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

160 - 1025 Dunaliella cells ml-1. Crystallizer pond brine samples of Eilat saltern

in 1993 April had density of 1.238 g cm-3 with 700 Dunaliella cells ml-1, 1250

cells ml-1 in June and 650 cells ml-1 in July (Oren, 1993; Oren et al., 1992). In

the brines of ChonBuri salt works with salinities of 132 g l-1, 294g.l-1 and 356

g l-1, D. salina were observed as 0.07 x 104, 1.77 x 104 and 0.37 x 104 cells ml-1

respectively. In province salterns, a cell density of 0.38 x 104 D. salina cells

ml-1 in a brine of 120 g l-1 salinity, while cell density of 0.88 x 104 D. salina cells

ml-1 in a brine of 212 g l-1 salinity, and it was 1.5 x 104 D.salina cells ml-1 in a

brine of 300 g l-1 salinity and in a brine of 320 g l-1 salinity a cell density of 0.38

x 104 D.salina cells ml-1. In Nakhon Ratchasima province, saltern brine with 230

g l-1, had 0.2 x 104 D. salina cells ml-1, while brine with that of 268 g l-1 salinity

had a cell density of 0.07 x 104 D. salina cells ml-1 (Powtongsook et al., 1995).

5.9 DUNALIELLA ISOLATION

D.salina isolated in pure culture from a salt marsh in Kuwait, was grown

in a medium containing following : 5% NaCl, 5mM KNO3, 5mM MgSO4, 0.2 mM

KH2PO4, 0.3 mM CaCl2, 1.5 µM FeCl3, 30 µM EDTA, pH 8.0 at 25ºC under a white

fluorescent light of 61 µmol m-2s-1 (Al-Hasan and Sallal, 1985). A unialgal stock

of Dunaliella viridis was produced by plating out material from Hutt lagoon,

and was used in laboratory experiments (Moulton, et al., 1987). A D.salina

5.10

strain was isolated (Marin et al., 1998) from the solar evaporation salt-ponds of

Araya (10º37’N; 64º17’W), Estads, Sucre, N.E. Venezula, using a micropipette

and serial dilution of a reddish water sample with high microalga

concentrations, a single cell was eventually isolated and cultivated using f/2

Guillard’s seawater medium (Guillard and Ryther,1962) under 80 µmol photons

m-2s-1 (Philips cool day light tubes) at 24 ºC, salinity of 3.6% (NaCl w/v) and a

photoperiod of 12:12. When grown under laboratory conditions the alga turned

green. It was identified as D.salina on the basis of physiological and

morphological characteristics of Dunaliella spp. (Loeblich, 1982; Borowitzka

and Borowitzka, 1988). A D.salina Strain W5 was isolated from salt works at

lake McLeod, Western Australia; and strain N43 from salt works at Bajool,

Queensland. The strains were selected by repeated plating on modified

Johnson’s medium at 20% w/v NaCl salinity at 26 ºC and at a photon flux

density of approximately 180µmol m-2 s-1. Growth was monitored by counting

the cells daily by haemocytometer (Borowitzka, 1988; Borowitzka et al, 1990).

Dunaliella viridis cells were isolated from the Anthellassic lake of

Fuente de Piedra (Málaga, Southern Spain; 37º06’N, 4º45’W). This lake has a

total drying cycle yearly with greatest variation in water temperature (-2 to

42ºC), salinity (2.96 to >30% of total dissolved solids) incident irradiance (up to

2500 µmol m-2s-1) and nitrogen concentration ranging from 6.3 – 3500 µMNO-3.

This population shows some differences from others described previously. It is

very small (3 - 5 µm wide 7 - 9 µm long ~ 50 µm3 volume), with two long

flagella almost twice as long as the cell and with a brown red eye spot in the

5.11

apex. As there is no good taxonomic basis for the description of Dunaliella

species, a provisional name Dunaliella viridis sensu Teodoresco (1905) was

chosen by Borowitzka and Borowitzka (1988).

The cells were cultured (Johnson, 1968) under 150µmol m-2 s-1 at a

temperature of 15ºC (±2) in 2M NaCl (pH 7.5) medium (Jiminez and Niell,

1990). The D.salina cells were isolated from brine (5.5 M total salts) salt works

from the island of Gran Canaria, Canary Islands, Spain (according to Massyuk,

1973). Cells exhibit a strong red colour (red form). The red form was step-wise

adapted during the two months from its natural medium (5.5M NaCl) to sea

water supplemented with 2M NaCl, 8mM KNO3, 2mM MgSO4, 1.9mM MgCl2,

0.01mM Ca(NO3)2, 4mM K2HPO4, and micronutrients (Surzycki, 1971). During this

period the chlorophyll content increased and as a consequence the alga took on

the green colour (green form). This process was reversible, when the green

form was re-adaptable to the initial growing conditions, the cells again took a

red colour. The green form was grown at 26 ± 1ºC under continuous

illumination with white light (50 W m-2) in 1 l glass bottles sparged with a CO2

:air (5 : 95, v /v) mixture (Gomez-Pinchetti et al., 1992).

Eight strains of D.salina, isolated from Salt ponds at Salar de Atacama

and Antofagasta, Chile, were grown as unialgal cultures in J/1 medium

(Borowitzka, 1988) supplemented with varying amounts of NaCl under a photon

flux density of approximately µmol m-2 s-1 (cool fluorescent lamp) under a 12:12

(L:D) photoperiod at a temperature of 20 ± 4 ºC. Cultures growing in 15% w/v

NaCl were used as inocula (Cifuentes et al., 1992). Various strains of Dunaliella

5.12

were isolated from different salterns by single cell isolation technique in ESM

enrichment medium (NaNO3 120 mg l-1, K2HPO4 5 mg l-1 , EDTA-Fe 0.26 mg l-1 ,

vit.B12 10 µg l-1 , biotin 1 µg l-1, Tris-buffer 1g l-1, 270µ mol m-2s-1 , at 30 – 37 ºC,

shaken gently in mechanical shaker at 90 rpm (Powtongsook et al.,1995).

Dunaliella singach was isolated from Singach salt works, Singach,

Gujarat, India. Cells were grown in modified artificial sea (AS) water medium

containing 15% NaCl at 28 ± 0.2 ºC under artificial light at 2000-3000 lux

provided by white fluorescent tube lamps. The cultures were grown in 100-1000

ml Erlenmeyer flasks or 4 - 20 litres open tubes in the growth room in ponds

(algal race ways fitted with paddle wheel, 10m3 culture volumes), maintaining

culture depth between 10 - 15 cm (Goyal et al., 1998). The cells of Dunaliella

strain R was isolated from salt works at Roquetas de Mar, Almeria, Spain;

Dunaliella L4 and L6 strains were isolated from a saline lake on Lanzarote,

Spain. Cells were grown on artificial medium containing 2M NaCl (food grade

salt type I, purchased from salinas de la Rosa, Marcia, Spain) other nutrients

were 5mM NaNO3; 2mM NaHCO3; 80 µM NaH2PO4; 10 µM FeCl3; 8 µM Na-EDTA;

0.37 µM vit.B12; 0.29 µM vit.B1, and essential micro-nutrients. For outdoor

cultures in 20m2 open ponds, sea water of 12.5% salinity was sterilized by

10ppm chlorine and neutralized and supplemented with 1.5 mM NaNO3, 100 µM

NaH2PO4 and 12 µM FeCl3.6H2O (Gonzalez, et al., 2003).

5.10 PIGMENT CONCENTRATION IN SALT WATERS

The pigment responsible for the brightly red colouration displayed by

D. salina has been recognized as a carotenoid. As such it was identified by

5.13

Blanchard (1891) and Teodoresco (1906). Lerche (1937) and Ruinen (1938)

confirmed this identification based on the solubility of the pigment in alcohol

and in ether and on the blue color formed in the presence of concentrated

sulfuric acid. The β-carotene occurs as granules between the thylakoids of the

cell's single chloroplast. Baas-Becking (1931) correctly located the red-orange

pigment in the chloroplast, and Lerche (1937) realized that the carotene masks

the chlorophyll, so that the chloroplast can assume all shades from orange-red

to yellow-green, olive and green. The major carotenoid accumulated by D.

salina and D. bardawil, is β-carotene, a valuable chemical, in high demand as a

natural food colouring agent, as pro-vitamin A (retinol), as additive to

cosmetics, and as a health food (Borowitzka, 1986). Some Dunaliella strains

can accumulate very large amounts of this carotenoid. Thus, as much as 13.8%

of the total dry organic matter in the D. salina community in Pink Lake,

Victoria, Australia, was estimated to be β-carotene (Aasen et al., 1969). Some

strains may contain up to 10% and more of β-carotene in their dry weight,

including a large percentage of the 9-cis isomer (Ben-Amotz et al., 1988). The

first pilot plant for Dunaliella cultivation for β-carotene production was

established in the USSR in 1966 (Massyuk, 1968; Drokova, 1961).

The commercial cultivation of Dunaliella for the production of β-

carotene throughout the world is now one of the success stories of halophile

biotechnology (Ben-Amotz, 1980; Ben-Amotz and Avron, 1983; Borowitzka et

al., 1984). Different technologies are used, from low-tech extensive cultivation

in lagoons to intensive cultivation at high cell densities under carefully

5.14

controlled conditions (Ben-Amotz and Avron 1989). Different methodologies

employed for the optimum production of β- carotene from Dunaliella species

has been discussed in detail by Raja et al. (2007).

5.10.1 PIGMENT CONTENT IN INDIVIDUAL CELLS OF DUNALIELLA

In Dunaliella either light or salinity of the medium or low nutrients in

the medium was used as an inducing/stimulating factor in pigment production

(Annexure 5.3.). In Dunaliella bardawil maximum concentration of chlorophyll

(5 - 10 pg chl. a. cell-1 ) as well as β-carotene (25-45 pg cell-1 ) was observed by

Ben-Amotz et al. (1982), where cells were grown in 3M NaCl under natural

illumination. But other workers (Ben-Amotz and Avron, 1983; Ben-Amotz, 1995;

Sanchez et al., 1996) have recorded a values ranging between 1.42 to 9.1 pg

chl. a. cell-1 and a β-carotene concentration ranged between 7.9 to 35.5 pg

cell-1. The total carotenoid content of D. bardawil ranged between 7.2 to 47.7

pg cell-1 (Gomez et al., 2003). In D. salina chl. a concentration ranged between

0.75 to 17.38 pg cell-1 (Jimenez and Pick, 1994; Orsett and Young, 2000) and β-

carotene concentration ranged between 0.60 to 96.00 pg cell-1 (Ben-Amotz et

al., 1982; Orsett and Young, 2000; Cifuentes et al., 1992; Phillips et al., 1995).

The total carotenoid content of D. salina ranged between 14 to 92.40 pg cell-1

(Gomez et al., 2003; Gomez and Gonzalez, 2005).

In Dunaliella viridis chlorophyll a concentration ranged between 0.78 to

1.59 pg cell-1. The concentration of β-carotene concentration ranged between

0.26 to 52.00 pg cell-1 (Aguilera et al., 1994; Moulton and Burford, 1990).

5.15

5.10.2 EFFECT OF NON-LIVING AND LIVING ADDITIVES ON SALT PRODUCTION

It is known that organic and inorganic substances can modify the crystal

shape of halite from cubic to other forms. For example, dendritic crystals

(Annexure table 5.4) were formed when NaCl solutions were mixed with

ferrocyanide (Ploss, 1964; Shumann, 1965), but this compound is not found in

natural evaporate environments (Javor, 1989).

Halite has a crystalline form based on cubic symmetry. Haloarcula

vallismortis is a triangular bacterium or square shaped bacterium. The

triangular or square shapes of bacterium provide a template that mimics the

crystal structure and thus serves as a means of mechanical nucleation, similar

to the way foreign particles acts as seeds or nuclei to promote crystal

formation in saturated solutions (Norton and Grant, 1988). The formation of a

greater number of cubic crystals in the halophilic archaebacteria was

confirmed at the micrometer level.

5.10.3 EMPIRICAL EXPERIENCE - PIGMENTED BRINE WITH APHANOTHECE BLOOM AFFECT SALT PRODUCTION

Dunaliella salina turns brick red in colour (Fig. 5.1F), while

A.halophytica turns turbid orange. Turbid orange pigmented A. halophytica

badly affects salt crystallization (Fig.5.1A and B) and salt scrapping whereas in

most of the literatures Aphanothece bloom is often mistaken for Dunaliella

bloom and mentioned that Dunaliella affects salt production.

M/s Alaguvel salt works of Alangara Thattu, Tuticorin region reported

the problem of lack of crystal formation in their salt ponds and poor yield. An

5.16

inspection by the author as consultant revealed that the turbid nature of the

brine rendered scrapping of salt difficult. The orange – yellow turbid brine in

the crystallizers was mainly due to the presence of Aphanothece halophytica

(8.6 x 5.16 µm) with a cell count of 726 x 104 cells mL-1 where the

concentration of total carotenoids was about 1.301 mg mL-1. At this

concentration totally brine’s oily consistency retarded the crystal formation. As

a remedial measure, application of sodium hypochlorite (1000ppm = 1mg l-1)

was recommended. Author had suggested this remedy after laboratory trials.

This algal bloom was mainly due to the poor brine management (retaining

bittern in crystallizers) and addition of nitrogen and phosphate sources to the

brine due to the human interference. One other possible way of elimination

was to let out the brine from reservoirs with 296 x 104 cells mL-1 forming

greenish yellow colored brine to prevent loading of further inoculums from the

reservoirs in to crystallizers.

According to Yopp et al., (1978) A. halophytica was cultured from

waters with salinities up to saturated NaCl (~30% w/v). It has an optimum

salinity for growth of about 16% NaCl, but can grow very slowly even in

saturated NaCl. The lowest salinity at which A. halophytica can grow is about

the same as that of Dunaliella. A. halophytica being unable to grow at

salinities <15% w/v and growing quite well at saturared NaCl. A. halophytica

utilize nitrate, ammonia, nitrite and urea almost equally well neither glycine

nor glycyl-glycine were not used effectively. Nitrate nitrogen is the preferred

source than ammonical nitrogen. The highest k value (14.5 h with a doubling

5.17

Fig. 5.1 Dunaliella

5.18

time) for A. halophytica in 2M NaCl medium, at 40-43°C present in a high-light

intensity. Elongated, filamentous and giant cells were observed in saturated

NaCl growth medium.

Turbid orange pigmented Aphanothece halophytica badly affects salt

crystallization and salt scrapping. In most of the instances Aphanothece bloom

exudes more polysaccharide is often mistaken as Dunaliella bloom and

mentioned that Dunaliella affects salt production.

5.11 BRINE COLOUR VERSUS EVAPORATION RATE

Certain hypersaline microscopic flagellate organisms and bacteria colour

the brine of crystallizing ponds red. Thus coloured brine will help absorb some

of the radiation and enhance evaporation, promoting salt production. Some salt

field operations have considered encouraging the growth of the organism by

feeding them with the nutrients and so reducing the evaporation losses. Some

actually add an organic dye to the brine with the object of entrapping all the

incoming radiation.

Table 5.1 Salt analysis of samples from Alaguvel salt works (personal observation, during the study)

Serial number

Salt/chemical constituents Percentage

1. Moisture content of salt 36.67

2. Calcium sulphate (CaSO4) 0.48

3. Magnesium sulphate (MgSO4) 1.45

4. Magnesium chloride (MgCl2) 5.35

5. Sodium chloride (NaCl) 56.05

The dye most commonly used is of the 2-naphthol green type. Green is

not ideal spectral colour for optimum radiation absorption but this particular

5.19

dye has adequate absorptive power, and has a combination of properties

leading to its choice for the duty (Bloch and Schuerb, 1945; Kane and Kulkarni,

1950; Garrett, 1966).

It is commercially available at a competitive price. It remains dispersed

in true solution in natural brine and is reasonably resistant to fading in sun

light. This dye has better fading resistance than other dyes.

The process of evaporation has been used on an industrial scale in many

arid and semi-arid regions. The effect of dyes (Methylene Blue, Congo Red,

Nigrosine, Bismark Brown, and 2-Naphthol Green dyes) on solar evaporation of

brine tested at the Roswell Saline Water Conversion Plant Effluent Ponds, near

Roswell, in Southeast New Mexico indicated that Blue dye increased the solar

evaporation of brine while the Congo Red dye had little or no effect on the

evaporation of brine (Winans, 1967).

At Dry Creek, South Australia, in the 1948-49 summer, a group of 58

acres of salt crystallizing ponds were treated with “Solivap Green 150” dye at a

concentration of 7000 µg l-1 (7.0 ppm) in a six-inch layer of brine over salt

crust, was compared with 87 acres of an untreated control group. Both groups

were supplied with saturated brine, during the season. The result was arrived

by weighing tonnages of salt recovered per acre. The dyed ponds showed an

increase in performance, salt thickness and yield of 15 - 20% over the control

crystallizers (Bonythan, 1965).

5.20

The results indicate that brine alone has a high absorption capacity, but

this is certainly enhanced by the addition of dyes (Solivap is more effective in

this respect than Rayvap). They also indicated that a suspension of red

halophilic bacteria at a carotenoid concentration of 0.30 mg l-1 (= 300 µg l-1) is

only slightly less effective than Solivap at a concentration of 20 mg l-1 (= 20000

µg l-1). Since Solivap concentration in the salt fields are usually only 5-10 mg l-1

(5000-10000 µg l-1), but 18-30 times more concentrated than halophilic

suspension (0.3 mg l-1 or 300 µg l-1 carotenoids). Even then, halophilic

suspension shows maximum absorbance and more effective than the dye. The

results imply that sufficient number of halophilic bacteria in crystallizer brines

(See annexure 5.5) could cause sufficient solar absorption to obviate the need

to use dyes Jones et al., 1981). Field testing of this implication is required.

Note that because of the exponential dependence of absorption with depth,

the presence of colour in brines should be of relatively greater benefit in

shallow areas of brine crystallizers.

5.12 UNIQUE ORGANISM DUNALIELLA IN THE EXTREME ENVIRONMENT AND ITS ROLE IN THE ENVIRONMENT

Dunaliella responds to high salinity by enhancement of photosynthetic

CO2 assimilation and by diversion of carbon and energy resources for synthesis

of glycerol, the osmotic element in Dunaliella. The ability of Dunaliella to

enhance photosynthetic activity at high salinity is remarkable because, in most

plants and cyanobacteria, salt stress inhibits photosynthesis (Liska et al.,

2004).

5.21

5.12.1 ALKALINITY CHANGES GENERATED BY PHYTOPLANKTON GROWTH

In the halotolerant green alga D. salina, deposits of poly phosphates

reach levels near 1 M in Pi equivalents. When stressed at alkaline pH, amines

enter the algal vacuoles and are neutralized by protons released by the

enzymatic hydrolysis of poly P (Pick and Weiss, 1991).

5.12.2 ROLE OF PHYTOCHELATIN IN ION CHELATION

The enhancement of tolerance to reactive oxygen species such as

hydrogen peroxide (H2O2) and superoxide radical anion (O2- ) was achieved in a

marine green alga Dunaliella tertiolecta (ATCC30929) in which phytochelatin

(PC-(-[-Glu-Cys-]-Gly-)n n= 2-10) synthesis was induced by treatment with Zinc

(Tsuji etal,2002). These studies suggest that PCs play an important role not

only in the chelation of heavy metals but also in the mitigation of oxidative

stress caused by heavy metals. Dunaliella PC synthase is expected to be a

useful tool for generating transformed plants practically applicable to phyto-

remediation of various toxic heavy metals. It would also be interesting to apply

PCs and Dunaliella cells containing high levels of PCs in the fields of functional

foods cosmetic and medicine (Hirata, et al., 2005).

5.12.3 UPTAKE OF CALCIUM AND POTASSIUM BY DUNALIELLA THROUGH DIFFERENT MECHANISMS

The presence of two distinct carriers for K+ and Ca2+ in D. salina cells

play, a vital role in hyper-saline environment. The K+ carrier is characterized

by a very high selectivity for K+ versus Na+ the ratio of flux/external

concentration for K+ and Na+ is over 5000 (K+ carrier - with a high selectivity

5.22

against Na+, Li+, and choline+ but not towards Rb+, K+, Cs+, or NH4+). This high

selectivity is necessary for an organism which survives in high extra cellular

NaCl concentrations while sustaining low intracellular Na+ levels. The Ca2+

carrier has a high selectivity against Mg2+ of about 100, suggesting that Mg2+

ions are taken up via a different system. Effects of permeable ions and

ionophores on K+ and Ca++ uptake suggest that the driving force for their uptake

is the transmembrane electrical potential generated by H+-ATPase (Pick et al.,

1986).

Hyper-osmotic shock induces an increase in cytosolic Ca2+ ion that

mediates ionic adaptation (Matsumoto et al., 2002). Dunaliella cells were

cultured at 1-4M NaCl, 5mM K+ and 0.3mM Ca2+ medium accumulated 100 to 200

mM of K+ (Balnokin et al., 1984; Ehrenfeld, and Cousin, 1982; Gimmler and

Schirling, 1978; Ginzburg, 1981).

The Ca2+ is accumulated (1-3mM) by D. salina, likely to play a specific

unknown role in the algae. Dunaliella can grow in the presence of a wide range

Ca2+ concentrations from a few µM to over 10mM. The intracellular

concentration of Ca2+ is not strictly regulated as the intracellular concentration

of Na+ or K+ in Dunaliella. So it is believed that most of the accumulated Ca2+ is

precipitated in a specific target organelle inside the Dunaliella cells (Pick et

al., 1986).

5.23

5.12.4. VACUOLES IN pH MAINTAINANCE

The acidic vacuoles in DunalielIa serve as a high-capacity buffering

system for amines, and as a safeguard against cytoplasmic alkalinization and

uncoupling of photosynthesis (Pick et al., 1991).

5.13 METHODS

5.13.1 BRINE SAMPLING FOR THE ESTIMATION OF DUNALIELLA POPULATION

Brine samples from 4 corners and center of the reservoirs were collected

and pooled and 1 litre from this pooled sample was used for estimating

Dunaliella population. Samples were preserved in 1% Lugol’s Iodine solution,

(final concentration) prepared in the medium/natural brine to prevent bursting

of cells due to hypoosmotic shock. Cells were counted in a standard

haemocytometer (1 mm2 lined grid with a depth of 0.02 mm). At least 5

replicates were taken for each cell number determination.

5.13.2 ISOLATION OF DUNALIELLA FROM BRINE SAMPLES THROUGH ENRICHMENT CULTURE TECHNIQUE

D.salina as well as D.viridis was seen in all the ponds of PSW. One

species turns orange on reaching condensers and other species remains green

even in the crystallizer stage but slightly smaller than the first species. The

zooplanktons, (Voracious feeders of Dunaliella) were filtered using suitable

Zooplankton nets. Though different medium compositions were tried;

successful enrichment was achieved only when seawater enrichment medium of

following composition was used.

5.24

5.13.2.1 Medium Composition

Sea water was heated to boiling temperature and allowed to cool, then

filtered through GF/C filter paper to prevent contamination. To this sea water,

various salts were added as mentioned in the table 5.2 and pH was adjusted to

8.5. It was found that some salts tend to deposit after autoclaving only clear

supernatant medium was used for the study.

Maximum concentration of D.salina and D.viridis were found in the 8th

(11.4-12.4ºBe) and 10th (11.6-13.4ºBe) reservoirs. This may be due to

accumulation of cells by brine movement or due to ideal Baume for growth.

To the Dunaliella containing reservoir brine, medium (1:1 v/v) was

added after filtering with zooplankton net in Ehrlen meyer conical flask and

placed under cool white light of 2000-3000 lux with 16:8h (Light: Dark) cycle,

at room temperature 30 ± 1°C. After one-week, good growth of both the

species was observed.

The two species were separated, by plating on a 0.6 % agar medium. The

moisture content of the medium, allows Dunaliella cells to freely swim about

and later develop into a non-motile, green coloured colony. The two species

were isolated and pure culture was obtained after repeated plating on

Penicillin and Streptomycin containing agar medium and by picking an isolated

clone from solid medium.

5.25

Table 5.2 Modified culture medium of Gomez-Pinchetti et al., 1992.

Ingredient Amount Unit NaCl 87.66 g l-1 KNO3 809.00 mg l-1 MgSO4 493.00 mg l-1 NaHCO3 2.10 g l-1 KH2PO4 697.00 mg l-1 FeCl3 50.00 mg l-1 Na-EDTA 0.26 mg l-1 vit.B12 10.00 µg l-1 biotin 1.00 µg l-1 Tris-buffer 1.00 g l-1 Penicillin G 150.00 µg/ml Streptomycin 1000.00 µg/ml Sea Water 1000.00 ml Trace element mix* 1.00 ml pH. 8.50 *(nutrients - ZnCl2 41µg l-1, H3BO3 610µg l-1, CoCl2 15µg l-1, CuCl2 41µg l-1, MnCl2 410µg l-1, Ammonium molybdate 380 µg l-1, VoCl2 41µg l-1).

After separation and purification on Penicillin G and Streptomycin

containing medium cells were transferred to 1.5 M NaCl liquid medium without

penicillin G and Streptomycin. Frequent shaking was done to get uniform

suspension of the culture. In order to obtain salt adapted cultures the algae

were grown for at least 6 days at one of the five salinities investigated: 1, 1.5,

2, 3, 4 M NaCl. The pH 8.5 was kept constant (after trying various pH 7.5, 8.0,

and 8.5).

5.13.3. GROWTH STUDIES

Specific growth rate was determined, following the methods of Guillard

(1973).

5.26

µ = 3.32• log (N2 – N1) / (t2 – t1)

where µ is the specific growth rate, N2 is the number of cells at time t2 and N1

is the number of cells at time t1.

5.13.4 CALCULATION OF CELL VOLUME

The formula for calculating the volume of prolate spheroids is

V = 4/3. π ab2

where V is the volume, a = ½ length, b = ½ diameter given by Dor (1985).

Chlorophyll and carotenoid estimation was carried out as described in

chapter three.

5.13.5 MASS CULTURE OF DUNALIELLA

Dunaliella viridis cells (Fig. 5.1D), were mass cultured to obtain required

concentration of culture that was used for the continuous experiments carried

out under field conditions. For mass culture, 1.5 M NaCl (pH 8.5) supplemented

Sea water growth medium was used, as almost this concentration matches the

initial bore well brine concentration. For experiments, the algal cells (3.1D)

were harvested by centrifugation (3200 rpm 10 min) and resuspended in fresh

culture medium and grown for another 24 hrs before using the cultures in the

logarithmic growth phase for the experiments.

5.27

5.13.6. CULTURE PREPARATION FOR TRAY EXPERIMENT

Stainless steel trays of the size of 20 x 20 x 7.5 cm were used (3.1E) to

simulate the reservoirs, condensers and crystallizer ponds. 1 litre of brine,

showed a depth of 25 mm and that of 2 litres brine 50mm depth.

The concentration of pigment was taken into consideration as lay man/

non-technical person can visibly see colouration due to the presence of

Dunaliella cells than counting the cells. The different concentrations of

pigment tried were 50, 100, 200 and 400 µg l-1. One litre and 2 litres trials

were carried out and in addition to that 3 different concentrations of brine

(8.2, 12, and 16 ºBe) were used.

Each concentration of pigment in single brine concentration in one

volume of brine was repeated thrice. Serially transferring the brine from Tray 1

(day 0) to Tray 3 (day 2). Likewise chain of trials for each concentration was

carried out continuously without break for each concentration. In toto, 72

trials were carried out [4 concentrations (50, 100, 200 and 400 µg l-1) x 2

volumes (1 and 2 litres) x 3 densities (8.2, 12 and 16ºBe) of brines x 3

repetitions].

After estimating the concentration of total chlorophylls / ml the volume

of culture required for the planned concentration was calculated and

centrifuged at 3200 rpm for 10 minutes. This ensures pelleting of cells.

Supernatant medium decanted and the pellet was resuspended in a small

5.28

volume of selected brine concentration. The brine containing cells were later

transferred to 500 ml standard flask (Borosil) and volume of culture was made

up to 500 ml, after thorough shaking to get uniform suspension and then

poured into the test tray. After repeated rinsing of culture vessel the volume

of standard flask was adjusted to 500 ml with the chosen brine and poured into

test tray, to make a litre of brine. Control tray was filled in first using the

standard flask.

5.14 MEASUREMENTS IN THE TRAY

As depth measurements give an idea about the amount of brine

evaporated and amount of brine remaining brine depths inside the trays were

measured (in mm) thrice in a day i.e., in the morning at 08:00 hours, at noon,

12:00 hrs and at evening 16:00 hrs. Trimmed polyethylene scale was used to

minimize brine loss during measurements. This gives an idea as how far

pigmented cells enhance the rate of evaporation of the brine.

(Note: Salt harvested from tray experiments were analysed following the

similar procedure adopted for brine analysis but reported as percentage).

5.15 SUMMARY

This chapter is exclusively about Dunaliella describing its taxonomy,

ecology, culture, isolation and its role in salt pans. Also discussed are growth

characteristics and field distribution of Dunaliella and brine analyses in terms

of chlorophyll and carotenoid concentrations. Sequel to this chapter is the

outcome of field experiments.