falvarezsego@gmail.com
https://orcid.org/0009-0009-9135-1386
Centro de Investigación y Desarrollo
Tecnológico en Algas (CIDTA)
Chile
ABSTRACT
Prorocentrum lima is highly investigated in the aquaculture of bivalve mollusks due to the production
of toxins. Currently, various studies with traditional autotrophic means are used to increase the
production of okadaic acid (OA) in P. lima culture, although the synthesis of its toxins occurs in small
quantities. The innovation of mixotrophic cultures with macroalgal polysaccharides allowed us to know
that the P. lima strains used were capable of assimilating organic carbon. Strain D008-1 grew better in
the medium with Agarophyton chilensis polysaccharides (0.07±0.06 day-1), however, strain D008-5 that
grew in the medium with Codium fragile polysaccharides synthesized the greatest amount of OA (13.2
pg OA cell-¹) on day 7, achieving more OA in a shorter time than the L1-Si autotrophic medium. This
production could be due to the presence of sulfated and glucose groups in the polysaccharides of C.
fragile. In both autotrophic and mixotrophic cultures, a high production of OA was obtained compared
to a reduced amount of Dinophysistoxin-1 (DTX-1). This study consisted of implementing a non-
traditional means through an organic carbon source to increase biomass growth and toxin synthesis of
P. lima.
Keywords: culture, benthic dinoflagellate, diarrheal toxin, macroalgal polysaccharides, mixotrophy
1 Autor principal.
Correspondencia: roberto.pino@up.ac.pa
DOI: https://doi.org/10.37811/cl_rcm.v9i3.17924
Production of Okadaic Acid in Prorocentrum Lima Through Mixotrophic
Cultures with Macroalgal Polysaccharides
Roberto Pino Selles1
roberto.pino@up.ac.pa
https://orcid.org/0000-0002-0782-2459
Universidad de Panamá
Panamá
Gonzalo Álvarez Vergara
gmalvarez@ucn.cl
https://orcid.org/0000-0001-5812-1559
Universidad Católica del Norte Sede Coquimbo
Chile
Eduardo Uribe
euribe@ucn.cl
https://orcid.org/0000-0003-4913-8038
Universidad Católica del Norte Sede Coquimbo
Chile
Geovanna Parra Riofrío
geovannaparrar@ug.edu.ec
https://orcid.org/0000-0001-5400-2181
Universidad de Guayaquil
Ecuador
Michael Araya
mmaraya@ucn.cl
https://orcid.org/0000-0001-6142-7823
Centro de Investigación y Desarrollo
Tecnológico en Algas (CIDTA)
Chile
Francisco Álvarez Segovia
falvarezsego@gmail.com
https://orcid.org/0009-0009-9135-1386
Centro de Investigación y Desarrollo
Tecnológico en Algas (CIDTA)
Chile
ABSTRACT
Prorocentrum lima is highly investigated in the aquaculture of bivalve mollusks due to the production
of toxins. Currently, various studies with traditional autotrophic means are used to increase the
production of okadaic acid (OA) in P. lima culture, although the synthesis of its toxins occurs in small
quantities. The innovation of mixotrophic cultures with macroalgal polysaccharides allowed us to know
that the P. lima strains used were capable of assimilating organic carbon. Strain D008-1 grew better in
the medium with Agarophyton chilensis polysaccharides (0.07±0.06 day-1), however, strain D008-5 that
grew in the medium with Codium fragile polysaccharides synthesized the greatest amount of OA (13.2
pg OA cell-¹) on day 7, achieving more OA in a shorter time than the L1-Si autotrophic medium. This
production could be due to the presence of sulfated and glucose groups in the polysaccharides of C.
fragile. In both autotrophic and mixotrophic cultures, a high production of OA was obtained compared
to a reduced amount of Dinophysistoxin-1 (DTX-1). This study consisted of implementing a non-
traditional means through an organic carbon source to increase biomass growth and toxin synthesis of
P. lima.
Keywords: culture, benthic dinoflagellate, diarrheal toxin, macroalgal polysaccharides, mixotrophy
1 Autor principal.
Correspondencia: roberto.pino@up.ac.pa
pág. 3054
Producción de Ácido Okadaico en Prorocentrum Lima Mediante Cultivos
Mixotróficos con Polisacáridos Macroalgales
RESUMEN
Prorocentrum lima es muy investigada en la acuicultura de moluscos bivalvos por la producción de
toxinas. Actualmente, diversos estudios con los tradicionales medios autotróficos son usados para
incrementar la producción de ácido okadaico (AO) en cultivo de P. lima, aunque la síntesis de sus
toxinas ocurre en pequeñas cantidades. La innovación de cultivos mixotróficos con polisacáridos
macroalgales permitió conocer que las cepas de P. lima utilizadas fueron capaces de asimilar carbono
orgánico. La cepa D008-1 creció mejor en el medio con polisacáridos de Agarophyton chilensis
(0,07±0,06 día-1), sin embargo, la cepa D008-5 que creció en el medio con polisacáridos de Codium
fragile sintetizó la mayor cantidad de AO (13,2 pg AO célula-¹) al día 7, logrando en menor tiempo más
AO que el medio autotrófico L1-Si. Esta producción pudo deberse a la presencia de los grupos
sulfatados y de glucosa en los polisacáridos de C. fragile. Tanto en cultivos autotróficos como en
mixotróficos se obtuvo una alta producción de AO en comparación con una reducida cantidad de
Dinophysistoxina-1 (DTX-1). Este estudio consistió en implementar un medio no tradicional a través
de una fuente de carbono orgánico para incrementar el crecimiento en biomasa y la síntesis de toxinas
de P. lima.
Palabras clave: cultivo, dinoflagelado bentónico, toxina diarreica, polisacáridos macroalgales,
mixotrofía
Artículo recibido 05 mayo 2025
Aceptado para publicación: 30 mayo 2025
Producción de Ácido Okadaico en Prorocentrum Lima Mediante Cultivos
Mixotróficos con Polisacáridos Macroalgales
RESUMEN
Prorocentrum lima es muy investigada en la acuicultura de moluscos bivalvos por la producción de
toxinas. Actualmente, diversos estudios con los tradicionales medios autotróficos son usados para
incrementar la producción de ácido okadaico (AO) en cultivo de P. lima, aunque la síntesis de sus
toxinas ocurre en pequeñas cantidades. La innovación de cultivos mixotróficos con polisacáridos
macroalgales permitió conocer que las cepas de P. lima utilizadas fueron capaces de asimilar carbono
orgánico. La cepa D008-1 creció mejor en el medio con polisacáridos de Agarophyton chilensis
(0,07±0,06 día-1), sin embargo, la cepa D008-5 que creció en el medio con polisacáridos de Codium
fragile sintetizó la mayor cantidad de AO (13,2 pg AO célula-¹) al día 7, logrando en menor tiempo más
AO que el medio autotrófico L1-Si. Esta producción pudo deberse a la presencia de los grupos
sulfatados y de glucosa en los polisacáridos de C. fragile. Tanto en cultivos autotróficos como en
mixotróficos se obtuvo una alta producción de AO en comparación con una reducida cantidad de
Dinophysistoxina-1 (DTX-1). Este estudio consistió en implementar un medio no tradicional a través
de una fuente de carbono orgánico para incrementar el crecimiento en biomasa y la síntesis de toxinas
de P. lima.
Palabras clave: cultivo, dinoflagelado bentónico, toxina diarreica, polisacáridos macroalgales,
mixotrofía
Artículo recibido 05 mayo 2025
Aceptado para publicación: 30 mayo 2025
pág. 3055
INTRODUCTION
In recent decades, some species of phototrophic dinoflagellates (e.g. Takayama helix, Karenia brevis,
Alexandrium catenella, A. minutum, Heterocapsa rotundata, P. minimum, P. donghaiense, P. micans,
etc.), have been identified as organisms that have the ability to obtain their energy through mixotrophy
(Jeong et al., 2005; 2010; 2015; 2016; Lee et al., 2016). These dinoflagellates that use mixotrophic
feeding represent a small proportion of the total populations of phototrophic dinoflagellates (Gómez,
2012; Lee et al., 2014; Jeong et al., 2016). These species commonly inhabit shallow waters, attached to
different biotic and abiotic substrates (Lee et al., 2020), allowing the development of a complex
epiphytic community, where they can interact with other microorganisms such as bacteria, fungi and
other types of microalgae (Florez et al., 2017; Zhang et al., 2009; Tiffany et al., 2011). The benthic
dinoflagellate Prorocentrum lima is frequently found attached to these substrates and due to this strong
adhesion, its dispersion in adjacent environments is very difficult, so its nutrition constantly depends
on these substrates. Furthermore, as these benthic species tend to be more flattened with respect to
planktonic dinoflagellates, their surface/volume ratio is favored, facilitating the absorption of nutrients
in oligotrophic conditions (Fraga, 2014) and being in direct contact with the coastal seabed and not
depending on the water column to obtain most of the nutrients, it can be considered a species with the
ability to develop under different nutritional conditions.
P. lima, has been the most researched microalgae with the purpose of being cultivated, since in addition
to being one of the first species confirmed as causing Diarrheal Poisoning Syndrome (DPS) in mollusks
(Murakami et al., 1982), it has been linked to various episodes of this poisoning in mollusks grown in
various farms (Lawrence et al., 2000; Levasseur et al., 2003) with case reports annually, which shows
its health risk worldwide (Nascimento et al., 2016).
Parsons & Preskitt, 2007; Tester et al., 2014, mentioned that P. lima is frequently found on various
species of macroalgae such as Codium fragile, Ulva spp., Agarophyton chilensis among others, in
addition to rocky substrates and sand, being able to take advantage of the nutrients coming from these
habitats. This ability to be able to use both autotrophic and possibly mixotrophic feeding; would
influence its physiology, growth and the increase in its secondary metabolites, such as okadaic acid
(OA) and its analogues (Mitra & Flynn, 2010; Zhang et al., 2013; Jeon et al., 2015), compounds
INTRODUCTION
In recent decades, some species of phototrophic dinoflagellates (e.g. Takayama helix, Karenia brevis,
Alexandrium catenella, A. minutum, Heterocapsa rotundata, P. minimum, P. donghaiense, P. micans,
etc.), have been identified as organisms that have the ability to obtain their energy through mixotrophy
(Jeong et al., 2005; 2010; 2015; 2016; Lee et al., 2016). These dinoflagellates that use mixotrophic
feeding represent a small proportion of the total populations of phototrophic dinoflagellates (Gómez,
2012; Lee et al., 2014; Jeong et al., 2016). These species commonly inhabit shallow waters, attached to
different biotic and abiotic substrates (Lee et al., 2020), allowing the development of a complex
epiphytic community, where they can interact with other microorganisms such as bacteria, fungi and
other types of microalgae (Florez et al., 2017; Zhang et al., 2009; Tiffany et al., 2011). The benthic
dinoflagellate Prorocentrum lima is frequently found attached to these substrates and due to this strong
adhesion, its dispersion in adjacent environments is very difficult, so its nutrition constantly depends
on these substrates. Furthermore, as these benthic species tend to be more flattened with respect to
planktonic dinoflagellates, their surface/volume ratio is favored, facilitating the absorption of nutrients
in oligotrophic conditions (Fraga, 2014) and being in direct contact with the coastal seabed and not
depending on the water column to obtain most of the nutrients, it can be considered a species with the
ability to develop under different nutritional conditions.
P. lima, has been the most researched microalgae with the purpose of being cultivated, since in addition
to being one of the first species confirmed as causing Diarrheal Poisoning Syndrome (DPS) in mollusks
(Murakami et al., 1982), it has been linked to various episodes of this poisoning in mollusks grown in
various farms (Lawrence et al., 2000; Levasseur et al., 2003) with case reports annually, which shows
its health risk worldwide (Nascimento et al., 2016).
Parsons & Preskitt, 2007; Tester et al., 2014, mentioned that P. lima is frequently found on various
species of macroalgae such as Codium fragile, Ulva spp., Agarophyton chilensis among others, in
addition to rocky substrates and sand, being able to take advantage of the nutrients coming from these
habitats. This ability to be able to use both autotrophic and possibly mixotrophic feeding; would
influence its physiology, growth and the increase in its secondary metabolites, such as okadaic acid
(OA) and its analogues (Mitra & Flynn, 2010; Zhang et al., 2013; Jeon et al., 2015), compounds
pág. 3056
currently in high demand as standards for chromatographic analysis (Suzuki et al., 2014), which makes
it a candidate to be cultivated under nutritional conditions typical of benthic organisms. These
conditions could offer certain advantages, such as growing with limited light, depleted nutrients or high
concentrations of organic matter (Salerno & Stoecker, 2009; Riebesell et al., 2017). This species could
use organic matter during periods of darkness, while inorganic matter would be in the presence of light
and it could even occur that both sources are assimilated concomitantly (Jeong et al., 2005).
Currently, there are several studies in which crops have been carried out with autotrophic media, to
increase the production of OA in P. lima, varying proportions of macronutrients such as nitrogen and
phosphorus (McLachlan et al., 1994; Vanucci et al., 2010; Varkitzi et al., 2010; Varkitzi et al., 2017),
thermal variations (McLachlan et al., 1994; Koike et al., 1998; Aquino-Cruz et al., 2018), salinities
(Wang et al., 2015), irradiation and photoperiod (Vanucci et al., 2010; Wang et al., 2015; Aquino-Cruz
et al., 2018; David et al., 2018), providing valuable information for understanding the cultivation of this
dinoflagellate.
There is little background on the effects of different organic compounds and extracellular products
exuded by macroalgae that can promote the growth and development of microalgal species (Carlson et
al., 1984; Bomber et al., 1989; Rains & Parsons, 2015). Wang et al., (2012), reported that the organic
matter from the Ulva macroalga proliferates in decomposition, produced the development, and increase
of species that cause Harmful Algal Blooms (Heterosigma akashiwo, Alexandrium tamarense, P.
donghaiense and Skeletonema costatum). Also, Santelices & Varela, (1993), mentioned that the
exudates of total polysaccharides of A. chilensis in a medium stimulate the settlement and recruitment
of species such as Ulva lactuca, which in P. lima could similarly favor its development. Likewise,
Shalaby & Amin, (2019), mentioned the possible nutritional value of the sulfated polysaccharide,
ulvano, obtained from different extracts of U. lactuca, which could be used by P. lima to increase its
cell density. This work aims to evaluate the growth and production of OA and derivatives of the benthic
dinoflagellate P. lima, in mixotrophic culture media based on the polysaccharides of the macroalgae C.
fragile, A. chilensis and Ulva spp., in comparison with the traditional autotrophic medium, L1 without
silicate (L1-Si).
currently in high demand as standards for chromatographic analysis (Suzuki et al., 2014), which makes
it a candidate to be cultivated under nutritional conditions typical of benthic organisms. These
conditions could offer certain advantages, such as growing with limited light, depleted nutrients or high
concentrations of organic matter (Salerno & Stoecker, 2009; Riebesell et al., 2017). This species could
use organic matter during periods of darkness, while inorganic matter would be in the presence of light
and it could even occur that both sources are assimilated concomitantly (Jeong et al., 2005).
Currently, there are several studies in which crops have been carried out with autotrophic media, to
increase the production of OA in P. lima, varying proportions of macronutrients such as nitrogen and
phosphorus (McLachlan et al., 1994; Vanucci et al., 2010; Varkitzi et al., 2010; Varkitzi et al., 2017),
thermal variations (McLachlan et al., 1994; Koike et al., 1998; Aquino-Cruz et al., 2018), salinities
(Wang et al., 2015), irradiation and photoperiod (Vanucci et al., 2010; Wang et al., 2015; Aquino-Cruz
et al., 2018; David et al., 2018), providing valuable information for understanding the cultivation of this
dinoflagellate.
There is little background on the effects of different organic compounds and extracellular products
exuded by macroalgae that can promote the growth and development of microalgal species (Carlson et
al., 1984; Bomber et al., 1989; Rains & Parsons, 2015). Wang et al., (2012), reported that the organic
matter from the Ulva macroalga proliferates in decomposition, produced the development, and increase
of species that cause Harmful Algal Blooms (Heterosigma akashiwo, Alexandrium tamarense, P.
donghaiense and Skeletonema costatum). Also, Santelices & Varela, (1993), mentioned that the
exudates of total polysaccharides of A. chilensis in a medium stimulate the settlement and recruitment
of species such as Ulva lactuca, which in P. lima could similarly favor its development. Likewise,
Shalaby & Amin, (2019), mentioned the possible nutritional value of the sulfated polysaccharide,
ulvano, obtained from different extracts of U. lactuca, which could be used by P. lima to increase its
cell density. This work aims to evaluate the growth and production of OA and derivatives of the benthic
dinoflagellate P. lima, in mixotrophic culture media based on the polysaccharides of the macroalgae C.
fragile, A. chilensis and Ulva spp., in comparison with the traditional autotrophic medium, L1 without
silicate (L1-Si).
pág. 3057
MATERIAL AND METHODS
Collection of macroalgae and extraction of macroalgal polysaccharides
A collection of macroalgae (A. chilensis, Ulva spp. and C. fragile) was carried out in La Herradura Bay,
Coquimbo, Chile (29°58'44"S and 71°21'8"W). The samples were immediately washed with distilled
water to remove microepibionts, remains of sand and salts. Subsequently, the algae were dried in an LS
Series Freeze Dryer and pulverized with a mortar. For the extraction of macroalgal polysaccharides
(PsMA), 20 g of dry powder of each species of macroalgae were taken and depigmented using 200 mL
of 96% ethanol (Sigma-Aldrich), shaking them in a Quimis Q261A21 shaking plate. The extract
obtained was centrifuged for 5 minutes at 20000× g and 8ºC, using a Centurion K2015R refrigerated
centrifuge (Centurion Scientific Limited). The pellet obtained was mixed with 200 mL of distilled water
and stirred for 30 minutes on a heating plate with stirring at 90ºC (Quimis, SH-3). The polysaccharides
(Ps) obtained in the extract were precipitated by adding 200 mL of 96% ethanol (v/v) for 24 hours (Sun
et al., 2014). Subsequently, the extract was centrifuged at 20000× g for 5 minutes at 8°C. This pellet
obtained was mixed with 100 mL of 4 M NaCl (Sigma-Aldrich) with stirring and a temperature of 90ºC
until completely dissolved. Once cooled, 200 mL of 96% ethanol in a (v/v) extract/alcohol ratio were
added for 24 h. They were then centrifuged at 20000× g for 5 minutes to 8°C. Subsequently, the pellet
containing the total Ps and the salts from the 4 M NaCl solution were placed on a dialysis membrane
(Sigma-Aldrich) and in a 0.5 M NaCl solution for 24 h. Finally, the solution was centrifuged at 20000×
g for 5 minutes at 8°C, the resulting pellets were dried in an oven at 40°C for 24 h and pulverized with
a mortar.
Determination of the concentration of treatments with macroalgal polysaccharide media (15, 50
and 100 mg L-1) in P. lima strain D008-5
Triplicate cultures of strain D008-5 were performed with approximate inocula of 4,000 cells mL-1 in
250 mL Erlenmeyer flasks with 100 mL-1 of the L1-Si media (Guillard & Hargraves, 1993) plus A.
chilensis polysaccharides (PsACH) at 15 mg. L-1, 50 mg L-1 and 100 mg L-1; polysaccharides from Ulva
spp. (PsULV) at 15 mg L-1, 50 mg L-1 and 100 mg L-1; C. fragile polysaccharides (PsCFR) at 15 mg L-
1, 50 mg L-1 and 100 mg L-1 and in L1-Si without PsMA as a control medium, for 18 days. All cultures
were maintained at a temperature of 20 °C, irradiation of 80 μmol of photons m-2s-1 and photoperiod of
MATERIAL AND METHODS
Collection of macroalgae and extraction of macroalgal polysaccharides
A collection of macroalgae (A. chilensis, Ulva spp. and C. fragile) was carried out in La Herradura Bay,
Coquimbo, Chile (29°58'44"S and 71°21'8"W). The samples were immediately washed with distilled
water to remove microepibionts, remains of sand and salts. Subsequently, the algae were dried in an LS
Series Freeze Dryer and pulverized with a mortar. For the extraction of macroalgal polysaccharides
(PsMA), 20 g of dry powder of each species of macroalgae were taken and depigmented using 200 mL
of 96% ethanol (Sigma-Aldrich), shaking them in a Quimis Q261A21 shaking plate. The extract
obtained was centrifuged for 5 minutes at 20000× g and 8ºC, using a Centurion K2015R refrigerated
centrifuge (Centurion Scientific Limited). The pellet obtained was mixed with 200 mL of distilled water
and stirred for 30 minutes on a heating plate with stirring at 90ºC (Quimis, SH-3). The polysaccharides
(Ps) obtained in the extract were precipitated by adding 200 mL of 96% ethanol (v/v) for 24 hours (Sun
et al., 2014). Subsequently, the extract was centrifuged at 20000× g for 5 minutes at 8°C. This pellet
obtained was mixed with 100 mL of 4 M NaCl (Sigma-Aldrich) with stirring and a temperature of 90ºC
until completely dissolved. Once cooled, 200 mL of 96% ethanol in a (v/v) extract/alcohol ratio were
added for 24 h. They were then centrifuged at 20000× g for 5 minutes to 8°C. Subsequently, the pellet
containing the total Ps and the salts from the 4 M NaCl solution were placed on a dialysis membrane
(Sigma-Aldrich) and in a 0.5 M NaCl solution for 24 h. Finally, the solution was centrifuged at 20000×
g for 5 minutes at 8°C, the resulting pellets were dried in an oven at 40°C for 24 h and pulverized with
a mortar.
Determination of the concentration of treatments with macroalgal polysaccharide media (15, 50
and 100 mg L-1) in P. lima strain D008-5
Triplicate cultures of strain D008-5 were performed with approximate inocula of 4,000 cells mL-1 in
250 mL Erlenmeyer flasks with 100 mL-1 of the L1-Si media (Guillard & Hargraves, 1993) plus A.
chilensis polysaccharides (PsACH) at 15 mg. L-1, 50 mg L-1 and 100 mg L-1; polysaccharides from Ulva
spp. (PsULV) at 15 mg L-1, 50 mg L-1 and 100 mg L-1; C. fragile polysaccharides (PsCFR) at 15 mg L-
1, 50 mg L-1 and 100 mg L-1 and in L1-Si without PsMA as a control medium, for 18 days. All cultures
were maintained at a temperature of 20 °C, irradiation of 80 μmol of photons m-2s-1 and photoperiod of
pág. 3058
8 h: 16 h (light/dark). Every three days, 1 mL samples were taken from each culture to record the
experimental cell densities achieved. Growth curves and standardization of growth rates for each
treatment were estimated by calculating cell densities every three days using the method described by
LeGeresley & McDermott, 2010. Once these values are obtained the daily growth rates (day-1) were
estimated (Levasseur et al., 1993), using the following formula:
μ = ln(𝑁2/𝑁1)
(𝑡2−𝑡1) (1),
where N2 is the final concentration (cells mL-1), N1 is the initial concentration (cells mL-1), t2 is the
final time (day) and t1 is the initial time (day).
Additionally, with the values of the experimental cell densities, the maximum growth rates and
maximum cell concentrations were estimated, using a mathematical model of exponential growth with
saturation, from the initial observation (day 0). The observed experimental data were fitted according
to the equation:
G=Gm-A*exp(-μm*(t-λ)) (2),
where G is the cell concentration (cells mL⁻¹), Gm is the maximum cell concentration (cells mL⁻¹), A
is the inoculum (cells mL⁻¹), μm is the maximum specific growth rate (day⁻¹), t is the cultivation period
(day) and 𝜆 is the lag phase (day).
Numerical modeling of the growth data was performed using the nonlinear least squares method of the
"Solver" macro of the Microsoft Excel spreadsheet and the error associated with the parameter estimates
(as confidence intervals) was calculated and evaluated using Student's t test. With the theoretical data
obtained in the program and with the experimental data, the growth curves were generated and
compared. The experimental results (observed) and the theoretical results (expected) of the
mathematical model were statistically analyzed with one-way analysis of variance (ANOVA) for each
strain in each culture, performing Fisher's post-hoc test using the statistical program Statistics (StatSoft,
Inc. 2005). The variability of the maximum cell densities, the theoretical maximum growth rates
obtained in the mathematical model, and the variability of the experimental cell densities and growth
rates were then evaluated.
8 h: 16 h (light/dark). Every three days, 1 mL samples were taken from each culture to record the
experimental cell densities achieved. Growth curves and standardization of growth rates for each
treatment were estimated by calculating cell densities every three days using the method described by
LeGeresley & McDermott, 2010. Once these values are obtained the daily growth rates (day-1) were
estimated (Levasseur et al., 1993), using the following formula:
μ = ln(𝑁2/𝑁1)
(𝑡2−𝑡1) (1),
where N2 is the final concentration (cells mL-1), N1 is the initial concentration (cells mL-1), t2 is the
final time (day) and t1 is the initial time (day).
Additionally, with the values of the experimental cell densities, the maximum growth rates and
maximum cell concentrations were estimated, using a mathematical model of exponential growth with
saturation, from the initial observation (day 0). The observed experimental data were fitted according
to the equation:
G=Gm-A*exp(-μm*(t-λ)) (2),
where G is the cell concentration (cells mL⁻¹), Gm is the maximum cell concentration (cells mL⁻¹), A
is the inoculum (cells mL⁻¹), μm is the maximum specific growth rate (day⁻¹), t is the cultivation period
(day) and 𝜆 is the lag phase (day).
Numerical modeling of the growth data was performed using the nonlinear least squares method of the
"Solver" macro of the Microsoft Excel spreadsheet and the error associated with the parameter estimates
(as confidence intervals) was calculated and evaluated using Student's t test. With the theoretical data
obtained in the program and with the experimental data, the growth curves were generated and
compared. The experimental results (observed) and the theoretical results (expected) of the
mathematical model were statistically analyzed with one-way analysis of variance (ANOVA) for each
strain in each culture, performing Fisher's post-hoc test using the statistical program Statistics (StatSoft,
Inc. 2005). The variability of the maximum cell densities, the theoretical maximum growth rates
obtained in the mathematical model, and the variability of the experimental cell densities and growth
rates were then evaluated.
pág. 3059
Once this concentration of PsMA was selected, strain D008-1 was also cultured in each of the media
with the three PsMA at the concentration selected as the best treatment in strain D008-5 and under the
same culture conditions for 15 days. Finally, both strains were acclimated to these media with inoculum
of approximately 10,000 cells mL-1 for the growth test of both strains in macroalgal polysaccharide
media (50 mg L-1).
Growth of P. lima strains D008-1 and D008-5 in macroalgal polysaccharide media (50 mg L-1)
For the cultivation of P. lima strains D008-1 and D008-5 in mixotrophic media with macroalgal
polysaccharides (PsMA), 25 mL of L1-Si media, plus PsACH at 50 mg L-1, PsULV 50 mg L-1 and
PsCFR 50 mg L-1, in addition to L1-Si without PsMA (control), were transferred into glass test tubes
with screw caps. In these media, both strains acclimated to the media with PsMA were inoculated in
triplicate, with an initial density of between 9,000 and 12,000 cells mL-1. These strains grew for 18 days
under the same culture conditions indicated in the previous section. From these cultures in the test tubes,
samples of 1 mL of each strain were taken in triplicate in the different media, on days 3, 5, 7, 12, 15
and 18, and the cell densities of each sample were obtained. The growth curves for each treatment and
strain were estimated with the mathematical growth model described above.
Production of Okadaic Acid (OA) and Dinophysistoxin-1 (DTX-1) of P. lima strains D008-1 and
D008-5 in culture media of 50 mg L-1 of macroalgal polysaccharides (PsMA)
The 24 mL samples of each of the cultures in the test tubes were concentrated by centrifuging at 20000×
g for 10 minutes, using a refrigerated centrifuge (4ºC) (Centurion K2015R, Centurion Scientific Ltd.,
Stoughton, West Sussex, UK). The supernatants were discarded, and each resulting cell pellet was
resuspended in 2 mL of 100% methanol, sonicated for two minutes in pulse mode (50% duty cycle, 375
watts) in a Branson 150D ultrasonic homogenizer (Branson Ultrasonic Corp), while cooling in an ice
bath. Each extract was separated by centrifugation at 18.973× g for 10 minutes. The supernatants were
filtered through syringes with a PTFE syringe filter (0.22 μm) (Jet Bio-Filtration Co., Ltd).
Subsequently, they were placed in amber chromatographic vials and stored at -20ºC until analysis.
Before analysis, each sample was hydrolyzed to esterify the toxins that were not free and thus obtain
the total concentrations of OA and DTX's, following the protocol of Mountfort et al., (2001) using 0.5
mL of the sample, adding 62.5 μL of NaOH4 (2.5 M) and mixing with vortex for 30 seconds.
Once this concentration of PsMA was selected, strain D008-1 was also cultured in each of the media
with the three PsMA at the concentration selected as the best treatment in strain D008-5 and under the
same culture conditions for 15 days. Finally, both strains were acclimated to these media with inoculum
of approximately 10,000 cells mL-1 for the growth test of both strains in macroalgal polysaccharide
media (50 mg L-1).
Growth of P. lima strains D008-1 and D008-5 in macroalgal polysaccharide media (50 mg L-1)
For the cultivation of P. lima strains D008-1 and D008-5 in mixotrophic media with macroalgal
polysaccharides (PsMA), 25 mL of L1-Si media, plus PsACH at 50 mg L-1, PsULV 50 mg L-1 and
PsCFR 50 mg L-1, in addition to L1-Si without PsMA (control), were transferred into glass test tubes
with screw caps. In these media, both strains acclimated to the media with PsMA were inoculated in
triplicate, with an initial density of between 9,000 and 12,000 cells mL-1. These strains grew for 18 days
under the same culture conditions indicated in the previous section. From these cultures in the test tubes,
samples of 1 mL of each strain were taken in triplicate in the different media, on days 3, 5, 7, 12, 15
and 18, and the cell densities of each sample were obtained. The growth curves for each treatment and
strain were estimated with the mathematical growth model described above.
Production of Okadaic Acid (OA) and Dinophysistoxin-1 (DTX-1) of P. lima strains D008-1 and
D008-5 in culture media of 50 mg L-1 of macroalgal polysaccharides (PsMA)
The 24 mL samples of each of the cultures in the test tubes were concentrated by centrifuging at 20000×
g for 10 minutes, using a refrigerated centrifuge (4ºC) (Centurion K2015R, Centurion Scientific Ltd.,
Stoughton, West Sussex, UK). The supernatants were discarded, and each resulting cell pellet was
resuspended in 2 mL of 100% methanol, sonicated for two minutes in pulse mode (50% duty cycle, 375
watts) in a Branson 150D ultrasonic homogenizer (Branson Ultrasonic Corp), while cooling in an ice
bath. Each extract was separated by centrifugation at 18.973× g for 10 minutes. The supernatants were
filtered through syringes with a PTFE syringe filter (0.22 μm) (Jet Bio-Filtration Co., Ltd).
Subsequently, they were placed in amber chromatographic vials and stored at -20ºC until analysis.
Before analysis, each sample was hydrolyzed to esterify the toxins that were not free and thus obtain
the total concentrations of OA and DTX's, following the protocol of Mountfort et al., (2001) using 0.5
mL of the sample, adding 62.5 μL of NaOH4 (2.5 M) and mixing with vortex for 30 seconds.
pág. 3060
Subsequently, it was heated to 76°C for 40 minutes, cooled to room temperature and neutralized with
62.5 mL of HCl (EU-RL-MB, (2015). The identification and quantification of okadaic acid (OA),
dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2) for these strains was carried out by Ultra
High Pressure Liquid Chromatography (UHPLC) and detection by high resolution mass spectrometry
(HRMS). The chromatographic analysis was developed following the methodology described by
(Regueiro et al., 2011) with small modifications. The chromatographic system corresponded to a
Dionex Ultimate 3000 chromatograph (UHPLC) (Thermo Fisher Scientific, Sunnyvale, CA, USA).
Chromatographic separation was performed using a Gemini-NX C18 reversed-phase column (100 x 2.0
mm, 3μm) with an Ultra Guard C18 precolumn (Phenomenex; Torrance, California, USA) maintained
at 40°C. The mobile phases consisted of 6.7 mM NH4OH in MilliQ water (phase A); and 100 % ACN
with 6.7 mM NH4OH (phase B). The flow of the mobile phases was maintained at 0.35 mL/min. The
gradient used was as follows: 15% phase B maintained for 1 minute, followed by a linear increase over
2.85 minutes until reaching 80% phase B, an increase to 85% Phase B in 0.15 minutes, 90% B for 0.75
minutes and 100% B for 3.25 minutes. Finally, the gradient returned to initial conditions in 2 minutes.
Detection of lipophilic toxins was carried out using a Q Exactive Focus mass spectrometer with HESI-
II electrospray interface (Thermo Fisher Scientific, Sunnyvale, CA, USA). The HESI was operated in
negative ionization mode with a spray voltage of 3 kV. The temperature of the transfer tube and HESI
vaporizer was 200 and 350°C, respectively. At the source, nitrogen (> 99.98%) was used as sheath gas
and auxiliary gas with pressures of 20 and 4 arbitrary units, respectively. Data were acquired in SIM
(Selected Ion Monitoring) mode and data acquisition in ddMS2 (data dependent) mode. In SIM mode,
the scanning range was from 100 – 1000 m/z with a resolution of 70,000, an automatic gain (AGC) of
5 x 104 and a maximum injection (IT) of 3,000 ms. For dds2 the mass resolution was 70,000, the AGC
was 5 x 104 and a maximum injection (IT) of 3,000 ms. In both cases the isolation window was 2 m/z.
For each compound of interest, the exact mass of the precursor ion, retention time, and collision energy
were included. Toxins were identified based on retention time and mass spectra in comparison with
calibration curves and mass spectra of external standards. The toxin concentration in the extracts of
each strain was quantified by comparing the area and peaks obtained in the chromatograms with those
of the certified reference materials obtained NCR, Canada. Finally, the toxin content was calculated per
Subsequently, it was heated to 76°C for 40 minutes, cooled to room temperature and neutralized with
62.5 mL of HCl (EU-RL-MB, (2015). The identification and quantification of okadaic acid (OA),
dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2) for these strains was carried out by Ultra
High Pressure Liquid Chromatography (UHPLC) and detection by high resolution mass spectrometry
(HRMS). The chromatographic analysis was developed following the methodology described by
(Regueiro et al., 2011) with small modifications. The chromatographic system corresponded to a
Dionex Ultimate 3000 chromatograph (UHPLC) (Thermo Fisher Scientific, Sunnyvale, CA, USA).
Chromatographic separation was performed using a Gemini-NX C18 reversed-phase column (100 x 2.0
mm, 3μm) with an Ultra Guard C18 precolumn (Phenomenex; Torrance, California, USA) maintained
at 40°C. The mobile phases consisted of 6.7 mM NH4OH in MilliQ water (phase A); and 100 % ACN
with 6.7 mM NH4OH (phase B). The flow of the mobile phases was maintained at 0.35 mL/min. The
gradient used was as follows: 15% phase B maintained for 1 minute, followed by a linear increase over
2.85 minutes until reaching 80% phase B, an increase to 85% Phase B in 0.15 minutes, 90% B for 0.75
minutes and 100% B for 3.25 minutes. Finally, the gradient returned to initial conditions in 2 minutes.
Detection of lipophilic toxins was carried out using a Q Exactive Focus mass spectrometer with HESI-
II electrospray interface (Thermo Fisher Scientific, Sunnyvale, CA, USA). The HESI was operated in
negative ionization mode with a spray voltage of 3 kV. The temperature of the transfer tube and HESI
vaporizer was 200 and 350°C, respectively. At the source, nitrogen (> 99.98%) was used as sheath gas
and auxiliary gas with pressures of 20 and 4 arbitrary units, respectively. Data were acquired in SIM
(Selected Ion Monitoring) mode and data acquisition in ddMS2 (data dependent) mode. In SIM mode,
the scanning range was from 100 – 1000 m/z with a resolution of 70,000, an automatic gain (AGC) of
5 x 104 and a maximum injection (IT) of 3,000 ms. For dds2 the mass resolution was 70,000, the AGC
was 5 x 104 and a maximum injection (IT) of 3,000 ms. In both cases the isolation window was 2 m/z.
For each compound of interest, the exact mass of the precursor ion, retention time, and collision energy
were included. Toxins were identified based on retention time and mass spectra in comparison with
calibration curves and mass spectra of external standards. The toxin concentration in the extracts of
each strain was quantified by comparing the area and peaks obtained in the chromatograms with those
of the certified reference materials obtained NCR, Canada. Finally, the toxin content was calculated per
pág. 3061
cell and then at the level of each 24 mL culture. The experimental results were statistically analyzed
with one-way analysis of variance (ANOVA) for the selected days in each crop, performing Tukey's
post-hoc test, from the Statistica statistical program (StatSoft, Inc. 2005) to evaluate the variability of
cell densities, growth rates and production of OA and DTX1 between treatments.
RESULTS AND DISCUSSION
Determination of the concentration of treatments with macroalgal polysaccharide media in P. lima
strain D008-5
The cultures of P. lima strain D008-5 in the L1-Si media, plus the three concentrations (15, 50 and 100
mg L-1) of polysaccharides (Ps) from A. chilensis (PsACH) presented similar growth the first 12 days
of cultivation (Fig. 1). Then they began to differentiate, reaching the highest experimental cell density
of 29,913 ± 1,255 cells mL-1 in the treatment of 50 mg L-1 of PsACH after 18 days. Their experimental
growth rates were between 0.1051 – 0.1152 day-1, obtaining close values when comparing the
experimental results with the theoretical ones in each treatment.
This D008-5 strain in cultures with L1-Si media, plus the three concentrations (15, 50 y 100 mg L-1) of
Ps from Ulva spp. (PsULV), also presented similar growth the first 12 days of culture. After that time,
no differentiation was observed in their growth, reaching a maximum experimental cell density of
27,043 ± 4,553 cells mL-1 in the treatment of 50 mg L-1 of PsULV at 18 days of culture (Fig. 1). These
cultures in PsULV obtained experimental growth rates between 0.1007 – 0.1061 day-1. Close values
were also obtained when comparing the experimental results with the theoretical results in each
treatment.
Finally, in the cultures with the L1-Si media, plus the three concentrations (15, 50 and 100 mg L-1) of
Ps from C. fragile (PsCFR), likewise, strain D008-5 presented similar growth in the first 12 days of
culture (Fig. 1). Subsequently, no differentiation was observed in their growth, with a maximum
experimental cell density of 27,253 ± 1,848 cells mL-1 in the treatment of 50 mg L-1 of PsCFR at the
end of the culture. The cultures in PsCFR obtained experimental growth rates between 0.0984 – 0.1062
day-1. Similarly, close values were obtained when comparing the experimental results with the
theoretical results in each treatment.
cell and then at the level of each 24 mL culture. The experimental results were statistically analyzed
with one-way analysis of variance (ANOVA) for the selected days in each crop, performing Tukey's
post-hoc test, from the Statistica statistical program (StatSoft, Inc. 2005) to evaluate the variability of
cell densities, growth rates and production of OA and DTX1 between treatments.
RESULTS AND DISCUSSION
Determination of the concentration of treatments with macroalgal polysaccharide media in P. lima
strain D008-5
The cultures of P. lima strain D008-5 in the L1-Si media, plus the three concentrations (15, 50 and 100
mg L-1) of polysaccharides (Ps) from A. chilensis (PsACH) presented similar growth the first 12 days
of cultivation (Fig. 1). Then they began to differentiate, reaching the highest experimental cell density
of 29,913 ± 1,255 cells mL-1 in the treatment of 50 mg L-1 of PsACH after 18 days. Their experimental
growth rates were between 0.1051 – 0.1152 day-1, obtaining close values when comparing the
experimental results with the theoretical ones in each treatment.
This D008-5 strain in cultures with L1-Si media, plus the three concentrations (15, 50 y 100 mg L-1) of
Ps from Ulva spp. (PsULV), also presented similar growth the first 12 days of culture. After that time,
no differentiation was observed in their growth, reaching a maximum experimental cell density of
27,043 ± 4,553 cells mL-1 in the treatment of 50 mg L-1 of PsULV at 18 days of culture (Fig. 1). These
cultures in PsULV obtained experimental growth rates between 0.1007 – 0.1061 day-1. Close values
were also obtained when comparing the experimental results with the theoretical results in each
treatment.
Finally, in the cultures with the L1-Si media, plus the three concentrations (15, 50 and 100 mg L-1) of
Ps from C. fragile (PsCFR), likewise, strain D008-5 presented similar growth in the first 12 days of
culture (Fig. 1). Subsequently, no differentiation was observed in their growth, with a maximum
experimental cell density of 27,253 ± 1,848 cells mL-1 in the treatment of 50 mg L-1 of PsCFR at the
end of the culture. The cultures in PsCFR obtained experimental growth rates between 0.0984 – 0.1062
day-1. Similarly, close values were obtained when comparing the experimental results with the
theoretical results in each treatment.
pág. 3062
The lowest experimental density among all cultures was obtained in L1-Si at the end of the experiment
(23,309 ± 796 cells mL-1) with experimental growth rates of 0.0975 ± 0.0270 day-1 (Fig. 1).
P. lima strain D008-5 was grown with mixotrophic media (L1-Si + 15, 50 and 100 mg L-1 of PsMA),
presented experimental average growth rates, like and higher (0.09 – 0.11 day-1) than the L1-Si control
(0.09 day-1). These growth rates were lower than those reported by Morton & Tindall, (1995) (0.20 day-
1) and similar to Nascimento et al., (2005) (0.11 day-1), which differed in the base culture medium.
The maximum experimental cell density of P. lima was recorded in the treatment with 50 mg L-1 of all
PsMA (A. chilensis, C. fragile and Ulva spp.), and now no studies have been found that relate the
cultivation of this species and the addition of PsMA to increase its biomass.
The crops to which PsMA were added showed better growth (> 13%) compared to L1-Si, which
indicates the ability of P. lima to assimilate an organic carbon source, pointing out that P. lima is not
an exclusively autotrophic species but rather mixotrophic. Glibert & Legrand, (2006), mention that
some dinoflagellates are capable of producing extracellular enzymes that hydrolyze macromolecules in
the aquatic environment, to be absorbed by these cells (Cembella et al., 1985), or through an enzymatic
action developed on its cell surface (Stoecker & Gustafson, 2003). In this context, little is known about
the enzymatic profiles in benthic dinoflagellates, but transcriptomic studies have shown that
Gambierdiscus caribaeus has and galactosidases that allow the growth of this species through the
use of galactose, in laboratory cultures (Price et al., 2016).
Matsuhiro & Urzúa, (1990), reported that A. chilensis is made up of galactose molecules with similar
percentages for 3,6-anhydrous galactose (33.5%) and sulfated galactans (2.3%), in addition to the
presence of galactose (32.2%), 6-0 Methylgalactose (6.5%) and pyruvic acid (0.16 %). These
polysaccharides would provide a source of organic carbon that would be assimilated by P. lima cells
through their enzymatic actions. The enzymatic reactions produced by agarases and carrageenases
would be very relevant during the mixotrophic metabolic process from polysaccharides, since they
would trigger the biosynthesis of biologically active components that would form their cellular
structures, promoting the growth of this dinoflagellate.
The highest theoretical cell density of P. lima obtained with the treatment of 50 mg L-1 of the Ps of A.
chilensis, C. fragile and Ulva spp., presented significant differences (p < 0.05) with the concentrations
The lowest experimental density among all cultures was obtained in L1-Si at the end of the experiment
(23,309 ± 796 cells mL-1) with experimental growth rates of 0.0975 ± 0.0270 day-1 (Fig. 1).
P. lima strain D008-5 was grown with mixotrophic media (L1-Si + 15, 50 and 100 mg L-1 of PsMA),
presented experimental average growth rates, like and higher (0.09 – 0.11 day-1) than the L1-Si control
(0.09 day-1). These growth rates were lower than those reported by Morton & Tindall, (1995) (0.20 day-
1) and similar to Nascimento et al., (2005) (0.11 day-1), which differed in the base culture medium.
The maximum experimental cell density of P. lima was recorded in the treatment with 50 mg L-1 of all
PsMA (A. chilensis, C. fragile and Ulva spp.), and now no studies have been found that relate the
cultivation of this species and the addition of PsMA to increase its biomass.
The crops to which PsMA were added showed better growth (> 13%) compared to L1-Si, which
indicates the ability of P. lima to assimilate an organic carbon source, pointing out that P. lima is not
an exclusively autotrophic species but rather mixotrophic. Glibert & Legrand, (2006), mention that
some dinoflagellates are capable of producing extracellular enzymes that hydrolyze macromolecules in
the aquatic environment, to be absorbed by these cells (Cembella et al., 1985), or through an enzymatic
action developed on its cell surface (Stoecker & Gustafson, 2003). In this context, little is known about
the enzymatic profiles in benthic dinoflagellates, but transcriptomic studies have shown that
Gambierdiscus caribaeus has and galactosidases that allow the growth of this species through the
use of galactose, in laboratory cultures (Price et al., 2016).
Matsuhiro & Urzúa, (1990), reported that A. chilensis is made up of galactose molecules with similar
percentages for 3,6-anhydrous galactose (33.5%) and sulfated galactans (2.3%), in addition to the
presence of galactose (32.2%), 6-0 Methylgalactose (6.5%) and pyruvic acid (0.16 %). These
polysaccharides would provide a source of organic carbon that would be assimilated by P. lima cells
through their enzymatic actions. The enzymatic reactions produced by agarases and carrageenases
would be very relevant during the mixotrophic metabolic process from polysaccharides, since they
would trigger the biosynthesis of biologically active components that would form their cellular
structures, promoting the growth of this dinoflagellate.
The highest theoretical cell density of P. lima obtained with the treatment of 50 mg L-1 of the Ps of A.
chilensis, C. fragile and Ulva spp., presented significant differences (p < 0.05) with the concentrations
pág. 3063
of 15 mg L-1, 100 mg L-1 and the autotrophic medium L1-Si, results that allowed selecting the treatment
of 50 mg L-1 for the media with PsMA in mixotrophic cultures.
Growth of P. lima strains D008-1 and D008-5 in macroalgal polysaccharide media (50 mg L-1)
On day 18, strain D008-1 presented the highest experimental cell densities in PsCFR (32,800 ± 1,924
cells mL-1) and in PsULV (32,320 ± 520 cells mL-1). The lowest experimental cell density (28,850 ±
926 cells mL-1) was obtained in L1-Si (p < 0.05). The highest experimental growth rate was obtained
in PsACH (0.0726 ± 0.0615 day-1) and the lowest in L1-Si (0.0560 ± 0.00537 day-1). Close values were
obtained when comparing the experimental results with the theoretical results in each treatment.
The D008-5 strain obtained the highest experimental densities in the cultures with PsACH (29,724 ±
254 cells mL-1) (p < 0.05), followed by PsULV (27,235 ± 1,310 cells mL-1), both on day 18. The lowest
experimental maximum cell density (22,250 ± 601 cells mL-1) was obtained in L1-Si. The maximum
experimental growth rate was obtained in PsACH (0.0682 ± 0.0448 day-1) and the lowest in L1-Si
(0.0498 ± 0.0397 day-1) Close values were also obtained when comparing the experimental results with
the theoretical ones in each treatment.
The maximum cell density obtained in strain D008-1 (32,800 ± 1,924 cells mL-1), was produced with
the PsCFR medium, coinciding with the fact that this strain was isolated from C. fragile, giving a
species-specific physiological condition (Bravo et al., 2001), of the host and the Ps. This relationship
did not occur with strain D008-5 since it obtained lower cell densities (23,182 ± 2,781 cells mL-1) in
this same culture medium. This strain was isolated from the surface of the valves of A. purpuratus.
When macroalgal sugars are added to the inorganic culture medium, they would provide a source of
organic carbon, whose absorption would be favored over that produced in photosynthesis when
mixotrophic conditions exist in a culture, synthesizing compounds typical of photosynthetic and
heterotrophic metabolisms (Smith & Gilmour, 2018), increasing growth rates and microalgal biomass
concentration (Abreu et al., 2012). In the present study, it was observed that mixotrophic cultures with
the addition of organic carbon sources from macroalgal polysaccharides favored (> 13%) the cellular
increase of P. lima strains D008-1 and D008-5.
of 15 mg L-1, 100 mg L-1 and the autotrophic medium L1-Si, results that allowed selecting the treatment
of 50 mg L-1 for the media with PsMA in mixotrophic cultures.
Growth of P. lima strains D008-1 and D008-5 in macroalgal polysaccharide media (50 mg L-1)
On day 18, strain D008-1 presented the highest experimental cell densities in PsCFR (32,800 ± 1,924
cells mL-1) and in PsULV (32,320 ± 520 cells mL-1). The lowest experimental cell density (28,850 ±
926 cells mL-1) was obtained in L1-Si (p < 0.05). The highest experimental growth rate was obtained
in PsACH (0.0726 ± 0.0615 day-1) and the lowest in L1-Si (0.0560 ± 0.00537 day-1). Close values were
obtained when comparing the experimental results with the theoretical results in each treatment.
The D008-5 strain obtained the highest experimental densities in the cultures with PsACH (29,724 ±
254 cells mL-1) (p < 0.05), followed by PsULV (27,235 ± 1,310 cells mL-1), both on day 18. The lowest
experimental maximum cell density (22,250 ± 601 cells mL-1) was obtained in L1-Si. The maximum
experimental growth rate was obtained in PsACH (0.0682 ± 0.0448 day-1) and the lowest in L1-Si
(0.0498 ± 0.0397 day-1) Close values were also obtained when comparing the experimental results with
the theoretical ones in each treatment.
The maximum cell density obtained in strain D008-1 (32,800 ± 1,924 cells mL-1), was produced with
the PsCFR medium, coinciding with the fact that this strain was isolated from C. fragile, giving a
species-specific physiological condition (Bravo et al., 2001), of the host and the Ps. This relationship
did not occur with strain D008-5 since it obtained lower cell densities (23,182 ± 2,781 cells mL-1) in
this same culture medium. This strain was isolated from the surface of the valves of A. purpuratus.
When macroalgal sugars are added to the inorganic culture medium, they would provide a source of
organic carbon, whose absorption would be favored over that produced in photosynthesis when
mixotrophic conditions exist in a culture, synthesizing compounds typical of photosynthetic and
heterotrophic metabolisms (Smith & Gilmour, 2018), increasing growth rates and microalgal biomass
concentration (Abreu et al., 2012). In the present study, it was observed that mixotrophic cultures with
the addition of organic carbon sources from macroalgal polysaccharides favored (> 13%) the cellular
increase of P. lima strains D008-1 and D008-5.
pág. 3064
Production of Okadaic acid (OA) and Dinophysistoxin-1 (DTX-1) of P. lima strains D008-1 and
D008-5 grown on macroalgal polysaccharides (PsMA)
▪ OA and DTX-1 content per cell
The D008-1 strain recorded its highest OA content per cell in the L1-Si medium (8.42 ± 2.33 pg OA
cell-¹), while the lowest was in PsACH (6.11 ± 0.92 pg OA cell-¹). P. lima strain D008-5 recorded its
highest OA content per cell with PsCFR (11.95 ± 1.98 pg OA cell-¹), while in L1-Si (6.12 ± 0.54 pg OA
cell-¹) the lowest value was obtained. During the study, P. lima strain D008-1 presented the highest
content of DTX-1 in the L1-Si medium (0.0198 ± 0.0034 pg DTX-1 cell-¹) and the lowest in PsACH
(0.0108 ± 0.0013 pg DTX-1 cell-¹). P. lima strain D008-5 obtained the highest content of DTX-1 in the
PsCFR medium (0.0298 ± 0.0016 pg DTX-1 cell-¹) (p ˂ 0.05) and the lowest in PsACH (0.0140 ±
00024 pg DTX-1 cell-¹).
▪ Amount of OA per culture
Strain D008-1 obtained the highest experimental value of OA production in the culture in L1-Si
(5,829.24 ng OA) on day 18, while the lowest experimental value of OA was obtained in the PsCFR
culture (4,358.74 ng OA) of the same day. Statistical tests showed no differences (p ˃ 0.05) between
the maximum experimental amounts of OA produced, in any of the treatments throughout the study
(Table 1). Cultivation in L1-Si for strain D008-1 obtained an experimental synthesis of OA, 1.28; 1.34
and 1.27 times higher than in PsULV, PsCFR and PsACH cultures, respectively; during the test. This
strain failed to produce high concentrations of OA when grown with PsMA, synthesizing the greatest
amount in the L1-Si autotrophic medium.
For its part, the strain D008-5 obtained the highest values in experimental OA production in mixotrophic
cultures with PsMA. PsULV began to produce the highest amounts of OA from day 12, obtaining the
highest experimental value (5,529.76 ng OA) on day 18. PsCFR also produced a higher value (4,769.63
ng OA) on day seven, while PsACH (4,160.36 ng OA), on day 12. This strain in the autotrophic culture
L1-Si obtained 2,959.04 ng OA (p ˂ 0.05), on day 15. Close values were recorded when comparing the
experimental results with the theoretical results in each treatment (Table 1). The cultures in PsULV,
PsCFR and PsACH for strain D008-5 obtained an experimental synthesis of OA, 1.87; 1.61 and 1.41
times higher, respectively, than in the culture in L1-Si, during the test.
Production of Okadaic acid (OA) and Dinophysistoxin-1 (DTX-1) of P. lima strains D008-1 and
D008-5 grown on macroalgal polysaccharides (PsMA)
▪ OA and DTX-1 content per cell
The D008-1 strain recorded its highest OA content per cell in the L1-Si medium (8.42 ± 2.33 pg OA
cell-¹), while the lowest was in PsACH (6.11 ± 0.92 pg OA cell-¹). P. lima strain D008-5 recorded its
highest OA content per cell with PsCFR (11.95 ± 1.98 pg OA cell-¹), while in L1-Si (6.12 ± 0.54 pg OA
cell-¹) the lowest value was obtained. During the study, P. lima strain D008-1 presented the highest
content of DTX-1 in the L1-Si medium (0.0198 ± 0.0034 pg DTX-1 cell-¹) and the lowest in PsACH
(0.0108 ± 0.0013 pg DTX-1 cell-¹). P. lima strain D008-5 obtained the highest content of DTX-1 in the
PsCFR medium (0.0298 ± 0.0016 pg DTX-1 cell-¹) (p ˂ 0.05) and the lowest in PsACH (0.0140 ±
00024 pg DTX-1 cell-¹).
▪ Amount of OA per culture
Strain D008-1 obtained the highest experimental value of OA production in the culture in L1-Si
(5,829.24 ng OA) on day 18, while the lowest experimental value of OA was obtained in the PsCFR
culture (4,358.74 ng OA) of the same day. Statistical tests showed no differences (p ˃ 0.05) between
the maximum experimental amounts of OA produced, in any of the treatments throughout the study
(Table 1). Cultivation in L1-Si for strain D008-1 obtained an experimental synthesis of OA, 1.28; 1.34
and 1.27 times higher than in PsULV, PsCFR and PsACH cultures, respectively; during the test. This
strain failed to produce high concentrations of OA when grown with PsMA, synthesizing the greatest
amount in the L1-Si autotrophic medium.
For its part, the strain D008-5 obtained the highest values in experimental OA production in mixotrophic
cultures with PsMA. PsULV began to produce the highest amounts of OA from day 12, obtaining the
highest experimental value (5,529.76 ng OA) on day 18. PsCFR also produced a higher value (4,769.63
ng OA) on day seven, while PsACH (4,160.36 ng OA), on day 12. This strain in the autotrophic culture
L1-Si obtained 2,959.04 ng OA (p ˂ 0.05), on day 15. Close values were recorded when comparing the
experimental results with the theoretical results in each treatment (Table 1). The cultures in PsULV,
PsCFR and PsACH for strain D008-5 obtained an experimental synthesis of OA, 1.87; 1.61 and 1.41
times higher, respectively, than in the culture in L1-Si, during the test.
pág. 3065
These results would indicate that cultivating P. lima under this type of mixotrophic conditions would
induce the production of high amounts of OA in a shorter cultivation time compared to traditional
autotrophic media. But the results could vary in some P. lima strains, as occurred in D008-1, which
synthesized a lower amount of OA compared to D008-5.
Although strain D008-1, when cultivated in PsMA, did not assimilate them in a similar way or obtain
high concentrations like D008-5, the results for each of the cultures were significantly similar (p ˃ 0.05)
throughout the test.
The lower OA production of strain D008-5 obtained in PsACH would be due to the lack of glucose in
the Ps of A chilensis, which does not occur in C. fragile and Ulva spp., (Matsuhiro & Urzúa, 1990;
Kaeffer et al., 1999; Yaich et al., 2014; Kolsi et al., 2017; Wang et al., 2020), a situation that could
indicate that glucose is an essential sugar within the metabolic pathways of this strain of P. lima to be
able to synthesize a greater amount of OA in culture. Another factor would be the sulfate groups that
are present in the Ps of C. fragile and Ulva spp., since they would provide a greater amount of sulfate
molecules available for the formation of sulfated diesters, which could subsequently be transformed to
diol-ester in a few minutes, as a precursor to OA (Quilliam & Ross, 1996; Suzuki et al., 2004).
According to Quilliam et al., (1996), these sulfated diesters would be a means of storing toxins within
the cell of dinoflagellates. Subsequently, Hu et al., (2017) point out a supposed hydrolysis of sulfated
diesters that would be carried out in two steps mediated by specific enzymes (esterases) of sulfated
diesters (such as DTX-4 and DTX-5), initially leading to the formation of diol esters and ultimately to
the release of free OA. This would favor the cellular integrity of P. lima, providing self-protection in
the main structure of the free acids of OA, through the insertion of oxygen in the extended side chain
of these sulfated dieters (Bravo et al., 2001), since the sulfated esters are essentially inactive against
phosphatases PP1 and PP2A.
What happened in these processes would occur in the mixotrophic cultures of P. lima, Ps of C. fragile
and Ulva spp., as observed in the present work. Furthermore, all of this will depend on each type of P.
lima strain, since each of them presents a certain type of variability to the different conditions in which
they are grown (Bravo et al., 2001).
These results would indicate that cultivating P. lima under this type of mixotrophic conditions would
induce the production of high amounts of OA in a shorter cultivation time compared to traditional
autotrophic media. But the results could vary in some P. lima strains, as occurred in D008-1, which
synthesized a lower amount of OA compared to D008-5.
Although strain D008-1, when cultivated in PsMA, did not assimilate them in a similar way or obtain
high concentrations like D008-5, the results for each of the cultures were significantly similar (p ˃ 0.05)
throughout the test.
The lower OA production of strain D008-5 obtained in PsACH would be due to the lack of glucose in
the Ps of A chilensis, which does not occur in C. fragile and Ulva spp., (Matsuhiro & Urzúa, 1990;
Kaeffer et al., 1999; Yaich et al., 2014; Kolsi et al., 2017; Wang et al., 2020), a situation that could
indicate that glucose is an essential sugar within the metabolic pathways of this strain of P. lima to be
able to synthesize a greater amount of OA in culture. Another factor would be the sulfate groups that
are present in the Ps of C. fragile and Ulva spp., since they would provide a greater amount of sulfate
molecules available for the formation of sulfated diesters, which could subsequently be transformed to
diol-ester in a few minutes, as a precursor to OA (Quilliam & Ross, 1996; Suzuki et al., 2004).
According to Quilliam et al., (1996), these sulfated diesters would be a means of storing toxins within
the cell of dinoflagellates. Subsequently, Hu et al., (2017) point out a supposed hydrolysis of sulfated
diesters that would be carried out in two steps mediated by specific enzymes (esterases) of sulfated
diesters (such as DTX-4 and DTX-5), initially leading to the formation of diol esters and ultimately to
the release of free OA. This would favor the cellular integrity of P. lima, providing self-protection in
the main structure of the free acids of OA, through the insertion of oxygen in the extended side chain
of these sulfated dieters (Bravo et al., 2001), since the sulfated esters are essentially inactive against
phosphatases PP1 and PP2A.
What happened in these processes would occur in the mixotrophic cultures of P. lima, Ps of C. fragile
and Ulva spp., as observed in the present work. Furthermore, all of this will depend on each type of P.
lima strain, since each of them presents a certain type of variability to the different conditions in which
they are grown (Bravo et al., 2001).
pág. 3066
▪ Amount of DTX-1 per culture
The highest experimental amount of DTX-1 produced by strain D008-1 was obtained in the L1-Si
culture (13.77 ± 2.67 ng DTX-1) on day 18, while the lowest amount was produced in PsACH (7.16 ±
2.17 ng DTX-1) on day 15 (Table 1). Strain D008-5 obtained the highest experimental amount of DTX-
1 by culture in L1-Si (12.22 ± 1.41 ng DTX-1) on day 15, while the lowest was obtained in culture in
PsACH (8.22 ± 0.91 ng DTX-1) (p < 0.05), on day 18 (Table 1).
Both strains (D008-1 and D008-5) in all mixotrophic treatments with PsMA obtained low DTX-1
contents, with values that fluctuated between 0.0114 and 0.0297pg DTX-1 cell-1, compared to values
from studies with treatments autotrophic such as those of Wang et al., (2015) in f/2, Nishimura et al.,
(2020) in Daigo IMK medium or Wu et al., (2020) in f/2-Si, which recorded higher contents between
16.58 – 70.73 pg DTX-1 cell-¹. The values of DTX-1 compared to those of OA can have very significant
variations at the cellular level, which will depend on the different strains of P. lima that produce them.
Luo et al., (2017) isolated eight strains of P. lima in Beihai, China and one in Kending, Taiwan, which
when grown at f/2, only two presented DTX-1 (0.91 – 1.81 pg DTX-1 cell-¹), while this toxin was not
detected in six of them, although OA was detected in all strains (0.55 – 10.26 pg OA cell-¹). This
variability between the concentrations of OA and DTX-1 of strains D008-1 and D008-5 could probably
be due to the influence of environmental, physical, physiological and genetic factors, as pointed out by
Aquino-Cruz et al., (2018) and Niu et al., (2019).
Nascimento et al., (2005) when analyzing 20 strains isolated in the south of England from the Fleet
Dorset Lagoon, found that DTX-1 values fluctuated between 0.41 – 11.29 pg cell-1, while those of OA
were between 0.42 and 17.13 pg cell-1. Similarly, in the study by Wu et al., (2020) carried out in China,
of five strains analyzed, three presented a ratio of lower amounts of total DTX-1 with respect to total
OA (14.45 – 32.54 pg of OA cell-¹ and 0.69 – 4.40 pg of OA cell-¹); relationship similar to that obtained
in the present study for strain D008-5 (0.0298 pg DTX-1 cell-¹ and 11.95 pg of OA cell-¹). Studies such
as those by de Pan et al., (1999); Nascimento et al., (2016) and Pan et al., (2017), have shown that the
highest production of these toxins (OA and DTXs) are related to the growth phases of P. lima, where
the highest production of these polyketides occurs in the stationary phase.
▪ Amount of DTX-1 per culture
The highest experimental amount of DTX-1 produced by strain D008-1 was obtained in the L1-Si
culture (13.77 ± 2.67 ng DTX-1) on day 18, while the lowest amount was produced in PsACH (7.16 ±
2.17 ng DTX-1) on day 15 (Table 1). Strain D008-5 obtained the highest experimental amount of DTX-
1 by culture in L1-Si (12.22 ± 1.41 ng DTX-1) on day 15, while the lowest was obtained in culture in
PsACH (8.22 ± 0.91 ng DTX-1) (p < 0.05), on day 18 (Table 1).
Both strains (D008-1 and D008-5) in all mixotrophic treatments with PsMA obtained low DTX-1
contents, with values that fluctuated between 0.0114 and 0.0297pg DTX-1 cell-1, compared to values
from studies with treatments autotrophic such as those of Wang et al., (2015) in f/2, Nishimura et al.,
(2020) in Daigo IMK medium or Wu et al., (2020) in f/2-Si, which recorded higher contents between
16.58 – 70.73 pg DTX-1 cell-¹. The values of DTX-1 compared to those of OA can have very significant
variations at the cellular level, which will depend on the different strains of P. lima that produce them.
Luo et al., (2017) isolated eight strains of P. lima in Beihai, China and one in Kending, Taiwan, which
when grown at f/2, only two presented DTX-1 (0.91 – 1.81 pg DTX-1 cell-¹), while this toxin was not
detected in six of them, although OA was detected in all strains (0.55 – 10.26 pg OA cell-¹). This
variability between the concentrations of OA and DTX-1 of strains D008-1 and D008-5 could probably
be due to the influence of environmental, physical, physiological and genetic factors, as pointed out by
Aquino-Cruz et al., (2018) and Niu et al., (2019).
Nascimento et al., (2005) when analyzing 20 strains isolated in the south of England from the Fleet
Dorset Lagoon, found that DTX-1 values fluctuated between 0.41 – 11.29 pg cell-1, while those of OA
were between 0.42 and 17.13 pg cell-1. Similarly, in the study by Wu et al., (2020) carried out in China,
of five strains analyzed, three presented a ratio of lower amounts of total DTX-1 with respect to total
OA (14.45 – 32.54 pg of OA cell-¹ and 0.69 – 4.40 pg of OA cell-¹); relationship similar to that obtained
in the present study for strain D008-5 (0.0298 pg DTX-1 cell-¹ and 11.95 pg of OA cell-¹). Studies such
as those by de Pan et al., (1999); Nascimento et al., (2016) and Pan et al., (2017), have shown that the
highest production of these toxins (OA and DTXs) are related to the growth phases of P. lima, where
the highest production of these polyketides occurs in the stationary phase.
pág. 3067
However, their results differ from those obtained by Wu et al., (2020), who recorded the highest
concentrations of OA and DTX-1 before the stationary stage, and also differ from the results of the
present study in mixotrophic culture (polysaccharides), where the highest OA values were obtained
from the exponential phase, which began on day seven of culture for strain D008-5. These quantities
remained stable throughout the study with very high values in all mixotrophic media with PsCFR (11.95
pg of OA cell-¹ and 0.0298 pg DTX-1 cell-¹), compared to those obtained by Nascimento et al., (2005)
that were between 0.14 – 3.20 pg of OA cell-¹ in the L1-Si autotrophic medium.
The L1-Si medium with polysaccharides from intertidal macroalgae, developed in this work, presented
better results, both in growth of P. lima and in OA production, than autotrophic media. It could be
expected that this mixotrophic medium could further raise the OA concentrations of the different strains
of P. lima grown with autotrophic mediums and that have been mentioned in the present study.
Especially the strain presented by Nishimura et al., 2020 that presented high amounts of OA and DTX-
1 (13.39 and 55.27 pg cell-¹, respectively).
Figura 1
.
Fig. 1. Experimental (lines with markers) and theoretical (gray markers) growth of Prorocentrum lima strain D008-5 grown
in 100 mL of the media at 15, 50 and 100 mg L-1 of the polysaccharides of Agarophyton chilensis, Ulva spp. and Codium
fragile, in addition to L1-Si. Different letters below the curves indicate differences with the other theoretical treatments (p <
0.05) (n=3) throughout the culture.
However, their results differ from those obtained by Wu et al., (2020), who recorded the highest
concentrations of OA and DTX-1 before the stationary stage, and also differ from the results of the
present study in mixotrophic culture (polysaccharides), where the highest OA values were obtained
from the exponential phase, which began on day seven of culture for strain D008-5. These quantities
remained stable throughout the study with very high values in all mixotrophic media with PsCFR (11.95
pg of OA cell-¹ and 0.0298 pg DTX-1 cell-¹), compared to those obtained by Nascimento et al., (2005)
that were between 0.14 – 3.20 pg of OA cell-¹ in the L1-Si autotrophic medium.
The L1-Si medium with polysaccharides from intertidal macroalgae, developed in this work, presented
better results, both in growth of P. lima and in OA production, than autotrophic media. It could be
expected that this mixotrophic medium could further raise the OA concentrations of the different strains
of P. lima grown with autotrophic mediums and that have been mentioned in the present study.
Especially the strain presented by Nishimura et al., 2020 that presented high amounts of OA and DTX-
1 (13.39 and 55.27 pg cell-¹, respectively).
Figura 1
.
Fig. 1. Experimental (lines with markers) and theoretical (gray markers) growth of Prorocentrum lima strain D008-5 grown
in 100 mL of the media at 15, 50 and 100 mg L-1 of the polysaccharides of Agarophyton chilensis, Ulva spp. and Codium
fragile, in addition to L1-Si. Different letters below the curves indicate differences with the other theoretical treatments (p <
0.05) (n=3) throughout the culture.
pág. 3068
Table 1. Maximum quantities of experimental and theoretical toxins produced by strains D008-1 and
D008-5 of Prorocentrum lima in the 24 mL cultures of macroalgal polysaccharide media (50 mg L-1)
and in L1-Si. Different letters indicate differences with the other crops (p < 0.05) (n=3) throughout the
crop.
Strain Medium Experimental toxin (ng culture) Theoretical toxin (ng culture)
R2
OA DTX-1 OA DTX-1 OA DTX-1
PsULV 4,568.09 ± 270.64 a 7.35 ± 0.22 b 4,348.33 ± 154.15 a5.23 ± 0.10 b 0.9386 0.5078
D008-1 PsCFR 4,358.74 ± 1,028.57 a 8.03 ± 1.19 b 4,136.03 ± 397.56 a4.11 ± 0.60 c 0.9906 0.4825
PsACH 4,603.61 ± 1,585.24 a 7.16 ± 2.17 b 4,184.00 ± 106.72 a4.31 ± 0.30 bc 0.942 0.0419
L1-Si
5,829.24 ± 1,644.63 a 13.77 ± 2.67 a
3,821.33 ± 113.45
ab
7.60 ± 0.46 a 0.7108 0.8763
PsULV 5,529.76 ± 1,415.40 a 11.69 ± 2.86 a 5,271.19 ± 153.02 a8.62 ± 0.05 b 0.9675 0.7706
D008-5 PsCFR 4,769.63 ± 932.30 a 11.82 ± 0.70 a 4,565.38 ± 716.16 a7.80 ± 0.45 c 0.8020 0.4322
PsACH 4,160.36 ± 1,732.47 a 8.22 ± 0.91 b 3,944.33 ± 95.21 ab 5.31 ± 0.41 d 0.9166 0.9408
L1-Si 2,959.04 ± 356.61 ab 12.22 ± 1.41 a 2,693.78 ± 98.22 c 9.57 ± 0.37 a 0.8587 0.3481
CONCLUSION
The addition of polysaccharides extracted from macroalgae to traditional P. lima culture media provides
a source of organic carbon that stimulates the increase in its biomass, in addition to increasing the
synthesis of the diarrheal toxins okadaic acid and dinophysistoxin-1. Regarding the growth of P. lima
strains, the highest cell densities were obtained in media with C. fragile and Ulva sp. polysaccharides
for D008-1, and in A. chilensis and Ulva sp. for D008-5. The lowest cell densities for both strains were
obtained in the autotrophic medium L1-Si. However, the high production of these diarrheal toxins
depends on the P. lima strain cultivated and the macroalgae species used to extract the polysaccharides.
It was found that strain D008-5, when grown in media with the polysaccharides of Ulva sp. and C.
fragile, obtained a high production of OA per culture, although in strain D008-1 it was higher in the Li-
Si culture. Synthesis of the diarrheal toxin DTX-1 was higher in strain D008-1 in L1-Si media, with
strain D008-5 showing increased production in media containing Ulva sp. and C. fragile
polysaccharides, although there were no significant differences with the Li-Si medium. It is expected
that mixotrophic cultures with macroalgal polysaccharides could increase OA concentrations in any
strain that produces high amounts of these toxins, and even with other macroalgal polysaccharides.
Table 1. Maximum quantities of experimental and theoretical toxins produced by strains D008-1 and
D008-5 of Prorocentrum lima in the 24 mL cultures of macroalgal polysaccharide media (50 mg L-1)
and in L1-Si. Different letters indicate differences with the other crops (p < 0.05) (n=3) throughout the
crop.
Strain Medium Experimental toxin (ng culture) Theoretical toxin (ng culture)
R2
OA DTX-1 OA DTX-1 OA DTX-1
PsULV 4,568.09 ± 270.64 a 7.35 ± 0.22 b 4,348.33 ± 154.15 a5.23 ± 0.10 b 0.9386 0.5078
D008-1 PsCFR 4,358.74 ± 1,028.57 a 8.03 ± 1.19 b 4,136.03 ± 397.56 a4.11 ± 0.60 c 0.9906 0.4825
PsACH 4,603.61 ± 1,585.24 a 7.16 ± 2.17 b 4,184.00 ± 106.72 a4.31 ± 0.30 bc 0.942 0.0419
L1-Si
5,829.24 ± 1,644.63 a 13.77 ± 2.67 a
3,821.33 ± 113.45
ab
7.60 ± 0.46 a 0.7108 0.8763
PsULV 5,529.76 ± 1,415.40 a 11.69 ± 2.86 a 5,271.19 ± 153.02 a8.62 ± 0.05 b 0.9675 0.7706
D008-5 PsCFR 4,769.63 ± 932.30 a 11.82 ± 0.70 a 4,565.38 ± 716.16 a7.80 ± 0.45 c 0.8020 0.4322
PsACH 4,160.36 ± 1,732.47 a 8.22 ± 0.91 b 3,944.33 ± 95.21 ab 5.31 ± 0.41 d 0.9166 0.9408
L1-Si 2,959.04 ± 356.61 ab 12.22 ± 1.41 a 2,693.78 ± 98.22 c 9.57 ± 0.37 a 0.8587 0.3481
CONCLUSION
The addition of polysaccharides extracted from macroalgae to traditional P. lima culture media provides
a source of organic carbon that stimulates the increase in its biomass, in addition to increasing the
synthesis of the diarrheal toxins okadaic acid and dinophysistoxin-1. Regarding the growth of P. lima
strains, the highest cell densities were obtained in media with C. fragile and Ulva sp. polysaccharides
for D008-1, and in A. chilensis and Ulva sp. for D008-5. The lowest cell densities for both strains were
obtained in the autotrophic medium L1-Si. However, the high production of these diarrheal toxins
depends on the P. lima strain cultivated and the macroalgae species used to extract the polysaccharides.
It was found that strain D008-5, when grown in media with the polysaccharides of Ulva sp. and C.
fragile, obtained a high production of OA per culture, although in strain D008-1 it was higher in the Li-
Si culture. Synthesis of the diarrheal toxin DTX-1 was higher in strain D008-1 in L1-Si media, with
strain D008-5 showing increased production in media containing Ulva sp. and C. fragile
polysaccharides, although there were no significant differences with the Li-Si medium. It is expected
that mixotrophic cultures with macroalgal polysaccharides could increase OA concentrations in any
strain that produces high amounts of these toxins, and even with other macroalgal polysaccharides.
pág. 3069
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