Production of carotene with chemostat cultures of Dunaliella

Transcription

Production of carotene
with chemostat cultures of
Dunaliella
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam,
op gezag van de Rector Magnificus
prof. dr. P.W.M. de Meijer
ten overstaan van een door het college van dekanen ingestelde
commissie in het openbaar te verdedigen in de Aula der Universiteit
op donderdag 19 oktober 1995 te 13.00 uur
door
Pieter Vorst
geboren te Zijpe
Promotiecommissie:
Prof. Dr. H. van den Ende (promotor)
Prof. Dr. L.R. Mur (promotor)
Prof. Dr. A. Gibor
Prof. Dr. K.J. Hellingwerf
Dr. F.M. Klis
Prof. Dr. J. W. de Leeuw
Dr. H.C.P. Matthijs
Prof. Dr. L.H.W. van de Plas
The research presented in this thesis was carried out at the Research School
BioCentrum Amsterdam, Institute for Molecular Cell Biology, section Plant
Physiology, University of Amsterdam, Kruislaan 318, 1098!SM Amsterdam, The
Netherlands.
Publication of this thesis was financially assisted by contributions of Apotheken
Almere Haven, the University of Amsterdam, family and friends.
The cover illustration and the cartoons, introducing each chapter, were made by
Sijko Florijn.
voor Laura, Jonathan & Tobias
Contents
page
Chapter 1
Introduction.
Chapter 2
Comparison of two species of Dunaliella with
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23
emphasis on the factors influencing the specific
growth rate.
Chapter 3
Carotene accumulation in Dunaliella bardawil is
43
brought about by growth arrest, using nitrate-limited
chemostat cultures.
Chapter 4
Regulation of carotene- and starch synthesis in
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Dunaliella bardawil, in relation to growth arrest.
Chapter 5
Effect of growth arrest on carotene accumulation and
85
photosynthesis in Dunaliella.
Chapter 6
Samenvatting
General Discussion.
101
105
Chapter 1
Introduction
INTRODUCTION
INTRODUCTION.
1. General Introduction
The unicellular green alga Dunaliella is becoming an important model system for
experimental biology. Especially its resistance to high salinity, high light intensities and other stresses, which are most important in plant biology, makes it a
popular object of research. This is strengthened by the fact that it is a quite
amenable organism to study the effects of environmental variables on various
cellular processes, such as photosynthesis, respiration, phospholipid metabolism
and secondary metabolism.
Dunaliella stands out as an organism that accumulates carotenoids in
response to stress. For that reason, it is successfully applied for carotene production on an industrial scale. This thesis deals with this specific property. One of the
principal aims was to explore possibilities for optimizing this carotenoid production
by Dunaliella.
Morphology.
Members of the genus Dunaliella are unicellular green algae, with two flagella of
equal length placed at the anterior side. The cell shape is usually ovoid, although
this can vary with growth conditions. The cell lacks a rigid cell wall but is covered
with a mucilaginous coat (Melkonian and Preisig, 1984). One large, cup-shaped
chloroplast is located at the posterior side of the cell. A pyrenoid is present in the
chloroplast. An eyespot (D. bioculata has two eyespots) is laterally located at the
anterior part of the chloroplast. Some species are able to accumulate large
amounts of carotene. These carotenoids are located in oily globules in the interthylakoid space of the chloroplast and they give the cells an orange or red appearance, depending on the amount of carotene.
Taxonomy.
As a member of the Chlorophyta the genus Dunaliella is placed in the class
Chlorophyceae, the order Volvocales and the family Polyblepharidaceae. Ettl
(1983) used a different classification and placed Dunaliella in the new order
Dunaliellales with the family Dunaliellaceae. Presently 28 species of Dunaliella are
recognized, of which 5 occur in freshwater and 23 in saline environments (Preisig,
1992). Distinction between species is primarily based on morphological character-
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istics. These characteristics are subject to environmental conditions and are
partly to blame for the many ill-defined and misnamed species presently used in
many laboratories around the world. Comparison of various reports can therefore
only be done with some caution.
Dunaliella is related to Asteromonas (Peterfi and Manton, 1968) and has
many characteristics in common with Chlamydomonas. However, Dunaliella cannot be simply regarded as a wall-less Chlamydomonas. Large differences are observed, e.g. in ultrastructure (Marano et al., 1985; Melkonian and Preisig, 1984).
Habitat.
The halophilic members of Dunaliella are mainly found in subtropical areas
throughout the world, but also in more moderate regions (Borowitzka and
Borowitzka, 1988). Due to their unique capability to cope with large differences in
salinity, they can be found in various environments. Depending on the species,
members of Dunaliella are able to survive in low salinities of < 1% NaCl to saturated solutions (> 35%). For comparison, the salinity of normal sea water is approximately 3% NaCl. In salt lakes, such as the Great Salt Lake in Utah, U.S.A.
and the salt lakes in Australia, in which the salinities are much higher, Dunaliella
can be the predominant species and their presence can give the water a green or
red colour (Borowitzka, 1981; Felix and Rushforth, 1979).
Vegetative and sexual reproduction.
Cell division in Dunaliella occurs by binary fission. The volume of the cell increases
and a longitudinal division plane is formed. The duplication of the flagella can be
completed before the cell division has finished, resulting in large cells with four
flagella (Lerche, 1937; Teodoresco 1905, 1906; Penn, 1937). The total cell cycle
can take place in 10 hours (this thesis). Using synchronized cultures, cell division
occurs in the dark period every 24 h (Wegmann and Metzner, 1971; Zachleder et
al., 1989).
Sexual reproduction in Dunaliella has been described by Teodoresco (1905,
1906) and Lerche (1937). This process is induced by adverse conditions such as nitrate depletion and resembles the sexual reproduction of Chlamydomonas (for review, see van den Ende, 1994). Two gametes fuse and a zygote is formed. In contrast to vegetative cells, this zygote has a cell wall. After a maturation period, the
zygote wall bursts and 2, 4, 8 or 16 daughter cells are released. Sexual reproduction has rarely been observed in nature, and has not been reported to occur under
laboratory conditions since the publications mentioned above. This lack of a sexual cycle, which impedes genetic research, has to some extent been circumvented
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INTRODUCTION
by using mutants (Brown et al., 1987; Chitlaru and Pick 1989; Hard and Gilmour
1991; Latorella et al., 1981; Shaish et al., 1991) and somatic fusion (Lee and Tan,
1988). A stable genetic system has not been attained.
Vegetative survival bodies are described by Loeblich (1969). These
aplanospores also contain a cell wall and are formed during adverse environmental
conditions.
2. Halotolerance
Dunaliella is able to withstand extremely low osmotic potentials in (hyper)saline
environments by accumulating glycerol as osmoprotectant. The amount of glycerol present in the cytoplasm is a linear function of the osmotic potential of the
environment (Avron, 1992). The production of glycerol at hyperosmotic shock
mainly takes place via photosynthetic CO2 fixation or by starch degradation,
whereas at hypoosmotic shock, for example by diluting the NaCl concentration in
the surrounding medium, the cells increase their volume within 2-3 min and
rapidly convert glycerol into osmotically inactive starch.
An interesting aspect of this property is how the cell senses such changes
in osmotic conditions and what the nature is of the transduction of the signal
leading to the physiological response. It appears that the responses to dilution are
accompanied by hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) within
2 min (Einspahr et al., 1988). The products of this reaction are inositol 1,4,5trisphosphate (IP3) and diacylglycerol. It is probable that the activation of the
responsible enzyme, phospholipase C, is mediated by a G-protein. Two candidate
G-proteins of 34 and 42 kDa have been identified in Dunaliella through the use of
antibodies raised against mammalian G-protein !-subunits (Thompson, 1994).
Possibly the G-protein is activated via a stretch-activated protein present in the
plasma membrane. IP3 is known to release Ca2+ from intracellular stores and
presumably the resulting increased Ca2+ concentration in the cytoplasm induces
the conversion of glycerol to starch. In line with this view is the observation that
several Ca2+ activated protein kinases have been identified in Dunaliella (Yuasa
and Muto, 1992). Diacylglycerol is expected to activate protein kinase C, but this
enzyme has not been detected in Dunaliella.
Cells subject to hyperosmotic shock react by increasing their content of
phosphoinositol 4-phosphate and phosphoinositol 4,5-bisphosphate, suggesting an
inhibition of phospholipase C or an increased polyphosphoinositol synthesis, or
both. Also a plasma membrane ATP-ase is activated, resulting in a decreased
ATP level and an increase of the inorganic phosphate level in the cytoplasm. This
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CHAPTER 1
is thought to result in increased glycerol synthesis (Bental et al., 1990).
It is notable that Dunaliella is one of the few plant systems in which several
of the steps associated with PIP2-mediated signalling have been detected. It is
possible that even more well-known second messenger systems are operative. For
example, Cowan et al. (1992) have obtained evidence that abscisic acid is involved
in hypertonic shock response, leading to increased carotenoid synthesis. We shall
return to this in the next section.
3. Carotene production
Historical.
It was Dunal (1838) who observed that it was an alga which gave the salt water
basins, used for sea salt production in southern France, their reddish colour.
Teodoresco (1905) described the type species and named it after Dunal: Dunaliella
salina. Later, a distinction was made between two species, D. salina and D. viridis
(Teodoresco, 1906). This was based on the ability of the first species to attain a
red colour under certain conditions. Lerche (1937) observed that stress conditions
like nutrient deprival or high light intensities induced the red colour in D. salina.
Fox and Sargent (1938) reported that a high carotene content was responsible for
the red colour of D. salina. Presently, we know that "-carotene is the major
carotenoid produced, besides !-carotene and several oxygenated carotenoids
(Chapter 5).
Occurrence of carotene.
Carotenoids are widely distributed in nature. They are mainly produced by plants,
but also by several fungi and bacteria (Goodwin, 1971). In plants the presence of
carotenoids is often masked by chlorophyll. Through the food-chain many animals,
such as tropical birds, insects and marine animals derive their characteristic
colours from carotenoids (Bauernfeind, 1981).
The highest concentration of "-carotene is found in the red fringe of the
corona of the pheasant’s eye narcissus, Narcissus majalis: a concentration up to
16% of the dry weight can be reached (Ong and Tee, 1992). For whole organisms,
the highest concentration of "-carotene ever reported is in Dunaliella: more than
10% of the dry weight can consist of "-carotene (Shaish et al., 1992).
More than 500 different carotenoids are presently recognized. They are
separated into two groups: the first group contains hydrocarbons such as !- and
"-carotene, the second group comprises the oxygenated carotenoids such as lutein,
zeaxanthin and astaxanthin. All carotenoids are derived from chains of (usually
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INTRODUCTION
eight) isoprene units, which consist of five carbon atoms each (Britton, 1976;
Goodwin, 1971, 1980; Gray, 1987; Jones and Porter, 1986; Porter and Anderson,
1967; Straub, 1971).
Functions of carotenoids.
In photosynthetic organisms, carotenoids exert an essential function in the photosynthetic apparatus. They serve as accessory pigment in light harvesting, but in
addition, they are important for protecting photosynthetic organisms from
destructive photooxidation which can occur in the presence of light, O2 and chlorophylls. Especially carotenoids are scavengers of triplet excitations, which prevents singlet oxygen formation. This is apparent from the fact that seedlings,
treated with norflurazon, by which carotenoid biosynthesis is inhibited, die quickly,
due to the degradation of their chloroplasts. Mutants of Rhodopseudomonas,
lacking carotenoids, perform photosynthesis in a normal manner in the absence of
O2; when O2 is introduced in the light, the bacteriochlorophyll is photooxidized and
the bacteria are killed.
Carotenoids have also been implicated in a second type of photoprotective
process: the regulation of absorbed light energy utilization in the photosystem II
antenna (Demmig-Adams, 1990). In this process, generally referred to as energyor #pH-dependent non-photochemical quenching, excess absorbed light energy in
the PS II antenna is dissipated as heat. The quenching process is strongly dependent on the conversion of the carotenoid violaxanthin to zeaxanthin.
In animals, "-carotene is mainly used as a natural source of vitamin A (see
Bauernfeind, 1981). It is also used for chemoprevention of some types of cancer
(Henderson et al., 1991). For example, suppression of the progression of spontaneous mammary tumours in SHN virgin mice has been reported (Nagasawa et
al., 1991). Due to its ability to scavenge triplet excitations, it is also used as
antioxidant (Mokady, 1992).
Carotenogenesis in Dunaliella.
Under adverse conditions, such as high light intensity and nutrient stress,
Dunaliella reacts by accumulating large amounts of "-carotene. The hormonal
signal involved in stress reactions is possibly abscisic acid. Recent evidence suggests that this hormone is also present in algae and cyanobacteria (Hirsch et al.,
1989; Tietz et al., 1989), and in D. bardawil (Cowan and Rose, 1991). According to
these authors, "-carotene accumulates in two stages after hypertonic shock, the
first being accompanied by an increase in abscisic acid synthesis, the second is
supposed to be the result of this abscisic acid signal. This is plausible in view of the
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CHAPTER 1
biosynthetic route of abscisic acid: the oxygenated carotenoid all-trans-violaxanthin, derived from "-carotene, is converted to 9-cis-neoxanthin, which is cleaved to
xanthoxin and results, via abscisic acid-aldehyde, in the C-15 terpenoid abscisic
acid (Cowan et al., 1992; Zeevaart et al., 1989).
Carotene accumulation not only occurs at hyperosmolarity but also in
conditions of high light intensity and nutrient stress. Here, another sensing
mechanism is investigated. In light induced carotenogenesis in moulds, light can be
replaced by substances such as H2O2, methyl blue and methyl viologen, which
generate active oxygen species (Theimer and Rau, 1970). A similar observation
was made in Haematococcus pluvialis, where active oxygen species enhanced the
formation of the oxocarotenoid astaxanthin (Kobayashi et al., 1993). In D.
bardawil carotenogenesis also was enhanced by active oxygen species (Shaish et
al., 1993). It was argued that, at high light intensity, photosynthetically produced
oxygen radicals are involved in triggering the "-carotene accumulation. Lers et al.
(1991) found that carotene accumulation in Dunaliella, induced by high light intensities, was accompanied by the expression of a specific protein termed "early
light induced protein". The corresponding gene was cloned. Recent evidence suggests that it binds zeaxanthin to form photoprotective complexes within the light
harvesting antennae of the photosystems and is therefore not closely related to
the mass accumulation of carotene (Levy et al., 1993).
At low light intensities, where carotene accumulation can take place by
nutrient stress, active oxygen species are presumably less abundant. Here, the
signal to accumulate "-carotene must probably be sought elsewhere.
Biotechnology.
Algal biotechnology is distinguishable in several topics. From algae such as
Spirulina the total biomass is used as end product in the (health) food market, but
algae can also be used to remove pollutants in waste water or as fuel. Another
application is the production of fine chemicals. "-Carotene production with
Dunaliella was first proposed by Masjuk in 1966, and later also glycerol production
was proposed by Ben-Amotz in 1980 (see Borowitzka and Borowitzka, 1988). To
date, Dunaliella is the most successful alga in applied algology.
Most "-carotene used is synthetically all-trans-"-carotene and was introduced commercially as food colorant in 1954 (Isler, 1971). "-Carotene from
Dunaliella is currently marketed as solution in edible oil in a range of concentrations between 1.5 and 30% (Ben-Amotz, 1993). For the health market capsules of
dried Dunaliella powder containing about 5% "-carotene are produced.
The production of "-carotene by Dunaliella is performed outdoors in sub-
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INTRODUCTION
tropical areas. The alga is grown at a high rate in batch cultures in race-way type
reactors, aerated with paddle wheels and / or CO2 bubbles. The culture is started
with small volumes which are used to inoculate larger volumes. When fully grown,
the biomass is harvested and the production process is restarted from the beginning. The production process is short to reduce the risk of predators (Ben-Amotz
and Avron, 1989). The carotene content of the algae is increased by high light intensity. Dunaliella is also grown continuously in large non-stirred volumes
(Curtain et al., 1987). Then the growth rate is much slower, so to avoid predation
the salt concentration is increased. This also stimulates the carotene content. In
both production systems the harvesting by centrifugation or bioflocculation is the
most costly part of the production process (Borowitzka and Borowitzka, 1988).
Extraction of "-carotene from the algal biomass is usually done with edible oil. At
least 10 companies are involved in the production of "-carotene with Dunaliella
(Gudin and Chaumont, 1991).
"-Carotene from D. bardawil consists of equal amounts of all-trans-"carotene and 9-cis-"-carotene. These isomers differ in physiological properties.
The 9-cis-"-carotene isomer has a higher solubility in hydrophobic solvents, does
not form crystals and is an oil at high concentrations. All-trans-"-carotene is
practically insoluble in oil and is easily crystallized (Ben-Amotz et al., 1989).
These properties influence the bioavailability of "-carotene. When both isomers
are supplied together, the uptake is enhanced compared to all-trans-"-carotene
alone.
4. Scope of this thesis
Irrespective of the fact that Dunaliella is successfully applied for mass production
of carotenoids, there are a number of questions that can be asked. The first one
concerns the regulation of carotenoid biosynthesis. What is the relationship between the biosynthesis of carotenoids which is a function of chloroplast biosynthesis and the mass production which apparently is stress-related? A second
question deals with a possible function of this mass production. One can imagine
that the accumulation of "-carotene protects the cell, and particularly the photosynthetic apparatus, against photodamage at high light intensities. This "sunglass" hypothesis is supported by the observation that strains unable to accumulate "-carotene show low photoprotection and die while "-carotene-rich D. bardawil
survives. However, as mentioned above, mass-production of carotenoids is evoked
by several stress treatments, even under low light conditions. Considering the
large amounts of "-carotene accumulated, it is worthwhile to investigate if "-
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CHAPTER 1
carotene functions as a sink for photosynthetically fixed carbon.
A third question concerns the taxonomy of Dunaliella. According to Shaish
et al. (1991) only a few members of the genus, namely D. salina and D. bardawil
are able to produce large amounts of "-carotene. However there are strains of D.
salina that do not mass-produce "-carotene, while according to Borowitzka and
Borowitzka (1988) D. bardawil is a strain of D. salina. The D. salina of Ben-Amotz
et al. (1982) is probably D. parva, to which all strains are assigned that accumulate leaner amounts of carotenoids. This confusion makes it difficult to compare
results of different authors and especially to judge whether results obtained with
one strain (e.g. "D. bardawil") is applicable to other strains.
A fourth question concerns the optimization of "-carotene production. Is it
possible to realize this by optimizing growth conditions? From the foregoing it is
clear that "-carotene accumulation is a stress response, often accompanied by
growth arrest (e.g. under conditions of saline stress). It is clear that for industrial
application it would be advantageous to create conditions under which large quantities of "-carotene are produced in strongly proliferating populations. A related
question is if it is possible to optimize carotene accumulation by strain selection
and recombination. However, since initial attempts to induce sexual reproduction
were not successful, this line was not pursued.
To obtain at least a partial answer to these questions, a number of experiments was performed in which a carotene-accumulating strain, D. bardawil was
compared with a non-accumulating strain of D. salina. In chapter 2 the two
strains are compared in terms of optimal growth conditions in chemostat cultures,
morphology, etc. In chapter 3 the accumulation of large amounts of "-carotene is
reported in D. bardawil in response to growth arrest under low light intensities.
This phenomenon was not exhibited by D. salina. This suggests that it is possible
to realize a production system for "-carotene in moderate regions, and also allows
one to investigate the regulation of carotene synthesis. Such investigations are
described in chapter 4. In chapter 5, finally, it is reported that after growth arrest
the photosynthetic efficiency shows a temporary increase in D. bardawil and a decrease in D. salina. This increase did not occur when carotenogenesis was inhibited
by diphenylamide, implying a causal relationship between enhanced carotenogenesis and the increase of photosynthetic efficiency.
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INTRODUCTION
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INTRODUCTION
beta-carotene-rich algae Dunaliella bardawil of the progression, but not the
development, of spontaneous mammary tumours in SHN virgin mice. Anticancer
Research 11, 713-717.
Ong, A. S. H. & Tee, E. S. (1992). Natural sources of carotenoids from plants and oils. Methods
in Enzymology 213, 142-167.
Penn, A. B. K. (1937). Die Cytologie der Zellteilung von Dunaliella (Teodoresco). Archiv für
Protistenkunde 90, 162-164.
Peterfi, L. S. & Manton, I. (1968). Observations with the electron microscope on Asteromonas
gracilis Artari emend. (Stephanoptera Gracilis (Artari) Wisl.), with some comparative
observations on Dunaliella sp. British Phycological Bulletin 3, 423-440.
Porter, J. W. & Anderson, D. G. (1967). Biosynthesis of carotenes. Annual Review of Plant
Physiology 18, 197-228.
Preisig, H. R. (1992). Morphology and taxonomy. In Dunaliella: Physiology, Biochemistry, and
Biotechnology, pp. 1-15. Edited by M. Avron & A. Ben-Amotz. Boca Raton: CRC Press.
Shaish, A., Avron, M., Pick, U. & Ben-Amotz, A. (1993). Are active oxygen species involved in
induction of "-carotene in Dunaliella bardawil? Planta 190, 363-368.
Shaish, A., Ben-Amotz, A. & Avron, M. (1991). Production and selection of high "-carotene
mutants of Dunaliella bardawil (Chlorophyta). Journal of Phycology 27, 652-656.
Shaish, A., Ben-Amotz, A. & Avron, M. (1992). Biosynthesis of "-carotene in Dunaliella. Methods
in Enzymology 213, 439-444.
Straub, O. (1971). List of natural carotenoids. In Carotenoids, pp. 771-850. Edited by O. Isler.
Basel: Birkhäuser Verlag.
Teodoresco, E. C. (1905). Organisation et développement du Dunaliella, nouveau genre de
Volvocacée-Polyblépharidée. Beihefte zum Botanischen Centralblatt 18, 215-232 & figs.
8-9.
Teodoresco, E. C. (1906). Observations morphologiques et biologiques sur le genre Dunaliella.
Revue Generale de botanique 18, 353-371 & 409-427 & figs. 1-75.
Theimer, R. R. & Rau, W. (1970). Untersuchungen über die lichtabhängige Carotinoidsynthese.
V. Aufhebung der lichtinduktion durch Reduktionsmittel und Ersatz des Lichts durch
Wasserstoffperoxid. Planta 92, 129-137.
Thompson, G.A. (1994). Special roles of inositol lipids in cell signaling and metabolic regulation.
Progress in Lipid Research 33, 129-135.
Tietz, A., Ruttkowski, U., Köhler, R. & Kasprik, W. (1989). Further investigations on the
occurrence and the effects of abscisic acid in algae. Biochemie und Physiologie der
Pflanzen 184, 259-266.
Wegmann, K. & Metzner, H. (1971). Synchronization of Dunaliella cultures. Archiv für
Mikrobiologie 78, 360-367.
Yuasa T. & Muto S. (1992). Ca(2+)-Dependent protein kinase from the halotolerant green alga
Dunaliella tertiolecta: partial purification and Ca(2+)-dependent association of the
enzyme in microsomes. Archives of Biochemistry and Biophysics 296, 175-182.
Zachleder, V., Kuptsova, E. S., Los, D. A., Cepák, V., Kubín, S., Shapizugov, J. M. &
Semenenko, V. E. (1989). Division of chloroplast nucleoids and replication of chloroplast
DNA during the cell cycle of Dunaliella salina grown under blue and red light.
Protoplasma 150, 160-167.
Zeevaart, J. A. D., Heath, T. G. & Gage, D. A. (1989). Evidence for a universal pathway of
abscisic acid biosynthesis in higher plants from 18O incorporation patterns. Plant
- 19 -
Chapter 2
Comparison of two species of Dunaliella with emphasis
on the factors influencing the specific growth rate.
FACTORS INFLUENCING THE GROWTH RATE
COMPARISON OF TWO SPECIES OF DUNALIELLA WITH EMPHASIS ON
THE FACTORS INFLUENCING THE SPECIFIC GROWTH RATE.
Abstract
The specific growth rate (µ) of two species, D.!salina and D.!bardawil, was established in a controlled environment. The effect of light intensity, temperature,
nitrogen source and salinity of the medium was investigated. For both species the
optimal light intensity was approximately 500 µmol!m-2!s-1 and the optimal temperature 28°C. D.!bardawil grew best at a salinity of 1!M NaCl, whereas the
growth of D.!salina was optimal at a salinity of 0.5!M NaCl. Nitrate or ammonium
were equally effective as nitrogen source. The maximum growth rate observed
was 1.7!d-1 for D.!salina and 1.2!d-1 for D.!bardawil. From the data presented, the
minimal requirements for a growth rate of 0.8!d-1 in chemostat cultures were determined: a temperature of 28°C, a light intensity of 100!µmol!m-2!s-1 and 1!M
NaCl for both species. Growth in nitrate-limited chemostat cultures was established for both species of Dunaliella using these conditions.
Cells from chemostat cultures were used to compare several characteristics of the two species. The cell volume of D.!bardawil was 3.7 times larger than
D.!salina. Related to this was a higher protein, chlorophyll, carotene- and starch
content per cell. However, when compared on protein content, D.!salina had a
higher chlorophyll-, carotene- and starch content.
Introduction
Carotene production by mass cultivation of the unicellular halotolerant green alga
Dunaliella is one of the major successes in applied algal biotechnology. It is carried
out in Israel, Australia and the United States (see Ben-Amotz and Avron, 1989;
Borowitzka and Borowitzka, 1988b; Curtain et al., 1987).
The total production of carotenoids is dependent on the amount of pigment
per cell and the total amount of cells produced. Therefore, research on carotene
production by Dunaliella has focused on mass cultivation techniques (Borowitzka
et al., 1984; Moulton et al., 1987; Thomas et al., 1984) and selection of strains accumulating large amounts of carotene (Burrascano and Spencer, 1988; Shaish et
al., 1991). The amount of carotene per cell depends on environmental factors such
as high temperature, high light intensity, high salinity and/or nutrient deprival
- 23 -
CHAPTER 2
(Loeblich, 1982). These factors are partly contrary to the conditions needed for a
high growth rate.
Using outdoor production plants, carotene production by Dunaliella can
easily be carried out in subtropical regions. In moderate regions, the rate of growth
of Dunaliella is low and outdoor carotene production is therefore less feasible.
Therefore, it is worthwhile to investigate whether carotene production can be accomplished using indoor growth. Although the production costs will probably be
higher, a major advantage could be the high level of control in an indoor production
system. Also, the risk of contamination by other micro-organisms can be avoided.
Indoor cultivation techniques might not be restricted to production of carotene by
Dunaliella alone, but may also be used with other algae for the production of a wide
range of fine chemicals (see Borowitzka and Borowitzka, 1988a; Cresswell et al.,
1989). In this thesis Dunaliella and the production of carotene, using chemostat
cultures, was chosen as a model system.
In order to reduce indoor production costs, it is necessary to know the
minimal set of conditions required for the cultivation of Dunaliella. Numerous data
on factors influencing the rate of growth of several Dunaliella species have been
reported (see Borowitzka and Borowitzka, 1988c; Ginzburg, 1987; Jiménez and
Niell, 1991, McLachlan, 1959, 1960, 1964; Van Auken and McNulty, 1973).
Optimal temperature, light intensity and salinity of the medium have been reported for several species of Dunaliella in outdoor and indoor cultivation systems.
We wanted to establish an indoor chemostat cultivation system in which the cells
can be grown at a high specific growth rate. In this chapter, the optimal conditions
for high specific growth rates are described in a system in which the cells receive
continuous illumination and aeration with 2% CO2. In general, cultures with continuous illumination and aeration (with elevated CO2 levels) have much higher
growth rates than cultures illuminated in a light dark regime and without aeration
(compare Cifuentes et al., 1992 and Ginzburg and Ginzburg, 1985).
The genus Dunaliella was divided in two groups by Teodoresco (1906):
D.!salina and D.!viridis. The distinction is based on the ability to accumulate
carotenoids. Up to the present 23 salt-water species are recognized (Preisig,
1992), which are divided into three sections: Tertiolecta, Dunaliella and Viridis.
Members of the section Tertiolecta show optimal growth at a salinity of 2-4%,
whereas members of the sections Dunaliella and Viridis show optimal growth at
higher salinities. Further determination is based on morphological characteristics
which is difficult because these can be subject to growth conditions. Members of
the section Dunaliella are able to accumulate carotenoids whereas members of
the other two groups are not. In this chapter, a carotene accumulating and, as a
- 24 -
control, a non-accumulating species are studied in a controlled environment:
D.!bardawil, able to accumulate large amounts of carotenoids, and D.!salina,
unable to accumulate carotenoids. Their growth rate was investigated in response
to salinity, temperature and light intensity. This resulted in a set of conditions
that can be employed in chemostat cultivation, for both species of Dunaliella. The
chemostat technology offers the advantage of providing a highly controlled growth
environment. It is in this environment that a comparison of characteristics of the
two species, used in this thesis, was made.
Material and Methods
Strains and cultivation methods. Dunaliella bardawil (strain 30861 of the
American Type Culture Collection) and D.!salina (strain 9 of the Institute of Plant
Physiology of the Russian Academy of Sciences, Moscow) were used.
The algae were cultivated in modified Bold's Basal Medium (Harris, 1989) or
modified TAP medium (Amrhein and Filner, 1973). BBM consisted of NaNO3,
2.94!mM; MgSO4.7H2O, 0.3!mM; CaCl2.2H20, 0.17!mM; K2HPO4.3H2O,
0.42!mM; KH2PO4, 1.29!mM; FeNaEDTA, 0.068!mM; NaHCO3, 5!mM; NaCl,
1!M (unless stated otherwise) and micro nutrients according to Wiese (1965):
H3BO3, 9.7!µM; MnSO4.4H2O, 1.79!µM; NaVO3, 0.52!µM; ZnSO4.7H2O,
0.15!µM; CuSO4.5H20, 0.06!µM; CoSO4.7H2O, 0.02!µM; (NH4)6Mo7O24.4H2O,
0.003!µM. The medium was sterilized by autoclaving at 120°C. The phosphate
components were autoclaved separately and the NaHCO3 solution was filtersterilized before being added to the sterilized medium. TAP medium consisted of
Tris(hydroxymethyl)-aminomethane, 20!mM; NH4Cl, 7.48!mM; MgSO4.7H2O,
0.41!mM; CaCl2.2H2, 0.34!mM; K2HPO4.3H2O, 1!mM; FeNaEDTA, 0.068!mM;
NaCl, 1!M and micro!nutrients as BBM. The medium was adjusted to pH 7.2 with
acetic acid glacial (±1 ml L-1) and sterilized by autoclaving at 120°C. The phosphate was adjusted to pH 7.2 with KOH and autoclaved separately.
Stock cultures were routinely maintained on 1.5% agar slants of BBM, except that the phosphate components and NaHCO3 were autoclaved together with
the medium. The cultures were kept at 19°C and illuminated with two TL 58 W/33
white fluorescent tubes together with a HPI/T 400 W daylight lamp (Philips,
Eindhoven, The Netherlands) at an average light intensity of 150!µmol!m-2 s -1, or
illuminated with one TL 58 W/33 white fluorescent tube at an average light intensity of 15!µmol!m-2!s-1. A 12!h light/12!h dark regime was applied. Stock cultures
were transferred to fresh agar slants every 6 to 8 weeks (150!µmol!m-2!s-1) or
every three months (15!µmol!m-2!s-1).
- 25 -
CHAPTER 2
Nitrate-limited chemostat cultures were carried out at 28°C in 1!L culture
vessels, containing 565!ml, or 2!L culture vessels, containing 1.5!L medium.
Continuous one-sided illumination was provided by a HPI/T 400 W daylight lamp
at an average light intensity of 100 µmol!m-2!s-1 in the center of the vessel, as
measured with a Li-cor model LI-185B photometer equipped with a LI-190SB
quantum sensor. Batch cultures with 50!ml volume, at the beginning of the stationary phase, were used as inoculum. The cultures were aerated with 2% CO2
and large bubbles were used because small bubbles derived from porous materials,
such as glass filters, caused destruction of cells. The cultures were continuously
stirred with a magnetic Teflon-coated rod. Cells that adhered to the glass wall were
regularly scraped off, using the magnetic Teflon-coated rod. Nitrate limitation was
imposed by reducing the nitrate content of the medium to 10% of the original
amount. The specific growth rate (µ) was maintained at 0.8!d-1. A steady-state
situation, in which the rate of growth can be regulated by manipulating the rate of
medium supply to the chemostat vessel, was easily achieved.
Cell densities. To determine cell densities, culture samples were fixed with
glutaraldehyde (final concentration 1.25% (v/v)) and counted in a haemocytometer. A minimum of 400 cells was counted for each sample.
Determination of specific growth rate. Cells of Dunaliella were pre-grown in
Erlenmeyer flasks as batch cultures at 21°C, continuously illuminated with two
TL 58 W/33 white fluorescent tubes, at an average light intensity of
100!µmol!m-2!s-1. Cells from the exponential growth phase were used to inoculate
test tubes (230 x 16!mm), containing 15!ml medium. The test tubes were placed in
a water bath of the desired temperature and continuously illuminated with a
HPI/T 400W daylight lamp (Philips, Eindhoven, The Netherlands), placed at appropriate distance to give the desired light intensity. The cultures were aerated
with 2% CO2. Four replicate cultures were used for each specific set of conditions.
The cultures were diluted with fresh medium every 24!h. Cultures of D. salina were
diluted to a density of 1*106 cells ml-1 and cultures of D. bardawil were diluted to a
density of 1*105 cells ml-1, to ensure that the cultures remained in the exponential growth phase during the experiment. The medium composition in the pre-cultures was the same as the medium in the tubes, in order to reduce the time of
adaptation. Cell density measurements started at day 2, allowing adaptation to
the new conditions. The measurements were continued for 3 days. The specific
growth rate was calculated using the following expression:
- 26 -
µ=
ln (Nt / No)
t
where µ is the specific growth rate, Nt is the cell density at time t and No is the
cell density at time 0.
Cell volume measurements. Culture samples of 10!ml were fixed with 1!ml 5%
glutaraldehyde solution (v/v) in 1!M NaCl. Cells were concentrated by centrifugation for 5!min at 1600!g. Cells were placed in a haemocytometer to avoid pressure
on the cells. In this manner, conditions influencing the cell size of Dunaliella cells
were avoided. Cells were measured using a Televal 3 reversal microscope, to which
a Philips CCD camera was fitted and connected to a Macintosh Quadra 700 computer equipped with a frame grabber (QuickCapture from Data Translation Inc.).
For each sample, the long and short axis from 500 cells were measured using image processing software from the National Institutes of Health, University of
Minnesota (NIH Image, version 1.49). Cell volume was calculated using the equation of an ellipsoid:
2
Volume = 4 ( 1 a) ( 1 b)
3 2
2
where a is the minor axis and b is the major axis. Measurements were finished
within 30 min after sampling.
Pigment analysis. Carotene and chlorophyll contents were estimated by extracting 10!ml cell culture with 80% acetone and the absorption was measured at
663.2, 646.8 and 470.0!nm in a Perkin-Elmer Hitachi 200 spectrophotometer.
Carotene and chlorophyll contents were calculated using the equations of
Lichtenthaler (1987).
Chemical analyses. Cellular starch content was determined as described by
Herbert et al. (1971), using a solution of 2!mg ml-1 anthrone in 98% sulphuric acid,
with D-glucose as standard. Cells were freeze-dried before analysis. Protein content was estimated according to Bradford (1976), with bovine serum albumin as a
standard. Cells were freeze-dried and boiled for 5!min in 1!M NaOH before analysis.
- 27 -
CHAPTER 2
Figure 1. Specific growth rate of D. salina during exponential growth.
The cells are cultivated using BBM (
) or TAP medium (
). The data are averages
of 8 to 12 experiments. Bars indicate the standard error. A. Light-dependent growth
(temperature, 28°C; salinity, 1 M NaCl). B. Temperature-dependent growth (light intensity, 100
µmol!m-2 s-1; salinity, 1 M NaCl).
- 28 -
Results
TAP medium versus BBM.
Dunaliella can be cultivated in artificial minimal salt media. In this chapter two
frequently used media were compared in their effects on the specific growth rate of
Dunaliella: TAP medium, with ammonia as the sole nitrogen source and BBM,
with nitrate as the sole nitrogen source. Only minor, but significant differences
were observed in the specific growth rate of D. salina in these two media (Fig.!1). A
somewhat higher specific growth rate was found using BBM at higher light intensities (Fig.!1a). With BBM, but not with TAP medium, growth occurred at 13.5°C
(Fig.!1b).
D. bardawil versus D. salina.
Two species of Dunaliella were used, D. bardawil, capable of accumulating
carotene and, as a control, D. salina, not capable of accumulating carotene. The
specific growth rate of D. salina was considerably higher than that of D.!bardawil,
when grown at various light intensities (Fig. 2a) and temperatures (Fig. 2b). The
maximum specific growth rate was reached at a light intensity of approximately
500 µmol m-2 s -1 for both D. salina and D.!bardawil (Fig. 2a). The optimal temperature was 28°C for both species (Fig.!2b). D. bardawil had a lower temperature
tolerance than D. salina: growth was absent at 13.5°C and strongly reduced at
34°C. This reduction could be overcome using a light intensity of 2500 µmol m-2
s -1 at this temperature, giving a specific growth rate of 1.0!d-1!±!0.1 (n=8).
Large differences were observed in the salt tolerance of the two species (Fig.
2c). Only D. salina tolerated very low concentrations of NaCl (50 mM) whereas D.
bardawil was able to grow even at 5 M NaCl. The optimal salt concentration was
1 M NaCl for D. bardawil and 0.5 M NaCl for D. salina.
The maximal specific growth rate in this set of experiments was for
D.!bardawil 1.2 d-1 ± 0.1 (n=11) at 28°C, 1 M NaCl and 2500 µmol!m-2 s -1 and for
D. salina 1.7 d-1 ± 0.1 (n=12) at 28°C, 1 M NaCl and 500 µmol!m-2 s-1.
- 29 -
CHAPTER 2
Figure 2. Specific growth rate of D. bardawil (
) and D. salina (
) during exponential growth.
The cells are cultivated using BBM. The data are averages of 8 to 12 experiments. Bars indicate
the standard error. A. Light-dependent growth (temperature, 28°C; salinity, 1 M NaCl). B.
Temperature-dependent growth (light intensity, 100 µmol!m-2 s -1; salinity, 1 M NaCl). C.
(opposite page) Salinity-dependent growth (light intensity, 100 µmol m-2 s -1; temperature,
28°C).
- 30 -
Cultivation in nitrate-limited chemostat cultures.
Nutrient-limited chemostat cultures are widely used for the cultivation of alga.
Nitrate limitation is the most common limitation practised. Besides nitrate, also
phosphate- and sulphate limitations were tried in chemostat cultivation of
Dunaliella, but were not established (see Chapter 3). However, both species of
Dunaliella could be easily cultivated in nitrate-limited chemostat cultures at identical conditions. At a temperature of 28°C, a light intensity of 100!µmol!m-2!s-1
and 1 M NaCl in the medium, a steady-state situation with a specific growth rate
of 0.8 d-1 was attained. More characteristics of chemostat culturing at these conditions are given in chapter 3.
In order to compare the characteristics of the two species, only cells from
the steady-state situation of nitrate-limited chemostat cultures were used.
Identical conditions were used to avoid differences attributed to changes in light intensity or temperature. A large difference in cell size of the two species was observed (Fig. 3). With a cell volume of 1007 µm3, D. bardawil was about 3.7 times
larger than D.!salina (Table 1a). This difference was also found in protein, chlorophyll and carotene contents per cell, but the starch content was only twice that in
D. salina. When based on protein content however, the starch content of D.!salina
was two times higher than that of D.!bardawil (Table 1b). The chlorophyll and
carotene contents were also higher in D.!salina than in D.!bardawil, when based on
protein content. The large difference in volume and protein content can be used to
support the distinction between the two species.
- 31 -
CHAPTER 2
Figure 3. Photographs of D. bardawil (A) and D. salina (B).
The cells were grown in batch cultures on BBM with 1 M NaCl at a light intensity of 20 µmol
m-2 s-1 and at 21°C. Samples of 10 ml taken during the exponential growth phase were fixed
with 1 ml 5% glutaraldehyde solution (v/v) in 1 M NaCl. The bar size is 10 µM.
- 32 -
Discussion
Maximum specific growth rate.
The maximum specific growth rate of 1.2 d-1 reported here for D.!bardawil corresponds with a generation time of 14 h and that of 1.7 d-1 for D. salina with a
generation time of 10 h. These specific growth rates are in agreement with those
of Ginzburg and Ginzburg (1981), who reported generation times for members of
the D. salina type of 18-30.5 h and 14-24 h for members of the D.!viridis type at
29°C, 2 M NaCl and a light intensity of 80 µmol!m-2!s-1. Jiménez and Niell (1991)
have reported a generation time for a D.!viridis species of 11.9!h at 30°C, 1 M
NaCl and a light intensity of 150 µmol m-2!s -1. Thus the specific growth rates reported in this chapter are in agreement with those in other reports. The maximum
growth rate, reported here, is among the highest reported for species of Dunaliella.
The considerable reduction in growth rate of D. bardawil at 34°C and
100!µmol m-2 s-1 could be overcome by increasing the light intensity to 2500!µmol
m-2 s-1. In contrast to the report of Eppley and Sloan (1966) this result indicates
that temperature and light intensity are not independent variables. Presumably,
growth at a fast rate at high temperature can only be maintained when sufficient
energy is provided by photosynthesis.
Choice of nitrogen source.
Both D. salina and D. bardawil can be cultivated on media with urea, nitrate or
ammonia as the sole nitrogen source (data not shown). This is in agreement with
reports of Ben-Amotz and Avron (1989) and Borowitzka and Borowitzka (1988b).
But urea can not be used by all species of Dunaliella (Gibor, 1956). Urea as nitrogen source was not further investigated.
No important differences were observed between the growth rates of
D.!salina growing on TAP medium or on BBM (Fig. 1), which is in agreement with
Goldman and Peavey (1979), who compared the growth rate of D.!tertiolecta on
media with different nitrogen sources. TAP medium has the disadvantage of containing Tris(hydroxymethyl)-aminomethane and acetate and is therefore more
susceptible to contamination (Fábregas et al., 1993). All other experiments were
therefore carried out using BBM. Cultivation of algae on media with nitrate as the
sole nitrogen source results in an increase of pH (Brewer and Goldman, 1976),
partly because BBM is poorly buffered. To prevent large pH changes the cultures
were aerated with 2%!CO2 (Ben-Amotz and Avron, 1989).
- 33 -
CHAPTER 2
Chemical composition.
Numerous reports have been published about the chemical composition of
Dunaliella (see for reviews: Ginzburg, 1987; Borowitzka and Borowitzka, 1988b).
However, comparison of these results is difficult because of ill-defined growth conditions, ill-defined species or methodological problems. The chemical composition of
Dunaliella can vary because of nutrient limitation and is also dependent on the intensity of illumination (Ben-Amotz, 1987). Not only the content of pigments involved in photosynthesis can vary, but also the protein/carbohydrate ratio varies
with growth phase and/or nutrient limitation. During nitrate starvation the
amount of protein decreases and the amount of starch increases. Another major
aspect involved in the chemical composition is the osmolarity of the medium in
which Dunaliella is cultivated. The higher the osmolarity, the higher is the amount
of glycerol, which functions as osmoprotectant in the cells. Lacking a rigid cell wall,
cells of Dunaliella are subject to swelling and shrinking upon changes in the osmolarity of the medium The cell volume is restored by subsequent increase or decrease in the amount of glycerol (see for review, Avron, 1992).
For these reasons it is important to standardize the environmental conditions during growth. Such an environment is found in the chemostat: in the
steady-state situation, all environmental factors are constant. Cells grow exponentially at a fixed rate. However, two drawbacks of the chemostat cultivation
system must be made in the comparison between D. bardawil and D. salina: (1)
The larger cells of D. bardawil will influence the pattern of light scattering in the
culture, compared to the smaller cells of D. salina. This, combined with the higher
pigment content of D. salina (Table 1), may result in a somewhat lower light intensity for D. salina, but this will have only a limited effect on cell growth. (2) The
stringency of the limitation, which is the ratio µ/µmax, is higher for D.!salina than
for D.!bardawil. The specific growth rate in the chemostat cultures was set at 0.8
d-1. This growth rate was close to the maximum growth rate of 0.9 d-1 for
D.!bardawil at these conditions, but the maximum growth rate under these conditions for D.!salina was much higher: 1.4 d-1.
The chemical composition of D. bardawil during cultivation in nitrate-limited
chemostat cultures is comparable with that of cells during the exponential growth
phase in batch culture, regarding protein, starch and chlorophyll content, but the
carotene content is somewhat higher in batch culture (data not shown). The similarity in chemical composition is probably accidental and might be due to counteracting environmental factors. Batch culturing was performed at 21°C without
aeration, which differs from the chemostat cultivation conditions. The chemical
composition shown here (Table 1) confirms the data of Ben-Amotz (1987) during
- 34 -
non-limited growth of this species, but are not comparable with the composition of
cells, grown under nitrate deficiency. The chemical composition during nitrate deficiency resembles the “growth arrest” phase in our experiments: cells are grown
in a chemostat culture and after terminating the medium supply the cells experience an immediate lack of nitrogen. This results in carotene- and starch accumulation (Chapter 3).
Table 1. Comparison of D. bardawil and D. salina, grown in nitrate-limited chemostat cultures.
In this table the values are given as average ± standard error of 4 samples taken during steadystate growth in the chemostat. The values are given per cell (A) or per mg protein (B).
A
D. bardawil
D. salina
Protein
(pg cell-1)
363 ± 21
82 ± 1
Chlorophyll
(pg cell-1)
6.3 ± 0.3
2.0± 0.1
Carotene
(pg cell-1)
2.0 ± 0.1
0.6 ± 0.0
Starch
(pg glucose cell-1)
150 ± 16
87 ± 6
1007 ± 42
273 ± 7
D. bardawil
D. salina
Chlorophyll
(µg mg protein-1)
17.5 ± 0.7
24.3 ± 0.5
Carotene
(µg mg protein-1)
5.5 ± 0.5
7.0 ± 0.2
0.41 ± 0.05
1.06 ± 0.07
Cell volume
(µm3)
B
Starch
(mg glucose mg protein-1)
- 35 -
CHAPTER 2
As to protein content, the chemical composition of D. salina is comparable
with that reported by Fabregas et al. (1989). This might indicate a cell volume in
the same order of magnitude. The chlorophyll content however, is two times higher
while the carbohydrate content is four times lower in the D.!tertiolecta they used.
The lipid content can be two to three times as high as the amount of carbohydrate
in this species (Fabregas et al., 1989).
As stated above, the chemical composition of the cells can vary largely with
cultivation conditions. This variability makes it necessary to use a stable reference method when comparing characteristics of cells and / or situations. Although
fluctuations can occur, protein seems to be the most constant component of the
cells. Comparisons are therefore routinely based on protein content throughout
this thesis. Especially when comparing cells differing in size, this choice of reference is convenient. But, depending on purpose, cell-based comparisons are also
made.
Measurements of biomass and cell volumes.
Biomass and cell volume measurements of Dunaliella are not without problems
due to the fragility of the wall-less cells. When estimating the fresh- or dry weights,
care must be taken with the high NaCl concentrations used in the growth media.
The NaCl present in the extra-cellular fluid will remain after the evaporation of
water and strongly influence the dry weight. To overcome this, cells can be washed
and re-suspended in ammonium acetate or ammonium carbonate to remove the
NaCl (Spektorov and Nazarenko, 1989). These inorganic osmotic agents will
evaporate while drying. But repeated washings increase the risk of cell rupture.
The packed-cell-volume method for determining cell volumes suffers also
from the non-rigid cell shape of the cells. By increasing the speed of centrifugation
the cells become deformed and the pellet will be more compact and exact cell volume measurements are therefore difficult. Katz and Avron (1985) have used a
method for determination of volumes of Dunaliella cells based on measuring the
total volume of the pellet with 3H2O of which the extra-cellular volume, measured
as Li+ concentrations, was subtracted. Blackwell and Gilmour (1989) have improved this method by circumventing the need for 3H2O.
Here, a microscopic determination method of cell volume was used. Caution
was exersized to prevent osmotic changes during sampling and measurements.
Although this is a more direct way to measure cell volume, compared to measuring with a particle-size analyzer or using the pellet method (Ginzburg, 1987), the
assumption that the cell shape is an ellipsoid can cause small deviations.
Because Dunaliella lacks a rigid cell wall, cell volume is not fixed.
- 36 -
Experiments with hypo- and hyper-osmotic shocks indicate that Dunaliella can
regulate its cell volume. This control is performed by regulating the amount of
compatible solutes, mainly glycerol (Ginzburg, 1987). Presumably the same regulation results in the large differences in cell size observed between different species
of Dunaliella.
Taxonomy.
In this chapter a comparison of characteristics between two species of the genus
Dunaliella is described. Dunaliella bardawil was originally isolated from a salt pond
near Bardawil Lagoon in the North Sinai in 1976 by Ben-Amotz and Avron.
Although this species is called D. bardawil, the characteristics indicate that it is in
fact D. salina Teod. (Ben-Amotz et al., 1982). Numerous reports have already
used the name D. bardawil and therefore this name will be used in this thesis.
Cowan and Rose (1991) have used the descriptive name D. salina var. bardawil to
avoid confusion. The characteristics of the D. salina species used here indicate
that also this species is misnamed. The cell size, the optimal salinity of the
medium for a maximum rate of growth and the inability to accumulate carotenoids indicate that this species should be defined as D. tertiolecta (see for taxonomic keys of Dunaliella: the monograph of Masjuk, 1973, which is in Ukrain or
the translation by Preisig, 1992).
Acknowledgements
I wish to thank Wies van den Briel for her large share in counting cell densities.
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Thomas, W. H., Seibert, D. L. R., Alden, M., Neori, A. & Eldridge, P. (1984). Yields,
photosynthetic efficiencies and proximate composition of dense microalgal cultures. II.
Dunaliella primolecta and Tetraselmis suecica experiments. Biomass 5, 211-225.
Van Auken, O. W. & McNulty, I. B. (1973). The effect of environmental factors on the growth of a
halophylic species of algae. Biological Bulletin 145, 210-222.
Wiese, L. (1965). On sexual agglutination and mating-type substances (gamones) in isogamous
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- 39 -
Chapter 3
Carotene accumulation in Dunaliella bardawil is
brought about by growth arrest, using nitrate-limited
chemostat cultures.
GROWTH ARREST BRINGS ABOUT CAROTENE ACCUMULATION
CAROTENE ACCUMULATION IN DUNALIELLA BARDAWIL IS BROUGHT
ABOUT BY GROWTH ARREST, USING NITRATE-LIMITED CHEMOSTAT
CULTURES.
Abstract
The cultivation of two Dunaliella species, D. bardawil and D. salina, in nitratelimited chemostat cultures at low fluence rates is described. D.!bardawil, derived
from such cultures, was able to accumulate large amounts of !-carotene after
growth arrest. This is in contrast to D.!salina, which did not show this phenomenon
under these conditions. Both species accumulated large amounts of starch. The effect of light intensity, temperature and CO2 concentration on !-carotene accumulation was studied. The cell volume of D. bardawil increased by 70% after growth
arrest, while the cell volume of D.!salina remained constant. While phosphate- or
sulphate limitation did not result in steady-state growth in chemostat cultures, a
deficiency in sulphate or phosphate did lead to carotene accumulation in D.
bardawil. The possibilities of a stable, two-stage carotene production system,
suitable for production in moderate climate regions, is discussed.
Introduction
Commercial production of !-carotene, using selected strains of Dunaliella, is performed in open ponds in subtropical regions of Australia, Israel and the United
States (see, e.g. Ben-Amotz and Avron, 1989; Borowitzka and Borowitzka, 1988;
Curtain et al., 1987). These regions have characteristics suitable for cultivation of
Dunaliella, such as a high temperature, and a high light intensity. These environmental conditions stimulate both growth and carotene accumulation in Dunaliella.
Carotene accumulation is also enhanced by a high salinity of the medium and
nutrient deprival (Loeblich, 1982; Borowitzka et al., 1990), but these factors decrease the rate of growth. Outdoor carotene production with Dunaliella in moderate regions is not feasible, because temperature and light intensity in these regions are not optimal for growth and carotene accumulation. In this chapter a stable indoor carotene production system is described, in which growth and carotene
accumulation are spatially and temporally separated. This system, based on
nutrient deprival, does not require high light intensities for growth or carotene accumulation.
- 43 -
CHAPTER 3
American Type Culture Collection) and D.!salina (strain 9 of the Institute of Plant
Physiology of the Russian Academy of Sciences, Moscow) were used. The algae
were cultivated in modified Bold's Basal Medium, as described (Chapter 2).
Nitrate-limited chemostat cultures were carried out at 28°C in 1 L culture
vessels, containing 565 ml or 2 L culture vessels, containing 1.5 L medium essentially as described (Chapter 2) with few modifications. The cultures were (1)
stirred every 6 h for 1 min, and also prior to sampling, with a magnetic Tefloncoated rod (experiments described in Fig. 3, 6 & 7) or (2) continuously stirred with
a magnetic Teflon-coated rod (other experiments). Nitrate limitation was imposed
by reducing the nitrate content of the medium to 10% of the original amount, unless stated otherwise. The bottle receiving the effluent was changed every 24 h
during experiments performed with cells from the effluent. The specific growth
rate (µ) was maintained at 0.8 d-1, unless stated otherwise.
Growth arrest was achieved in two ways. (1) Growth arrest was applied by
transferring cells from a nitrate-limited chemostat culture to Erlenmeyer flasks
at 21°C. The flasks were shaken manually once a day and illuminated with 4 TL
58!W/33 white fluorescent tubes at a light intensity of 80-100 µmol m-2 s -1, unless stated otherwise. (2) Cells were taken from the 24 h effluent of the chemostat
and transferred to Erlenmeyer flasks (same conditions as in 1).
Phosphate- and sulphate limitations were essentially performed as
described for the nitrate limitation. All glass material was rinsed with 5% HCl before use. The phosphate concentration of the medium was reduced to 0.5% in order
to achieve phosphate limitation; in the sulphate limitation MgSO4.7H2O was replaced by an equal molarity of MgCl2.6H2O, the sulphate concentration was reduced to 4% and sulphate was supplied as Na2SO4. Fresh medium was prepared
daily and was continuously stirred with a magnetic Teflon-coated rod to reduce
precipitation. The specific growth rate (µ) was 0.6 d-1 for both limitations.
Cell densities. Cell densities were estimated as described (Chapter 2).
Cell volume measurements. Cell volume measurements were performed as
described (Chapter 2).
Pigment analysis. Carotene content was estimated as described (Chapter 2).
- 44 -
Chemical analyses. Cellular starch- and protein content were determined as
described (Chapter 2). The phosphate concentration in the phosphate limitation
experiment was analyzed as described by Healey (1978) using 5 cm path length
cuvettes and with KH2PO4 as standard.
Figure 1. Effect of growth arrest on starch and carotenoid accumulation in nitrate-limited cells of
D.!bardawil (
) and D.!salina (
).
Growth arrest was applied by transferring cells from nitrate-limited chemostat cultures to
Erlenmeyer flasks at time zero. A. !-Carotene content. B. Starch content.
- 45 -
CHAPTER 3
Results
Cultivation in chemostat cultures.
Two species of Dunaliella, D. bardawil and D. salina, were cultivated in chemostat
culture using nitrate limitation. Both species were cultivated using the same conditions: a temperature of 28°C, continuous illumination at a light intensity of 100
µmol!m-2!s-1 and aeration with 2% CO2. The nitrate concentration of the medium
supplied to the chemostat was 0.30 mM. The extracellular nitrate concentration
in the culture vessel was below the detection limit (0.2 µM, Chapter 5). A steadystate situation, in which the rate of growth can be regulated by manipulating the
rate of medium supply to the chemostat vessel, was easily achieved for both
species. The specific growth rate was set at 0.8 d-1. This growth rate was close to
the maximum growth rate of 0.9!d-1 for D.!bardawil at these conditions, but this
was much higher for D. salina: 1.4 d-1 (Chapter 2). Although all environmental
factors were equal, the stringency of the nitrate limitation, which is the ratio
µ/µmax, was therefore higher for D. salina than for D. bardawil.
Figure 2. Photograph of D. bardawil after 8 days of carotene accumulation.
Erlenmeyer flasks at time zero. A sample of 10 ml was taken and fixed with 1 ml 5%
glutaraldehyde solution (v/v) in 1 M NaCl. The bar size is 10 µM.
- 46 -
Growth arrest and carotene accumulation.
When cells were transferred from the growing situation in the culture vessel of a
nitrate-limited chemostat to Erlenmeyer flasks and placed in continuous light of
100!µmol m-2 s-1 and a temperature of 21°C, the cells experienced growth arrest
because of immediate lack of nitrate. In this situation, cells of D. bardawil accumulated large amounts of carotene, in contrast to D. salina (Fig. 1a, and Fig.!2). A
carotene content of 145 µg mg protein-1 (50 pg cell-1) was reached in 9!days in
D.!bardawil.
Carotene accumulation in D. bardawil, after growth arrest, was dependent
on light (Fig. 3). The limiting light intensity, required for fast carotene accumulation, was approximately 45 µmol m-2 s -1. When cells were illuminated with 1500
µmol m-2 s-1 after growth arrest, the rate of carotene accumulation was not significantly enhanced (data not shown).
Figure 3. Effect of light intensity on carotene accumulation of D. bardawil after growth arrest.
Light intensity:
,0;
,45;
,60;
,100;
,200
µmol!m-2!s-1. Growth arrest was applied by transferring cells from the 24 h effluent of a
nitrate-limited chemostat culture to Erlenmeyer flasks.
- 47 -
CHAPTER 3
Other characteristics influenced by growth arrest.
After growth arrest, both species accumulated large amounts of starch, exceeding
those of carotene in D.!bardawil: A starch content of 3 mg mg protein-1 for D.
salina and 2.6 mg mg protein-1 for D. bardawil was reached in 9!days (Fig. 1b).
During the first days of growth arrest, starch accumulation was fast, after which
the rate of accumulation decreased.
In both species the chlorophyll content decreased after growth arrest to
approximately half of the content during growth (Fig. 4a). The decrease was
gradual and stopped after approximately 10 days of growth arrest.
The cell volume of D. bardawil was 1007 µm3 during steady-state growth,
which is 3.7 times larger than the cell volume of D. salina (Table 1). After growth
arrest the cell volume of D. salina remained the same, while there was an increase
of 70% in cell volume of D.!bardawil (Fig. 4b).
Table 1. Comparison of nitrate-limited chemostat cultures of D. bardawil and D. salina, during
steady-state growth.
In this table the values are given as average ± standard error of 4 samples taken during steadystate growth in the chemostat. The values are given per ml cell culture.
D. bardawil
D. salina
Protein
(µg ml-1)
67.2 ± 2.0
76.1 ± 1.1
Chlorophyll
(µg ml-1)
1.17 ± 0.03
1.85 ± 0.05
Carotene
(µg ml-1)
0.37 ± 0.03
0.53 ± 0.01
Starch
(µg glucose ml-1)
27.8 ± 2.8
80.6 ± 6.0
Cell number
(ml-1)
18.6*104 ± 0.5*104
93.0*104 ± 1.1*104
Cell volume
(µm3)
1007 ± 42
273 ± 7
- 48 -
Figure 4. Effect of growth arrest on chlorophyll content and cell volume of nitrate-limited cells of
D.!bardawil (
) and D.!salina (
).
Erlenmeyer flasks at time zero. A. Chlorophyll content. B. Cell volume.
- 49 -
CHAPTER 3
Performance of the cultivation system.
During steady-state growth, cells were proliferating with a doubling time of 20.8!h.
The cell density in the chemostat vessel was approximately 1*106 cells ml-1 for
D. salina and 2*105 cells ml-1 for D.!bardawil. This difference is probably correlated with the difference in cell size (Table 1). The protein level in the chemostat
cultures was approximately the same for both D. bardawil and D. salina (Table!1).
This reflects the nature of the nitrate limitation: a protein limitation. Upon transfer from the chemostat vessel to the Erlenmeyer flasks the protein content did not
increase (Fig 5a). This means that growth arrest was immediately attained.
However, after growth arrest, the cell density in the Erlenmeyer flasks increased
slightly, but this reflects only the number of cells being in the stage of cell division
at the moment of transfer and does not indicate proliferation (Fig.!5b).
The nitrate concentration of the medium supplied to the chemostat was
0.30 mM. When the amount of nitrate was doubled to 0.60 mM, the nitrate limitation was lost. The cell density increased, but had not doubled, as was expected.
When the nitrate concentration in the medium was reduced to 0.15!mM nitrate, a
corresponding decrease in cell density, protein content and chlorophyll content of
the chemostat vessel occurred. However, the carotene content of the cells in this
situation was doubled compared to the 0.30 mM situation. Apparently accumulation of carotene already took place. Although this might indicate that only at 0.15
mM nitrate the cells experienced actual nitrate limitation, the low substrate concentration in the chemostat vessel at 0.30 mM nitrate (below 0.2 µM) confirms
that at this concentration the chemostat was truly nitrate limited. The higher
amount of carotene during growth had no significant effect on carotene accumulation after growth arrest (data not shown).
Lowering the growth rate of D. bardawil increased the carotene content of
the cells in a linear manner (Table 2). The increased amount of carotene during
growth had no effect on the rate of carotene accumulation after growth arrest of
these cells (Chapter 4). The starch content of D. bardawil only increased at the
lowest specific growth rate.
Conditions required for carotene accumulation.
Growth arrest can be brought about by transferring cells from the chemostat
vessel to Erlenmeyer flasks, but also by transferring cells from the 24 h effluent of
the chemostat to Erlenmeyer flasks. The cells of D. bardawil, obtained by the latter method, usually had a higher carotene content at the start of growth arrest.
During gathering of the 24 h effluent, carotene accumulation already took place.
However, carotene accumulation after growth arrest, although starting from dif-
- 50 -
ferent levels, took place at the same rate in both situations.
Table 2. Carotene- and starch content of D. bardawil during steady-state growth in nitratelimited chemostat cultures, differing in specific growth rate.
In this table the values are given as average ± standard error of samples, taken during steadystate growth. The number of samples (n) is indicated.
µ (day-1)
Carotene content
(µg mg protein-1)
Starch content
(mg glucose mg protein-1)
0.2
56 (n=1)
5.0 (n=1)
0.4
44 ± 3 (n=5)
1.9 ± 0.3 (n=5)
0.6
27 ± 3 (n=4)
2.2 ± 0.3 (n=4)
0.8
14 ± 2 (n=6)
1.6 ± 0.3 (n=6)
Table 3. Comparison of properties of D. bardawil cultivated in nitrate-limited chemostat culture
and sulphate - or phosphate-limiting continuous cultures.
In this table the values for the nitrate limitation are given as average ± standard error of 4
samples taken during steady-state growth in the chemostat; averages of the values of 2 samples
during sulphate-limited growth in continuous culture and 1 value of phosphate-limited growth in
continuous culture.
Nitrate
Sulphate
Phosphate
Protein
(pg cell-1)
363 ± 21
481
534
Chlorophyll
(pg cell-1)
6.3 ± 0.3
7.6
5.6
Carotene
(pg cell-1)
2.0 ± 0.1
9.7
6.3
Starch
(pg glucose cell-1)
150 ± 16
820
600
1007 ± 42
2342
2057
Cell volume
(µm3)
- 51 -
CHAPTER 3
Figure 5. Effect of growth arrest on protein content and cell density of nitrate-limited chemostat
cultures of D.!bardawil (
) and D.!salina (
).
Erlenmeyer flasks at time zero. A. Protein content. B. Cell density.
- 52 -
Temperature was found not to be an important factor in carotene accumulation after growth arrest in D. bardawil (Fig. 6). Although the rate at 10°C was
initially slower than at 21°C or 30°C, the total amount of accumulated carotene
after 7 days of growth arrest at all three temperatures tested, was comparable.
Supply of CO2 was essential in order for D. bardawil to accumulate
carotene after growth arrest (Fig. 7a). However, it was not necessary to aerate
these cultures with air or 2% CO2. Cells of D.!bardawil accumulated carotene at a
fast rate in cultures without aeration. But when CO2 was removed from the cultures, carotene accumulation was slowed down. The remaining carotene accumulation was starch dependent. When growth-arrested cells were incubated for 6
days in the dark, the starch content of the cells was lowered and, upon transfer to
light under conditions of CO2 removal, carotene accumulation was almost completely abolished (Fig. 7b).
Figure 6. Effect of temperature on carotene accumulation of D. bardawil after growth arrest.
Temperature:
,10°C;
,21°C;
,30°C. Growth arrest was applied by
transferring cells from a nitrate-limited chemostat culture to Erlenmeyer flasks.
- 53 -
CHAPTER 3
Figure 7. Effect of CO2 supply on carotene accumulation of D. bardawil after growth arrest.
A. CO2 supply:
,no aeration;
,aeration with 2% CO2 ;
,aeration;
,aeration without CO2 , CO2 was removed by KOH traps. Growth arrest was applied
by transferring cells from the 24 h effluent of a nitrate-limited chemostat culture to Erlenmeyer
flasks. The 24 h effluent was aerated without CO2 during gathering of the effluent. B. CO2
supply and the influence of starch:
,aeration;
,aeration without CO2 ;
,aeration after 6 d darkness;
,aeration without CO2 after 6 d darkness.
Conditions as in A. During the 6 d dark period the starch content decreased from 215 to 44 pg
cell-1.
- 54 -
Effect of other nutrient limitations.
Not only nitrate deprival brought about carotene accumulation in D.!bardawil,
also phosphate- and sulphate deprival had this effect, although not as extensive
as nitrate deprival (Fig. 8). Phosphate- and sulphate limitations were performed in
the same manner as the nitrate-limited chemostat cultures, but steady states
were not easily achieved. Although the extracellular phosphate concentration in
the chemostat vessel was less than 10% of the phosphate concentration supplied
with the medium, a steady-state cell density during growth was not attained.
Nevertheless, when cells were transferred from the culture vessels to Erlenmeyer
flasks, growth was considerably reduced, compared to control cultures which received a dosage of phosphate or sulphate at the time of transfer. Growth arrest in
these two limitations can therefore be regarded as nutrient deprival situations, in
which cells had a lowered amount of the limiting compound as reserve at the start
of nutrient deprival. The results indicate that in order to produce carotene in
D!bardawil in a growth-arrest situation, nitrate limitation is the best choice as limiting nutrient in chemostat culturing to cause growth arrest and subsequently
carotene accumulation.
Figure 8. Effect of nutrient limitation on carotenoid accumulation in nitrate (
), sulphate(
) and phosphate-limited cells (
) of D.!bardawil.
Nutrient limitation was applied by transferring cells from nutrient-limited cultures to Erlenmeyer flasks at time zero.
- 55 -
CHAPTER 3
Cells of D. bardawil grown under phosphate- or sulphate limitation had a
higher protein content than cells grown in nitrate-limited chemostat cultures
(Table 3). Whereas the chlorophyll contents of the cells were comparable under all
three limitations, the carotene- and starch contents were higher in the sulphate
and phosphate limitation. The cell volume of cells in the sulphate- or phosphate
limitation was enlarged more than 2 times, compared to the nitrate limitation. The
cell volume of nitrate-limited cells increased after growth arrest, but the volume of
the cells during several days of sulphate- or phosphate deprival decreased (data
not shown). The cell volume after 7 to 9 days of nutrient deprival was equal for all
three nutrient deprival situations.
Growth arrest verses nitrate starvation.
When cells of D. bardawil were grown in complete BBM as batch cultures, harvested during exponential growth and resuspended in BBM without nitrate, they
showed carotene accumulation comparable with that caused by growth arrest of
nitrate-limited cells (Fig. 9a). They also accumulated starch (Fig. 9b). This suggests a similarity between chemostat and batch cells: in both situations nitrate
stress is causing carotene- and starch accumulation. However a difference was
observed in the increase in cell density. Whereas after growth arrest of nitratelimited cells the cell density was only slightly increased, that of the nitrate-starved
batch culture was more than doubled in four days. This suggests that cells grown
in batch culture have a larger pool of nitrogen compounds compared to cells grown
in nitrate-limited chemostats, but this has apparently no effect on carotene accumulation. From this it can be concluded that it is the lack of nitrate in the
medium that causes the accumulation of carotene and starch, independent of the
conditions experienced during growth by the cells. The regulation of carotene synthesis in this system will be discussed later (Chapter 4).
Discussion
Production of carotenoids.
Carotene production using Dunaliella is mainly performed in subtropical regions,
where light and high temperatures are available in abundance. Carotene accumulation is largely enhanced at high light intensity and high temperatures and therefore these environmental factors make commercial production of carotene with
Dunaliella possible. In this chapter a system is described in which cells of D.
bardawil accumulate carotenoids, using only one stress condition: nutrient deprival. Carotene accumulation in D. bardawil using growth arrest requires no extra-
- 56 -
ordinary environmental demand. A moderate light intensity and temperature are
sufficient and extensive aeration is not necessary. Therefore carotene production
can also be performed in moderate climate zones.
Figure 9. Comparison of carotene- and starch accumulation in D. bardawil, induced by growth
arrest (
) or nitrate-starved cells (
).
Growth arrest was applied by transferring cells from a nitrate-limited chemostat culture to
Erlenmeyer flasks. Batch cultures were cultivated in complete BBM at 21°C and continuous
light of 80-100 µmol m-2 s -1, harvested during the exponential growth phase and resuspended
in BBM without nitrate at time 0. A. !-Carotene content. B. Starch content.
- 57 -
CHAPTER 3
The processes of growth and carotene accumulation are separated in this
system. Growth is established using nitrate-limited chemostat cultures, while
carotene accumulation is brought about by growth arrest. Indoor growth of
Dunaliella requires energy in the form of light, while the modest temperature and
aeration used in this system are sufficient for a fast growth rate. The second stage
of this production system, the accumulation of carotene, is less demanding. Here,
neither an elevated temperature, nor aeration is required. Even the light intensity
can be reduced during the process of carotene accumulation.
Growth arrest is applied by arresting the nutrient supply to the cells. This
can be accomplished in three ways: (1) By arresting the medium supply to the
chemostat vessel. This is the most direct way of applying growth arrest
(Chapter!4), but at the expense of a chemostat culture, each time when practised.
Although this is the preferred way to study the effects of growth arrest, since no
environmental factors are changed, it is not useful as a production system. (2)
Transferring cells from the culture vessel to Erlenmeyer flasks. (3) Transferring
cells from the 24 h effluent to Erlenmeyer flasks. As a carotene production system, it is preferable to place the total effluent each day under conditions in which
carotene accumulation can take place.
The amount of carotenoids produced in this two-staged system is approximately 145 µg mg protein-1 (50 pg cell-1) after 9 days of growth arrest and is
comparable with other reports concerning nutrient limitation or carotene accumulation stimulated by high salinity (Ben-Amotz, 1987; Ben-Amotz et al., 1982;
Cifuentes et al., 1992; Loeblich, 1982) but less than the carotene accumulation
induced by high light intensities (Lers et al., 1990), where a carotene content of 80
pg cell-1 was reached in 9 days or the carotene accumulation by salinity increase
as described by Borowitzka et al. (1990), reaching carotene concentrations as high
as 400 µg mg protein-1 in 8 days at 30% NaCl (Table 4). A mutant of D. bardawil
reached a carotene content of 103 pg cell-1 under nitrate stress (Shaish et al.,
1991).
Chemostat technology.
In a nitrate-limited chemostat culture all environmental conditions must be present in non-limiting amounts, except the concentration of nitrate. The cell density
can be manipulated by lowering the amount of nitrate in the medium. By increasing the amount of nitrate (above 0.30 mM), however, the limitation was lost.
Another environmental condition became the limiting factor in cell growth. This
was most likely the light intensity, due to the moderate fluence rate of 100
µmol!m-2 s-1 used during chemostat cultivation. By increasing the nitrate concen-
- 58 -
tration of the medium the cell density increased, increasing self shading.
Table 4. Comparison of carotene content of Dunaliella.
In this table are the maximum values of carotene content presented of several strains of
Dunaliella, reported in literature plus the results obtained in this investigation.
Amount
(approximately)
Strain
Environmental
condition
Report
50 pg cell-1 (145µg
mg protein-1)
D. bardawil
growth arrest by
nitrate stress
this chapter
0.6 pg cell-1 (9.5µg
mg protein-1)
D. salina
growth arrest by
nitrate stress
this chapter
35 pg cell-1
D. salina
(UTEX 1644)
high salinity
Loeblich, 1982
42 pg cell-1
D. salina
(CONC-007)
high salinity
Cifuentes et al.,
1992
45 pg cell-1
D. bardawil
sulphate stress and
high salinity
Ben-Amotz, 1987
45 pg cell-1
D. bardawil
outdoor cultivation
Ben-Amotz et al.,
1982
80 pg cell-1
D. bardawil
high light intensity
Lers et al., 1990
103 pg cell-1
D. bardawil
(mutant DB1)
nitrate stress
Shaish et al., 1991
400 µg mg protein-1
D. salina
(N41, N43 and W5)
high salinity
Borowitzka et al.,
1990
By using nitrate as the limiting component, chemostat cultivation can be
easily accomplished and parameters, such as the specific growth rate and cell
density, can be controlled. This contrasts with phosphate- and sulphate limitations, where the conditions required for nutrient-limited growth are not met.
Differences between nitrate limitation compared to sulphate- and phosphate limitation were also found by Ben Amotz (1987). Nutrient limitations in batch cultures of D. bardawil were performed, growth was normal in the nitrate limitation
but was inhibited in the sulphate- and phosphate limitation. This resulted in large
stationary phase cells. A large increase in cell volume during sulphate starvation
was also noted by Shaish et al. (1991). Sulphate- and phosphate limitation seem
to interfere with normal growth whereas this is not so during nitrate limitation.
- 59 -
CHAPTER 3
These observations correspond only partly to the natural environment of
Dunaliella. In evaporating saline waters nitrate and phosphate are only available
in limiting amounts, while sulphate is a major component of the environment. So
growth in the natural habitat is therefore never sulphate limited and applying this
condition in laboratory experiments is a rather unusual environment for
Dunaliella.
Influence of light.
Carotene accumulation was exhibited by high light intensity in D. bardawil (Lers
et al., 1990). The light intensity in these experiments was 1650!µmol!m-2!s-1 but
the cells continued to grow. In the growth-arrest system carotene accumulation
took place already at low fluence rates (45 µmol m-2 s -1). Increasing the light intensity, however, did not result in faster carotene accumulation. Light-dependent
synthesis of carotenoids occurs also in several non photosynthetic bacteria and
moulds (Goodwin, 1971; Harding and Shropshire, 1980). Oxygen is required for
carotenoid accumulation in species such as Neurospora crassa, Mycobacterium
marinum, Flavobacterium dehydrogans, Fusarium aquaeductuum and
Phycomyces blakesleeanus (e.g. Bejarano et al., 1990). In general: in light-dependent carotenogenesis light can act as (1) inductor or, (2) activator of enzymes involved in carotene biosynthesis or (3) energy supply. In a number of organisms,
the induction by light can be replaced by active oxygen species, as will be discussed later (Chapter 4).
Cell volume.
Most remarkable is the increase in cell volume of 70% of D.!bardawil after growth
arrest. This contrasts with D. salina which does not show this feature. The increase in cell volume in D. bardawil coincides with carotene accumulation. Cells of
D. bardawil grown under sulphate- or phosphate limitation show an even higher
cell volume. Already during cultivation are these cells restricted in proliferation.
The increase in cell volume in D. bardawil seems therefore to correlate with an inability to sustain growth. The reason for the increased cell volume is not known.
Dunaliella has no rigid cell wall, and the cell volume depends therefore largely upon
the amount of compatible solute used to withstand the osmotic pressure of the
surrounding medium. Although glycerol is the most abundant component used as
osmoprotectant (Avron, 1992), sucrose was also found in increasing quantities in
Dunaliella tertiolecta during adverse conditions (Müller and Wegmann, 1978). It
can therefore be reasoned that accumulation of low molecular metabolites might
cause an increased cell volume in D. bardawil during situations of restrained or in-
- 60 -
hibited growth. Differences in the regulation of the amount of low molecular
metabolites might also cause the difference in cell size between the species.
However, the difference between D. bardawil and D. salina in this respect cannot
be explained by the accumulation of carotene, because there is also a difference in
cell size when D.!bardawil has not accumulated carotene.
Conclusion.
By using nitrate-limited chemostat cultures of D. bardawil, growth can easily be
controlled and manipulated. Growth arrest can be applied and carotene accumulation takes place under moderate conditions. The carotene accumulation does not
require processing and does not strictly depend on well-defined environmental conditions: carotene accumulation brought about by growth arrest is a stable production system. This two-stage carotene production system might be applied in
moderate regions where conditions normally essential for carotene production
such as high light intensity and high temperature are not present.
References
Bejarano, E. R., Avalos, J., Lipson, E. D. & Cerdá-Olmedo, E. (1990). Photoinduced
accumulation of carotene in Phycomyces. Planta 183, 1-9.
Ben-Amotz, A. (1987). Effect of irradiance and nutrient deficiency on the chemical composition of
Dunaliella bardawil Ben-Amotz and Avron (Volvocales, Chlorophyta). Journal of Plant
Press.
Borowitzka, M. A., Borowitzka, L. J. & Kessly, D. (1990). Effects of salinity increase on
carotenoid accumulation in the green alga Dunaliella salina. Journal of Applied
Phycology 2, 111-119.
Cifuentes, A. S., González, M., Conejeros, M., Dellarossa, V. & Parra, O. (1992). Growth and
carotenogenesis in eight strains of Dunaliella salina Teodoresco from Chile. Journal of
Applied Phycology 4, 111-118.
Curtain, C. C., West, S. M. & Schlipalius, L. (1987). Manufacture of !-carotene from the salt
Goodwin, T. W. (1971). Biosynthesis. In Carotenoids, pp. 577-636. Edited by O. Isler. Basel:
- 61 -
CHAPTER 3
Birkhäuser Verlag.
Harding, R. W. & Shropshire Jr., W. (1980). Photocontrol of carotenoid biosynthesis. Annual
Review of Plant Physiology 31, 217-238.
Healey, F. P. (1978). Phosphate uptake. In Handbook of Phycological Methods. Physiological
and Biochemical Methods, pp. 412-417. Edited by J. A. Hellebust & J. S. Craigie.
Cambridge: Cambridge University Press.
Lers, A., Biener, Y. & Zamir, A. (1990). Photoinduction of massive !-carotene accumulation by
the alga Dunaliella bardawil. Plant Physiology 93, 389-395.
Müller, W. & Wegmann, K. (1978). Sucrose biosynthesis in Dunaliella. I. Thermic and osmotic
regulation. Planta 141, 155-158.
Shaish, A., Ben-Amotz, A. & Avron, M. (1991). Production and selection of high !-carotene
- 62 -
Chapter 4
Regulation of carotene- and starch synthesis in
Dunaliella bardawil, in relation to growth arrest.
REGULATION OF CAROTENE SYNTHESIS
REGULATION OF CAROTENE- AND STARCH SYNTHESIS
DUNALIELLA BARDAWIL, IN RELATION TO GROWTH ARREST.
IN
Abstract
The halotolerant green alga Dunaliella bardawil is known to accumulate carotene
in response to stress factors such as high light intensity, high salt concentrations
and nutrient stress. In this chapter, the accumulation of carotene is described as
studied in cells from nitrate-limited chemostat cultures and is compared with the
accumulation of starch. D.!bardawil responded to growth arrest of nitrate-limited
cells by accumulating large amounts of carotene and starch. Carotene accumulation was dependent on protein synthesis. The transcriptional inhibitor actinomycin D did not affect carotene accumulation. However, actinomycin D did inhibit
carotene accumulation after transfer of cells to high light conditions. In contrast,
starch accumulation after growth arrest was not inhibited by actinomycin D, nor
by the translational inhibitor cycloheximide. Its rate was two times slower after
growth arrest than during growth, but was 50 to 100 times faster than that of
carotene synthesis. The rate of carotene accumulation was the same both during
growth and after growth arrest. These results suggest that (1) carotene synthesis
during growth arrest is not subject to down-regulation like starch synthesis and (2)
carotene synthesis after growth arrest might be regulated differently from that at
high light intensity. The implications to the origin of carotene accumulation in D.
bardawil as a result of growth arrest are discussed.
Introduction
D. bardawil is capable of synthesizing carotene. In plants this process is regulated
with chloroplast development (Kleinig, 1989), but the nature of this regulation is
unknown. However, under specific conditions the cells of D.!bardawil accumulate
large amounts of this pigment (Ben-Amotz et al., 1989), implying that the strict
regulation is somehow released. Carotenoid accumulation is brought about by high
light intensities, nutrient stress or high salt conditions. Combinations of these factors enhance carotene accumulation (Loeblich, 1982). In the system described
here, carotene accumulation is brought about by growth arrest. Cells are grown in
nitrate-limited chemostat cultures and by arresting the medium supply they experience growth arrest, which is accompanied by immediate accumulation of
- 65 -
CHAPTER 4
carotenoids and starch (Chapter 3). The regulation of carotenoid accumulation
brought about by high light conditions has been investigated by Lers et al. (1990).
They found that carotene accumulation is regulated at the transcriptional level.
This led to the question whether in cells subjected to growth arrest a similar regulation is operative. In this chapter a number of experiments about the regulation
of carotene accumulation are described and the results are compared with those of
starch accumulation.
American Type Culture Collection) was used. The algae were cultivated in modified Bold's Basal Medium, as described (Chapter 2). Nitrate-limited chemostat cultures were grown at 28°C in 1 L culture vessels, containing 565 ml or 2 L culture
vessels containing 1.5 L medium, as described (Chapter 2). The specific growth
rate (µ) was maintained at 0.8 d-1, unless stated otherwise. Growth arrest was
achieved in 3 ways. (1) Arresting the medium supply to the chemostat culture resulted in immediate growth arrest. All other conditions remained constant. (2)
Growth arrest was applied by transferring cells from a nitrate-limited chemostat
culture to Erlenmeyer flasks at 21°C. The flasks were shaken manually once a
day and illuminated with 4 TL 58 W/33 white fluorescent tubes at a light intensity
of 80-100 µmol m-2 s -1, as measured with a Li-cor model LI-185B photometer
equipped with a LI-190SB quantum sensor. (3) Cells were taken from the 24 h effluent of the chemostat and transferred to Erlenmeyer flasks (conditions as 2).
Cell densities. Cell densities were estimated as described (Chapter 2).
Light induction of carotene accumulation. Cells were grown in batch culture at
21°C, illuminated with 1 TL 58!W/33 white fluorescent tube at a light intensity of
20 µmol m-2 s -1, until a cell density of 2*105 cells ml-1 was reached. Batch cultures were transferred to a water bath at 21°C, illuminated with a HPI/T 400!W
daylight lamp (Philips, Eindhoven, The Netherlands) at an average light intensity
of 750 µmol m-2 s-1. Cultures were shaken manually once a day.
Pigment analysis. Carotene content was estimated as described (Chapter 2).
Total cell extracts, denaturing gel electrophoresis and immunoblot analysis. Total
cell extracts and denaturing gel electrophoresis were performed as described by
- 66 -
Levy et al. (1992) and immunoblot analysis employing anti-CBR (Carotene
Biosynthesis Related) antibodies as described by Levy et al. (1993).
Figure 1. Carotene (
) and starch accumulation (
) during the first 10 h of
growth arrest.
Growth arrest was applied by arresting the nutrient supply of nitrate-limited chemostat
cultures of D. bardawil. The data are averages of 3 independent experiments. Bars indicate the
standard error. A. Carotene accumulation. B. Starch accumulation.
- 67 -
CHAPTER 4
Chemical analyses. Cellular starch and protein content were determined as described (Chapter 2). Cycloheximide (Sigma, St.!Louis, Mo.) was used as inhibitor of
protein synthesis. Actinomycin!D (Merck, Darmstadt, Germany) was used as inhibitor of RNA synthesis. When the cultures were illuminated with high light intensities (750 µmol!m-2!s-1), they were placed behind a yellow cellophane sheet to
reduce photo-destruction of actinomycin!D.
Results
Cultivation of D. bardawil.
D. bardawil was cultivated in chemostat cultures using nitrate limitation. The nitrate concentration inside the culture vessel was below the detection limit (0.2 µM,
Chapter 5). A steady-state situation, in which the rate of growth can be regulated
by manipulating the rate of medium supply to the chemostat vessel, was easily
achieved.
Growth arrest results in carotene- and starch accumulation.
When growth arrest was applied by terminating the nutrient supply, proliferation
of D. bardawil stopped immediately and the cells started to accumulate
carotenoids and starch. The accumulation of carotenoids showed a delay of 4 h and
subsequently the rate of synthesis increased continuously during the first 9 h (Fig.
1a). This is in contrast to the accumulation of starch which had a constant rate
during the first 10 h of growth arrest (Fig. 1b). During growth, cells were subject to
a nitrate-limited environment but after the transfer they immediately experienced
a complete lack of nitrate, which somehow resulted in carotene accumulation.
Starch accumulation did not show a lag period. The immediate start of starch accumulation suggests that it is the result of an unchanged rate of synthesis: we
hypothesize that the starch accumulated in the cells because the process of
balanced growth is interrupted and its synthesis was not down regulated.
Effect of a translational inhibitor.
When the protein-synthesis inhibitor cycloheximide was added to growth-arrested
cells, carotene accumulation was inhibited in a concentration-dependent manner
(Fig. 2a). Starch accumulation in growth-arrested cells was not inhibited by cycloheximide (Fig. 2b). Even when cycloheximide was added 24 h after the start of
growth arrest, further carotene accumulation was inhibited (Fig. 3). This again
had no effect on starch accumulation (data not shown). These results clearly show
the involvement of proteins with a high rate of turn-over in carotene synthesis (or
- 68 -
in its regulation), in comparison with starch synthesis.
Figure 2. Effect of cycloheximide on carotene- and starch accumulation after growth arrest.
Growth arrest was applied by transferring cells from a nitrate-limited chemostat culture of D.
bardawil to Erlenmeyer flasks. A. Carotene accumulation (
,control;
,25;
,50;
,100;
,200 ng cycloheximide ml-1). B. Starch accumulation
(
,control;
,25;
,50;
,100;
,200 ng cycloheximide ml-1).
- 69 -
CHAPTER 4
Figure 3. Effect of cycloheximide on carotene accumulation, added at various times during
growth arrest.
Growth arrest was applied by transferring cells from a nitrate-limited chemostat culture of D.
bardawil to Erlenmeyer flasks. Cycloheximide was added in a concentration of 200 ng ml-1.
Arrows indicate the time of addition. (
,control: no cycloheximide;
,0;
,4;
,8;
,24 h after growth arrest).
Effect of a transcriptional inhibitor.
When the transcriptional inhibitor actinomycin D was added to growth-arrested
cells, starch accumulation was not influenced and carotene accumulation was
only reduced when applied at the highest concentrations ("15 µg ml-1, Fig. 4a).
But when it was applied for more than 2 days at 30 µg ml-1, the number of living
cells decreased rapidly. Therefore the reduction in carotene accumulation here
might be caused by severe damage to the metabolism of the cells. When 15
µg!ml-1 actinomycin D was added to growth-arrested cells, carotene accumulation
was not significantly inhibited during the first 10 h of growth arrest (Fig. 4b).
These results suggest that cells derived from nitrate-limited cultures are capable
of accumulating carotene without prior formation of specific mRNA.
When cells of D.!bardawil were grown in complete BBM as batch cultures,
harvested during exponential growth and resuspended in BBM without nitrate,
they showed a carotene- and starch accumulation which was comparable with
that caused by growth arrest of nitrate-limited cells (Chapter 3). When actinomycin D was added at the time of resuspension, this had hardly an effect on
starch and carotene accumulation (data not shown). Carotene- and starch accu- 70 -
mulation in growth-arrested cells seems therefore also in this respect highly similar to that of batch-cultured cells in complete BBM, which have subsequently
been deprived of nitrate.
Figure 4. Effect of actinomycin D on carotene accumulation after growth arrest.
A. Growth arrest was applied by transferring cells from a nitrate-limited chemostat culture of D.
bardawil to Erlenmeyer flasks. (
,control;
,5;
,10;
,15;
,30 µg actinomycin D ml-1). B. Growth arrest was applied by arresting the nutrient
supply of a nitrate-limited chemostat culture of D. bardawil. (
,control;
,15
µg!actinomycin D ml-1).
- 71 -
CHAPTER 4
Rate of synthesis.
By varying the rate of medium supply to the chemostat vessel, it is possible to
regulate the specific growth rate (µ). Cells were routinely grown at a µ of 0.8 d-1
which was close to the maximum growth rate (µ = 0.9 d-1) for D. bardawil
(Chapter 2). At this rate, the amount of carotene present in the chemostat cells
was comparable to the amount of carotene present in batch cells in the exponential growth phase (data not shown). When the growth rate was reduced, the
carotene concentration increased in a linear manner (Chapter 3). The starch concentration increased only at the lowest specific growth rate. From these data it
was possible to calculate the rates of carotene- and starch synthesis (Table 1).
The rate of starch synthesis was 50-100 times higher than that of carotene synthesis. Apparently there is a negative correlation between specific growth rate
and the rate of carotene synthesis, but this is not the case with starch accumulation. While the rate of carotene synthesis after growth arrest remained in the
same order of magnitude compared to the steady-state growth situation, the rate
of starch synthesis decreased with a factor 2. These results suggest that both
carotene- and starch accumulation are the result of ongoing synthesis while cell
proliferation has stopped, but that starch synthesis exhibits down-regulation, as
can be expected in a non-growing situation, while the rate of carotene synthesis is
not suppressed. The rate of carotene synthesis after growth arrest was not correlated with the specific rate of growth preceding growth arrest.
Discussion
Regulation of carotene accumulation.
Using a nitrate limitation, cells of D. bardawil were cultivated at a growth rate,
close to the maximal growth rate of a batch culture, using comparable conditions
(Chapter 2). During growth, carotenoids were synthesized in amounts, related to
the photosynthetic machinery of the cells. When growth arrest was applied to the
culture, the carotene- and starch content of cells of D. bardawil rose dramatically.
This extra amount of carotenoids was not related to the photosynthetic demand.
It was located in oily globules in the stroma of the chloroplast (Ben-Amotz et al.,
1982; 1987). While the starch accumulation started immediately, the carotene
accumulation gradually increased (Fig. 1). The adaptation of cells directing
carotene not to the thylakoid membrane but to the oily globules might explain the
lag period of a few hours in carotene accumulation after growth arrest. However,
the lag period could also point to an inductive process. Therefore, the regulation of
carotene- and starch synthesis after growth arrest was investigated using a
- 72 -
translational and a transcriptional inhibitor.
Table 1. Rate of carotene (A) and starch (B) synthesis in D. bardawil during steady-state growth
and during growth arrest.
In this table the average ± standard error is given of samples, taken during steady-state growth.
The number of samples (n) is indicated. The number of samples during growth arrest is 2 for µ
= 0.4 and 0.8 and 1 for µ = 0.6 for both carotene- and starch synthesis. During steady-state
growth, the rates of synthesis are calculated by multiplying the carotene- and starch contents of
cells from nitrate-limited chemostats with the cell division factor: eµ . The carotene- and starch
contents of cells are derived from Chapter 3, Table 2. The rates of synthesis, during growth
arrest, are calculated using linear regression of the first 2 d after growth arrest. Growth arrest
was applied to cells, taken from the effluent of nitrate-limited chemostats cultivated at the indicated specific growth rate (µ). The steady-state growth and the growth arrest situation are derived from corresponding chemostat cultures.
Note the difference in order of quantity of the units used in part A and B of the table.
A
B
Rate of carotene synthesis (µg mg protein-1 day-1)
µ (day-1)
steady-state growth
growth arrest
0.4
66 ± 4 (n=5)
65
0.6
49 ± 5 (n=4)
39
0.8
31 ± 4 (n=6)
57
Rate of starch synthesis (mg glucose mg protein-1 day-1)
µ (day-1)
steady-state growth
growth arrest
0.4
2.8 ± 0.4 (n=5)
1.8
0.6
4.0 ± 0.5 (n=4)
0.9
0.8
3.5 ± 0.6 (n=6)
1.8
- 73 -
CHAPTER 4
Protein synthesis is necessary for carotene accumulation.
When the translational inhibitor cycloheximide was added to growth-arrested cells,
carotene accumulation was completely inhibited. This result indicates that protein
synthesis is necessary for carotene accumulation to take place. When cycloheximide was added at a time when carotene accumulation already took place, further
carotene accumulation was inhibited. This indicates that there is a high turnover
of one (or more) enzymes necessary for carotene accumulation.
Lers et al. (1990) reported that in D. bardawil, cells accumulated
carotenoids when transferred from low light to high light intensity. In this case,
carotene accumulation was also inhibited by cycloheximide. In our experiments
complete inhibition of carotene synthesis took place at lower concentrations of
cycloheximide than in Lers’ experiments, probably due to the lower cell densities
applied in our experiments. In all experiments starch accumulation continued
when cycloheximide was added, indicating that 1) starch accumulation is not induced; 2) starch accumulation depends on enzymes which have a low turnover.
Semenenko and Abdullaev (1980) reported that carotene accumulation in
D. salina occurred after a transfer of the cells from 28°C to 22°C at 1700
µmol!m-2 s -1. This accumulation was also inhibited by cycloheximide. Carotene
accumulation in Dunaliella seems therefore generally dependent on protein synthesis.
Effect of actinomycin D on carotene accumulation.
Adding actinomycin D to growth-arrested cells did not lead to a complete inhibition
of carotene or starch accumulation (Fig. 4). Even at the highest concentration
used, at which the cells died after 2 days of incubation, no complete inhibition of
carotene accumulation was observed. This result suggests that carotene accumulation can only be influenced at the translational level. In contrast to these results, Lers et al. (1990) showed that carotene accumulation, induced by high light
intensity, was inhibited by actinomycin D in a concentration-dependent manner.
In these experiments actinomycin D was added 12 h before the transfer from low
light to high light intensity. It appeared, however, when we repeated Lers’ experiment that when actinomycin D was added at the time cells were transferred from
low light to high light conditions, carotene accumulation was not inhibited (Fig.!5a).
However, when actinomycin D was added 12 h before the start of the experiment,
carotene accumulation was inhibited in a concentration-dependent manner (Fig.
5b). This suggests that actinomycin D does not inhibit mRNA synthesis immediately, but that an incubation period before the start of an experiment is necessary. It is evident that such an incubation period is impossible in the chemostat -
- 74 -
growth arrest system. Adding actinomycin D to a chemostat culture would induce
growth arrest by itself, by inhibiting cell proliferation. Therefore the nitrate-limitation would be lost during the incubation period and growth arrest could not be applied properly. An alternative, in which at the same time actinomycin D and an
amount of nitrate, sufficient for 12 h of growth, are supplied to growth-arrested
cells, has the same disadvantage. The nitrate would not be used by the cells during
this incubation period because cell proliferation is inhibited by actinomycin D
(Table 2) and therefore the cells are not subject to a complete lack of nitrate and
no carotene accumulation will take place.
Actinomycin D is widely used in experiments determining the half-life of
mRNA’s. In these experiments the assumption is made that actinomycin D inhibits transcription immediately (Chen et al., 1993; Fogel-Petrovic et al., 1993;
Zhang et al., 1993). This gives support to the idea that carotene accumulation after growth arrest is the result of ongoing normal synthesis during the process of
interrupted cell division.
Comparison of the chemostat system with high light induction.
The question remains if the two different carotene accumulation systems can be
compared with each other, one using light and the other using nutrient deprival as
a stimulus. Is the signal to accumulate carotenoids in both systems the same signal? It is argued that in Dunaliella such a signal might involve active oxygen
species (Shaish et al., 1993). Also in the green alga Haematococcus pluvialis and
the mould Fusarium aquaeductuum active oxygen species are involved in carotene
accumulation (Kobayashi et al., 1993; Theimer & Rau, 1970). Using high light
conditions, photo-inhibition can occur and active oxygen species are generated
(Mishra et al., 1993). But in the chemostat - growth arrest system, cells receive a
light intensity of 100 µmol m-2 s-1, which is far below the intensity where the photosynthetic activity is maximal. Consequently photo-inhibition does not occur
(Chapter 5). However, cells are not proliferating after growth arrest and in this
situation it might be conceivable that active oxygen species are produced by the
photosystems. Thus the signal to accumulate carotenoids in both the light induction system and the chemostat - growth arrest system might be the same.
The next question is if the regulation of carotene accumulation in both systems is the same. In the chemostat - growth arrest system it seems that it is the
result of a complete lack of regulation that effects carotene accumulation, in contrast to the light induction system, where it is regulated at the transcriptional
level (Lers et al., 1990). Using this system, an early light induced protein-like gene
was cloned and its product was termed CBR for carotene biosynthesis related pro-
- 75 -
CHAPTER 4
tein by Lers et al. (1991). This gene was co-activated with the induction of
carotene accumulation. The product of this gene is also present in increasing
amounts during growth arrest in cells derived from a nitrate-limited chemostat
culture (Fig. 6). The protein has been localized in the thylakoid fraction of the
chloroplast (Levy et al., 1992) and is thought to bind zeaxanthin to form photoprotective complexes within the light-harvesting antennae of the photosystems
(Levy et al., 1993). CBR seems therefore not closely related to induction of
carotene accumulation.
- 76 -
Figure 5. Effect of actinomycin D on carotene- and starch accumulation after transfer of
D.!bardawil cells from low light to high light intensities.
(
,control;
,10;
,15;
,30 µg actinomycin D ml-1) A. (Opposite
page) Carotene accumulation. Actinomycin D was added at the time of transfer. B. (Opposite
page) Carotene accumulation. Actinomycin D was added 12 h before transfer. C. Starch accumulation. Actinomycin D was added at the time of transfer.
Table 2. Effect of actinomycin D on cell proliferation of D. bardawil.
In this table the increase in cell density after 2 days is given. Actinomycin D was added at the
indicated concentration to cells taken from the chemostat and at the same time 3 mM nitrate
was added. The cells were incubated using the same conditions as in the growth-arrest experiments. The cell density at the start of the experiment was 21*104 ml-1.
Concentration actinomycin D
(µg ml-1)
Increase in cell density
(*104 ml-1)
0
5
10
15
30
25
26
18
4
0
Recently, additional evidence regarding the regulation of carotene synthesis
in D. bardawil was presented by Jiménez and Pick (1994). The isomeric composition of !-carotene in the oily globules consists of 9-cis- and all-trans !-carotene in
- 77 -
CHAPTER 4
about equal amounts, the thylakoids however contain 85% of the total !-carotene
in the all-trans form. Upon a transfer from low to high light conditions the isomeric
composition changes in the oily globules in favour of the all-trans configuration,
but it does not change in the thylakoid fraction. These different (and differently
changing) isomeric ratios suggest a different kind of regulation of the two carotene
pools in D. bardawil: the first is involved in carotene synthesis of the photosynthetic machinery in the thylakoids and the second is involved in massive production of carotenoids eventually localized in the oily globules. Whether those two
carotene pools are indeed regulated separately, is still not known. More experiments are therefore necessary to determine the differences between the two systems.
Figure 6. Immunoblot analysis with anti-CBR antibodies of cell extracts of D.!bardawil during
growth (0) and after growth arrest (1, 2 and 4 days).
Growth arrest was applied by arresting the nutrient supply of a nitrate-limited chemostat
culture.
Stringency of the limitation.
The cells of D. bardawil in nitrate-limited chemostat cultures are routinely grown
at a µ of 0.8 d-1. This is close to the maximum growth rate (µ = 0.9 d-1) under the
same environmental conditions. In this situation the stringency of the limitation is
low. By lowering the growth rate the stringency was increased. The cells reacted
by increasing the rate of carotene synthesis during growth (Table 1). But the
stringency of the limitation during growth had no effect on the carotene accumulation after growth arrest: although variations were observed, a similar maximal
level was obtained at all growth-rates.
When cells of D.!bardawil are grown in batch cultures on complete BBM,
harvested and resuspended in medium without nitrate, carotene- and starch accumulation is comparable to that in growth-arrested cells (Chapter 3). Carotene
accumulation after growth arrest and in nitrate-free batch cultures seems there-
- 78 -
fore to depend on the total lack of nitrate, experienced by the cells after growth arrest, and is independent of the stringency of the limitation during growth. But
carotene accumulation in growing cells is dependent on the stringency of the limitation. When the amount of nitrate in the medium was reduced from 0.30 mM to
0.15 mM, the rate of carotene synthesis of growing cells in the chemostat increased (Chapter 3, Table 1). Here, again, the availability of nitrate influences the
rate of synthesis.
It can be hypothesized that energy (ATP), which can be used in carotene
synthesis, is more available in cells, growing at a slow rate, than in cells growing at
a fast rate. Or the sensor mechanism to account for the different rates of carotene
synthesis might be found in the nitrogen stress - response system found in bacteria, where glutamine synthetase has a key position (for review see Reitzer and
Magasanik, 1987). This enzyme, which is also present in Dunaliella, functions in
ammonium assimilation but has also a central role in the cellular nitrogen
metabolism (Seguineau et al., 1989). Regulatory mechanisms such as feedback
inhibition and covalent adenylation of the glutamine synthetase are found. This
enzyme is not only involved in the production of glutamine, but also in genetic control of catabolic nitrogen uptake. This system becomes activated when growth is
ammonium limited. However, in bacteria growing on all other organic nitrogen
sources (including nitrate), growth is nitrogen limited, because the uptake and assimilation systems of these nitrogen sources are rate-limiting. Growth of
Dunaliella on nitrate or ammonia as nitrogen source is almost equal (Chapter 2).
Here, it can not be ruled out that nitrate stress is regulating carotene synthesis
by this system. An observation with Chlamydomonas monoica illustrates the role
of small amounts of nitrate in signalling (van den Ende, 1994). Gametic induction
in nitrate starved cells of C.!monoica only takes place when a small amount of nitrate is added. This resulted in a synchronous cell division and immediate mating
of the daughter cells. Here, an optimum was found in the amount of nitrate (0.1
µmol nitrate per 105 cells): increasing the amount of nitrate resulted in the loss of
gametogenesis and promotion of vegetative cell division, while lowering it inhibited
the synchronous cell division, necessary for mating.
Comparison of carotene- and starch synthesis.
Several differences have been noticed between the synthesis of carotene and
starch. (1) During growth, the rate of starch synthesis is approximately 50-100
times the rate of carotene synthesis (Table 1). (2) The rate of starch synthesis after growth arrest is decreased by half, whereas the rate of carotene synthesis remains at the same rate. This decrease in rate of starch synthesis can be at-
- 79 -
CHAPTER 4
tributed to down-regulation, which is probably due to the general decrease in
metabolism of the cells upon the transfer to growth arrest. (3) During steadystate growth, the rate of carotene synthesis is correlated with the specific growth
rate. Starch synthesis is not. (4) Although both carotene- and starch accumulation correlate strongly with the cessation of cell proliferation, starch accumulation
is more influenced by this. When actinomycin D was added at the time of light induction to cells of D.!bardawil, starch accumulation was strongly enhanced in a
concentration-dependent manner (Fig. 5c). Here, the cessation of cell proliferation
by actinomycin D effects immediate starch accumulation. In general: starch accumulation seems correlated with cessation of growth, whereas carotene accumulation seems correlated with the stringency of the nitrate-limitation. A maximal
stringency results of course in growth arrest.
These differences underline the different roles of carotene and starch in the
metabolism of cells of D. bardawil; starch being a storage polymer, while the function for accumulated carotene is not clear. However, three hypotheses are suggested for the function of the accumulated carotenoids. (1) Protection against high
light intensities was reported by Ben-Amotz et al. (1989). Accumulated carotene
is localized in oily globules in the interthylakoid space of the chloroplast. The accumulated carotenoids operated as a screen to protect against excess irradiation.
(2) The possibility of carotene acting as a storage polymer is not likely, because,
upon transfer to a normal growth condition, the amount of carotene only gradually
decreases (Borowitzka et al., 1984). In a similar experiment we observed that the
rate of carotene decrease was as fast as the cell density increased, but not faster.
Also Ben-Amotz et al. (1982) reported that D.!bardawil did not utilize accumulated
carotene on transfer to darkness or to CO2-free medium in the light. (3) The possibility that carotene accumulation acts as an overflow metabolism is reported by
Borowitzka and Borowitzka (1988). The neutral compound carotene might act as
a “carbon sink” in a non-growth situation, allowing photosynthesis to continue.
Effects of growth arrest on carotene accumulation and photosynthetic parameters are reported in chapter 5 and the possible involvement of carotene as carbon
sink is discussed.
Acknowledgements
I wish to thank Dr. David Govezensky of the Biochemistry department of the
Weizmann Institute of Science, Rehovot, Israel for performing the immunoblot
analysis.
- 80 -
References
Ben-Amotz, A., Gressel, J. & Avron, M. (1987). Massive accumulation of phytoene induced by
norflurazon in Dunaliella bardawil (Chlorophyceae) prevents recovery from
photoinhibition. Journal of Phycology 23, 176-181.
Ben-Amotz, A., Shaish, A. & Avron, M. (1989). Mode of action of the massively accumulated !carotene of Dunaliella bardawil in protecting the alga against damage by excess
irradiation. Plant Physiology 91, 1040-1043.
Borowitzka, L. J., Borowitzka, M. A. & Moulton, T. P. (1984). The mass culture of Dunaliella
salina for fine chemicals: From laboratory to pilot plant. Hydrobiologia 116/117, 115121.
Press.
Chen, M., Schnermann, J., Smart, A. M., Brosius, F. C., Killen, P. D. & Briggs, J. P. (1993).
Cyclic AMP selectively increases renin mRNA stability in cultured juxtaglomerular
granular cells. The Journal of Biological Chemistry 268, 24138-24144.
Ende, H. van den (1994). Vegetative and gametic development in the green alga
Chlamydomonas. Advances in Botanical Research 20, 125-161.
Fogel-Petrovic, M., Shappell, N. W., Bergeron, R. J. & Porter, C. W. (1993). Polyamine and
polyamine analog regulation of spermidine/spermine N1-acetyltransferase in MALME3M Human melanoma Cells. The Journal of Biological Chemistry 268, 19118-19125.
Jiménez, C. & Pick, U. (1994). Differential stereoisomer compositions of !,!-carotene in
thylakoids and in pigment globules in Dunaliella. Journal of Plant Physiology 143, 257263.
Kleinig, H. (1989). The role of plastids in isoprenoid biosynthesis. Annual Review of Plant
Physiology and Plant Molecular Biology 40, 39-59.
Kobayashi, M., Kakizono, T. & Nagai, S. (1993). Enhanced carotenoid biosynthesis by oxidative
stress in acetate-induced cyst cells of a green unicellular alga, Haematococcus pluvialis.
the alga Dunaliella bardawil. Kinetics and dependence on gene activation. Plant
genes in higher plants and !-carotene biosynthesis in the alga Dunaliella bardawil. The
Levy, H., Gokhman, I. & Zamir, A. (1992). Regulation and light-harvesting complex II
association of a Dunaliella protein homologous to early light-induced proteins in higher
plants. The Journal of Biological Chemistry 267, 18831-18836.
Levy, H., Tal, T., Shaish, A. & Zamir, A. (1993). Cbr, an algal homolog of plant early lightinduced proteins, is a putative zeaxanthin binding protein. The Journal of Biological
Chemistry 268, 20892-20896.
Mishra, N. P., Mishra, R. K. & Singhal, G. S. (1993). Involvement of active oxygen species in
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photoinhibition of photosystem II: protection of photosynthetic efficiency and inhibition of
lipid peroxidation by superoxide dismutase and catalase. Journal of Photochemistry and
Photobiology. B: Biol 19, 19-24.
Reitzer. L. J. & Magasanik, B. (1987). Ammonia assimilation and the biosynthesis of
glutamine, glutamate, aspartate, asparagine, L- alanine, and D-alanine. In Escherichia
coli and Salmonella typhimurium: Cellular and Molecular Biology, Vol 1, pp. 302-321.
Edited by F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter &
H. E. Umbarger. Washington, D.C.: American Society for Microbiology.
Seguineau, C., Batrel, Y. & Le Gal, Y. (1989). Glutamine synthetase of Dunaliella primolecta.
Partial characterization and possible adenylation control in relation to nitrogen nutrient
levels. Biochemical Systematics and Ecology 17, 503-508.
Semenenko, V. E. & Abdullaev, A. A. (1980). Parametric control of !-carotene biosynthesis in
Dunaliella salina cells under conditions of intensive cultivation. Soviet Plant Physiology
27, 22-30.
Shaish, A., Avron, M., Pick, U. & Ben-Amotz, A. (1993). Are active oxygen species involved in
induction of !-carotene in Dunaliella bardawil? Planta 190, 363-368.
Theimer, R. R. & Rau, W. (1970). Untersuchungen über die lichtabhängige Carotinoidsynthese.
V. Aufhebung der lichtinduktion durch Reduktionsmittel und Ersatz des Lichts durch
Wasserstoffperoxid. Planta 92, 129-137.
Zhang, S., Sheng, J., Liu, Y. & Mehdy, M. C. (1993). Fungal elicitor-induced bean proline-rich
protein mRNA down-regulation is due to destabilization that is transcription and
translation dependent. The Plant Cell 5, 1089-1099.
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Chapter 5
Effect of growth arrest on carotene accumulation and
photosynthesis in Dunaliella.
(Pieter Vorst, Robert L. Baard, Luuc R. Mur, Harry J. Korthals and Herman van
den Ende. 1994. Published in: Microbiology 140: 1411-1417.)
CAROTENE ACCUMULATION AND PHOTOSYNTHESIS
EFFECT OF GROWTH ARREST ON CAROTENE ACCUMULATION AND
PHOTOSYNTHESIS IN DUNALIELLA.
Abstract
The halotolerant green alga Dunaliella bardawil is known to accumulate !carotene in response to stress factors such as high light intensity, high salt concentrations and nutrient limitation. In this report, the accumulation of !-carotene
was studied in cells from nitrate-limited chemostat cultures, in comparison with
those of D. salina, a strain that does not accumulate !-carotene under stress conditions. D. bardawil responded to growth arrest by accumulating !-carotene and,
to a lesser degree, lutein and zeaxanthin. A substantial fraction of !-carotene and
all the lutein and zeaxanthin was associated with the thylakoid fraction. The accumulation of carotenoids in D. bardawil occurred only in the light, but the light intensities were far below those where the photosynthetic rate is maximal. After
growth arrest, the amount of chlorophyll (Chl) decreased in both strains. However,
in D. bardawil Chl a decreased to a lesser extent in comparison with Chl b, which
resulted in an increased Chl a/b ratio. The maximum photosynthetic capacity declined rapidly in both strains after growth arrest. In contrast, the photosynthetic
efficiency showed a temporary increase in D. bardawil and a decrease in D. salina.
This increase did not occur when carotenogenesis was inhibited by diphenylamine,
implying a causal relationship between enhanced carotenogenesis and increase of
photosynthetic efficiency. The possible involvement of stress-accumulated
carotenoids in photosynthetic activity is discussed.
Introduction
In plants, carotenoids are essential components of the light-harvesting complexes
and reaction centres of the photosynthetic apparatus. They are involved in light
harvesting, energy transduction and protection against damage by photo-oxidation (Koyama, 1991). Their synthesis is co-ordinatedly regulated with chloroplast
development (Kleinig, 1989), but the mechanism of regulation is unknown. It is of
interest to study chloroplast-associated carotenogenesis in a system in which the
co-ordination with chloroplast development is released under specific conditions.
Such a system is Dunaliella salina Teod., a halotolerant unicellular green alga,
containing a single chloroplast. This species synthesizes carotenoids which are
- 85 -
CHAPTER 5
typical of green plants, but some isolates, designated as Dunaliella bardawil, have
the potential to accumulate carotenoids to as much as 8% of the dry weight (BenAmotz et al., 1989). Carotenoid accumulation is brought about by high light intensities, nutrient stress or high salt conditions. Each of these factors individually
has an effect on carotenoid biosynthesis, but when applied together, their effects
are additive (Loeblich, 1982).
We show here that growth arrest of nitrate-limited D. bardawil cells under
moderate light intensities also results in the accumulation of carotenoids, especially !-carotene. This property was studied in comparison with a carotenoid nonaccumulating strain of D. salina. Growth arrest also led to changes of photosynthetic characteristics. The interaction between carotenoid accumulation and photosynthesis in these two strains was examined.
Materials and Methods
American Type Culture Collection) and D. salina (strain 9 of the Institute of Plant
Physiology of the Russian Academy of Sciences, Moscow) were used.
The algae were cultivated in modified Bold's Basal Medium (Harris, 1989)
which consisted of NaNO3, 2.94 mM; MgSO4.7H2O, 0.3 mM; CaCl2.2H20, 0.17
mM; K2HPO4.3H2O, 0.42 mM; KH2PO4, 1.29 mM; FeNaEDTA, 0.068 mM;
NaHCO3, 5 mM; NaCl, 1 M and micro nutrients according to Wiese (1965):
H3BO3, 9.7 µM; MnSO4.4H2O, 1.79 µM; NaVO3, 0.52 µM; ZnSO4.7H2O, 0.15
µM; CuSO4.5H20, 0.06 µM; CoSO4.7H2O, 0.02 µM; (NH4)6Mo7O24.4H2O, 0.003
µM. The medium was sterilized by autoclaving at 120°C. The phosphate components were autoclaved separately and the NaHCO3 solution was filter-sterilized
before being added to the sterilized medium.
Stock cultures were routinely maintained on 1.5% (w/v) agar slants of the
same medium, except that the phosphate components and NaHCO3 were autoclaved together with the medium. The cultures were kept at 19°C and illuminated
with two TL 58 W/33 white fluorescent tubes together with a HPI/T 400 W daylight lamp (Philips, Eindhoven, The Netherlands) at an average light intensity of
150 µmol m-2 s-1. A 12 h light/12 h dark regime was applied. Stock cultures were
transferred to fresh agar slants every 6 to 8 weeks.
Chemostat cultures were carried out at 28°C in 1 l culture vessels, containing 565 ml medium. Continuous one-sided illumination was provided by a HPI/T
400 W daylight lamp at an average light intensity of 100 µmol m-2 s -1 in the centre of the culture, as measured with a Li-cor model LI-185B photometer equipped
- 86 -
with a LI-190SB quantum sensor. Batch cultures (50 ml) at the beginning of the
stationary phase, were used as inoculum. The cultures were aerated with 2% (v/v)
CO2 and stirred every 6 h for 1 min, and also prior to sampling with a magnetic
Teflon-coated rod. Cells that adhered to the glass wall were regularly scraped off,
using the magnetic Teflon-coated rod. Nitrate limitation was imposed by reducing
the nitrate content of the medium to 10% of the original amount. The specific
growth rate was maintained at 0.8 d-1. This resulted in a cell density of 2*105 cells
ml-1 for D. bardawil and 106 cells ml-1 for D. salina. Interrupting the medium
supply to the culture resulted in immediate growth arrest.
Thylakoid Isolation. Thylakoids were isolated according to Ben-Amotz et al.
(1982), with slight modifications. Cells were harvested at 1600 g for 5 min. The algal pellet was resuspended in 25 ml 30 mM NaCl and centrifuged at 1600 g for 5
min. The pellet was resuspended in 10 ml water and after mixing thoroughly for 1
min, centrifuged at 44 g for 5 min. The supernatant was centrifuged at 12000 g for
10 min. The pellet fraction was washed once in water and contained the thylakoids
while the supernatant fraction contained carotene globules.
Pigment analysis. Pigments were analyzed by HPLC (system I) as described by
Van der Staay et al. (1992). Lutein and zeaxanthin were separated by HPLC
(system II) as described by Korthals & Steenbergen (1985), with some modifications. A linear gradient was used. Phase A (hexane with 0.02% water) and phase B
(acetone with 0.5% methanol) were mixed from an initial value of 20% B to 30% B
in 12 min. The flow rate was 1.5 ml min-1. Pigments were detected with a HP
1040 diode array detector.
Photosynthesis. Photosynthesis versus illumination (P-I) curves were made by
measuring oxygen production in whole cells using a Clark-type electrode as
described by Dubinsky et al. (1987) at 28°C. From these curves maximal photosynthetic rates (PMAX) and light utilization efficiencies (") were derived
(Falkowski, 1984). During the transfer from the culture vessel to the reaction vessel the cell samples were kept in the dark. The rate of oxygen production by isolated thylakoids was measured using a Clark-type electrode at 20°C with red light
illumination (# > 600 nm) at saturated light intensity and K3Fe(CN)6 or benzyl
viologen as electron acceptor.
- 87 -
CHAPTER 5
Table 1. Effect of growth arrest on pigment content of nitrate-limited D. bardawil, D. salina and
D. bardawil with DPA.
Experimental conditions as in Fig. 1. The data at day zero are means ± standard error (n= 4 for
D. bardawil and D. salina, and n = 3 for D. bardawil + DPA) of the analyses done during
steady-state growth in the chemostat. The standard error is derived from the data of one experiment.
Pigments (µg mg protein-1)
Time
(d)
!-Carotene
Lutein
and zeaxanthin
Antheraxanthin
Neoxanthin
Violaxanthin
Chl a
Chl b
Chl a/b
34.6 ± 0.1
7.9 ± 0.1
4.4
D. bardawil
0
10.3 ± 0.4 3.1 ± 0.1
0.5 ± 0.0
0.6 ± 0.0 1.2 ± 0.1
4
116.8
8.1
0.7
0.7
0.5
24.6
5.2
4.7
7
190.9
12.3
0.9
0.8
0.9
29.2
5.5
5.3
47.7 ± 2.2
12.0 ± 0.5
4.0
D. salina
0
4.5 ± 0.2
7.4 ± 0.3
0.7 ± 0.0
1.0 ± 0.0 3.1 ± 0.1
3
6.5
11.0
1.7
0.9
3.6
38.2
10.4
3.7
6
6.9
10.9
1.7
0.8
4.2
21.8
7.2
3.0
29.7 ± 1.8
7.1 ± 0.4
4.2
D. bardawil + DPA
0
9.6 ± 0.5
3.0 ± 0.1
0.5 ± 0.0
0.4 ± 0.0 1.1 ± 0.0
3
10.3
4.1
0.5
0.4
0.2
24.7
7.7
3.2
4
14.7
4.2
0.5
0.5
0.2
24.7
7.5
3.3
Excitation fluorescence of isolated thylakoids. Fluorescence of Chl a was measured on a SLM-aminco spf-500 spectrofluorometer at room temperature. The
thylakoid suspension was diluted to an OD663 of 0.2 and placed in a 1 cm pathlength four-sided quartz cuvette. Excitation ranged from 350 to 680 nm with a
step size of 1 nm; emission of Chl a was measured at 690 nm.
Chemical analyses. Cellular starch content was determined as described by
Herbert et al. (1971), using a solution of 2 mg anthrone ml-1 in 98% sulphuric acid,
with D-glucose as standard. Cells were freeze-dried before analysis. Protein content was estimated according to Herbert et al. (1971), with bovine serum albumin
as a standard. Cells were freeze-dried and boiled for 5 min in 1 M NaOH before
analysis. Nitrate levels in culture liquids were measured according to Kempers &
Luft (1988). Nitrate was reduced to nitrite, which was assayed using 1% sulphanilamide in 35% HCl and 0.3% N-(1-naphtyl)-ethylenediamine dihydrochloride
- 88 -
in 1% HCl as reagents. Diphenylamine (DPA) (Merck) was used as an inhibitor of
carotene biosynthesis. It was added to the culture as a solution in ethanol (0.12%),
at a final concentration of 40.6 µM.
Figure 1. Effect of growth arrest on (A) starch and (B) carotenoid accumulation in nitrate-limited
cells of D. bardawil (
), D. salina (
) and D. bardawil + DPA (
).
At time zero the nutrient supply to the chemostat was terminated. At the indicated times the
content of !-carotene was analyzed, using HPLC (system I). The data are single determinations.
The data of D. bardawil with DPA were normalized to the data of D. bardawil without DPA by
multiplying by a factor of 1.14, based on the slight difference in protein content per cell during
the steady-state before growth arrest.
- 89 -
CHAPTER 5
Results
Two species of Dunaliella, D. bardawil and D. salina, were cultivated under identical conditions. D. bardawil is capable of accumulating large amounts of carotene
(Ben-Amotz et al., 1982), in contrast to D. salina. The two species differ in size, D.
bardawil being approximately four times larger than D. salina. This resulted in a
difference in cell-based protein and Chl content. Comparisons were therefore made
on a protein basis. The genetic relationship between D. bardawil and D. salina was
not investigated.
The two species were cultivated in chemostat cultures with nitrate limitation. The nitrate level of the culture liquid inside the culture vessel was below the
detection limit (0.2 µM). A steady-state situation, in which the rate of growth can
be regulated by manipulating the rate of medium supply to the chemostat vessel,
was easily achieved. Although nitrate limitation was used, the carotenoid content
of D. bardawil was comparable to that of exponential phase cells, grown on complete medium in batch cultures (data not shown). When the medium supply was
arrested, cell proliferation stopped immediately, which was accompanied by an increase of starch content in both species (Fig. 1a). Only D. bardawil exhibited in
addition a considerable increase in !-carotene content (Fig. 1b). This could be inhibited by DPA, which was added at the time that growth arrest was imposed.
After four days, this treatment was no longer effective, indicating a possible inactivation of DPA. The inhibitor had no significant effect on the accumulation of
starch.
!-Carotene was the major carotenoid that accumulated in D. bardawil after
growth arrest (Table 1). There was also a marked increase in the content of lutein
and zeaxanthin (Table 1). This is in agreement with previous studies of stress-induced carotenogenesis in this organism (Borowitzka et al., 1990). In comparison to
lutein and zeaxanthin, the other xanthophylls are less abundant in both species of
Dunaliella (Table 1). The contents of neoxanthin and violaxanthin in both strains
hardly changed after growth arrest while antheraxanthin increased more in D.
salina than in D. bardawil. In the DPA-treated cells of D. bardawil the content of
violaxanthin decreased considerably.
Ben-Amotz et al. (1987) showed that the accumulated !-carotene was, to a
large extent, deposited in oily globules in the stroma fraction of the chloroplasts.
Table 2 shows, nevertheless, that a substantial fraction of the accumulated !carotene was associated with the thylakoid fraction. The ratio of !-carotene / Chl
a in the thylakoids increased by a factor of eight during growth arrest and lutein
and zeaxanthin were quantitatively recovered in the thylakoid fraction.
- 90 -
Figure 2. Light dependency of carotenogenesis in D. bardawil after 5 days of growth arrest.
Cells were taken from the 24 h effluent of the chemostat at day 0 and incubated at room temperature. Carotene content was estimated by extracting 10 ml of cell culture with 80% acetone
and the absorption was measured at 663.2, 646.8 and 470.0 nm in a Perkin-Elmer Hitachi 200
spectrophotometer. Carotene content was calculated using the equations of Lichtenthaler
(1987). The data are combined from two independent experiments with a different range of light
intensity. The average carotene concentration at t = 0 was 8 pg cell-1.
Figure 3. Photosynthesis versus illumination curves of D. bardawil (
(
) during steady-state growth in the chemostat.
- 91 -
) and D. salina
CHAPTER 5
The accumulation of carotenoids in D. bardawil was enhanced in the light,
as shown in Fig. 2. The light stimulation was saturated at approximately 100
µmol m-2 s-1, and was far below the light intensity where the photosynthetic rate
is maximal (PMAX) which was about 1500 µmol m-2 s -1 for D. bardawil and 250
µmol m-2 s -1 for D. salina (Fig. 3). Thus, this system was considered suitable to
study the interference of carotenogenesis with photosynthesis that occurs at low
light intensity.
Figure 4. Effect of growth arrest in nitrate-limited cells of D. bardawil (
), D. salina
(
) and D. bardawil + DPA (
) on photosynthetic parameters.
A. Photosynthetic capacity (PMAX); B. Photosynthetic efficiency ("). Experimental conditions
were as in Fig. 1.
- 92 -
After growth arrest, the amount of Chl decreased in both strains (Table 1).
However, in D. bardawil Chl a decreased to a lesser extent in comparison with Chl
b, which resulted in an increased Chl a/b ratio.
PMAX, measured as oxygen production, declined rapidly in both strains after
growth arrest, with or without DPA (Fig. 4a). The light utilization efficiency ("), on
the other hand, showed a rapid decrease in D. salina, but a slight increase in D.
bardawil (Fig. 4b), which was maintained for about 4 days. This increase was completely inhibited by DPA, implying a causal relationship between enhanced
carotenogenesis and the temporary increase of photosynthetic efficiency.
However, even though it was demonstrated that a proportion of the accumulated
carotenoids (especially lutein and zeaxanthin) was located in the thylakoid membrane, it seems improbable that they took part in the light-harvesting process.
Excitation fluorescence spectra of isolated functional thylakoids of D. bardawil before and after growth arrest showed no difference in the contribution of light-harvesting pigments to the excitation profile of Chl a (Fig. 5).
Discussion
In nitrogen-limited chemostat cultures of two Dunaliella species, grown under
moderate light intensities, growth arrest resulted in an immediate and dramatic
rise in carotenoid content in D. bardawil, but not in D. salina. The maximum
amount of carotenoids in D. bardawil was attained after about 6 days. The predominant accumulated pigment was !-carotene, but lutein and zeaxanthin also increased. The increase of !-carotene and the minor carotenoids lutein and zeaxanthin in D. bardawil under stress conditions has been reported previously, for example after transfer from low-light to high-light (Lers et al., 1990) or low-salt to
high-salt conditions (Gómez-Pinchetti et al., 1992; Borowitzka et al., 1990). In the
D. bardawil strain used here, the levels of "-carotene were extremely low, in contrast to studies by other groups reporting "-carotene levels of 2.5% or higher (BenAmotz et al., 1988; Erazo et al., 1989).
The ratio total carotene over thylakoid-associated carotene in Table 2 is
normalized to Chl content, because the isolation and purification of thylakoids is
so lengthy that considerable losses occurred and budgeting was impractical. This
could be done because it is reasonable to assume that all Chl is in the thylakoids.
Although most of the stress-produced !-carotene was located in oily globules inside
the stroma fraction of the chloroplast, a substantial fraction of !-carotene and essentially all the lutein and zeaxanthin were bound to the thylakoid fraction of the
chloroplast. The amount of carotene in the thylakoid fraction reached a maximum
- 93 -
CHAPTER 5
at day 3 after growth arrest and did not increase further. Although the amount of
accumulated carotene in the oily globules is considerably higher than in the thylakoid fraction and even continued to increase after day 3 of growth arrest, it cannot be excluded a priori that the accumulated carotene in the thylakoid fraction is
involved in the light-harvesting process. This observation is interesting in view of
the finding by Lers et al. (1991) that induction of carotenogenesis is accompanied
by the increase of a transcript that is closely related to early light-induced genes of
higher plants and whose translational product might function as a pigment-binding protein, whose synthesis is co-ordinately regulated with carotenogenic enzymes.
Table 2. Distribution of carotenoids in whole cells and isolated thylakoids in nitrate-limited D.
bardawil after growth arrest.
Data for the ‘cells’ fraction represent total pigments; ‘thyl’ is the thylakoid fraction from the cells,
isolated as described in Material and Methods. Pigments were extracted from cells and freezedried thylakoids with acetone and hexane and analyzed by HPLC (system II). n.d. = not detected.
Time (d)
Pigments/Chl a (molar ratio)
Fraction
!-Carotene
Lutein
Zeaxanthin
Chl b
0
Cells
Thyl
1.14
0.39
0.18
0.20
n.d.
n.d.
0.39
0.45
3
Cells
Thyl
12.06
3.13
0.91
1.06
0.32
0.36
0.40
0.44
4
Cells
Thyl
8.69
2.75
0.61
0.59
0.21
0.20
0.32
0.41
10
Cells
Thyl
22.24
2.55
0.60
0.47
0.19
0.23
0.26
0.32
In most higher plants and green algae, the reaction centres and light-harvesting complexes decrease more or less proportionally during nutrient stress;
therefore the Chl a/b stoichiometry remains rather constant (Anderson, 1986;
Siefermann-Harms, 1985; Thornber et al., 1991). Dunaliella is exceptional in
having a high Chl a/b ratio (Sukenik et al., 1987). Growth arrest results in a de- 94 -
crease of Chl a and b content, which might reflect a decrease in the number and/or
size of the photosystems and light-harvesting complexes. This is accompanied by
a decrease in photosynthetic O2 evolution. However, compared to D. salina, D.
bardawil exhibits a temporary increase in the Chl a/b ratio, which implies a slower
decrease in the number of photosystems. This increase in the Chl a/b ratio is not
present when cells are incubated with DPA. Since DPA is quite specific in inhibiting the increase of carotenoid content, this implies that the altered Chl a/b ratio is
connected with the increase of carotenoids after growth arrest. This phenomenon
is accompanied by a temporary apparent increase of photosynthetic efficiency,
reflecting an increased efficiency of electron transport.
Irrespective of the mechanism by which the temporary increase of photosynthetic efficiency is brought about, it is possible that a relatively large demand
of reducing equivalents required for carotenoid biosynthesis might influence the
photosynthetic efficiency. This contention is supported by the fact that DPA also
blocks the increase in photosynthetic efficiency. In this way, the accumulated
carotenoids, of which the bulk is located in oily globules, might serve as an electron
sink. Another possibility is that the stress-induced carotenoids function as a
screen to protect the photosystems from photochemical degradation (Ben-Amotz
et al., 1987, 1989). The low-light intensities used in our experiments make such a
hypothesis less probable. Zeaxanthin is more directly involved in protection of the
photosystems, but only in situations where photo-inhibition of photosynthesis occurs (Demmig-Adams, 1990; Neubauer & Yamamoto, 1992). Zeaxanthin, which is
formed by de-epoxidation of violaxanthin via antheraxanthin in the xanthophyll
cycle, is involved in the radiationless dissipation process of excess excitation energy. By protecting the photosystems it is possible that the temporary increase in
photosynthetic efficiency in D. bardawil could be maintained for a prolonged period
of time. It is unlikely that this is the mechanism by which the temporary increase
in photosynthetic efficiency in D. bardawil can be explained because: (1) the cells
were grown under moderate light intensities (2) the amounts of violaxanthin and
antheraxanthin were much lower than the amount of zeaxanthin, and (3) their
abundances did not change significantly.
The question arises whether the stress-accumulated carotenoids, in particular lutein, become involved in the light-harvesting process, which is implied by
the fact that they are associated with the thylakoid membrane fraction. Lutein
has been reported to be an integral part of LHCII (Sukenik et al., 1988;
Siefermann-Harms, 1985). Recently Bassi et al. (1993) reported lutein to be
bound to the proteins CP47 and CP43 of photosystem II. However, the fact that
excitation spectra of isolated thylakoids of D. bardawil before and after growth ar-
- 95 -
CHAPTER 5
rest did not show an increased contribution of light harvesting pigments to the Chl
a fluorescence contradicts this possibility.
Figure 5. Excitation fluorescence spectra and absorption spectra of isolated thylakoids.
A & B. Excitation fluorescence of thylakoids at day 0 (A) and day 4 (B); C & D. Absorption spectra of isolated thylakoids at day 0 (C) and day 4 (D). Isolated thylakoids were extracted in 80%
acetone and absorption spectra were measured in a Perkin-Elmer Hitachi 200 spectrophotometer.
Acknowledgements
We wish to thank Hans Matthijs (Department of Microbiology, University of
Amsterdam) for helpful discussions, Martin Kooijman (Department of Biophysics,
Free University, Amsterdam) for his help with the excitation fluorescence spectra
and the Department of Plant Physiology of the Free University for the use of their
spectrofluorometer.
References
Anderson, J. M. (1986). Photoregulation of the composition, function, and structure of thylakoid
membranes. Annual Review of Plant Physiology 37, 93-136.
Bassi, R., Pineau, B., Dainese, P. & Marquardt, J. (1993). Carotenoid-binding proteins of
photosystem II. European Journal of Biochemistry 212, 297-303.
- 96 -
Ben-Amotz, A., Gressel, J. & Avron, M. (1987). Massive accumulation of phytoene induced by
norflurazon in Dunaliella bardawil (Chlorophyceae) prevents recovery from
photoinhibition. Journal of Phycology 23, 176-181.
Ben-Amotz, A., Lers, A. & Avron, M. (1988). Stereoisomers of !-carotene and phytoene in the
alga Dunaliella bardawil. Plant Physiology 86, 1286-1291.
Ben-Amotz, A., Shaish, A. & Avron, M. (1989). Mode of action of the massively accumulated !carotene of Dunaliella bardawil in protecting the alga against damage by excess
irradiation. Plant Physiology 91, 1040-1043.
Dubinsky, Z., Falkowski, P. G., Post, A. F. & Van Hes, U. M. (1987). A system for measuring
phytoplankton photosynthesis in a defined light field with an oxygen electrode. Journal
of Plankton Research 9, 607-612.
Erazo, S., Proust, P., Viani, M. & Müller, K. (1989). Estudio de la biomasa y de los pigmentos
carotenoides contenidos en una especie nativa de la microalga Dunaliella salina sp.
Revista de Agroquímica y Tecnologia de Alimentos 29, 538-547.
Falkowski, P. G. (1984). Kinetics of adaptation to irradiance in Dunaliella tertiolecta.
Photosynthetica 18, 62-68.
Gómez-Pinchetti, J. L., Ramazanov, Z., Fontes, A. & García-Reina, G. (1992). Photosynthetic
characteristics of Dunaliella salina (Chlorophyceae, Dunaliellales) in relation to !carotene content. Journal of Applied Phycology 4, 11-15.
Harris, E. H. (1989). Culture and storage methods. In The Chlamydomonas Sourcebook, pp. 2564. Edited by E. H. Harris. London: Academic Press.
Herbert, D., Phipps, P. J. & Strange, R. E. (1971). Chemical analysis of microbial cells. In
Methods in Microbiology, Vol. 5B, pp. 209-344. Edited by J. R. Norris & D. W. Ribbons.
London: Academic Press.
Kempers, A. J. & Luft, A. G. (1988). Re-examination of the determination of environmental
nitrate as nitrite by reduction with hydrazine. Analyst 113, 1117-1120.
Kleinig, H. (1989). The role of plastids in isoprenoid biosynthesis. Annual Review of Plant
Physiology and Plant Molecular Biology 40, 39-59.
Korthals, H. J. & Steenbergen, C. L. M. (1985). Separation and quantification of pigments from
natural phototrophic microbial populations. FEMS Microbiological Ecology 31, 177-185.
Koyama, Y. (1991). Structures and functions of carotenoids in photosynthetic systems. Journal
of Photochemistry and Photobiology B - Biology 9, 265-280
genes in higher plants and !-carotene biosynthesis in the alga Dunaliella bardawil.
Lichtenthaler, H. K. (1987). Chlorophylls and carotenoids: pigments of photosynthetic
biomembranes. Methods in Enzymology 148, 350-382.
- 97 -
CHAPTER 5
Neubauer, C. & Yamamoto, H. Y. (1992). Mehler-peroxidase reaction mediates zeaxanthin
formation and zeaxanthin-related fluorescence quenching in intact chloroplasts. Plant
Siefermann-Harms, D. (1985). Carotenoids in photosynthesis. I. Location in photosynthetic
membranes and light-harvesting function. Biochimica et Biophysica Acta 811, 325-355.
Sukenik, A., Wyman, K. D., Bennett, J. & Falkowski, P. G. (1987). A novel mechanism for
regulating the excitation of photosystem II in a green alga. Nature 327, 704-707.
Sukenik, A., Bennett, J. & Falkowski, P. G. (1988). Changes in the abundance of individual
apoproteins of light-harvesting chlorophyll a/b-protein complexes of Photosystem I and II
with growth irradiance in the marine chlorophyte Dunaliella tertiolecta. Biochimica et
Biophysica Acta 932, 206-215.
Thornber, J. P., Morishige, D. T., Anandan, S. & Peter, G. F. (1991). Chlorophyll-carotenoid
proteins of higher plant thylakoids. In Chlorophylls, pp. 549-585. Edited by H. Scheer.
London: CRC Press.
Van der Staay, G. W. M., Brouwer, A., Baard, R. L., van Mourik, F. & Matthijs, H. C. P. (1992).
Separation of Photosystems I and II from the oxychlorobacterium (prochlorophyte)
Prochlorotrix hollandica and association of chlorophyll b binding antennae with
Photosystem II. Biochimica et Biophysica Acta 1102, 220-228.
Wiese, L. (1965). On sexual agglutination and mating-type substances (gamones) in isogamous
heterothallic Chlamydomonads. I. Evidence of the identity of the gamones with the
surface components responsible for sexual flagellar contact. Journal of Phycology 1, 4654.
- 98 -
Chapter 6
General Discussion
GENERAL DISCUSSION
GENERAL DISCUSSION.
At the end of this study, I would like to return to the questions that were raised in
the introduction.
1. Regulation of carotenoid biosynthesis.
In green cells or tissues, carotenoids are synthesized in chloroplasts, they are essential components of the light-harvesting reaction center complexes. The final
concentration of carotenoids within one cell is determined by the availability of
binding sites in these complexes. Thus the rate of their synthesis in a growing
population of cells is directly dependent on the growth rate.
Nevertheless, I found a negative correlation between the specific growth
rate and the rate of !-carotene synthesis. This may be related to the function of
carotenoids, to protect the photosynthetic complexes against photo-oxidation
damage. It can be argued that, even at relatively low light intensities, photo-oxidative stress, accompanied by photoinhibition, may occur due to sink limitation
(Harbinson, 1994). This results in a response in which the carotenoid content of
the complexes is increased. There are many recent reports in plant science literature about oxidative cell damage at non-saturating light intensities, caused by
chilling, drought, pathogen invasion, etc. (e.g. Bladier et al., 1994). This is well documented for the components of the xanthophyll cycle, particularly zeaxanthin
(Demmig-Adams and Adams, 1993).
However, after growth arrest of D. bardawil this picture has changed, because the accumulation of carotenoids is continued at more or less the same rate,
while cell and chloroplast biogenesis have stopped. It then seems that a situation
occurs which has been described in flower or fruit development (e.g. Deruère et al.,
1994). For example, in bell pepper fruits, undergoing colour changes associated
with ripening, carotenoids accumulate inside the chloroplasts in discrete
plastoglobules. This process is often, but not always, accompanied by a decrease
of chlorophyll content. This post-growth accumulation of carotenoids seems to be
of another nature than the well-documented response to high light stress (Lers et
al., 1990) or salinity (e.g. Borowitzka et al., 1990). The latter seems to be typically
the result of specific gene expression, while in our study the use of transcription
inhibitors did not yield evidence that this is the case for post-growth carotene
synthesis in D. bardawil (Chapter 4). This is in contrast to the D. salina strain
that was studied in comparison and in which carotenoid synthesis stopped
- 101 -
CHAPTER 6
together with growth. It is also in contrast to other Dunaliella strains, collected
from the field (e.g. Borowitzka et al., 1990) which only accumulated carotenoids
under severe stress conditions. So it may be that D. bardawil is a special case
among carotenoid-accumulating Dunaliellas, characterized by its inability to down
regulate carotenogenesis after growth arrest. Interestingly, this behaviour is also
exhibited by Haematococcus lacustris. In this organism the astaxanthin content
of the cells in nitrogen-limited cultures was found to be inversely related to the
growth rate (Lee and Soh, 1991). In non-growing cultures, the specific rate of
astaxanthin accumulation was determined by the growth rate of the culture
during the growth phase. However, the specific rate of astaxanthin accumulation
was a function of the light intensity, suggesting an effect of photo-oxidative stress.
In D. bardawil, the effect of light intensity during growth on carotenogenesis was
not investigated, so the connection with photo-oxidative stress remains to be
established.
2. The function of carotenoid accumulation.
In chapter 5 it is shown that !-carotene accumulation in growth-arrested cells results in a transient enhancement of photosynthetic efficiency. This is consistent
with the idea that !-carotene can function as a carbon sink. This is reinforced by
the finding that !-carotene accumulation was strongly dependent on the CO2
availability (Chapter 3). The idea that accumulated carotenoids can function as a
screen to protect the photosynthetic apparatus was not investigated. However,
the methodology presented in this thesis, particularly the use of a carotenoid accumulating and a non-accumulating strain, should allow the investigation of possible protection properties of accumulated carotenoids in non-growing cells, even
under non-saturating light conditions.
3. The taxonomy of Dunaliella.
As mentioned above, it seems that D. bardawil is a special strain (mutant?) in the
saline group, because it is apparently deficient in down-regulating carotenoid
biosynthesis after growth arrest. Apparently, there are large differences in
response among different Dunaliellas in this respect, so this physiological characteristic is unsuitable for taxonomic purposes. Clearly, there is some need for a
molecular approach, such as the RFLP analysis of 18S ribosomal RNA genes (e.g.
Scholfield et al., 1991), to clarify the taxonomic relations between various isolates
of Dunaliella.
- 102 -
GENERAL DISCUSSION
4. Optimization of !-carotene production.
I hope to have shown that carotene production in growth-arrested cells may in
principle be suitable for biotechnological application at low light conditions. A further study concerning the cost / yield ratio, in comparison with other manufacturing methods therefore seems worthwhile.
References
Bladier, C., Carrier, P. & Chagvardieff, P. (1994). Light stress and oxidative cell damage in
photoautotrophic cell suspension of Euphorbia characias L. Plant Physiology 106, 941947
Demmig-Adams, B. & Adams, W. W. III (1993). The xanthophyll cycle, protein turnover and the
high light tolerance of sun-acclimated leaves. Plant Physiology 103, 1413-1420.
Deruère, J., Römer, S., d'Harlingue, A., Backhaus, R. A., Kuntz, M. & Camara, B. (1994). Fibril
assembly and carotenoid over-accumulation in chromoplasts: a model for supramolecular
lipoprotein structures. The Plant Cell 6, 119-133.
Harbinson, J. (1994). The responses of thylakoid electron transport and light utilization
efficiency to sink limitation of photosynthesis. In Photoinhibition of photosynthesis. pp.
273-290. Edited by N. R. Baker & J. R. Bowyer. Oxford: Bios.
Lee, Y. K. & Sohl, C. W. (1991). Accumulation of astaxanthin in Haematococcus lacustris
(Chlorophyta). Journal of Phycology 27, 575-577.
Scholfield, C. I., Gacesa, P., Price, J. H., Russell, S. J. & Bhoday, R. (1991). Restriction fragment
length polymorphisms of enzymically-amplified small-subunit rRNA-coding regions from
Gracilaria and Gracilariopsis (Rhodophyta) - a rapid method for assessing 'species'
limits. Journal of Applied Phycology 3, 329-334.
- 103 -
SAMENVATTING
SAMENVATTING: PRODUCTIE VAN CAROTEEN MET CHEMOSTAATCULTUREN VAN DUNALIELLA.
De ééncellige groene alg Dunaliella is één van de meest succesvolle algen die in de
biotechnologie worden gebruikt. Een aantal soorten heeft namelijk de opmerkelijke
eigenschap om veel caroteen te kunnen maken en op te hopen. Deze eigenschap is
voor verscheidene bedrijven aanleiding om op grote schaal Dunaliella te kweken,
om op deze manier caroteen van natuurlijke oorsprong te kunnen produceren.
Caroteen is een stof waaruit het menselijk lichaam vitamine A kan maken en
komt voor in alle groene planten. De oranje/rode kleur van deze stof wordt gebruikt
als kleurstof bij de productie van diverse levensmiddelen, waaronder margarine.
Hiervoor wordt hoofdzakelijk op chemische wijze geproduceerd caroteen gebruikt.
Het op natuurlijke wijze geproduceerde caroteen wordt voornamelijk in de veevoederindustrie en de gezondheidssector gebruikt. De laatste jaren is er een groeiende
belangstelling voor caroteen vanwege de mogelijke preventieve werking van deze
stof tegen het ontstaan van een aantal soorten kanker.
De productie van dit natuurlijke caroteen met Dunaliella vindt tot nu toe
alleen plaats in subtropische klimaatzones in open, kunstmatige kweekvijvers of
in meren van natuurlijke oorsprong. In deze gebieden komt Dunaliella van nature
reeds voor in meren met een zoutgehalte dat dat van zeewater kan overtreffen.
Het is echter de vraag of productie van caroteen met Dunaliella beperkt is tot
subtropische gebieden. In dit proefschrift wordt een productiesysteem beschreven
dat toepasbaar kan zijn in gematigde klimaat gebieden zoals in Nederland. De factoren die caroteenophoping in Dunaliella veroorzaken zijn: een hoge lichtintensiteit,
een hoge temperatuur, een hoog zoutgehalte van het kweekmedium en een tekort
aan nutriënten. Deze factoren veroorzaken elk afzonderlijk caroteenophoping
maar in combinatie versterken zij elkaar. In het in dit proefschrift beschreven
productieproces wordt gebruik gemaakt van één factor, namelijk een tekort aan
het nutriënt nitraat. Behalve de omstandigheden waarbij Dunaliella wordt gekweekt en caroteen wordt opgehoopt, behandelt dit proefschrift verscheidene wetenschappelijk interessante aspecten omtrent het ophopen van caroteen door
Dunaliella.
Hoofdstuk 2 beschrijft de omstandigheden waaronder D. bardawil, een
Dunaliella-soort met de eigenschap om caroteen te kunnen ophopen, een hoge
- 105 -
SAMENVATTING
groeisnelheid bereikt. Tevens wordt een vergelijking gemaakt met D. salina, een
soort die geen caroteen kan ophopen. Deze soort wordt in het hele proefschrift als
controle gebruikt.
Hoofdstuk 3 beschrijft het kweken van de twee Dunaliella-soorten in
nitraat-gelimiteerde chemostaatculturen. Cellen in het effluent van deze
kweekopstelling ervaren “groei-arrest”. In deze situatie is geen groei meer mogelijk
door het ontbreken van nitraat. Deze situatie leidt ertoe dat D. bardawil grote hoeveelheden caroteen gaat ophopen, dit in tegenstelling tot D. salina. Bij beide soorten vindt tevens een ophoping van zetmeel plaats.
Hoofdstuk 4 beschrijft verscheidene aspecten rond de regulatie van
caroteenophoping door D. bardawil als gevolg van groei-arrest. Een vergelijking
wordt gemaakt met de regulatie van zetmeelophoping. Tevens wordt er een vergelijking gemaakt tussen caroteenophoping als gevolg van groei-arrest enerzijds en
dat als gevolg van een hoge lichtintensiteit anderzijds. De verkregen gegevens wijzen erop dat in beide systemen de caroteenophoping op een verschillende manier
gereguleerd wordt.
Hoofdstuk 5 beschrijft het effect van groei-arrest op caroteenophoping en
fotosynthese. Ofschoon het opgehoopte caroteen geen directe interactie aangaat
met het fotosynthese-systeem van de cel, valt de ophoping samen met de handhaving van de efficiëntie van de fotosynthese gedurende de eerste dagen van groeiarrest. In cellen die geen caroteen kunnen ophopen, zoals D. salina, of als gevolg
van een specifieke remmer van caroteensynthese, daalt de efficiëntie onmiddellijk
tijdens groei-arrest. Een model wordt gepresenteerd om dit verschil te verklaren.
In hoofdstuk 6 worden, aan de hand van de vragen die zijn gesteld in de inleiding van dit proefschrift, een aantal interessante aspecten besproken met betrekking tot het onderzoek aan Dunaliella. Tevens wordt duidelijk gemaakt dat het
onderzoek, waarvan dit proefschrift het resultaat is, slechts een klein begin is.
Vele vragen zijn tijdens dit onderzoek opgeroepen en deze kunnen een leidraad zijn
voor toekomstig onderzoek aan Dunaliella.
- 106 -

Production of carotene with chemostat cultures of Dunaliella

Transcription

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