5.1 CHAPTER 5 DUNALIELLA: TAXONOMY, MORPHOLOGY, ISOLATION, CULTURE, AND ITS ROLE IN SALT PANS. 5.1 INTRODUCTION Dunaliella was first noticed in the saltern evaporation ponds in 1838 by Michael Felix Dunal and named after it’s discoverer by Teodoresco in 1905. After discovery Dunaliella has become a convenient model organism for the study of salt adaptation. The establishment of concept of organic compatible solutes to provide osmotic balance was largely based on the study of Dunaliella species. More over the massive accumulation of β-carotene by some strains under suitable growth conditions has led to interesting biotechnological application (Oren, 2005). In this backdrop, this chapter addresses taxonomy, ecology, isolation, and culture of Dunaliella and its role in salt pans. 5.2 TAXONOMY Dunaliella is a genus of unicellular alga belonging to the family Polyblepharidaceae. Its cells lack a rigid cell wall. Teodoresco (1905) described two species D. salina and D. viridis. D. salina cells are larger and under suitable conditions it synthesizes massive amounts of carotenoid pigments colouring the cells brightly red, while D. viridis are smaller than D. salina, and
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5.1
CHAPTER 5
DUNALIELLA: TAXONOMY, MORPHOLOGY, ISOLATION,
CULTURE, AND ITS ROLE IN SALT PANS.
5.1 INTRODUCTION
Dunaliella was first noticed in the saltern evaporation ponds in 1838 by
Michael Felix Dunal and named after it’s discoverer by Teodoresco in 1905.
After discovery Dunaliella has become a convenient model organism for the
study of salt adaptation. The establishment of concept of organic compatible
solutes to provide osmotic balance was largely based on the study of Dunaliella
species. More over the massive accumulation of β-carotene by some strains
under suitable growth conditions has led to interesting biotechnological
application (Oren, 2005). In this backdrop, this chapter addresses taxonomy,
ecology, isolation, and culture of Dunaliella and its role in salt pans.
5.2 TAXONOMY
Dunaliella is a genus of unicellular alga belonging to the family
Polyblepharidaceae. Its cells lack a rigid cell wall. Teodoresco (1905) described
two species D. salina and D. viridis. D. salina cells are larger and under
suitable conditions it synthesizes massive amounts of carotenoid pigments
colouring the cells brightly red, while D. viridis are smaller than D. salina, and
5.2
remain green. Lerche (1937) and Butcher (1959) added more species to the
genus Dunaliella. Of all the species only Dunaliella salina and D. viridis are
halotolerant and are found in hypersaline brines. An in-depth Taxonomic
treatment of the genus is available in Massyuk’s monograph (1973).
5.3 MORPHOLOGY
The cell shape in species of Dunaliella varies from ellipsoid, ovoid,
cylindrical, pyriform, and fusiform to almost spherical. The cell symmetry is
radial (sections Dunaliella, Tertiolectae, and virides), bilateral or slightly
asymmetrical (flattened, dorsiventrally curved, and slightly asymmetrical cells
exist in section Peirceinae). Cells of a given species may change shape with
changing conditions, often becoming spherical under unfavourable conditions.
Cell size may also vary to some degree with growth conditions and light
intensity (Marano, 1976; Riisgård, 1981; Einsphar et al., 1988). The general
cell organization has been studied in most detail (Light microscope and
electron microscope) in Dunaliella salina ( Teoderesco, 1905; Lerche, 1937;
Butcher, 1959; Masjuk, 1973; Melkonian and preisig, 1984; Hamburger, 1905;
Trezzi et al., 1964; Vladimirova, 1978; Anghel et al., 1980). A rigid cell wall is
lacking, but there is a distinctive mucilaginous cell coat. The two flagella are
apically inserted, equal in length, and usually exhibit homodynamic pattern of
beating. The fine structure of the flagellar apparatus is complex and is of the
type found in other Chlorophyceae (Melkonian, 1989). The single chloroplast
occupies most of the cell body. It is cup-, dish-, or bell-shaped and has a
thickened basal portion containing a pyrenoid. The chloroplasts are sometimes
5.3
arranged in dense stacks of up to 10 units. Stacking of thylakoids was found to
be particularly pronounced in cells grown at high light intensity and high salt
concentration (Hoshaw and maluf, 1981; Pfeifhofer and Belton, 1975). Starch
grains usually surround the pyrenoid, but may also be found at other places of
the chloroplast. The chloroplast may also accumulate large quantities of β-
carotene within oily globules in the inter-thylakoid spaces, so that the cells
appear orange-red rather than green. The β-carotene globules of Dunaliella
salina were found to be composed of practically only neutral lipids, more than
half of which were β-carotene (Ben-Amotz et al., 1982). The eyespot (stigma)
has an anterior peripheral location in the chloroplast. It consists of one or two
rows of lipid globules. The nucleus is generally obscured in life by a number of
granules. It occupies most of the anterior part of the cell and is often
surrounded by anterior lobes of the chloroplast. Ultrastructural studies show
that it has a porous envelope and a single prominent nucleolus, which is often
surrounded by clumped heterochromatin.
5.4 LOCOMOTION OR SWIMMING BEHAVIOUR
The cells swam forward with sinusoidal tracks and rotated around their
longitudinal axis. The cells always rotated counter-clockwise, similar to
Chlamydomonas. In normal conditions of medium, we didn’t see collision
between cells. They can avoid one another by passing around or by modifying
the orientation of the track. The mean velocity from a population of 480 cells
was 105 ± 10 µm s-1 (Kamiya and Witman, 1984; Ruffer and Nultsch, 1987).
Flagellate algae regulate their exposure to light by swimming toward and away
5.4
from the light source and they are able to detect a gradient of distribution of
light. In natural conditions, the light is diffused coming from different
directions, and the cells must orient towards the resultant light direction.
Phototactic algae solve the problem of finding the light direction by scanning
their environment with an antenna sensitive to light. The photoreceptor
pigment (rhodopsin) is located between the eye spot and the adjacent cell
surface. Rhodopsin could provide quick communication of the signal to the
flagella via ion currents or potential charges (Litvin et al., 1978).
5.5 HIGH SALINITY SUSTENANCE
Osmoregulation in plants and in Dunaliella has been reviewed
extensively (Avron, 1991). The genus Dunaliella contains several species which
stand out as being the only eukaryotic and photosynthetic organisms which are
able to grow in media containing an extremely wide range of salt
concentrations, from 0.05M (0.3%) to saturation (~5.5 M or 35%). Dunaliella
adapts to high extra cellular osmotic stress by synthesis of intracellular
glycerol. Glycerol is produced either photosynthetically or by degradation of
starch reserves. The induction of glycerol synthesis or reassimilation is
triggered by volume changes. Glycerol phosphate dehydrogenase and
phosphofructokinase are probably the check point enzymes which control
glycerol synthesis. Changes in the plasma membrane, inorganic phosphate, and
pH following osmotic shocks suggest that plasma membrane sensors as well as
soluble metabolites are involved in the activation of glycerol synthesis (Avron,
1991).
5.5
5.6 REPRODUCTION
5.6.1 VEGETATIVE REPRODUCTION
Vegetative reproduction is by lengthwise division in the motile state
(Labbé, 1925; Hamburger, 1905). Mitosis and cytokinesis of Dunaliella cells
exhibit the characteristics of Chlorophyceae (Marano, 1976; Melkonian, 1989).
A division of chloroplast starts at preprophase by the division of the pyrenoid,
but the complete fission of the chloroplast only takes place during cytokinesis
(Marano, 1976). In D.viridis cell division was found (Jimenez et al., 2007) to be
affected by the processes such as, hyper-osmotic shock, nitrogen starvation or
sub-lethal UV radiations that induce dephosphorylation of signal – regulated
kinases (ERKs). D.viridis cell cultures exposed to PD98059, a very specific
inhibitor of the ERK signalling pathway, resulted in a total arrest of cell
proliferation and a complete dephosphrylation of ERK.
5.6.2 SEXUAL REPRODUCTION IN DUNALIELLA
They reproduce by longitudinal division of the motile cell or by fusion of
two motile cells to form a zygote. Fusion of two equally sized gametes to form
a zygote was documented in many of the early studies (Hamburger, 1905;
Teodoresco 1906; Hamel, 1931). Lerche (1937), who reported sexual zygote
formation in five of the six species studied (D. salina, D. parva, D. peircei, D.
euchlora, and D. minuta), also reported zygote formation in D. salina induced
by a reduction in salt concentration from 10 to 3%. In the process, first the
flagella touch, and then the gametes form a cytoplasmic bridge and fuse. The
zygote has a thick outer layer. It can withstand exposure to freshwater and also
5.6
survive prolonged periods of dryness. These zygotes germinate with the release
of up to 32 haploid daughter cells through a tear in the cell envelope under
favorable climatic conditions. It is well possible that the cyst-like structures
observed by Oren et al., (1992) at the end of a bloom of green Dunaliella cells
in the Dead Sea in 1992 were actually such zygotes. In this case, however, the
formation of these rounded, thick-walled cells took place at a time of an
increase in water salinity. Loeblich (1969) has reported formation of such cysts
in media of reduced salinity.
5.7 GROWTH CHARACTERISTICS OF DUNALIELLA
The growth rate of Dunaliella varies based on the factors such as light
intensity, temperature and salinity of the medium (See annexure 5.1). In
Dunaliella bardawil the growth rate range from 0.51 to 2.00 div. day-1 was
observed (Ben-Amotz, 1996; Sanchez et al., 1996; Gomez and Gonzalez, 2005).
A maximum growth rate of 2.00 div. day-1 occurred (Ben-Amotz, 1996) when
grown under a light intensity of 25 W. m-2, at 10°C, in a 1.5M NaCl medium.
In D. salina growth rate ranged from 0.14 to 1.407 div. day-1 (Orsett and
young, 2000; Cifuentes et al., 1992; Chang et al., 1986; Moulton and Burford,
1990; Moulton et al., 1987; Gomez and Gonzalez, 2005; Aguilar et al., 2004)
with a maximum growth rate of 1.407 div. day-1 observed (Chang et al.,1986)
when grown under 54 µE m-2 s-1 at 26 °C in a medium of 260 x 10-3 salinity.
In D. viridis growth rate ranged from 0.46 to 1.40 div. day-1 (Jimenez
and Niell, 1991; Moulton and Burford, 1990; Moulton et al., 1987). The
5.7
maximum growth rate of 1.40 div. day-1 was observed (Jimenez and Niell,
1991), when D. viridis cells were grown under 150 µmol. m-2 s-1, at 30 °C in 1M
NaCl medium.
5.8 DISTRIBUTION OF DUNALIELLA
5.8.1 IN SALT LAKES
Interesting studies on population dynamics of Dunaliella spp. of inland
salt lakes such as Great Salt Lake, Utah and Dead Sea are available in the
literatures (Kaplan and Friedman, 1970). Dunaliella Spp. population is limited
by i) nutrients availability and ii) predation by Artemia Spp. Dunaliella
Population is fluctuating with the salinity variation of salt lakes in response to
seasonal vagaries. In South basin of Great Salt Lake Utah, Dunaliella viridis
reached its peak with a population density of 24 x 106cells l-1 in April 1974 and
declined to less than 1x106 cells l-1 in June, probably as a consequence of
rapidly expanding Artemia salina population. An optimum predation rate of
D. viridis by Artemia was about 1000 cells day-1. Dense populations (up to
2 x 105 cells ml-1) of red D. salina and green D. viridis have been reported
(Stephens, 1974; Post, 1977).
Dead Sea, a salt lake of Mediterranean region, when salinity drops below
1.21 g/ml, favoured the development of Dunaliella Sp. Several authors
attempted on population dynamics of Dunaliella Spp. of Dead Sea. A bloom of
Dunaliella with a cell density of 40000 cells ml-1 was reported from Dead Sea in
1964 (Kaplan and Friedman, 1970). A mass development of the green
5.8
unicellular alga Dunaliella parva (up to 8800 cells ml-1) was observed in 1980.
Bloom was present for more than two years and disappeared at the end of 1982
as a result of complete mixing of the water column. During the period 1983-
1991, the lake was holomictic and no Dunaliella was observed. Due to heavy
rain and floods during the winter of 1991-1992, the upper water column
became diluted to 70% of their normal salinity. In this water layer, a bloom of
Dunaliella parva (15x103 cells ml-1) developed in the upper 5m of the water
column of the Dead Sea in May - June 1992. A small secondary bloom (1850
cells ml-1) developed between 6-10m depth at the end of summer (Oren and
Shilo, 1985; Oren et al., 1995). Simulation studies have shown that at higher
salinities (1.22-1.23 g ml-1), growth of Dunaliella is negligible.
In the pilot plant of Salt Lake Hut lagoon, a cell density of 1 x 104
D. salina cells ml-1 was observed during May 1982, and 3 x 104 D. salina cells
ml-1 was observed during September 1982. In 1983 May, it was 5 x 104 D. salina
cells ml-1. The cell density of D. viridis was observed to be 0.08 x 104 and 3 x
104 during May and September 1982, respectively. In April 1983, it was 1 x 104
cells ml-1 (Moulton et al., 1987).
5.8.2 IN SALTERNS
The highest number of Dunaliella was found in multi-pond salterns
(Annexure 5.2) between 20 and 30% of salts (103-105 cells ml-1). They then
decreased, reaching very low numbers in the NaCl saturated ponds where they
5.9
appeared inactive. Protozoans, other green algae and diatoms were seen in
ponds up to 15% total salts (Rodriguez-Valera et al., 1985). Brine samples from
red crystallizer ponds varied in salinity between 34 and 36 % and red Dunaliella
cells were present in all the samples examined and their numbers varied from