Journal of Applied Phycology 10: 481501, 1999. 1999 Kluwer Academic Publishers. Printed in the Netherlands.

481

Cryopreservation of eukaryotic algae a review of methodologies

Rebecca Taylor1, & Robert L. Fletcher2
1 Department 2 Institute

of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK of Marine Sciences, University of Portsmouth, Ferry Road, Eastney, Portsmouth, Hampshire, PO4 9LY,

UK ( Author for correspondence: e-mail: r.taylor@dundee.ac.uk)

Received 10 July 1998; revised 28 August 1998; accepted 29 August 1998

Key words: cryopreservation, microalgae, macroalgae, cryoprotectant, cooling rate, freezing tolerance, storage

Abstract Since pioneering work in the early 1960s, there has been growing interest and numerous experimental investigations into the cryopreservation of algal material. Mostly, these studies relate to the requirement for long term preservation and storage of algal material contained in culture collections or used in the seaweed mariculture industry. The present review deals with techniques used in the cryopreservation of biological samples and their application to both micro- and macroalgae. Methods for the prevention of cell damage and freezing injury during the cooling and low-temperature storage of algal material are discussed with reference to the effect on viability of such variables as cooling rates, nal temperatures attained, the use of various types and concentrations of cryoprotectants, thawing rates, and storage times and temperatures. Some consideration is also given to the various methods used for increasing cell viability, including the induction of freezing tolerance. Cryopreservation protocols employed by numerous workers in this eld are detailed, and concluding remarks are made on those techniques and conditions providing optimum viability of cryopreserved algae.

Introduction

The general effects of freezing The major effect of reduced temperatures on any system is the reduction of molecular motion which, for biological systems, has important consequences. The inhibitory effects of low temperatures on chemical and physical processes provide the basic means for achieving long-term preservation of cells, tissues and organs. This ultimate goal, however, is not easy to achieve and workers attempting to freeze algal material, particularly those working with macroalgae, have often had limited success. This is perhaps unsurprising, since the cooling of cells below 0 C brings about dramatic changes in biological systems, often resulting in irreversible cell damage. Due to the complexity of biological systems, cell damage during the freezing process occurs due to a number of different mechanisms (Karlsson & Toner, 1996). The high concentration of electrolytes and

Cryopreservation refers to the maintenance of biological samples in a state of suspended animation at low (i.e. cryogenic) temperatures (Karlsson & Toner, 1996). The main aim of this process is to preserve organisms without change in their morphological, physiological, biochemical and genetic properties (Saks, 1978). Since the pioneering work of Polge et al. (1949) many organisms, and indeed parts of organisms, from both plant and animal kingdoms have been successfully cryopreserved. In general, however, comparitively little attention has been given to the storage of algae at cryogenic temperatures. The following, therefore, presents a review of cryopreservation studies carried out on both micro and macroalgae, with particular respect to discussing the methodologies used.

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Table 1. Cryopreservation studies on microalgae. Alga RHODOPHYTA Bangiophyceae Porphyridium sp. HETEROKONTOPHYTA Xanthophyceae Chlorocloster engadinensis Heterococcus caespitosus Tribonema sp. Eustigmatophyceae Monodopsis subterranea Nannochloropsis sp. Bacillariophyceae Actinocyclus subtilis Asterionella formosa Attheta decora Aulacoseira italica Chaetoceros sp. Cyclotella cryptica Cylindrotheca sp. Ditylum brightwelli Fragilaria crotonensis Fragilaria pinnata Nitzschia sp. Phaeodactylum tricornutum Reference

Leibo & Jones, 1963; Holm-Hansen, 1963

Day, 1998 Day, 1998 Holm-Hansen, 1963 Day, 1998 Caavate & Lubian, 1994, 1997a; Hirata et al., 1996 McLellan, 1989 McLellan, 1989 McLellan, 1989 McLellan, 1989 McLellan, 1989; Caavate & Lubian, 1994; 1997a; Cordero & Voltolina, 1997 McLellan, 1989; Day, 1998 Saks, 1978 McLellan, 1989 McLellan, 1989 McLellan, 1989 Tsuru, 1973; Saks, 1978; McLellan, 1989 Tsuru, 1973; Saks, 1978; Gilboa & Ben-Amotz, 1979; Ben-Amotz & Gilboa, 1980a, b; McLellan, 1989; Cordero & Voltolina, 1997; Day, 1998 McLellan, 1989

Stephanodiscus sp. HAPTOPHYTA Haptophyceae Cricosphaera roscoffensis Isochrysis galbana CRYPTOPHYTA Cryptophyceae Chroomonas placoidea Rhodomonas sp. EUGLENOPHYTA Euglenophyceae Euglena sp.

Hirata et al., 1996 Caavate & Lubian, 1995 a, b; Day, 1998

Day, 1998 Caavate and Lubian, 1994, 1997a; Hirata et al., 1996

Hwang & Horneland, 1965; Morris & Canning, 1978; Day et al., 1997; Day, 1998

CHLOROPHYTA Prasinophyceae Tetraselmis sp.

Gilboa & Ben-Amotz, 1979; Ben-Amotz & Gilboa, 1980a, b; Fenwick & Day, 1992; Day & Fenwick, 1993; Caavate & Lubian, 1994, 1997a; Day et al., 1997; Day, 1998

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Table 1. Continued. Alga Chlorophyceae Ankistrodensmus sp. Brachiomonas submarina Bracteacoccus sp. Carteria inversa Chlamydomonas reinhardtii Reference

Chlamydomonas sp. Chlorella sp.

Chlorococcales (selection) Chlorococcum infusionum Coccomyxa sp. Dunaliella sp. Hormidium accidum Nannochloris sp. Neochloris sp. Selenastrum capricornutum Scenedesmus sp. Stigeoclonium sp. Volvox aureus Klebsormidiophyceae Stichococcus bacillaris

Holm-Hansen, 1963; Hwang & Horneland, 1965; Morris, 1978; Bodas et al., 1995; Day, 1998 Hirata et al., 1996 Holm-Hansen, 1963 Hirata et al., 1996 Hwang & Horneland, 1965; Hwang & Hudock, 1971; Gresshoff, 1977; McGrath & Daggett, 1977; Bodas et al., 1995; Hirata et al., 1996 Bodas et al., 1995; Hirata et al., 1996 Holm-Hansen, 1963; Hwang & Horneland, 1965; Tsuru, 1973; Morris, 1976a, b; Gilboa & Ben-Amotz, 1979; Ben-Amotz & Gilboa, 1980a, b; Bodas et al., 1995; Hirata et al., 1996; Day et al., 1997 Morris, 1978 Holm-Hansen, 1963 Hwang & Horneland, 1965; Bodas et al., 1995 Saks, 1978; Ben-Amotz & Gilboa, 1980a, b; Mortain-Bertrand et al., 1996; Hirata et al., 1996 Holm-Hansen, 1963 Saks, 1978; Ben-Amotz & Gilboa, 1980a, b; Caavate & Lubian, 1994, 1997a; Hirata et al., 1996 Holm-Hansen, 1963; Bodas et al., 1995 Day, 1998 Hwang & Horneland, 1965; Tsuru, 1973; Morris, 1978; Benhra et al., 1994; Bodas et al., 1995 Holm-Hansen, 1963 Hirata et al., 1996 Holm-Hansen, 1963

other solutes in the extracellular medium and the resulting dehydration through the removal of water as ice has been proposed as a source of cell damage (Lovelock, 1953; Mazur et al., 1972), whilst other theories have focused on the potentially damaging effect of cell shrinkage as a response to a highly concentrated extracellular solution (Meryman, 1970; Steponkus et al., 1983). The osmotically induced ow of water through the cell membrane has also been proposed as a potential source of damage (Muldrew & McGann, 1990). It is generally believed, however, that the formation of ice crystals during the freezing process is the fundamental cause of severe cell damage, with differences in water activity, nucleation site viscosity, membrane permeability and many other factors sig-

nicantly affecting the nucleation and growth of ice in cells and tissues (Meryman, 1966; Snell, 1991). Based on observation, Mazur et al. (1972) proposed the two factor hypothesis of freezing damage, according to which there are two independent mechanisms of damage during freezing, one active at slow cooling rates, the other at fast cooling rates (Karlsson & Toner, 1996). Mazur et al. (1972) suggest that slow cooling rates tend to encourage the formation of large, mechanically damaging, external ice crystals, whilst rapid cooling rates tend to encourage the formation of damaging intracellular ice crystals. The mechanical effects of freezing, and causes of freezing injury are further discussed by Karlsson and Toner (1996).

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Table 2. Cryopreservation studies on macroalgae. Alga RHODOPHYTA Bangiophyceae Porphyra dentata Porphyra haitanensis Porphyra linearis Porphyra miniata Porphyra pseudolinearis Porphyra tenera Porphyra yezoensis Florideophyceae Chondrus crispus Devaleraea ramentacea Gracilaria foliifera Gracilaria tikvahiae Palmaria palmata HETEROKONTOPHYTA Phaeophyceae Fucus edentatus Laminaria digitata Undaria pinnatida CHLOROPHYTA Ulvophyceae Enteromorpha intestinalis Ulva lactuca Reference

Kuwano et al., 1994 Kuwano et al., 1994 Arbault & Delanoue, 1994 Day, 1998 Kuwano et al., 1994 Migita, 1964, 1966, 1967; Kuwano et al., 1994, 1996 Kuwano et al., 1992, 1993, 1994, 1996 Van der Meer & Simpson, 1984 Van der Meer & Simpson, 1984 Van der Meer & Simpson, 1984 Van der Meer & Simpson, 1984 Van der Meer & Simpson, 1984

Bird & McLachlan, 1974 Vigneron et al., 1997 Arbault et al., 1990; Ginsburger-Vogel et al., 1992; Renard et al., 1992

Terumoto, 1961 Van der Meer & Simpson, 1984

Cryopreservation studies on algae The effect of subzero temperatures and freezing on algae has been the subject of relatively few reports since Cohn (1871) rst reported the tolerance of the green alga Nitella to a temperature of 20 C, and there are still wide gaps in our knowledge. Tables 1 and 2 list both the microalgae and macroalgae respectively which have been investigated to date. (Taxonomic arrangement is according to van den Hoek et al., 1995). Fully documented reports on the freezing of algal cells were rst published in the early 1960s, HolmHansen (1963), Leibo and Jones (1963) and Hwang and Horneland (1965), for example, reporting on the successful cryopreservation of a range of microalgae. During this period, Terrumoto (1961) similarly reported the successful freezing of a macroalga, the green alga Enteromorpha intestinalis. Subsequently, however, most of the work on algal cryopreservation has involved the use of unicellular species, and a wide

range of groups have been studied (Table 1) with variable success rates reported. In comparison, research into the cryopreservation of macroalgae is not as extensive, and as such there are relatively few literature reports pertaining to macroalgal freezing (Table 2). Genera from each of the three macroalgal groups have been studied, with the Rhodophyta being the most well represented. Species of the commercially important genus Porphyra have been particularly well studied, both by early workers and in more recent reports.

Freezing methodology Over the years, a variety of organisms and cells have been successfully cryopreserved and although there are similarities, the techniques used to freeze and recover various taxa are almost as varied as the organisms themselves (Bajaj & Reinert, 1977; Leibo & Mazur, 1978; Maurer, 1978; Karha, 1981). Response to freezing varies from organism to organism,

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Table 3. Freezing techniques and methodologies used in the cryopreservation of microalgae. Reference Alga Freezing procedure Storage time and temp. Momentary exposure to end temperature Reported viability 030% 0100% 082% 10 to 100 min 1 month to 36 months at 196 C some recovery up to 100%

Holm-Hansen, 1963

Extensive selection of unicellular Chlorophyceae

Leibo & Jones, 1963 Hwang & Horneland, 1965

Porphyridium cruentum Ankistrodesmus sp., Chlamydomonas pseudagloe, Chlorella sp., Coccomyxa sp., Euglena sp., Scenedesmus sp.,

Hwang & Hudock, 1971

Chlamydomonas reinhardtii

Tsuru, 1973

Morris, 1976a, b McGrath & Daggett, 1977 Gresshoff, 1977

Chlorella sp. Scenedesmus sp. Nitzschia sp. Phaeodactylum tricornutum Chlorella sp. Chlamydomonas reinhardtii Chlamydomonas reinhardtii

1. Uncontrolled fast freezing by plunging to 196 C 2. Uncontrolled freezing to a range between 10 C & 70 C 3. Uncontrolled freezing to 25 C 1. Rapid cooling to 76 C 2. Slow cooling to 76 C 1. Slow cooling to 30 C, then plunging to 79 C 2. Uncontrolled fast freezing to 79 C 3. Slow cooling to 30 C then plunging to 196 C 1. Slow cooling to 40 C, then plunging to 196 C 2. Rapid cooling to 25 C, holding for 20 min then plunging to 196 C Uncontrolled fast freezing by plunging to 196 C

6 to 17 months at 196 C

viable colonies recovered

24 h at 196 C

7590%

Morris, 1978

Extensive selection of Chlorococcales Euglena gracilis

Morris & Canning, 1978

Saks, 1978

Nitzschia sp. Cylindrotheca sp. Phaeodactylum tricornutum Nannochloris sp. Dunaliella salina

Slow cooling to 60 C then plunging to 196 C Slow cooling to 55 C, then plunging to 196 C 1. Slow cooling to 20 C, then plunging to 75 C 2. Slow cooling to 180 C, then plunging to 196 C 3. Slow cooling to 20 C, 75 C then plunging to 196 C Slow cooling to between 8 C and 30 C, then plunging to 196 C 1. Controlled cooling to a range between 0 C and 20 C 2. Range of cooling rates to 196 C Controlled cooling to 100 C, then plunging to 196 C

3 months at 196 C Momentary exposure to 196 C 7 days at 196 C 10 days to 5 wks at 75 C 10 days to 4 wks at 196 3 days at 196 C up to 2 yr at 196 C not detailed

6085% 0.1> 90% 0.00310% 0.017% 0.051% 0.015% up to 96.7%

at least 30%

12 months at 196 C

up to 87%

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486

Table 3. Continued. Reference Alga Freezing procedure Slow cooling to 30 C, then plunging to 196 C 1. Uncontrolled fast freezing by plunging to 196 C 2. Slow cooling to 30 C then plunging to 196 C 1. Cooling to 40 C 2. Cooling to 40 C then plunging to 196 C Slow cooling to 30 C, then plunging to 196 C Selection of cooling rates to 30 C, then plunging to 196 C 1. Slow cooling to 30 C, then fast freezing to 80 C 2. Fast freezing to 80 C Controlled cooling to 50 C then plunging to 196 C Storage time and temp. 24 h at 196 C Reported viability 0.10.7%

Gilboa & BenAmotz, 1979 Ben-Amotz & Gilboa, 1980a, b

McLellan, 1989

Chlorella sp. Tetraselmis suecica Phaeodactylum tricornutum Chlorella sp., Dunaliella salina Nannochloris sp. Tetraselmis suecica Phaeodactylum tricornutum Selection of diatoms

Momentary exposure to 196 C Momentary exposure to 196 C Not detailed

064.4%

20.087.1% 0100%

Fenwick & Day, 1992 Day & Fenwick, 1993 Benhra et al., 1994 Caavate & Lubian, 1994

Tetraselmis suecica Tetraselmis suecica Tetraselmis chui Scenedesmus subspicatus

24 h at 196 C Not detailed 10 days at 80 C

774% up to 70% up to 40% 060%

Bodas et al., 1995

Chaetoceros gracilis Nannochloris gaditana Nannochloropsis atomus Rhodomonas baltica Tetraselmis chuii Extensive selection of unicellular Chlorophyceae

Momentary exposure to 196 C

017.1%

Caavate & Lubian, 1995a, b

Hirata et al., 1996 Mortain-Bertrand et al., 1996 Caavate & Lubian, 1997a

Chaetoceros gracilis Nannochloris gaditana Nannochloropsis atomus Rhodomonas baltica Tetraselmis chuii Isochrysis galbana Extensive selection of (a) marine microalgae (b) freshwater microalgae Dunaliella salina Chaetoceros gracilis Nannochloris gaditana Nannochloropsis atomus Thodomonas baltica Tetraselmis chuii Chaetoceros sp. Phaeodactyllum ricornutum

1. Cooling to 70 C, then plunging to 196 C 2. Cooling to 20 C, Cooling to 70 C, then plunging to 196 C 1. Controlled cooling (range of rates) to 51 C then plunging to 196 C 2. Controlled cooling to 50 C then plunging to 196 C Encapsulation-Dehydration then plunging to 196 C Slow cooling to 40 C then plunging to 196 C Controlled cooling to 50 C then plunging to 196 C

5 days at 196 C 5 days at 196 C 3 to 7 weeks at 196 C 4 to 8 weeks at 196 C Several days at 196 C 24 h at 196 C 3 to 5 weeks at 196 C

58100%

0.1095.3%

7.397.9%

(a) 067% (b) 030% up to 78.6% up to 100%

Cordero & Voltolina, 1997

1. Cooling to 10 C

7 to 30 days at 20 C or 60 C

high survival after 7 days

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487
Table 3. Continued. Reference Alga Freezing procedure Storage time and temp. Reported viability

Day et al., 1997

1. Chlorella emersonii

2. Tetraselmis suecica 3. Euglena gracilis Day, 1998 Extensive selection of microalgal strains

2. Freezing by immersion in liquid N2 then transferred to 60 C 1. Uncontrolled fast freezing by plunging to 196 C 2. Slow cooling to 30 C, then plunging to 196 C 3. Slow cooling to 30 C, then plunging to 196 C 1. Cooling to 30 C then plunging to 196 C 2. Controlled cooling to 30, 40 or 60 C then plunging to 196 C

1 year at 196 C

70%

1 year at 196 C 1 year at 196 C Long-term storage at 196 C

73% growth recorded < 10100% 3470%

Refer to Table 1 for further details of species.

and a general method of cryopreservation applicable to all organisms or cell types is thought to be impossible to achieve (Van der Meer & Simpson, 1984). Methods suitable for the cryopreservation of animal tissues, culture cells or bacteria give poor results when applied directly to algae (Morris, 1976a), with complex interactions between (i) the rate of cooling, (ii) cryprotective additive and (iii) warming rate having a profound effect on the survival of algal cells (Leibo & Jones, 1963; Hwang & Horneland, 1965; Morris, 1976a, b). (i) Rate of cooling the manipulation of freezing rates A range of freezing protocols have been developed for the cryopreservation of algae, though optimum methods appear to vary from species to species. Tables 3 and 4 summarise the techniques and methodologies used in cryopreservation experiments on microalgae and macroalgae respectively. In these studies, two main techniques have been utilised (1) rapid cooling and (2) two-step cooling. The rapid cooling technique consists mostly of simply plunging the algal material, suspended in culture medium, rapidly into liquid nitrogen at 196 C. At such rapid cooling rates, however the internal solution of the material becomes supercooled, increasing the possibility of damaging intracellular ice formation (Meryman, 1966; Karlsson & Toner, 1996). Most

freezing protocols avoid this by utilizing a two-step cooling process, with controlled or semi-controlled cooling from room temperature (generally at a rate of ca. 1 C min1 ) to a holding temperature of around 30 C before the material is plunged into liquid nitrogen to complete the freezing process. Damage is thought to be prevented using this method because the reduced cooling rate allows sufcient time for osmotic equilibrium to be maintained by shrinkage of the cell. Variations in these two more conventional freezing methods have also be used in an attempt to further limit cell damage during the cryopreservation of plant material. The process of vitrication, for example, attempts to reduce cell damage by preventing ice formation. Using standard freeze-thaw methods, the extracellular solution is frozen, but steps must be taken to minimize the probability of intracellular ice formation (both the intrinsic parameters characteristic of the specimen and the cryopreservation protocol must be optimised in order to avoid cell damage). In vitrication procedures, however, there is an attempt to prevent ice formation throughout the entire sample (Karlsson & Toner, 1996) and this is achieved by dehydration of the cells prior to cooling. The procedure, rst described by Rall and Fahy (1985), basically involves loading the specimen with an appropriate cryoprotectant and then placing the cells in an extremely concentrated solution (known as the vitrication solution). This results in extreme dehydration, with

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Table 4. Freezing techniques and methodologies used in the cryopreservation of macroalgae. Reference Alga Freezing procedure Storage time and temp. 3 days at 20 C 3 days at 15 C 5 to 20 days at 20 C Momentary exposure to end temperature Reported viability 50% 0% 4593% 70100%

Terrumoto, 1961

Enteromorpha intestinalis Ulva pertusa Porphyra tenera (thallus) Porphyra tenera Prophyra yezoensis Porphyra suborbiculata

Slow cooling to temps between 5 C and 25 C Slow cooling to 20 C 1. Rapid cooling to 20 C or 40 C 2. Slow cooling to 20 C 3. Slow cooling to 20 C, held for 24 h, then freezing to 75 C Slow cooling to 20 C Slow cooling to: 1. 2 C, 5 C 2. 10 C 3. 15 C to 25 C Slow cooling to 40 C, then plunging to 196 C

Migita, 1964 Migita, 1966

70100% up to 55%

Migita, 1967 Bird & McLachlan, 1974

Porphyra tenera (conchocelis phase) Fucus edentatus

10 days at 20 C 2 h to 7 days

up to 30%

Van der Meer & Simpson, 1984

Arbault et al., 1990

Gracilaria foliifera Palmaria palmata Devaleraea ramentacea Chondrus crispus Ulva lactuca Gracilaria tikvahiae Undaria pinnatida (gametophytes)

1 h at 196 C

100% 492% 0100% 35100%

1. Cooling to 80 C 2. Cooling to 30 C, then plunging to 196 C Slow cooling to 40 C, then plunging to 196 C 1. Slow cooling to 30 C 2. Slow cooling to 80 C Slow cooling to 30 C or 40 C then plunging to 196 C Slow cooling to range of 20 C to 80 C, then plunging to 196 C Controlled cooling to 40 C, then plunging to 196 C 1. Controlled cooling to range between 20 C to 60 C, then plunging to 196 C

GinsburgerVogel et al., 1992 Kuwano et al., 1992 Renard et al., 1992 Kuwano et al., 1993 Arbault & Delanoue, 1994 Kuwano et al., 1994

Undaria pinnatida

Porphyra yezoensis Undaria pinnatida

1 h to 39 days at 196 C 1 h to 14 days at 196 C Momentary exposure to 196 C 24 h at 30 C 24 h at 80 C 15 min at 196 C 1 to 300 days at 196 C 15 to 20 min at 196 C 24 h at 196 C

Survival for 9 days Survival for 4 days High Mortality up to 63.7% up to 1.7% up to 50%

Porphyra yezoensis

up to 60%

Porphyra linearis (conchocelis phase) Porphyra yezoensis Porphyra tenera Porphyra pseudolinearis Porphyra dentata Porphyra haitanensis

up to 70%

38.472.5%

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489
Table 4. Continued. Reference Alga Freezing procedure 2. Slow cooling to 40 C, then plunging to 196 C 1. Controlled cooling to range between 20 C to 60 C, then plunging to 196 C 2. Slow cooling to 40 C, then plunging to 196 C EncapsulationDehydration then slowcooling to 40 C, before plunging to 196 C Controlled cooling to 30 C, then plunging to 196 C Storage time and temp. Reported viability 44.377.9% 24 h at 196 C up to 100%

Kuwano et al., 1996

Porphyra yezoensis Porphyra tenera

12.096.2%

Vigneron et al., 1997

Laminaria digitata (gametophytes)

3 days

2575%

Day, 1998

Porphyra miniata

Long-term storage

Cells Viable

solutions in excess of 8M typically used, resulting in more than 90% of the osmotically active water being removed from the cells (Steponkus et al., 1992). Specimens are then cooled and stored in liquid N2 as in conventional cryopreservation procedures. Care is necessary upon thawing, however, and because of the extreme conditions used for vitrication, dilution of the suspending medium should be conducted as rapidly as possible. Although not widely used to date, vitrication does appear to have some potential use in the cryopreservation of algal material a more detailed description of vitrication methods can be found in Steponkus et al. (1992) and Karlsson and Toner (1996). A further method recently employed in the successful cryopreservation of both microalgae (Hirata et al., 1996) and macroalgae (Vigneron et al., 1997) is that of encapsulation-dehydration. This technique involves the dehydration of encapsulated algal cells by means of sterile air drying followed by immersion and storage in liquid N2 . The method has the advantage that no toxic cryoprotectants are required hence upon thawing there can be direct cultivation without the need for repeated washing of the sample. Practical details on the protocols routinely employed for the freezing of microalgae at the Centre for Culture of Algae and Protozoa, UK (CCAP) are given by Day and DeVille (1995). Further details on

cryostorage units and freezing equipment widely used in the freeze-preservation of algal material are given by McLellan et al. (1991). From the data presented in Tables 3 and 4, the plunging of material directly into liquid N2 at 196 C appears to be the most simple method implemented in the freezing of algae to date. Holm-Hansen (1963), Ben-Amotz and Gilboa (1980a, b) and Hirata et al. (1996) all report success using this uncontrolled, fast-freezing method. Although these reports refer to the viability of samples that have been stored at 196 C for relatively short periods, it is likely that at low subzero temperatures (i.e. <135 C) no further deterioration of stored material will occur (Warren et al., 1997), viability generally being independent of storage duration (Morris, 1981). Indeed, Day et al. (1997) suggest that with use of this rapid freezing technique selected microalgae may be successfully frozen and stored for time periods of at least a year. In general, however, increased viabilities are often achieved using a two-step cooling procedure. Reports generally document the slow-cooling of microalgal material to between 25 C and 70 C followed by a plunging directly to 196 C by immersion in liquid N2 . The controlled cooling of samples to an initial temperature of around 30 C to 50 C immediately prior to storage in liquid N2 has produced high viabilities in the long-term storage of many

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490 species (see Table 3 for details). Controlled cooling of Chlamydomonas to the lower temperature of 55 C before liquid N2 freezing, however, led to very poor recovery being recorded by McGrath and Daggett (1977), and similarly by Gresshoff (1977) who slow-cooled the alga to 180 C before plunging into liquid N2 . Success rates do appear to be extremely variable, however, and between these two temperature extremes is a report by Saks (1978) who demonstrates the successful long-term storage of a range of microalgae by controlled cooling to 100 C prior to freezing at 196 C. Success with short-term storage after using the two-step freezing method has similarly been reported by several authors cited in Table 3. Again, there are exceptions, however, with a report by Caavate and Lubian (1994) showing only 0 to 17% viability in a range of microalgae control cooled to 50 C before a momentary exposure to 196 C. Storage at non-liquid N2 temperatures has also been investigated by workers attempting to successfully freeze microalgae. Holm-Hansen (1963), for example, reports viabilities of up to 100% in microalgae frozen to a range of temperatures between 10 C and 70 C. Samples were, however, frozen for only a short time period, in this case experiencing only a momentary exposure to the end temperature. Benhra et al. (1994) report the successful storage of Scenedesmus subspicatus for up to 10 d at a 80 C end temperature, whilst some recovery of algal material has been noted by Leibo and Jones (1963) following short-term storage at 76 C. Gresshoff (1977), on the other hand, reports little success with the storage of Chlamydomonas reinhardtii at similar end temperatures. Cordero and Voltolina (1997) used a slightly different two-step technique, in that algal material was rst frozen in liquid N2 and subsequently transferred to the higher temperature of 60 C but again, however, algal recovery decreased with increasing storage time. Brown and Day (1993) suggest, therefore, that although some organisms can be successfully cryopreserved and stored at higher sub-zero temperatures, viability levels fall rapidly during storage and these temperatures may be suitable for only short-term maintenance. With respect to the freezing of macroalgae, the storage of material at non-cryogenic temperatures has yielded some favourable results. Viabilities of up to 100% have been reported by Migita (1966) for the storage of Porphyra at 20 C to 75 C, although the study involved only the momentary exposure of the test inoculum to the end temperature. Success has similarly been reported by Kuwano et al. (1992) in the freezing of Porphyra yezoensis for 24 h at 30 C, although viabilities were very much reduced after storage at 80 C. Investigations involving the storage of macroalgae for longer time periods at noncryogenic temperatures generally result in lower viabilities, but several authors have reported success. Bird and McLachlan (1974) demonstrated the successful storage of Fucus at a range of temperatures between 5 C and 25 C for up to 7 d, whilst Terrumoto (1961) successfully stored both Enteromorpha and Ulva thalli at similar temperatures for 3 d. Longerterm storage of up to 20 d at temperatures around 20 C has also been reported by Migita (1964, 1976) for Porphyra tenera. Successful storage of macroalgal material at cryogenic temperatures has been achieved by several workers, but as reported for microalgae, viability and recovery of the test inoculum vary both with the alga and the freezing technique employed. The technique of rapid freezing by simply plunging the alga into liquid N2 at 196 C has not been employed for macroalgae. Grout and Morris (1987) suggest that in larger cells with low surface area:volume ratios, water is lost less effectively during freezing than in smaller cells with high surface area:volume ratios. This makes larger cells more prone to intracellular ice formation during freezing, hence slower cooling rates should be selected in order to prevent this (McLellan, 1989). In general, most workers report the two-step method to be the most appropriate technique in the cooling of macroalgae to very low temperatures. Several authors report viabilities of up to 100% after the short-term storage of macroalgae at 196 C, whilst Kuwano et al. (1993) and Day (1998) suggest that cryogenic storage of some Porphyra species may also be feasible in the long-term. With respect to other algal genera, however, Arbault et al. (1990) report the survival of Undaria pinnatida gametophytes at 196 C for just 4 d compared with a survival duration of 9 d at 80 C. Limited success using the two-step cooling method has similarly been reported by GinsburgerVogel et al. (1992) working with Undaria pinnatida, though Renard et al. (1992) reported viabilities of up to 50% using similar techniques. (ii) The use of cryoprotective additives Many of the early successes in recovering viable material after freezing were achieved with the aid of protective additives (Meryman, 1966). A variety of

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491
Table 5. Previous investigations involving cryoprotectants, their application concentration and the duration of pretreatment. Reference Alga Cryoprotectant concentration Duration of pre-treatment

MICROALGAE Holm-Hansen, 1963 Leibo & Jones, 1963 Hwang & Horneland, 1965

Hwang & Huddock 1971 Tsuru, 1973

Extensive selection of unicellular Chlorophyceae Porphyridium cruentum Anistrodesmus sp. Chlamydomonas reinhardtii Chlorella sp., Coccomyxa sp., Euglena sp., Scenedesmus sp. Chlamydomonas reinhardtii Chlorella sp., Scenedesmus sp., Nitzshia sp., Phaeodactylum tricornutum Chlorella sp.

No Protectant No Protectant 10% glycerol

N/A N/A

5% DMSO 10% DMSO 10% glycerol 10% DMSO

30 min 30 min

Morris, 1976a, b

McGrath & Daggett, 1977 Gresshoff, 1977 Morris, 1978

Chlamydomonas reinhardtii

Morris & Canning, 1978

Chlamydomonas reinhardtii Chlorella sp., Scenedesmus sp., Ankistrodesmus sp., Euglena gracilis

2.55% glycerol 2.55% DMSO 1015% PVP 10% glycerol 5% DMSO 1% Tween 80 15% DMSO 5% DMSO 10% PVP 10% DMSO 10% ethanol 10% methanol 10% PVP 7.5% sucrose 5% glycerol 5% glycose 5% glycerol 5% DMSO

5 min

4570 min

18 h 5 min

15 min

Saks, 1978

Gilboa & BenAmotz, 1979; BenAmotz & Gilboa, 1980a, b McLellan, 1989

Nitzshia sp., Cylindrotheca sp. Phaeodactylum tricornutum Nannochloris sp., Dunaliella salina Chlorella sp., Tetraselmis suecica, Phaeodactylum tricornutum Selection of diatoms

30 min

520% DMSO

5 min

Fenwick & Day, 1992

Tetraselmis suecica

515% DMSO 515% glycerol 515% methanol 515% DMSO 515% ethanol 515% glycerol 515% methanol

30 min

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492
Table 5. Continued. Reference Alga Cryoprotectant concentration 510% DMSO 510% glycerol 10% sucrose 5% PVP, 10% sucrose & 3% methanol mix 10% DMSO 5% methanol Duration of pre-treatment 30 min 30 min

Day & Fenwick, 1993 Benhra et al., 1994

Tetraselmis suecica Tetraselmis chui Scenedesmus subspicatus

Caavate & Lubian, 1994

Bodas et al., 1995 Caavate & Lubian, 1995a, b

Hirata et al., 1996

Mortain-Bertrand et al., 1996 Caavate & Lubian, 1997a

Chaetoceros gracilis, Nannochloris gaditana, Nannochloropsis atomus, Rhodomonas baltica, Tetraselmis chuii Extensive selection of unicellular Chlorophyceae Chaetoceros gracilis, Nannochloris gaditana, Nannochloropsis atomus, Rhodomonas baltica, Tetraselmis chuii, Isochrysis galbana Chlamydomonas reinhardtii Extensive selection of marine microalgae Dunaliella salina Chaetoceros gracilis, Nannochloris gaditana, Nannochloropsis atomus, Rhodomonas baltica, Tetraselmis chuii Chaetoceros sp., Phaeodactylum tricornutum Chlorella emersonii, Tetraselmis suecica Extensive selection of microalgal strains

30 min

5% methanol 58% DMSO 515% DMSO 15% methanol

45 min

0.5 M sucrose

3.5 M glycerol 515% DMSO

60 min

Cordero & Voltolina, 1997 Day et al., 1997

Day, 1998

110% DMSO 110% glycerol 510% DMSO 10% glycerol 10% methanol 5% DMSO 10% glycerol 10% methanol DMSO (no conc. details) No protectant 2.520% glycerol 2.520% glucose 0.53 M DMSO

15 min

MACROALGAE Terrumoto, 1961 Migita, 1964, 1966, 1967 Van der Meer & Simpson, 1984

Enteromorpha intestinalis, Ulva pertusa Porphyra tenera

Gracilaria foliifera, Palmaria palmata, Devaleraea ramentacea, Chondrus crispus, Ulva lactuca, Gracilaria tikvahiae

5 to 90 min

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493
Table 5. Continued. Reference Alga Cryoprotectant concentration 510% glycerol 28% glycerol 520% glycerol 5% DMSO, glycerol, ethyleneglycol, proline, hydrochloride betaine, skimmed milk 1 M sucrose, glucose, sorbitol mannitol 1530% glycerol 520% DMSO and 0.5 M sorbitol mix 15% DMSO 10% sucrose 10% DMSO & 0.5 M sorbitol mix Duration of pre-treatment up to 30 min 10 min 45 min

Arbault et al., 1990 Ginsburger-Vogel et al., 1992 Kuwano et al., 1992

Undaria pinnatida Undaria pinnatida Porphyra yezoensis

Renard et al., 1992 Kuwano et al., 1993 Arbault & Delanoue, 1994 Kuwano et al., 1994

Undaria pinnatida Porphyra yezoensis Porphyra linearis Porphyra yezoensis, Porphyra tenera, Porphyra pseudolinearis, Porphyra dentata, Porphyra haitanensis Porphyra yezoensis, Porphyra tenera

45 min 45 min up to 1 h 45 min

Kuwano et al., 1996

Day, 1998

Porphyra miniata

510% DMSO 5% dextran, PVP, Ficoll, PEG, propyleneglycol, ethyleneglycol, glycerol, sorbitol, sucrose, glucose 5% DMSO

45 min

15 min

= not documented in report.

simple neutral solutes have been shown to protect living cells against damage during freezing, thawing and storage at low temperatures, with sugars such as glucose and lactose among the rst solutes to receive extensive study and application (Woodcock et al., 1941; Florio et al., 1943). Theories put forward to explain the protective actions of these compounds and others are discussed in detail by Meryman (1966) and Mazur (1960, 1970). In general terms, cryoprotectants are used to limit ice formation (Andersen, 1996) and as is the case for rate of cooling, the best cryoprotectant for a particular alga is generally determined empirically (Morris,

1981; Lee & Soldo, 1992). The three most widely used cryoprotectants to date are dimethyl sulphoxide (DMSO), glycerol and methanol, with DMSO often the most popular choice. The latter compound has the ability to pass through cell membranes more freely than glycerol (Meryman, 1966), and, in addition, due to its rapid transfer DMSO is much more easily removed from cells than glycerol, hence less care in resuspension is necessary. Because of its viscous nature, glycerol may be difcult to remove from algal cells, with incomplete removal of the protectant upon thawing causing bacterial blooms and contamination

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494 during subsequent culture periods (Van der Meer & Simpson, 1984). In addition to the nature of the cryoprotectant itself, its concentration may also affect the recovery of cells. Once again, suitable concentrations are often found by trial-and-error, Morris (1981) even suggesting that for a particular alga different cryoprotectant concentrations may be necessary at different freezing rates. In general, the relevant concentration of cryoprotectant is added to the culture medium as a pretreatment, allowing the algal cells to soak up the protective chemical prior to freezing. Pretreatment is generally most successful at room temperature (2023 C) and, at the optimum cryoprotectant concentration, the duration of pretreatment from 5 to 90 min is not thought to be crucial (Van der Meer & Simpson, 1984). It should be noted, however, that despite the protection they afford cells during freezing and thawing, cryoprotective chemicals can themselves be damaging especially when used in high concentrations (Fahy, 1986). Toxicity is generally increased by long term exposure of the cells to the protectant, though different algae demonstrate different tolerance levels (Caavate & Lubian, 1994). Table 5 shows a selection of the cryoprotectants and pretreatment conditions investigated by previous workers concerned with the cryopreservation of algae. Early studies on the cryopreservation of algae, microalgae in particular, do not report the use of a cryoprotectant. Both Holm-Hansen (1963) and Leibo and Jones (1963) report recoveries varying from some to up to 100% in the absence of any protective compound. It is pertinent to note, however, that in both these studies, exposure to the end temperature was only for a short time period. For more successful freezing, particularly for the long-term storage of algal cells, a cryoprotectant is generally necessary. It can be seen from Table 5 that in addition to the three most wisely used protectants (i.e. DMSO, glycerol and methanol) an extensive range of protective compounds have been used in algal cryopreservation. These include PVP, Tween 80, ethyleneglycol, proline, hydrochloride betaine and ethanol along with sugar compounds such as sucrose, sorbitol, mannitol and glucose. Application concentrations range from 1% to 30% protectant, with 510% and 520% being the most widely used concentrations for the freezing of microalgae and macroalgae respectively. Generally, the compounds are applied singly, though Kuwano et al. (1993, 1994, 1996) report high success rates after using DMSO in combination with other protective agents. The duration of algal pretreatment varies from report to report, several authors recording high viabilities in material frozen after just a 5 min exposure to the protective compound. Gresshoff (1977), on the other hand, reports the use of a 18 h pretreatment, with only limited success after longer-term microalgal storage. Van der Meer and Simpson (1984) are among the surprisingly few authors that have investigated a range of pre-treatment times in order to maximise algal viability upon recovery. Most workers appear to choose an appropriate protectant and pre-treatment according to those previously reported in the literature. McGrath and Daggett (1977) and Mortain-Dertrand et al. (1996) have reported varying degrees of success using a pre-treatment time of between 45 and 90 min, but the optimum exposure time reported by the majority of authors is shorter than this, namely between 30 and 45 min, and successful freezing of both microalgae and macroalgae has been achieved using this pre-treatment time as part of the overall freezing protocol.

Algal tolerance to freezing Resistance in nature In general, microbes are those organisms most resistant to physical trauma including freezing. Many viruses and bacteria appear to be resistant at least to moderate freezing with dehydrated forms, such as spores, being particularly hardy. More complex organisms are substantially more susceptible to freezing injury, though direct observation of tissues in nature has shown many algal species to possess the ability to survive temperatures well below 0 C. Certain algae are able to grow on the surface of snow, some even being ecologically restricted to this habitat (Marr, 1962), and many littoral and sublittoral algae are annually subjected to air and/or water temperatures below 0 C (Marr, 1962; Biebl, 1962). Some forms, e.g. Fucus vesiculosus may even spend six months or more embedded in ice at temperatures below 40 C (Kanwisher, 1957; Parker, 1960). The ability of algae to survive such temperature extremes suggests the development of cold-hardiness within plants (Kylin, 1917; Biebl, 1958). This is of great signicance to cryobiological research and several workers have found the natural frost resistance

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495
Table 6. Techniques for increasing the viability of cryopreserved algae. Treatment IMMEDIATELY PRIOR TO FREEZING Reduction of salinity Reference

Reduction of water content Alteration of chlorinity DURING CULTURE PERIOD PRIOR TO FREEZING Acclimation to reduced temperatures

Holm-Hansen, 1973; Saks, 1978; Van der Meer & Simpson, 1984 Kuwano et al., 1992, 1994, 1996; Caavate & Lubian, 1995a, b 1997b; Mortrain-Bertrand et al., 1996 Migita, 1964, 1966; Hirata et al., 1996 Migita, 1964 Migita, 1966 Bird & McLachlan, 1974 Morris, 1976b; Ben-Amotz & Gilboa, 1980b Mortain-Bertrand et al., 1996 Ben-Amotz & Gilboa, 1980b; Fenwick & Day, 1992

Alteration of nutrient regime

and cold-hardiness of certain algae to be advantageous during laboratory freezing procedures. Holm-Hansen (1963) for example, reported successful freezing of strains of green algae from the Antartic, with lower viabilities for species from warmer areas. Dudgeon et al. (1990) conducted a similar study on Mastocarpus stellatus from the northern Gulf of Maine, where, under eld conditions, the macroalga may be frozen up to three times within a 24 h period. The repeated exposure of this alga to temperatures below 20 C during the winter months may well explain its tolerance of chronic freezing exposures applied under laboratory conditions. The induction of freezing tolerance Workers have shown that whilst some species are naturally frost resistant, cold-hardiness and freezing tolerance can be successfully induced in some algal species. The freezing resistance of some algae has been shown to increase with a decrease in ambient culture temperature, this phenomenon being analogous to the cold-hardening of higher plants during the cooler autumn months (Leeson et al., 1984). Morris (1976b) investigated the cryopreservation of Chlorella after culturing the cells at low temperatures prior to freezing. His study showed that following a 24 d incubation at 4 C, the recovery following freezing and thawing was 38%, this be-

ing equivalent to results obtained with cells grown at 25 C and frozen in the presence of cryoprotectant. Similar ndings were reported in a subsequent study (Morris, 1981) and the author concluded that culturing cells at low temperatures provides a simple and effective method for cryopreservation. BenAmotz and Gilboa (1980b) similarly found that a variety of marine unicellular algae grown at 4 C for 4 weeks showed increased viabilities when cryopreserved, whilst Mortain-Bertrand et al. (1996) report cold-adaptation in the dark to increase post-thaw viability in Dunaliella salina. The induction of freezing tolerance has also been documented with respect to macroalgae. Bird and McLachlan (1974) reported that the storage of Fucus edentatus zygotes at 0 C increased cold-hardiness upon freezing, whilst the pre-freezing of Porphyra thalli at 20 C has been shown to induce increased cold tolerance when the material is subsequently frozen at 75 C (Migita, 1966). Such reports suggests that low temperature acclimation can induce freezing tolerance in at least some algae, though, of course, there are exceptions to be found. These include certain members of the genus Tetraselmis, with Day and Fenwick (1993) observing that following thawing, cells previously grown at a sub-optimal temperature of 10 C had lower survival

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496 rates than those grown at ambient temperatures in the range 1525 C. In addition to studies on low temperature acclimation, workers have investigated several other methods of inducing freezing tolerance in algae. Techniques include alteration of salinity, nutrient levels, chlorinity and water content prior to freezing. A summary of those methods found to be successful in increasing viability is shown in Table 6. The treatments shown in Table 6 all serve to produce unfavourable growth conditions. Such conditions slow algal division rate and reduce metabolic activity. Ben-Amotz and Gilboa (1980b) suggest that freezing acclimation is induced by the reduction of metabolic rate, whilst Leeson et al. (1984) propose that the susceptibility of algal cells to injury is associated with the presence of a large vacuole. A reduction in growth rate reduces the degree of vacuolation following the accumulation of storage lipid, thus reducing the risk of freezing injury. Moreover, algal resistance to freezing injury may also be related to cellular dehydration and intracellular solute accumulation (Steponkus et al., 1983). Whatever the mechanism, all data indicate that algae grown under unfavourable conditions develop the intrinsic chemical and physical properties which endow them with tolerance to freezing. cells during many years storage at 196 C. Indeed the shelf-life of certain cells stored at liquid nitrogen temperature has been estimated to be of the order of 103 y (Mazur, 1984). Success has been achieved by many workers investigating the long term storage of microalgae at 196 C, though less favourable results are reported for the freezing of macroalgae at such low temperatures. Storage of macroalgae at non-cryogenic temperatures in the range of 20 to 80 C appears more appropriate, but at these higher sub-zero temperatures, storage time is not extensive. Reports in the literature tend to suggest that storage of cells for periods of more than a year requires cryogenic refrigeration equipment to maintain the temperature below 135 C, but for relatively short term storage of algae the use of a deep-freeze at ca. 25 C might be satisfactory (Holm-Hansen, 1973). (iii) The inuence of thawing rates The effect of thawing rates on cell survival has not received the same degree of attention as have freezing rates, probably because their inuence is less striking. Literature concerning the effect of warming rate on cryopreserved materials is largely limited to the study of animal tissue, and papers dealing with the effect of warming rate on algal material are relatively few (e.g. Mayer, 1985). For most algal cultures it has been found satisfactory to merely plunge the frozen sample into warm culture medium at a temperature in the range 20 to 41 C. Caavate and Lubian (1995b) suggest post-thaw viability in marine microalgae is improved when thawing rates are in the range of 0.25 to 16 C min1 , and further work by these authors (Caavate & Lubian, 1997b) reports signicantly higher viabilities observed in algae thawed rapidly. With respect to macroalgae, similar ndings are reported by Renard et al. (1992) who found the survival of Undaria pinnatida gametophytes to vary according to thawing rate. Recovery of rapidly-thawed gametophytes was between 50 and 100% higher than that reported for slowly-thawed material. It should again be noted, however, that upon thawing of frozen material, cryoprotectant additives are potentially cytotoxic (Fahy, 1986). Immediate dilution of the medium is generally necessary in order to avoid toxicity to the cells. High concentrations or extensive exposure to both DMSO and glycerol prove toxic to algal cells, but damage by DMSO is generally prevented by its rapid dilution upon thawing. Repeated

Long term storage of material Degradative enzymatic reactions continue to occur in all frozen cells resulting in a continual loss of viability with time. The rates of most chemical reactions decrease exponentially with decreasing temperatures, so that great differences in metabolic activity can be expected in cells stored at 20 C as compared to 100 C. Nearly all available data indicate that the lower the storage temperature, the greater the viability of the cells. For very long periods of storage, therefore, it is desirable to use temperatures below the eutectic point (lowest solidifying point of any component) of the mixture, with Warren et al. (1997) suggesting that temperatures of at least 135 C are necessary in order to prevent cell deterioration. At temperatures as low as 196 C, there is insufcient thermal energy for signicant chemical changes to occur in a biological sample (McGee & Martin, 1962). The only deterioration that can occur is DNA damage by background radiation and cosmic rays (Mazur, 1984), hence most evidence indicates that there is little or no detectable decline of viability of

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497
Table 7. Viability tests used for algae after thawing. Method of determining viability MICROALGAE Ability of preserved cells to actively divide / form colonies Reference

Cell counts / Growth

Measurement of protein Measurement of chlorophyll Most probable number technique Uptake of stain

Motility of cells

Oxygen evolution MACROALGAE Staining and/or examination of cells for ultrastructural effects

Holm & Hansen, 1963 Hwang & Horneland, 1965 Hwang & Hudock, 1971 Morris, 1976a, b; 1978 Morris & Canning, 1978 McGrath & Daggett, 1977 Gilboa & Ben-Amotz, 1979 Ben-Amotz and Gilboa, 1980a, b Fenwick & Day, 1992 Day & Fenwick, 1993 Bodas et al., 1995 Day et al., 1997 Day, 1998 Holm-Hansen, 1963 Leibo & Jones, 1963 Hwang & Horneland, 1965 Tsuru, 1973 Saks, 1978 Gilboa & Ben-Amotz, 1979 Ben-Amotz & Gilboa, 1980a, b Fenwick & Day, 1992 Benhra et al., 1994 Caavate & Lubian, 1994; 1995a, b; 1997a, b; Mortain-Bertrand et al., 1996 Cordero and voltolina, 1997 Leibo & Jones, 1963 Mortain-Bertrand et al., 1996 Morris, 1978 Hirata et al., 1996 McLellan, 1989 Fenwick & Day, 1992 McLellan, 1989 Fenwick & Day, 1992 Day & Fenwick, 1993 Bodas et al., 1995 Tsuru, 1973 Saks, 1978 Fenwick & Day, 1992 Day & Fenwick, 1993 Mortain-Bertrand et al., 1996 Gilboa & Ben-Amotz, 1979 Ben-Amotz & Gillboa, 1980a, b Terumoto, 1961 Migita, 1964; 1966 Arbault et al., 1990 Ginsburger-Vogel et al., 1992 Kuwano et al., 1992, 1993, 1994, 1996 Arbault & Delanoue, 1994

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498
Table 7. Continued. Method of determining viability Measurement of photosynthetic rate Ability of preserved cells to exhibit cell division and re-growth Reference

Dudgeon et al., 1989, 1990 Van der Meer & Simpson, 1984 Renard et al., 1992 Kuwano et al., 1994 Day, 1998 Migita, 1967 Vigneron et al., 1997 Van der Meer & Simpson, 1984 Hirata et al., 1996 Vigneron et al., 1997

Measurement of spore liberation / gamete release Measurement of cell pigmentation

and thorough washing of algal material with culture medium is generally considered sufcient to remove the protectant. Viability assaying following cell thawing Following thawing and the removal of cryoprotectants, algal cells are generally placed in fresh culture medium for a recovery phase. After a time period allowing some kind of measurable response, viability can be ascertained. Some of the most widely used techniques for measuring viability are shown in Table 7. Both qualitative and quantitative methods have been employed to assess viability following freezing and thawing. The most widely employed methods for measuring viability in microalgae involve either determining the ability of preserved cells to actively divide and form colonies or monitoring growth by means of cell counts (for methodology, see: Day & DeVille, 1995; Leeson et al., 1984; McLellan et al., 1991). Increase in a particular protein or pigment content of a culture is also used, as is the estimation of numbers by most probable number techniques (Swaroop, 1938, cited in Leeson et al. (1984)). Indirect methods of estimating viability such as uptake of a particular stain, cell motility and oxygen evolution are also used, but these simple methods may signicantly over-estimate the recovery potential (Day et al., 1998). Leeson et al. (1984) hence suggest that regrowth and division are the denitive indicators of viability and are the methods that should be used whenever practical.

Ultrastructural damage and the ability for regrowth are characteristics often used in the viability testing of macroalgae, though recovery can also be based on some kind of physiological function such as photosynthetic rate. Other methods of viability testing in macroalgae include examination for the presence of, or changes in, cell pigmentation and the liberation of spores or gametes upon thawing. Indeed, it appears that any response indicative of recovery and subsequent growth and propagation can potentially be used to assess cell viability following the freezing process. This, in turn, means that one of the fundamental problems in comparing cryopreservation methods, and hence assessing the relative efciency of each, is the wide variation in methods for assessing viability (McLellan et al., 1991). Caution must, therefore, be exercised when attempting to compare measurements made by different types of viability assay. Indeed, even the comparison of seemingly straighforward records of cell recovery and growth may be further complicated by the use of a viability index (e.g. Caavate & Lubian, 1994, 1995a, b, 1997a, b) rather than the measurement of absolute viability.

Summary and general conclusions When algal cells are subjected to freezing, low temperature storage and subsequent thawing, they are exposed to a sequence of chemical and physical stresses, any one of which may result in loss of cell viability. Cellular damage may be caused during freezing by the formation of ice crystals, whilst decreasing tempera-

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499 tures down to and even a few degrees below the suspension freezing point may alter overall metabolic patterns due to differential effects of temperature on rates of various enzyme reactions (Holm-Hansen, 1973). In addition, the thawing of a frozen sample exposes the cells to the potentially lethal effects of changing electrolyte concentrations and temperature effects on enzymatic rates (Holm-Hansen, 1973). For algal cryopreservation to be successful, therefore, these stresses and potential causes of cellular damage must be overcome. This has often been achieved by trial and error. A particular alga may be tolerant to momentary exposure to low temperature using one freezing method, for example, but employment of a different method may be necessary for successful long-term storage. In addition, a method found to produce high viabilities in one alga may be totally unsuitable for the freezing of another. The majority of cryopreservation protocols in current use are developed empirically, with prefreezing culture regimes, cryoprotectant choice and concentration, and thawing regimes often manipulated in order to minimise cell damage and maximise viability levels (Day & DeVille, 1995). Leeson et al. (1984), however, suggest that since different techniques are generally required for different specimens, improvement may be achieved by an understanding of the basic principles of cryobiology rather than by following recipes. The use of the relatively new technique of light cryomicroscopy, for example, may be a great aid in understanding the events associated with chilling and freezing of algal material, and may ultimately be used to develop successful cryopreservation protocols (Fleck et al., 1997). Long-term storage of algal material is generally the ultimate aim of workers investigating cryogenic techniques. To this end, the two-step method is reported as the most successful cooling protocol, although, as previously discussed, there are exceptions. Microalgae, in particular, have been successfully frozen using the two-step method and stored at 196 C for time periods ranging from several weeks to several years. Indeed, some 694 strains of microalgae are currently cryopreserved as a means of long-term storage at the Culture Centre for Algae and Protozoa (Day, 1998). Storage of algal cultures in liquid N2 has the advantage of requiring little routine maintenance, whilst the low temperature also guarantees genetic stability (Warren et al., 1997). The main disadvantage, particularly in individual laboratories where the number of samples to be maintained may be relatively low, is that in addition to relatively expensive storage equipment, specialised freezing equipment and consumables are required (McLellan et al., 1991). Andersen (1996) further suggests that both limited cryopreservation research on many algal groups and the lack of consistent success in some algal groups may also be reasons why cryopreservation techniques are not widely employed for algal storage. With respect to macroalgae, long-term survival at liquid N2 temperatures is not well documented. Several reports suggest that macroalgae may be tolerant of cryogenic temperatures for comparatively short time periods only, and, in general, greatest success has been reported at non- cryogenic temperatures. At these high sub-zero temperatures, however, both biochemical and biophysical processes may still be occurring, often resulting in a reduction in cellular viability with increasing storage time (Leeson et al., 1984). For long-term cryopreservation of algae to be successful, therefore, storage must be at a temperature below 139 C, where no growth of ice crystals can occur and the rate of other biophysical processes are too slow to affect cell survival. In addition to the employment of appropriate end temperatures and rates of cooling, the current review has shown that for the freezing of algal material to be a success, many other factors must also be taken into consideration. Viability of the test inoculum may be increased by the application of the appropriate concentration of a suitable cryoprotectant, for example, whilst the introduction of unfavourable conditions prior to freezing may be used to induce some degree of freezing tolerance. Indeed, as Steponkus et al. (1992) point out, there is a complex set of interacting variables that must be taken into consideration when attempting the long-term freeze preservation of algal material in the laboratory. Acknowledgements We gratefully acknowledge support, in part, by a grant from Hempels Marine Paints, Copenhagen. References
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