World Journal of Microbiology & Biotechnology (2006) 22:1003–1006 Ó Springer 2006

DOI 10.1007/s11274-006-9145-1

Pilot culture of three strains of Dunaliella salina for b-carotene production in open
ponds in the central region of Iran

Ali Hosseini Tafreshi and Mansour Shariati*

Received 19 November 2005; accepted 9 February 2006

Keywords: b-Carotene, climatic conditions of Iran, Dunaliella salina, outdoor culture

Summary

Three strains of Dunaliella salina (I, G and A) were cultivated under the climatic conditions of Iran, in open ponds
to compare the b-carotene production and the specific rate of growth. The experiments were accomplished in two
separate stages. In the first stage, the cells were grown in ponds on nutrient-rich medium containing 2 M NaCl to
obtain the necessary biomass. In the second stage, cells were stressed on nutrient-poor medium containing 2.5 M
NaCl for b-carotene induction. The results showed that the specific growth rate of strain I was the highest during the
first stage, whereas during the second stage, the growth rates of three strains were approximately the same. The
overall results indicated that strain G had the highest potential for b-carotene accumulation of the strains tested and
hence it was concluded that this strain is more suitable for outdoor cultivation under the climatic conditions of Iran
than the other two.

Introduction We hope that the present study will increase the interest
in the mass culture of Dunaliella for b-carotene pro-
Dunaliella salina is the best commercial source of natural duction and other biotechnological purposes in Iran.
b-carotene in the world (Borowitzka 1995). This alga
accumulates large amounts of b-carotene (up to 14% of
dry weight) in conditions such as high light intensity, Materials and methods
increased temperature, high salinity and nutrient defi-
ciency (Ben-Amotz & Avron 1983; Ben-Amotz & Shaish The strains of Dunaliella utilized in this experiment
1992). Dunaliella b-carotene is used in the food, cos- were: strain I, isolated from the salt marsh of Gavkhoni,
metic, and pharmaceutical industries as a colorant, anti- Iran; strain G, a kind gift from the biotechnology center
oxidant, anti-tumor agent, and heart disease preventive of Gheshm, Iran; and strain A, a kind gift from the Prof.
in addition to its characteristic as precursor of Vitamin Lilley, s laboratory at the University of Wollongong,
A (Ben-Amotz & Avron 1990; Davison et al. 1993). Australia. At first, cells were grown on modified John-
Nowadays, most commercial and common systems son’s medium as previously described (Shariati & Lilley
for mass culture of Dunaliella are open-air ponds 1994) containing 2 M NaCl in 2-l flasks. Outdoor
(Borowitzka 1999). Among the parameters that can experiments were performed near Kashan, almost in the
significantly influence the production of biomass and center of Iran (33°58 N, 51°29 E). Three similar paddle-
b-carotene in open ponds is the selection of the area. wheel ponds of concrete, with a surface area of 3.5 m2,
Dunaliella production plants are located in areas having and a linear flow rate of 15 cm s)1 were constructed for
a hot and dry climate with minimal cloudiness and each strain. Middle and lateral ponds were connected to
commonly situated at, or near a suitable source of brine. each other by valves. Outdoor cultures were performed
The climatic conditions in Iran make it into one of the in two stages (Ben-Amotz 1995; Enes & Saravia 1996).
most suitable areas not only for mass culture of D. salina In stage one (in mid-June), cells were grown in the
but also for other microalgae. In addition, in Iran the middle ponds (depth:15 cm). Fresh water containing
costs of salt and energy are very low. In order to survey 2 M NaCl was enriched with 5 mM KNO3, 2 mM
the possibility of mass culture of Dunaliella at a com- MgSO4, 0.1 mM KH2PO4, 50 mM NaHCO3, and
mercial level in Iran, pilot ponds were constructed for 0.01 mM FeCl3Æ6H2O to attain the necessary biomass.
primary cultivation of three existing strains of this alga. At the beginning of the second stage, the medium in the
1004 A.H. Tafreshi and M. Shariati
middle and lateral ponds were adjusted in such a way have the maximum growth rate and b-carotene content.
that each pond contained 2.5 M NaCl, 0.1 mM KNO3 The time dependence of cell growth and the specific
and 0.2106 cells ml)1 up to 12 cm depth after opening growth rates of three strains of Dunaliella are shown in
the valves. In both stage 1 (24 days) and stage 2 Figures 1 and 2, respectively. As indicated, during the
(20 days), evaporative losses were compensated by the first stage (Figure 1a), strain I reached the logarithmic
addition of fresh water and the samples were taken to phase faster with a higher specific growth rate (Figure 2)
determine the cell concentrations and/or of the quanti- than the other two strains. Thus this strain avoided the
tative changes in b-carotene every 4 days. exposure of high-intensity irradiation by decreasing the
The specific growth rate (l) was calculated by cell duplication time (Ben-Amotz & Avron 1983). Of three
counting using a light microscope and haemocytometer. strains, strain G had lowest specific growth rate (Fig-
Pigments were determined spectrophotometrically in ure 2) and hence were exposed longer to high light
85% acetone extracts using equations previously derived intensity and was therefore probably more tolerant to
(Eijckelhoff & Dekker 1997). The mean and standard severe irradiation than strain I.
deviation values of the parameters were calculated. All
statistical analyses of variance (one-way ANOVA) were Stage 2
performed using SPSS Software.
Mode of b-carotene induction
Several strategies have been used to maximize the pro-
Results and discussion duction of b-carotene per unit time and per culture
volume (Borowitzka & Borowitzka 1990; Ben-Amotz
Stage 1 1995; Garcia-Gonzalez et al. 2003). These strategies are
based on the observation that severe conditions such as
Stage one was necessary to reach the necessary cell high salinity, low nutrient levels and high temperature
concentration. The best strain for outdoor culture would induce b-carotene production in the cell. However, these

(a) 450
A
Cell Number(×10 Cell . mL )

400 G
-1

I
350
300
250
4

200
150
100
50
0

(b) 450
400 A
Cell number(×10 Cell . mL )
-1

G
350 I

300
250
4

200
150
100
50
0
0 4 8 12 16 20 24 28
Time (days)
Figure 1. Cell concentrations of three strains of Dunaliella salina (A, G and I) cultured in open ponds as a function of time in (a) stage 1, nutrient-
rich medium, containing 2 M NaCl; (b) stage 2, nutrient-poor medium, containing 2.5 M NaCl. The values are means of three replicates±SD.
Dunaliella growth in Iranian ponds 1005

0.6
stage 1
stage 2

Specific growth rate(day )

-1
0.4

0.3

0.2

0.1

0
A G I
strain
Figure 2. Comparison of the specific growth rates of three strains of Dunaliella salina (A, G and I) cultured in open ponds determined for the two
stages of the experiment. The values are means of three replicates±SD.

conditions at the same time decrease the cell number per tense light other than by b-carotene accumulation. The
culture volume by affecting cell viability. For this rea- b-carotene contents per cell (Figure 3a), per unit volume
son, one of the most efficient strategies has been the of the suspension (Figure 3b) and the ratio of b-carotene
cultivation of D. salina in two separate nursery and to chlorophyll of strain G (data not shown) were the
production stages by which, the productivity up to highest. Therefore, it seems that strain G is more suit-
450 mg m2 day)1 in stage one and to 300 mg m2 day)1 able for b-carotene production under the climatic con-
in stage two have been reported, compared to the rela- ditions of Iran than the two other strains.
tively low productivity of below 200 mg m2 day)1, using The maximum specific growth rate and the highest
a one-stage method of cultivation (Ben-Amotz 1995). b-carotene content per unit volume of strain G were
This study was therefore carried out using a two-stage 0.16±0.02 day)1 and 7.1±0.1 mg l)1, respectively. The
strategy (Ben-Amotz 1995; Enes & Saravia 1996) which values for strain G were not as good as those of strains
means that the culture in stage 2 was not only diluted of D. salina that were tested in Spain (Garcia-Gonzalez
with medium poor in nitrate but that it was also more et al. 2003). In that study, the growth rate ranged from
saline than the medium of stage 1. It seems that the 0.16 to 0.20 day)1, the highest value being for strain
selection of the best strategy for commercial b-carotene UTEX 2538; the carotenoid content ranged from 8.1 to
production in open ponds should be tested by consid- 15.1 mg l)1, the maximum again being for UTEX 2538.
ering different strains, the climatic conditions of area These results were similar to those published elsewhere
and the total cost of each strategy. (Cifuentes et al. 1992; Ben-Amotz 1995). Although the
maximum specific growth rate (16±0.3 day)1) and
Growth rate b-carotene content (8.1±1.2 mg l)1) of the strain L6
As indicated in Figures 1b and 2, there were no signifi- (Garcia-Gonzalez et al. 2003) are similar to those of
cant differences between the growth curves and specific strain G in our experiment, the productivity of the L6 is
growth rates of the three strains during stage two. The higher than that of the G (1.15 versus 0.35 mg l)1 day)1,
general decrease in the specific growth rate of the three respectively). This lag in b-carotene production in the
tested strains during stage 2, specially in strain I can lead mass culture of strain G needs a further optimization of
to the induction of b-carotene synthesis in Dunaliella, the parameters that affect b-carotene accumulation. In
because the cells would be subjected to more light per addition, other commercial strains of this alga should be
division cycle (Ben-Amotz & Avron 1983). tested in Iran to compare them in terms of maximum
growth rate and b-carotene production to select the best
b-Carotene production strain for mass culture.
The most important part of the results is formed by the
time dependences of b-carotene production by the three
strains shown in Figure 3. It has previously been sug- Acknowledgements
gested that b-carotene accumulation protects cells
against the deleterious effects of high intensity irradia- This work was supported by the Faculty of Post-
tion (Ben-Amotz et al. 1987). In spite of the good graduate Studies of University of Isfahan, Isfahan,
growth of strain I in this experiment, the b-carotene Iran. We thank to Dr Gert Schansker and Professor
content of this strain was very low. Thus this strain Reto Strasser (University of Geneva) for their linguistic
probably uses a different strategy to cope with the in- help.
1006 A.H. Tafreshi and M. Shariati

(a) 25
A

Beta-carotene content (pg . Cell )

15

10

(b) 8
A
Beta-carotene content (mg . mL )
-1

7 G
I
6

0
0 4 8 12 16 20 24
Time(days)
Figure 3. Quantitative changes in b-carotene as a function of time per cell (a) and per unit volume (b) of three strains of Dunaliella salina (A, G
and I) in open ponds during the stage 2 (nutrient-poor medium, containing 2.5 M NaCl). The values are means of three replicates±SD.

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