The Effect of Various Environmental Factors on the Growth of a Red Pigmented Dunaliella Species from the Great Salt Lake

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5-1978

The Effect of Various Environmental Factors on the Growth of a Red Pigmented Dunaliella Species From the Great Salt Lake

Sam Oeun May Utah State University

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Recommended Citation May, Sam Oeun, "The Effect of Various Environmental Factors on the Growth of a Red Pigmented Dunaliella Species From the Great Salt Lake" (1978). All Graduate Theses and Dissertations. 3381. https://digitalcommons.usu.edu/etd/3381

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ACKNOWLEDGEMENTS

The author greatly appreciates the generous encouragement, patience, advice, able and ideal guidance of his major professor, Dr. Frederick J. Post. He also wishes to express his gratitude to the members of his committee, Dr. Dean V. Adams and Dr. Raymond Lynn, for their suggestions and review of this manuscript. Lastly, the author wishes to thank his parents, Mr. Chau May and Mrs. Keo Sa Ith for giving him their profound spirit of encouragement in obtaining a Master of Science degree . Sam Oeun May

ii TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS i i LIST OF TABLES . iv

LIST OF FIGURES v ABSTRACT .. vi INTRODUCTION REVIEW OF LITERATURE 4 t1ATERIALS AND METHODS 10 Bas a 1 Medi urn ...... 10 Modified Gaines Medium Composition 10 Organism and Condition of Growth 14 Growth Measurements ...... 16 Chlorophyll and Carotenoid Assay 16

RESULTS 18 Growth of the red Dunaliella species at various sa 1i niti es 18 Growth of the red Dunaliella species at various temperatures 21 Growth of the red Dunaliella species at different light intensities ...... 24 Growth of the red Dunaliella species in different sources of N 27 DISCUSSION . . . . 33 SUMMARY AND CONCLUSIONS 44 LITERATURE CITED .. . . 45

iii LIST OF TABLES

Table Page l . Modified Gaines Medium, Ingredients 11 2. Artificial Salt Lake Water 12 3. Modified Gaines Medium, Final Composition 13 4. The effect of NaCl concentration on the growth of the red Dunaliella species. Growth conditions: 750 lux, initial pH 7.62, 28 ± l 0 C, ammonium carbonate as N- source, and a 22-6 hr light-dark cycle ...... 19 5. The effect of temperature on the growth of red Duna liella species. Growth conditions: 7500 lux, initial pH 7.62, NaCl 10%, ammonium carbonate as N-source, and a 22-6 hr light-dark cycle ...... 22 6. The influence of light intensity on the growth of tae red Dunaliella species. Growth conditions: 28 ± l C, initial pH 7.62, NaCl 10%, ammonium carbonate as N- source, and a 22-6 hr light-dark cycle 25 7. The effect of N-source on the chlorophyll and carotenoid concentrations of the red Dunaliella species. Growth conditions : 7500 lux, initial pH as indicated, NaCl 10%, and a 22-6 hr light-dark cycle ...... 29 8. Th e effect of different N-sources on the growth of the reg Dunaliella species. Growth conditions: 28 ± l C, 7500 lux, NaCl 10%, pH as indicated, and a 22-6 hr light-dark cycle ...... 30

iv LIST OF FIGURES

Figure Page la. The effect of NaCl concentration on the number of cells per ml at stationary phase of the red Duna liella species. Growth conditions: 7500 lu~ initial pH 7.62, 28 ± 1° C, ammonium carbonate as N-source, and a 22-6 hr light-dark cycle 20 lb. Growth rate expressed as generation time in hours 20 2a. The effect of temperature on the number of cells/ ml at stationary phase of the red Dunaliella species. Growth conditions: 7500 lux, initial pH 7.62, NaCl 10%, ammonium carbonate as N-source, and a 22-6 hr light-dark cyc le ...... 23 2b . Growth rate expressed as generation time in hours 23 3a . Th e effect of light intensity on the number of cells/ ml at stationary phase of ~he red Dunaliella species. Growth conditions: 28 ± 1 C, initial pH 7.62, NaCl 10%, ammonium carbonate as N-source, and a 22-6 hr light-dark cycle ...... 26 3b. Growth rate expressed as generation time in hours 26 4a. The effect of N-source on the number of cells/ml at stationary phase of the red Dunaliella species . Growth conditions: 28 ± 10 C, 7500 lux, initial pH 7.62, NaCl 10%, and 22-6 hr light-dark cycle 31 4b. Growth rate expressed as generation time in hours 31

v ABSTRACT The Effect of Various Environmental Factors on the Growth of a Red Pigmented Dunaliella Species From the Great Salt Lake by Sam Oeun May, Master of Science Utah State University, 1978 Major Professor: Dr. Frederick C. Post Department : Biology

A red, obligately halophilic Dunaliella species believed to be D. salina was isolated from the North Arm of the Great Salt Lake, Utah, and its optimum growth conditions were determined. The red pigmented Dunaliella species required an optimum NaCl concentration of 10%, temperature 28° C, and an illuminance of 7500 lux. Ammonium carbonate was preferred over potassium nitrate and ammonium chloride. Potassium nitrate was preferred over ammonium chloride. The average division (generation) time under the above con ditions was 46.5 hours. The alga grown in ammonium carbonate showed a lower content of chlorophyll a compared to those grown in potassium nitrate and in ammonium chloride. The concentration of carotenoids of the alga grown in ammonium carbonate was higher than those in potassium nitrate and in ammonium chloride.

(57 pages)

vi INTRODUCTION

The first discovery of the organism Dunaliella was due to the

i nterest of French scientists in the coloration of the red salt mar

shes along the Mediterranean Sea.

The red Dunaliella species used in the present study was isolated

from the North Arm of the Great Salt Lake, Utah . The Great Salt Lake

is located in the arid Great Basin region of northern Utah. The Lake

is 122 km long, 50 km wide, and 10 m depth . The Great Salt Lake is the

remnant of freshwater Lake Bonneville originally 20 times the size of the present Great Salt Lake . Fifty thousand years ago, Lake Bonneville covered western Utah and parts of Nevada and Idaho. Its remnant has about 3,900 km2 surface area against 52,000 km 2 of the original.

The diminution in size and depth of the lake has resulted in an increase of the total salt content which is 10-12 times that of sea water. The Great Salt Lake receives 2 to 3 million metric tons of dissolved minerals from its 3 main watershed rivers : the Bear, the

Weber, and the Jordan. It has been reported that 60% of the dissolved solids which flow into the Great Salt Lake are sodium and chloride. 2 The Great Salt Lake in contrast to the Dead Sea, another extremely saline water body, has been found to contain a variety of organisms in cluding a number of protozoa, a brine shrimp, 2 or 3 species of brine fly, and several bacteria (Stephens, 1974; Neissenbaum, 1975; Post, 1977) . The main primary producers in the lake are algae of the divisions Chlorophyta and Cyanophyta (Blue green algae). The former is charac terized by 2 flagellated species of the order Volvocales, Q. salina and D. viridis. The construction of a rock fill railroad causeway across the lake in 1959 created two ecologically distinct lakes which not only affect lake chemistry and community metabo li sm but also the biota. Depending on the season, the south arm may be as much as a meter higher than the north arm. The north arm contains higher concentrations of dissolved solids (330-350 mg/1) than the south arm (120-130 mg/l) with the former reported to be populated mainly by Q. salina, and the latter by the green pigmented Q. viridis (Post, 1977). The red strain of Dunaliella from the Great Salt Lake typically lacks a cell wall but is larger in diameter and contains much more carotenoid pigment than the Q. salina strains received from the Indiana University Algae Collection. The chlorophyll pigment of the Great Salt Lake red pigmented Dunaliella is completely masked by the abundant carotenoid pigment. The vegetative cell of the Great Salt Lake Q. salina in its nat ural habitat is about 16-21 ~m in diameter which is larger than the strain of Q. salina, 14 ~m x ll ~m, described by Loeblich in 1972. 3 The present study of the effect of environmental factors on the growth of the Great Salt Lake red Dunaliella species aims to determine its growt h characteristics which is necessary for the study of its fundamenta l biology, and to provide data for model studies on the ecol ogy of the Great Salt Lake. The primary objective of this investigation was to determine the optimum growth conditions of the Great Salt Lake algal species tenta tively identified as Q. salina in respect to: l. salt concentration 2. temperature 3. light intensity 4. nitrogen source preference 4 REVIEW OF LITERATURE

The genus Dunaliella· is worldwide in distribution. Butcher (1959) included 12 species in this ge nus which may occur in diverse habitats from almost freshwater to salt brine (Smith, 1950; Teodoresco, 1905, 1906). The obligately halophilic flagellate Dunaliella was first described by Dunal (1838) as Haematococcus salinus, then Monas dunalii (Joly, 1840), Duselmis dunalii by Dujardin in 1841, Chlamydomonas dunalii by Cohn in 1865, and finally Dunaliella sa lina by Teodoresco (1905). Duna liella species in the Great Sa lt La ke were first reported by Daines (1917) as Chlamydomonas species. Electron microscopy showed that Q. salina lacked a cell wall, being surrounded only by a cytoplasmic membrane (Trezzi et al., 1965). Van Au ken and McNulty (1973) reported considerable difficulty in culturing Dunaliella species on solid media and concluded this was probably due to the fragility of the naked cells. Some studies have reported that Dunaliella cells tolerate a wide range of salt from low to almost saturated NaCl solution and are able to withstand sudden substantial changes in salinities (Melville, 1968; Marre et al., 1958; Marre and Servettaz, 1959; Trezzi et al., 1965; Loebli ch, 1972; Ben-Amotz and Avron, 1973) . The nature of this tolerance has been disputed. Some early workers believed that osmotic balance in Dunaliella was mediated by the occurrence of a high concentration of salt inside the algal cel l s. The cytoplasmic membrane of Q. parva was reported 5 permeable to inulin or sucrose, and even to tris or phosphate (Ginz burg, 1969; Ben-Amotz and Ginzburg, 1969) . Kwon and Grant ( 1971) found that Q. tertiolecta fails to use glucose due to membrane impermeabili ty, not because of the lack of hexokinase which is an enzyme necessary for the breakdown of reduced carbon compounds (Hutner and Provasoli, 1955). However, it might be due to the lack of a specific transport system required for glucose uptake . They found that only acetate and pyruvate could enter the cell. Melville (1968) found that the Q. salina membrane was not instantaneously permeable to Na+, Cl- and K+; a time factor was involved for the uptake. Marre and Servettaz (1959) found that the internal NaCl concentration of Q. salina cells increases in proportion to that of the growth medium. They determined that the alga is highly permable to NaCl. However, a number of more recent studies indicate that the inside of the Dunaliel la cell maintains a considerably lower salt concentration than that present in the surrounding medium (Johnson et a 1. , 1968; Ben-Amotz and Avron, 1972). The production of glycerol in the genus Dunaliella has been studied by a number of workers. Some suggested that the alga produces glycerol in response to salt concentration of the medium (Craigie and Mclachlan, 1964; Brown, 1976). The concentration of intracellular glycerol corre sponded with the increase or the decrease of the extracellular salt concentration of the alga Q. parva (Ben-Amotz and Avron, 1973). It has been suggested that cytopl asmic osmotic balance relies on the degrada tion or the synthesis of intracellular glycerol in response to the external concentration of salt (Ben-Amotz and Avron, 1973; Ben-Amotz, 1975) . 6

Wegmann (1971) showed that upon increasing the concentration of external osmotic substances, Q. tertio1ecta first decreased photosyn thesis, then respiration. He considered glycerol formation in Duna liella as a protective mechan i sm for the survival of the alga in its natural habitat. Masyuk and Yurchenko (1962) reported that Q. salina tolerated pH in the range from 5 to 10.5 and possibly higher. Davie (1976) found that tolerance to mercury in Q. tertiolecta is largely due to the de toxification of this element within the cell, possibly caused by the precipitation of insoluble Hg compounds. Dunaliella species were found to produce H2S and excrete a high level of organic compounds com pared to the prasinophycean alga Tetraselmis species (Craigie et al., 1966). Huntsman (1972) found that the percent excretion of fixed carbon as organic compounds by Q. tertiolecta in healthy cultures was related to cell density and was independent of light and preconditioning of the medium. Many studies have been reported on optimal growth of members of the genus Dunaliella. At pH 9-9.1, Gibor (1956) found maximum growth of Q. salina at 30°C, while Q. viridis grew best at l4-l6°C. Mil 'ko (l963b) reported that the maximum growth rate of Q. salina occurred at 25°C at 6000 lux light. Mclachlan (1960) reported the necessity of all the elements of sea water, except chloride, for the growth of Q. tertiolecta (Butcher). The optimum salt co ncentration for growth of Q. salina has been found to be different in several reports. Many workers found that 5-15% NaCl was the range preferred by Dunaliella 7 species (Gibor, 1956 ; Mil 'ko , 1963a, 1963b; Johnson et al., 1968; Me lvi lle, 1968; Loeblich, 1972; Brock, 1975). Loe blich (1970) showed that with Q. sa lina high temperature and acidic pH reduced the growth rat e. VanAuken and McNu lty (197 3) determined the optimum conditions for growth of an unidentified Dunaliella spec i es isolated from the Great

Sa lt Lake to be: 32°C, 19.2% NaCl , 1-2% co 2, 25 -35 Klux, pH 5.8-6.5. Dun aliella salina contains large amo unts of a-carotene, traces of a-carotene and lutein (Fox and Sargent, 1938; Strain, 1958; Dales, 1960; Drokova et al., 196 4; Mil 'ko, 1963a, 1963b ) . The alga i s al so a valu- ab le so urce of vitamin A. It has bee n found that a change in co lor from green to red i s induced by high light i ntensity, high salinity, nitrogen or phosphorus deficiency and an acidic pH all of which lead to stimula tion of carotenoid production (Mil 'ko, 1963a, l963b; Nelville, 1968; Mironyuk and Einor, 1968; Mushak, 1968; Loeblich, 1972). Dunaliella salina was found capable of surviving at low tempera tures (Kaniwisher, 1957). Po st (1977) found that there was no change in algal moti lity for Q. viridis and Q. salina in the Great Sa l t Lake even wh en the lake temperature dropped to 0°C or below. Duna liel la salina was capable of surviving at -10°C for three months (Melville , 1968) . The tolerance of low temperature in darkness also has been re ported (Antia, 1976; Post, 1977 ). Great Salt Lake Q. viridis i s reported to exhibit a bloom in the late spring (Wirick, 1972; Stephens and Gillespie, 1972, 1976) while Q. salina does not show predictable appearance of blooms (Post, 1977). When the Great Sa lt Lake temperature i s above 6°C, Dunaliella species 8 are actively grazed by the brine shrimp zooplankter Artemia salina (Wirick, 1972). Grant (1967) studied the action of light on nitrate and nitrite assimilation by Q. tertiolecta (Butcher). He indicated that C0 2 in the presence of light increases assimilation of nitrate or nitrite. Duna liella salina of the Great Salt Lake preferred ammonia in one study (Stube, 1976), while Q. viridis was reported to be stimulated by nitrate (Stephens and Gillespie, 1976). s-10

MATERIALS AND ~1ETHODS

Basa l Medium

All glassware used for the preparation of nutrients, purifica tion of the organism and storage of all solutions was cleaned in the following manner: a) washed with hot detergent solution b) rinsed 3X with deionized water

c) washed and soaked for 24 ho urs in 1:3 (v/v) HCl :H 20 to dissolve salts and displace metal ions. Cleaning solution containing dichromate was not used because the reagents are diffi cu lt to remove and are toxic to some organisms d) rinsed 5X with a large volume (3-5 1) of deionized water . All med ia and stock solutions were prepared with glass dis tilled water.

Modified Gaines Medium Composition

The alga was grown as a stock cu l ture in 1000 ml of modified Gaines medium made with 12.5% (w/v) NaCl in an artificial Salt Lake water. The compositions of modified Gaines med ium, artificial Salt Lake water and the final composition of the mod ified Gaines medium are shown in Tables l, 2 and 3, respectively. Solution 1 was added to the artificial Salt Lake water and auto claved. After cooling the indicated amount of separately autoclaved phosphate solution 2 and meta l solution 3 were added folloVJed by 11

Table 1. Modified gaines mediuma.

Ingredients Solution 1 1 molar

or KN0 , 1 molar ) 3 NH 1 or 4CL, molar ) Solution 2 1 molar

Solution 3 - Metals (g/1)

Na EDTA 2 6. 00 FeCL 3·6H20 0.288 H}03 6.84 Mncl 2·4H 20 0.864 znc1 2 0.0624 CoC1 2·6H20 0.0258 pH 7.5 Solution 4

Vitamin B12 100 11g/ml Solution 5 Thiamin 0.002 g/ml Solution 6 Biotin 0.0001 g/ml

~/ Post, personal communication 12

Table 2. Artificial salt lake watera.

ARTIFICIAL SA LT LAKE WATERa

KCl 5 g

MgS0 4·7H 20 10 g CaCl 2·2H 20 0.2 g NaCl 125 g Distilled water 1000 ml

~ Post, personal communication 13

Table 3. Modified gaines mediuma.

Final Composition Solutions Volume (ml) 2.06 (or 2.66) (orl.41) 2 0.266 3 6.66 4 1.32 5 1.32 6 1.32 Artificial Salt Lake Water (Table 2) 1000 pH as is

~ Post, personal communication 14 solutions 4, 5 and 6 which had been filter sterilized. Autoclaving was at 15 lbs. for 15 minutes. The amount of NaCl in Table 2 was varied depending on the percen tage of sodium chloride needed in the experiment. The pH of the medium was not adjusted.

Organism and Condition of Growth The red Dunaliella species used in this study was isolated in ax enic culture by M. A. Kasnicka at Utah State University from the North Arm of the Great Salt Lake, Utah. A stock culture of cell s was grown in modified Gaines medium for 2-3 weeks until it had reached the stationary phase. Cells were then harvested aseptically by centrifugation at 2000-3000 rpm for 10 minutes. Prior to inoculation into the experimental media, the cells were washed repeatedly with steril e artificial Salt Lake water of the same concentration to remove the original nutrients. Five ml of modified Gaines medium in 13 x 100 mm screw capped test tubes served as the test medium. Six to eight replicates of each test condition were made. Each tube was inoculated with washed cells so that the initial concentration of cells was 1 x 104/ml. Five different NaCl concentrations were studied: 5%, 10%, 15%, 20% and 25 % (w/v) in the Artificial Salt Lake Water. To determine the optimal temperature for growth, the alga was grown at 10 different temperatures. The culture tubes were incubated on a temperature gradient bar. This bar consisted of an aluminium block 75 x 10 x 3 cm 3 with aluminium legs 10 x 10 x 3 cm3 attached 15

to the block with aluminium bolts. The l ength of the bar was covered on all sides by 15 mm thick sytrofoam. The styrofoam on the top of the bar had 10 holes cut about 6 em in diameter. Aluminium foil covered the styrofoam and was pressed into the holes to be in contact with the sur face of the bar. The aluminium foil in the holes was filled with distilled water. The 13 x 100 mm screw capped tubes were incubated in an incl i ned position within the holes. A thermometer was placed in the water in each of the 10 holes of the styrofoam to record their tempera ture. One end of the temperature gradient bar was put in a water bath with flowing cold tap water adjusted to produce a temperature in the

first culture tube of 13 ± 1°C. The other end of the bar was put in a second water bath adjusted so that the temperature of the last culture

tube was 35 ± 1°C. Th e tubes between the two ends of the bar formed a gradient between 13 and 35°C. To determine the preference of the alga for a particular source of nitrogen, nitrogen was supplied in 3 forms: potassium nitrate, ammonium carbonate or ammonium chloride (Table 1) . The composition of the modified Gaines medium shown in Tabl e 1 was varied only in that the source of nitrogen was changed. The red Dunaliella was grown under 8 different light intensities ranging from BOO to 10,700 lux. Illumination was supplied by 2 General Electric cool-white 40 Wfluorescent lamps No. C 240 ACL. Intensity was measured with a Gossen Panlux Electronic Footcandle Meter. The test tubes were kept in an inclined position under the light. All tubes -were shaken daily at least 1 minute with a Vortex Jr. mixer. 16

Growth Measurements The cell population in each culture was determined at daily in- tervals by making 4 to 8 replicate cell counts per treatment using a hemacytometer counting chamber (Stein, 1973). Cells were killed before counting with a 7% formaldehyde solution (Wegmann and Metzner, 1971). Average counts were made from 8 squares of 1 mm2 Cells per ml was then calcul ated by using the following formula (Stein, 1973): Cells/ml = Q x 104 where Q is the average number cells over 1 mm 2 of the hemacytometer grid. The division (generation) time was calculated following the equation by Stein (1973). During counting work the chambers were washed with distilled water, then acetone, then dried with cheese cloth. Th e chambers were filled with sterile Pasteur pipettes. The cel l numbers were plotted against time to determine the growth curves of the organism. A calibrated ocular micrometer was used to measure cell diameters. Averages were determined from 50-100 cells.

Chlorophyll and Carotenoid Assay Fifteen ml samp les of algal culture were centrifuged in tubes at 5000 rpm for 10 minutes. Th e pellet was resuspended in 5 ml of 90% acetone (Stein, 1973; APHA, 1975); the tubes were then refrigerated in the dark. After 24 hours, the acetone tubes were centrifuged at 5000 rpm for 10 minutes. The acetone extract was decanted into a cuvette for chlorophyll and carotenoid determination. The absorption of acetone extract was then determined on a Beckman DU-2 spec trophotometer at 17 at 480 nm for carotenoid, at 630, 645 and 663 nm for chlorophyll and at 750 nm for a turbidity correction (APHA, 1975 ; Stein, 1973; Parson and Strickland, 1963; Strickland and Parson, 1972). The 0. D. reading at 750 nm was subtracted from each of the other readings. The chlorophyll concentrations were calculated using the following equation (APHA, 1975; Strickland and Parson, 1972) :

ca 11.64 o663 2.16 o645 + 0.10 o630 cb 20.97 o645 3.94 o663 3.66 o630 where: Ca and Cb were concentrations of chlorophyll a and b respectively in the extract, in mg/1, and 0 , D and were optical densities at the respective wave 663 645 o630 l engths after subtraction of the reading at 750 nm. The carotenoid concentrations were determined by usi ng the follow ing equation (Strickland and Parson, 1972):

C = 4.0 E 480 where E was extinction at the wavelength indicated, and the concentra tion of carotenoid wa s calculated in millispecific pigment units (mSPU) by using the formula (Strickland and Parson, 1972):

mSPU/1 = (C/V)/N where C was the concentration of carotenoid from the above equation and V was the original volume of culture in liters, and N was the number of cells in millions. 18

RESULTS

Growth of the red Dunaliella species at various sa linities. The red Dunaliella was grown in 5 different salt concentrations in modified Gaines medium: 0.85 M or 5% (w/v) NaCl, 1.71 M or 10%, 2.56 M or 15%, 3.42 M or 20%, and 4.27 M or 25% (w/v) NaCl. The growth in cell numbers of the alga at different salinities is shown in Figure la. Figure lb and Table 4 show the growth rate expressed as division (generation) time in hours. In our present study, the fastest rate of growth and the maximum biomass of the red Dunaliella species occurred in 1.71 M or 10% (w/v) NaCl with a generation time at about 42 hours (Table 4), almost two days. The generation times at 15 and 20% (w/v) NaCl were about 53 and 57 hours respectively, and at 5% (w/v) NaCl, 66 hours. When the salt concentration was increased to 25 % (w/v) NaCl, the generation time, 92 hours, was more than double the 10% NaCl time . After 3 weeks of incubation, the cells of the alga looked green yel l owish in 5, 10 and 15% (w/v) NaCl, the cells in 20 and 25% NaCl appeared red. Measurements of algal cells were made in each salt condition at the stationary phase of growth. The cells at all concentrations were spherical but larger in 5-15% NaCl than those grown in 20-25% NaCl (Table 4). The diameter of the algal cells in 20-25% NaCl was 17.3-

17.5 ~m. in 5-15% NaCl, 19.1-19.5 ~m . 19

Table 4. The effect of NaCl co ncentration on the growth of the red Dunal i ell a species. Growth conditions: 750 lux, initial pH 7.62, 28 ± l°C, ammonium carbonate as N-source, and a 22-6 hr light-dark cycle.

% NaCl Stati onary phase Stationary phase Generation time (w/v) ce 11 diameter ~ m Ce ll s/ml x 10 hrs

5 19.5 46 66 10 19.1 82 42 15 19.3 71 53 20 17 .5 55 57 25 17. 3 25 92 20

a 10

X 6

2 0 b 100

20 0 5 10 15 20 25 PERCENT (w/v) CONCENTRATION NaC l Figure l a. The effect of NaCl concentration on the number of cells/ ml at stationary phase of the red Dunaliella species. Growth conditions: 7500 lux, initial pH 7.62, 28 ~ 1° C, ammon ium carbonate as N-source, and a 22 -6 hr li ght-dark cyc le . Figure lb. Growth rate expressed as generation time in hours . 21 At the stationary phase, the cells in 20% and higher salinity moved slower than those in 5-15% NaCl. It shou ld be noted that when the modified Gaines medium in 25% NaCl became salt saturated through evaporation, the alga was still active.

Growth of the red Dunaliella species at va rious temperatures. The optimum temperature for growth was determined for the red Dunaliella species over a temperature range from 13 to 35°C in 10% (w/v) NaCl-modified Gaines medium, initial pH 7.62, and at 7500 lux. The effect of temperature on the growth of the alga is shown in Figures 2a and 2b , and in Table 5. Maximum cell yield was at 28 ± l°C with a generation time of about 53 hours. When the temperature was increased by 7 degrees (35°C), the generation time increased about 6 times that at the optimum temperature (from 53 hrs to 300 hrs). When the temperature was lowered to 13° C, the generation time increased about 3.2 times that at the optimum temperature. But the increase was considerably less than at high temperature. As indicated in Fig ure 2b, when the temperature was increased 4 or 5 degrees from the optimum temperature (from 28 to 32° C), it was apparent that growth decreased drastically. Th e generation time was increased 2 or 3 times (from 53 hrs to 133 hrs). The red Dunaliella is much less tolerant of temperatures, above optimum than temperatures below optimum. The cells of the red Dunaliella grown at 13° C, and 32-35° C appeared relatively larger than those grown between 17 to 30° C (Table 5). The range of diameter of the algal cells at 17 to 30° C was 18.3 22

Table 5. The effect of temperature on the growth of red Dunaliella spec ies. Growth conditi ons: 7500 lux, initial pH 7 .52, NaCl 10%, ammoni um carbonate as N- so urce , and a 22-6 hr li ght-dark cyc le .

In cubation Stati onary phase Stationary phase Generation time ~ m cell s/ml x 105 hrs oc ± 1o cell di ameter

13 21.5 20 171 17 19.3 30 11 4 19 18.5 34 96 21 19.1 40 85 24 19 .5 45 80 26 l B.3 51 56 28 19. 1 81 53

30 19.9 72 50 32 21. 1 25 133 35 21.5 ll 300 23

8 a

6 "'0 X 5 'E '- V) 4 _) _) w u 3

300 b '- ::l"' 0 :r: 200 w :5 160 f- z: 0 120 f- 0 <( c:: w z: 80 w "' 40 10 15 20 25 30 35 TEr·1PERATURE °C Figure 2a. The effect of temperature on the number of cells/ml at stationary phase of the red Dunaliella species. Growth conditions: 7500 lux, initial pH 7.62, NaCl 10%, ammon ium carbonate as N-source, and a 22 -6 hr light-dark cycle. Fi gu re 2b. Growth rate expressed as generation time in hours . 24 to 19.9 ~m , and 21 .1 to 21.5 ~m at 13°C and 32-35° C. Only the ce ll s at 13° C appeared yellow-orange, at the other t emperatures the ce lls appea red red .

Growt h of the red Dunaliella speci es at di fferent light intensities. Dunaliella was grown at B different light intensities varying be tween BOO to 10,700 lux. The results of the growth of the organ ism are shown in Figures 3a, 3b, and Table 6. From the Figure 3b, the maximum growth of the red Dunaliella spe cies at 10% NaCl and at its optimum temperature 2B° C occurred at about 700 lux with a generation t ime of 40 hours (Table 6). Th e organism showed sl owe r growth at l ow intensity. Increasing the light intens ity from BOO lux, also made the growth rate faster. Li ght sa turation seemed to be at 10,700 lux or hi gher. Increasi ng the illumina tion from 7500 lux to 10,700 lux slowed growth of the red Ounaliella somewhat and intensities below 7500 lux became increasingly limiting (Fig. 3a an d 3b). The generation time was considerably long er at low intensity, for example at 800 lux (Table 6) it was 109 hours. Light intensi ty beyond 7500 lux was growth-inhibitory (Fig. 3b) and t he cell counts decreased (Tabl e 6). Th e cells of Dunaliella at 7500 lux and higher were brick red, and were co nsiderably smaller and rounder than the cells at low intensities (Table 6) which were larger and tended to appear pear shape. At low intensities, for example at 1200 lux, the cells of the alga were greener than those in higher intensities. 25

Table 6. The influence of light intensity on the growth of the red

Dunaliella species. Growth conditions: 28 ± 1° C, initial pH 7.62, NaCl 10%, ammonium carbonate as N-source, and a 22-6 hr light-dark cycle.

Illuminance Stationary phase Stationary pha§e Generation time lux cell diameter ~m ce11s/ml x 10 hrs

800 19.1 23 109 1200 19 .3 30 96 1800 20.1 42 85 3000 19.5 60 66 3600 20.1 80 57 5500 20.3 95 52 7500 17.3 120 40 10 ,700 17.1 110 47 26

a 12

10 0

<.0 0 8

X 6 0 ...... 'E V1 --' 4 --'w u 2

0 b 120

V> s.. 100 0 :r:"' w 80

1--"" z: 60 0 l et: 40 0:: w z w <.!l 20

0 1000 5000 10,000 LIGHT INTENSITY, LUX Figure 3a. The effect of lioht intensity on the number of cells/ml at stationary phase of the red Dunaliella species. Growth conditions: 28 t 1° C, initial pH 7.62, NaCl 10% ammonium carbonate as N-source, and a 22-6 hr light-dark cycle. Figure 3b. Growth rate expressed as generation time in hours. 27 The difference in co lor of the red Duna li ella species in different light intensities could be observed in the culture tubes. The change in color strongly suggests that increasing the light intensity also increased the production of carotenoid and the reddening of the alga. The al ga l cells were measured at each light intensity. From Tabl e 6, it is apparent that high light intensity reduces the size of the alga. From this study, it appea rs that light intensity influence the growth rate, the color and the size and/or shape of the red Dunaliella species. Cells were transferred from low light intensity, 800 lux, to high light intensity, 10,700 lux, to see if there wou ld be a change in color due to the increase in light intensity. The cu lture tubes which had been incubated under 800 lux were transferred and incubated under 10,700 lux. After the first week, the algal cells appeared microscop ically from green to yellowish. After the second week, the cu lture in the tube looked more yellow compared to the tubes incubated under 800 lux, about 50-60% of the cells were micro scopically red. The algal growth in the tubes showed a red-orange color but not until the fourth or the fifth week.

Growth of the red Dunaliella species in different sources of N.

From the Figure 4a, the oxidized form of nitrogen, KN0 3, was pre ferred over its reduced form (NH 4Cl). But when the reduced form of nitrogen was supplied along with carbonate co3- (ammonium carbonate), the growth rate was faster than in potassium nitrate. 28 As shown in Figures 4a and 4b, the maximum cell yield occurred with ammonium carbonate as the N-source with a generation time of about

51 hours. The generation times of the alga in KN0 3 and NH 4Cl were 77 and 82 hours respectively. It should be noted that after the third week of culture, the growth of the alga in ammonium chloride was de pressed, and it was found that the pH had decreased to 5.52 (from 6.95). The algal cells were bleached during this period as well. The utilization of the ammonium apparently released HCl causing the pH to decline with subsequent damage to the cells. The pH of the algal growth in ammonium carbonate decreased from 7.62 to 7.25, while in potassium nitrate it increased from 7.67 to 9. 34 (Table 8). In the present study, ammonium carbonate seemed to be preferred by the red Dunaliella. During the exponential phase, the cells of the red Dunaliella species in the three different source of nitrogen were similar in size, (Table 8). The effect of the nitrogen source on the growth of the red Duna liella in modified Gaines medium is shown in Tables 7 and 8. The amount of chlorophyll a and b, and carotenoid in the cells wa s measured from the cells grown Vlith three different nitrogen sources. Cells from potassium nitrate showed a higher content of chlorophyll a (0.534 mg/1/ 106cells) than the cells grown in ammonium carbonate (0 . 494 mg/l/106 cells) or in ammonium chloride (0.525 mg/1/106 cells) (Table 6). The concentration of chlorophyll bin cells from ammonium carbonate (0.110 mg/l/106 cells) was higher than in those from ammonium chloride (0.095 mg/l/106 cells) or potassium nitrate (0. 108 mg/ l /106 cel l s) . Table 7. The effect of N-source on the chlorophyll and carotenoid concentrations of the red Dunaliella species. Growth conditions: 7500 lux, initial pH as indicated, NaCl 10%, and a 22-6 hr light-dark cycle.

N-source Ch 1orophy11 a Ch 1orophy11 b Carotenoids 1 M mg/1 mg/106 cells mg/l mg/l o6 ce 11 s mSPU/l mSPU/1 06 ce 11 s

(NH 4)2C0 3·H 20 3.952 0.494 0.880 0. 11 0 17.800 2.225 KN0 3 3.471 0.534 0.702 0.108 9.529 1 .466 NH 4Cl 2.205 0.525 0.399 0.095 6.547 1 .559

N .0 Table 8. The effect of different N-sources on the growth of the red Dunaliella species.

Growth conditions: 28 + 1° C, 7500 lux, NaCl 10%, pH as indicated, and a 22-6 hr light dark cycle.

N-source Stationary phase Stationary phase pH Generation time 1 r~ cell diameter ~m cells/ml x 10 original final hrs

(NH 4)2C0 3·H 20 18.3 80 7.62 7.25 51 KN0 3 18.5 65 7.62 9.34 77 NH 4Cl 18.1 42 6.95 5.62 82

w 0 31

10 a

~ D 8 r-- X r--- E 6 ~ ~ r--- ~ 4 w~ u 2

0 2 3 ammoni um carbonate

~ ~ ~ b 2 potassi um nitrate 0 100 I 3 ammonium chloride w 90 ~ r z 70 8 r ~ 50 ~w z w 0 30 2 3 SOURCE OF NI TROGEN, 1M

Figure 4a. The effect of N-source on the number of ce ll s/ml at stationary phase of the red Dunaliella species. Growth conditi ons: 28 ± 1° C, 7500 l ux, ini tial pH 7.62, NaC l 10%, and 22 -6 hr light-dark cycle. Figu re 4b. Growth rate expressed as generation time in hours. 32 However, the algal cells of red Dunaliella species grown in ammo nium carbonate gave the highest yield of carotenoids (2.225 mSPU/l/106 cells) compared to the algal cel l s grown in potassium nitrate (1.466 mSPU/l/106 cells) and ammonium chlorid& (1.559 mSPU/l/106 cells). The carotenoid concentration of the alga in ammonium carbonate was i ncreas - ed by a factor of 1.42-1.51 . The cells of the alga appeared red in ammonium carbonate medium while they appeared green in both potassium nitrate and ammon ium ch l oride. Th is is probab ly due to the change in the arne 1nt of the carotenoids in the algal cells. 33

DISCUSSION

Mil 'ko (l963a) grew Q. salina in a NaND 3-medium without vitamins with salinities in the range of 6 to 18% NaCl, and found that the op timum salt concentration for growth was at 6% NaCl. The algal cells

had a green color. Loeblich (1972} cultured Q. salina with KN0 3-MH medium at 25° C, pH 8 under 21,000 lux, and found a minimum generation time of 21 hours at 10-15% NaCl . The algal cells appeared red green at 5 to 15% NaCl, yellow at 20% and red at 25 %. The alga did change color to yellow-orange when green cells were transferred from low salinity to higher salinities. Melville {1968} reported that under 2700 lux, pH 8, 22-25° C with

NaN03, the best growth of Q. salina from the Great Salt Lake was ob tained at 10-15% NaCl. The maximum yield was at 15% NaCl (or 2.55 M). Gibor (1956} reported that the optimum salinity for growth of. a Duna liella species was 10 to 15% NaCl, Johnson et al. (1968) between 5-12%

(w/v) NaCl . Van Auken and McNulty (1973} isolated an unidentified Dunaliella species resembling Q. viridis from the Great Salt Lake and under optimum conditions found that maximum growth occurred in 2.75 M or 19 .2% (w/v) NaCl with a generation time of 30 hours. Brock {1975} found an optimum salinity for growth and photosynthesis of a Dunaliella species from the Great Salt Lake at 10 to 15% (w/v) NaCl. Our present study indicates similar results. We found that the red Dunaliella isolated from the North Arm of the Great Salt Lake showed best growth at 10-15% (w/v} NaCl, the maximum cell yield was at 10% (w/v) NaCl or 1.71 M with a generation time of about 42 hours. 34 Our strain had a generation time longer than the strain of Loeblich (1972). The algal cells of our strain appeared green in low salinities, for examp l e 5-15% (w/v) NaCl, and red at 20 - 25% (w/v) NaCl. Dunaliella species have been reported to survive in a wide varia tion of salinities. Lower or higher concentrations than 10-20% (w/v) NaCl have been reported not to favor growth of Dunaliella species. Some organisms which tolerate extreme salinities do so by forming osmo tically active organic substances in the cells (Soeder and Stengel, 1974). It has been found that at low salt concentration, the osmoreg ulation might be overloaded, or the plasma membrane may be leaky; or it increases secretion of metabolic constituents into the medium (Van Auken and McNulty, 1973). Baas-Becking (1930) transferred Q. viridis from a medium contain ing 15% NaCl into more concentrated and less concentrated NaCl media, and he found the alga capable of tolerating the transfer. Dunaliella salina, Q. viridis and Q. tertiolecta have been reported to withstand considerabl e salinity variations (Gibor, 1956; Mclachlan, 1960; Jo hn son et al., 1967) . Melville (1968) transferred Q. salina from a solution containing 2. 04 M and 2.72 M NaCl into a range of salt concen trations from 0 to 5.61, and he found no growth or survival from 0 to 0.17 M NaCl. A lag in growth occurred when Q. tertiolecta was trans ferred from 0.5 M NaCl to higher salinities. When adapted to higher salinities they had increased carbonic anhydrase in the cells (Later ella and Vadas, 1973) . . The adaptation capability of the algae to change of osmotic pressure has been reported. It was found that there is an increase i n 35 isofloridoside, an osmotically active a-galactoglyceride, when Ochromo

~ (Kauss, 1967} and red algae (Kauss, 1968} were transferred to higher salinities. Dedio {1966, 1968) reported that Chlorella responds to osmotic stress by accumulation of oligosaccharides. Mushak (1968) found that salinity-induced color changes from green to red in Duna liella were accompanied by a decrease in RNA content and the accumula tion of protein. Antonyan and Pinevich (1967) observed that the adap tation of Chlorella to high-salinity stress is a rapid loss of TCA-soluble organic phosphate into the medium. The transfer of Duna liella to higher sa linity shows an inhibition or a decrease of photosynthesis (Mironyuk and Einar, 1968; Loeblich, 1972}. The temperature of the Great Sa lt Lake ranges from -5° to 35° C (up to 45° C in the shallow margin) (Post, 1977). Dunaliella species have been reported to grow at temperatures between 5 and 40° C {Gibor, 1956; Mil 'ko, l963a, 1963b; Mclachlan, 1960; Johnson et al . , 1968; Van Auken and McNulty, 1973; Brock, 1975}. Gibor (1956} found that .Q_. sali na and .Q_. viridis grew best at 30° and 14-1 6° C respectively with light intensity of 100 ft-c (about 1076 lux). Mil 'ko (1963b} reported the maximum biomass of .Q_. salina at 25° C. Loeblich (1972) found 27° C the optimum temperature for growth of .Q_. sa lina isolated from the sa lt evaporating ponds in Punta Colorado Sa lina, Baja California, Mexico. Van Auken and McNulty (1973} found the optimum temperature for growth of an undescribed Dunaliella of the Great Sa lt Lake was at 32° C with a generation time of 23.8 hours which is shorter than what we found with our strain red Duna- liella isolated from the Great Salt Lake. We found the maximum cell 36 yield at 28 t 1° C with a generation time of 53 hours under a light intensity of 7500 lux. The algal cells of our strain appeared larger in lower temperature, for example at 13°C, and in higher temperature, 35°C. J¢rgensen (1968} studied Skeletonema costatum and found that the concentration of pro tein per cell and the length of the cells increase with decreasing temperature. It was found that decreasing temperature from the opti mum for Skeletonema and Chlorella also decreases the rate of the light-saturated photosynthesis (Wassink et al., 1938; Aruga, 1965; Steemann and J¢rgensen, 1968}. The growth of our algal strain was slower at temperatures lower and higher than 28° C. J¢rgensen (1968} found that the growth rate of~- costatum decreases with the decrease of temperature but there is the increase of production of organic matter. Th e cells of Scenedesmus obliguus (Turp.) Kuetz. were found to change size and shape at 11 and 34°C (Martinez and Krauss, 1977). They found that increasing or de creasing the temperature from 30° C in this species shown the decrease of growth. Higher temperature i s be lieved to affect growth by denatur ing the enzyme-protei n complex. And lower temperatures may suppress growth by inactivating the physico-chemical processes within the cells

(J¢rgensen, 1968; Martine~ and Krauss, 1977). Van Auken and McNulty (1973) suggested that the cells became distorted and abnormally large at higher temperature due to damage to the cell enzymes responsible for the osmoregulation system. Post (1977) reported that there is no apparent cha nge i n moti lity of two species of alga, Q. salina and Q. viridis, when the temperature of the Great Salt Lake drops to 0° C or below. 37 In earlier studies, it has been shown that light variation affects algal physiology. Sorokin and Krauss (1965) found that increasing the light intensity at first favors cell division and after the optimum is reached, inhibits. Low intensities affect cell division by limit ing the available photosynthetate while light of higher intensity has an inhibitory effect. Mil 'ko (1963b) found that the growth of Q. salina increased when the illumination increases from 1000 to 6000 lux. The maximum biomass occurred at 6000 lux. Melville (1968) showed that an increase in light intensity resulted in an increase of biomass of Q. salina when NaCl concentration was held constant at 14%. Loeblich (1972) obtained the shortest generation time of Q. salina at 850 ft-c (approximately 9000 lux) or greater in 15% NaCl. The yield of our red strain of Dunaliella reached its maximum biomass at 7500 l ux. At 10,700 lux the generation time increased again. VanAuken and McNulty (1973) reported that the growth rate decreases when the light intensity is higher than 35,000 lux for the growth of an unidentified Dunali ella (Q. viridis?) species which was isolated from the Great Salt Lake. They found that the maximum growth took place between 25,000 and 35,000 lux with a genera tion time of 16 hours. The growth rate decreased upon increasing the light intensity above 35,000 lux due probably to pigment bleaching or photo-oxidation. High light intensity has been found to inhibit respiration of actively photosynthesizing cell s (Brown and Tregunna, 1967; Epel and Butler, 1970). Watt (1969) found that the extracellular release of photosynthetic carbon products reaches its maximum at light intensities 38

which inhibit photosynthesis slightly. Fogg et al., (1965) stated that there is an inhibition of extracellular photosynthetate release at even higher light intensities. The release of extracellular carbon was also found to be high at very low light intensities (Watt, 1966, 1969 ; Watt and Fogg, 1966 ). Most studies of light intensity show direct correlation with specific composition of algal pigmentation. It has been found that higher light intensity resulted an increase in carotenoid production which may be responsible a part for the reddening of algal cells (Mil 'ko, l963b; Lo eb lich, 1972). The strain of our red Dunaliella became orange-red and red when the light intensity was higher than 1000 lux. The trans fer of cells grown at low light intensity, for example 800 lux, to higher light intensity (10,700 lux) changed the color of the cells from green to red. It has been reported that the light adaptation of the "Chlorella type" is completed mainly by changes in pigment concentra- ti on ( J¢rgensen, 1969; Be a 1e and App 1eman, 1971). Mi 1 'ko ( l963b) found that at 1200 lux there is little chlorophyll, but a maximum amount of s-carotenoids. The a-carotenoid concentration rises steadily with increasing light intensity from 500 to 12,000 lux. Melville (1968) found that at 350 ft-c (3700 lux), the cells of Q. salina turned and remained yellow, but at 2000 ft-c (about 21,500 lux) all cells turned red. Loe blich (1972) reported that at 15% NaCl, the carotenoid content was foun d to increase in a linear fashion, from 4.6 ~g/ 10 6 cells at 53 ft-c (about 570 lux) to about 21 ~g/ 10 6 cells at 2000 ft-c (about 21,500 lux), an increase of about four and a half fold. 39 Ziegler and Schanderl {1969) explained that the destruction of ch lorophy ll in Chlorella by high light intensity is favored by 02 and involves an increase of chlorophyllase activity. The autotrophic Euglena during adaptation to higher light intensity shows a decrease of chlorophyll and lipid content (Constantopoulos and Bloch, 1967). Brown et al. (1967) found that the synthesis of red carotenoids in Chlorococcum wimmeri is a function of light intensity. Light has been also found to affect the size or the shape of the algae. We found that the algal cells of red Dunaliella grown at high light intensity, for example at 7500 lux and higher, appeared smal ler than in lower light intensities . Martinez and Krauss (1977) reported that when grown at low intensity of light at 53 ft-c (about 570 lux), cells of euryhaline green alga Scenedesmus obliguus (Turq.) Kue tz are relatively larger than at higher intensity. In the al gae, the mode of utilization of nitrogen varies from species to species. In natural habitats, the main nitrogen sources are nitrate and ammo nium salts. In artificial cultures, nitrogen is genera lly supplied as ammon ium salts or nitrate. Most al gae can assimilate either but there is much evidence that ammonium is preferred.

In the North Arm of the Great Salt Lake, N0 3 was not detected, and ammonia was found to be the main source of nitrogen, but it is found relatively very low in concentration, 1080 ~g/ 1 at its highest (Post, 1977), and it can be detected mainly in the early spring and during summer . Post (1977) suggested that the dormant stage of Q. salina observed during winter may be initiated by the low ammonia levels in the l ake rather than the cold of wi nter. 40

Gibor (1956) tested Q. salina and Q. viridis with KN0 3 and NH 4Cl in media buffered to pH 8.1 , and found that the growth of both species wa s induced by the reduced form of nitrogen, NH 4Cl. However, when grown in an unbuffered medium, it has found that the oxidized form of nitrogen (KN03) was superior to ammonium for growth of both species. But Gibor (1956) found that the organic compounds of nitrogen, for example urea, were preferred over the inorganic ones as an N-source for -D. -salina.- - In our study, ammonium carbonate was found to be a superior nitrogen source for the red Dunaliella over potassium nitrate (KN03) and ammonium chloride (NH 4Cl). This is probably because of there is no added C02 in the other 2 sources of nitrogen, KN0 3 and NH 4Cl, and ammonium carbonate supplied 2M of N, ((NH4)2co3-H 20). Growth was better in potassium nitrate than in ammonium chloride. It has been reported that the utilization of ammonium and nitrate is function of pH (Grant et al., 1967). The pH of the North Arm of the Great Salt Lake was found to be about 7.7 (Post, 1977). In our study, the growth of the alga using nitrogen from potassium nitrate shown a tremendous increase in pH apparently due to the production of base in the medium during growth. However, the pH of the medium was better maintained in ammonium carbonate than in other two sources of nitrogen (Table 7). Over a period of time, the growth of red Dunali ella in ammonium chloride was totally depressed and the final pH was 5.52 which just was 0.1 less than its pH when showing growth of the alga at stationary phase (Table 8). The decrease in the pH of the medium is due -probably to the uptake of ammonium ions by the cells with the release of HCl 41 lowering the pH. Loeblich (1970) reported that the growth rate of D. salina was reduced in acidic pH. It seems apparently that the growth of the red Dunaliella species is better in high pH . Stube {1976) and Stube et al. (1976) reported that the growth of Q. sa lina from the Great Salt Lake is better with ammonium than with nitrate, and Stephen (1976 ) and Stube et al. (1976) found that Q. viridis prefers nitrate. Urea-N and glutamic acid-N were not directly used by the al gae (Stube et al., 1976). Over a 2 year period of study in the Great Salt Lake, Post (1977) reported no nitrite or nitrate was found in the North Arm of the Great Salt Lake, but has been observed in the South Arm .. As mentioned earlier, in the North Arm of the Great Sa lt Lake, ammon ia was the main source of nitrogen (Post, 1977). The predominance of Q. salina in the North Arm might explain the preference of this species for ammonium as reported by Stube {1976) and Stube et al. (1976). The utilization of ammonium and nitrate is found to be not only a function of pH bu t is also a function of their ambient concentration (Grant et al., 1967). According to Michaelis-Menton kinetics, their coassimilation does not occur at identical rates. When available to gether , ammonium is assimi lated preferentially to nitrate at least by some algae (Gu ill ard, 1963; Syrett, 1962; Grant et al., 1967 ; Strick land et al., 1969). Martinez and Krauss (1977) shown that the growth of Scenedesmus obliguus (Turp.) Kuetz is better in potassium nitrate {pH increases from an initial 6.7 to a final 7.2) than in ammonium chloride (pH 6.7 to 5.35) and in ammonium nitrate (NH 4N03) {pH 6.7 to 6.63). However, they found that ammonium nitrate was preferred over ammo nium chloride. 42

This indicates that the preference of the algae for the N03 form of nitrogen is not obligatory. The reddening of Q. salina was found due to several conditions in cluding high sali nity, acidic pH, phosphorus or nitrogen deficiency, and high light intensity (Mil'ko, 1963a, 1963b; Melville, 1968; Miro nyuk and Einor, 1968; Mushak, 1968; Loeblich, 1972). In our present study, the algal cells of red Dunaliella appeared red in ammonium carbonate, and green in both potassium nitrate and ammonium chloride. The analysis of the pigment content shown that the content of chloro- phyl l a of the alga grown in potassium nitrate is higher than in the other sources of nitrogen (Table 6). The alga grown in ammonium car bonate contained 0.494 mg/106 cells, while in potassium nitrate and ammonium chloride were 0.534 mg/106 ce lls and 0.525 mg/106 ce ll s re spective ly . While the amount of chlorophyll a of the algae grown in ammonium carbonate is lower than in ammoni um chloride and potassium nitrate, the amo unt of carotenoids is the highest (Table 6). The carotenoid concentration of the alga in ammonium carbonate was 2.225 6 6 mSPU/1/10 cells, and in KN0 3 and NH 4Cl were 1.466 mSPU/l/10 cells 6 and 1.559 mSPU/1/10 cells respectively. The concentration of carot enoids i s higher by a factor of 1.42-1.51. This explains why the algal cells grown in ammonium carbonate appeared red. Presumably the more neutral pH with the extra C02 availability might stimulate the produc tion of carotenoid in the organism. Drokova and Dovgoruka {1966) found that C02, NaHC0 3 and dimethylvinylcarbonol stimulate a-carotene production in Q. salina. However, Melville {1968) found increasing the 43

concentration of co3 did not increase a-carotene production but greater biomass of Q. salina. Loeblich (1969) found relatively very sma ll amount of carotenoids, 5.6 ~g/10 6 cells, when grown Q. salina in 10% salinity, and Mironyuk and Einar (1968) only found 4.9 ~g/10 6 cells at 11.7% NaCl. Our red strain Dunaliella seems to contain much more carotenoids than those strain (2.225 mSPU_2.2 mg/106 cells). Mil 'ko (l963b) reported that wh en the medium was deficient in nitrogen and phosphorus, the amounts of chlorophyll and lutein were reduced, and the amount of a-carotene was increased by a factor 2.0-2 .5. Lo eblich (1972) transferred the algal cells of Q. sal ina from N- free medium toN-free or 0.198 mM KN0 3, they reddened; if transferred to 1.98-40 mM KN0 3, they become green. Similar results were shown with phosphorus deficiencies. According to Mil'ko (l 963a, l963b), Melville (1968), Mironyuk and Einar (1968), Mushak (1968) and Loeblich (1969, 1972) the ce ll s of Dunaliella reddened at high sa linities, high light intensity, in nitrogen or phosphorus deficiency due to the increase in the synthesis of the amount of carotenoids and decomposition of chlorophyll. When the nutrient is l im iti ng the growth of photosynthesis of the algae at high salinity and high light intensity, it is believed that the excess amount of carotenoid production may play the role as pro - tective mec han ism against photo-oxidation sensitized by chlorophyll (Griffiths et al., 1955 ; Cohen-Bazire and Stanier, 1958; Mil'ko, l963a). 44

SUMMARY AND CONCLUSIONS

The red Dunaliella species which is believed to be Q. salina was isolated from the Great Salt Lake to determine its growth characteristics in respect to NaCl concentration, temperature, light intensity, and nitrogen source preference. When compared to other strains of Q. salina studied by previous workers, the red Dunaliella species from the Great Salt Lake showed a similar requirement for NaCl concentration. The optimum temperature for growth of Q. salina which was investigated by some workers was between 25-30° C, and the optimum light intensity was between 6000- 9000 lux (ours was 28° C and 7500 lux). Our red Dunaliella strain

preferred NH 4+ supplied with co3 in unbuffered medium. This pre ference for NH 4+ by Q. salina has been also reported by a number of workers. Other sources of nitrogen were not studied and the pH-nitrogen source interaction needs to be further studied. The red pigmented Dunaliella of the Great Salt Lake lacks a cell wall, but its size is larger than strains previously described, and it contained a large amount of red carotenoid in the cell. From the above factors, it might be concluded that our red Duna- liella species isolated from the North Arm of the Great Salt Lake is Q. salina which has been investigated by previous workers. 45

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