J. Phycol. 35, 215–226 (1999)                                                            REVIEW                 COMMERCIAL...
216                                    KIRK E. APT AND PAUL W. BEHRENSwith 22 carbon atoms and 6 methylene-interrupted    ...
MICROALGAL BIOTECHNOLOGY                                             217growing interest in aquaculture. Total global aqua...
218                                    KIRK E. APT AND PAUL W. BEHRENS               FLUORESCENT PIGMENTS                 ...
MICROALGAL BIOTECHNOLOGY                                           219highly valued organic compounds. For unicellular    ...
220                                      KIRK E. APT AND PAUL W. BEHRENSanti-HIV, antiviral, and various neurological acti...
MICROALGAL BIOTECHNOLOGY                                           221uct. A common limitation to all these systems is the...
222                                       KIRK E. APT AND PAUL W. BEHRENSthe production of high biomass levels can make th...
MICROALGAL BIOTECHNOLOGY                                                             223   Several parameters are generall...
224                                                KIRK E. APT AND PAUL W. BEHRENSCannell, R. J. 1990. Algal biotechnology...
MICROALGAL BIOTECHNOLOGY                                                                225     uction of docosahexaenoic ...
226                                                 KIRK E. APT AND PAUL W. BEHRENS    T. 1996. DHA enrichment of rotifers...
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Aptand behrens1999

  1. 1. J. Phycol. 35, 215–226 (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 the term ‘‘algae’’ refers to a polyphyletic, artificialin microalgal biotechnology in recent years that are assemblage of organisms.slowly moving the field into new areas. New prod- It has been proposed that at least one nontradi-ucts are being developed for use in the mass com- tional organism, Schizochytrium, could be consideredmercial markets as opposed to the ‘‘health food’’ an alga (Barclay 1992). The genus Schizochytrium ismarkets. These include algal-derived long-chained classified in the Thraustochytrids (Chamberlain andpolyunsaturated fatty acids, mainly docosahexaenoic Moss 1988), which is traditionally considered to beacid, for use as supplements in human nutrition and part of the lower fungi. Molecular phylogenies haveanimals. Large-scale production of algal fatty acids indicated that the Thraustochytrids are allied withis possible through the use of heterotrophic algae the Chromophytic algae (Cavalier-Smith et al.and the adaptation of classical fermentation systems 1994). Because Schizochytrium is currently marketedproviding consistent biomass under highly con- as an algal product, it is included here.trolled conditions that result in a very high quality This article first summarizes developments in nu-product. New products have also been developed tritional products used for direct supplementationfor use in the development of pharmaceutical and by humans and as aquaculture feeds. It then dis-research products. These include stable-isotope bio- cusses products for pharmaceutical and researchchemicals produced by algae in closed-system pho- uses. Finally, it discusses advances in productiontobioreactors and extremely bright fluorescent pig- techniques and then the progress that has beenments. Cryopreservation has also had a tremendous made in the critical area of maintaining the thou-impact on the ability of strains to be maintained for sands of strains required for biotechnological re-long periods of time at low cost and maintenance search.while preserving genetic stability. NUTRITIONAL PRODUCTSKey index words: aquaculture; cryopreservation; do-cosahexaenoic acid; heterotrophic algae; infant for- Nutritional supplements produced from microal-mula; phycobilisomes; stable isotopes gae have been the primary focus of microalgal bio- technology for many years. Dried biomass or cell extracts produced from Chlorella (Lee 1997, Yama- In recent years, numerous general reviews on var- guchi 1997), Dunaliella (Avron and Ben-Amotzious aspects of ‘‘algal biotechnology’’ have appeared 1992), and Spirulina (Vonshak 1997) (also see the(Borowitzka and Borowitzka 1988, Cresswell et al. reviews listed previously) have dominated the com-1989, Cannell 1990, Avron and Ben-Amotz 1992, Bo- mercial opportunities. These products are directedrowitzka 1992, Becker 1994, Parker 1994, Allnutt mainly at the nutraceutical or health food market1996, Metting 1996, Borowitzka 1997, Renn 1997, and collectively are likely worth many hundreds ofVonshak 1997). This review is intended not to be million dollars. These products, which have beencomprehensive but to focus on recent applications discussed in many reviews, are not discussed here.and new developments that are diversifying the di- In recent years, considerable attention has beenrections for commercial exploitation of microalgae. directed at unicellular algae for the production ofIt also focuses on products that are at or near com- oils and fatty acids. Initially, much of the effort wasmercial availability. conducted at the Solar Energy Research Institute Traditionally, the definition of ‘‘algae’’ includes and focused on utilizing algal oils as biofuels (Neen-the cyanobacteria; green, red; and brown algae; an et al. 1986). Although this work did not provecharophytes; cryptophytes; chrysophytes; diatoms; commercially viable, it did stimulate research on al-dinoflagellates; and others, encompassing photosyn- gal oils. Recent efforts have focused on the use ofthetic prokaryotic and eukaryotic organisms as well algal oils containing long-chain polyunsaturated fat-as their heterotrophic derivatives. On the basis of ty acids (LCPUFAs) as nutritional supplements (re-molecular phylogenies, many of these groups are viewed in Cohen et al. 1995 and Behrens and Kylewidely scattered on the ‘‘tree of life.’’ As a result, 1996). The most prominent of these are the omega- 3 LCPUFAs: docosahexaenoic acid (DHA) and ei- 1 Received 30 April 1997. Accepted 2 December 1998. cosapentaenoic acid (EPA). 2 Author for reprint requests; email kirkapt@aol.com. Docosahexaenoic acid is an omega-3 LCPUFA 215
  2. 2. 216 KIRK E. APT AND PAUL W. BEHRENSwith 22 carbon atoms and 6 methylene-interrupted its mother’s breast milk, is the primary source ofcis-double bonds (abbreviated 22:6). It is a dominant DHA.fatty acid in neurological tissue, constituting 20%– Infants fed commercial formulas typically do not25% of the total fatty acids in the gray matter of the receive any DHA in their diet, and so a number ofhuman brain and 50%–60% in retina rod outer seg- nutritional and professional organizations, includ-ments. It is also abundant in heart muscle tissue and ing the World Health Organization (FAO/WHO Ex-sperm cells (reviewed in Salem et al. 1986, Salem pert Committee 1994) have recommended the in-1989, and Gill and Valivety 1997). Humans are not clusion of supplementary DHA in infant formula.capable of synthesizing DHA de novo, and their ca- Although fish oils are rich in many LCPUFAs, theypacity to synthesize DHA from its precursor, ␣ lin- are typically not suitable for infant formulas becauseolenic acid, is relatively poor. Thus, adequate sup- of the presence of EPA, which can significantly low-plies of DHA must be obtained from dietary sources er growth rates and cause other developmental dif-(Emken et al. 1994). ficulties (Carlson et al. 1994). In addition, serious Fish and fish oils have long been recognized as questions remain about the possible contaminationgood sources of LCPUFAs; they are enriched with of fish oils with heavy metals and other toxins. Forboth DHA and EPA. However, safety issues have these reasons, a manufacturing method that pro-been raised repeatedly about the contamination lev- duced a high-quality DHA-containing oil (with noels of various toxins that accumulate in fish and are EPA) from the dinoflagellate Crypthecodinium hasconcentrated in the fish oils. As a result, alternative been used in infant formulas (Kyle 1996).sources of high-quality LCPUFAs have been sought. Docosahexaenoic acid oil produced from Crypthe- codinium (see the section ‘‘Production Tech-Like humans, fish receive much of their LCPUFAs niques’’) is currently available worldwide (includingfrom dietary sources, which in this case are the pri- Europe, Australia, Asia, and the Middle East) inmary producers in the oceanic environment: the al- both pre- and full-term formulas (Brower 1998). Atgae. present, infant formula containing DHA is not com- A number of algal groups have been identified mercially available on the U.S. market.that produce high levels of LCPUFAs, including di- A number of algae have been proposed for pro-atoms, chrysophytes, cryptophytes, dinoflagellates, duction of EPA, including Nitzschia sp. (Boswell etand others (reviewed in Cohen et al. 1995 and Beh- al. 1992), Nannochloropsis (Sukenik 1991), Navicularens and Kyle 1996). Dinoflagellates are especially sp. (Tan and Johns 1996), Phaeodactylum (Molina etwell suited for the production of DHA. The dinofla- al. 1995), and Porphyridium (Cohen 1990). In addi-gellate Crypthecodinium cohnii can produce most of tion, EPA is a LCPUFA, but with 20 carbons and 5its fatty acid as DHA (Behrens and Kyle 1996) with double bonds (abbreviated 20:5). Changes in EPAno other detectable LCPUFAs, such as EPA or ar- levels can significantly change an individuals coro-achidonic acid (ARA). Docosahexaenoic acid accu- nary vascular status because the products of EPA me-mulates mainly in the form of triaclyglycerides. A tabolism are eicosanoids with antithrombotic andDHA-enriched vegetarian oil derived from Crypthe- antiaggregatory effects (Salem 1989).codinium is currently widely distributed in the U.S. Unfortunately most algae do not accumulate largefor the health food market (Brower 1998). A DHA- amounts of EPA in the form of triacylglyceride, andenriched product derived from Schizochytrium has re- those that do are obligate phototrophes, makingcently become available for use as an animal feed their commercial use economically limited (see the(see also the section ‘‘Aquaculture Feeds’’). In one section ‘‘Production Techniques’’). At present, aapplication, DHA is used to supplement the diet of purified algal oil containing EPA is not commer-chickens to increase the LCPUFA content of the cially available, although the dried biomass from sev-eggs. These DHA-enriched eggs are available in Eu- eral algae is marketed as a source of EPA (Yama-rope and the U.S. (Anonymous 1996, Riordan guchi 1997).1997). Plans have also been announced to introduce Algae can also serve as a source of genes involveda line of Schizochytrium-derived products for the in PUFA synthesis. Once the genes are isolated andhealth food market (Anonymous 1998a, Brower characterized, they could be evaluated for suitability1998). for transfer into other organisms, such as higher Docosahexaenoic acid is also a conditionally es- plants (Yaun and Knauf 1997). A number of marinesential nutrient during infancy (reviewed in Innis bacteria have recently been discovered that also pro- duce DHA and EPA (reviewed in Singh and Ward1994 and Makrides et al. 1995). During fetal growth 1997). The genes encoding EPA biosynthesis fromin utero, the mother supplies the DHA required for a marine bacteria have been partially characterizedneurological development through the placental cir- and successfully transferred into other prokaryoticculatory system. There is also a large requirement organisms (Takeyama et al. 1997).for DHA for postnatal brain development. Undernormal conditions, DHA accretion in the brain tis- AQUACULTURE FEEDSsues continues for at least the first 2 years of life. Decreasing natural catches and increasing worldThe natural nutritional source for a human infant, dependence on fish as a food source has led to a
  3. 3. MICROALGAL BIOTECHNOLOGY 217growing interest in aquaculture. Total global aqua- estimated to be an order of magnitude higher (Wil-culture production in 1995 was estimated to be over kinson 1998), and actual production costs for pho-25 million tons and accounted for nearly one-fifth totrophic algae at aquaculture facilities are often twoof the world’s fish consumption. This industry is orders of magnitude higher (Benemann 1992). Aprojected to grow at a rate of 8% per year for the number of the commonly used aquaculture algae,foreseeable future (Tacon 1998). Central to sustain- including Chlorella, Nitzschia, Cyclotella, and Tetrasel-able aquaculture is the need to establish and main- mis (Day et al. 1991, Gladue and Maxey 1994), aretain a food chain to support the animals until they able to grow heterotrophically.achieve market size. Another challenge for aquaculture that is being Aquaculture animals must obtain all their nutri- addressed by phycologists is improvement of larvalents (except minerals) through the food chain, and nutrition to achieve higher larval survival ratesbecause algae are the basis of the food chain, the (Brett and Mu ¨ller-Navarra 1997). Given the substan-nutrient properties of the algae are critical for the tial cost of maintaining the food chain for larvae,growth and survival of larvae and adults. In a typical any increase in larval survival can have a significantfood chain, algae are consumed by zooplankton (ro- impact of the economics of an aquaculture facility.tifers and Artemia), which in turn are consumed by Improving the nutritional properties of the rotifersfish larvae (DeSilva and Anderson 1995). The algal and Artemia by feeding them more nutritionally bal-species commonly cultured for aquaculture fed in- anced algae is a simple way to improve larval nutri-clude Chlorella, Tetraselmis, Isochrysis, Pavlova, Phaeo- tion (Coutteau and Sorgeloos 1997). Much researchdactylum, Navicula, Dunaliella, Amphora, Nitzschia, Cy- has focused on the importance of polyunsaturatedclotella, Chaetoceros, Nannochloropsis, and Skeletonema fatty acids in larval growth and development (Sar-(De Roeck et al. 1993, Renaud et al. 1994, Takeyama gent et al. 1994, Barclay and Zeller 1996, Takeyamaet al. 1996). et al. 1996). In particular, DHA, EPA, and more re- Although algae are an important part of any aqua- cently arachidonic acid (AA) have been recognizedculture facility, the reliability of the algal supply is a as important nutrients for larvae (Estevez et al. 1997,major problem in attaining a profitable operation Harel et al. 1998). Schizochytrium, Crypthecodinium,(Borowitzka 1997). If there is an interruption in the and other algae that contain high levels of DHAsupply of algae, the entire food chain could be bro- have been used as a source of DHA for the aqua-ken, resulting in loss of fish larvae and eventually culture food chain (Kashiwakura et al. 1994, Barclaydecreased production of adult fish. This need for and Zeller 1996). Schizochytrium has been shown toreliability to support zooplankton and larvae has led enrich and boost the fatty acid and DHA content into a number of different designs for algal culturing rotifers and Artemia and to improve larval growthsystems, ranging from ponds to tanks and to sophis-ticated photobioreactors (Chaumont 1993, Qiang (Barclay and Zeller 1996). Schizochytrium- basedand Richmond 1994, Borowitzka 1997, Spektorova products are now commercially available from sev-et al. 1997). Photobioreactors are generally de- eral distributors. In addition, EPA is recognized assigned and constructed with input from engineers an important fatty acid in larval nutrition, and theas well as biologists, so they can be very efficient at ratio of DHA/EPA is critical for larval developmentgrowing algae. However, even in technologically ad- (Brett and Mu ¨ller-Navarra 1997). Recently, AA isvanced photobioreactors, the maximum algal cell also receiving attention as a potentially importantdensities attained are relatively low. Low densities nutrient for larval nutrition, and it is possible thatnecessitate large-volume cultures and can result in the ratio of these three fatty acids might be morea substantial cost for harvesting the algae. On the important than their absolute levels (Harel et al.other hand, photobioreactors are generally more re- 1998).liable than ponds or tanks, but almost always at a Alterations in pigmentation can also be an im-higher cost. Thus, reliability must be balanced portant criteria for organisms grown in culture be-against cost to achieve the best algal system for a cause they can affect commercial acceptability. Ar-particular application. tificial diets typically lack the natural sources of pig- An alternative to photobioreactors and a potential ments that give organisms such as salmon and troutmeans to substantially reduce the growth costs is to their characteristic coloration. As a result, the ca-use heterotrophic algae and grow them in conven- rotenoid astaxanthin is used to supplement feedtional fermentors (Day et al. 1991, Orus et al. 1991, (Shahidi et al. 1998). In the natural food chain, al-Barclay et al. 1994, Gladue and Maxey 1994). In this gae are the primary source of astaxanthin and othercase, algae are cultured using glucose (or other car- pigments. For artificial diets, synthetic sources arebon compounds) as both a carbon and an energy commonly used because of reduced costs. The algasource (see the section ‘‘Production Techniques’’). Hematococcus has been found to be an abundant pro-The cost of producing heterotrophic algal biomass ducer of astaxanthin (Johnson and An 1991), andis estimated to be less than $5 per kilogram (Gladue several companies have successfully commercializedand Maxey 1994), whereas the theoretical cost of Hematococcus as a source of natural astaxanthin forproducing algae phototrophically in bioreactors is animal feeds (Anonymous 1998b, Lynch 1998).
  4. 4. 218 KIRK E. APT AND PAUL W. BEHRENS FLUORESCENT PIGMENTS structural, spectral, and energy transfer characteris- Many algal photosynthetic systems have been well tics of the phycobilisomes (Sidler 1994). Phycobili-characterized (reviewed in Grossman et al. 1995), somes are unstable in low salt buffer and at diluteand a number of pigments present in these systems protein concentrations and were thus consideredare being utilized for commercial applications. The unsuitable for most biological detection systems. Re-most widely used are the phycobiliproteins. cently, methods have been developed to stabilize the Phycobiliproteins are a family of light-harvesting phycobilisomes by chemical crosslinking (Cubicciot-macromolecules that function as components of the ti 1997). Stabilized phycobilisomes have the same advantages as individual biliproteins but contain upphotosynthetic apparatus in cyanobacteria and sev- to 1400 chromophores, making them the most pow-eral groups of eukaryotic algae, including the red erful fluorescent pigments currently available on aalgae, cryptomonads, and glaucophytes (MacColl per-binding-event basis. They have broad wave-and Guard-Friar 1987, Sidler 1994). Their main length adsorption characteristics, with the promi-function is to trap light energy in the 495–650-nm nent absorption peaks corresponding to each of thewavelength range and transfer it to chl a of the pho- phycobiliproteins present. This is well suited for ex-tosynthetic reaction centers. citation by both argon and helium-neon lasers. They Phycobiliproteins can be divided into three major can also have an extraordinary Stokes shift of up togroups on the basis of their spectral properties: phy- 178 nm. The emission wavelength of approximatelycoerythrin (PE) Amax ϭ 560 nm, emission ϭ 580; 670 nm provides minimal overlap with mammalianphycocyanin (PC) Amax ϭ 620 nm, emission ϭ 650; cell autofluorescence. They are easily conjugated toand allophycocyanin (AP) Amax ϭ 650 nm, emis- the same materials as the individual biliproteins,sion ϭ 660 nm. Each of the different phycobilipro- which include antibodies, peptides, streptavidin, bi-teins assemble into high-molecular-mass complexes otin, and DNA. By using various source organisms,composed of two nonidentical polypeptide subunits a variety of different forms of phycobilisomes can be(␣ and ␤). The number of chromophores present isolated. Some have a high proportion of PE, where-in these complexes ranges from 6 to 34, and these as others have high levels of PC and no PE, whichcomplexes have extremely high absorbance coeffi- can be desirable for specific applications.cients. When excited with light energy at the maxi- Stabilized phycobilisomes are commercially avail-mal absorbance, greater than 90% of the absorbed able as secondary labels for a wide variety of uses.energy can be emitted as fluorescence (Sidler 1994). Phycobilisomes are well suited for direct fluorescent As mentioned previously, many characteristics detection in immunoblots, in which they are capablemake phycobiliproteins well suited for commercial of detecting subpicogram levels of protein (Morse-applications: (1) they have large numbers of chro- man et al. 1998). In microplate immunoassays, phy-mophores and high quantum yields, (2) they are cobilisomes are capable of detecting 40-femtomolarcapable of large Stokes shifts (displacement of ab- levels of antigenic protein, with a linear assay rangesorption and emission wavelengths) with the fluo- of four orders of magnitude (Zoha et al. 1998). Forrescence emission at wavelengths with minimal au- use in flow cytometry, they are five-fold brightertofluorescence from biological materials, (3) they than PE and thus well suited for detection of low-form very stable conjugates with many materials, (4) density cell surface markers, which were previouslythey are fully water soluble, and (5) they can be ef- undetectable through conventional fluors.ficiently excited by argon or helium-neon lasers. Phycoiliproteins from cryptomonads, which pro- The ability to form stable conjugates with anti- vide unique absorption and emission characteristicsbodies, strepavidin, biotin, and so on has been es- (Wedemayer et al. 1996) along with relatively lowpecially important for developing valuable applica- molecular mass (Ͻ50 kDa), are also commerciallytions for the phycobiliproteins. This allows the phy- available. These have possible applications for use ascobiliproteins to function as fluorescent tags for la- intracellular markers or in cases in which specializedbeling highly specific probes to identify cell types or absorption and emission requirements are desired.proteins (reviewed in Glazer 1994). Some of the Dinoflagellates also produce a pigment that hasmore significant applications are in flow cytometry found limited applications as an additional color inand fluorescence-activated cell sorting. In these ap- flow cytometry (Afar et al. 1991). The peridininplications, PE is 20 times brighter on a molar ratio chlorophyll proteins are water-soluble pigments con-than FITC and provides an important additional col- taining carotenoids and chl a.or for multicolor detection systems in conjunctionwith other fluorescent pigments. In addition, APC STABLE-ISOTOPE BIOCHEMICALSis a significant pigment for flow cytometry applica- Microalgae are ideally suited as sources of stabletions. Biliproteins have also been widely used in im- isotopically labeled compounds. They are easily han-munohistochemistry. dled and cultured, and their ability to perform pho- Phycobiliprotein complexes assemble into ex- tosynthesis allows them to incorporate 13C, 15N, andtremely large macromolecular complexes called 2 H from relatively inexpensive inorganic com-phycobilisomes. Previous studies have elucidated pounds (i.e. 13CO2, 15NO3, and 2H2O) into more
  5. 5. MICROALGAL BIOTECHNOLOGY 219highly valued organic compounds. For unicellular tabolism. For example, 13C-labeled linoleic acid andmicroalgae, each cell is exposed to the isotope, re- linolenic acid have been useful in studying the syn-sulting in uniform labeling of compounds. Closed thesis of polyunsaturated fatty acids in infants (Car-photobioreactor systems make it possible to have a nielli et al. 1996).very high conversion of 13CO2 into biomass, thus Breath tests for the diagnosis of medical diseaseminimizing the cost associated with producing 13C- and dysfunction represent another application forlabeled substrates (see following discussion). Finally, the use of microalgal-derived stable isotopically la-microalgae are metabolically very flexible and can beled products. In the broadest sense, a breath testbe made to overproduce a variety of different prod- is simply the determination and quantitation of theucts through simple manipulations of the culture compounds in human breath. The principle ofenvironment (Behrens et al. 1989a, b, 1994, Beh- these tests is that a substrate labeled with 13C is in-rens and Kyle 1996). gested, absorbed from the small intestine, and ulti- One application for algal-produced stable isoto- mately metabolized to carbon dioxide. The magni-pically labeled complex organic compounds is form- tude and the rate of the appearance of 13CO2 in theing the basis of culture media of bacteria, yeast, and exhaled breath is used to diagnose the subject’smammalian cells. Stable isotopes provided in the physiological state.media are incorporated into cellular components Several different approaches to measuring 13CO2and, in particular, proteins. Proteins of interest can have been developed. These include the use of iso-be produced in large quantity using molecular tech- tope ratio-mass spectrometry, infrared spectroscopy,nology, and, coupled with recent developments in and laser-based systems (Schadewaldt et al. 1997).multidimensional NMR technology and stable-iso- The costs of 13CO2 analysis is a consideration, andtope-editing techniques (Kainosho 1997), the pri- continued development of these systems will un-mary, secondary, and tertiary structures of small- doubtedly continue to reduce those costs.and medium-sized proteins can be determined Several Chlamydomonas species are known to pro-(Lustbader et al. 1996, Weller et al. 1996). Structural duce high levels of a galactose containing polysac-information can be used to predict the interactions charides (Behrens et al. 1996), which can be hydro-of substrates with the active sites of proteins and to lyzed to produce monosaccharides; 13C-galactose hassite specifically modify the protein to alter biological been used to measure liver function (Shreeve et al.activity (Bertini et al. 1996, Enokizono et al. 1997). 1976, Caspary and Schaffer 1978, Behrens et al. Two commonly used stable isotopically labeled 1996), and its noninvasive nature gives it an advan-compounds for cell culture are glucose and glycerol. tage over liver biopsy.Many algae (especially chlorophytes) are known to In addition, 13C-xylose has been produced fromaccumulate high levels of glucose in the form of Chlamydomonas, which can produce nearly 25% ofstarch (Behrens et al. 1989a). When these organisms its biomass as xylose; 13C-xylose has been used toare grown in the presence of 13CO2, they will pro- diagnose bacterial overgrowth of the small intestineduce labeled starch that can then be easily hydro- (Dellert et al. 1997) because xylose is poorly ab-lyzed and purified as crystalline 13C-glucose. Like- sorbed from the small intestine and is metabolizedwise, Dunaliella produces high levels of glycerol and largely by colonic microflora.has been used for 13C-glycerol production. On pro- Finally, 13C-labeled mixed triglycerides (known aslonged culturing, much of the glycerol synthesized Hiolein) have been produced from Neochloris andby Dunaliella leaks into the culture medium, greatly used to diagnose fat malabsorption (Lembcke et al.simplifying its purification. 1996). Hiolein is a triglyceride oil that contains over In addition to the use of glucose and glycerol as 50% oleic acid, and it is functionally equivalent tocell culture nutrients, other stable isotopically la- other triglycerides that have been used as breath testbeled compounds derived from algae are being substrates (Watkins et al. 1982, Kyle 1995).used to study macromolecular interactions and theelucidation of metabolic pathways. For example, 13C- DRUG SCREENINGglucose has been included in growth media, en- Algae are a very diverse group of organisms thatabling the algae to produce 13C-DHA-containing tri- occupy a wide variety of ecological niches. As such,glyceride, which is used to study the metabolism and they have the potential to be a rich source of bio-turnover of DHA (Brossard et al. 1994). Algal-de- active compounds. In many ways, algae are similarrived stable isotopically labeled compounds have to higher plants. However, they also possess manyalso been used as metabolic tracers to elucidate var- of the same characteristics as other microorganisms.ious metabolic pathways (Cunnane and Likhodii Both higher plants and microorganisms have prov-1996, Hellerstein et al. 1997); 13C-palmitic acid has en to be rich sources of bioactive compounds, so inbeen used to measure palmitic acid flux in the principle it is reasonable to expect that the algaeblood (Guo et al. 1997), and labeled galactose has might also serve as an important resource for usefulbeen used to follow carbohydrate metabolism in the molecules.liver (Hellerstein et al. 1997). A variety of labeled A large number of bioactivities have been report-fatty acids have been used to monitor fatty acid me- ed in algae, including anticancer, antimicrobial,
  6. 6. 220 KIRK E. APT AND PAUL W. BEHRENSanti-HIV, antiviral, and various neurological activi- et al. 1994, Xiaoqiang et al. 1997). Cyanobacteriaties (Schwartz, et al. 1990, Cannell 1993, Codd 1995, are a food source for mosquito larvae, and the BtMoore 1996, Sivonen 1996). Despite the activities toxin is capable of inhibiting larval development. Inthat have been reported, algae are perhaps best principle, recombinant cyanobacteria could be dis-known for the highly potent toxins that some spe- persed in areas of high mosquito infestation, and ascies of blue green algae and dinoflagellates can pro- the larvae consume the cyanobacteria, larval devel-duce (Codd 1995). For example, the microcystins opment would be inhibited. An attempt is beingare a group of circular peptides produced by blue- made to commercialize cyanobacteria containing Btgreen algae, and some of the more potent deriva- toxin (Watanabe 1996), but this venture faces obvi-tives have an LD50 of 50 ␮g/kg (Rinehart et al. ous difficulties because of the potential for wide-1994). Saxitoxin and the brevetoxins are produced spread dispersal of recombinant organisms thatby dinoflagellates, and each has significant bioactive might rapidly lose their effectiveness because of theeffects on humans and fish (Yasumoto and Murata development of resistant larvae.1993). In addition to toxins, many other bioactive Advances in eukaryotic algal recombinant tech-compounds have been found in algae (Schwartz et niques have recently been extensively reviewed (Ste-al. 1990, Cannell 1993, Moore 1996). vens and Purton 1997). Chlamydomonas has devel- The National Cancer Institute (NCI) has played a oped into a sophisticated molecular system that hasmajor role in the search for bioactive compounds made important contributions to the understandingfrom algae through both their in-house and their of photosynthetic processes. Although recombinantintramural programs. Early work at the NCI dem- Chlamydomonas does not at this time have directonstrated that sulfolipids had in vitro activity against commercial applications, the technology developedthe HIV virus (Gustafson et al. 1989). More recently, for Chlamydomonas has provided direction for the de-NCI scientists have discovered cyanovirin from the velopment of transformation techniques in other al-blue-green alga Nostoc ellipsosporum (Boyd et al. gae. Recently developed transformation techniques1997). This compound is a low-molecular-weight for Chlorella (Dawson et al. 1997, Bingham, unpubl.)protein that can be produced as a recombinant mol- and diatoms (Dunahay et al. 1995, Apt et al. 1996)ecule in E. coli. Cyanovirin irreversibly inactivates have potential use in direct commercial applicationsHIV without adversely affecting the host cells, and (see previous discussion). At the very least, recom-work on this compound is being actively pursued by binant techniques in economically valuable algaethe NCI. The discovery of cyanovirin has provided provide an important tool for elucidating and un-additional incentive to continue searching for new derstanding the biochemical pathways responsiblecompounds in algae. for the synthesis of products of interest (e.g. biosyn- Despite the growing number of novel bioactive thesis of PUFAs).compounds that have been identified in algae, none At this point, recombinant techniques have nothas yet become a commercially useful pharmaceu- contributed directly to a commercial product. How-tical. A possible explanation is that algae have re- ever, with public and government acceptance of re-ceived considerably less attention as a source for combinant and continued progress in developingpharmaceutical screening than other microorgan- methodologies for algal systems, significant contri-isms or higher plants. Because the probability of de- butions could be realized in the near future.veloping a successful pharmaceutical directly corre-lates with the amount of screening that is done, the PRODUCTION TECHNIQUESlack of a commercial product could be due to the Most algal species are obligate phototrophs andrelatively limited amount of screening of algal sam- thus require light for their growth. The requirementples. for light, coupled with the high extinction coeffi- cient of chlorophyll in these organisms, has neces- MOLECULAR BIOLOGY sitated the design and development of novel systems Recently, tremendous advances have been made for large-scale growth. A few algal species are capa-in the tools available to study a variety of algae. ble of heterotrophic growth, and for these organ-Highly sophisticated molecular systems are being isms conventional fermentation technology can beused to dissect biological processes in many cyano- used for large-scale cultivation.bacteria (reviewed in Thiel 1994). The cyanobacte-ria can be readily transformed with autonomously Phototrophic systemsreplicating plasmids, and endogenous genes can be Commercial growth of photosynthetic algae hasdisrupted by homologous recombination. Although been achieved in different ways: (1) open cultivationa number of commercial possibilities have been pro- using natural sunlight, (2) closed cultivation usingposed for recombinant cyanobacteria (Elhai 1994, natural sunlight, and (3) closed cultivation using ar-Vermaas 1996), the potential has yet to be realized. tificial illumination. Each system has advantages andA novel application of recombinant techniques was disadvantages, and the choice of system depends onto transfer the cryIVC gene for producing Bt toxin the degree of parameter control needed to produceto Synechococcus (Murphy and Stevens 1992, Stevens the desired product and on the value of the prod-
  7. 7. MICROALGAL BIOTECHNOLOGY 221uct. A common limitation to all these systems is the tors, and in principle they are similar to convention-need to supply light to the culture, making it advan- al fermentor, the major difference being that theytageous to maximize the surface-to-volume ratio of are driven by light rather than by an organic carbonthe culture. source. These vessels provide the ability to control Many configurations of open cultures using nat- and optimize culture parameters, and, coupled withural sunlight have been proposed and constructed the closure that they provide, photobioreactors are(Oswald 1988, Chaumont 1993, Pushparaj et al. suitable for culturing many different types of algae1997). These systems are generally large, open (Ratchford and Fallowfield 1992). Photobioreactorsponds or raceways, and the principle advantage of are generally more expensive to build than outdoorthese configurations is that the light energy is free. systems, but the cost can be justified, depending onHowever, this advantage is more than offset by sev- the application of the algal biomass. Photobioreac-eral significant disadvantages. In open systems, it is tors have been an important tool for small-scale pro-very difficult to prevent contamination of the algal duction of high-value products, such as stable-iso-culture by other organism (i.e. algae and other mi- tope-labeled biochemicals (see previous discussion).croorganisms). This problem has been addressed byculturing algae that require or tolerate unique Heterotrophic systemsgrowth conditions that would exclude contaminat- The most significant advance in closed culture sys-ing organisms; however, this restricts the usefulness tems is the adaptation of fermentation technologyof these systems to a limited number of organisms. that allows for the heterotrophic growth of microal-Suitable species include Dunaliella, which can be gae and eliminates the problem of light limitationgrown at very high salinity, and Spirulina, which will (Barclay et al. 1994, Kyle 1996, Chen 1997). A sig-grow at high pH. Open cultures attain cell densities nificant number of microalgae are capable of het-leading to the need to process large quantities of erotrophic growth and potentially suitable forwater to harvest the algae. Outdoor phototrophic growth in fermentors (Droop 1974, Gladue andgrowth systems are also subject to daily and seasonal Maxey 1994). Heterotrophic fermentor culturesvariations in light intensity and temperature, mak- have a number of important advantages over cultureing it difficult to control or reproduce specific cul- systems requiring light for photosynthesis. Fermen-ture conditions.. Nevertheless, for specific algal tation technology is preexisting, highly sophisticat-products this technology has proven very successful, ed, and utilized worldwide on a massive scale.producing many thousands of tons of dried biomass The basic principle of fermentor growth is to pro-per year (Lee 1997). This is especially true for Spi- vide highly controlled optimal growth conditions torulina, which is extensively cultured in the U.S., maximize productivity. The culture vessels range inMexico, Thailand, and China (Metting 1996, Li and volume from 1 to 500,000 L and are operated underQi 1997, Vonshak 1997). sterile conditions. A motorized shaft with a series of Several different closed systems using natural sun- impellers provide the mixing. Sterile air is pumpedlight have been described (Richmond et al. 1993, into the system at high pressure and flow rates toQiang and Richmond 1994, Molina Grima et al. ensure proper gas exchange, with continuous mon-1995, Spektorova et al. 1997). In these systems, the itoring and adjustment of dissolved O2 and CO2 lev-algae are enclosed in a transparent material (either els. Heating or cooling coils regulate temperature,glass or plastic) and the vessels placed outdoors for and the automatic addition of acid or base main-illumination. The closure of the vessels minimizes tains pH. The culture medium for algal fermentativecontamination by other algal species. These systems growth is similar to that used for phototrophichave been designed to provide a higher surface-to- growth, except that glucose or a similar carbohy-volume ratio than is possible with the ponds and drate provides both fixed carbon and energy. Otherraceways, so cell densities are often higher than in nutrient levels (i.e. nitrogen and phosphorus) arethe open systems. Closed, outdoor systems are still also continuously monitored and adjusted.subject to variations in light intensity and tempera- As a result of the high level of process control,ture that make cultivation reproducibility problem- culture conditions and biomass yields are consistentatic. In addition, a major problem with closed sys- and reproducible, with algal cell densities reachingtems is the removal of oxygen from the culture and 50 g dry biomass per liter (Gladue and Maxey 1994)the provision of adequate temperature control. Al- to as high as 100 g dry biomass per liter (Runningthough both of these issues can be resolved, the cost et al. 1994). These biomass levels are at least 10-foldof doing so can more than offset the cost advantage higher then those achieved by photosynthesis-basedof using natural sunlight. culture systems (Radmer and Parker 1994). The As with the outdoor systems, numerous designs high biomass levels also greatly decrease the volumehave been constructed for the indoor, closed cul- of water that must be processed during harvesting.ture of algae using electric lights for illumination Because cultures can be routinely run in fermenters(Ratchford and Fallowfield 1992, Wohlgeschaffen et with volumes greater than 100,000 L, several thou-al. 1992, Iqbal et al. 1993, Lee and Palsson 1994). sand kilograms of dried biomass can be producedThese vessels are often referred to as photobioreac- per run. The effectiveness of large-scale cultures and
  8. 8. 222 KIRK E. APT AND PAUL W. BEHRENSthe production of high biomass levels can make the STRAIN MAINTENANCEcost of fermentative growth an order of magnitude Maintenance of organisms is very important forless expensive than photobioreactors (Radmer and the future of biotechnology (Hunter-Cevera andParker 1994). Belt 1996). With an increasing number of applica- The ability to provide complete control over the tions and potential applications for algae, it is criti-culture is also critical for maintaining food industry cal that the organisms be preserved. For those or-standard Good Manufacturing Practices (GMP), as ganisms that form the basis of a product, it is notdesignated by the U.S. Food and Drug Administra- only sufficient that the organism be preserved buttion, which are required for the production of high- also important that the special and unique charac-quality food- or pharmaceutical-grade materials. teristics of that organism be maintained. Thus, Larger-scale production of the dinoflagellate Cryp- strain maintenance is not limited to preservation ofthecodinium by fermentative growth for the produc- the organism; it must also ensure that it is geneti-tion of the polyunsaturated fatty acid DHA has been cally stable.under way for several years (Kyle 1996). Productionof Crypthecodinium begins with a certified seed stock Various methods have been used to preserve al-that was cryopreserved under liquid nitrogen con- gae, including serial transfer, freeze-drying, andditions to maintain genetic stability. A simple cul- cryopreservation (Andersen 1996, Day et al. 1997).ture medium containing NaCl, CaCl, MgSO4, glu- Each method has advantages and disadvantages, andcose, and yeast extract is utilized for all culture sizes. it is possible that no one method is ideal or usableThe cultures are progressively transferred from a for all algal strains. By far, the most widely usedshake flask through a series of scale-up fermenters, method for maintenance is serial transfer (Andersenterminating in a production fermenter of 120,000 L 1996). This method requires no expensive equip-volume. The cultures are continuously monitored, ment and is generally very satisfactory for the main-and when a predetermined cell density and fatty tenance of a small number of noncritical cultures.acid level is reached, the culture is harvested and Good microbial technique can greatly minimizespray-dried. The oil is extracted from the dried bio- problems of contamination, but genetic drift is notmass using procedures similar to those for conven- minimized and might even be increased through se-tional vegetable oil processing, which involve extrac- rial transfer. Freeze-drying is generally considered ation with hexane, refining, bleaching, and deodor- better technique than serial transfer, and the equip-izing. Following blending, it is sold as a pure vege- ment required is minimal. Unfortunately, freeze-dry-table oil containing 20% or 40% DHA for ing is unreliable, often giving survival rates of lessapplications in human nutrition as described previ- than 5% (McGrath et al. 1978), and recent successesously. Crypthecodinium has been reported to produce with cryopreservation has provided even less incen-approximately 30% of their dry weight as total fatty tive to pursue and optimize this technique. Cryo-acid (Kyle et al. 1992), with DHA making up close preservation (maintenance at temperatures colderto 50% of the total fatty acid (Behrens and Kyle than Ϫ120Њ C) offers low maintenance of cultures1996). and the virtual elimination of genetic drift. Of the Schizochytrium is also produced using microbial various preservation methods that are available,fermentation techniques (Barclay et al. 1994) for cryopreservation is generally regarded as the singleuse in the health food market and as an animal feed best method for the long-term preservation of or-supplement (see previous sections). Special strains ganisms and their properties (Andersen 1996).of the organism have been reported that are capable At least some degree of success with cryopreser-of producing 30%–40% of their fatty acids as long- vation techniques has been reported with several dif-chain omega-3 fatty acids. When cultured at low sa- ferent algal groups, including green algae, red al-linities with glucose as the carbon source culture, gae, euglenophytes, diatoms, and cyanobacteria al-densities of 20 g·LϪ1 dry biomass and yields of ap- gae (Morris 1978, Canavate and Lubian 1997, Dayproximately 1.0 g·LϪ1·dayϪ1 can be achieved. New et al. 1997), and with macroalgae as well as microal-strains have recently been isolated that are capable gae (Kuwano et al. 1994, Kono et al. 1997). Beatyof producing Ͼ70% of their biomass as fatty acids, and Parker (1991) attempted cryopreservation on aof which 35% is DHA, with yields under laboratory large number of different genera and found anconditions exceeding 3 g DHA·LϪ1·day (Nakahara overall success rate of almost 80%. Likewise, Morriset al. 1996). (1978) found a very high success rate with a large Chlorella is also extensively grown in large quanti- number of green algae. Canavate and Lubianties by fermentation techniques. Production levels in (1995a, b) have done several detailed studies ofJapan are estimated to exceed 500 T per year, ac- cryopreservation variables with several genera andcounting for 50% of the country’s total production have obtained survival rates as high as 98% with Te-(Lee 1997). Plans have also been announced in Ko- traselmis. Although survival rates vary somewhat be-rea to begin production of heterotrophically grown tween genera, the results to date on cryopreserva-Chlorella at a level exceeding 1000 T per year (Lee tion suggest that this preservation technique should1997). be applicable to many algal genera.
  9. 9. MICROALGAL BIOTECHNOLOGY 223 Several parameters are generally considered very n-6 fatty acids in rotifers and Artemia nauplii by feeding spray-important in cryopreservation, including choice of dried Schizochytrium sp. J. World Aquacult. Soc. 27:314–22. Beaty, M. H. & Parker, B. C. 1991. Investigations of cryo-preser-cryoprotectant, cryoprotectant concentration, freez- vation and storage of eukaryotic algae and protozoa. J. Phycol.ing rate, physiological status of the culture, and 26S: P1.thawing procedure. A wide variety of cryoprotec- Becker, E. W. [Ed.]. 1994. Microalgae: Biotechnology and Microbiol-tants have been tried, including dimethyl sulfoxide ogy. Cambridge Studies in Biotechnology, vol. 10, Cambridge University Press, Cambridge, 239 pp.(DMSO), glycerol, methanol, polyvinylpyrrolidone, Behrens, P. W. & Kyle, D. J. 1996. Microalgae as a source of fattyproline, propylene glycol, ethylene glycol, sorbitol, acids. J. Food Lipids 3:259–72.glucose, sucrose, dextran, and betaine (Canavate Behrens, P. W., Bingham, S. E., Hoeksema, S. D., Cohoon, D. L.and Lubian 1995a, Andersen 1996, Kono et al. & Cox, J. C. 1989a. Studies on the incorporation of CO2 into starch by Chlorella vulgaris. J. Appl. Phycol. 1:123–30.1997). Glycerol, DMSO, and methanol are the most Behrens, P. W., Hoeksema, S. D., Arnett, K. L., Cole, M. S., Heub-widely used cryoprotectants, and each has been ner, T. A., Rutten, J. M. & Kyle, D. J. 1989b. Eicosapentaenoicshown to give good success rates (Beaty and Parker acid from microaglae. In Demain, A. L., Somkuti, G. A.,1991, Canavate and Lubian 1995b). The successes Hunter-Cevera, J. C. & Rossmoore, H. W. [Eds.]. Novel Micro- bial Products for Medicine and Agriculture. Society of Industrialand failures experienced by different workers could Microbiology, pp. 253–9.likely be due to other differences in their protocols Behrens, P. W., Piechocki, J. A., Purdon, P. A. & Delente, J. J.or the physiological state of the algae used. Various 1996. Microalgal production of 13C-galactose and its use as afreezing rates have also been tried. Generally, un- measure of liver function. J. Phycol. (Suppl.)32: 6. Behrens, P. W., Sicotte, V. J. & Delente, J. J. 1994. Microalgae ascontrolled freezing gives poor results, and it is nec- a source of stable isotopically labeled compounds. J. Appl.essary to have equipment to control the freezing Phycol. 6:113–22.rate. Successful cryopreservation has been demon- Benemann, J. R. 1992. Microalgae aquaculture feeds. J. Appl. Phy-strated with freezing rates ranging from 0.5Њ C to col. 4:233–45. Bertini, I., Couture, M. M., Donaire, A., Eltis, L. D., Felli, I. C.,16Њ C per minute (Canavate and Lubian 1995b, Day Luchinat, C., Piccioli, M. & Rosato, A. 1996. The solutionet al. 1997), and some organisms appear to revive structure refinement of the paramagnetic reduced high-po-better with a slow thawing rate, although most pre- tential iron-sulfur protein I from Ectothiorhodospira halophilafer rapid thawing (Canavate and Lubian 1997). by using stable isotope labeling and nuclear relaxation. Eur.Higher revival rates were found when agar-grown J. Biochem. 241:440–52. Borowitzka, M. A. 1992. Algal biotechnology products and pro-rather than liquid-grown algae were used (Beaty and cesses-matching science and economics. J. Appl. Phycol. 4:267–Parker 1991), and perhaps the osmotic stress of 79.growth on agar might precondition the cells to bet- 1997. Microalgae for aquaculture: opportunities and con-ter withstand cryopreservation. The results of these straints. J. Appl. Phycol. 9:393–401. Borowitzka, M. A. & Borowitzka, L. J. [Eds.]. 1988. Micro-Algalstudies can now serve as a basis for increasing the Biotechnology. 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