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Growth kinetics and fucoxanthin production of


Phaeodactylum tricornutum and Isochrysis
galbana cultures at different light and agitation
conditions
ARTICLE in JOURNAL OF APPLIED PHYCOLOGY JANUARY 2015
Impact Factor: 2.49 DOI: 10.1007/s10811-015-0635-0

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J Appl Phycol
DOI 10.1007/s10811-015-0635-0

Growth kinetics and fucoxanthin production of Phaeodactylum


tricornutum and Isochrysis galbana cultures at different light
and agitation conditions
Alma Gmez-Loredo 1 & Jorge Benavides 1 &
Marco Rito-Palomares 1

Received: 15 December 2014 / Revised and accepted: 24 May 2015


# Springer Science+Business Media Dordrecht 2015

Abstract Fucoxanthin is a carotenoid that exerts multiple


beneficial effects on human health. However, reports comparing microalgae culture conditions and their effect on growth
and fucoxanthin production are still limited. Isochrysis
galbana and Phaeodactylum tricornutum cultures in different
light (62.0, 25.9, 13.5, or 9.1 mol photons m-2 s-1), mixing
conditions (1 vvm aeration or 130 rpm agitation), and media
compositions (F/2 and Conway medium) were studied for
comparison of cellular growth and fucoxanthin production
on F/2 medium. I. galbana showed a better adaptation to
tested culture conditions in comparison with P. tricornutum,
reaching 2.15 107 4.07 106 cells mL-1 and a specific
growth rate () of 1.120.05 day-1 under aerated conditions
and 62.0 mol photons m-2 s-1 light intensity. Fucoxanthin
concentration was about 25 % higher in P. tricornutum cultures under 13.5 mol photons m-2 s-1 light intensity and aerated conditions, but the highest fucoxanthin total production
was higher in I. galbana, where 3.32 mg can be obtained from
1 L batch cultures at the 16th day under these conditions.
Moreover, higher cell densities (~32.41 %), fucoxanthin concentration (~42.46 %), and total production (~50.68 %) were
observed in I. galbana cultures grown in Conway medium, if
compared with cultures grown in F/2 medium. The results
show that the best growth conditions did not result in the best
fucoxanthin production for either microalgae, implying that
there is not a direct relationship between cellular growth and
fucoxanthin production. Moreover, the results suggest that
* Marco Rito-Palomares
mrito@itesm.mx
1

Centro de Biotecnologa-FEMSA, Tecnolgico de Monterrey,


Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur,
Monterrey, NL 64849, Mexico

I. galbana cultures on Conway medium are strong candidates


for fucoxanthin production, where 1.2 to 15 times higher fucoxanthin concentration are observed in comparison to
macroalgal sources.
Keywords Microalgae . Growth kinetics . Isochrysis
galbana . Phaeodactylum tricornutum . Fucoxanthin

Introduction
Fucoxanthin is an allenic carotenoid that has shown important
nutraceutical bioactivity, exerting antioxidant, anti-cancer, anti-diabetic, anti-obesity, anti-photoaging, anti-angiogenic, and
anti-metastatic effects (Miyashita et al. 2011; DOrazio et al.
2012; Chung et al. 2013) on a variety of biological models.
This carotenoid has been proven to be safe for animal consumption (DOrazio et al. 2012), opening up the opportunity
of using this bioactive compound in the treatment of different
pathologies. Moreover, the carotenoid can be separated and
purified through a series of novel separation technologies such
as ethanol-phosphate aqueous two-phase systems (GmezLoredo et al. 2014a) followed by ultrafiltration (GmezLoredo et al. 2014b), where the use of chromatographic steps
is avoided.
Fucoxanthin is naturally found in marine organisms, like
brown algae (Miyashita et al. 2011) and microalgae, where it
is coupled to the thylakoid membrane to transfer excitation
energy to the photosynthetic electron transport chain via chlorophyll a (Jin et al. 2003; Mulders et al. 2014). Fucoxanthin
concentration ranges from 2.24 to 18.23 mg Fucoxanthin
gDry Weight-1 in microalgae, which is one to three orders of
magnitude greater than that found in macroalgae (Xia et al.
2013). Two of the main microalgal producers of fucoxanthin

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are Phaeodactylum tricornutum and Isochrysis galbana,


where concentrations up to 18.23 mgFucoxanthin gDry Weight-1
have been reported for I. galbana (Kim et al. 2012a, b).
Since wild microalgae and macroalgae harvesting may
have a negative environmental impact due to overexploitation
of these natural sources, it is expected that carotenoid extraction from cultured organisms will have a major role to play in
the coming years (Murray et al. 2013). Although both
microalgae and macroalgae can be grown in controlled environments, carotenoid production in macroalgae cultures is still
not commercially feasible. The methods employed in seaweed
tissue culture involve axenic cell culture techniques enriched
with nutrients and growth regulators for explant generation,
which generally yields small and slow growing calli (Reddy
et al. 2008) and can lead to low production yields. On the other
hand, microalgae are organisms that have simpler growth requirements. Moreover, due to their simpler structure, energy is
directed into photosynthesis, growth and reproduction processes instead of the maintenance of differentiated structures
(Walker et al. 2005), making microalgae organisms of interest
for production of biomass and bioactive compounds.
Carotenoid composition in microalgae depends greatly on
environmental factors such as temperature, pH, nutrient concentration, light intensity, cell density, aeration, and physiological state of the culture (Klein 1988; Durmaz et al. 2008;
Murray et al. 2013). Light is the primary source of energy that
drives all biochemical processes in microalgae, but it can also
cause damage when present in excessive quantities (Mulders
et al. 2014). On the other hand, agitation during microalgae
culture is needed to promote an adequate light-dark cycle regime, to increase oxygen mass transfer, and to help avoid
detrimental conditions such as cell sedimentation or accumulation of oxygen in the medium (Brindley Alas et al. 2004).
Since light and agitation are important process parameters for
culture growth, it is important to determine the optimal parameters for cellular growth and fucoxanthin production in
microalgae. However, no studies so far have investigated the
simultaneous effect of light intensity and mixing conditions on
both cellular growth and fucoxanthin production in
microalgae. The objective of the present study was to evaluate
culture growth of P. tricornutum and I. galbana under different growth conditions (light intensity and mixing/agitation)
and medium compositions to determine optimal conditions
for the production of fucoxanthin.

Materials and methods


Chemicals Analytical grade solvents and chemicals such
as ethanol, hexane, hydrochloric acid, potassium hydroxide, ammonium acetate, BD Bioxon agar, and salts for
media compositions were from DEQ (Monterrey,
Mexico). Fucoxanthin commercial standard was from

Sigma-Aldrich (USA). Deionized water was used for all


experiments, and HPLC-grade solvents such as water,
methanol, acetonitrile, and ethyl acetate were purchased
from VWR (Symphony, PA, USA).

Biological material
Isochrysis galbana (ISG-1) and Phaeodactylum tricornutum
(PHT-1) were acquired from Centro de Investigaciones
Biolgicas del Noroeste, S.C. (CIBNOR, Mexico) and maintained in F/2 medium (Guillard and Ryther 1962; Guillard
1975) as previously reported (Boczar and Prezelin 1989;
Suzuki et al. 1993; Lavaud et al. 2003). For cultivation, pH
was adjusted to 7.3 for I. galbana and 7.6 for P. tricornutum in
both solid and liquid media.
Microalgae pre-inoculum, inoculum, and cultivation
conditions In accordance to previous experiences of our
research group, solid media cultures (pre-inoculum) were
maintained for 6 days in F/2 solid medium (BD Bioxon
agar, 15 % strength) at 50 mol photons m-2 s-1 light
intensity and 25 C. Biomass was scraped from the solid
medium and transferred to 250 mL Erlenmeyer flasks
with 100 mL of F/2 liquid medium to grow the inoculum
for 4 days. Inoculum was kept at 283 C under continuous shaking (130 rpm, 19.0 mm amplitude) and continuous light (40 mol photons m-2 s-1) provided by 39 W
cold light lamps (Phillips, Netherlands). For preparation
of experimental cultures, 1 L of culture medium was inoculated to have a concentration of 5.00104 cells mL-1
in 2 L flasks, and cultivated for 18 days at 283 C under
aeration or passive agitation. Aeration (1 vvm) was done
through a 29-cm air curtain (Hagen Elite, Canada), while
orbital agitation was at 130 rpm on a 19.0-mm amplitude
orbital shaker. The different incident irradiances tested
(9.1, 13.5, 25.9, or 62.0 mol photons m-2 s-1 continuous
light intensity) were achieved by varying the number of
lamps turned on or off, and the light intensity was quantified with a light meter (LI-COR Biosciences, USA). In
this way, the effect of aeration and light intensity on the
cell growth kinetics, fucoxanthin concentration, and fucoxanthin productivity of both microalgae species was evaluated. In order to determine if an increase of fucoxanthin
can be achieved through a change in medium composition, further experiments were carried out only on the
microalgae species and conditions that provided the
highest total production. Cell growth and fucoxanthin production of I. galbana was characterized in F/2 and
Conway (or Walnes) medium (Walne 1970). All media
compositions used in the present study were prepared
with artificial seawater. The composition of the two media
is presented in Table 1.

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Table 1 Chemical composition
of Conway (Walnes) medium
and F/2 medium

Conway (Walnes) medium

F/2 medium

Nutrient

Molar concentration

Nutrient

Molar concentration

NaH2PO4

1.2310-4

NaH2PO4

3.1310-5

NaNO3
Na2SiO3 9H2O
ZnSO4 7H2O
MnSO4 H2O
Na2MoO4 2H2O
CoSO4 7H2O
CuCl2 2H2O
Fe(NH4)2(SO4)2 6H2O
Na2EDTA2H2O
B12
B1
Biotin

8.8210-4
1.0610-4
1.6010-7
1.8010-6
6.0310-8
9.9610-8
7.9810-8
5.3410-6
1.6810-5
9.9710-7
1.6410-6
1.0210-7

NaNO3
Na2SiO3 9H20
ZnCl2
MnCl2 4H2O
(NH4)Mo7O24 4H2O
CoCl2 6H20
CuSO4 5H2O
FeCl3 H2O
Na2EDTA2H2O
B12
B1
Biotin

-3

1.1810
4.7110-4
1.5310-7
1.6010-6
7.2810-9
8.4110-8
8.0110-8
4.8110-6
1.2110-4
7.3910-8
2.9710-7

Conway and F/2 medium were prepared in artificial sea water solution which comprised 0.485 mM H3BO3,
2.381 mM NaHCO3, 0.840 mM KBr, 10.466 mM CaCl2, 54.616 mM MgCl2, 9.390 mM KCl, 0.420 M NaCl,
and 0.029 M Na2SO4)

Growth kinetics In order to monitor culture growth, culture


sampling was done every 2 days for 18 days. Cell counts were
performed using a hemocytometer (Hausser Scientific, USA).
The specific growth rate () of the linear growth phase was
calculated from Eq. 1, where t is the culture time (in days) after
inoculation, and N0 and Nt are the cell densities after inoculation and after t days, respectively.
!
LnN t=N 0

1
t
Reynolds (Re) number calculation For Reynolds (Re) number calculation, the culture was seen as an agitated (130 rpm
rotational speed, N) vessel of diameter (D) without baffles
(Eq. 2) as reported by Bchs et al (2000). Densities and dynamic viscosities were calculated with F/2 medium without biomass
at 25, 28, and 31 C. Density () was obtained by experimental
weighting 1 mL of medium on an analytical balance. Dynamic
viscosity () was obtained in an Ostwald viscometer and Eq. 3,
taking as reference distilled water at the desired medium temperature. Time (t) required for the test liquid to flow through a
capillary of a known diameter between the two marked points
of the viscometer was measured through a timer.
N D2

t medium
medium medium

water t water water


Re

2
3

Fucoxanthin extraction Fucoxanthin extraction was performed in accordance to the optimal conditions previously
reported (Gmez-Loredo et al. 2014a). Briefly, culture

samples (2030 mL) were taken on day 8 and 16 of


growth, and the biomass harvested by centrifugation
(30 min, 4 C, 2400 g). Ethanol was added in a 2.5
and 5 % weight volume ratio for I. galbana and
P. tricornutum biomass, respectively. Extraction was performed in orbital agitation at 250 rpm for 1 h at 20 C.
Depleted biomass was removed by centrifugation (25 min,
4 C, 7000g) and filtration with 0.2 m acrodisc syringe
filters. In order to remove lipophilic compounds that
could interact with the analysis, the extract was
partitioned in a separation funnel with 1 volume of hexane. Subsequently, the alcoholic phase (containing the fucoxanthin) was separated for quantification. During
growth media comparison experiments, samples on day
4, 8, 12, and 16 were taken for fucoxanthin extraction,
conducted as previously described. In order to avoid oxygen or light-induced degradation of fucoxanthin, all extraction procedures were performed under dimmed light
and nitrogen headspace.
Analytical methods Fucoxanthin concentration and total
production values are reported on a fresh weight (fresh biomass) basis. Fucoxanthin concentration is defined as the mg of
fucoxanthin contained per gram of fresh biomass, and is reported as mgFucoxanthin gFresh Biomass-1. Likewise, fucoxanthin
total production is defined as the fucoxanthin production obtained from 1.0 L batch cultures, and is reported as
mgFucoxanthin batch-1.
A purified fucoxanthin standard was used for quantification and identification of fucoxanthin in the alcoholic phase by
means of a calibration curve and comparison of retention time

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and ultraviolet-visible absorption spectra, respectively.


Total fucoxanthin concentration was analyzed by HPLCPDA (1200 Series, Agilent Technologies, USA).
Chromatograms were obtained after injection of 10 l of
sample, and detection was done at 448 nm. Separation
was performed in a COGENT C30 column (2504.6 mm
i.d. with a 5-m particle size; MicroSolv, USA) with
1 mL min-1 flow. The mobile phase used was (A) 80 %
methanol, 20 % 0.5 M ammonium acetate, (B) 90 % acetonitrile, and (C) 100 % ethyl acetate. Separation was
achieved through a series of linear gradients as previously
reported (Gmez-Loredo et al. 2014a).

Results
Growth kinetics of I. galbana and P. tricornutum cultures
in F/2 medium

Statistical analysis All experiments were performed at


least in triplicate, and results were expressed as the
meanstandard error. Data was analyzed by analysis of
variation (ANOVA) procedures, and differences among
means were compared using Tukey test with a level of
significance of p< 0.05. JMP 11 software was used for
all statistical analyses.

Cultures subjected to orbital agitation showed lower cell densities than aerated cultures at any light intensity tested for
I. galbana (Fig. 1a) and P. tricornutum (Fig. 1b). However,
no cell growth was observed for P. tricornutum cultures under
orbital agitation at the highest light intensities tested (62.0 and
25.9 mol photons m-2 s-1). For both microalgae species, a
light intensity of 62.0 mol photons m-2 s-1 and aerated conditions resulted in the highest cell densities. Linear growth
phase in all aerated cultures started at day 4 and lasted for 8
to 10 days. All cultures under orbital agitation took 2 more
days to enter the linear growth phase, which lasted for 6 to
8 days. Moreover, aerated cultures under all light intensities
presented a wave-like behavior in some points of the growth
culture, starting as early as day 8 on P. tricornutum cultures
under 13.5 mol photons m-2 s-1 light intensity, or as late as

Fig. 1 Growth curves of Isochrysis galbana (a) and Phaeodactylum


tricornutum (b) batch cultures under different continuous light intensity
and agitation conditions on F/2 medium. Results are the average of a
triplicated experiment. Growth curves with aeration,
); aeration, 25.9 mol photons m62.0 mol photons m-2 s-1 (
2 -1
s (
); aeration, 13.5 mol photons m-2 s-1 (
); aeration,

); orbital agitation,
9.1 mol photons m-2 s-1 (
); orbital agitation,
62.0 mol photons m-2 s-1 (
); orbital agitation,
25.9 mol photons m-2 s-1 (
); and orbital agitation,
13.5 mol photons m - 2 s - 1 (
) conditions are presented. Error bars
9.1 mol photons m-2 s-1 (
show standard error of n=3 replicates

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day 12 on I. galbana cultures under 25.9 mol photons m-2 s-1


light intensity.
As expected from the growth curves in Fig. 1, greater and
cellular densities were achieved by I. galbana cultures
(Table 2). Moreover, aerated cultures for each microalgae species showed the best and cell density at high light intensity
conditions, while the worst culture conditions were presented
in orbital agitation and high light intensity. According to the
experimental measurements, turbulent flow was achieved on
cultures under orbital agitation (Table 3), where 52,800<Re>
74,700 values were observed for the temperatures and vessel
diameters tested.
Fucoxanthin production of I. galbana and P. tricornutum
cultures in F/2 medium
The culture conditions that rendered the highest cell densities
were not the same conditions where the highest fucoxanthin
concentration was observed. Contrary to cell growth, the
highest values for fucoxanthin concentration in I. galbana
cultures were obtained in aerated conditions under mediumlow light intensity (13.5 mol photons m-2 s-1). Under such
conditions, 0.14 mgFucoxanthin gFresh Biomass-1 was produced on
the 16th day of growth (Fig. 2), but said fucoxanthin concentration was not significantly different from the one obtained in
the majority of the tested conditions in both microalgae species. However, P. tricornutum cultures provided the highest
concentration of fucoxanthin (about 0.2 mg Fucoxanthin
gFresh Biomass-1) on day 8 of cultivation in the same light and
agitation conditions (Fig. 2).
Although P. tricornutum had the highest fucoxanthin concentration (about 0.2 mg Fucoxanthin gFresh Biomass -1), this
microalgae achieved lower cell densities, and usually presents

higher cell size (10 to 20 m) if compared to I. galbana (5 m).


For that reason, it is necessary to take into account the total
fucoxanthin production in the batch culture in order to compare
the performance of both microalgae species. It is clear from
Fig. 3 that the total fucoxanthin production for I. galbana is
higher than for P. tricornutum. Again, aeration and mediumlow light intensity conditions (13.5 mol photons m-2 s-1) provided the highest production of fucoxanthin for both
microalgae species. According to this information, I. galbana
biomass should be harvested at day 16 of growth in order to
obtain the highest fucoxanthin production in practically all light
intensities and aeration conditions (except for cultures with
62 mol photons m-2 s-1). On the other hand, P. tricornutum
biomass harvesting time seems to vary in accordance to mixing
conditions, since aerated cultures had greater fucoxanthin production on day 8, and cultures grown in orbital agitation had a
higher production on day 16.
Comparison of I. galbana growth and fucoxanthin
production in F/2 and Conway medium
Culture media can have an impact on both cell density
and production of metabolites in microalgae. Since
I. galbana showed higher fucoxanthin total production,
further study on the impact of media composition in fucoxanthin production was investigated in 1 L batch cultures under
13.5 mol photons m -2 s -1 light intensity and both
aerated and orbital agitation conditions. For said study, F/2
and Conway (or Walnes) medium were used as described in
Table 1. It was observed that growth of I. galbana cultures in
F/2 and Conway medium is very similar for every agitation
condition on the first 10 days of culture (Fig. 4). Moreover,
aerated conditions provided better conditions for cell growth

Table 2 Specific growth rate () during the linear growth phase and maximum cell density on the stationary phase observed on Isochrysis galbana and
Phaeodactylum tricornutum batch cultures in F/2 medium
Culture parameter

Isochrysis galbana

Phaeodactylum tricornutum

Agitation

Light intensity
(mol photons m-2 s-1)

(day-1)

Maximum cell density


(107 cells mL-1)

(day-1)

Maximum cell density


(107 cells mL-1)

Aeration
Aeration
Aeration

9.1
13.5
25.9

0.9170.019b
0.7160.062c
1.0810.023a

1.010.22b
1.880.16a
1.430.31a

0.6530.016b,c
0.8350.023a,b
0.4870.068d

0.990.13c
1.330.09a,b
0.190.16d

Aeration
Orbital
Orbital
Orbital
Orbital

62.0
9.1
13.5
25.9
62.0

1.1220.050a
0.5940.003c,d
0.5330.017d
0.5880.005c,d
0.4920.016d

2.150.41a
0.280.02d
0.250.06d
0.410.04 c
0.340.06d

1.0110.030a
0.4930.026c
0.5570.029c
ng
ng

1.710.03a
0.680.07c
1.230.10b
ng
ng

Levels in the same column with different superscript letter are significantly different (p<0.05)
Valuesstandard error of n=3 replicates
ng no growth

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Table 3 Reynolds number (Re),
density (), and dynamic
viscosity () determinations for
F/2 medium at 25, 28, and 31 C
at different vessel diameters (D)

Temperature (C)

(kg m-3)

(kg m-1 s-1)

D (cm)

Reynolds number

31

1041.41.4

8.2710-4 3.5110-6

28

1038.02.8

8.6610-4 2.0610-6

0.15
0.16
0.17
0.15
0.16

61,048.46
69,090.61
74,728.40
58,107.90
65,762.68

0.17
0.15
0.16
0.17

71,128.91
52,854.83
59,817.60
64,698.71

25

-4

1034.80.2

-6

9.4910 2.4610

Valuesstandard error of n=3 replicates

in both media compositions, since longer linear growth phases


were observed under said conditions (4 days longer than cultures in orbital agitation). However, from day 12 of growth,
aerated cultures in Conway medium present about 13.43 %
higher cell densities, and up to 18.126 gfresh biomass L-1 on day
16 of growth. As shown in Table 4, the maximum cell densities presented in stationary phase are statistically different between agitation conditions, but are not different if comparing
media composition. Regarding , no differences were found
between cultures.
Higher fucoxanthin concentrations were obtained in aerated
cultures at day 8 of culture in both media cultures (Fig. 5), but
Conway medium provided a significantly greater fucoxanthin
concentration (0.2960.033 mgFucoxanthin gFresh Biomass-1) if
compared to the concentration obtained in F/2 medium
(0.1710.042 mgFucoxanthin gFresh biomass-1). Although fucoxanthin concentration was not the highest on day 16 of cultivation,
total fucoxanthin production was greater on the 16th day due to
Fig. 2 Fucoxanthin
concentration on Isochrysis
galbana and Phaeodactylum
tricornutum batch cultures on F/2
medium. Results are the average
of a triplicated experiment and are
expressed wet weight basis.
Product concentration is shown
for cultures agitated by aeration
( ) or by orbital agitation ( ).
Error bars show standard error of
n=3 replicates

high cell densities presented on aerated cultures (Fig. 6). In this


sense, biomass harvesting on day 16 of growth (during stationary phase of growth) provided higher fucoxanthin total production in either media, where cultures grown in F/2 medium
(1.5890.048 mgFucoxanthin batch-1) produced about 50.68 %
less fucoxanthin than cultures grown in Conway medium.

Discussion
Growth kinetics of I. galbana and P. tricornutum cultures
in F/2 medium Two main process parameters were studied
for cultivation of microalgae in F/2 medium: light intensity
within the culture vessel and agitation. Light intensity inside
the cultures is affected by the light scattering effects of the
biomass (mutual cell shading) and by the intracellular pigments that absorb light. Such conditions lower the light intensity inside the culture, and most importantly, affect the growth

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Fig. 3 Total fucoxanthin
production in 1 L Isochrysis
galbana and Phaeodactylum
tricornutum batch cultures on F/2
medium. Results are the average
of a triplicated experiment and are
expressed wet weight basis.
Fucoxanthin production is shown
for cultures agitated by aeration
( ) or by orbital agitation ( ).
Error bars show standard error of
n=3 replicates

of cultured cells (Rosello Sastre et al. 2007). For I. galbana


growth, high light intensities (200 to 280 mol photons m-2 s1
) have been shown to provide cell densities up to 2.12
107 cells mL-1 (Sun and Wang 2009), which is similar to the
values found at a 62.0 mol photons m-2 s-1 light intensity.
This goes in accordance with other reports, where the resulting

Fig. 4 Growth curves of Isochrysis galbana batch cultures under


different m e di a composi tio n a nd agita tion conditi ons a t
13.5 mol photons m-2 s-1. Results are the average of a triplicated
); aeration,
experiment. Growth curves in aeration, F/2 medium (
); orbital agitation, F/2 medium (
); and
Conway medium (
) conditions are presented.
orbital agitation, Conway medium (
Error bars show standard error of n=3 replicates

was not significantly different in a wide range of light intensities (25 to 200 mol photons m-2 s-1) (Alkhamis and Qin
2013). However, said study was performed at a temperature of
24 C in F/2 enriched with organic carbon, providing a 12
times higher than the one found in our study. With this information along with our findings, it can be inferred that further
increasing light intensity values would not render a significant
increase to I. galbana cell density.
P. tricornutum cell growth is reduced as light intensity decreases from 72 to 18 mol photons m-2 s-1 during the exponential phase (Kaixian and Borowitzka 1993), which is in
accordance with our findings in Fig. 1b. However,
P. tricornutum has efficient photoprotective mechanisms to
use excess energy available in light conditions up to
500 mol photons m-2 s-1 (Nymark et al. 2009), but in our
study, the microalgae did not grow in cultures under the
highest light intensities (25.9 and 62.0 mol photons m-2 s1
) and under 130 rpm orbital agitation. With this information,
it can be implied that P. tricornutum growth on high light
intensities is dependent on the agitation parameter, where aeration of culture is needed in order to obtain a growing culture
at the highest light intensities.
Light intensity is a source of energy that drives biochemical
processes in microalgae, particularly photosynthesis. Thus, it
would be expected that phototropic organisms would generate
more biomass as light intensity increases. I. galbana and
P. tricornutum showed a different behavior in cell growth at
different light intensities, where P. tricornutum showed lower
light adaptation since cell growth on orbital agitation was not
possible under high light intensities; while I. galbana cultures
showed no significant increment in cell densities and . The
difference in the results could be caused by the different
photoadaptation efficiencies on the studied microalgae species, which seem to be higher on I. galbana cultures. Said

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Table 4 Specific growth rate () during the linear growth phase, maximum biomass generated and maximum cell density on the stationary phase
observed on Isochrysis galbana batch cultures in F/2 and Conway media grown at 13.5 mol photons m-2 s-1 light intensity
Agitation

Medium

(day-1)

Maximum biomass generated (g L-1)

Maximum cell density (107 cells mL-1)

Aeration
Orbital
Aeration
Orbital

F/2
F/2
Conway
Conway

0.7260.039a
0.6410.010a
0.7400.035a
0.6650.021a

10.8771.910a,b
8.96181.4375c
18.1263.2292a
8.38971.0565b,c

1.140.20a,b
0.580.06b
1.690.18a
0.810.00b

Levels in the same column with different superscript letter are significantly different (p<0.05)
Valuesstandard error of n=3 replicates

adaptation responses include the synthesis or degradation of


light harvesting complexes that help to balance the absorption
of excitation energy for growth and cell maintenance (Torzillo
et al 2003); therefore, a synthesis of harvesting complexes
would be beneficial for biomass and pigment (fucoxanthin)
production.
As a part of the study, two different mixing mechanisms
were explored, where orbital agitation rendered the lower cell
densities. Orbital agitation generated a turbulent flow
(Table 3) with Re numbers that were higher than those reported for stirred vessels without baffles (Re<50,000) (Bchs et al
2000). The differences between the values previously reported
could be due to the higher rotation frequency (130 min-1)
employed in our study, as well as the density of culture
media (1038.131.45 kg m-3) since the referred study used
demineralized water as sample. Turbulent flow provided by
orbital agitation might be one of the causes behind the lower
I. galbana growth at all light conditions, while P. tricornutum
growth was only achieved under low light conditions under
the same agitation regime. However, P. tricornutum growth
was similar under both agitation regimes (aeration and orbital
agitation) at low light conditions (Table 2), so orbital agitation
can come in handy as a substitute of aerated conditions for
agitation. This is of importance since agitation by aeration is
Fig. 5 Fucoxanthin
concentration in Isochrysis
galbana batch cultures under
different media composition and
agitation conditions at
13.5 mol photons m-2 s-1.
Results are the average of a
triplicated experiment and are
expressed wet weight basis.
Product concentration is shown
for cultures agitated by aeration
( ) or by orbital agitation ( ).
Error bars show standard error of
n=3 replicates

detrimental on P. tricornutum growth, where a greater than 2vvm aeration rate had a negative effect on cultures grown in
narrow vessels (D/H=0.2) (Brindley Alas et al. 2004).
On the other hand, aeration caused a higher (but not
significantly different) in cultures under 62 and
25.9 mol photons m-2 s-1 light intensities, while lower intensities led to significantly smaller (Table 2). A flow rate
intensity of 2.5 vvm resulted in higher I. galbana cell densities
than that at 0.75-vvm. (Molina Grima et al. 1992). Also, flow
rate intensities greater than 2.5 vvm did not significantly increase cell growth rate at 22 mol photons m-2 s-1 light intensity (Molina Grima et al. 1992), suggesting that an increase in
aeration rate would not be necessary for enhancing cellular
densities. In the same study, a comparison of growth under
different light intensities was also provided. At 2.5 vvm airflow, the linear growth rate at 22 mol photons m-2 s-1 was
higher in comparison to the rate presented at 11.2, 24.8, and
43.6 mol photons m-2 s-1. The differences between Molina
Grima et al (1992) the results and the ones presented in our
study might emerge from the aeration intensity used, since a
different aeration regime (1 vvm) was employed in our experiments. These observations suggest that under a specific range
of air flow conditions, the maximum cellular density might be
obtained using different light intensities. Although several

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J Appl Phycol
Fig. 6 Total fucoxanthin
production in 1 L Isochrysis
galbana batch cultures under
different media composition and
agitation conditions at
13.5 mol photons m-2 s-1.
Results are the average of a
triplicated experiment and are
expressed wet weight basis.
Fucoxanthin production is shown
for cultures agitated by aeration
( ) or by orbital agitation ( ).
Error bars show standard error of
n=3 replicates

light intensities have been proven to have a positive effect


on I. galbana cellular growth, it can be inferred that a range
from 22 to 65 mol photons m-2 s-1 should produce high
cellular densities on aerated (0.75 to 2.0 vvm) batch
cultures.
Agitation is necessary to prevent cell sedimentation and to
enhance gas exchange. This parameter permits the cells to
follow with the medium and experience permanent changes
in environmental conditions, principally light conditions.
However, agitation can generate high shear stress that inhibits
either growth or product generation (Rosello Sastre et al.
2007). Shear stress due to orbital agitation in combination
with high light intensities showed to produce low cell densities on the studied microalgae. This could be due to a possible
combination of overexposure of the microalgae cells to light.
On the other hand, P. tricornutum aerated cultures could have
experienced a similar high shear, where the air flow may have
lead to injury of cells through bubble breakup at the liquid-gas
interface, which is a common mechanism of cell damage in
sparged cultures (Carvalho et al 2006).
Despite light and mixing conditions, another cause of low
cell densities and (Table 2) observed in the present study
might be the carbon limitation conditions of the experimental
cultures. In cultures mixed by orbital agitation, the only carbon source was HCO3- obtained from the NaHCO3- component of artificial seawater media. On the other hand, aerated
cultures could have experienced a higher input of carbon
through media composition and CO2 present in the air that
was bubbled through the culture. However, atmospheric
CO 2 alone might not be sufficient for enhancing cell
production as observed by Sutherland et al (2015) in algal
ponds, where CO2 enrichment was necessary for maintaining
pH balance and increasing light absorption and biomass production. P. tricornutum cultures have also been proven to approximately double biomass production under 2 % CO2 incubation (Peng et al 2014). On the other hand, I. galbana

cultures have also been shown to increase culture cell density


with increasing concentrations of inorganic carbon (Clark et al
1999). This information suggests that the cell densities observed in the present study could be further incremented with
the addition of carbon sources. However, in order to obtain
cost-effective production of algae, CO2 supply should be done
through waste industrial streams, which are low-cost carbon
sources that are not always available due to transfer and use
constrains (Christenson and Sims 2011). Moreover, largescale production designs such as raceway ponds and some
closed reactors present poor mixing, inefficient use of CO2
supply, or adverse CO2 gradients, which could hinder biomass
production (Mata et al 2010).
There are a number of additional factors that can decrease
cell density on microalgal cultures, being temperature, CO2
transport, O2 excess, pH changes, and nutrient consumption
(Mata et al 2010) uncontrolled parameters that must have
changed over culture time in our study. Since no quantitative
data were obtained for any of said parameters, it cannot be
defined if the change of one of those parameters caused decreases of cell density at any point of cultivation. However, an
unnoticed change in temperature, pH, and carbon depletion
could be the reason behind the wave-like fluctuations observed on the growth curves, where a parameter modification
between time points could have affected growth negatively.
Since microalgae can adapt to environment fluctuations by
storage or utilization of resources (Mata et al 2010), an increase of cellular density can be accomplished if sufficient
adapted cells get to divide and grow. Taking this information
into account, it could be possible to observe wave-like fluctuations on cellular growth on batch cultures.
Fucoxanthin production of I. galbana and P. tricornutum
cultures in F/2 medium Fucoxanthin content of stirred nonaxenic P. tricornutum cultures (80 rpm, 23.8 mol photons m2 -1
s , 20 C) decrease as the culture enters the stationary phase

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J Appl Phycol

(Carreto and Catoggio 1976). However, our data showed that


under orbital agitation at low light intensities, a higher production of fucoxanthin was achieved on day 16 of growth (stationary phase). Moreover, fucoxanthin content in aerated
P. tricornutum cultures was similar in both linear growth phase
and stationary phase at 9.1, 25.9, and 62 mol photons m-2 s-1
light intensities, while a 13.5 mol photons m-2 s-1 light intensity caused a higher fucoxanthin production in the linear
growth phase (day 8). This information suggests that fucoxanthin production in P. tricornutum is not only dependent on the
age of the culture, but that different culture conditions (perhaps
a higher agitation intensity as done in our investigation) can
delay the peak of its production to the stationary phase of
growth.
Likewise, fucoxanthin concentration in I. galbana cultures
under 13.5 mol photons m-2 s-1 light intensity was higher in
the stationary phase (16 days) in comparison with the
contents detected in mid-linear growth phase (8 days).
However, I. galbana cultures, grown in 100 L bags in
Wallerstein and Miquel nutrient medium at 18 C and continuous light, reached its highest carotenoid concentration
(4.14 mgFucoxanthin gDry Weight-1) at the exponential phase,
while a decline in carotenoid composition was observed in
the stationary and decay phases (Durmaz et al. 2008). The
Durmaz et al (2008) results depict the overall carotenoid composition, not the specific concentration of fucoxanthin, and the
mixing conditions were quite different from those employed
in the present work, so differences on fucoxanthin production
can arise from those factors. The different composition
patterns could also be due to culture media composition and
temperature used in both studies. To this effect, Durmaz et al
(2008) also reported that the carotenoid content was lower
(about 1.75 mgFucoxanthin gDry Weight-1) when cultured at
26 C, suggesting that the temperature used in our study could
have affected in some degree the fucoxanthin production
obtained.
The effect of light is of great importance for carotenoid
production in microalgae. In our study, both I. galbana and
P. tricornutum presented higher fucoxanthin concentration
(1.2 to 10.9 times increase, respectively) and total fucoxanthin
production (2.2 to 16.4 times increase, respectively) at
medium-low light conditions in comparison to the values obtained at high light intensities, which goes in agreement with
literature. An increment in pigment concentration has been
previously attributed to the light harvesting role that the pigments posses. Low irradiances and self-shading of cells due to
cell growth have been shown to be responsible for a fivefold
increase in carotenoid composition (Snchez Mirn et al.
2002). It has also been proposed that higher carotenoid compositions are reached when cells grow under subsaturating
light conditions for a prolonged time period and acclimate to
that light regime. Supposedly, by increasing the number of
photosystems, the cellular content of the primary pigments

associated also increase in low light regimes (Mulders et al.


2014). For instance, it has been recently reported that
I. galbana cultured under low light intensity
(27 mol photons m-2 s-1) in an aerated 5 L bubble column
exhibited higher levels of proteins, sugars, lipids, and carotenoids (Guermazi et al. 2014); although according to our results in Figs. 2 and 3, an even lower light density could provide a greater carotenoid composition.
Media comparison for I. galbana cultures at optimal fucoxanthin production conditions Since I. galbana showed
higher fucoxanthin total production, a media comparison
was made in order to study the effect of a different media
composition (Table 1) on cell density and fucoxanthin production in 1 L batch cultures. Nutrient concentration often has a
dramatic effect on the biochemical profile of microalgae with
variations dependent on the species and the nutrients provided. In terms of nutrient concentration, it has been reported for
I. galbana that growth rate was limited at initial concentrations below 0.5 mM NaNO3, a component that often plays an
important role in the lipid content of I. galbana (Molina Grima
et al. 1992). Although NaNO3 concentration in both media
compositions studied are greater than the limiting concentration, Conway medium concentration is 1.33 times higher than
F/2 medium, which might explain the differences in
I. galbana growth at stationary phase.
Likewise, concentrations between 50 and 500 M of phosphorus and 10 M of Zn2+ have been reported to improve
I. galbana cell growth (Sun and Wang 2009). F/2 medium
does not contain the optimal concentrations for phosphorus
(31 M), while Conway medium offers about 4 times the
concentration of said element and falls in the range of the
concentration suggested by Sun and Wang (2009). Other elements that are found in greater proportions in Conway medium are sodium (2.1 times), chlorine (111.7 times), phosphorous (3.9 times), and silica (4.5 times), while metal trace elements can be found in lower concentrations than in F/2 medium. However, Zn2+ concentration in both media is about 65
times lower than the suggested concentration, so enrichment
would be necessary in order to further improve cell growth.
A previous comparison of the growth of an Isochrysis species isolated from the South China Sea grown in Conway
medium demonstrated a slightly higher maximum cell density
(1.45 %) than cultures grown in F/2 medium, and in both
media conditions, the maximum cell density was reached at
day 17 of growth (Lananan et al. 2013). However, the study
does not report culture conditions, such as aeration/agitation,
temperature or light; the study was solely focused on biomass
production. In the present study, cultures grown in both media
reached the maximum cell density at day 14 of growth, where
a concentration of 1.69107 cells mL-1 was reached using
Conway medium. The said concentration was 32.41 % higher
than the cell density obtained by F/2 medium under the same

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J Appl Phycol

conditions, surpassing the difference reported for Isochrysis


sp. by Lananan et al (2013). Moreover, fucoxanthin concentration and total production was higher in Conway medium
aerated cultures (~42.46 and ~50.68 %, respectively), which
proves the importance of medium composition and nutrient
concentration. As described earlier, several component concentrations are different in Conway medium, so an increase in
fucoxanthin concentration and total production could be derived from the different concentration of media components.
It is important to note that I. galbana cultures grown
at the optimal conditions (agitation by aeration in
13.5 mol photons m-2 s-1 on Conway medium) had higher
fucoxanthin concentration than those reported for several
macroalgae species. According to Xia et al. (2013),
macroalgae possess a fucoxanthin production in a range from
0.02 to 0.58 mgFucoxanthin gFresh Weight-1. Then, the fucoxanthin
concentration observed on I. galbana cultures resulted to be
1.2 to 15 times higher than species such as Eisenia bicyclis,
Hizikia fusiformis, Saccharina japonica, and Scytosiphon
lomentaria, while species such as Petalonia binghamiae have
been reported to have higher concentrations than I. galbana.
In conclusion, fucoxanthin microalgal production is of
great interest due to the vast number of beneficial effects that
the carotenoid can exert on human health and the commercial
feasibility that microalgae cultivation offers against
macroalgae cultures. In order to determine the best parameters
for fucoxanthin production, the study of simultaneous culture
conditions (light intensity, mixing/agitation, harvesting time,
and media composition) is of great importance. In the present
study, I. galbana cultures had a better adaptation than
P. tricornutum to the tested light and agitation growth conditions in F/2 medium, and exhibited the highest total fucoxanthin production in 1 L batch cultures, making I. galbana a
strong candidate for fucoxanthin microalgal production.
Moreover, the best growth conditions did not result in the best
fucoxanthin production conditions for either species, making
the study of culture process parameters and their relationship
with metabolite production of great importance. Also,
Conway medium composition resulted in even greater cell
density and fucoxanthin concentration and total production
in I. galbana cultures grown at optimal conditions (agitation
by aeration in 13.5 mol photons m-2 s-1). Furthermore, fucoxanthin production resulted in 1.2 to 15 times the ones previously reported in macroalgae on fresh biomass basis. With this
information, adequate conditions for fucoxanthin production
can be employed, where further separation and purification of
the compound can be achieved through a series of novel separation technologies.

Acknowledgments The authors would like to thank the financial support of Consejo Nacional de Ciencia y Tecnologa (CONACyT, Grant
333554), the ITESM Research Chair (Grant CAT020), the Bioprocess

and Synthetic Biology Strategic Focus Group of Tecnolgico de Monterrey (0821C01004), and the Zambrano-Hellion Fund (CDB090).

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