The CSIRO Collection of Living Microalgae: an Australian Perspective on Microalgal Biodiversity and Applications

The CSIRO Collection of Living Microalgae: An Australian Perspective on Microalgal Biodiversity and Applications

S.I. Blackburn, I.D. Jameson, D. Frampton, M. Brown, M. Mansour, A. Negri, N.S. Parker, S. Robert, C.J. Bolch, P.D. Nichols and J.K. Volkman

CSIRO Marine Research, Hobart, Tasmania, Australia [email protected] [email protected]

ABSTRACT The CSIRO Collection of Living Microalgae maintains over 800 strains from 140 genera representing the majority of marine and some freshwater microalgal classes. The Collection is incorpoarted within the CSIRO Microalgal Research Centre (CMARC) which provides a supply service of microalgal strains to industry, government and university organizations. Research within CMARC and in partnership with collaborators spans a wide base within the three themes of Environment, Aquaculture and Biotechnology. Some of the research projects undertaken include physiological studies of different life-history stages of microalgae, toxin production in harmful algal bloom (HAB) species, phylogenetic studies of different populations of HAB species, optimizing the nutritional benefit of microalgal diets in larval and broodstock aquaculture species, including important nutrients such as vitamins and polyunstaurated fatty acids. Research is also being undertaken into optimizing high biomass production systems through the use of photobioreactors.

1. THE CSIRO COLLECTION OF LIVING MICROALGAE

The CSIRO Collection of Living Microalgae maintains over 800 strains from 140 genera representing the majority of marine and some freshwater microalgal classes (see Fig.1 for summary, list of strains available from the authors or downloadable from http://www.marine.csiro.au). There are also some selected micro-heterotrophic strains. This collection is the largest and most diverse microalgal culture collection in Australia and, with NIES-Collection (National Institute for Environmental Studies, Environment Agency) in Japan, ranks as a major microalgal collection in the the Asia-Pacific region.

1-1. Development of the CSIRO Collection of Living Microalgae

The collection originated in the 1960s from fundamental research into the pigment composition of marine microalgae, a theme which has continued through to the present and which has culminated in the incorporation of microalgal pigments as essential oceanographic reference standards (Jeffrey et al.1997). The aquisition of microalgal strains has focused on isolates from Australian waters - over 80% of strains have been isolated from diverse localities and climatic zones, from tropical northern Australia to the Australian Antarctic Territory, from oceanic, inshore coastal, estuarine, intertidal and freshwater environments (Fig. 2). Additionally, emphasis has also been placed on representation of

39 different populations of a single species, usually by more than one strain. This is particularly the case for harmful bloom-forming species where there are up to 20 strains of a single species from a single locality.

250 40 strains N= 803 35 200 genera N=139 30

150 25 20 100 15 10 50 5 number of strains per Class number of genera per Class 0 0 Dinophyceae Cyanophyceae Cryptophyceae Chlorophyceae Rhodophyceae Chrysophyceae Xanthophyceae Prasinophyceae Euglenophyceae Raphidophyceae Bacillariophyceae Dictyochophyceae Prymnesiophyceae Eustigmatophyceae Fig. 1. Number of strains and genera in different algal classes held in the CSIRO collection of living microalgae.

250 Australian Overseas 200

150

100

Number of strains 50

0 Other Dinophyceae Cyanophyceae Cryptophyceae Rhodophyceae Chlorophyceae Chrysophyceae Prasinophyceae Euglenophyceae Raphidophyceae Bacillariophyceae Dictyochophyceae Prymnesiophyceae Eustigmatophyceae

Fig. 2. Number of strains of Australian and non-Australian origin in different classes held in the CSIRO collection of living microalage.

40 All strains in the culture collection are unialgal and the majority are clonal. A subset of strains, including particular strains used by the aquaculture industry and some used for genetic and toxin profiles, are axenic. Since the mid 1980s research has focused on the rapidly developing aquaculture industry in Australia and key environmental issues such as harmful and toxic algal blooms. Since 1995 the potential for biotechnological development of bioactive and novel compounds, particularly lipids, from microalgae has been explored. In parallel, the issue of efficient, high production of quality-controlled microalgae using photobioreactors has been a research focus. Such systems will allow the full potential of microalgae for biotechnology and aquaculture applications to be realized.

1-2. Genetic Diversity

Microalgae are known for their cosmopolitanism at the morphological species level, with very low endemicity being shown (Norton et al.1996). However this morphological cosmopolitanism can hide a plethora of diversity at the intra-specific level. There have been a number of studies of genetic diversity on different microalgae using various approaches such as interbreeding, isozymes, growth rates and a range of molecular genetic techniques. The diversity identified by these studies ranges from large regional and global scales (Chinain et al.,1997) to between and within populations at small scales (Gallagher, 1980; Medlin et al.,1996; Bolch et al.,1999a,b). Variation at the intra-specific level, between morphologically indistinguishable microalgae, can usually only be identified using strains isolated from the natural environment and cultured in the laboratory. For selected microalgae we have demonstrated the distinctness of Australian populations compared with populations from outside Australia. The dinoflagellate Gymnodinium catenatum has formed recurrent toxic blooms in south-eastern Tasmanian waters since 1986. Cargo vessel ballast water from Japan or Spain has been proposed as one likely vector of introduction. Using both interbreeding and molecular approaches we have shown considerable within-population variation in G. catenatum as well as a complex multi-group mating system. The interbreeding data showed a slightly closer relationship between Tasmanian and Spanish populations than between Tasmanian and Japanese populations (Blackburn et al.2001), whereas multidimensional scaling analysis (MDS) of Randomly Amplified Polymorphic DNA (RAPD) indicated that Australian strains were almost equally related to both the Spanish/Portuguese population and the Japanese population (Bolch et al.1999b). Analysis of molecular variance (AMOVA) found that genetic variation was partitioned mainly within the Tasmanian populations (87%) compared to the variation between the regions (8%) and between populations within regions (5%). The potential source population for Tasmania’s introduced G. catenatum remains equivocal; however, strains from the more recently discovered mainland Australian population (Port Lincoln, South Australia, 1996) clustered with Tasmanian strains, supporting the notion of a secondary relocation of Tasmanian G. catenatum populations to the mainland via a shipping vector. Geographic and temporal clustering of strains was evident among the Tasmanian strains, indicating that genetic exchange between neighboring estuaries is limited and that Tasmanian G. catenatum blooms are composed of localized, estuary-bound subpopulations (Fig. 3). Another phylogenetic study of the toxic cyanobacterial bloom-forming species Nodularia

41 spumigena highlighted some similar findings. Australian populations of N. spumigena were distinctly different from Nodularia strains from Canada and Europe including the proposed type strain for the species. Within Nodularia strains, genetic variation could be resolved at several levels, from intra-generic through to intra-population levels, clearly defining generic, species and population limits of genetic variation in these clonal organisms (Bolch et al.1999a). Unlike G. catenatum, within population heterogeneity was not high in Australian N. spumigena. This may reflect a difference in genetic complexity and life history between these eutcaryotic and prokaryotic microalgae. Culture collections maintain living and characterised biodiversity for research and comparison with new strain acquisitions as well as with natural biodiversity. It is essential to have identifiable and stable genotypes. While there are recorded instances of change or loss of particular characteristics in long term culture (Coleman, 1977), in general, culturing guarantees genetic continuity and stability of a particular strain. We are now developing cryopreservation strategies to limit the potential for genetic drift, beginning with our cyanobacterial subset of strains. Cryopreservation is also attractive because it decreases the demanding task of regular subculturing.

HU JP

TRA DE '93

)

3 0

(

D DE '87 SP PTL02 PTL01-4 -1

-2 -1012 DIM (1) Fig. 3. Three-dimensional MDS analysis of RAPD data from G. catenatum strains, not including the two outgroup species. Kruskal stress = 0.193. (a) Plot of first and second dimension of the three-dimensional MDS analysis of G. catenatum strains. Region/population clusters (bounded by solid line): Huon Estuary and Hastings Bay (HU/HA), Derwent Estuary 1987 (DE”87) and 1993 (DE’93), Triabunna (TRA), and Japan, Spain and Portugal (JP/SP/PT). Port Lincoln, South Australia strains (PTL) marked by strain number. (b) Plot of the first and third dimension of the three-dimensional MDS analysis of G. catenatum strains. G. catenatum clusters bounded by solid or shaded lines: Huon Estuary and Hastings Bay (HU/HA, shaded), Derwent Estuary 1987 (DE”87) and 1993 (DE’93), Triabunna (TRA, shaded), and Japan (JP), Spain (SP), and Portugal (PT). Port Lincoln, South Australia strains (PTL) marked by strain number after Bolch et al.1999b.

42 1-3. Global Diversity of Microalgal Culture Collections

There are 471 culture collections in 62 countries registered in the World Federation For Culture Collection’s (WFCC) World Data Centre for Microorganisms (WDCM) as of September 2002. Thirty-eight of these collections are registered as holding more than 10 strains of microalgae (i.e. collections holding <10 strains have been excluded), 11 of which are in the Asia-Pacific region. A wider survey based on the WDCM, UNESCO Manual on Harmful Marine Microalgae (1995) and internet sources records 124 collections holding microalgae from 40 countries and 40 collections from 13 countries in the Asia-Pacific region. Appendix 1 displays all the culture collections found in the Asia-Pacific region and only those in the rest of the world where the number of strains is available. The WDCM records a total of 1,081,791 microbial strains in its registered culture collections with 1,687 type of species or sub-species of algae. The number of microalgal collections grouped by major geographical region is shown in Figure 4a. An example of the diversity within algal classes between 4 major microalgal collections is given in Figure 4b. CSIRO and the Culture Collection of Marine Phytoplankton (CCMP) are relatively similar in the diversity of strains within Classes, for example relatively high in marine diatoms (Bacillariophyceae) and dinoflagellates (Dinophyceae). In contrast Sammlung von Algenkulturen, University Goettingen (SAG) and the Culture Collection of Algae and Protozoa (CCAP) are low in these groups but much higher in green algae groups such as Chlorophyceae and the Euglenophyceae. Comparisons between collections need to be mindful of the differences in taxonomic classification used. CSIRO classifies all diatoms into Bacillariophyceae whereas CCMP classifies diatoms into three classes. Accordingly, in Fig. 4b, the 3 classes of CCMP diatoms have been consolidated into Bacillariophyceae. These collections demonstrate the biodiversity of microalgae held in collections, the value of which has been stressed by Norton et al.,1996.

10 # of microalgal collections collections microalgal # of 0 Africa Americas Asia-Pacific Europe MiddleEast

Fig. 4a. Number of microalgal collections grouped by geographic region as registered with the World Federation for Culture Collection’s World Data Centre for Microorganisms.

43 300 CSIRO CCMP 250

200

150

100

Number of strains 50

0 other Dinophyceae Cyanophyceae Cryptophyceae Chlorophyceae Rhodophyceae Pelagophyceae Xanthophyceae Chrysophyceae Prasinophyceae Euglenophyceae Raphidophyceae Bacillariophyceae Dictyochophyceae Trebouxiophyceae Prymnesiophyceae Zygnematophyceae Eustigmatophyceae

1050 SAG CCAP 900 750 600 450 300 Number of strains 150 0 other Dinophyceae Cyanophyceae Cryptophyceae Rhodophyceae Chlorophyceae Pelagophyceae Xanthophyceae Chrysophyceae Prasinophyceae Euglenophyceae Raphidophyceae Bacillariophyceae Dictyochophyceae Trebouxiophyceae Prymnesiophyceae Zygnematophyceae Eustigmatophyceae Fig. 4b. Diversity of strain holdings per algal class in the CSIRO, CCMP, SAG and CCAP Collections.

2. HARMFUL AND TOXIC ALGAL BLOOM RESEARCH

Strains from the CSIRO Collection have been fundamental to a wide range of toxic and harmful algal bloom research since the mid 1980s. In particular the focus has been on toxic species from Australian waters, both paralytic shellfish toxin (PST) producing dinoflagellates and also PST and hepatotoxin-producing cyanobacteria. Ecophysiological and life history studies including mating systems of G. catenatum were described by Blackburn et al.(1989, 2001) and, more recently, extensive studies on mating systems and factors affecting encystment and excystment in G. catenatum, Alexandrium minutum and A. catenella have been completed (Parker 2002b). For the cyanobacteria, factors affecting growth and akinete production and germination in Anabaena circinalis (Blackburn et

44 al.2000) and the effect of salinity on growth and toxin production in N. spumigena (Blackburn et. al 1996) were investigated. The results from controlled culturing studies using clonal or unitrichrome strains were related with field ecological studies, thus giving a strong feedback between the precise experimentation in the laboratory and the ‘bigger picture’ ecological studies e.g. see Jones et. al (1994) for N. spumigena and CSIRO (2000) and Parker (2002b) for bloom dynamics of G. catenatum in the Huon Estuary, south-east Tasmania. As described in Section 1, the toxic algal strains in the CSIRO Collection have formed the basis of considerable phylogenetic studies examining the generic diversity within and between populations on local, regional and global scales. Details on toxins and toxic algae are given in Section 4.6.

3. NUTRITIONAL PROPERTIES OF MICROALGAE AND THEIR USE IN AQUACULTURE

Microalgae are utilized in aquaculture as live feeds for all growth stages of bivalve molluscs (e.g. oysters, scallops, clams and mussels), for the larval/early juvenile stages of abalone, crustaceans and some fish species, and for zooplankton used in aquaculture food chains. Microalgae must possess a number of key features to be useful aquaculture species. They must be of an appropriate size for ingestion, e.g. from 1 to 15 µm for filter feeders; 10 to 100 µm for grazers (Webb and Chu, 1983; Jeffrey et al.,1992) and readily digested. They must have rapid growth rates, be amenable to mass culture, and also be stable in culture to any fluctuations in temperature, light and nutrients as may occur in hatchery systems. Finally, they must have a good nutrient composition, including an absence of toxins that might be transferred up the food chain. Strains fulfilling these attributes and used widely in aquaculture include northern hemisphere strains such as Isochrysis sp. (T.ISO) CS-177, Pavlova lutheri CS-182, Chaetoceros calcitrans CS-178, C. muelleri CS-176, Skeletonema costatum CS-181, Thalassiosira pseudonana CS-173, Tetraselmis suecica CS-187 and Nannochloropsis oculata CS-189. These strains are also used by Australian industry, but following requests by industry for endemic strains more suitable to local conditions, CSIRO research has focused on the isolation and characterisation of Australian strains over the last decade. Today, approximately 10-15% of microalgae requested by Australia hatcheries are local isolates, examples including Pavlova pinguis CS-375, Skeletonema sp. CS-252, Nannochloropsis sp. CS-246, Rhodomonas salina CS-24 and Navicula jeffreyi CS-46. As part of our studies on microalgae for aquaculture, during the last decade we have assessed the nutritional performance and biochemical profiles of a range of algal strains (including Australian isolates) and alternatives to live algae such as algal concentrates. Feeding experiments with Crassostrea gigas oyster spat established that the local strains Pavlova pinguis CS-375, Rhodomonas salina CS-24, Attheya septentrionalis CS-425 and Entomoneis cf punctulata CS-426 had good food value (McCausland et al.,1999; Knuckey et al.,2002). Experiments with algal concentrates of Chaetoceros spp. prepared by centrifugation (McCausland et al.,1999) or using a novel flocculation procedure developed at CSIRO (Brown and Robert, 2002) demonstrated that they had equivalent

45 nutritional value for larval or juvenile C. gigas as the fresh algal counterpart. Our current focus is the assessment of high-biomass algal cultures as aquaculture feeds. Our biochemical assessment of over 50 strains of microalgae used (or of potential use) in aquaculture found that cells grown to late-logarithmic growth phase typically contained 30 to 40% protein, 10 to 20% lipid and 5 to 15% carbohydrate (Brown et al.,1997). When cultured through to stationary phase, the proximate composition of microalgae changed significantly; for example when nitrate was limiting in cultures of Isochrysis sp. (T.ISO) and N. oculata, protein levels reduced by up to 50% with corresponding increases in carbohydrate (Brown et al.,1993a). There does not appear to be a strong correlation between the proximate composition of microalgae and nutritional value, although algal diets with high levels of carbohydrate are reported to produce the best growth for juvenile oysters (Ostrea edulis; Enright et al.1986) and larval scallops (Patinopecten yessoensis; Whyte et al.,1989) provided polyunsaturated fatty acids (PUFAs) are also present in adequate proportions. In contrast, high dietary protein provided best growth for juvenile mussels (Mytilus trossulus; Kreeger and Langdon 1993) and Pacific oysters (Crassostrea gigas; Knuckey et al.,2002). PUFA derived from microalgae, e.g. docosahexanoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (AA) are known to be essential for various larvae (Sergent et al.,1997). The proportion and content of these important PUFAs in 46 strains of microalgae showed systematic differences according to taxonomic group (Fig. 5; see also Section 4.1), although there were also examples of significant differences between microalgae from the same class. Pertinent to aquaculture operations, chlorophytes (Dunaliella spp. and Chlorella spp.) are deficient in both C20 and C22 PUFAs, although some species have small amounts of EPA (up to 3.2%). Because of this PUFA deficiency, chlorophytes generally have low nutritional value and are not suitable as a single species diet (Brown et al.,1997).

AAAA

i EPAEPA

a DHADHA

Chlorophytes CryptomonadsCryptomonads PennatePenate Diatoms EuEustigmatophytes st i g m a t o p h y t e s Ha pHaptophytes to p hyte s Prasinophytes Centric Diatoms Rhodophtyes Hapttophytes(Iso (Isochrysis) c hrysi s) (Paviova) Dinoflagellates Pra si n o p h y t e s Centric Diatoms Rhodophytes Ha p t o p h yt e s Dinoflagellates Algal Classes (Pavlova) Algal classes Fig. 5. Distribution of long-chain polyunsaturated fatty acids (LC-PUFA) in microalgal classes. Arachidonic acid [AA, 20:6(n-3)], Eicosapentaenoic acid [EPA, 20:5(n-3)] and Docosahexaenoic acid [DHA, 22:6(n-3)].

46 While the importance of PUFA is recognized, the quantitative requirements of larval or juvenile animals feeding directly on microalgae is not well established (Knauer and Southgate, 1999). Thompson et al.(1993) found that the growth of Pacific oyster C. gigas larvae was not improved by feeding them microalgae containing higher than 2% (total fatty acids) of DHA; moreover the percentage of dietary EPA was negatively correlated to larval growth. However, the authors found a correlation between the percentage composition of the short chain fatty acids 14:0 + 16:0 in microalgae, and larval growth rates. They reasoned that diets with higher percentages of the saturated fats were more beneficial for the rapidly growing larvae, because energy is released more efficiently from saturated fats than unsaturated fats. In late-logarithmic phase, prymnesiophytes, on average, contain the highest percentages of saturated fats (33% of total fatty acids), followed by diatoms and eustigmatophytes (27%), prasinophytes and chlorophytes (23%) and cryptomonads (18%) (Brown et al.1997). The content of saturated fats in microalgae can also be improved by culturing under high light conditions (Thompson et al.,1993). The content of vitamins can vary between microalgae. Ascorbic acid shows the greatest variation, i.e. 16-fold (1 to 16 mg g-1 dry weight; Fig. 6 and Brown and Miller 1992). Other vitamins typically show a two- to four-fold difference between species, i.e. β-carotene 0.5 to 1.1 mg g-1, niacin 0.11 to 0.47 mg g-1, α-tocopherol 0.07 to 0.29 mg g-1, thiamin 29 to 109 µg g-1, riboflavin 25 to 50 µg g-1, pantothenic acid 14 to 38 µg g-1, folates 17 to 24 µg g-1, pyridoxine 3.6 to 17 µg g-1, cobalamin 1.8 to 7.4 µg g-1, biotin 1.1 to 1.9 µg g-1, retinol ≤ 2.2 µg g-1 and vitamin D < 0.45 µg g-1 (Seguineau et al.,1996; Brown et al.,1999). To put the vitamin content of microalgae into context, data should be compared with the nutritional requirements of the consuming animal. Unfortunately, nutritional

µ µ

Fig. 6. The concentrations of ascorbic acid, riboflavin and thiamin in selected microalgae. Microalgae were grown at 10 µmol photon.m-2s-1(12:12 h light:dark). Grey bars denote log phase, black bars denote stationary phase.

47 requirements of larval or juvenile animals that feed directly on microalgae are, at best, poorly understood. However, the requirements of the adult are often far better known e.g. for marine fish and prawns (Tacon 1991; Conklin, 1997) and, in the absence of information to the contrary, can provide a guide for the larval animal. These data suggest that a carefully selected, mixed-algal diet should provide adequate concentrations of the vitamins for aquaculture food chains. The amino acid composition of the protein of microalgae was very similar between species (Brown, 1991) and relatively unaffected by the growth phase and light conditions (Brown et al.,1993a, b). Further, the composition of essential amino acids in microalgae is very similar to that of protein from oyster larvae (C. gigas; see Fig.7). This finding indicates that it is unlikely the protein quality is a factor contributing to the differences in nutritional value of microalgal species. % of total amino acids acids amino % of total

Fig. 7. Comparison of the percentages of essential amino acids in microalgae (grey bars) and Pacific oyster (C. gigas) larvae (black bars). Bars for algae represent averages of 47 species; error bars represent the range (after Brown 1997).

4. CHEMICAL DIVERSITY AND BIOACTIVITY 4-1. Polyunsaturated Fatty Acids (PUFA)

There is considerable interest in microalgae containing a high content of the nutritionally important long-chain polyunsaturated fatty acids (LC-PUFA), EPA [eicosapentaenoic acid, 20:5(n-3)] and DHA [docosahexaenoic acid, 22:6(n-3)] as these are essential for the health of both humans and aquacultured animals (see Section 3). While these PUFA are available in fish oils, microalgae are the primary producers of EPA and DHA. Microalgae offer a renewable, plant source of these LC-PUFA using high production techniques (see Section 5). Screening of CSIRO microalgal extracts for novel and nutritionally important bioactive molecules, including PUFA, is an ongoing area of research. As part of this procedure, C18-C22 PUFA

48 composition of microalgal strains from different algal classes has been analysed (e.g. Dunstan et. al. 1994, Volkman et al.,1989; Mansour et al.,1999a and see Fig. 5). Diatoms and eustigmatophytes are rich in EPA and produce small amounts of the less common PUFA, AA [arachidonic acid, 20:4(n-6)] with negligible amounts of DHA. In addition, diatoms make unusual C16 PUFA such as 16:4(n-1) and 16:3(n-4). In contrast, dinoflagellates have high concentrations of DHA and moderate to high proportions of EPA and precursor C18 PUFA [18:5(n-3) and 18:4(n-3)]. Prymnesiophytes also contain

EPA and DHA, with EPA the dominant PUFA. Cryptomonads are a rich source of the C18 PUFA 18:3(n-3) (ALA β-linolenic acid) and 18:4(n-3) (STA, stearidonic acid), as well as EPA and DHA.

Green algae (e.g. Dunaliella salina) typically contain low concentrations of C20 and C22 PUFA, have abundant 18:3(n-3) and 18:2(n-6), and are also able to make 16:4(n-3). The biochemical or nutritional significance of uncommon C16 PUFA [e.g. 16:4(n-3), 16:4(n-1), 16:3(n-4)] and C18 PUFA (e.g.

18:5(n-3) and 18:4(n-3)] is unclear. However there is current interest in C18 PUFA such as stearidonic acid which is now being increasingly recognized as a precursor for the beneficial EPA and DHA, unlike β-linolenic acid which has only limited conversion to EPA and DHA.

As a result of our search for novel PUFA, two very-long-chain highly unsaturated (C28) VLC-HUFA: octacosaheptaenoic acid [28:7(n-6)(4,7,10,13,16,19,22)] and octacosaoctaenoic acid [28:8(n-3)(4,7,10,13,16,19,22,25)] were discovered in seven marine dinoflagellates (Mansour et al.1999b). Argentation TLC and reverse phase preparative HPLC were used to isolate and purify these fatty acids and electron-impact GC-MS of their dimethyl oxazoline (DMOX) derivatives was used to determine the positions of the double bonds. These fatty acids accounted for less than 2.3 % of the total fatty acids. The main VLC-HUFA in Symbiodinium microadriaticum was 28:7(n-6), but in the other species 28:8(n-3) was the major or only C28 PUFA present. Both 28:7(n-6) and 28:7(n-3), as well as other VLC-HUFA, had been detected previously in fish such as Baltic herring (Rezanka, 1990); our results suggest that they may have originated from microalgae. The biosynthesis of VLC-HUFA is still unclear although presumably they are formed by elongation of shorter-chain PUFA. It seems likely that 28:7(n-6) can be desaturated to 28:8(n-3), and because there is room for only one more methylene-interrupted double bond, by necessity it must be located in the n-3 position. These VLC-HUFA were not detected in other algal classes and to date seem to be specific to dinoflagellates. They can therefore be regarded as chemotaxonomic markers. Leblond and Chapman (2000) recently analysed the polar lipid fraction in 16 strains of dinoflagellates; 28:8(n-3) was found exclusively in the phospholipid fraction suggesting a membrane function. The possible biochemical effect of these novel fatty acids is as yet unknown.

4-2. Unusual Lipids

For several decades organic and petroleum geochemists have discovered new and unusual lipid components in studies of Recent and ancient sediments. Information obtained from distribution of components within a variety of lipid classes in such samples has led to searches for the same compounds in possible source organisms, in particular microalgae. We highlight below some of our own studies that have revealed a variety of unusual lipid classes derived from microalgae.

49 OH O

4αMe 24Et 3β,4β dihydroxy sterol C32:0 monohydroxy fatty acid C30:0 pavlovol

HO H OH HO O CH3

OH OH

C32:0 dihydroxy fatty acid C30:1 4α,23,24 tri Me sterol dinosterol

OH HO H CH 3 C26:1* n-alkenol

C30:1 4α,23,24 tri Me steroidal ketone dinosterone OH OH C28:0 1,15 dihydroxy alkane long chain diol

O H CH3 O (CH2)5 R

O C37:3 & C38:3 long chain alkenones (R=CH3, C2H5) & alkenoates (R=OCH3, OC2H5) OH 28:8(n-3) VLC-HUFA

O C25:5 Highly branched Isoprenoid alkene (HBI) 22:6(n-3) LC-PUFA OH

Fig. 8. Chemical structures of novel lipids found in some microalgae (* Position of double bond in the chain was not determined).

4-3. Long-chain Unsaturated Ketones

Very long-chain (C35-C40 with C37 and C38 most abundant) unsaturated methyl and ethyl ketones termed alkenones, (Fig. 8) are found in certain prymnesiophytes including the coccolithophores Emiliania huxleyi and Gephyrocapsa oceanica (Volkman et al.,1980; 1995). Alkenones are straight-chain lipids and contain 2 to 4 double bonds having the trans geometry. Our recent work with French collaborators has demonstrated the presence of several new alkenones in these microalgae as well as the corresponding alkenols (Rontani et al.,2001). Alkenones are ubiquitous in marine sediments (e.g. Nichols et al.1986; Volkman et al.,1997), and the ratio of components has been found to vary systematically with the seawater temperature in which the microalgae grow. This has prompted many paleoceanographic studies that have used the ratio of concentrations of tri- to di-unsaturated C37 ketones in sediments up to 100 million years old to estimate the paleotemperature when the sediments were deposited (e.g. Brassell et al.,1986; Sikes et al.,1997 and references therein).

4-4. Highly Branched Isoprenoid Alkenes

Highly branched isoprenoid alkenes (HBIs) (Fig. 8) have an unusual coupling of C5 isoprene units and typically have 2-6 double bonds. The parent C20 alkane was first noted in the Rozel Point

50 crude oil (Yon et al.,1982) and this was followed by identification of the C20, C25 and C30 alkenes in algal mats, seawater and sediments with bacteria, microalgae and macroscopic algae amongst the first suggested sources. A microalgal source seemed likely when we showed that sea-ice diatom communities contained high concentrations of a C25:2 HBI with the 2,6,10,14-tetramethyl-7-(3-methylpentenyl)-pentadecane skeleton (Nichols et al.,1988). However, it was only after a number of microalgae from our collection were screened that we were able to confirm a diatom source for these compounds (Volkman et al.,1994). Recent studies have extended the number of diatom species known to contain these compounds and we have identified some of the factors that influence the distribution of the different isomers that have been elucidated (e.g. Rowland et al.,1998; 2001). Of particular interest has been the finding that some diatom-derived HBI isomers have weak cytostatic activity towards cells of non-small-cell lung cancer. The pharmaceutical potential of HBIs has therefore attracted recent interest (Rowland et al.,1998).

4-5. Sterols

Like fatty acid distributions, sterol profiles vary markedly across the various algal classes and a great range of C26-C30 sterols have been reported (e.g. Volkman 1986, Volkman et al.1998 and references therein). Some sterols are found in only a few algal classes (e.g. 4α,23,24-trimethylcholest-22E-en-3β-ol (dinosterol) occurs mainly in dinoflagellates, 24-n-propylidenecholesterol seems to be a marker for some chrysophytes), whereas others such as cholesterol, 24-ethylcholesterol and 24-methylcholesta-5,22E-dien-3β-ol are quite widely distributed. Steroidal ketones are found in dinoflagellates, but have not been commonly reported in other microalgae. For example, Harvey et al.(1988) identified 21 steroidal ketones in Scrippsiella trochoidea and we found the same three main steroid ketones (dinosterone, dinostanone and 4α,23,24-trimethyl-5α-cholest-8(14)-en-3β-one) in Scrippsiella sp. (Mansour et al.,1999a). An interesting observation was that species from the genus Pavlova (prymnesiophyte) contain novel 3,4-dihydroxy-4α-methylsterols termed pavlovols (Fig. 8) as well as 4-methyl sterols and 5α(H)-stanols (Volkman et al.,1990; 1995). This finding indicates that 4-methyl sterols are not restricted to dinoflagellates as had been proposed earlier by some organic geochemists. The more recent isolation of pavlovols in other closely related species suggests that they may be useful taxonomic markers for species from the order Pavlovales (Volkman et al.,1997).

4-6. Microalgal Toxins A range of Australian marine and freshwater microalgae species are known to produce at least six classes of toxin (see Table 1 for summary). The most widely studied are the hepatotoxic microcystins and nodularins (Fig. 9) from freshwater and estuarine cyanobacteria and the neurotoxic paralytic shellfish toxins (PST) that are produced by freshwater cyanobacteria and marine dinoflagellates (Fig. 9). Toxins have the potential to affect drinking water supplies for human consumption and stock, along with several sectors of the seafood industry.

(A)Toxic cyanobacteria

51 Although toxic cyanobacterial blooms in freshwater supplies were recorded in Australia as far back as the 19th century (Francis, 1878), it was not until 1988 that the hepatotoxic compounds were identified as the nodularin group of peptides freshwater cyanos (Rinehart et al.,1988). Microcystis aeruginosa produces larger related peptide toxins, the microcystins which are commonly detected in lakes and rivers. Later still, PST were found to be the causative neurotoxic factor associated with stock deaths due to consumption of Ananaena circinalis (Humpage, et al.,1994; Negri and Jones, 1995). The alkaloid hepatotoxin cylindrospermopsin is associated with two freshwater cyanobacteria, Cylindrospermopsis raciborskii (Baker and Humpage, 1994) and Aphanizomenon ovalisporum (Shaw et al.,1999). Regular toxic events due to M. aeroginosa and A. circinalis in particular have resulted in the development of regulatory levels of microcystins and nodularins at 10 µg per litre and PSTs at 3

µg STXeq per litre respectively (Fitzgerald et al.,1999). Although, microcystins and PST are common and highly toxic, few significant human poisonings from these toxins have been reported in Australia. Regional and state monitoring programs are now a standard feature of drinking water management in Australia. Toxins from the marine cyanobacterium Lyngbya majuscula (Fig. 9) induce symptoms such as skin, eye and respiratory irritation (Osborne et al.,2001).

(B)Toxic marine microalgae Toxins produced by microalgae are also common in the marine environment, where they are best known for accumulating in shellfish and have the potential to poison humans who consume contaminated flesh (Sommer et al.,1937). In Australia, several species, including Gymnodinium catenatum (Oshima et al.,1993), Alexandrium catenella, A. tamarense (de Salas et al.,2001) and A. minutum (Oshima et al.,1989) are known to produce PST. These species are all bloom-forming and are most abundant in the temperate waters of coastal Australia. In general, the maximum allowable level of PST is 80 µg STXeq per 100 g shellfish flesh (AOAC, 1990). Shellfish containing higher levels are usually quarantined and may be fit for consumption after depuration of the toxins. The Tasmanian shellfish industry was the first Australian industry to suffer in the mid 1980s as a result of PST contamination from G. catenatum (Hallegraeff and Summer, 1986) and closures have been commonplace since this time. The benthic dinoflagellate Gambierdiscus toxicus is responsible for contamination of finfish with the toxin ciguatera in tropical waters. The gambiertoxins produced by the algae are converted to ciguatoxins (Fig. 9) in the food web (Lewis and Holmes, 1993). These toxins are extremely difficult to detect in contaminated fish and can accumulate in the human body, causing a wide range of neurological and gastrointestinal symptoms. Domoic acid (Fig. 9) from the diatom Pseudonitzschia sp. has been blamed for illness in about 40 consumers of pipis in a single event in New South Wales (Zantiotis-Linton, 1988).

(C)Toxic microalgae and the CSIRO collection of living microalgae The Collection maintains over 200 strains of toxic microalgal species and closely related non-toxic species from Australian fresh and coastal waters as well as some representatives from populations outside Australia. In many cases there are multiple strains isolated both within and

52 between populations. These include the PST producers G. catenatum, A. catenella and A. minutum as well as non-toxic A. tamarense strains. Strains held in the Collection have been used to identify toxin profiles of these species as well as factors influencing them e.g. G. catenatum (Oshima et al.1987, 1993.) and A. minutum (Oshima et al.1989, Negri et al.,2002).

O HO

H O H O N H2N HN NH H O H H N H H O HN HO O O N+ HN NH H H H H N N H + N H N N H H OH NH O H H O O O H OH HO H2N N microcystin LR H saxitoxin (PST) O

O H

OH - O N N OH H O H H HH O S O O O O NH H H H H NNH O O OH H NNHNNH H nodularin O O

H + N OH NH cylindrospermopsin H

HN NH2

H O N O N OH OH O

O O O O N H

O debromoaplsiatoxin OH lyngbiatoxin A

O HO

HO H H O H O OH H HO N O H H O O O domoic acid OH H H H H O H OOH H H OH HHH H O O

O ciguatoxin H H O O H H H H OH HO OH Fig. 9. Examples of toxins produced by microalgae.

53 The CSIRO Collection holds 150 strains of cyanobacteria including Microcystis aeruginosa, Anabaena circinalis and Nodularia spumigena. For the majority of these strains toxicity, or lack thereof, has been verified. Anabaena circinalis strains have been important in characterization of the PST produced by this species and the factors affecting toxicity (Negri et al., 1997). Alexandrium minutum from the CSIRO Collection has been used as a model organism to improve scale and yield in culture. This species is more robust than many other dinoflagellates, achieves high biomass in static culture, and has a well-characterised toxin composition. Its growth, mating system, and life cycle have been characterised from small-scale laboratory culture (e.g. Parker 2000a). We are able to achieve high densities of around 3.2 x 105 cells mL-1 and a productivity of 2.1 x 104 cells mL-1day-1, while maintaining the same level of cellular toxicity as static cultures (Parker et al.,2002a). It is also possible to use intelligent nutrient manipulation to increase biotoxin yield. In experiments also conducted on A. minutum, we were able to increase the cellular concentration of PSTs 10-fold by phosphate (P) limitation (Lippemeir et al.2002). Limiting P also reduced cell density; however subsequent spiking of the cultures with P stimulated cell division and resulted in an overall increase in toxin production. Under nutrient limitation, the fluorescence yields of A. minutum were reduced and a significant inverse correlation with cellular toxin content was evident. The use of in-line fluorometry may be valuable in managing efficient biotechnological production of valuable marine biotoxins from microalgae.

(D)Microalgal culture for biotoxin production The physiological and ecological roles of the toxins discussed remain unknown. However, other marine toxins such as tetrodotoxins and conotoxins are proving valuable in medical research and as pharmaceutical leads (Kem, 1997; Jones and Bulaj, 2000). As blooms of toxic algae are becoming more widespread, possibly due to factors such as increasing eutrophication and transportation via ballast water, toxin testing is becoming mandatory, requiring the supply of pure toxin standards for bioassays and chemical analyses. At present there are very few reliable sources of these pure toxin compounds that currently have a value of up to $2500 US per mg. Cultured cyanobacteria and dinoflagellates have the potential to supply commercial quantities of biotoxins for regulatory testing and research. The challenges are to increase the scale of production and maximise the yield of toxin.

4-7. Pigments

Microalgae provide a diverse array of carotenoid and chlorophyll pigments, including a number of newly discovered components. The CSIRO Collection of Living Microalgae has a prestigious history in microalgal pigment research due to Dr. Shirley Jeffrey’s esteemed career in pigment discovery, characterisation and applications (see Table 1 for some examples). The recent SCOR-UNESCO publication on phytoplankton pigments in oceanography (Jeffrey et al.1997) reviewed the state of the science and made it available to oceanographers and other scientists worldwide. The CSIRO Collection plays an important role in supplying the SCOR-UNESCO pigment reference cultures to researchers internationally. Another valuable development has been the software program CHEMTAX (Mackey et al.1996) that uses specific pigment ratios to ascertain the abundance of microalgal groups in seawater samples.

54 4-8. Long-chain Alkyl Diols

C30-C32 alkyl diols (Fig. 7) were first discovered in Black Sea sediments (de Leeuw et al.1981), with numerous reports then occurring on their occurrence in other sediments (e.g. Nichols and Johns, 1986; Versteegh et al.,2000). After initial suggestions that the diols were derived from cyanobacteria, work in our laboratories showed that these components occur in marine eustigmatophytes from the genus Nannochloropsis (Volkman et al.,1992). Subsequent work established that they are also present in freshwater eustigmatophytes (Volkman et al.,1999a) and that these compounds are probably building blocks for the biopolymeric algaenans found in these microalgae (Gelin et al.,1997). The modes of biosynthesis of these diols remain unknown although some clues are provided by the occurrence of long-chain alcohols and hydroxy fatty acids in these species (e.g. Volkman et al.,1999b).

4-9. UV Absorbing Compounds

Increasing levels of Ultraviolet-B radiation (UVB) reaching the earth’s surface as a result of anthropomorphic damage to the stratospheric ozone layer may be a threat to marine ecosystems. Shirley Jeffrey and collaborators (Jeffrey et al.,1999) examined 152 species (206 strains, 12 classes) of marine microalgae from the CSIRO Collection for UVA and UVB-absorbing compounds and were able to delineate three groups of species on the basis of their ratios of UV absorbance (280 to 390nm) to chlorophyll a (665nm); Low UV:CHL a ratios (0.18 to 0.9, diatoms, green algae, cyanophytes, euglenophytes, eustigmatophytes, rhodophytes, some dinoflagellates, some prymnesiophytes), intermediate ratios (0.9 to 1.4, chrysophytes, some prasinophytes, some prymnesiophytes), very high ratios (1.4 to 6.75, surface bloom forming dinoflagellates, crytomonads, prymnesiophytes and raphidophytes). These UV-screening pigments varied across species of the same algal class and strains of the same species. The highest UV-screening capacity was found in surface bloom forming species of dinoflagellates, particularly strains of G. catenatum, Alexandrium, Heterocapsa, Scrippsiella and Wolozynskia. Four out of five species analysed for mycosporine-like amino acids, compounds that have been shown to protect against UV damage in other marine plants and animals, tested positive (Jeffrey et al.,1999). The above examples highlight the range of lipids including unusual compounds, toxins, pigments and other bioactive molecules found across the various microalgal classes. We expect that discovery of such components will continue, and that the biotechnological application of these will likely provide important research and development opportunities. The role of many of the unusual lipids within microalgae remains to be determined, and research in this area will also be fruitful as will application of these unusual components as signatures in environmental research.

5. MICROALGAL PRODUCTION AND HARVESTING

Microalgae are widely recognized as having great potential for biotechnological exploitation. Owing to their extensive biological and chemical diversity (examples of which are shown above), microalgal application areas include aquaculture and livestock feeds, high value products in the

55 pharmaceutical, nutraceutical, cosmetics and biomedical industries (Table 1) and as model organisms in biosynthesis and energy transfer processes. Despite this potential, examples of the successful long-term commercial application of microalgae are rare. A major constraint for commercial application of microalgae is their amenability to mass cultivation, the successes and failures of which have been reviewed on several occasions. These reviews, including reasons for the low number of commercially successful species, show the evolution from photoautotrophic high volume/low density open pond systems (Goldman, 1979), to low volume/high density photobioreactor systems (e.g. Tredici, 1999; Richmond, 2000; Pulz, 2001) and more recently to photoheterotrophic (mixotrophic) and heterotrophic cultivation systems (Lee, 2001; Ogbonna and Tanaka, 2001). The efficient utilization of light as well as the degree of culture perturbation and oxygen saturation are identified as essential generic considerations in the transition from laboratory scale systems through to commercially viable production. Less emphasized, yet no less important, is the species-specific nature in which these considerations must be applied as part of the culture system design and testing process. For a greater number of microalgal applications to be explored, species-specific approaches to high-density mass cultivation are required. Using various laboratory scale photobioreactors, the high biomass production potential of several microalgae species from the CSIRO Collection has been studied. Based on photobioreactors from Tredici’s (e.g. Tredici et al.,1991; Chini Zittelli et al.,2000) and Richmond’s (e.g. Hu and Richmond, 1996; Cheng-Wu et al.,2001) research groups, reactor types including plastic vertical alveolar panel (VAP), vertical annular column (VAC) and vertical glass panel (VGP) designs were used. Species tested include a paralytic shellfish toxin (PST) producing dinoflagellate (Alexandrium minutum), a tropical diatom (Skeletonema sp.) and an unidentified tropical cryptophyte (Table 2). With limited culture optimization, the most successful semi-continuous trials to date (based on long-term productivity and reliability) have been with Skeletonema production in VAC photobioreactors, giving an average areal productivity over a 60 day period of 4.05 gm-2d-1. Production of PUFA, e.g. EPA production by Skeletonema sp. (Table 1), has also been compared between traditional carboy culturing methods and photobioreactor trials, with VAC- and VGP-grown biomass producing at least three times more EPA per equivalent dry biomass. The greatest areal biomass and EPA productivities were achieved by VGP-grown Skeletonema with 6.25 gm-2d-1 and 42.7 mgm-2d-1, respectively, although individual trials did not last for more than 15 days. An important component of microalgal production research is the need for comparison between photobioreactor function and application, photosynthetic productivity and efficiency, species’ capability and cost effectiveness amongst research groups. Making these comparisons, however, has proven difficult due to historical and regional inconsistencies in reporting production “success”. For example, methods used thus far to express production achievements range from maximum biomass in gL-1 to volumetric productivity in gL-1d-1 and areal productivity in gm-2d-1 (in which case either illuminated surface area or ground area needs to be specified), and more recently to photosynthetic efficiency – expressed either as a percentage of photosynthetically active radiation, gmol photon-1 or gMJ-1. Furthermore, few investigators have gone so far as to attempt an estimation of the cost

56 effectiveness of microalgal production (e.g. US$ kg-1 dry weight biomass): the ultimate indication of production “success” as applied to commercial applications. Reasons for the latter are many fold, although significant contribution relies on the fact that many research groups have not performed investigations beyond the laboratory scale and/or outdoors (Richmond, 2000) (a complicated issue in itself) and also because of the location-specific cost of labour and input energy – two vital ingredients in microalgal mass cultivation. Preparation of concentrates by a flocculation method has been investigated for CSIRO microalgae, representing several microalgal classes (Knuckey, 1998), with success at medium to large scales (10-300 L) being achieved using diatom species in particular (Brown and Robert, 2002; DeSouza et al.,2002). Cell recovery efficiencies have ranged from approximately 75-95%, depending on the algal species, with an associated reduction in time required to produce concentrates (cells concentrated ca. 100 times) compared with traditional harvesting techniques e.g. centrifugation and/or filtration. Consequently, recent Pacific oyster (Crassostrea gigas) feeding trials, using VAC-grown fresh and flocculated Skeletonema as supplementary diets, have been conducted for validation of high biomass production and harvesting methods as well as biomass quality (data not shown). Future microalgae high density and scale-up work within CSIRO’s Microalgae Research Centre includes: high biomass production testing of new species; growth optimization of species already tested; development of microalgal cell recovery systems with greater efficiency; feeding trials on current and new target organisms using novel and/or optimized microalgae and their products.

6. RESEARCH ON COMMERCIAL MICROALGAE Australia is a major supplier of β-carotene isolated from the halotolerant green microalga Dunaliella salina grown in open ponds in South and Western Australia (Borowitzka, 1999). In a recent collaboration with industry, we examined seasonal changes in the abundance of fatty acid, sterol and hydrocarbon profiles within the open ponds. Polar lipid was the main lipid class present in all algal concentrates. Hydrocarbon (predominately β-carotene) was the next most abundant class, with considerable variation occurring over time and to a lesser extent between ponds sampled on the same date. The highest hydrocarbon levels were generally observed over spring and summer. The main fatty acids in pond samples were 18:3(n−3), 16:0, the unusual 16:4(n-3) and 18:2(n-6); these four components accounted for 82% to 87% of the total fatty acids. In comparison to the fatty acid profile of D. tertiolecta grown at 20oC (Volkman et al.1989), D. salina contained higher relative levels of saturated fatty acids (SFA) and lower levels of polyunsaturated fatty acids (PUFA). As levels of PUFA are generally inversely related to temperature, these findings are consistent with the higher temperatures used to grow D. salina. The main two sterols in all pond samples were 24-ethylcholesta-5,7,22-trien-3β-ol (7-dehydroporiferasterol) and 24-methylcholesta-5,7,22-trien-3β-ol (ergosterol). For the lipid class, hydrocarbon, fatty acid and sterol profiles, only limited variation occurred between and within ponds; exceptions were the level of total hydrocarbons and free fatty acids, and vitamin E. This information is pertinent to examining how such changes, when occurring, may influence processing of both algal concentrates and products.

57 Table 1. Research and application areas concerning microalgae from the CSIRO Collection of Living Microalgaea.

Application area CSIRO strain Australian Compound Species example Referencea Target organism code isolate Pigments Molecular & medical Chlorophylls science; human health Ditylum brightwellii CS–131 + 1 Carotenoids Dunaliella salina CS–265 + 2 Emiliania huxleyi CS–57 - 3 Biliproteins Rhodomonas salina CS-24 + 2 Polyunsaturated fatty acids (PUFA) Aquaculture; human 18:3(n-3) health Dunaliella tertiolecta CS–175 - 4 18:4(n-3) Rhodomonas salina CS–24 - 4 20:5(n-3) Nannochloropsis oculata CS–216 - 5 22:6(n-3) Fragilidium sp. CS–382 + 6 Toxins Environmental and Cylindrospermopsin human health Cylindrospermopsis raciborskii CS–505 - 7 Microcystins Microcystis aeruginosa CS–563 + 8 Nodularins Nodularia spumigena CS–591 + 9 PST – marine Alexandrium minutum CS–324 + 10 PST – freshwater Anabaena circinalis CS-337 + 11 Vitamins Aquaculture; human Ascorbic acid health Chaetoceros muelleri CS–176 - 12, 13 α -tocopherol Nannochloropsis sp. CS–246 - 13 Retinol Tetraselmis sp. CS–362 + 13 Whole cell biomass Aquaculture Juvenile abalone Navicula jeffreyi CS–46 + 14 Larval pearl oysters Pavlova lutheri CS-182 - 15 Zooplankton Rhodomonas salina CS–24 + 14 Larval prawns Skeletonema sp. CS–252 + 14 Larval pacific oysters Thalassiosira psuedonana CS–20 + 14 Unusual compounds Various Hydrocarbons (e.g. HBI alkenes) (e.g. Environmental Haslea ostrearia CS–250 + 16 biomarkers) Rhizosolenia setigera CS–389/A + 16, 17 Long-chain alkyl diols Nannochloropsis oculata CS–179 - 5 Vischeria punctata CS–142 - 18 Emiliania huxleyi CS–57 - 19, 20

58 Long-chain unsaturated ketones Gephyrocapsa oceanica S–335 + 19, 20 Diacronema vlkianum CS–266 - 21 Pavlovols Pavlova pinguis CS–375 + 21 Scrippsiella sp. CS–295 + 6 Steroidal ketones Gymnodinium catenatum CS–301/09 + 22 Novel PST Fibrocapsa sp. CS–220 + 23 UV-absorbing compounds Gymnodinium catenatum CS–309/01 + 23 Woloszynskia sp. CS–341 + 23 Fragilidium sp. CS–382 + 6, 24 Very long-chain highly unsaturated Gymnodinium catenatum CS–389/01 + 6, 24 fatty acids (VLC-HUFA) Prorocentrum mexicanum CS-292 + 6, 24 Symbiodinium microadriaticum CS–155 - 6, 24

a References: 1, Stauber and Jeffrey (1988); 2, Jeffrey and Vesk (1997); 3, Jeffrey and Wright, 1994; 4, Volkman et al. (1989); 5, Volkman et al. (1992); 6, Mansour et al. (1999a); 7, Saker et al. (1999); 8, Bolch and Blackburn (1996); 9, Jones et al. (1994); 10, Parker et al. (in press); 11, Negri et al. (1997); 12, Brown & Miller (1992); 13,

59 Brown et al. (1999); 14, Brown et al. (1997); 15, Johnston (pers. comm.); 16, Volkman et al. (1994); 17, Rowland et al. (2001); 18, Volkman et al. (1999b); 19, Volkman et al. (1980); 20, Volkman et al. (1995a); 21, Volkman et al. (1997); 22, Negri et al. (2001); 23, Jeffery et al. (1999); 24, Mansour et al. (1999b)

Table 2. Photobioreactor growth trials using microalgae from the CSIRO Collection of Living Microalgaea

Light Path Biomass Volumetric Arealc Average EPA Photobioreactor Average EPA production Species Length Concentration Productivity Productivity Productivity Designb (mg.100g dry biomass) (cm) (g.L-1) (g.L-1.d-1) (g.m-2.d-1) (mg.m-2.d-1)

Skeletonema sp. Carboy (batch) 25 0.25 - - 200 -

Alexandrium minutum VAP 1.2 1.78 0.06 0.73 - -

Skeletonema sp. VAP 1.2 1.45 0.17 2.07 235 49 Unidentified cryptomonad VAP 1.2 0.81 0.11 1.32 - -

Skeletonema sp. VAC 2.0 1.36 0.26 4.05 600 24.3

Skeletonema sp. VGP 2.5 1.40 0.25 6.25 683 42.7

a All cultures harvested semi-continuously unless otherwise stated. Duration of trials range from 15 to 60 days after first harvest b VAP = Vertical alveolar panel (4.5 L capacity); VAC = Vertical annular column (13 L capacity); VGP = Vertical glass panel (5 L capacity). c Data based on illuminated surface area.

59 7. MICROALGAL GENE AND COMPOUND DISCOVERY

Whilst microalgae are producers of important PUFA (e.g. long-chain n-3) and other biomolecules, our nascent ability to cultivate them on a large scale combined with their relatively low yields of product, necessitate the investigation of other strategies. Terrestrial crop plants, the product of thousands of years of artificial selection to be efficient abundant producers, would serve as useful hosts in which to produce microalgal biomolecules at high levels. As far as lipids are concerned, most crop plants make neither long nor n-3 desaturated PUFA. By isolating elongase and desaturase encoding genes from microalgae it should be possible to introgress these traits into oilseed crops. Similar methodology would enable the production of other microalgal biomolecules, such as certain carotenoids. Although microalgae as we know them are diverse in their production of biomolecules, there is an adundance of biodiversity that remains as yet unexplored. The challenge is to discover which microalgae make interesting compounds. One approach that we are taking is to devise mass screenings of microalgal samples that select for strains that carry novel biochemistries.

8. THE FUTURE

In 2002 Environment Australia, through the Australian Biological Resources Study (ABRS), commissioned a project to develop a 10 year workplan for nationally integrated research strategies to advance knowledge of algae, protista and fungi. This is due to the recognition that information about the taxonomy of these groups is essential for the conservation and sustainable use of Australia’s biodiversity. The CSIRO Collection of Living Microalgae, through fundamental isolation and culture maintenance, continues to provide much insight into the genetic variation and ecological dynamics within and between populations of a variety of economically important Australian microalgae. As well, the research areas of aquaculture, biotechnology and environmental issues demonstrate the fundamental importance of microalgae and the value of the Collection as a living library and research resource.

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Appendix 1 Culture Collections maintaing living microalgae

65 WDCM #, (Collection Acronym) Number of strains held Country Collection Name and Organization (# endemic to country)

WDCM532 (CS) CSIRO Culture Collection of Australia >800 strains (500), 140 Microalgae, CSIRO Marine Laboratories, Hobart, Tas genera, ~350 species, all major algal groups. WDCM598 (MUACC) Murdoch University Algal Australia ~250 (225) strains of Culture Collection, School of Biological Sciences and algae, of some Biotechnology commercial interest. (UTAS) Harmful Algae Culture Collection, School of Australia Plant Science, University of Tasmania, Hobart, Tasmania. (ACCM) The Australian Collection of Marine Australia Microorganisms, Dept of Biomedical & Tropical Veterinary Science, James Cook University, Townsville, QLD (ACM) Australian Collection of Microorganisms, Center Australia for Bacterial Diversity and Identification, Dept of Microbiology, University of Queensland, Brisbane, Queensland (AWQC) Australian Water Quality Centre, Salisbury, Australia ~400 from 10 genera of South Australia cyanobacteria (MUCC) Melbourne University Culture Collection, Australia School of Botany, University of Melbourne, Parkville, Melbourne, Victoria WDCM794 (CMBGCAS) Collection of Marine China 138 (97) Biological Germplasm, Institute of Oceanology, Chinese Academy of Sciences, Qingdao,Shandong General Microbiological Culture Collections Center, China Institute of Microbiology, Zhongguancun, Beijing China Center for Type Culture Collection, Wuhan China University, Luo Jia Shan, Wuhan, Hubei, (CAP) Collection of Asian Phytoplankton, Institute of China Oceanology Academia Sinica, Qinddao

(FACHB) Freshwater Algae Collection, Institute of China Hydrobiology, The Chinese Academy of Sciences, Wuhan (JINAN) Institute of Hydrobiology Algal Collection, China Jinan University, Guangzhou

(MACC) Marine Algal Culture Collection, Ocean China University, Aquaculture Food Organism Research Laboratory, Qingdao (CSIR) National Collection of Industrial India 15

66 Microorganisms, Biochemistry Division, National Chemical Laboratory, Poona Mahasahtra

WDCM632 (BTCC) Biotechnology Culture Collection Indonesia 7 (5) Institution Pusat, Research and Development Center for Biotechnology, Indonesian Institute of Sciences, Cibinong, West Java

(ITBCC) Institute of Technology Bandung Culture Indonesia 12 (12) Collection, Laboratory for Microbiology & Fermentation Technology, Bandung,

WDCM591 (NIES) Microbial Culture Collection, Japan Algae 840 (694), National Institute for Environmental Studies, Tsukuba, Protozoa 2 (2) Ibaraki

WDCM190 (IAM) Institute of Applied Microbiology, Japan University of Tokyo

Marine Biotechnology Institute Japan

(FAUT) Faculty of Agriculture, University of Tokyo Japan

(KAGAWA) Akashiwo Research Institute of Kagawa Japan Prefrecture, Yashima-Higashi-machi, Takamatsu

(KUCC) Kyuto University Culture Collection, Japan Laboratory of Microbiology, Department of Fisheries, Kyoto

(MBI) Marine Biotechnology Institute, Kamaishi Japan Laboratories, Kamaishi-shi, Iwate

(MSTU) Department of Marine Science, School of Japan Marine Science and Technology, Tokai University, Orido, Shimizu, Shizuoka

WDCM765 (DBUM;IPT) Department of Biochemistry, Malaysia 300 Faculty of Medicine, University of Malaya, Kuala Lumpur

(CAW) Cawthron Micro-algae Culture Collection. New Zealand 157, 98 strains of Cawthron Institute, Nelson. dinoflagellates from 38 species and 15 genera Pakistan Type Culture Collections, Biotechnology & Pakistan Food Research Center, Pakistan Council of Scientific & Industrial, Lahore, Punjab

WDCM39 (MCC-UPLB) Microbial Culture Collection, Philippines 235 (235) Museum of Natural History, University of the Philippines Los Banos

WDCM444 (DBUP) Algal Culture Collection, Museum Philippines 20 of Natural History, Laguna

(IRRI) Blue-green Algal Culture Collection, Philippines

67 International Rice Research Institute, Soil Microbiology Division, Manila

(MSIUP) Marine Science Institute, University of the Philippines Philippines, Diliman, Quezon City

University of Jaffna, Dept. of Botany, Jaffna Sri-Lank (TML) Tungkang Marine Laboratory, Taiwan Fisheries Taiwan Research Institute, Tungkang, Pingtung

WDCM676 (IFRPD) Institute of Food Research and Thailand 200 Product Development, Kasetsart University, Bankok

WDCM783 (BCC) BIOTEC Culture Collection, Thailand 150 (150) National Center for Genetic Engineering and Biotechnology, Bangkok

WDCM692 (MSCMU) Microbiology Section, Chiang Thailand 18 Mai University Department of Biology, Faculty of science, Thailand

WDCM491 (BSMB) Bacteriology and Soil Thailand 15 Microbiology Branch, Div. Plant Pathology, Dept. of Agriculture, Bangkok

(TISTR) Thailand Institute of Scientific and Thailand Technological Research Culture Collection, Bangkok MIRCEN, Chatuchak, Bangkok,

(VIETNAM) National Scientific Research Centre of Vietnam Vietnam, Institute of Biology, Tu Liem, Ha Noi

WDCM505 (ASIB) Algensammlung am Institut fur Austria 1570 Botanik, Universitat Innsbruck

WDCM728 (IOUSP) Marine Microalgae Culture Brazil 159 strains, 24 genera Collection, Instituto Oceanografico da USP, Sao Paulo from main algal groups

WDCM535 (NEPCC) North-East Pacific Culture Canada Several hundred isolates Collection, Dep’t of Botany, University of British from all major algal Columbia Vancouver, B.C. groups

WDCM605 (UTCC) University of Toronto Culture Canada ~400 isolates, primarily Collection of Algae and Cyanobacteria, Department of freshwater algae and Botany, Ontario cyanobacteria WDCM348 (CCALA) Culture Collection of Autotrophic Czech 315 algae, 207 Organisms Czechoslovak Acad’ of Sci’, Institute of cyanophytes Botany, Department Hydrobotany, Dukelska Trebon,

WDCM486 (CCA, CAUP) Culture Collection of Algae Czech 200 Department of Botany, Faculty of Science, Charles University, Prague

WDCM792 (ALCP) Algotheque du Laboratoire de France 600

68 Cryptogamie, Museum National d'Histoire Naturelle, Paris

WDCM829 (RCC) Roscoff Culture Collection, Station France 500 marine strains, Biologique de Roscoff Phytoplancton emphasis on picoplankton, Prochlorococcus, Synechococcus and picoeucaryotes

Culture Collection of Conjugatophyceae (Chloropyta) Germany 490 (SVCK), Institut f. Allgemeine Botanik University of Hamburg

WDCM192 (SAG) Sammlung von Algenkulturen, Germany 2141 strains, 1228 Albrecht-v.Haller-Institut, Gottingen species and 486 genera from all main groups (67% Green algal line) CCAC Culture Collection of Algae at the University of Germany 100 (41) Cologne

WDCM147 (CSMA) Centro di Studio dei Italy 58 Algae, 69 Microorganismi Autotrofi - CNR, Instituto di Cyanobacteria Microbiologia Agraria e Tecnica Universita degli Studi -Firenze

WDCM500 (CDBB) Coleccion del Departmento de Mexico 22 Biotechnologia y Bioingenieria, Centro de Investigacion y de Estudios Avanzados-IPN

WDCM498 (NIVA) Culture Collection of Algae, Norway 242 Algae, 450 Norwegian Institute for Water Research, Kjelsas, Oslo Cyanobacteria

WDCM461 (CALU) Collection of Algal Cultures of Russia 704 strains (145) Leningrad University

WDCM641 (PGC) Peterhof Genetic Collection of Russia 700 Microalgae, Biological Research Institute of Leningrad

WDCM596 (IPPAS) Collection of Microalgae of the Russia 300 (250) Institute of Plant Physiology, K.A. Timirjazev Institute of Plant Physiology, Moscow

WDCM602 (BOROK) The Collection of Algae, Institute Russia 15 strain names for Biology of Inland Waters Academy of Sciences of Russia

WDCM657 (DGUB) Department of Genetics, Slovakia 120 (40) University of Bratislava, Faculty of Natural Sciences Comenius University, Bratislava

WDCM522 (CCAP) Culture Collection of Algae and UK 540 marine strains

69 Protozoa, Dunstaffnage Marine Laboratory, Oban, Argyll

WDCM128 (PLYMOUTH) Plymouth Culture UK 475 Collection, Marine Biological Association of the UK, Devon

WDCM140 (CCAP) Culture Collection of Algae and UK 1200 Freshwater strains, Protozoa, Institute of Freshwater Ecology, Windemere, (800) Cumbria

WDCM1 (ATCC) American Type Culture Collection, USA 88 algal strains Virginia

WDCM533 (ACF) Algal Culture Facility University of USA 25 South Carolina

WDCM606 (UTEX) Culture Collection of Algae at the USA 2000 from all major University of Texas at Austin, Dept Botany Austin, Texas groups (diversity greatest in Chlorophytes) WDCM530 (LMS) Carolina Biological Supply Co. USA 165

WDCM2 (CCMP) Provasoli-Guillard National Center USA 1450 strains, mainly for Culture of Marine Phytoplankton, Bigelow Lab, planktonic, some benthic

(FDCC) The Loras College Freshwater Diatom Culture USA 1200 cultures, 64 genera, Collection, Dept Biology, Dubuque, IA 350 species

All collections in the Asia-Pacific region followed by only those collections in rest of world where number of strains is available. Information sourced from the WFCC-MIRCEN World Data Centre for Microorganisms (WDCM), {http://wdcm.nig.ac.jp/}, UNESCO Manual on Harmful Marine Microalgae (1995) and internet sources.