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215

J. Phycol. 35, 215226 (1999)


REVIEW
COMMERCIAL DEVELOPMENTS IN MICROALGAL BIOTECHNOLOGY
1
Kirk E. Apt
2
and Paul W. Behrens
Martek Biosciences Corporation, 6480 Dobbin Road, Columbia, Maryland 21045
A number of important advances have occurred
in microalgal biotechnology in recent years that are
slowly moving the eld into new areas. New prod-
ucts are being developed for use in the mass com-
mercial markets as opposed to the health food
markets. These include algal-derived long-chained
polyunsaturated fatty acids, mainly docosahexaenoic
acid, for use as supplements in human nutrition and
animals. Large-scale production of algal fatty acids
is possible through the use of heterotrophic algae
and the adaptation of classical fermentation systems
providing consistent biomass under highly con-
trolled conditions that result in a very high quality
product. New products have also been developed
for use in the development of pharmaceutical and
research products. These include stable-isotope bio-
chemicals produced by algae in closed-system pho-
tobioreactors and extremely bright uorescent pig-
ments. Cryopreservation has also had a tremendous
impact on the ability of strains to be maintained for
long periods of time at low cost and maintenance
while preserving genetic stability.
Key index words: aquaculture; cryopreservation; do-
cosahexaenoic acid; heterotrophic algae; infant for-
mula; phycobilisomes; stable isotopes
In recent years, numerous general reviews on var-
ious aspects of algal biotechnology have appeared
(Borowitzka and Borowitzka 1988, Cresswell et al.
1989, Cannell 1990, Avron and Ben-Amotz 1992, Bo-
rowitzka 1992, Becker 1994, Parker 1994, Allnutt
1996, Metting 1996, Borowitzka 1997, Renn 1997,
Vonshak 1997). This review is intended not to be
comprehensive but to focus on recent applications
and new developments that are diversifying the di-
rections for commercial exploitation of microalgae.
It also focuses on products that are at or near com-
mercial availability.
Traditionally, the denition of algae includes
the cyanobacteria; green, red; and brown algae;
charophytes; cryptophytes; chrysophytes; diatoms;
dinoagellates; and others, encompassing photosyn-
thetic prokaryotic and eukaryotic organisms as well
as their heterotrophic derivatives. On the basis of
molecular phylogenies, many of these groups are
widely scattered on the tree of life. As a result,
1
Received 30 April 1997. Accepted 2 December 1998.
2
Author for reprint requests; email kirkapt@aol.com.
the term algae refers to a polyphyletic, articial
assemblage of organisms.
It has been proposed that at least one nontradi-
tional organism, Schizochytrium, could be considered
an alga (Barclay 1992). The genus Schizochytrium is
classied in the Thraustochytrids (Chamberlain and
Moss 1988), which is traditionally considered to be
part of the lower fungi. Molecular phylogenies have
indicated that the Thraustochytrids are allied with
the Chromophytic algae (Cavalier-Smith et al.
1994). Because Schizochytrium is currently marketed
as an algal product, it is included here.
This article rst summarizes developments in nu-
tritional products used for direct supplementation
by humans and as aquaculture feeds. It then dis-
cusses products for pharmaceutical and research
uses. Finally, it discusses advances in production
techniques and then the progress that has been
made in the critical area of maintaining the thou-
sands of strains required for biotechnological re-
search.
NUTRITIONAL PRODUCTS
Nutritional supplements produced from microal-
gae have been the primary focus of microalgal bio-
technology for many years. Dried biomass or cell
extracts produced from Chlorella (Lee 1997, Yama-
guchi 1997), Dunaliella (Avron and Ben-Amotz
1992), and Spirulina (Vonshak 1997) (also see the
reviews listed previously) have dominated the com-
mercial opportunities. These products are directed
mainly at the nutraceutical or health food market
and collectively are likely worth many hundreds of
million dollars. These products, which have been
discussed in many reviews, are not discussed here.
In recent years, considerable attention has been
directed at unicellular algae for the production of
oils and fatty acids. Initially, much of the effort was
conducted at the Solar Energy Research Institute
and focused on utilizing algal oils as biofuels (Neen-
an et al. 1986). Although this work did not prove
commercially viable, it did stimulate research on al-
gal oils. Recent efforts have focused on the use of
algal oils containing long-chain polyunsaturated fat-
ty acids (LCPUFAs) as nutritional supplements (re-
viewed in Cohen et al. 1995 and Behrens and Kyle
1996). The most prominent of these are the omega-
3 LCPUFAs: docosahexaenoic acid (DHA) and ei-
cosapentaenoic acid (EPA).
Docosahexaenoic acid is an omega-3 LCPUFA
216 KIRK E. APT AND PAUL W. BEHRENS
with 22 carbon atoms and 6 methylene-interrupted
cis-double bonds (abbreviated 22:6). It is a dominant
fatty acid in neurological tissue, constituting 20%
25% of the total fatty acids in the gray matter of the
human brain and 50%60% in retina rod outer seg-
ments. It is also abundant in heart muscle tissue and
sperm cells (reviewed in Salem et al. 1986, Salem
1989, and Gill and Valivety 1997). Humans are not
capable of synthesizing DHA de novo, and their ca-
pacity to synthesize DHA from its precursor, lin-
olenic acid, is relatively poor. Thus, adequate sup-
plies of DHA must be obtained from dietary sources
(Emken et al. 1994).
Fish and sh oils have long been recognized as
good sources of LCPUFAs; they are enriched with
both DHA and EPA. However, safety issues have
been raised repeatedly about the contamination lev-
els of various toxins that accumulate in sh and are
concentrated in the sh oils. As a result, alternative
sources of high-quality LCPUFAs have been sought.
Like humans, sh receive much of their LCPUFAs
from dietary sources, which in this case are the pri-
mary producers in the oceanic environment: the al-
gae.
A number of algal groups have been identied
that produce high levels of LCPUFAs, including di-
atoms, chrysophytes, cryptophytes, dinoagellates,
and others (reviewed in Cohen et al. 1995 and Beh-
rens and Kyle 1996). Dinoagellates are especially
well suited for the production of DHA. The dinoa-
gellate Crypthecodinium cohnii can produce most of
its fatty acid as DHA (Behrens and Kyle 1996) with
no other detectable LCPUFAs, such as EPA or ar-
achidonic acid (ARA). Docosahexaenoic acid accu-
mulates mainly in the form of triaclyglycerides. A
DHA-enriched vegetarian oil derived from Crypthe-
codinium is currently widely distributed in the U.S.
for the health food market (Brower 1998). A DHA-
enriched product derived from Schizochytrium has re-
cently become available for use as an animal feed
(see also the section Aquaculture Feeds). In one
application, DHA is used to supplement the diet of
chickens to increase the LCPUFA content of the
eggs. These DHA-enriched eggs are available in Eu-
rope and the U.S. (Anonymous 1996, Riordan
1997). Plans have also been announced to introduce
a line of Schizochytrium-derived products for the
health food market (Anonymous 1998a, Brower
1998).
Docosahexaenoic acid is also a conditionally es-
sential nutrient during infancy (reviewed in Innis
1994 and Makrides et al. 1995). During fetal growth
in utero, the mother supplies the DHA required for
neurological development through the placental cir-
culatory system. There is also a large requirement
for DHA for postnatal brain development. Under
normal conditions, DHA accretion in the brain tis-
sues continues for at least the rst 2 years of life.
The natural nutritional source for a human infant,
its mothers breast milk, is the primary source of
DHA.
Infants fed commercial formulas typically do not
receive any DHA in their diet, and so a number of
nutritional and professional organizations, includ-
ing the World Health Organization (FAO/WHOEx-
pert Committee 1994) have recommended the in-
clusion of supplementary DHA in infant formula.
Although sh oils are rich in many LCPUFAs, they
are typically not suitable for infant formulas because
of the presence of EPA, which can signicantly low-
er growth rates and cause other developmental dif-
culties (Carlson et al. 1994). In addition, serious
questions remain about the possible contamination
of sh oils with heavy metals and other toxins. For
these reasons, a manufacturing method that pro-
duced a high-quality DHA-containing oil (with no
EPA) from the dinoagellate Crypthecodinium has
been used in infant formulas (Kyle 1996).
Docosahexaenoic acid oil produced from Crypthe-
codinium (see the section Production Tech-
niques) is currently available worldwide (including
Europe, Australia, Asia, and the Middle East) in
both pre- and full-term formulas (Brower 1998). At
present, infant formula containing DHA is not com-
mercially available on the U.S. market.
A number of algae have been proposed for pro-
duction of EPA, including Nitzschia sp. (Boswell et
al. 1992), Nannochloropsis (Sukenik 1991), Navicula
sp. (Tan and Johns 1996), Phaeodactylum (Molina et
al. 1995), and Porphyridium (Cohen 1990). In addi-
tion, EPA is a LCPUFA, but with 20 carbons and 5
double bonds (abbreviated 20:5). Changes in EPA
levels can signicantly change an individuals coro-
nary vascular status because the products of EPA me-
tabolism are eicosanoids with antithrombotic and
antiaggregatory effects (Salem 1989).
Unfortunately most algae do not accumulate large
amounts of EPA in the form of triacylglyceride, and
those that do are obligate phototrophes, making
their commercial use economically limited (see the
section Production Techniques). At present, a
puried algal oil containing EPA is not commer-
cially available, although the dried biomass from sev-
eral algae is marketed as a source of EPA (Yama-
guchi 1997).
Algae can also serve as a source of genes involved
in PUFA synthesis. Once the genes are isolated and
characterized, they could be evaluated for suitability
for transfer into other organisms, such as higher
plants (Yaun and Knauf 1997). A number of marine
bacteria have recently been discovered that also pro-
duce DHA and EPA (reviewed in Singh and Ward
1997). The genes encoding EPA biosynthesis from
a marine bacteria have been partially characterized
and successfully transferred into other prokaryotic
organisms (Takeyama et al. 1997).
AQUACULTURE FEEDS
Decreasing natural catches and increasing world
dependence on sh as a food source has led to a
217 MICROALGAL BIOTECHNOLOGY
growing interest in aquaculture. Total global aqua-
culture production in 1995 was estimated to be over
25 million tons and accounted for nearly one-fth
of the worlds sh consumption. This industry is
projected to grow at a rate of 8% per year for the
foreseeable future (Tacon 1998). Central to sustain-
able aquaculture is the need to establish and main-
tain a food chain to support the animals until they
achieve market size.
Aquaculture animals must obtain all their nutri-
ents (except minerals) through the food chain, and
because algae are the basis of the food chain, the
nutrient properties of the algae are critical for the
growth and survival of larvae and adults. In a typical
food chain, algae are consumed by zooplankton (ro-
tifers and Artemia), which in turn are consumed by
sh larvae (DeSilva and Anderson 1995). The algal
species commonly cultured for aquaculture fed in-
clude Chlorella, Tetraselmis, Isochrysis, Pavlova, Phaeo-
dactylum, Navicula, Dunaliella, Amphora, Nitzschia, Cy-
clotella, Chaetoceros, Nannochloropsis, and Skeletonema
(De Roeck et al. 1993, Renaud et al. 1994, Takeyama
et al. 1996).
Although algae are an important part of any aqua-
culture facility, the reliability of the algal supply is a
major problem in attaining a protable operation
(Borowitzka 1997). If there is an interruption in the
supply of algae, the entire food chain could be bro-
ken, resulting in loss of sh larvae and eventually
decreased production of adult sh. This need for
reliability to support zooplankton and larvae has led
to a number of different designs for algal culturing
systems, ranging from ponds to tanks and to sophis-
ticated photobioreactors (Chaumont 1993, Qiang
and Richmond 1994, Borowitzka 1997, Spektorova
et al. 1997). Photobioreactors are generally de-
signed and constructed with input from engineers
as well as biologists, so they can be very efcient at
growing algae. However, even in technologically ad-
vanced photobioreactors, the maximum algal cell
densities attained are relatively low. Low densities
necessitate large-volume cultures and can result in
a substantial cost for harvesting the algae. On the
other hand, photobioreactors are generally more re-
liable than ponds or tanks, but almost always at a
higher cost. Thus, reliability must be balanced
against cost to achieve the best algal system for a
particular application.
An alternative to photobioreactors and a potential
means to substantially reduce the growth costs is to
use heterotrophic algae and grow them in conven-
tional fermentors (Day et al. 1991, Orus et al. 1991,
Barclay et al. 1994, Gladue and Maxey 1994). In this
case, algae are cultured using glucose (or other car-
bon compounds) as both a carbon and an energy
source (see the section Production Techniques).
The cost of producing heterotrophic algal biomass
is estimated to be less than $5 per kilogram (Gladue
and Maxey 1994), whereas the theoretical cost of
producing algae phototrophically in bioreactors is
estimated to be an order of magnitude higher (Wil-
kinson 1998), and actual production costs for pho-
totrophic algae at aquaculture facilities are often two
orders of magnitude higher (Benemann 1992). A
number of the commonly used aquaculture algae,
including Chlorella, Nitzschia, Cyclotella, and Tetrasel-
mis (Day et al. 1991, Gladue and Maxey 1994), are
able to grow heterotrophically.
Another challenge for aquaculture that is being
addressed by phycologists is improvement of larval
nutrition to achieve higher larval survival rates
(Brett and Muller-Navarra 1997). Given the substan-
tial cost of maintaining the food chain for larvae,
any increase in larval survival can have a signicant
impact of the economics of an aquaculture facility.
Improving the nutritional properties of the rotifers
and Artemia by feeding them more nutritionally bal-
anced algae is a simple way to improve larval nutri-
tion (Coutteau and Sorgeloos 1997). Much research
has focused on the importance of polyunsaturated
fatty acids in larval growth and development (Sar-
gent et al. 1994, Barclay and Zeller 1996, Takeyama
et al. 1996). In particular, DHA, EPA, and more re-
cently arachidonic acid (AA) have been recognized
as important nutrients for larvae (Estevez et al. 1997,
Harel et al. 1998). Schizochytrium, Crypthecodinium,
and other algae that contain high levels of DHA
have been used as a source of DHA for the aqua-
culture food chain (Kashiwakura et al. 1994, Barclay
and Zeller 1996). Schizochytrium has been shown to
enrich and boost the fatty acid and DHA content in
rotifers and Artemia and to improve larval growth
(Barclay and Zeller 1996). Schizochytrium- based
products are now commercially available from sev-
eral distributors. In addition, EPA is recognized as
an important fatty acid in larval nutrition, and the
ratio of DHA/EPA is critical for larval development
(Brett and Muller-Navarra 1997). Recently, AA is
also receiving attention as a potentially important
nutrient for larval nutrition, and it is possible that
the ratio of these three fatty acids might be more
important than their absolute levels (Harel et al.
1998).
Alterations in pigmentation can also be an im-
portant criteria for organisms grown in culture be-
cause they can affect commercial acceptability. Ar-
ticial diets typically lack the natural sources of pig-
ments that give organisms such as salmon and trout
their characteristic coloration. As a result, the ca-
rotenoid astaxanthin is used to supplement feed
(Shahidi et al. 1998). In the natural food chain, al-
gae are the primary source of astaxanthin and other
pigments. For articial diets, synthetic sources are
commonly used because of reduced costs. The alga
Hematococcus has been found to be an abundant pro-
ducer of astaxanthin (Johnson and An 1991), and
several companies have successfully commercialized
Hematococcus as a source of natural astaxanthin for
animal feeds (Anonymous 1998b, Lynch 1998).
218 KIRK E. APT AND PAUL W. BEHRENS
FLUORESCENT PIGMENTS
Many algal photosynthetic systems have been well
characterized (reviewed in Grossman et al. 1995),
and a number of pigments present in these systems
are being utilized for commercial applications. The
most widely used are the phycobiliproteins.
Phycobiliproteins are a family of light-harvesting
macromolecules that function as components of the
photosynthetic apparatus in cyanobacteria and sev-
eral groups of eukaryotic algae, including the red
algae, cryptomonads, and glaucophytes (MacColl
and Guard-Friar 1987, Sidler 1994). Their main
function is to trap light energy in the 495650-nm
wavelength range and transfer it to chl a of the pho-
tosynthetic reaction centers.
Phycobiliproteins can be divided into three major
groups on the basis of their spectral properties: phy-
coerythrin (PE) Amax 560 nm, emission 580;
phycocyanin (PC) Amax 620 nm, emission 650;
and allophycocyanin (AP) Amax 650 nm, emis-
sion 660 nm. Each of the different phycobilipro-
teins assemble into high-molecular-mass complexes
composed of two nonidentical polypeptide subunits
( and ). The number of chromophores present
in these complexes ranges from 6 to 34, and these
complexes have extremely high absorbance coef-
cients. When excited with light energy at the maxi-
mal absorbance, greater than 90% of the absorbed
energy can be emitted as uorescence (Sidler 1994).
As mentioned previously, many characteristics
make phycobiliproteins well suited for commercial
applications: (1) they have large numbers of chro-
mophores and high quantum yields, (2) they are
capable of large Stokes shifts (displacement of ab-
sorption and emission wavelengths) with the uo-
rescence emission at wavelengths with minimal au-
touorescence from biological materials, (3) they
form very stable conjugates with many materials, (4)
they are fully water soluble, and (5) they can be ef-
ciently excited by argon or helium-neon lasers.
The ability to form stable conjugates with anti-
bodies, strepavidin, biotin, and so on has been es-
pecially important for developing valuable applica-
tions for the phycobiliproteins. This allows the phy-
cobiliproteins to function as uorescent tags for la-
beling highly specic probes to identify cell types or
proteins (reviewed in Glazer 1994). Some of the
more signicant applications are in ow cytometry
and uorescence-activated cell sorting. In these ap-
plications, PE is 20 times brighter on a molar ratio
than FITC and provides an important additional col-
or for multicolor detection systems in conjunction
with other uorescent pigments. In addition, APC
is a signicant pigment for ow cytometry applica-
tions. Biliproteins have also been widely used in im-
munohistochemistry.
Phycobiliprotein complexes assemble into ex-
tremely large macromolecular complexes called
phycobilisomes. Previous studies have elucidated
structural, spectral, and energy transfer characteris-
tics of the phycobilisomes (Sidler 1994). Phycobili-
somes are unstable in low salt buffer and at dilute
protein concentrations and were thus considered
unsuitable for most biological detection systems. Re-
cently, methods have been developed to stabilize the
phycobilisomes by chemical crosslinking (Cubicciot-
ti 1997). Stabilized phycobilisomes have the same
advantages as individual biliproteins but contain up
to 1400 chromophores, making them the most pow-
erful uorescent pigments currently available on a
per-binding-event basis. They have broad wave-
length adsorption characteristics, with the promi-
nent absorption peaks corresponding to each of the
phycobiliproteins present. This is well suited for ex-
citation by both argon and helium-neon lasers. They
can also have an extraordinary Stokes shift of up to
178 nm. The emission wavelength of approximately
670 nm provides minimal overlap with mammalian
cell autouorescence. They are easily conjugated to
the same materials as the individual biliproteins,
which include antibodies, peptides, streptavidin, bi-
otin, and DNA. By using various source organisms,
a variety of different forms of phycobilisomes can be
isolated. Some have a high proportion of PE, where-
as others have high levels of PC and no PE, which
can be desirable for specic applications.
Stabilized phycobilisomes are commercially avail-
able as secondary labels for a wide variety of uses.
Phycobilisomes are well suited for direct uorescent
detection in immunoblots, in which they are capable
of detecting subpicogram levels of protein (Morse-
man et al. 1998). In microplate immunoassays, phy-
cobilisomes are capable of detecting 40-femtomolar
levels of antigenic protein, with a linear assay range
of four orders of magnitude (Zoha et al. 1998). For
use in ow cytometry, they are ve-fold brighter
than PE and thus well suited for detection of low-
density cell surface markers, which were previously
undetectable through conventional uors.
Phycoiliproteins from cryptomonads, which pro-
vide unique absorption and emission characteristics
(Wedemayer et al. 1996) along with relatively low
molecular mass (50 kDa), are also commercially
available. These have possible applications for use as
intracellular markers or in cases in which specialized
absorption and emission requirements are desired.
Dinoagellates also produce a pigment that has
found limited applications as an additional color in
ow cytometry (Afar et al. 1991). The peridinin
chlorophyll proteins are water-soluble pigments con-
taining carotenoids and chl a.
STABLE-ISOTOPE BIOCHEMICALS
Microalgae are ideally suited as sources of stable
isotopically labeled compounds. They are easily han-
dled and cultured, and their ability to perform pho-
tosynthesis allows them to incorporate
13
C,
15
N, and
2
H from relatively inexpensive inorganic com-
pounds (i.e.
13
CO
2
,
15
NO
3
, and
2
H
2
O) into more
219 MICROALGAL BIOTECHNOLOGY
highly valued organic compounds. For unicellular
microalgae, each cell is exposed to the isotope, re-
sulting in uniform labeling of compounds. Closed
photobioreactor systems make it possible to have a
very high conversion of
13
CO
2
into biomass, thus
minimizing the cost associated with producing
13
C-
labeled substrates (see following discussion). Finally,
microalgae are metabolically very exible and can
be made to overproduce a variety of different prod-
ucts through simple manipulations of the culture
environment (Behrens et al. 1989a, b, 1994, Beh-
rens and Kyle 1996).
One application for algal-produced stable isoto-
pically labeled complex organic compounds is form-
ing the basis of culture media of bacteria, yeast, and
mammalian cells. Stable isotopes provided in the
media are incorporated into cellular components
and, in particular, proteins. Proteins of interest can
be produced in large quantity using molecular tech-
nology, and, coupled with recent developments in
multidimensional NMR technology and stable-iso-
tope-editing techniques (Kainosho 1997), the pri-
mary, secondary, and tertiary structures of small-
and medium-sized proteins can be determined
(Lustbader et al. 1996, Weller et al. 1996). Structural
information can be used to predict the interactions
of substrates with the active sites of proteins and to
site specically modify the protein to alter biological
activity (Bertini et al. 1996, Enokizono et al. 1997).
Two commonly used stable isotopically labeled
compounds for cell culture are glucose and glycerol.
Many algae (especially chlorophytes) are known to
accumulate high levels of glucose in the form of
starch (Behrens et al. 1989a). When these organisms
are grown in the presence of
13
CO
2
, they will pro-
duce labeled starch that can then be easily hydro-
lyzed and puried as crystalline
13
C-glucose. Like-
wise, Dunaliella produces high levels of glycerol and
has been used for
13
C-glycerol production. On pro-
longed culturing, much of the glycerol synthesized
by Dunaliella leaks into the culture medium, greatly
simplifying its purication.
In addition to the use of glucose and glycerol as
cell culture nutrients, other stable isotopically la-
beled compounds derived from algae are being
used to study macromolecular interactions and the
elucidation of metabolic pathways. For example,
13
C-
glucose has been included in growth media, en-
abling the algae to produce
13
C-DHA-containing tri-
glyceride, which is used to study the metabolism and
turnover of DHA (Brossard et al. 1994). Algal-de-
rived stable isotopically labeled compounds have
also been used as metabolic tracers to elucidate var-
ious metabolic pathways (Cunnane and Likhodii
1996, Hellerstein et al. 1997);
13
C-palmitic acid has
been used to measure palmitic acid ux in the
blood (Guo et al. 1997), and labeled galactose has
been used to follow carbohydrate metabolism in the
liver (Hellerstein et al. 1997). A variety of labeled
fatty acids have been used to monitor fatty acid me-
tabolism. For example,
13
C-labeled linoleic acid and
linolenic acid have been useful in studying the syn-
thesis of polyunsaturated fatty acids in infants (Car-
nielli et al. 1996).
Breath tests for the diagnosis of medical disease
and dysfunction represent another application for
the use of microalgal-derived stable isotopically la-
beled products. In the broadest sense, a breath test
is simply the determination and quantitation of the
compounds in human breath. The principle of
these tests is that a substrate labeled with
13
C is in-
gested, absorbed from the small intestine, and ulti-
mately metabolized to carbon dioxide. The magni-
tude and the rate of the appearance of
13
CO
2
in the
exhaled breath is used to diagnose the subjects
physiological state.
Several different approaches to measuring
13
CO
2
have been developed. These include the use of iso-
tope ratio-mass spectrometry, infrared spectroscopy,
and laser-based systems (Schadewaldt et al. 1997).
The costs of
13
CO
2
analysis is a consideration, and
continued development of these systems will un-
doubtedly continue to reduce those costs.
Several Chlamydomonas species are known to pro-
duce high levels of a galactose containing polysac-
charides (Behrens et al. 1996), which can be hydro-
lyzed to produce monosaccharides;
13
C-galactose has
been used to measure liver function (Shreeve et al.
1976, Caspary and Schaffer 1978, Behrens et al.
1996), and its noninvasive nature gives it an advan-
tage over liver biopsy.
In addition,
13
C-xylose has been produced from
Chlamydomonas, which can produce nearly 25% of
its biomass as xylose;
13
C-xylose has been used to
diagnose bacterial overgrowth of the small intestine
(Dellert et al. 1997) because xylose is poorly ab-
sorbed from the small intestine and is metabolized
largely by colonic microora.
Finally,
13
C-labeled mixed triglycerides (known as
Hiolein) have been produced from Neochloris and
used to diagnose fat malabsorption (Lembcke et al.
1996). Hiolein is a triglyceride oil that contains over
50% oleic acid, and it is functionally equivalent to
other triglycerides that have been used as breath test
substrates (Watkins et al. 1982, Kyle 1995).
DRUG SCREENING
Algae are a very diverse group of organisms that
occupy a wide variety of ecological niches. As such,
they have the potential to be a rich source of bio-
active compounds. In many ways, algae are similar
to higher plants. However, they also possess many
of the same characteristics as other microorganisms.
Both higher plants and microorganisms have prov-
en to be rich sources of bioactive compounds, so in
principle it is reasonable to expect that the algae
might also serve as an important resource for useful
molecules.
A large number of bioactivities have been report-
ed in algae, including anticancer, antimicrobial,
220 KIRK E. APT AND PAUL W. BEHRENS
anti-HIV, antiviral, and various neurological activi-
ties (Schwartz, et al. 1990, Cannell 1993, Codd 1995,
Moore 1996, Sivonen 1996). Despite the activities
that have been reported, algae are perhaps best
known for the highly potent toxins that some spe-
cies of blue green algae and dinoagellates can pro-
duce (Codd 1995). For example, the microcystins
are a group of circular peptides produced by blue-
green algae, and some of the more potent deriva-
tives have an LD
50
of 50 g/kg (Rinehart et al.
1994). Saxitoxin and the brevetoxins are produced
by dinoagellates, and each has signicant bioactive
effects on humans and sh (Yasumoto and Murata
1993). In addition to toxins, many other bioactive
compounds have been found in algae (Schwartz et
al. 1990, Cannell 1993, Moore 1996).
The National Cancer Institute (NCI) has played a
major role in the search for bioactive compounds
from algae through both their in-house and their
intramural programs. Early work at the NCI dem-
onstrated that sulfolipids had in vitro activity against
the HIV virus (Gustafson et al. 1989). More recently,
NCI scientists have discovered cyanovirin from the
blue-green alga Nostoc ellipsosporum (Boyd et al.
1997). This compound is a low-molecular-weight
protein that can be produced as a recombinant mol-
ecule in E. coli. Cyanovirin irreversibly inactivates
HIV without adversely affecting the host cells, and
work on this compound is being actively pursued by
the NCI. The discovery of cyanovirin has provided
additional incentive to continue searching for new
compounds in algae.
Despite the growing number of novel bioactive
compounds that have been identied in algae, none
has yet become a commercially useful pharmaceu-
tical. A possible explanation is that algae have re-
ceived considerably less attention as a source for
pharmaceutical screening than other microorgan-
isms or higher plants. Because the probability of de-
veloping a successful pharmaceutical directly corre-
lates with the amount of screening that is done, the
lack of a commercial product could be due to the
relatively limited amount of screening of algal sam-
ples.
MOLECULAR BIOLOGY
Recently, tremendous advances have been made
in the tools available to study a variety of algae.
Highly sophisticated molecular systems are being
used to dissect biological processes in many cyano-
bacteria (reviewed in Thiel 1994). The cyanobacte-
ria can be readily transformed with autonomously
replicating plasmids, and endogenous genes can be
disrupted by homologous recombination. Although
a number of commercial possibilities have been pro-
posed for recombinant cyanobacteria (Elhai 1994,
Vermaas 1996), the potential has yet to be realized.
A novel application of recombinant techniques was
to transfer the cryIVC gene for producing Bt toxin
to Synechococcus (Murphy and Stevens 1992, Stevens
et al. 1994, Xiaoqiang et al. 1997). Cyanobacteria
are a food source for mosquito larvae, and the Bt
toxin is capable of inhibiting larval development. In
principle, recombinant cyanobacteria could be dis-
persed in areas of high mosquito infestation, and as
the larvae consume the cyanobacteria, larval devel-
opment would be inhibited. An attempt is being
made to commercialize cyanobacteria containing Bt
toxin (Watanabe 1996), but this venture faces obvi-
ous difculties because of the potential for wide-
spread dispersal of recombinant organisms that
might rapidly lose their effectiveness because of the
development of resistant larvae.
Advances in eukaryotic algal recombinant tech-
niques have recently been extensively reviewed (Ste-
vens and Purton 1997). Chlamydomonas has devel-
oped into a sophisticated molecular system that has
made important contributions to the understanding
of photosynthetic processes. Although recombinant
Chlamydomonas does not at this time have direct
commercial applications, the technology developed
for Chlamydomonas has provided direction for the de-
velopment of transformation techniques in other al-
gae. Recently developed transformation techniques
for Chlorella (Dawson et al. 1997, Bingham, unpubl.)
and diatoms (Dunahay et al. 1995, Apt et al. 1996)
have potential use in direct commercial applications
(see previous discussion). At the very least, recom-
binant techniques in economically valuable algae
provide an important tool for elucidating and un-
derstanding the biochemical pathways responsible
for the synthesis of products of interest (e.g. biosyn-
thesis of PUFAs).
At this point, recombinant techniques have not
contributed directly to a commercial product. How-
ever, with public and government acceptance of re-
combinant and continued progress in developing
methodologies for algal systems, signicant contri-
butions could be realized in the near future.
PRODUCTION TECHNIQUES
Most algal species are obligate phototrophs and
thus require light for their growth. The requirement
for light, coupled with the high extinction coef-
cient of chlorophyll in these organisms, has neces-
sitated the design and development of novel systems
for large-scale growth. A few algal species are capa-
ble of heterotrophic growth, and for these organ-
isms conventional fermentation technology can be
used for large-scale cultivation.
Phototrophic systems
Commercial growth of photosynthetic algae has
been achieved in different ways: (1) open cultivation
using natural sunlight, (2) closed cultivation using
natural sunlight, and (3) closed cultivation using ar-
ticial illumination. Each system has advantages and
disadvantages, and the choice of system depends on
the degree of parameter control needed to produce
the desired product and on the value of the prod-
221 MICROALGAL BIOTECHNOLOGY
uct. A common limitation to all these systems is the
need to supply light to the culture, making it advan-
tageous to maximize the surface-to-volume ratio of
the culture.
Many congurations of open cultures using nat-
ural sunlight have been proposed and constructed
(Oswald 1988, Chaumont 1993, Pushparaj et al.
1997). These systems are generally large, open
ponds or raceways, and the principle advantage of
these congurations is that the light energy is free.
However, this advantage is more than offset by sev-
eral signicant disadvantages. In open systems, it is
very difcult to prevent contamination of the algal
culture by other organism (i.e. algae and other mi-
croorganisms). This problem has been addressed by
culturing algae that require or tolerate unique
growth conditions that would exclude contaminat-
ing organisms; however, this restricts the usefulness
of these systems to a limited number of organisms.
Suitable species include Dunaliella, which can be
grown at very high salinity, and Spirulina, which will
grow at high pH. Open cultures attain cell densities
leading to the need to process large quantities of
water to harvest the algae. Outdoor phototrophic
growth systems are also subject to daily and seasonal
variations in light intensity and temperature, mak-
ing it difcult to control or reproduce specic cul-
ture conditions.. Nevertheless, for specic algal
products this technology has proven very successful,
producing many thousands of tons of dried biomass
per year (Lee 1997). This is especially true for Spi-
rulina, which is extensively cultured in the U.S.,
Mexico, Thailand, and China (Metting 1996, Li and
Qi 1997, Vonshak 1997).
Several different closed systems using natural sun-
light have been described (Richmond et al. 1993,
Qiang and Richmond 1994, Molina Grima et al.
1995, Spektorova et al. 1997). In these systems, the
algae are enclosed in a transparent material (either
glass or plastic) and the vessels placed outdoors for
illumination. The closure of the vessels minimizes
contamination by other algal species. These systems
have been designed to provide a higher surface-to-
volume ratio than is possible with the ponds and
raceways, so cell densities are often higher than in
the open systems. Closed, outdoor systems are still
subject to variations in light intensity and tempera-
ture that make cultivation reproducibility problem-
atic. In addition, a major problem with closed sys-
tems is the removal of oxygen from the culture and
the provision of adequate temperature control. Al-
though both of these issues can be resolved, the cost
of doing so can more than offset the cost advantage
of using natural sunlight.
As with the outdoor systems, numerous designs
have been constructed for the indoor, closed cul-
ture of algae using electric lights for illumination
(Ratchford and Falloweld 1992, Wohlgeschaffen et
al. 1992, Iqbal et al. 1993, Lee and Palsson 1994).
These vessels are often referred to as photobioreac-
tors, and in principle they are similar to convention-
al fermentor, the major difference being that they
are driven by light rather than by an organic carbon
source. These vessels provide the ability to control
and optimize culture parameters, and, coupled with
the closure that they provide, photobioreactors are
suitable for culturing many different types of algae
(Ratchford and Falloweld 1992). Photobioreactors
are generally more expensive to build than outdoor
systems, but the cost can be justied, depending on
the application of the algal biomass. Photobioreac-
tors have been an important tool for small-scale pro-
duction of high-value products, such as stable-iso-
tope-labeled biochemicals (see previous discussion).
Heterotrophic systems
The most signicant advance in closed culture sys-
tems is the adaptation of fermentation technology
that allows for the heterotrophic growth of microal-
gae and eliminates the problem of light limitation
(Barclay et al. 1994, Kyle 1996, Chen 1997). A sig-
nicant number of microalgae are capable of het-
erotrophic growth and potentially suitable for
growth in fermentors (Droop 1974, Gladue and
Maxey 1994). Heterotrophic fermentor cultures
have a number of important advantages over culture
systems requiring light for photosynthesis. Fermen-
tation technology is preexisting, highly sophisticat-
ed, and utilized worldwide on a massive scale.
The basic principle of fermentor growth is to pro-
vide highly controlled optimal growth conditions to
maximize productivity. The culture vessels range in
volume from 1 to 500,000 L and are operated under
sterile conditions. A motorized shaft with a series of
impellers provide the mixing. Sterile air is pumped
into the system at high pressure and ow rates to
ensure proper gas exchange, with continuous mon-
itoring and adjustment of dissolved O
2
and CO
2
lev-
els. Heating or cooling coils regulate temperature,
and the automatic addition of acid or base main-
tains pH. The culture medium for algal fermentative
growth is similar to that used for phototrophic
growth, except that glucose or a similar carbohy-
drate provides both xed carbon and energy. Other
nutrient levels (i.e. nitrogen and phosphorus) are
also continuously monitored and adjusted.
As a result of the high level of process control,
culture conditions and biomass yields are consistent
and reproducible, with algal cell densities reaching
50 g dry biomass per liter (Gladue and Maxey 1994)
to as high as 100 g dry biomass per liter (Running
et al. 1994). These biomass levels are at least 10-fold
higher then those achieved by photosynthesis-based
culture systems (Radmer and Parker 1994). The
high biomass levels also greatly decrease the volume
of water that must be processed during harvesting.
Because cultures can be routinely run in fermenters
with volumes greater than 100,000 L, several thou-
sand kilograms of dried biomass can be produced
per run. The effectiveness of large-scale cultures and
222 KIRK E. APT AND PAUL W. BEHRENS
the production of high biomass levels can make the
cost of fermentative growth an order of magnitude
less expensive than photobioreactors (Radmer and
Parker 1994).
The ability to provide complete control over the
culture is also critical for maintaining food industry
standard Good Manufacturing Practices (GMP), as
designated by the U.S. Food and Drug Administra-
tion, which are required for the production of high-
quality food- or pharmaceutical-grade materials.
Larger-scale production of the dinoagellate Cryp-
thecodinium by fermentative growth for the produc-
tion of the polyunsaturated fatty acid DHA has been
under way for several years (Kyle 1996). Production
of Crypthecodinium begins with a certied seed stock
that was cryopreserved under liquid nitrogen con-
ditions to maintain genetic stability. A simple cul-
ture medium containing NaCl, CaCl, MgSO
4
, glu-
cose, and yeast extract is utilized for all culture sizes.
The cultures are progressively transferred from a
shake ask through a series of scale-up fermenters,
terminating in a production fermenter of 120,000 L
volume. The cultures are continuously monitored,
and when a predetermined cell density and fatty
acid level is reached, the culture is harvested and
spray-dried. The oil is extracted from the dried bio-
mass using procedures similar to those for conven-
tional vegetable oil processing, which involve extrac-
tion with hexane, rening, bleaching, and deodor-
izing. Following blending, it is sold as a pure vege-
table oil containing 20% or 40% DHA for
applications in human nutrition as described previ-
ously. Crypthecodinium has been reported to produce
approximately 30% of their dry weight as total fatty
acid (Kyle et al. 1992), with DHA making up close
to 50% of the total fatty acid (Behrens and Kyle
1996).
Schizochytrium is also produced using microbial
fermentation techniques (Barclay et al. 1994) for
use in the health food market and as an animal feed
supplement (see previous sections). Special strains
of the organism have been reported that are capable
of producing 30%40% of their fatty acids as long-
chain omega-3 fatty acids. When cultured at low sa-
linities with glucose as the carbon source culture,
densities of 20 gL
1
dry biomass and yields of ap-
proximately 1.0 gL
1
day
1
can be achieved. New
strains have recently been isolated that are capable
of producing 70% of their biomass as fatty acids,
of which 35% is DHA, with yields under laboratory
conditions exceeding 3 g DHAL
1
day (Nakahara
et al. 1996).
Chlorella is also extensively grown in large quanti-
ties by fermentation techniques. Production levels in
Japan are estimated to exceed 500 T per year, ac-
counting for 50% of the countrys total production
(Lee 1997). Plans have also been announced in Ko-
rea to begin production of heterotrophically grown
Chlorella at a level exceeding 1000 T per year (Lee
1997).
STRAIN MAINTENANCE
Maintenance of organisms is very important for
the future of biotechnology (Hunter-Cevera and
Belt 1996). With an increasing number of applica-
tions and potential applications for algae, it is criti-
cal that the organisms be preserved. For those or-
ganisms that form the basis of a product, it is not
only sufcient that the organism be preserved but
also important that the special and unique charac-
teristics of that organism be maintained. Thus,
strain maintenance is not limited to preservation of
the organism; it must also ensure that it is geneti-
cally stable.
Various methods have been used to preserve al-
gae, including serial transfer, freeze-drying, and
cryopreservation (Andersen 1996, Day et al. 1997).
Each method has advantages and disadvantages, and
it is possible that no one method is ideal or usable
for all algal strains. By far, the most widely used
method for maintenance is serial transfer (Andersen
1996). This method requires no expensive equip-
ment and is generally very satisfactory for the main-
tenance of a small number of noncritical cultures.
Good microbial technique can greatly minimize
problems of contamination, but genetic drift is not
minimized and might even be increased through se-
rial transfer. Freeze-drying is generally considered a
better technique than serial transfer, and the equip-
ment required is minimal. Unfortunately, freeze-dry-
ing is unreliable, often giving survival rates of less
than 5% (McGrath et al. 1978), and recent successes
with cryopreservation has provided even less incen-
tive to pursue and optimize this technique. Cryo-
preservation (maintenance at temperatures colder
than 120 C) offers low maintenance of cultures
and the virtual elimination of genetic drift. Of the
various preservation methods that are available,
cryopreservation is generally regarded as the single
best method for the long-term preservation of or-
ganisms and their properties (Andersen 1996).
At least some degree of success with cryopreser-
vation techniques has been reported with several dif-
ferent algal groups, including green algae, red al-
gae, euglenophytes, diatoms, and cyanobacteria al-
gae (Morris 1978, Canavate and Lubian 1997, Day
et al. 1997), and with macroalgae as well as microal-
gae (Kuwano et al. 1994, Kono et al. 1997). Beaty
and Parker (1991) attempted cryopreservation on a
large number of different genera and found an
overall success rate of almost 80%. Likewise, Morris
(1978) found a very high success rate with a large
number of green algae. Canavate and Lubian
(1995a, b) have done several detailed studies of
cryopreservation variables with several genera and
have obtained survival rates as high as 98% with Te-
traselmis. Although survival rates vary somewhat be-
tween genera, the results to date on cryopreserva-
tion suggest that this preservation technique should
be applicable to many algal genera.
223 MICROALGAL BIOTECHNOLOGY
Several parameters are generally considered very
important in cryopreservation, including choice of
cryoprotectant, cryoprotectant concentration, freez-
ing rate, physiological status of the culture, and
thawing procedure. A wide variety of cryoprotec-
tants have been tried, including dimethyl sulfoxide
(DMSO), glycerol, methanol, polyvinylpyrrolidone,
proline, propylene glycol, ethylene glycol, sorbitol,
glucose, sucrose, dextran, and betaine (Canavate
and Lubian 1995a, Andersen 1996, Kono et al.
1997). Glycerol, DMSO, and methanol are the most
widely used cryoprotectants, and each has been
shown to give good success rates (Beaty and Parker
1991, Canavate and Lubian 1995b). The successes
and failures experienced by different workers could
likely be due to other differences in their protocols
or the physiological state of the algae used. Various
freezing rates have also been tried. Generally, un-
controlled freezing gives poor results, and it is nec-
essary to have equipment to control the freezing
rate. Successful cryopreservation has been demon-
strated with freezing rates ranging from 0.5 C to
16 C per minute (Canavate and Lubian 1995b, Day
et al. 1997), and some organisms appear to revive
better with a slow thawing rate, although most pre-
fer rapid thawing (Canavate and Lubian 1997).
Higher revival rates were found when agar-grown
rather than liquid-grown algae were used (Beaty and
Parker 1991), and perhaps the osmotic stress of
growth on agar might precondition the cells to bet-
ter withstand cryopreservation. The results of these
studies can now serve as a basis for increasing the
number of genera that can be successfully cryopre-
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