In VitroCell.Dev.Biol.

--Plant35:127-136,March-April1999
© 1999Societyfor In VitroBiology
1054-5476/99 $05.00+ 0.00

IN VITRO CULTURE AND CONSERVATION OF MICROALGAE: APPLICATIONS FOR

AQUACULTURE, BIOTECHNOLOGY AND ENVIRONMENTAL RESEARCH

JOHN G. DAY,ERICA E. BENSON, 1 AND ROLANDA. FLECK2

Culture Collection of Algae and Protozoa, NERC Institute of Freshwater Ecology, Windermere Laboratory, Far Sawrey, Ambleside,
Cumbria, LA22 OLP, UK (J. G. D., R. A. E ), and Plant Conservation Biotechnology Group, Division of Molecular and Life Sciences,
School of Science and Engineering, University of Abertay Dundee, Bell Street, Dundee, DD1 1HG, Scotland, UK (E. E. B.)

(Accepted 5 March 1999; editor T. A. Thorpe)

SUMMARY
Microalgae are a highly diverse group of unicellular organisms comprising the eukaryotic protists and the prokaryotic
cyanobacteria or blue-green algae. The microalgae have a unique environmental status; being virtually ubiquitous in
euphoric aquatic niches, they can occupy extreme habitats ranging from tropical coral reefs to the polar regions, and they
contribute to half of the globe's photosynthetic activity. Furthermore, they form the basis of the food chain for more than
70% of the world's biomass. Microalgae are a valuable environmental and biotechnological resource, and the aim of this
review is to explore the use of in vitro technologies in the conservation and sustainable exploitation of this remarkable
group of organisms. The first part of the review evaluates the importance of in vitro methods in the maintenance and
conservation of microalgae and describes the central role of cuhure collections in applied algal research. The second part
explores the application of microalgal in vitro technologies, particularly in the context of the aquaculture and biotechnology
industries. Emphasis is placed upon the exploitation of economically important algal products including aquaculture feed,
biomass production for the health care sector, green fertilizers, pigments, vitamins, antioxidants, and antimicrobial agents.
The contribution that microalgae can make to environmental research is also appraised; for example, they have an important
role as indicator organisms in environmental impact assessments. Similarly, designated culture collection strains of mi-
croalgae are used for ecotoxicity testing. Throughout the review, emphasis is placed on the application of in vitro techniques
for the continued advancement of microalgal research. The paper concludes by assessing future perspectives for the novel
application of microalgae and their products.
Key words: algae; microalgae; aquaculture; cuhure collections; environment; cryopreservation.

INTRODUCTION
The algae comprise one of the most diverse plant groups. A species
range of 40 000 to 10 million has been estimated, with the majority
being the microalgae (Hawksworth and Mound, 1991; Metting,
1996). Algae are represented by both eukaryotic and prokaryotic
forms, ranging in size from giant kelps to the unicellular microalgae
(a group of protists which are only a few microns in size) and the
prokaryotic cyanobacteria (blue-green algae). They can be distin-
guished from higher plants in terms of their reproductive character-
istics. Unicellular algal cells function as gametes, whereas the mul-
ticellular forms produce reproductive structures which are either
special unicellular gametangia or which are muhicellular, with each
cell being fertile. This contrasts with vascular plants, which bear
reproductive structures containing both asexual and sexual ceils. Eu-
karyotic algae have been classified into three groups: the Rhodophy-
tes, Chromophytes, and the Chlorophytes, and there is considerable
debate regarding their subclassification, (Cavalier-Smith, 1993; An-
dersen, 1996). Molecular biology will assist to clarify future taxo-
nomic classification.
~Towhom correspondence should be addressed.
2present address: Department of Soil, Crop and Atmospheric Sciences,
Cornell University, Ithaca, New York 14853.
Algae are one of the earth's most important natural resources. They
contribute to approximately 50% of global photosynthetic activity
(Wiessner et al., 1995) and form the basis of the food chain for over
70% of the world's biomass (Andersen, 1996). A diverse group of
photosynthetic organisms, the algae have successfully adapted their
metabolism to occupy different habitat extremes ranging from the
polar regions to tropical coral reefs. The ability to withstand envi-
ronmental stress is matched by the capacity of algae to produce a
vast array of secondary metabolites, which are of considerable value
in biotechnology programs including the aquaculture, health, and
food industries (Andersen, 1996). As algae have a unique status in
terms of their environmental importance and their phytochemical
production capabilities are considerable, it is imperative that algal
diversity is protected with complementary ex situ and in situ con-
servation methods. In this respect, in vitro techniques have a key
role.
The objective of this review is to evaluate the application of in
vitro technologies for the maintenance, conservation, and sustainable
exploitation of microalgae. The first part of the review discusses in
vitro culture and maintenance methods and the second part explores
the value of algal culture techniques in environmental research,
aquaculture, and the biotechnology industries. The review will con-

127
128 DAY ET AL.

clude by offering some future perspectives on the importance of ap-

plied and fundamental microalgal research.

IN VITRO CULTUREAND MAINTENANCEOF MICROALGAE

Any in vitro conservation or maintenance strategy should guar-

(a) UNSTIRRED OPEN POND (b) CIRCULAR POND
antee long-term stability of the morphological, physiological, and ge-
netic characteristics of the preserved organism. Many algal isolates
have been successfully maintained ex situ for decades (Day, 1999):
however, for some strains this is problematic, as important physio-
logical and morphological characteristics may be unstable on pro-
longed in vitro culture. Examples of instability include the size of
diatoms' frustule (Jaworski et al., 1988), retention of spines in Mi-
cractinium pusillum, and loss of "normal" pigment composition in a (c) PADDLE-WHEEL RACEWAY (d) SLOPING CASCADE
number of algae (Warren et al., 1997). As with other groups of or-
ganisms, two basic options are available for the long-term conser-
vation of algae: in vitro culture (i.e., routine serial subcuhure under
controlled environmental conditions) or approaches that depend on
the removal or limitation of water and storage at low or uhralow 00000
temperatures. In microorganisms this normally involves drying, 000000
freeze-drying, or cryopreservation techniques (Kirsop and Doyle,
1991; Day and McLellan, 1995a). However, neither drying or freeze-
(e) TUBULAR REACTOR (f) TUBULAR REACTOR (g) TUBULAR REACTOR
drying have been extensively used to preserve microalgae, as levels (helix) (plane) (two layers)
of postpreservation may be extremely low and are reduced further on
storage (McGrath et al., 1978; Day et al., 1987; Malik, 1993). There-
fore, cryopreservation has been adopted by a number of the larger
algal culture collections (Morris, 1978; Watanabe et al., 1992; Bodas
et al., 1995).

In Vitro Culture

Under natural conditions, most algae grow as mixed communities,

which include various species and genera of algae and other micro-
organisms. On isolation, an algal strain requires a suitable environ-
ment for its growth, There is an extensive literature on culture tech-
niques and maintenance conditions (e.g., Stein, 1973; McLellan et
al., 1991; Becker, 1994). Medium composition depends both on the
requirements of the algae (e.g., diatoms require the inclusion of a
silica source: many marine algae require vitamins) and the prefer-
ences of the researcher. Information on medium suitability and full
recipes are listed in the catalogues of all the major protistan cuhure
collections (see Nerad, 1993; Starr and Zeikus, 1993; Schl~sser,
1994; Tompkins et al., 1995; Andersen et al., 1997; Watanabe and
Hiroki, 1997). In general, master stock-cultures maintained by rou-
tine serial subcuhure are grown under suboptimal temperature and
light regimes [<20 ° C and <50 p~mol photon m 2 s 1 on shorter
than normal daylight (12 h or less light : 12 h or more dark)], This
type of regime maximizes the interval between subcultures and thus
minimizes handling and transfers. Additionally, the use of medium
containing organic carbon for maintaining axenic strains capable of
heterotrophic or mixotrophic growth and solidified rather than liquid
medium may be used to maximize the period between transfers to
fresh medium.
For many applications, relatively small quantities of algae are re-
quired and scaling up may entail the use of a larger glass vessel
either with or without additional aeration to reduce carbon limitation
and keep the ceils in suspension. However, light limitation will re-
strict the growth rate and productivity in such systems and may in-
fluence the formation of any desired product. A variety of options are
(h) LAMINAR REACTOR (i) HANGING SLEEVE
FIG. 1. Typical examples of different algal mass culture systems used in
the aquaculture and biotechnology industries.
available for scaling up, with the choice of production method largely
depending on the amount of biomass required and value of any prod-
uct. Systems used may be divided into three categories: open sys-
tems, closed photobioreactors, and closed fermenters (heterotrophic
culture).
Open systems include the use of managed lakes which may be up
to 300 hectares (Schlipalius, 1991), unstirred open ponds like the
~-carotene production plant at Hutt Lagoon in Western Australia
(Borowitzka, 1991), circular ponds, paddle-wheel raceways, and
sloping cascades (Oswald, 1988; Becker, 1994). All of these gener-
ally have the advantage of being relatively cheap to construct. How-
ever, problems associated with excessive shear forces damaging the
algal ceils and the need for environmental control including pH,
temperature, nutrient levels, osmotic potential, contamination by
other algae, and grazing have restricted their use.
A large number of closed photobioreactor systems have been de-
veloped (Fig, 1) and these avoid some of the problems connected
with open system use, most notably better environmental control and
fewer problems associated with contamination and grazing. The least
complex and probably most widely used is the Hanging Sleeve. These
are commonly used in aquaculture for the production of food for
 IN VITRO CULTURE OF MICROALGAE
shrimp and mollusc larvae (McLellan et al., 1991) and also for poly-
saccharide production from Porphyridium (Becker, 1994). Other
types of bioreactors include tubular type reactors (Gudin and Chau-
mont, 1983; Torzillo et al., 1986), laminar types (Tredici et al., 1991)
and fermenter type reactors (Pohl et al., 1986). Fermenter type re-
actors offer the greatest degree of control and may be fitted with light-
diffusing optical-fiber systems to avoid the necessity of external il-
lumination and the problems associated with light limitation (Takano
et al., 1992).
The final type of culture system utilizes the capability of some
algae to take up and metabolize fixed carbon, i.e., to grow heterotro-
phically. Although this restricts the range of algae that may be grown
and their products, the system has been successfully used to produce
c~-tocopherol (Ogbonna et al., 1998), ascorbic acid (Running et al.,
1994), aquaculture organisms (Day and Tsavalos, 1996), fatty acids
(Barclay et al., 1994), and leutin (Shi et al., 1997).
Cryopreservation
Cryopreservation is the maintenance of living cells, tissues, organs
and microorganisms at uhralow temperatures (in general at - 196 °
C) and liquid nitrogen is used as the refrigerant. As long as liquid
nitrogen levels are maintained, living cells can be maintained in
cryogenic storage indefinitely. At liquid nitrogen temperatures all
metabolic activity ceases and the ceils do not undergo the genetic
changes which can occur when they are maintained by serial sub-
culturing. Furthermore, cryopreserved cells are not continuously ex-
posed to the risks of contamination and operator error as would be
the case during culture maintenance. Thus, cryopreservation is the
method of choice for the long-term conservation of in vitro culture
collections of microalgae. Investigations of cryogenic storage proce-
dures for algal cultures (Bodas et al., 1995) have been performed
and a number of robust protocols are now available for a range of
species (Morris, 1978; Day and DeVille, 1995: Day, 1998); however,
a large number of macro- and microalgae still remain recalcitrant to
cryopreservation. This is largely due to the fact that they comprise a
diverse group of organisms which display many different physiolog-
ical and morphological characteristics, and this makes the develop-
ment of a universally applicable cryopreservation method most dif-
ficult. Successful cryopreservation depends upon the application of
a cryoprotective strategy and the manipulation of freezing, thawing,
and recovery conditions. Differences between procedures can usually
be attributed to the use of alternative cryoprotection strategies. Algae
can be sensitive to the chemical cryoprotectants which are commonly
applied to higher plants, and cryoprotectant toxicity trials are an
essential prerequisite before researchers embark on developing a
cryopreservation protocol for microalgae. Canavate and Lubian
(1994), assessed the toxicity of dimethyl sulfoxide (DMSO) and meth-
anol on six taxonomically diverse marine microalgae and found all
to be sensitive to both cryoprotectants, and sensitivity in some cases
was concentration-dependent. The traditional approach to cryopre-
serving microalgae involves the application of chemical cryoprotec-
tants such as DMSO or methanol before freezing, followed by expos-
ing the cells to a controlled temperature gradient (e.g., - 0.5 to - 10 °
C min-1), then to a terminal transfer temperature (e.g., - 30 to - 60 °
C) after which the cells are plunged into liquid nitrogen (Morris,
1976; Day and McLellan, 1995b). This procedure is termed "two-
step" freezing and the majority of microalgae that have been placed
in cryogenic storage have been cryopreserved by this technique. Suc-
129
cessful applications of two-step freezing to algae which have previ-
ously proved difficult to freeze include Dunaliella tertiolecta (Hirata
et al., 1996); Laminaria digitata (Vigneron et al., 1997); Chaetoceros
gracilis, Tetraselmis chui, Nannochloris atomus, Nannochoropsis gad-
itana, Rhodominas baltica, and Isochyris galbana (Canavate and Lu-
bian, 1995a, 1995b) and Cylidrocystis brebissonii (Morris et al.,
1986).
During the last few years, new approaches to cryopreservation have
been developed for higher plants (for reviews see Benson, 1994,
1999) and now these are being successfully applied to macro- and
microalgae, some of which have previously proved recalcitrant to
freezing with traditional two-step methods (Fleck, 1998). More recent
cryopreservation methodology is based on the cryoprotective strategy
of vitrification. This is a process in which the cells are manipulated
such that, on exposure to liquid nitrogen, the water molecules form
an amorphous glass which lacks crystalline structure. Under these
conditions, ice is not formed and its damaging effects are thus
avoided. Glasses are, however, unstable and it is important that the
cryopreserved cells are rewarmed carefully to avoid destabilization
of the glass, as water can nucleate and form ice as it is warmed to
the glass transition temperature and beyond. Vitrification can be in-
duced in cryopreserved systems by either reducing the moisture con-
tent (e.g., via osmotic dehydration, silica gel desiccation, and/or air
drying) of the ceils and tissues or by the application of high concen-
trations of chemical cryoprotectants (e.g., plant vitrification solution
number 2, "PVS2", Yamada and Sakai, 1996). Both of these strate-
gies increase cellular viscosity to a critical level at which ice for-
mation is inhibited on exposure to uhralow temperatures. One novel
application of vitrification involves encapsulating cells and tissues
in a calcium alginate matrix (Fabre and Dereuddre, 1990) and ex-
posing the encapsulated cells to osmotic dehydration (usually by the
application of highly concentrated sucrose solutions) followed by air
or silica gel desiccation. This approach has been applied to algal
species including Dunaliella tertiolecta (Hirata et al,, 1996), Euglena
gracilis (Fleck, 1998) and Laminaria digitata (Vigneron et al., 1997).
Future cryoconservation research involving algae must evaluate
the basis of freeze sensitivity in microalgae. Understanding both the
biochemical and physical effects of freezing will be especially im-
portant, and recent studies have included the characterization of
freeze recalcitrance in the coenocytic alga Vaucheria sessilis with
cryomicroscopy (Fleck et al., 1997) and the role of oxidative stress
and antioxidant protection in Euglena gracilis and Haematococcus
pluvialis (Benson et al., 1998; Fleck, 1998).
Role of Algal Genetic Resource Centers and Culture Collections
In recent years, the trend has not been towards conservation per
se, but rather towards sustainable utilization and exploitation for
national benefit (Hawksworth and Ritchie, 1993; Hawksworth,
1996). Culture collections satisfy a multitude of purposes in any
conservation and sustainable exploitation strategy, In addition, the
collection and preservation of living specimens is essential for the
elucidation of an organism's life history through systematic and tax-
onomic investigation. Conserved specimens allow the effects of hu-
man demands and climatic changes on biodiversity to be studied
through the maintenance of accurate and verifiable taxonomic re-
sources (Roper, 1993; Cotterill, 1995).
The primary role of an algal culture collection is the same as any
other collection of living material, that is, to be a repository for cul-
130 DAY ET AL.

TABLE 1 TABLE 2

DISTRIBUTIONOF ALGAL CULTURE COLLECTIONS MICROALGALUSE IN AQUACULTURE

Use Collection
No. of Collection Internet Patent Cryopres- Species strain no." Molluscs Crustaceans Rotifers
Continent collections" acronym b Country Web site depository ~ ervation e
Chaetoceros calcitrans CCAP 1010/5 + +
Africa 3 CCMP 1315
Asia 36 NIES Japan - - + Chaetoceros muelleri CCMP 1316 +
Australia (and NZ) 12 Chaetoceros neogracile CCMP 1010/5 +
Europe 27 ASIB Austria - - + lsochrysis galbana CCAP 927/1 + +
CCAP UK + + + CCMP 1323
SAG Germany - - - Nanochlorophsis oculata C C A P849/1 +
N. America 27 CCMP USA + - + Nanochloropsis gaditana CCAP849/5 +
UTEX USA + - + Nannochloris atomus CCAP 251/4A +
S. America 2 Pavlova lutheri CCAP 931/1 + +
CCMP 1325
"No. of algal culture collections on the continent. Rhinomonas reticulata CCAP 978/28 +
bCollection holding > 1000 algal strains. Rhodomonas salina CCMP 1319 +
~Under the terms of the 1977 Budapest treaty. Skelatonema costatum CCAP 1077/5 + +
dUse cryopreservation to maintain some of their holdings (Data from An- Tetraselmis chui CCAP 8/6 + +
derson, 1996; WDCM, (World Data Centre on Microorganisms), 1994; Ma et Tetraselmis suecica CCAP 66/4 + +
al., 1995; and this paper). Thalassiosira pseudonoana CCAP1085/3 + +
Collection acronyms are NIES (National Institute of Environmental Stud- CCMP 1335
ies), ASIB (Algensammlung am Institut ftir Botanick, University of Inns- Thalassiosira weissflogii CCMP1336 + +
bruck), SAG (Sammlung yon Algenkulturen), CCMP (Provasoli Guillard Na-
tional Centre for Marine Phyloplankton), CCAP (Culture Collection of Algae °Acronyms for strain cultures are Culture Collection of Algae and Protozoa
and Protozoa), and UTEX (Culture Collection of Algae at the University of (CCAP) and Provasoli-Guillard National Centre for Marine Phyloplankton
Texas). (CCMP). For details of CCAP strains, see Tompkins et aI. (1995), and for
CCMP strains, see Andersen et al. (1997).

tures. In service collections, this role is often associated with other

products and services including provision of authentic specimens for
research, education, training, bioassay use, identification, and use as
aquaculture starter cultures. Importantly culture collections act as
depositories for patent purposes, consultancy, and other commercial
applications. All of these require the maintenance of viable, healthy,
physiologically and genetically stable cultures. There are more than
11 000 strains of algae including representatives of approximately
1600 different species retained in the algal culture collections reg-
istered with the World Data Centre on Microorganism's (WCDM) da-
tabase (Miyachi et al., 1989; WDCM, 1994). However, it is worth
noting that many of these collections hold very few algae (<50
strains) and that more than 80% of the algal strains listed are main-
tained in the six largest algal culture collections (Table 1).
APPLICATIONS OF IN V I T R O CULTURED MICROALGAE
In vitro technologies have a central role in applied microalgae
research and Culture Collections are particularly important for the
exploitation of algae and their products. Similarly, in vitro cultures
support environmental monitoring programs, allowing time-related
studies of pollution and climate change. Comparative studies of con-
served cultures held in repositories and natural algal populations
provide a means of conducting environmental impact assessments.
Culture collections assist taxonomic studies as they can be used to
verify evolutionary relationships and genetic change. This section
will evaluate the role of in vitro technologies in applied microalgal
research.
Aquaculture
From an economic perspective, aquaculture is the most important
applied use of algae. Products from macroalgae which have signifi-
cant financial value include Porphyra (Nori) harvested in Japan and
worth US$1 billion annually (Mumford and Miura, 1988) and algal
polysaccharides, primarily agars and carrageenans, with an annual
value of approximately US$500 million (Jensen, 1993). However, this
section focuses primarily on the use of microalgae as food in aqua-
culture.
Algae are the biological "starting point" or primary producers for
energy flow through aquatic food chains, it is therefore not surprising
that microalgae are so important in the aquaculture industries. The
choice of algal strains for aquaculture use is constrained by a number
of factors as outlined below. The first is the toxicity of the alga, as
many strains produce potent toxins (Addison and Stewart, 1989;
Shumway, 1989) and these are clearly unsuitable. Nutritional profile
is a key factor, with levels of vitamins, proteins, and unsaturated fatty
acids being of particular importance (De Pauw and Persoone, 1988;
Cahu et al., 1994; Borowitzka, 1997). Size and palatability also in-
fluence choice; large particles may be too big for ingestion and spe-
cies with thick cell walls, including Chlorella, may be indigestible.
The final criterion is that the selected algae should be relatively
robust and easy to culture. In in vitro culture systems, the range of
species used is much less than in natural environments; however,
these strains and a few others are the basis of a muhimillion dollar
industry.
Microalgae are the primary food for larvae and spat of bivalve
molluscs and penaeid prawn (shrimp) larvae, and they are live food
for rotifers which in turn are used to feed the larvae of marine finfish
and crustaceans (Table 2). The production of microalgae in aqua-
culture is commonly considered to be the major constraint on pro-
ductivity and expansion of a farm. Furthermore, it is the most ex-
pensive part of the process, accounting for 30M,0% of hatchery costs
(Borowitzka, 1997). The actual cost of producing algae varies from
 IN VITRO CULTUREOF MICROALGAE 131

site to site but may be in the range US$50-1000 kg -1 (Fulks and TABLE 3
Main, 1991; Benemann, 1992).
MICROALGALPRODUCTS
Production systems vary from farm to farm but can be categorized
as extensive, semi-intensive, or intensive systems. The first category Alga Use/product Reference
depends on natural phytoplankton, with algae and consumers coex-
Botryococcus braunii Fuel oil Murray and Thompson, 1977
isting in large volume (>1000 m3) lakes, lagoons, ponds, or in the
Chlorella Stable isotope biochemicals Behrens et al,, 1989
sea. Semi-intensive systems involve the addition of fertilizer to enrich Chlorella Ascorbic acid Running et al., 1994
the water, inducing a natural bloom of algae in systems with slightly Chlorella Leutin Shi et al., 1997
smaller volume (>100 mS). The algae and consumers may coexist, Euglena gracilis a-tocopherol Ogbonna et al., 1998
or alternatively, the "bloom" may be transferred to tanks containing Haematococcus spp. Astaxanthin Johnson and An, 1991
Porphyridium Polysaccharides Becker, 1994
the consumers. Although both of these approaches are relatively Spirulina spp. Phycobiliproteins Glazer. 1994
cheap, they have the disadvantage of being relatively unproductive Dunaliella spp, [3-carotene Ben-Amotz and Avron, 1980
and are uncontrolled or uncontrollable with respect to production, Various Oraega-3 fatty acids Barclay et al., 1994
species control, and the growth of interfering consumers. Further- Various Anticancer activity Gerwiek et al., 1994
Various Antimicrobial activity Patterson et al., 1994
more, in areas which are prone to toxic blooms, the production of
Various Hydrogen Benemann, 1990
algal toxins could result in total loss of the farmed animals. Various Vitamins Baker et al., 1981
Intensive systems involve the use of in vitro culture to provide a
more managed and controllable supply of algae. These are far more
regulated, as unialgal cultures are grown in batch or continuous cul-
ture systems separate from the consumer. They also have the advan- salmonid diets with astaxanthin derived from the freshwater mi-
tage of producing a more reliable and biochemically consistent sup- croalga Haematococcus pluvialis or synthetic pigments results in sig-
ply of algae, and problems associated with pathogens, toxic algae, nificant deposition of carotenoids including astaxanthin, visually en-
and undesired grazing organisms are avoided. hancing flesh coloration of the salmonids (Sommer et al., 1991).
In hatcheries, use of artificially illuminated bag or tank culture Other algae can also be used for the pigmentation of fish and crus-
vessels (up to 500 L) is standard. A variety of algae are normally taceans. Spirulina has been used to pigment shrimp (Liao et al.,
cultured in separate vessels and an algal mix is fed to the consumers 1993) and Dunaliella has been used to pigment other crustaceans
(normally molluscs or rotifers). However, algal productivity is low (Sommer et al., 1991). Spirulina is also a common feed component
and these systems may be unreliable, thus requiring very careful for ornamental fish such as Koi carp, as it enhances pigmentation
management (Sato, 1991). To overcome the problems intrinsic to this (Borowitzka, 1997).
approach, closed photobioreactor systems are used. One system, a
helical tubular photobioreactor developed by Biotechna Ltd. (Rob- Algal Biotechnology
inson et al., 1988) has been extensively tested for the production of
aquaculture strains in the UK and Australia (Borowitzka, 1996, Microalgae are extremely efficient solar energy converters and they
1997). With this type of culture system, environmental conditions can produce a great variety of metabolites. This capacity and their
(temperature, pH, light, nutrients) may be optimized and the bio- ubiquitous distribution have led to their exploitation by man for a
chemical composition of the algae manipulated to improve their nu- diverse range of purposes. This review can only touch on a few ap-
tritional value (Chrismadha and Borowitzka, 1994). These bioreac- plications and fuller information can be obtained from other sources
tots may be automated and run continuously, thus reducing labor (Borowitzka and Borowitzka, 1988; Boussiba et al., 1992; Wilde and
costs and other costs. Benemann, 1993; Johnson and Schroeder, 1995; Borowitzka, 1996;
As aquaculture has developed and expanded, there has been a Dixon et al., 1997; Duncan et al., 1997; Renn, 1997; Vilchez et al.,
significant increase in both the number and size of the farms. This 1997) which appraise methods for the cultivation of microalgae and
has resulted in a demand for removing or minimizing bottlenecks the potential commercial applications of microalgal biotechnology.
associated with culturing algae and the insufficient supply of algae However, the following sections present the main biotechnological
at key times. Improvements in algal culture systems, particularly the applications of microalgae.
more widespread use of photobioreactors, will continue to reduce
supply problems. Alternative approaches that have been taken with Useful Metabolites From Algae
varying degrees of success include the use of encapsulated nonalgal
The range of metabolites produced from algae is extremely exten-
food particles (Jones et al., 1987), and sun-dried algae (Millamena
sive with many having significant commercial value. In most cases,
et al., 1990), the heterotrophic production of algae to produce a
with the exception of extremophile algae including Dunaliella and
spray-dried powder (Day et al., 1991), and the storage and distri-
Spirulina, photobioreactors or other closed systems are likely to form
bution of algal pastes (Watson et al,, 1986; Brown, 1995). It seems
the basis of any economically viable culture system. Some products
probable that an algal product that could be stored would find wide-
are listed in Table 3; most are of commercial significance or have
spread application. However, to date, no alternative to a mix of live
been developed to pilot-plant scale processing.
algal species has been found to be fully satisfactory.
Microalgae can also be used to pigment fish and shrimps, as
Biomass and Health Food Production
farmed animals which lack the ability to synthesize carotenoids can
be supplied with algal pigment supplements in their feeds. This is Approximately 500 species of algae, including macroalgae, are
usually at considerable expense to the farmer and may contribute 10 used as human food or food products, and about 160 species are
to 15% of total feed costs (Johnson and An, 1991). Supplementing considered commercially valuable (Abbott, 1988). From a practical
132
point of view, growing of biomass is the most obvious and easiest
approach to producing algal foods. Mostly these are now produced
for the health food market and the organisms grown tend to be those
which grow in extreme environments, e.g., Spirulina and Dunaliella,
or those with fast growth rates, e.g., Chlorella and Scenedesmus. The
largest market is in Japan and the Far East, but in increasingly
health-conscious Europe and the USA, there is a niche market in
health food shops. The production processes for all of the commercial
production processes are photoautotrophic, with the exception of
some Chlorella production systems which are mixotrophic or which
utilize heterotrophically cultivated algae as an inoculum for the pro-
duction phase (Endo et ah, 1977).
Green Fertilizers
Cyanobacteria including Scytonema, Nostoc, and Anabaena com-
monly form the basis of "green" fertilizers (Venkataraman, 1986).
The largest usage of cyanobacterial fertilizers is in India where two
million hectares were fertilized in 1979 (Roger and Kalasooriya,
1980). The system used involves the central produetion of a mixed
inoculum of cyanobacteria which are then air dried and mixed with
soil. This material forms dried flakes which are then distributed and
used as an inoeulum which is grown in 40-m z ponds. This inoculum
in turn is used in the cultivated rice fields. An alternative approach
is to immobilize eyanobacteria in polyurethane foam matrices and
then add these to the rice fields (Kannaiyan et al., 1997). Develop-
ment of algal biofertilizers for use in temperate environments has
been investigated (Metting, I981), and commercial products have
been developed including an agricultural fertilizer produced by Soil
Technologies and a lawn and garden fertilizer produced by the Cy-
anoteeh Corporation (Day and Turner, 1992).
Ecotoxicity Testing
All new chemical products likely to be released into the environ-
ment must undergo standard algal growth inhibition toxicity tests
which have been internationally agreed upon (OECD,1984). Cul-
tures of microalgae used in these tests are maintained in the major
culture collections [Culture Collection of Algae and Protozoa (CCAP)
Sammlung von Algenkulturen, (SAG); Provasoli-Guillard National
Centre for Marine Phytoplankton, (CCMP); Culture Collection of Al-
gae at the University of Texas (UTEX), and the American Type Cul-
ture Collection (ATCC)] and include the following strains:
Chlorella vulgaris CCAP 211/llB SAG 211-11B UTEX259
Chlorella vulgaris CCAP 211/12 SAG 211-12 UTEX30
Selenastrum capricornutum CCAP284/4 UTEX 1648 ATCC22662
Scenedesmu~ subspicatus CCAP 276/22 SAG 86.81 U T E X2594
Phaeodactylum tricornutum CCAP 1052/1A
Skeletonema costatum CCAP 1077/3
Skelatonema costatum CCAP 1077/5 CCMP1332
In addition, a number of other organisms are routinely used as
bioassay organisms including Ochromonas danica for the bioassay of
biotin (Baker et al., 1962); Euglena gracilis for the bioassay of vi-
tamin B12 (Parker, 1977), and Trentepohlia aurea for paint biocide
testing.
Pollution Control
Microalgae are a key component in many pollution control sys-
tems, particularly those that use ponds or impounds. These can be
DAY ET AL.
divided into two categories: facultative ponds, which are over 1 m
deep and have algae restricted to the surface waters and high rate
oxidation ponds (HROP) which are generally shallow and mechani-
cally agitated. In the case of HROP, the algae are important as not
only do they provide 02 which is used by the oxidative bacteria
present, they are also predominantly facultative heterotrophs includ-
ing Euglena, Chlorella, and Scenedesmus (Abeliovich, 1980, 1986),
with up to 50% of their carbon assimilation by direct heterotrophic
nutrition (Abeliovich, 1978). The yields of algae in these systems
can be relatively high at 1-2 g 1 1 (Abeliovich, 1980). This has led
to the development of a number of pilot-scale processes which com-
bine waste treatment with the production of algal biomass for animal
feed (Fallowfield and Garrett, 1985; Pouliot and De la Noue, 1985).
Algae also perform a number of secondary functions in wastewater
treatment, which include disinfection of the effluent. They increase
the water temperature by converting light to heat, thus increasing the
death rate of enteric bacteria (Pharhad, 1970), and metabolize bi-
carbonate, increasing the pH. They thereby induce flocculation, ef-
fectively increasing the sedimentation rate of the effluent being
treated. Algae also have negatively charged cell walls, which in con-
junction with the pH, removes heavy metals from the effluent (Oswald
et ah, 1957; Becker, 1983). This phenomenon has been used to de-
velop filters that can remove heavy metal ions and refractory organic
compounds from industrial effluents (Wilson et al., 1991).
Environmental Research
In evolutionary terms, the algae have existed for 3.8 billion years
(Bold and Wynne, 1985). They are considered to be one of the first
group of organisms to colonize the earth and have a broad habitat
range. The role of algae in maintaining the stability of the earth's
ecosystems is considerable, and this can be largely attributed to the
major contribution that they make to photosynthesis and supporting
the food chains of the majority of the world's biomass (Andersen,
1996). However, one of the most significant, contemporary applica-
tions of algal culture collections is in impact assessment and the
monitoring of environmental change. The importance of microalgae
in these studies is considerable and their use must be underpinned
by the application of validated cultures which can be used to assist
taxonomic identification and the study of genetic drift. For example,
microalgae are used in paleolimnology, the study of cumulative
changes in the remains of plants and animals which are deposited
as time-related layers in soils and sediments. Diatoms are particu-
larly important, as the silicon components of these microalgae pro-
ATCC 16487
vide almost permanent markers of the changes that have occurred in
diatom populations. By analyzing the remains of diatoms sampled
from sedimentary cores of terrestrial and aquatic environments, it is
possible to gain an understanding of the history associated with pol-
lution events and climatic change (Haworth et al., 1996).
In vitro cultures of microalgae are also important in the study of
microbial ecology, as these organisms can be used to assist studies
of pollution in industrial, domestic, urban, and agricultural environ-
ments. Assessments of microbial diversity are particularly important
in environmental impact assessments, and the microalgae are sen-
sitive indicators of ecological changes (Kelley et al., 1998). Climatic
change and its impact on natural ecosystems is now a major global
issue and the algae, (especially the microalgae) make a significant
contribution to the earth's environmental stability through biomass
production, food chains, and the cycling of photosynthetic gases. It
 IN VITRO CULTUREOF MICROALGAE
is thus imperative that ecological studies are supported by ex situ
conservation programs which ensure the safe storage and mainte-
nance of algal genetic resources which can safeguard against species
loss as well as providing validated organisms for study. Current re-
search on climatic change can be largely related to the effects of light
and temperature. High light, oxygen, and nutrients are essential for
the support of planktonic algae; thus, climate changes which influ-
ence light quality (e.g., turbidity or UV) and temperature will influ-
ence the population dynamics of phytoplankton. Reduction in the
stratospheric ozone layers has resulted in an increase in UV-B which
has harmful effects on ecosystems, and phytoplankton are especially
vulnerable (Maberly and Pettitt, 1997). Algal populations are also
sensitive to changes in nutrient status, for example, as is the case
with phosphate enrichment (Gibson et al., 1996). An important future
role of microalgae and protozoa culture collections will be in risk
assessment studies related to the release of genetically modified or-
ganisms. Future activities will thus include monitoring the fate of
pathogenic and genetically engineered microorganisms in the aquatic
environment.
Novel Applications of In Vitro Technologies to Microalgal
Research
Cuhure collections provide essential resources for laboratory-
based experimental research involving microalgae, and in vitro tech-
nologies will continue to be essential to the aquaculture and bio-
technology industries. Future applications of in vitro technologies to
microalgae research will involve two main areas: (1) their value as a
biological resource of useful products and (2) their role in ecological
research.
There is a great deal of further potential for algal biotechnology.
Product areas that will undoubtedly continue to be developed include
lipids (specifically unsaturated fatty acids), pigment production, and
bioactive compounds. In addition, significant investment is being
made to develop algal technologies to remove carbon dioxide and
other pollutants from effluent gases, particularly in Japan under the
RITE program (Karube, pers. comm.). However, the key to economic
success will be improved productivity. This will require development
of both photobioreactors and conventional fermentation plant in ad-
dition to other aspects of process and product optimization. An im-
portant aspect of this which has not been extensively investigated as
yet is the selection and development of overproducing strains, either
by genetic manipulation, conventional mutagenesis, or direct selec-
tion techniques. In addition, modern molecular approaches may be
used to increase the range of products. A current application of this
is to clone the genes for the insecticidal Bacillus thuringiensis toxin
into the eyanobacterium Synechococcus (Stevens et al., 1994).
Global climate change is undoubtedly one of the most important
environmental problems of the future, and the study of microalgae
in their natural habitats will provide an important means of assessing
the long-term impact of pollution. A good example of how funda-
mental, generic research can be integrated into applied environmen-
tal science is in the use of remote sensing techniques to identify
different taxonomic groups of phytoplankton in aquatic environments
(George and Taylor, 1995). Such an approach can greatly assist water
quality testing, and for example, could aid the study of toxic algal
blooms. Remote sensing studies are based upon the fact that spectral
differences can be observed and detected in algae which have dif-
ferent photosynthetic pigments. However, spectral signatures of dif-
133
ferent freshwater plankton species must first be recorded. It is im-
portant to note that these spectra were initially derived and calibrated
with pure in vitro cultures of algae obtained from the Culture Col-
lection of Algae and Protozoa (Tompkins et al., 1995).
The ability of microalgae to survive in many different, and fre-
quently extreme environments can be attributed to their complex life
cycles and metabolic strategies, therefore, studies of algal production
characteristics are especially important. For example, iron is a key
limiting factor in phytoplankton found in high nutrient-low chloro-
phyll regions, and Fe amendment of these communities offers the
potential of relieving the nutrient constraints imposed on phytoplank-
ton photosynthetic efficiency and biomass accumulation. Novel in
vitro methods are becoming increasingly important in these studies.
McKay et al. (1997) have used trace metal-buffered culture tech-
niques to explore the potential use of flavodoxin as an in situ bio-
chemical marker for Fe limitation in marine diatoms. A further ap-
plication of in vitro technologies is in the increasingly important field
of bioremediation in which the capacity of plants and animals to
metabolize and detoxify xenobiotics is being explored in vitro. Such
studies will allow the development of specific algal systems which
can then be used in mitigating pollution episodes. Cytochrome P450
monooxygenases for fatty acids and xenobiotics have recently been
found in three families of macroalgae (Pflugmacher and Sandei~an,
1998), and Cytochrome P450 has been detected in Euglena gracilis
(Briand et al., 1993) and a range of unicellular green algae (Thies et
al., 1996). The capacity to use marine macroalgae to clean up off-
shore pollution and freshwater microalgae to detoxify polluted in-
shore sites could provide promising new approaches to solving
aquatic pollution problems.
In conclusion, in vitro technologies have made major contributions
to the study and exploitation of microalgae. Expanding our current
knowledge of basic algal physiology with strains derived from culture
collections can greatly enhance our capacity to exploit microalgae
not only as a bioresource but also as an investigative tool for envi-
ronmental monitoring. In the future, fundamental and applied re-
search targeted at improving culture and cryopreservation methods
will be particularly important if the full economic potential of mi-
croalgae is to be realized. Similarly. as microalgae have such a
unique ecological status, in vitro methods must continue to support
environmental and algal diversity research.
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