Full Text

In Vitro Cell. Dev. Biol.--Plant 35:127-136, March-April 1999 © 1999 Society for In Vitro Biology 1054-5476/99 $05.00 + 0.00

IN VITRO CULTURE AND CONSERVATION OF MICROALGAE: APPLICATIONS FOR AQUACULTURE, BIOTECHNOLOGY AND ENVIRONMENTAL RESEARCH

JOHN G. DAY, ERICA E. BENSON, 1 AND ROLAND A. FLECK 2

Culture Collection of Algae and Protozoa, NERC Institute of Freshwater Ecology, Windermere Laboratory, Far Sawrey, Ambleside, Cumbria, LA22 OLP, UK (J. G. D., R. A. E ), and Plant Conservation Biotechnology Group, Division of Molecular and Life Sciences,

School of Science and Engineering, University of Abertay Dundee, Bell Street, Dundee, DD1 1HG, Scotland, UK (E. E. B.)

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

SUMMARY

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

Key words: algae; microalgae; aquaculture; cuhure collections; environment; cryopreservation.

INTRODUCTION

The algae comprise one of the most diverse plant groups. A species range of 40 000 to 10 million has been estimated, with the majority being the microalgae (Hawksworth and Mound, 1991; Metting, 1996). Algae are represented by both eukaryotic and prokaryotic forms, ranging in size from giant kelps to the unicellular microalgae (a group of protists which are only a few microns in size) and the prokaryotic cyanobacteria (blue-green algae). They can be distin- guished from higher plants in terms of their reproductive characteristics. Unicellular algal cells function as gametes, whereas the mul- ticellular forms produce reproductive structures which are either special unicellular gametangia or which are muhicellular, with each cell being fertile. This contrasts with vascular plants, which bear reproductive structures containing both asexual and sexual ceils. Eu- karyotic algae have been classified into three groups: the Rhodophy- tes, Chromophytes, and the Chlorophytes, and there is considerable debate regarding their subclassification, (Cavalier-Smith, 1993; An- dersen, 1996). Molecular biology will assist to clarify future taxonomic classification.

~To whom correspondence should be addressed. 2present address: Department of Soil, Crop and Atmospheric Sciences,

Cornell University, Ithaca, New York 14853.

Algae are one of the earth's most important natural resources. They contribute to approximately 50% of global photosynthetic activity (Wiessner et al., 1995) and form the basis of the food chain for over 70% of the world's biomass (Andersen, 1996). A diverse group of photosynthetic organisms, the algae have successfully adapted their metabolism to occupy different habitat extremes ranging from the polar regions to tropical coral reefs. The ability to withstand environmental stress is matched by the capacity of algae to produce a vast array of secondary metabolites, which are of considerable value in biotechnology programs including the aquaculture, health, and food industries (Andersen, 1996). As algae have a unique status in terms of their environmental importance and their phytochemical production capabilities are considerable, it is imperative that algal diversity is protected with complementary ex situ and in situ conservation methods. In this respect, in vitro techniques have a key role.

The objective of this review is to evaluate the application of in vitro technologies for the maintenance, conservation, and sustainable exploitation of microalgae. The first part of the review discusses in vitro culture and maintenance methods and the second part explores the value of algal culture techniques in environmental research, aquaculture, and the biotechnology industries. The review will con-

127

128 DAY ET AL.

clude by offering some future perspectives on the importance of applied and fundamental microalgal research.

IN VITRO CULTURE AND MAINTENANCE OF MICROALGAE

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

In Vitro Culture

(a) UNSTIRRED OPEN POND (b) CIRCULAR POND

(c) PADDLE-WHEEL RACEWAY (d) SLOPING CASCADE

0 0 0 0 0 0 0 0 0 0 0

(e) TUBULAR REACTOR (f) TUBULAR REACTOR (g) TUBULAR REACTOR (helix) (plane) (two layers)

Under natural conditions, most algae grow as mixed communities, which include various species and genera of algae and other microorganisms. On isolation, an algal strain requires a suitable environment for its growth, There is an extensive literature on culture techniques and maintenance conditions (e.g., Stein, 1973; McLellan et al., 1991; Becker, 1994). Medium composition depends both on the requirements of the algae (e.g., diatoms require the inclusion of a silica source: many marine algae require vitamins) and the prefer- ences of the researcher. Information on medium suitability and full recipes are listed in the catalogues of all the major protistan cuhure collections (see Nerad, 1993; Starr and Zeikus, 1993; Schl~sser, 1994; Tompkins et al., 1995; Andersen et al., 1997; Watanabe and Hiroki, 1997). In general, master stock-cultures maintained by routine serial subcuhure are grown under suboptimal temperature and light regimes [<20 ° C and <50 p~mol photon m 2 s 1 on shorter than normal daylight (12 h or less light : 12 h or more dark)], This type of regime maximizes the interval between subcultures and thus minimizes handling and transfers. Additionally, the use of medium containing organic carbon for maintaining axenic strains capable of heterotrophic or mixotrophic growth and solidified rather than liquid medium may be used to maximize the period between transfers to fresh medium.

For many applications, relatively small quantities of algae are required and scaling up may entail the use of a larger glass vessel either with or without additional aeration to reduce carbon limitation and keep the ceils in suspension. However, light limitation will re- strict the growth rate and productivity in such systems and may influence the formation of any desired product. A variety of options are

(h) LAMINAR REACTOR (i) HANGING SLEEVE

FIG. 1. Typical examples of different algal mass culture systems used in the aquaculture and biotechnology industries.

available for scaling up, with the choice of production method largely depending on the amount of biomass required and value of any product. Systems used may be divided into three categories: open systems, closed photobioreactors, and closed fermenters (heterotrophic culture).

Open systems include the use of managed lakes which may be up to 300 hectares (Schlipalius, 1991), unstirred open ponds like the ~-carotene production plant at Hutt Lagoon in Western Australia (Borowitzka, 1991), circular ponds, paddle-wheel raceways, and sloping cascades (Oswald, 1988; Becker, 1994). All of these generally have the advantage of being relatively cheap to construct. How- ever, problems associated with excessive shear forces damaging the algal ceils and the need for environmental control including pH, temperature, nutrient levels, osmotic potential, contamination by other algae, and grazing have restricted their use.

A large number of closed photobioreactor systems have been developed (Fig, 1) and these avoid some of the problems connected with open system use, most notably better environmental control and fewer problems associated with contamination and grazing. The least complex and probably most widely used is the Hanging Sleeve. These are commonly used in aquaculture for the production of food for

IN VITRO CULTURE OF MICROALGAE 129

shrimp and mollusc larvae (McLellan et al., 1991) and also for polysaccharide production from Porphyridium (Becker, 1994). Other types of bioreactors include tubular type reactors (Gudin and Chau- mont, 1983; Torzillo et al., 1986), laminar types (Tredici et al., 1991) and fermenter type reactors (Pohl et al., 1986). Fermenter type reactors offer the greatest degree of control and may be fitted with light- diffusing optical-fiber systems to avoid the necessity of external il- lumination and the problems associated with light limitation (Takano et al., 1992).

The final type of culture system utilizes the capability of some algae to take up and metabolize fixed carbon, i.e., to grow heterotrophically. Although this restricts the range of algae that may be grown and their products, the system has been successfully used to produce c~-tocopherol (Ogbonna et al., 1998), ascorbic acid (Running et al., 1994), aquaculture organisms (Day and Tsavalos, 1996), fatty acids (Barclay et al., 1994), and leutin (Shi et al., 1997).

Cryopreservation

Cryopreservation is the maintenance of living cells, tissues, organs and microorganisms at uhralow temperatures (in general at - 196 ° C) and liquid nitrogen is used as the refrigerant. As long as liquid nitrogen levels are maintained, living cells can be maintained in cryogenic storage indefinitely. At liquid nitrogen temperatures all metabolic activity ceases and the ceils do not undergo the genetic changes which can occur when they are maintained by serial sub- culturing. Furthermore, cryopreserved cells are not continuously ex- posed to the risks of contamination and operator error as would be the case during culture maintenance. Thus, cryopreservation is the method of choice for the long-term conservation of in vitro culture collections of microalgae. Investigations of cryogenic storage procedures for algal cultures (Bodas et al., 1995) have been performed and a number of robust protocols are now available for a range of species (Morris, 1978; Day and DeVille, 1995: Day, 1998); however, a large number of macro- and microalgae still remain recalcitrant to cryopreservation. This is largely due to the fact that they comprise a diverse group of organisms which display many different physiological and morphological characteristics, and this makes the development of a universally applicable cryopreservation method most difficult. Successful cryopreservation depends upon the application of a cryoprotective strategy and the manipulation of freezing, thawing, and recovery conditions. Differences between procedures can usually be attributed to the use of alternative cryoprotection strategies. Algae can be sensitive to the chemical cryoprotectants which are commonly applied to higher plants, and cryoprotectant toxicity trials are an essential prerequisite before researchers embark on developing a cryopreservation protocol for microalgae. Canavate and Lubian (1994), assessed the toxicity of dimethyl sulfoxide (DMSO) and methanol on six taxonomically diverse marine microalgae and found all to be sensitive to both cryoprotectants, and sensitivity in some cases was concentration-dependent. The traditional approach to cryopre- serving microalgae involves the application of chemical cryoprotectants such as DMSO or methanol before freezing, followed by expos- ing the cells to a controlled temperature gradient (e.g., - 0.5 to - 10 ° C min-1), then to a terminal transfer temperature (e.g., - 30 to - 60 ° C) after which the cells are plunged into liquid nitrogen (Morris, 1976; Day and McLellan, 1995b). This procedure is termed "two- step" freezing and the majority of microalgae that have been placed in cryogenic storage have been cryopreserved by this technique. Suc-

cessful applications of two-step freezing to algae which have previously proved difficult to freeze include Dunaliella tertiolecta (Hirata et al., 1996); Laminaria digitata (Vigneron et al., 1997); Chaetoceros gracilis, Tetraselmis chui, Nannochloris atomus, Nannochoropsis gaditana, Rhodominas baltica, and Isochyris galbana (Canavate and Lu- bian, 1995a, 1995b) and Cylidrocystis brebissonii (Morris et al., 1986).

During the last few years, new approaches to cryopreservation have been developed for higher plants (for reviews see Benson, 1994, 1999) and now these are being successfully applied to macro- and microalgae, some of which have previously proved recalcitrant to freezing with traditional two-step methods (Fleck, 1998). More recent cryopreservation methodology is based on the cryoprotective strategy of vitrification. This is a process in which the cells are manipulated such that, on exposure to liquid nitrogen, the water molecules form an amorphous glass which lacks crystalline structure. Under these conditions, ice is not formed and its damaging effects are thus avoided. Glasses are, however, unstable and it is important that the cryopreserved cells are rewarmed carefully to avoid destabilization of the glass, as water can nucleate and form ice as it is warmed to the glass transition temperature and beyond. Vitrification can be in- duced in cryopreserved systems by either reducing the moisture content (e.g., via osmotic dehydration, silica gel desiccation, and/or air drying) of the ceils and tissues or by the application of high concentrations of chemical cryoprotectants (e.g., plant vitrification solution number 2, "PVS2", Yamada and Sakai, 1996). Both of these strategies increase cellular viscosity to a critical level at which ice formation is inhibited on exposure to uhralow temperatures. One novel application of vitrification involves encapsulating cells and tissues in a calcium alginate matrix (Fabre and Dereuddre, 1990) and ex- posing the encapsulated cells to osmotic dehydration (usually by the application of highly concentrated sucrose solutions) followed by air or silica gel desiccation. This approach has been applied to algal species including Dunaliella tertiolecta (Hirata et al,, 1996), Euglena gracilis (Fleck, 1998) and Laminaria digitata (Vigneron et al., 1997).

Future cryoconservation research involving algae must evaluate the basis of freeze sensitivity in microalgae. Understanding both the biochemical and physical effects of freezing will be especially important, and recent studies have included the characterization of freeze recalcitrance in the coenocytic alga Vaucheria sessilis with cryomicroscopy (Fleck et al., 1997) and the role of oxidative stress and antioxidant protection in Euglena gracilis and Haematococcus pluvialis (Benson et al., 1998; Fleck, 1998).

Role of Algal Genetic Resource Centers and Culture Collections

In recent years, the trend has not been towards conservation per se, but rather towards sustainable utilization and exploitation for national benefit (Hawksworth and Ritchie, 1993; Hawksworth, 1996). Culture collections satisfy a multitude of purposes in any conservation and sustainable exploitation strategy, In addition, the collection and preservation of living specimens is essential for the elucidation of an organism's life history through systematic and taxonomic investigation. Conserved specimens allow the effects of human demands and climatic changes on biodiversity to be studied through the maintenance of accurate and verifiable taxonomic resources (Roper, 1993; Cotterill, 1995).

The primary role of an algal culture collection is the same as any other collection of living material, that is, to be a repository for cul-

130 DAY ET AL.

TABLE 1 TABLE 2

DISTRIBUTION OF ALGAL CULTURE COLLECTIONS

Use No. of Collection Internet Patent Cryopres-

Continent collections" acronym b Country Web site depository ~ ervation e

Africa 3 Asia 36 NIES Japan - - + Australia (and NZ) 12 Europe 27 A S I B Austria - - +

CCAP UK + + + SAG Germany - - - CCMP USA + - + UTEX USA + - +

N. America 27

S. America 2

"No. of algal culture collections on the continent. bCollection holding > 1000 algal strains. ~Under the terms of the 1977 Budapest treaty. dUse cryopreservation to maintain some of their holdings (Data from An-

derson, 1996; WDCM, (World Data Centre on Microorganisms), 1994; Ma et al., 1995; and this paper).

Collection acronyms are NIES (National Institute of Environmental Stud- ies), ASIB (Algensammlung am Institut ftir Botanick, University of Inns- bruck), SAG (Sammlung yon Algenkulturen), CCMP (Provasoli Guillard Na- tional Centre for Marine Phyloplankton), CCAP (Culture Collection of Algae and Protozoa), and UTEX (Culture Collection of Algae at the University of Texas).

MICROALGAL USE IN AQUACULTURE

Collection Species strain no." Molluscs Crustaceans Rotifers

Chaetoceros calcitrans CCAP 1010/5 + + CCMP 1315

Chaetoceros muelleri CCMP 1316 + Chaetoceros neogracile CCMP 1010/5 + lsochrysis galbana CCAP 927/1 + +

CCMP 1323 Nanochlorophsis oculata CCAP 849/1 + Nanochloropsis gaditana CCAP 849/5 + Nannochloris atomus CCAP 251/4A + Pavlova lutheri CCAP 931/1 + +

CCMP 1325 Rhinomonas reticulata CCAP 978/28 + Rhodomonas salina CCMP 1319 + Skelatonema costatum CCAP 1077/5 + + Tetraselmis chui CCAP 8/6 + + Tetraselmis suecica CCAP 66/4 + + Thalassiosira pseudonoana CCAP 1085/3 + +

CCMP 1335 Thalassiosira weissflogii CCMP 1336 + +

°Acronyms for strain cultures are Culture Collection of Algae and Protozoa (CCAP) and Provasoli-Guillard National Centre for Marine Phyloplankton (CCMP). For details of CCAP strains, see Tompkins et aI. (1995), and for CCMP strains, see Andersen et al. (1997).

tures. In service collections, this role is often associated with other products and services including provision of authentic specimens for research, education, training, bioassay use, identification, and use as aquaculture starter cultures. Importantly culture collections act as depositories for patent purposes, consultancy, and other commercial applications. All of these require the maintenance of viable, healthy, physiologically and genetically stable cultures. There are more than 11 000 strains of algae including representatives of approximately 1600 different species retained in the algal culture collections reg- istered with the World Data Centre on Microorganism's (WCDM) database (Miyachi et al., 1989; WDCM, 1994). However, it is worth noting that many of these collections hold very few algae (<50 strains) and that more than 80% of the algal strains listed are maintained in the six largest algal culture collections (Table 1).

APPLICATIONS OF IN VITRO CULTURED MICROALGAE

In vitro technologies have a central role in applied microalgae research and Culture Collections are particularly important for the exploitation of algae and their products. Similarly, in vitro cultures support environmental monitoring programs, allowing time-related studies of pollution and climate change. Comparative studies of conserved cultures held in repositories and natural algal populations provide a means of conducting environmental impact assessments. Culture collections assist taxonomic studies as they can be used to verify evolutionary relationships and genetic change. This section will evaluate the role of in vitro technologies in applied microalgal research.

Aquaculture

From an economic perspective, aquaculture is the most important applied use of algae. Products from macroalgae which have signifi-

cant financial value include Porphyra (Nori) harvested in Japan and worth US$1 billion annually (Mumford and Miura, 1988) and algal polysaccharides, primarily agars and carrageenans, with an annual value of approximately US$500 million (Jensen, 1993). However, this section focuses primarily on the use of microalgae as food in aquaculture.

Algae are the biological "starting point" or primary producers for energy flow through aquatic food chains, it is therefore not surprising that microalgae are so important in the aquaculture industries. The choice of algal strains for aquaculture use is constrained by a number of factors as outlined below. The first is the toxicity of the alga, as many strains produce potent toxins (Addison and Stewart, 1989; Shumway, 1989) and these are clearly unsuitable. Nutritional profile is a key factor, with levels of vitamins, proteins, and unsaturated fatty acids being of particular importance (De Pauw and Persoone, 1988; Cahu et al., 1994; Borowitzka, 1997). Size and palatability also influence choice; large particles may be too big for ingestion and species with thick cell walls, including Chlorella, may be indigestible. The final criterion is that the selected algae should be relatively robust and easy to culture. In in vitro culture systems, the range of species used is much less than in natural environments; however, these strains and a few others are the basis of a muhimillion dollar industry.

Microalgae are the primary food for larvae and spat of bivalve molluscs and penaeid prawn (shrimp) larvae, and they are live food for rotifers which in turn are used to feed the larvae of marine finfish and crustaceans (Table 2). The production of microalgae in aquaculture is commonly considered to be the major constraint on productivity and expansion of a farm. Furthermore, it is the most ex- pensive part of the process, accounting for 30M,0% of hatchery costs (Borowitzka, 1997). The actual cost of producing algae varies from


site to site but may be in the range US$50-1000 kg -1 (Fulks and Main, 1991; Benemann, 1992).

Production systems vary from farm to farm but can be categorized as extensive, semi-intensive, or intensive systems. The first category depends on natural phytoplankton, with algae and consumers coex- isting in large volume (>1000 m 3) lakes, lagoons, ponds, or in the sea. Semi-intensive systems involve the addition of fertilizer to enrich the water, inducing a natural bloom of algae in systems with slightly smaller volume (>100 mS). The algae and consumers may coexist, or alternatively, the "bloom" may be transferred to tanks containing the consumers. Although both of these approaches are relatively cheap, they have the disadvantage of being relatively unproductive and are uncontrolled or uncontrollable with respect to production, species control, and the growth of interfering consumers. Further- more, in areas which are prone to toxic blooms, the production of algal toxins could result in total loss of the farmed animals.

Intensive systems involve the use of in vitro culture to provide a more managed and controllable supply of algae. These are far more regulated, as unialgal cultures are grown in batch or continuous culture systems separate from the consumer. They also have the advantage of producing a more reliable and biochemically consistent supply of algae, and problems associated with pathogens, toxic algae, and undesired grazing organisms are avoided.

In hatcheries, use of artificially illuminated bag or tank culture vessels (up to 500 L) is standard. A variety of algae are normally cultured in separate vessels and an algal mix is fed to the consumers (normally molluscs or rotifers). However, algal productivity is low and these systems may be unreliable, thus requiring very careful management (Sato, 1991). To overcome the problems intrinsic to this approach, closed photobioreactor systems are used. One system, a helical tubular photobioreactor developed by Biotechna Ltd. (Rob- inson et al., 1988) has been extensively tested for the production of aquaculture strains in the UK and Australia (Borowitzka, 1996, 1997). With this type of culture system, environmental conditions (temperature, pH, light, nutrients) may be optimized and the biochemical composition of the algae manipulated to improve their nutritional value (Chrismadha and Borowitzka, 1994). These bioreac- tots may be automated and run continuously, thus reducing labor costs and other costs.

As aquaculture has developed and expanded, there has been a significant increase in both the number and size of the farms. This has resulted in a demand for removing or minimizing bottlenecks associated with culturing algae and the insufficient supply of algae at key times. Improvements in algal culture systems, particularly the more widespread use of photobioreactors, will continue to reduce supply problems. Alternative approaches that have been taken with varying degrees of success include the use of encapsulated nonalgal food particles (Jones et al., 1987), and sun-dried algae (Millamena et al., 1990), the heterotrophic production of algae to produce a spray-dried powder (Day et al., 1991), and the storage and distribution of algal pastes (Watson et al,, 1986; Brown, 1995). It seems probable that an algal product that could be stored would find widespread application. However, to date, no alternative to a mix of live algal species has been found to be fully satisfactory.

Microalgae can also be used to pigment fish and shrimps, as farmed animals which lack the ability to synthesize carotenoids can be supplied with algal pigment supplements in their feeds. This is usually at considerable expense to the farmer and may contribute 10 to 15% of total feed costs (Johnson and An, 1991). Supplementing

TABLE 3

MICROALGAL PRODUCTS

Alga Use/product Reference

Botryococcus braunii Fuel oil Murray and Thompson, 1977 Chlorella Stable isotope biochemicals Behrens et al,, 1989 Chlorella Ascorbic acid Running et al., 1994 Chlorella Leutin Shi et al., 1997 Euglena gracilis a-tocopherol Ogbonna et al., 1998 Haematococcus spp. Astaxanthin Johnson and An, 1991 Porphyridium Polysaccharides Becker, 1994 Spirulina spp. Phycobiliproteins Glazer. 1994 Dunaliella spp, [3-carotene Ben-Amotz and Avron, 1980 Various Oraega-3 fatty acids Barclay et al., 1994 Various Anticancer activity Gerwiek et al., 1994 Various Antimicrobial activity Patterson et al., 1994 Various Hydrogen Benemann, 1990 Various Vitamins Baker et al., 1981

salmonid diets with astaxanthin derived from the freshwater mi- croalga Haematococcus pluvialis or synthetic pigments results in significant deposition of carotenoids including astaxanthin, visually en- hancing flesh coloration of the salmonids (Sommer et al., 1991). Other algae can also be used for the pigmentation of fish and crustaceans. Spirulina has been used to pigment shrimp (Liao et al., 1993) and Dunaliella has been used to pigment other crustaceans (Sommer et al., 1991). Spirulina is also a common feed component for ornamental fish such as Koi carp, as it enhances pigmentation (Borowitzka, 1997).

Algal Biotechnology

Microalgae are extremely efficient solar energy converters and they can produce a great variety of metabolites. This capacity and their ubiquitous distribution have led to their exploitation by man for a diverse range of purposes. This review can only touch on a few applications and fuller information can be obtained from other sources (Borowitzka and Borowitzka, 1988; Boussiba et al., 1992; Wilde and Benemann, 1993; Johnson and Schroeder, 1995; Borowitzka, 1996; Dixon et al., 1997; Duncan et al., 1997; Renn, 1997; Vilchez et al., 1997) which appraise methods for the cultivation of microalgae and the potential commercial applications of microalgal biotechnology. However, the following sections present the main biotechnological applications of microalgae.

Useful Metabolites From Algae

The range of metabolites produced from algae is extremely extensive with many having significant commercial value. In most cases, with the exception of extremophile algae including Dunaliella and Spirulina, photobioreactors or other closed systems are likely to form the basis of any economically viable culture system. Some products are listed in Table 3; most are of commercial significance or have been developed to pilot-plant scale processing.

Biomass and Health Food Production

Approximately 500 species of algae, including macroalgae, are used as human food or food products, and about 160 species are considered commercially valuable (Abbott, 1988). From a practical

132 DAY ET AL.

point of view, growing of biomass is the most obvious and easiest approach to producing algal foods. Mostly these are now produced for the health food market and the organisms grown tend to be those which grow in extreme environments, e.g., Spirulina and Dunaliella, or those with fast growth rates, e.g., Chlorella and Scenedesmus. The largest market is in Japan and the Far East, but in increasingly health-conscious Europe and the USA, there is a niche market in health food shops. The production processes for all of the commercial production processes are photoautotrophic, with the exception of some Chlorella production systems which are mixotrophic or which utilize heterotrophically cultivated algae as an inoculum for the production phase (Endo et ah, 1977).

Green Fertilizers

Cyanobacteria including Scytonema, Nostoc, and Anabaena commonly form the basis of "green" fertilizers (Venkataraman, 1986). The largest usage of cyanobacterial fertilizers is in India where two million hectares were fertilized in 1979 (Roger and Kalasooriya, 1980). The system used involves the central produetion of a mixed inoculum of cyanobacteria which are then air dried and mixed with soil. This material forms dried flakes which are then distributed and used as an inoeulum which is grown in 40-m z ponds. This inoculum in turn is used in the cultivated rice fields. An alternative approach is to immobilize eyanobacteria in polyurethane foam matrices and then add these to the rice fields (Kannaiyan et al., 1997). Develop- ment of algal biofertilizers for use in temperate environments has been investigated (Metting, I981), and commercial products have been developed including an agricultural fertilizer produced by Soil Technologies and a lawn and garden fertilizer produced by the Cy- anoteeh Corporation (Day and Turner, 1992).

Ecotoxicity Testing

All new chemical products likely to be released into the environment must undergo standard algal growth inhibition toxicity tests which have been internationally agreed upon (OECD, 1984). Cul- tures of microalgae used in these tests are maintained in the major culture collections [Culture Collection of Algae and Protozoa (CCAP) Sammlung von Algenkulturen, (SAG); Provasoli-Guillard National Centre for Marine Phytoplankton, (CCMP); Culture Collection of Al- gae at the University of Texas (UTEX), and the American Type Cul- ture Collection (ATCC)] and include the following strains:

Chlorella vulgaris CCAP 211/llB SAG 211-11B UTEX 259 Chlorella vulgaris CCAP 211/12 SAG 211-12 UTEX 30 ATCC 16487 Selenastrum capricornutum CCAP 284/4 UTEX 1648 ATCC 22662 Scenedesmu~ subspicatus CCAP 276/22 SAG 86.81 UTEX 2594 Phaeodactylum tricornutum CCAP 1052/1A Skeletonema costatum CCAP 1077/3 Skelatonema costatum CCAP 1077/5 CCMP 1332

In addition, a number of other organisms are routinely used as bioassay organisms including Ochromonas danica for the bioassay of biotin (Baker et al., 1962); Euglena gracilis for the bioassay of vitamin B12 (Parker, 1977), and Trentepohlia aurea for paint biocide testing.

Pollution Control

Microalgae are a key component in many pollution control systems, particularly those that use ponds or impounds. These can be

divided into two categories: facultative ponds, which are over 1 m deep and have algae restricted to the surface waters and high rate oxidation ponds (HROP) which are generally shallow and mechani- cally agitated. In the case of HROP, the algae are important as not only do they provide 02 which is used by the oxidative bacteria present, they are also predominantly facultative heterotrophs including Euglena, Chlorella, and Scenedesmus (Abeliovich, 1980, 1986), with up to 50% of their carbon assimilation by direct heterotrophic nutrition (Abeliovich, 1978). The yields of algae in these systems can be relatively high at 1-2 g 1 1 (Abeliovich, 1980). This has led to the development of a number of pilot-scale processes which com- bine waste treatment with the production of algal biomass for animal feed (Fallowfield and Garrett, 1985; Pouliot and De la Noue, 1985).

Algae also perform a number of secondary functions in wastewater treatment, which include disinfection of the effluent. They increase the water temperature by converting light to heat, thus increasing the death rate of enteric bacteria (Pharhad, 1970), and metabolize bi- carbonate, increasing the pH. They thereby induce flocculation, ef- fectively increasing the sedimentation rate of the effluent being treated. Algae also have negatively charged cell walls, which in con- junction with the pH, removes heavy metals from the effluent (Oswald et ah, 1957; Becker, 1983). This phenomenon has been used to develop filters that can remove heavy metal ions and refractory organic compounds from industrial effluents (Wilson et al., 1991).

Environmental Research

In evolutionary terms, the algae have existed for 3.8 billion years (Bold and Wynne, 1985). They are considered to be one of the first group of organisms to colonize the earth and have a broad habitat range. The role of algae in maintaining the stability of the earth's ecosystems is considerable, and this can be largely attributed to the major contribution that they make to photosynthesis and supporting the food chains of the majority of the world's biomass (Andersen, 1996). However, one of the most significant, contemporary applications of algal culture collections is in impact assessment and the monitoring of environmental change. The importance of microalgae in these studies is considerable and their use must be underpinned by the application of validated cultures which can be used to assist taxonomic identification and the study of genetic drift. For example, microalgae are used in paleolimnology, the study of cumulative changes in the remains of plants and animals which are deposited as time-related layers in soils and sediments. Diatoms are particularly important, as the silicon components of these microalgae provide almost permanent markers of the changes that have occurred in diatom populations. By analyzing the remains of diatoms sampled from sedimentary cores of terrestrial and aquatic environments, it is possible to gain an understanding of the history associated with pollution events and climatic change (Haworth et al., 1996).

In vitro cultures of microalgae are also important in the study of microbial ecology, as these organisms can be used to assist studies of pollution in industrial, domestic, urban, and agricultural environments. Assessments of microbial diversity are particularly important in environmental impact assessments, and the microalgae are sensitive indicators of ecological changes (Kelley et al., 1998). Climatic change and its impact on natural ecosystems is now a major global issue and the algae, (especially the microalgae) make a significant contribution to the earth's environmental stability through biomass production, food chains, and the cycling of photosynthetic gases. It


is thus imperative that ecological studies are supported by ex situ conservation programs which ensure the safe storage and maintenance of algal genetic resources which can safeguard against species loss as well as providing validated organisms for study. Current research on climatic change can be largely related to the effects of light and temperature. High light, oxygen, and nutrients are essential for the support of planktonic algae; thus, climate changes which influence light quality (e.g., turbidity or UV) and temperature will influence the population dynamics of phytoplankton. Reduction in the stratospheric ozone layers has resulted in an increase in UV-B which has harmful effects on ecosystems, and phytoplankton are especially vulnerable (Maberly and Pettitt, 1997). Algal populations are also sensitive to changes in nutrient status, for example, as is the case with phosphate enrichment (Gibson et al., 1996). An important future role of microalgae and protozoa culture collections will be in risk assessment studies related to the release of genetically modified organisms. Future activities will thus include monitoring the fate of pathogenic and genetically engineered microorganisms in the aquatic environment.

Novel Applications of In Vitro Technologies to Microalgal Research

Cuhure collections provide essential resources for laboratory- based experimental research involving microalgae, and in vitro technologies will continue to be essential to the aquaculture and biotechnology industries. Future applications of in vitro technologies to microalgae research will involve two main areas: (1) their value as a biological resource of useful products and (2) their role in ecological research.

There is a great deal of further potential for algal biotechnology. Product areas that will undoubtedly continue to be developed include lipids (specifically unsaturated fatty acids), pigment production, and bioactive compounds. In addition, significant investment is being made to develop algal technologies to remove carbon dioxide and other pollutants from effluent gases, particularly in Japan under the RITE program (Karube, pers. comm.). However, the key to economic success will be improved productivity. This will require development of both photobioreactors and conventional fermentation plant in addition to other aspects of process and product optimization. An important aspect of this which has not been extensively investigated as yet is the selection and development of overproducing strains, either by genetic manipulation, conventional mutagenesis, or direct selection techniques. In addition, modern molecular approaches may be used to increase the range of products. A current application of this is to clone the genes for the insecticidal Bacillus thuringiensis toxin into the eyanobacterium Synechococcus (Stevens et al., 1994).

Global climate change is undoubtedly one of the most important environmental problems of the future, and the study of microalgae in their natural habitats will provide an important means of assessing the long-term impact of pollution. A good example of how fundamental, generic research can be integrated into applied environmental science is in the use of remote sensing techniques to identify different taxonomic groups of phytoplankton in aquatic environments (George and Taylor, 1995). Such an approach can greatly assist water quality testing, and for example, could aid the study of toxic algal blooms. Remote sensing studies are based upon the fact that spectral differences can be observed and detected in algae which have different photosynthetic pigments. However, spectral signatures of dif-

ferent freshwater plankton species must first be recorded. It is important to note that these spectra were initially derived and calibrated with pure in vitro cultures of algae obtained from the Culture Col- lection of Algae and Protozoa (Tompkins et al., 1995).

The ability of microalgae to survive in many different, and fre- quently extreme environments can be attributed to their complex life cycles and metabolic strategies, therefore, studies of algal production characteristics are especially important. For example, iron is a key limiting factor in phytoplankton found in high nutrient-low chloro- phyll regions, and Fe amendment of these communities offers the potential of relieving the nutrient constraints imposed on phytoplankton photosynthetic efficiency and biomass accumulation. Novel in vitro methods are becoming increasingly important in these studies. McKay et al. (1997) have used trace metal-buffered culture techniques to explore the potential use of flavodoxin as an in situ biochemical marker for Fe limitation in marine diatoms. A further application of in vitro technologies is in the increasingly important field of bioremediation in which the capacity of plants and animals to metabolize and detoxify xenobiotics is being explored in vitro. Such studies will allow the development of specific algal systems which can then be used in mitigating pollution episodes. Cytochrome P450 monooxygenases for fatty acids and xenobiotics have recently been found in three families of macroalgae (Pflugmacher and Sandei~an, 1998), and Cytochrome P450 has been detected in Euglena gracilis (Briand et al., 1993) and a range of unicellular green algae (Thies et al., 1996). The capacity to use marine macroalgae to clean up off- shore pollution and freshwater microalgae to detoxify polluted in- shore sites could provide promising new approaches to solving aquatic pollution problems.

In conclusion, in vitro technologies have made major contributions to the study and exploitation of microalgae. Expanding our current knowledge of basic algal physiology with strains derived from culture collections can greatly enhance our capacity to exploit microalgae not only as a bioresource but also as an investigative tool for environmental monitoring. In the future, fundamental and applied research targeted at improving culture and cryopreservation methods will be particularly important if the full economic potential of microalgae is to be realized. Similarly. as microalgae have such a unique ecological status, in vitro methods must continue to support environmental and algal diversity research.

REFERENCES

Abbott, I. A. Food and food products from seaweeds. In: Lembi, C. A.; Waa- land, J. R., ed. Algae and human affairs. Cambridge: Cambridge Uni- versity Press; 1988:135-147.

Abeliovich, A.; Weisman, D. The role of heterotrophic nutrition in the growth of the alga Scenedesmus obliquous in high rate oxidation ponds. Appl. Environ, Microbiol. 35:32-36; 1978.

Abeliovich, A. Factors limiting algal growth in high rate oxidation ponds. In: Shief, G.; Soeder, C. J., ed. Algae biomass production and use. Am- sterdam: Elsevier North Holland Biomedical Press; 1980:205-216.

Abeliovich, A. Algae in wastewater oxidation ponds. In: Richmond, A., ed. Handbook of microalgal mass culture. Boca Raton: CRC Press; 1986:331-338.

Addison, R. E; Stewart, J. E. Domoic acid and the eastern Canadian mollus- can shellfish industry. Aquaculture 77:263-269; 1989.

Andersen, R. A. Algae. In: Hunter-Cevera, J. C.; Belt, A., ed. Maintaining cultures for biotechnology and industry. London: Academic Press, Inc.; 1996:29~64.

Andersen, R. A.; Morton, S. C.; Sexton, J. P. Culture collection of marine phytoplankton catalogue of strains. J. Phycol. 33, Supplement 1; 1997.

134 DAY ET AL.

Baker, H.; Frank, 0.; Pasher, I.; Matovitch, B.: Aaronsons, S.; Hutner, H.; Sobotka, H. A new bioassay method for biotin in blood, serum, urine and tissue. Ann. Biochem. 3:31-39; 1962.

Baker, E. R.; MeLaughlin, J. J. A.: Hutner, S. H.; DeAngelis, B.; Feingold, S.; Frank, 0.; Baker, H. Water soluble vitamins in cells and spent eulture supernatants of Poteriochromonas stipitata, Euglena gracilis and Tetrahymena thermophila. Arch. Microbiol. 129:310-313; 1981.

Barclay, W. R.; Meager, K. M.; Abril, J. R. Heterotrophic produetion of long- chain omega-3 fatty acids utilizing algae and algae-like organisms. J. Appl. Phycol. 6:123-130; 1994.

Becker, E. W. Limitations of heavy removal fi'om waste water by means of algae. Water Res. 17:458--466: 1983.

Becker, E. W. Cambridge studies in biotechnology. Vol. 10. Microalgae: biotechnology and microbiology. Cambridge: Cambridge University Press; 1994.

Behrens, P. W.; Bringham, S. E.; Hoeksema, S. D.; Cohoon, D. L.; Cox, J. C. Studies on the incorporation of CO 2 into starch by Chlorella vulgaris. J. Appl. Phycol. 1:123-130; 1989.

Ben-Amotz, A.: Avron, M. Glycerol, fl-carotene and dry algal meal production by commercial cultivation of Dunaliella. In: Shelf, G.: Soeder, C. J., ed. Algae biomass. Amsterdam: Elsevier/North Holland Biomedical Press; 1980:603~M0.

Benenmun, J. R. Hydrogen biotechnology: progress and prospects. Nat. Bio- technol. 14:1101-1103; 1990.

Benemann, J. R. Microalgae aquaculture feeds. J. Appl. Phycol. 4:233-245; 1992.

Benson, E. E. Cryopreservation. In: Dixon, R. A; Gonzalez, R. A., ed. Plant cell culture; a practical approach. Oxford: IRL Press: 1994:147-166.

Benson, E. E. Cryopreservation. In: Benson, E. E., ed. Plant conservation biotechnology. London: Taylor and Francis Ltd.; 1999:83-95.

Benson, E. E.; Fleck, R. A.: Bremner, D. A.; Day, J. G. Assessments of hydroxyl activity and antioxidant status in freeze-recalcitrant and freeze-sensitive algae: implications for cryopreserved culture collections. In Vitro Cell. Dev. Biol. Plant 34:P-1039, 52A; 1998.

Bodas, K.; Brennig, C.; Diller, K. R.; Brand, J. J. Cryopreservation of blue- green and eukaryotic algae in the culture collection at the University of Texas at Austin. Cryo. Lett. 16:267-274: 1995.

Bold, H. C.; Wynne, M. J. Introduction to the algae: structure and reproduc- tion. Englewood Cliffs, NJ: Prentice Hall Inc.; 1985.

Borowitzka, L. J. Development of Western Biotechnology's algal-carotene plant. Bioresour. Technol. 38:251-252: 1991.

Borowitzka, M. A. Closed algal photobioreactors: design considerations for large-scale systems. J. Mar. Biotechnol. 4:185-191: 1996.

Borowitzka, M. A. Microalgae for aquaculture: opportunities and constraints. J. Appl. Phycol. 9:393~t01; 1997.

Borowitzka, M. A.: Borowitzka, L. J. Micro-Algal biotechnology. New York: Cambridge University Press; 1988.

Boussiba, S.; Fan, L.: Vonshak, A. Enhancement and determination of astaxanthin accumulation in green alga Haematococcus pluvialis. In: Packer, L., ed. Methods in enzymology. Vol. 213. Carotenoids, Part A: chemistry, separation, quantitation, and antioxidation. San Diego, CA; London. England: Academic Press, Inc.; 1992:386-391.

Briand, J.; Julistiono, H.: Beaune, P.; Flinois, J. P.; Dewaziers, I.; Leroux, J. P. Presence of proteins recognized by mammalian cytochrome P-450 antibodies in Euglena gracilis. Biochem. Biophys. Acta 1203:199- 204; 1993.

Brown, M. R. Effects of storage and processing on the ascorbic acid content of concentrates prepared from Chaetoceros calcitrans. J. Appl. Phycol. 7:495-500; 1995.

Cahu, C.; Guillaume, J. C.; Stephan, G.; Chim, L. Influence of polypholid and highly unsaturated fatty acids on spawning rate and egg and tissue composition in Penaeus vannamei fed semi-purified diets. Aquacul- ture 126:159-170; 1994.

Cavalier-Smith, T. Kingdom Protozoa and its 18 phyla. Microbiol. Rev. 57:953-994; 1993.

Canavate, P. J.; Lubian, L. M. Tolerance of six marine microalgae to the cryoprotectants dimethyl sulfoxide and methanol. J. Phycol. 30:559- 565; 1994.

Canavate, J. P.; Lubian, L. M. Relationship between cooling rates, cryoprotectant concentrations and salinities in the cryopreservation of marine microalgae. Mar. Biol. (Berlin) 124:325-334; 1995a.

Canavate, J. E; Lubian, L. M. Some aspects on the cryopreservation of microalgae used as food for marine species. Aquaculture 136:277-290: 1995b.

Chrismadha, T.; Borowitzka, M. A. Effect of cell density and irradiance on growth, proximate composition and eicosapentaenoic acid production of Phaeodactylum tricornutum grown in a tubular bioreactor. J. Appl. Phycol. 6:67-74: 1994.

Cotterill, F. P. D. Systematic, biological knowledge and environmental conservation. Biodivers. Conserv. 4:183-205; 1995.

Day, J. G. Cryo-conservation of microalgae and cyanobacteria, Cryo. Lett. Suppl. 1:7-14; 1998.

Day, J. G. Conservation strategies for algae. In: Plant conservation biotechnology. Benson, E. E., ed. London: Taylor and Francis Ltd.; 1999:111-124.

Day, J. G.; DeVille, M. M. Cryopreservation of algae. Methods Mob Biol. 38:81-90; 1995.

Day, J. G.; Edwards, A. P.; Rodgers, G. A. Development of an industrial scale process for the heterotrophic production of a micro-algal mollusc feed. Bioresour. Technol. 38:245-250; 1991.

Day, J. G.; McLellan, M. R., ed. Cryopreservation and freeze-drying protocols. Methods in Molecular Biology 38: Totowa, NJ: Humana Press Inc.; 1995a.

Day, J. G.; McLellan, M. R. Conservation of algae. In: Grout, B. W. W., ed. Genetic preservation of plant cells In vitro. Heidelberg: Springer Ver- lag; 1995b:75-98.

Day, J. G.: Priestley, I. M: Codd, G. A. Storage reconstitution and photosynthetic activities of immobilized algae. In: Webb, C.: Mavituna, E, ed. Plant and animal ceils, process possibilities. Chichester: Ellis Har- wood Ltd.; 1987:257-261.

Day, J. G.; Tsavalos, A. J. An investigation of the heterotrophic culture of the green alga Tetraselmis. J. Appl. Phycol. 8:73-77; 1996.

Day, J. G.; Turner, M. F. Algal culture collections and biotechnology. In: Watanabe, M. M., ed. Proceedings of the Symposium on Culture Col- lections of Algae. Tsukuba, Japan: NIES; 1992:11-27.

De Pauw, N.; Persoone, G. Microalgae for aquaculture. In: Borowitzka, M. A.: Borowitzka, L. J., ed. Micro-Algal biotechnology. New York: Cam- bridge University Press; 1988:97-221.

Dixon, R.; Cheng, Q.; Shen, G.-F.; Day, A.: Dowson-Day, M. Nif gene transfer and expression in chloroplasts: prospects and problems. Plant Soil 194:193-203; 1997.

Duncan, J. R.; Brady, D.; Wilhelmi, B. Immobilization of yeast and algal cells for bioremediation of heavy metals. In: Sheehan, D., ed. Methods in biotechnology, 2. Bioremediation Protocols. Totowa, NJ: Humana Press Inc.; 1997:91-97.

Endo, H.; Sanasawa, H.; Nakajima K. Studies on Chlorella regularis, heterotrophic fast growing strain II. Mixotrophic growth in relation to light intensity and acetate concentration. Plant Cell Physiol. 18:199-205; 1977.

Fabre, J.; Dereuddre, J. Encapsulation-dehydration: a new approach to cryopreservation of potato shoot-tips. Cryo. Lett. 11:413~t26; 1990.

Fallowfield, H. J.: Garrett, M. K. The photosynthetic treatment of pig slurry in temperate climatic conditions: a pilot-plant study. Agric. Wastes 12:111-136; 1985.

Fleck, R. A. 1998. Mechanisms of cell damage and recovery in cryopreserved freshwater protists, Ph.D. Thesis, University of Abertay Dundee, Scot- land.

Fleck, R. A.; Day, J. G.: Rana, K. J.; Benson, E. E. Visualization of cryoinjury and freeze events in the coenocytic alga Vaucheria sessilis using cryomicroscopy. Cryo. Lett. 18:343-355; 1997.

Fulks, W.; Main, K. L. The design and operation of commercial-scale live feed production systems. In: Fulks, W.; Main, K. L., ed. Rotifer and microalgal culture systems. The Oceanic Institute, Honolulu; 1991:3- 52.

George, D. G.; Taylor, A. H. UK lake plankton and the Gulf Stream. Nature (Lond) 378:39; 1995.

Gerwick, W. H.; Roberts, M. A.; Proteau, P. J.: Chen, J. L. Screening cultured marine microalgae for anti-cancer-type activity. J. Appl. Phycol. 6:143-150; 1994.

Gibson, C.; Foy, R. H.: Bailey-Watts, A. E. An analysis of total phosphorus cycle in some temperate lakes; the response to enrichment. Fresh- water Biol. 35:525-532; 1996.


Glazer, A. N. Phycobilliproteins--a family of valuable, widely used fluoro- probes. J. Appl. Phycol. 6:105-112; 1994.

Gudin, C.; Chaumont, D. Solar biotechnology study and development of tubular solar receptors for controlled production of photosynthetic cellular biomass. In: Palz, W.; Pirrwitz, D., ed. Proceedings of the Work- shop and EC Contractors' meeting in Carri. Dordrecht: Reidel Publishing Co.; 1983:184-189.

Hawksworth, D. L. Microbial collections as a tool in biodiversity and biosystematic research. In: Samson, R. A.; Stalpers, J. A.; van der Mei, D.; Stouthamer, A. H., ed. Culture collections to improve the quality of life. Veldhoven, The Netherlands, August 25-29, 1996. Wagenin- gen, The Netherlands: Ponsen & Looyen; 1996:26-35.

Hawksworth, D. I.; Mound. L. A. Diversity data-bases: the crucial significance of collections. In Hawksworth, D. L., ed. The biodiversity of microorganisms and insects. Wallingford: CAB International; 1991:17-29.

Hawksworth, D. L.; Ritchie, J. M. Biodiversity and biosystematic priorities: microorganisms and invertebrates. Wallingford: CAB International; 1993.

Haworth, E. Y.; Pinder, L. C.; Lishman, J. P.; Duigan, C. A. The Anglesey lakes, Wales, UK--a palaeolimnological study of the eutrophication and nature conservation status. Aquat. Conserv. 6:61-80; 1996.

Hirata, K.; Phuchindawan, M.; Tukamoto, J.; Goda, S.; Miyamoto. K. Cryo- preservation of microalgae using encapsulation-dehydration. Cryo. Lett. 17:321-328; 1996.

Jaworski, G. H. M.; Wiseman, S. W.; Reynolds, C. S. Variability in sinking rate of the freshwater diatom Asterionella formosa: the influence of colony morphology. Br. Phycol. J. 23:167-176; 1988.

Jensen, A. Present and future needs for algae and algal products. Hydro- biologia 261:15-24; 1993.

Johnson, E. A.; An, G. H. Astaxanthin from microbial sources. Crit. Rev. Biotechnol. 11:297-326; 1991.

Johnson, E. A.; Schroeder, W. A. Microbial carotenoids. In: Fiechter, A., ed. Advances in biochemical engineeting-biotechnology. Vol. 53. Down- stream processing bioosurfactants/carotenoids. Berlin: Springer-Ver- lag; 1995:119-178.

Jones, D. A.; Kurmaly, K.; Arshard, A. Paneid shrimp hatchery trials using microencapsulated diets. Aquaculture 64:133-164; 1987.

Kannaiyan, S.; Aruna, S. J.; Kumari, S. M. P.; Hall, D. O. Immobilized cy- anobactetia as a biofertilizer for rice crops. J. Appl. Phycol. 9:167- 174; 1997.

Kelley, M. G.; Cazaubon, A.; Coting, E.; Dell"Uomo, A.; Ector, L. Recom- mendations for the routine sampling of diatoms for water quality assessments in Europe. J. Appl. Phycol. 10:215-224; 1998.

Kirsop, B.; Doyle, A. Maintenance of microorganisms and cultured cells. London: Academic Press Ltd.; 1991.

Liao, W. L.; Nureborhan, S. A.; Okada, S.; Matsui, T.; Yamaguchi, K. Pig- mentation of cultured black tiger prawn by feeding with a Spirulina- supplemented diet. Bull. Jpn. Soc. Sci. Fish. 59:165-169; 1993.

Ma, J.; Miyazaki, S.; Sugawara, H. A handy database for culture collections worldwide: CCINFO-PC. Comput. Appl. Biosci. 11:209-212; 1995.

Maberly, S. C.; Pettitt, M. The effects of ultraviolet radiation on Asterionella formosa. Phycologist 46:22-23; 1997.

Malik, K. A. Preservation of unicellular green-algae by liquid-drying. J. Mi- crobiol. Methods 18:41-46; 1993.

McGrath, M. S.; Daggett, P.-M.; Dilworth, S. Freeze-drying of algae: Chloro- phyta and Chrysophyta. J. Phycol. 14:521-525; 1978.

McKay, R. M.; Geider, R. J.; LaRoche, J. Physiological and biochemical response of the photosynthetic apparatus of two marine diatoms to Fe stress. Plant Physiol. 114:615-622; 1997.

McLellan, M. R.; Cowling, A. J.; Turner, M. E; Day, J. G. Maintenance of algae and protozoa. In: Kirsop, B.; Doyle, A., ed. Maintenance of microorganisms and eultnred cells. London: Academic Press Ltd.; 1991:183-208.

Metting, B. The systematics and ecology of soil algae. Bot. Rev. 147:195- 312; 1981.

Metting, E B., Jr. Micro-algae in agriculture. In: Borowitzka, M. A.;. Borow- itzka, L. J., ed. Microalgal biotechnology. Cambridge: Cambridge Univ. Press; 1988:228-304.

Metting, E B., Jr. Biodiversity and application of microalgae. J. Ind. Micro- biol. Biotechnol. 17:477-489; 1996.

Millamena. O. M.; Aujero, E. J.; Borlongan, I. G. Techniques on algal har- vesting and preservation for use in culture and as a lal"val food. Aqua- cult. Eng. 9:295-304; 1990.

Miyachi, S.; Nakayama, O.; Yokohama, Y.; Hara, Y.; Ohmori, M.; Komogata, K.; Sugawara, H.; Ugawa, Y., ed. World catalogue of algae. Tokyo: Japan Scientific Societies Press; 1989.

Morris, G. J. The cryopreservation of Chlorella I. Interactions of rate of cooling, protective additive and warming rate. Archly. Microbiol. 107:57- 62; 1976.

Morris, G. J. Cryopreservation of 250 strains of Chlorococcales by the method of two step cooling. Br. Phycot. J. 13:15-24; 1978.

MmTis, G. J.; Coulson, G. E.; Engels, M. A cryomicroscopic study of Cylin- drocystis brebissonii and two species ofMicrasterias Conjugatophyceae Chlorophyta during freezing and thawing. J. Exp. Bot. 37:842-856; 1986.

Mumford, T. E; Miura, A. Porphyra as food. In: Lembi, C. A.; Waaland, J. R., ed. Algae and human affairs. Cambridge: Cambridge University Press; 1988:87-117.

Murray, J.; Thompson, A. Hydrocarbon production in Anacystis montana and Botryococcus braunii. Phytochemistry 16:465-468; 1977.

Nerad, T. A. ATCC Catalogue of protists. Rockville: American Type Culture Collection; 1993.

OECD. OECD guidelines for testing chemicals. Section 2--Effects on biotic systems, No. 201, Alga, growth inhibition test. Paris: Organization for Economic Development; 1984.

Ogbonna, J. C.; Tomiyama, S.; Tanaka, H. Heterotrophic cultivation of Ea- glena gracilis Z for efficient production of d-tocopherol. J. Appl. Phy- col. 10:67-74; 1998.

Oswald, W. J. Micro-algae and waste-water treatment. In: Borowitzka, M.; Borowitzka, L. J., ed. Micro-algal biotechnology. Cambridge: Cam- bridge University Press; 1988:305-328.

Oswald, W. J.; Goluke, C. G.; Gee, H. K. Wastewater reclamation through production of algae. Univ. California Water Resources Center, Con- tribution 22; 1957.

Parker, M. Vitamin B12 in Lake Washington USA concentration and uptake. Limnol. Oceanogr. 22:527-538; 1977.

Patterson. G. M. L.; Larsen, L. K.; Moore, R. E. Bioactive natural products from blue-green algae. J. Appl. Phycol. 6:151-158; 1994.

Pflugmacher, S.; Sanderman, H. Cytochrome P450 monooxygenases for fatty acids and xenobiotics in marine macroalgae. Plant Physiol. 117:123- 128; 1998.

Pharhad, N. M. Studies on microbial flora in oxidation ponds. Ph.D. Thesis, Central Public Health Engineering Research Institute, Nagpur, India; 1970.

Pohl, M.; Kohlhase, M.; Martin, M. Pilot scale axenic mass cultivation of microalgae. I. Development of the biotechnology. Planta Med. 52:416-417; 1986.

Pouliot, Y.; De la Noue, J. Development of a pilot-scale facility for wastewater treatment and microalgal production. Rev. Fr. sci. eau 4:207-222; 1985.

Renn, D. Biotechnology and the red seaweed polysaccharide industry: status. needs and prospects. Trends Biotechnol. 15:9-14; 1997.

Robinson, L. E; Morrison, A. W.; Bamforth, M. R. Improvements relating to biosynthesis. European Patent No. 261872; 1988.

Roger, P. A.: Kulasooriya, S. A.. Blue green algae and rice. International Rice Research Institute, Los Banos, The Philippines; 1980.

Roper, M. M. Biological diversity of micro-organisms: An Australian perspective. Pac. Conserv. Biol. 1:21-28: 1993.

Running, J. A.; Huss, R. J.; Olson. P. T. Heterotrophic production of ascorbic acid by microalgae. J. Appl. Phycol. 6:99-104; 1994.

Sato, V. The development of a phytoplankton production system as a support base for finfish larval rearing research. In: Fulks, W.; Main, K. L., ed. Rotifer and microalgal culture systems. The Oceanic Institute, Hon- olulu; 1991:257-273.

Schlipalius, L. The extensive commercial cultivation of Dunaliella salina. Bioresour. Technol. 38:241-243; 1991.

Schl6sser, U. G. SAG Sammlung yon Algenkulturen at University of G~ttin- gen. Bot. Acta 107:3-186; 1994.

Shi, X.-M.; Chert, E; Yuan. J.-P.; Chen, H. Heterotrophic production of leutin by selected Chlorella strains. J. Appl. Phycol. 9:445--450; 1997.

Shumway, S. E. Toxic algae a serious threat to shellfish aquaculture. World Aquacult. 20:5-74; 1989.

136 DAY ET AL.

Sommer, T. R.; Potts, W. T.; Morrissy, N. M. Utilization of mieroalgal astaxanthin by rainbow trout (Oncorhynchus mykiss). Aquaculture 94:79- 88; 1991.

Starr, R. C.; Zeikus, J. A. UTEX-The culture collection of algae at the Uni- versity of Texas at Austin. J. Phycol. 29:1-106; 1993.

Stein, J., ed. Handbook of phycological methods: culture methods and growth measurements. Cambridge: Cambridge University Press; 1973.

Stevens, S. E.; Murphy, R. C.; Lamoreaux, W. J.; Coons, L. B. A genetically engineered mosquitocidal cyanobacterium. J. Appl. Phycol. 6:187- 198: 1994.

Takano, H.; Takeyama, H.; Nakamura, N.; Sode, K.; Burges, J. G.; Manabe, E.; Hirano, M.; Matsunaga, T. CO2 removal by high-density culture of a marine cyanobacterium Synechococcus sp. using an improved photobioreactor employing light-diffusing optical fibers. Appl. Biochem. Biotechnol. 34/35:449-458; 1992.

Thies, E: Backhaus, T.; Bossmann, B.; Grimme, L. H. Xenobiotic biotrans- formation in unicellular green algae. Involvement of cytochrome P450 in activation and selectivity of the pyridazinone pro-herbicide meta- flurazon. Plant Physiol. 112:361-370; 1996.

Tompkins, J.; DeVille, M. M.; Day, J. G.; Turner, M. E, ed. Culture collection of algae and Protozoa catalogue of strains. Ambleside: Culture Col- lection of Algae and Protozoa; 1995.

Torzillo, G.; Pushparaj, B.; Bocci, E; Balloni, W.; Materassi, R.; Florenzano, G. Production of Spiralina biomass in closed photobioreactors. Bio- mass 11:61-74; 1986.

Tredici, M. R.; Carlozzi, P.; Chini Zittelli, G.; Materassi, R. A vertical alveolar panel (VAP) for outdoor mass cultivation of microalgae and cyanobacteria. Bioresour. Technol. 38:153-159; 1991.

Venkataraman, L. V. Blue-green algae as biofertilizer. In: Richmond, A., ed. CRC handbook of microalgal mass culture. Boca Raton: CRC Press Inc.; 1986:455-472.

Vigneron, T.; Arbauh, S.; Kaas, R. Cryopreservation of gametophytes of Lam- inaria digitata (L.) by encapsulation dehydration. Cryo Lett. 18:117- 126; 1997.

Vilchez, C.; Garbayo, I.; Lobato, M. V.; Vega, J. M. Microalgae-mediated chemicals production and wastes removal. Enzyme Microb. Technol. 20:456-572; 1997.

Warren, A.; Day, J. G.; Brown, S. Cultivation of protozoa and algae. In: Hurst, C. J.; Knudsen, G. R.: Mclnerney, M. J.; Stezenbach, L. D.; Walter, M. V., ed. Manual of environmental microbiology. Washington DC: ASM Press; 1997:61-71.

Watanabe, M. M.; Hiroki, M. NIES-Collection List of strains. Tsukuba: NIES; 1997.

Watanabe, M. M.; Shimizu, A.; Satake, K. NIES-Microbial Culture Collection at the National Institute of Environmental Studies: cryopreservation and database of culture strains of microalgae In: Watanabe, M. M., ed. Proceedings of the Symposium on Culture Collections of Algae. Tsukuba, Japan: NIES; 1992:33-41.

Watson, R. H.; Jones, G. G.; Jones, B. L. Using centrifuged algae for feeding oyster larvae. J. Shellfish Res. 5:136; 1986.

WDCM. http://wdcm.nig.ac.jp/. 1994. Wiessner, W.; Schnepf, E.; Starr, R. C. Algae, environment and human affairs.

Bristol, UK: Biopress Ltd.; 1995. Wilde, E. A.; Benemann. J. R. Bioremoval of heavy metals by the use of

microalgae. Biotechnol. Adv. 11:781-812; 1993. Wilson, M. M.; Jackson, M. M.; Edyvean, R. G. J. The use of algae to de-

colourise and remove metal ions from industrial effluents. Br. Phycol. J. 26:99; 1991.

Yainada, T.; Sakai, A. Cryopreservation of cells and tissues using simplified methods. In: Normah, M. N.; Narimah, M. K.; Clyde, M. M., ed. Pro- ceedings of the International Workshop on the In Vitro Conservation of Plant Genetic Resources, July 4~5, 1995. Kuala Lumpur, Malaysia: Universiti Kebangsaan Malaysia Publishers; 1996:89-103.

Full Text

Documents

Transcript of Full Text