J Appl Phycol (2008) 20:737–742
DOI 10.1007/s10811-008-9331-7

Effects of temperature, photosynthetic photon flux density,
photoperiod and O2 and CO2 concentrations on growth ratesof the symbiotic dinoflagellate, Amphidinium sp.
Y. Kitaya & L. Xiao & A. Masuda & T. Ozawa & M. Tsuda &K. Omasa

Received: 11 May 2007 / Revised and accepted: 14 March 2008 / Published online: 18 June 2008
# Springer Science + Business Media B.V. 2008

Abstract Symbiotic dinoflagellates of the species Amphidinium are expected to be pharmaceutically useful microalgae
because they produce antitumor macrolides. A microalgae
production system with a large number of cells at a highdensity has been developed for the efficient production ofmacrolide compounds. In the present study, the effects ofculture conditions on the cellular growth rate of dinoflagellates were investigated to determine the optimum culture
conditions for obtaining high yields of microalgae. Amphidinium species was cultured under conditions with six
temperature levels (21–35°C), six levels of photosynthetic
photon flux density (15–70 μmol photons m−2 s−1), threelevels of CO2 concentration (0.02–0.1%), and three levels of
O2 concentration (0.2–21%). The number of cells cultured ina certain volume of solution was monitored microscopicallyand the cellular growth rate was expressed as the specific

Y. Kitaya (*) : L. Xiao
Osaka Prefecture University,
1-1, Gakuen-cho,
Sakai, Osaka 599-8531, Japane-mail: kitaya@envi.osakafu-u.ac.jp
A. Masuda : T. Ozawa
Yanmar Co.,
3286, Itohara, Musashi-machi,
Kunisaki, Oita 873-0421, Japan
M. Tsuda
Kochi University,
Monobe B200, Nankoku,
Kochi 783-8502, JapanK. Omasa
The University of Tokyo,
Yayoi 1-1-1, Bunkyo,
Tokyo 113-8657, Japan

growth rate. The maximum specific growth rate was
0.022 h−1 at a temperature of 26°C and O2 concentration of
5%, and the specific growth rate was saturated at a CO2
concentration of 0.05%, a photosynthetic photon flux densityof 35 μmol photons m−2 s−1 and a photoperiod of 12 h day−1
upon increasing each environmental parameter. The results
demonstrate that Amphidinium species can multiply efficiently under conditions of relatively low light intensity and low
O2 concentration.
Keywords Algae . Amphidinium sp. . Photosynthetic photon
flux density . Specific growth rate . Symbiotic dinoflagellate .
Temperature

Introduction
The amphidinolides are a series of unique cytotoxic
macrolides isolated from the dinoflagellate Amphidinium
sp. that has been separated from marine acoel flatworms ofthe genus Amphiscolops (Kobayashi and Tsuda 2004;
Kobayashi and Kubota 2007). Up to now, 39 amphidinolides have been reported (Oguchi et al. 2007). Amphidinolides H (Kobayashi et al. 1991, 2000) and B (Ishibashi et al.
1987; Kobayashi et al. 1989; Bauer et al. 1994), whichwere initially isolated from species of the marine dinoflagellate Amphidinium (strains Y-25 and Y-5, respectively),are 26-membered macrolides possessing unique structures
such as an allyl epoxide and an S-cis-diene, and exhibit
potent cytotoxicity (IC50 0.0045–0.00014 mg mL−1) against
cultured tumor cells in vitro and antitumor activities invivo. More recently, a new series of potently cytotoxic
Amphidinium macrolides, designated iriomoteolides, havebeen isolated from the benthic Amphidinium strain HYA024(Tsuda et al. 2007a, b). These macrolides are, therefore,

738
Fig. 1 a Amphiscolops species
containing dinoflagellate cells. b
Dinoflagellates of Amphidinium
sp. separated fromAmphiscolops

J Appl Phycol (2008) 20:737–742

0.5 mm(x 5)(a)expected to be good candidates for antitumor drugs with anovel mechanism of action because their chemical structureis unlike that of conventional anticancer materials. Nevertheless, development of these compounds as anticancer
drugs has not been smooth due to limited sample amounts.
Development of a production system for Amphidinium
cells separated from Amphiscolops is desirable in order toproduce macrolide compounds efficiently. However, theconstruction of a mass culture system for Amphidinium
production has not yet been established because environmental control of the mass culture of Amphidinium cells at
a high density is difficult using conventional culture
methods. Microalgal cell growth rates are affected byenvironmental parameters such as temperature, light intensity and period, and gas composition in the culture system,
as well as the combination of all these factors. Few papers
have focused on environmental control of cell cultures of
symbiotic Amphidinium. In order to establish a symbiotic
Amphidinium production system with a large number ofFig. 2 Apparatus of the culture
vessel with water droplets(b)cells and a rapid turnover rate, the appropriate combinationof environmental parameters needs to be determined.
This research was initiated to optimize culture
condition for symbiotic Amphidinium production. The
present study is a starting point for the optimization of theculture. Effects of variable culture conditions on the cell
growth rate of dinoflagellates were investigated todetermine the potentially optimum culture conditions for
obtaining high yields of this microalga. Effects oftemperature, photosynthetic photon flux density, photoperiod and CO2 and O2 concentrations on growth rates ofdinoflagellate cells were assessed using a hanging-drop
culture system.

Materials and methods
The dinoflagellate Amphidinium sp. (strain HYA002) was
separated from the internal cells of the marine acoel

PhotosTop view

Water droplets
includingdinoflagellate cells
Wet paper
Cross sectional view

Silicon rubber
for sealing
10 mm

J Appl Phycol (2008) 20:737–742

Enrichment concentrations (0–2%) and four levels of
NaHCO3 concentrations (0–10 mmol) to determine thechemical composition of the basal culture solution.
In the main experiment, Amphidinium was cultured at six
different temperatures (21–35°C), four photosynthetic
photon flux densities (PPFD, 15–70 μmol photons m−2
s−1; measured on the inner surface of plastic cover of thehanging-drop culture device), three photoperiods (6–24 h),three CO2 concentrations (0.02–0.1%), and three O2
concentrations (0.2–21%). The light source was fluorescentlamps (FPL55EX-N, Matsushita Electric, Osaka, Japan).
PPFD was controlled by covering each vessel withneutral shading films.

Results
Cells multiplied almost exponentially by cell division inboth the conventional culture and the hanging-drop culture(Fig. 3). The cell density reached about 6.6×104 cells mL−1
in the conventional culture, while the cell density and total
number of cells per hanging droplet were about 3.3×104
cells mL−1 and 98 cells per hanging droplet, respectively,
144 h after starting the culture. Specific growth rates
determined with the hanging-drop culture and the conventional culture methods showed good correlation (r=0.97) asshown in Fig. 4. It was confirmed that the specific growthrate can be estimated accurately with the water droplet
method. Specific growth rates increased significantly withincreasing concentration of Provasoli’s Marine Water
Enrichment solution from 0 to 0.5% and then gradually

8
Conventional culture
7

cell density (x10 4 mL-1)

flatworm Amphiscolops sp. (Fig. 1), which was collected
off Sunabe beach, Okinawa, Japan (Oguchi et al. 2007).
The voucher specimen and the SSU rDNA gene aredeposited at the Center for Advanced Marine Core
Research, Kochi University.
The experimental system for evaluating the effects ofenvironmental variables on the growth rate of microalgal
cells is shown in Fig. 2. The culture system was set up witha hanging-drop culture device according to the procedurereported by Kitaya et al. (1998, 2005). Amphidinium cells
were cultured in water droplets (3 μL in volume each) in a
vessel (100 mL in volume). Each water droplet is held on thesurface of a translucent plastic plate and contains initiallyabout ten Amphidinium cells. The culture vessel is completely sealed after placing water droplets with Amphidinium cells
and controlling the internal CO2 and O2 concentrations at setlevels. The vessel was sealed in a box in which the gas
composition had been controlled beforehand. Changes in thegas composition in the vessel were almost negligible during
the experiment. The number of water droplets in eachtreatment was at least five. The vessel was submerged inwater at constant temperature. The water layer above thevessel cut out long-wave radiation from the light source,which would otherwise cause a heat load inside the vessel.
Wet paper inside the vessel kept the relative humidity at
almost 100% and thus inhibited evaporation from the water
droplets. The basal culture solution was sea water. Provasoli’s
Marine Water Enrichment solution (Provasoli 1963) as asupplemental nutrition, and NaHCO3 as a carbon source forphotosynthesis, were mixed with the basal culture solution.
The initial pH in the culture solution was adjusted to 7.5.
The salinity of the culture solution was adjusted to 3.3%.
The number of dinoflagellate cells in each water droplet
was counted with a microscope at 70x magnification. The
specific growth rate was used as a standardized index forthe growth rate of cells. The specific growth rate (μ) was
determined in the logarithmic multiplication stage by thefollowing equation; μ = (lnN2−ln N1) / (T2−T1), where N1
and N2 are the cell numbers at times T1 and T 2,
respectively. In the present experiment, T1 and T2 were 48
and 144 h, respectively, after the start of the culture. The
doubling time or the mean generation time (td), is given by
the equation td = ln2 / μ.
Cell growth rates determined in the hanging-drop culture
method were compared with those in a conventional culture
method to certify the hanging-drop culture method. In theconventional culture method, Amphidinium was cultured in
aerated containers (210×140×70 mm depth and 800 mL
medium volume) with the same culture condition as that in
the water droplet method except culture vessel size andmedium volume.
In a preliminary experiment, Amphidinium was cultured
in solutions with four levels of Provasoli’s Marine Water

739

6

y = 5.4555e0.0252xµ=0.025

5

Hanging-drop
culture432

y = 5.3315e0.0202xµ=0.023

10

0

20 40 60

80 100 120 140 160 180

Time after starting the culture (h)Fig. 3 Examples of cell densities over time with the hanging-drop
culture and the conventional culture methods

J Appl Phycol (2008) 20:737–742

0.03

0.05
Specific growth rate (h-1)µ determined with the container method (h-1)

740

0.04
0.03
0.02

0
0

0.01

0.02

0.03

0.04

0.05

µ determined with the hanging-drop method (h-1)
Fig. 4 Specific growth rates (μ) of Amphidinium cells determinedwith the hanging-drop culture and the conventional container culture
methods. Volumes of culture media were 3 μL and 800 mL in the
hanging-drop culture method and the conventional culture method,respectively. Means with standard deviations are shown (n=3–10)

increased at concentrations higher than 0.5%, although
there were no significant differences among the treatments(Fig. 5). Specific growth rates slightly increased withincreasing NaHCO3 concentration from 0 to 2.5 mmol
and then decreased significantly at NaHCO3 concentrations
higher than 5 mmol (Fig. 6). The optimum NaHCO3
concentration for cell division was thus in the range of
2.5–5 mmol. Therefore, 1% Provasoli’s Marine Water
Enrichment solution and 5 mmol NaHCO3 were used as
basic supplements in the experiment for assessing appropriate culture conditions.

5
10
NaHCO3 concentration (mmol)

15

Specific growth rates doubled with increasing temperature from 21 to 26°C, and decreased with increasing
temperature from 29 to 35°C (Fig. 7). The optimum
temperature was 24–29°C .
Specific growth rates increased significantly by 50%
with increasing PPFD from 15 to 35 μmol photons m−2 s−1
and reached a constant value at PPFD values greater than
35 μmol photons m−2 s−1 under continuous light (Fig. 8).
Specific growth rates increased significantly by 80% withincreasing photoperiod from 6 to 12 h day−1 and reached analmost constant value at photoperiods of 12 to 24 h day−1(Fig. 9).
Specific growth rates tended to increase slightly withincreasing CO2 concentration from 0.002 to 0.05%, and

0.03

0.02

0.01
LSD (p<0.05)

0

0.5

1

1.5

2

2.5

Concentration of PMWE (%)
Fig. 5 Effects of Provasoli’s Marine Water Enrichment concentration
on specific growth rates of Amphidinium cells. Vertical bars on plots
indicate standard deviations (n=10). Temperature: 28°C, O2 concentration: 5%, CO2 concentration: 0.05%, photosynthetic photon flux
density (PPFD): 50 μmol photons m−2 s−1, photoperiod: 24 h day−1

Specific growth rate (h-1)

Specific growth rate (h-1)

0Fig. 6 Effects of NaHCO3 concentrations on specific growth rates ofAmphidinium cells. Vertical bars on plots indicate standard deviations(n=10). Temperature: 28°C, O2 concentration: 5%, CO2 concentration: 0.05%, PPFD: 50 μmol photons m−2 s−1, photoperiod: 24 h
day−1

0.03

0

0.01

0

y = 0.8984x + 0.009
r2 = 0.936

0.01

LSD (p<0.05)

0.02

0.02

0.01
LSD (p<0.05)

0
20

22

24

26

28

30

32

34

36

o

Temperature ( C)
Fig. 7 Effects of medium solution temperatures on specific growth
rates of Amphidinium cells. Vertical bars on plots indicate standard
deviations (n=10). O2 concentration: 5%, CO2 concentration: 0.05%,
PPFD: 50 μmol photons m−2 s−1, photoperiod: 24 h day−1

J Appl Phycol (2008) 20:737–742

741

0.03
Specific growth rate (h-1)

Specific growth rate (h-1)

0.03

0.02

0.01

0

LSD (p<0.05)

LSD (p<0.05)

0.02

NaHCO3 conc. (mmol)

0.01

0
5
0

0

20

40
60
-2
-1
PPFD (µmol m s )

0

80

0.05

0.10

0.15

CO2 concentration (%)

Fig. 8 Effects of PPFD on specific growth rates of Amphidinium
cells. Vertical bars on plots indicate standard deviations (n=10).
Temperature: 26°C, O2 concentration: 5%, CO2 concentration: 0.05%,
photoperiod: 24 h day−1

Fig. 10 Specific growth rates of Amphidinium cells as affected by
CO2 concentrations with or without NaHCO3 in the medium solution.
Vertical bars on plots indicate standard deviations (n=10). Temperature: 26°C, O2 concentration: 5%, PPFD: 40 μmol photons m−2 s−1,
photoperiod: 24 h day−1

reached a constant value at CO2 concentrations of 0.05 to
0.1% regardless of NaHCO3 concentration, although there
were no significant differences among the 0.002 to 0.1%
treatments (Fig. 10). Specific growth rates tended toincrease slightly with increasing O2 concentration from
0.3 to 5%, and then decreased with increasing O2
concentration from 5 to 21% regardless of CO2 concentration, although there were no significant differences among
the treatments of 0.3–21% (Fig. 11).
The maximum specific growth rate was 0.022 h−1 at atemperature of 26°C and O2 concentration of 5%, and thespecific growth rate was saturated at a CO2 concentration of
0.05%, a photosynthetic photon flux density of 35 μmol
photons m−2 s−1 and a photoperiod of 12 h day−1.

Discussion
The symbiotic dinoflagellates used in this study exhibited amaximum specific growth rate equivalent to that of asymbiotic dinoflagellates, e.g., Gyrodinium aureolum (μ=0.015)(Dixon and Syrett 1988), Ceratium furca (μ=0.03) andCeratium fusus (μ=0.023) (Baek et al. 2008). The conditions
that provided the highest growth rate of the symbiotic
dinoflagellates in this study would be similar to those inside
the body of the host animal, Amphiscolops. The doublingtime (td) was 31 h at a specific growth rate of 0.022 h−1. The
results demonstrate that symbiotic Amphidinium species canmultiply efficiently under conditions of relatively low lightintensity and low O2 concentration.

0.03
Specific growth rate (h-1)

Specific growth rate (h-1)

0.03

0.02

0.01

0

LSD (p<0.05)

LSD (p<0.05)

0.02

0

0

6

12

18

24

30

Photoperiod (h d-1)
Fig. 9 Effects of photoperiods on specific growth rates of Amphidinium
cells. Vertical bars on plots indicate standard deviations (n=10).
Temperature: 26°C, O2 concentration: 5%, CO2 concentration: 0.05%,
PPFD: 50 μmol photons m−2 s−1

CO2 conc. (%)
0.002
0.05
0.1

0.01

0

5

10

15

20

O2 concentration (%)
Fig. 11 Specific growth rates of Amphidinium cells as affected by O2
concentrations at different CO2 concentrations. Vertical bars on plots
indicate standard deviations (n=10). Temperature: 28°C, PPFD:
50 μmol photons m−2 s−1, photoperiod: 24 h day−1

742This experiment was conducted with algal cultures at
low cell densities. Cell productivity is dependent not
only on the growth rate but also on the population
density. Moreover, the fragility of cells represents one of
the biggest obstacles to the cultivation of dinoflagellates(e.g., Dixon and Syrett 1988). In order to establish asystem to produce secondary metabolites such as amphidinolides, it is necessary to determine appropriate conditions for cellular multiplication at high cell densities andalso for the efficient production of secondary metabolites
in cells.
Acknowledgment This work was supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan.

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