1. Introduction. Marine microalgal species are widely used as a food source in the artificial propagation ..... Brown, M.R., Jeffrey, S.W. and Garland, C.D., 1989.
Aquaculture Aquaculture 136 (1995) 277-290
Some aspects on the cryopreservation of microalgae used as food for marine species J. Pedro Cafiavate a**, Luis M. Lubirin b a Centro de Investigacidn y Cultivo de Especies Marinas “El Torwio”, Junta de Andalucia, PO Box 16, 11500 El Puerto de Santa Maria Cddiz, Spain b Institute de Ciencias Marinas de Andalucia. CSlC, Apartado Ojcial. 11510 Puerto Real, C&z, Spain
Accepted 30 April 1995
Abstract The response of marine microalgae to different ctyopreservation methods was described. Of the six species evaluated, only Chaetoceros grucifis depended on faster cooling rates to increase its postthaw viability (7.2% at 0.2X n-tin-‘, 29.3% at 4°C mm-‘) when using 15% dimethyl sulphoxide in 36 p.p.t. salinity seawater. Tetraselmis chuii, Nannochloris atomus and Nannochloropsis gaditana were the most tolerant species to biological freezing, achieving mean viabilities of 97.9%, 80.5% and 61.6% respectively. Rhodomonas baltica and Isochrysis galbana, T-IS0 strain, showed the lowest viability (means of 7.3% and 15.1% respectively) after cryopreservation under the same conditions of salinity, cryoprotectant concentration and cooling rates. Avoidance of undercooling by inducing ice nucleation when reaching the freezing point did not change viability in comparison to all the procedures that did not include seeding in any of the tested species. Five species showed similar viabilities when a single controlled cooling step to -50°C procedure was compared to a two-step cooling process, in which algae were plunged into liquid nitrogen (LN) after the first step. Isochrysis galbana represented an exception. A mean viability of 25.8% was achieved when cooled to - 5O”C, whereas viability decreased to 4.4% when the second cooling step to - 196°C was used. Replacing the special biological freezing equipment by a - 20°C freezer to perform the first cooling step resulted in a steady cooling rate after the commencement of ice formation. This was due to the fact that samples reached that temperature in a liquid state. Solidification occurred spontaneously at variable times once - 20°C was reached. A cooling rate of - 14°C min- ’ during the change from liquid to solid state was achieved when a - 80°C freezer was used to perform the first cooling step. The performance of the first cooling step in both type of freezers resulted in similar viabilities after thawing from liquid nitrogen, in comparison to the use of special equipment for controlling cooling rates in T. chuii (90.8% for - 20°C and 89.7% for - 8O”C), N. gadituna (44.6% for - 20°C and 42.6% for - 80°C) and N. atomus (85.9% for - 20°C and 85.6% for - 80°C). Lower viabilities were recorded for R. baltica and Ch. gracilis cooled to - 20°C and in LN (2.2% and 2.9% respectively) * Corresponding author. 0044-8486/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIOO44-8486(95)01056-4
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but no difference were found with respect to the first technique, when both species were cooled to - 80°C and in LN (6.3% and 19.9% respectively). I. gulbana showed no viability when cooled to - 80°C. Keywords:
Microalgae; Cryopreservation; Seeding ice formation; Cooling rate; DMSO
1. Introduction Marine microalgal species are widely used as a food source in the artificial propagation of hatchery-reared molluscs, crustacea and fish (De Paw and Persoone, 1988; Ylifera and Lubian, 1990). Intensive cultures of microalgae have now become a routine in most hatcheries where different strains are selected according to reared organisms and nutritional value (Brown et al., 1989). Although techniques for intensive phytoplanktonic mass production are well known (Richmond, 1986; Laing and Ayala, 1990)) problems derived from loss of quality stock cultures still occur in commercial hatcheries that have to rely on the supply of new strains from culture collections. Recent advances in cryobiology have produced different techniques, which enable the viable storage for extended periods of time of quite a diverse group of biological resources. The application of some of the cryopreservation techniques to marine microalgae of commercial interest is, however, little documented, and for some strains cryopreservation has not yet been attempted. The development of marine microalgae cryostorage might contribute to improve hatcheries efficiency by assuring the permanent and immediate availability of high-quality and stable stock cultures. Setting up simpler techniques for cryopreservation of small volume cultures is an important step towards future research on freezing of phytoplanktonic mass cultures. In this respect, knowing whether very simple cryopreservation procedures produce similar results to those obtained with special cryogenic equipments is of great importance, before attempting research on mass scale algal cryostorage. Conventional methods for cryopreserving living organisms require the application of specific procedures in order to reduce the injuries induced by freezing (Morris, 1987). Thus, the incubation with cryoprotective compounds, the cooling and warming rates and conditioning factors such as temperature and salinity are used to facilitate cryopreservation. Two conventional cryopreservation techniques are used. The first is the one-step cooling technique, in which algae are directly plunged into liquid nitrogen (LN) at - 196°C (Tsuru, 1973; Ben-Amotz and Gilboa, 1980). The other is the two-step cooling technique (Morris, 1976; Ben-Amotz and Gilboa, 1980), in which samples are first cooled under controlled conditions in order to reduce damage produced by the formation of ice. After complete solidification, samples are then transferred to LN. Depending on the requirements for cryopreserving the different types of biological materials, the transition from liquid to solid stage can be achieved either with the use of special cryogenic equipment, low melting point compounds baths, or simply with laboratory freezers from - 20°C to - 80°C. One specific procedure needed for optimum cryopreservation of some organisms is the seeding of ice formation when the samples reach their freezing point (Fuku et al., 1992). Ice seeding is achieved by using sophisticated cryogenic machines (Miralles et al., 1994). Samples are normally placed in straws or tubes of small volume to which local cold shocks
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are given when the temperature reaches the freezing point. Knowledge on whether this factor improves phytoplankton cryopreservation is an elemental question in devising successful methods for cryopreservation of microalgae. Literature on the cryopreservation of phytoplankton has not included up to now any study on the effects of ice seeding nucleation. This question is the main goal of this work, in which this factor has been studied at different cooling rates for algae frozen either to - 50°C or - 196°C. Results on alternative cooling methods, replacing the specific cryogenic equipment by laboratory freezers, are also described.
2. Materials and methods Six algae species from the culture collection of the Instituto de Ciencias Marinas de Andalucia (Lubian and Yufera, 1989) at Cadiz, Spain, were studied. Selection of species was done in order to achieve a wide taxonomic diversity and according to their importance in marine hatcheries. The algae were the following: Rhodomonas baltica Karsten (Cryptophyceae) , Tetraselmis chuii Butcher (Prasynophyceae), Zsochrysis galbana Parke (Haptophyceae) , T-IS0 strain, Nunnochloropsis g&tuna Lubian (Eustigmatophyceae), Chaetoceros grucilis Schiitt (Bacillariophyceae) and Nannochloris atomus Butcher (Chlorophyceae) . Batch cultures were performed in 3-litre beakers of autoclaved seawater (36 p.p.t.) enriched with Guillard’s medium (Thompson et al., 1988) with double the amount of nitrate and phosphate, and including 0.35 mM sodium silicate (Laing, 1985) for Ch. grucilis. Beakers were placed under permanent illumination of 130 pmol photons m-* s-i at 20 + 1°C in an algal room where 1% CO,-enriched air was supplied. Cultures were always harvested after 7 days of growth. 2.1. Thermokinetics
of seawater-cryoprotectant
systems
The changes taking place in temperature during liquid to solid change were characterized for seawater of two salinities (20 p.p.t. and 36 p.p.t.), each containing lo%, 15% or 20% (v/v) of the cryoprotective compounds dimethyl sulphoxide (DMSO), methanol and glycerol (All pure-grade reagents were from Sigma). Samples were introduced in 0.5 ml straws ( 135 mm length, Minitub GmBh, Germany)) and placed in a Cryoson BV-25 biological freezer (Cryoson GMBH, Postfach 1104, 8752 Schollkrippen, Germany). The freezer was fitted with a platinum sensor for temperature control in the freezing chamber, and two copper thermocouples: one forming the connection between the sample and the central processing unit, and another recording the environmental temperature in the chamber. Samples were then cooled from + 20°C to - 30°C at a constant rate of 1°C min- ‘. During that time, temperatures were recorded every 6 s in the computer attached to the biological freezer and a thermokinetic graphic produced. Seeding ice nucleation was achieved by means of a local cold shock given with LN at one end of the straws. This shock was controlled from the central processing unit as established in the experimental design. The effect of seeding ice formation on the sample temperature during change to the solid state was evaluated for 36 p.p.t. seawater. Seeding was applied at three different points of
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the temperature curve (freezing point and one degree below or above it) and for seawater with or without addition of 15% DMSO. 2.2. Cryopreservation trials
Depending on species, between 200 and 400 ml of cultures were harvested and concentrated by refrigerated ( 17°C) centrifugation at rotation speeds of 1000 Xg for R. baltica and T. chuii, 1800 X g for I. galbana and Ch. gracilis, and 2700 X g for N. gaditana and N. atomus. Concentrates were resuspended in 0.22 pm filtered seawater of 36 p.p.t. and mixed with seawater of the same salinity containing DMSO, resulting in a final concentration of 15% (v/v). Cryoprotectant incubation lasted 45 min at 17-20°C. After incubation, algae were introduced in 0.5 ml straws and frozen according to the experimental design. A total of four replicates were obtained after performing duplicated experiments in which two replicates were used. The freezing programmes used for the first cooling step initially lowered the temperature at a rate of - 3°C min- ’ from + 20°C to - 6.5”C (freezing point for 15% DMSO in full-strength seawater) when the seeding function was not used. The same cooling rate was used to reach a temperature of - 5.5”C when seeding ice formation. In this latter instance, the lowering from -5.5”C to -6.5”C was achieved at a rate of - 0.33”C min- ‘. On reaching the freezing point, the seeding function was used for 10 s, allowing ice to form for 5 min. Five different cooling rates (0.25,0.5, 1,2 and 4°C min- ‘) were employed to lower the temperature from the freezing point to - 30°C both for seeded and unseeded samples. On reaching - 30°C algae were finally cooled to - 50°C at a rate of -3°C min-’ . After this first cooling step, straws were either thawed by adirect immersion in a 20°C water bath, or introduced in LN and thawed between 4 and 8 weeks later. A second set of experiments was carried out with the only difference of using conventional - 20°C and - 80°C laboratory freezers to perform the first step of cooling. 2.3. Viability assessment After thawing, the cryoprotectant was removed by a lOO-fold dilution of algal concentrates with autoclaved culture medium. Algae were grown for 7 days in 10 ml tubes which were placed in an illuminated incubator under constant conditions of radiation ( 100 pm01 photons me2 s-’ on an 18:6 LD cycle) and temperature (19°C). Initial and final cell densities of these cultures were determined either microscopically in a haemocytometer or using a ZM Coulter Counter fitted with a 30 pm orifice tube. Viability was estimated by measuring both cell recovery and the capacity of those cells to grow in respect to unfrozen algae. These unfrozen controls were also carried out under the above conditions. Multiplicative regressions ( Y = axb) for cell density after 7 days of culture ( Y) against initial cell density (X) were calculated for a wide range of initial cell densities in the control cultures. Cell densities measured in 7-day-old cultures of thawed algae were compared to the corresponding theoretical cell densities predicted by the regression equation for any initial cell density. The viability index was calculated from the relationship between observed and predicted growth multiplied by the proportion of recovered cells.
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3. Results The changes in temperature with time for single events during phase change in seawater of 20 and 36 p.p.t. salinity containing no cryoprotectant and those with lo%, 15% and 20% (v/v) of DMSO, methanol or glycerol are shown in Fig. 1. The freezing point decreased proportionally to the osmolality of each combination of seawater-cryoprotectant and had no variability within any given process. Since methanol possessed the lowest molecular weight, its osmolality was higher than DMSO and glycerol in identical percentages (v/v). Although not represented in Fig. 1, a significant variability was found within each combination of salinity-cryoprotectant for undercooling occurring before the release of the latent heat of fusion. The extent of such undercooling was, however, similar (P> .Ol) for media of very different osmolalities such as distilled water, 36 p.p.t. seawater and 15% DMSO in 36 p.p.t. seawater (Table 1). It is also noticeable from Fig. 1 that both salinity and the addition of cryoprotectants eliminated the temperature plateau detected after pure freshwater solidification. Microscopic observations were carried out on algae which had been undercooled but not frozen. These revealed that all species tolerated very well undercooling temperatures when suspended in 36 p.p.t. seawater with 15% DMSO for periods of up to 60 min. Samples taken from concentrates at - 18°C ( which were still in a liquid state) and diluted lOO-fold in sterilized seawater showed that the whole population of R. baltica was swimming very actively. Tetruselmis chuii also exhibited normal swimming activity, but this was limited only to a period of 2-3 h after dilution of the cryoprotectant. Zsochrysis galbana lost its motility after the centrifugation process and was found to recover normal swimming activity on the following day. The six species responded normally when cultured after the undercooling process. The effects of seeding ice formation for durations of 10,20,30 and 40 s, on the temperature changes of 36 p.p.t. seawater during solidification, are shown in Fig. 2. The shortest seeding time of 10 s, done at the right freezing point, was enough to induce ice formation and avoid undercooling of seawater. Increasing seeding times resulted in increased degrees of undercooling, due to excessive temperature reduction produced by the longer application of the cold shock. The addition of 15% DMSO decreased the time taken for ice to form. This can be noticed after comparing left- and right-hand columns of Fig. 3. The column on the left (36 p.p.t. seawater without cryoprotectant) shows sample temperature curves which needed longer times to adjust to the programmed temperature after commencement of ice formation. It can also be noticed from Fig. 3(A) that the application of seeding ( 10 s) one degree above the freezing point has no effect on initiation of ice formation. Seeding samples at the right freezing point, together with the use of 15% DMSO, resulted in the best performance. This can be noticed from the absence of deviations from the programmed temperature during phase change (Fig. 3B). Seeding 1°C below the freezing point produced undercooling of samples (Fig. 3C). The viability of thawed algae after being frozen either to - 50°C (single step) or - 196” (two cooling steps), with or without seeding ( 10 s), and at five cooling rates is shown in Fig. 4. Important differences existed between species. Tetraselmis chuii showed the best capacity to recover from cryopreservation, always giving viabilities around the maximum (average of 97.9%). Good results (80.5% average) were also obtained for N. atomus,
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0 -6 -10 -16 -20 -25 -30
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Fig. 1. Thermokinetics during phase change in seawater of 36 p.p.t. (left) and 20 p_p.t. (right) without cryoprotectant (top) and containing three concentrations of DMSO, methanol or glycerol. Top graphs also include temperature changes for freshwater.
whereas N. gaditana ranged from 53.8% to 61.6%. The other three species did not perform so well, giving mean viabilities of 20.9% (Ch. gracilis), 15.1% (I. galbana) and 7.3% (R. baltica). The most important fact found in this work was the lack of significance (P > .05)
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Table 1 Mean undercooling values ( f standard phoxide (n = sample size)
deviation)
found for three combinations
Undercooling 0 Salinity no cryoprotectant 36 Salinity no cryoprotectant 36 Salinity 15% DMSO
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of salinity and dimethyl
values
sul-
n
10.1 f 1.58 10.5 f 1.48 ll.Ok 1.58
7 14 17
for the seeding of ice nucleation, at any rate, with all algal species tested (Table 2). Seeding of ice did not improve the cryopreservation of microalgae. A similar lack of significance was found when comparing the single cooling step to - 50°C with the corresponding twostep cooling to - 196°C. Zsocrhysis galbana was the only exception since its viability after cryopreservation was significantly different (P < .Ol ) when frozen to - 50°C (mean viability of 25.8%) in comparison to algae frozen to - 196°C (mean viability of 4.4%). The five cooling rates tested did not affect (P > .OS) the cryopreservation of R. balticu, T. chuii, I. gulbunu or N. guditunu. On the other hand, Ch. grucilis viability was significantly higher (P < .05) at faster cooling rates. A Scheffe multiple comparison test (Zar, 1974) revealed two homogeneous groups (P > .05): one for 0.25 and 0.5”C min- ’ (average viabilities of 7.26% and 10% respectively) and another for 1, 2 and 4°C min-’ (average viabilities of 29.7%, 28.2% and 29.3% respectively). The difference detected by the ANOVA for the cooling rate in N. utomus is due to the lower viability (P < .Ol) recorded for the cooling rate of 0.25”C mitt- ’ (7 1.4%) against the others (78.8-86.6%). 0,
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Fig. 2. Deviation (dashed line) from programmed temperature (solid line) after seeding at the freezing point of seawater of 36 p.p.t. without any cryoprotectant. Seeding was performed for: 10 s (A), 20 s (B), 30 s (C) and 40 s (D) An arrow indicates end of undercooling.
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-_ m -20
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Fig. 3. Deviation (dashed line) from programmed temperature (solid line) after ice seeding ( 10 s) in 0.5 ml straws containing seawater of 36 p.p.t. without any cryoprotectant (left) and with 15% DMSO (right). Seeding was performed at the freezing point (B), one degree below (A) and one degree above (C) the freezing point.
Tetraselmis chuii, N. gaditana and N. atomus showed similar viabilities after cryopreservation whether the cooling procedures included as the first step controlled cooling to - 50°C or use of - 20°C and - 80°C laboratory freezers. Fig. 5 compares the average viabilities obtained for the six algal species to those obtained for controlled cooling to - 50°C. Cooling first to - 20°C resulted in a slightly reduced viability for R. baltica (3.1% for reaching only - 20°C and 2.2% when continuing to - 196°C) with respect to the procedures using cooling to - 50°C and - 80°C. The latter were not different from each other. Viability of Ch. gracilis also decreased when a - 20°C freezer (7.3% and 2.9% for one- or two-step toolings, respectively) was used for cryopreservation. Cooling to - 20°C reduced the viability of I. galbana, but only when using the single cooling step (9.3% at - 20°C against 25.8% averaged for all cooling rates at - 50°C). However, when a second cooling step to - 196°C was applied to this alga, viability decreased to 4.5% and did not differ depending on controlled or uncontrolled first cooling step. No viability was achieved when attempting to cryopreserve this species using - 80°C as the first step. Viability of T. chuii, N. gaditana and N. atomus was not affected by any of the freezing processes under evaluation (Fig. 5).
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Tetraselmis
baltica
chuii
T_
_
120 100 80 60 40 20 Isochrysis
galbana
Chaetoceros
Nannochloropsis
Nannochloris
gracilis
gaditana
atomw
100 60 60 40 20 0.25
0.5
10
2.0
4.0
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rate
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Fig. 4. Mean viability ( *SD) of algae frozen at five cooling rates after thawing from - 196°C with (solid columns) or without (cross-hatch columns) seeding ice formation, and after thawing from - 50°C with (fine horizontal columns) or without (coarse horizontal columns) seeding ice formation (N=4).
Table 2 Significance level for algal viability of a multifactorial ANOVA applied to three factors considered (see Materials and methods) in the cryopreservation of microalgae. The last column on the right indicates the significance of a one-way ANOVA used to compare between the - 20°C and - 80°C cooling procedures. Probabilities below 0.05 are in italic Factors
R. baltica T. chuii I. galbana N. gaditana Ch. gracilis N. atomus
Interactions
Cooling to - 20°C or - 80°C
A: cooling rate
B: seeding
C: end lststep
A*B
A*C
B*C
0.282 0.210 0.316 0.095
0.093 0.820 0.067 0.498
0.068 0.870 0.000 0.398
0.682 0.750 0.348 0.964
0.656 0.793 0.002 0.395
0.976 0.815 0.027 0.691
0.008 0.806 0.000 0.028
0.000
0.191
0.441
0.000
0.328
0.022
0.000
0.001
0.987
0.530
0.884
0.535
0.210
0.524
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136 (1995) 277-290
-80
(“C) of first
-50
cooling
-20
-80
step
Fig. 5. Viability ( + SD) of algae frozen only in one step to - 50°C (average for all cooling rates), - 20°C and - 80°C (solid columns). Cross-hatch columns indicate the corresponding viabilities when a second cooling step to - 196°C was used.
The cooling rates achieved for 0.5 ml straws introduced at -20°C or - 80°C were estimated with the cryogenic equipment, in which both types of cooling were easily simulated. Recorded temperatures for six replicates of 15% DMSO in 36 p.p.t. seawater showed that samples reached -20°C in a liquid state. Once this undercooling temperature was reached, the commencement of ice formation was detected by the releasing of the latent heat of fusion, which caused the temperature to increase to the freezing point. Ice formation was initiated at variable times of 19, 25, 28, 3 1, 39 and 54 min after reaching - 20°C. A cooling rate of - 14°C min- ’ was found to occur between + 10°C and - 50°C for the - 80°C cooling process.
4. Discussion Increasing salinity and cryoprotectant concentration lead to a depression in the freezing point, as expected ( 1.86”C per osmol solute). However, neither salinity nor cryoprotectants could significantly increase the extent of undercooling for freshwater (Table 1) . The lack of any clear pattern for the release of the latent heat of fusion in all cases registered in Fig. 1 reflected the randomness for the commencement of ice formation. Undercooled seawater with cryoprotectants has been used for short-term preservation of live rotifers (Berghahn et al., 1990) but there is no information on its use with microalgae. A general good tolerance of marine microalgae to undercooling temperatures for short periods of time can be concluded from this work. However, nothing is known on algal tolerance to longer periods of undercooling. Investigations on this topic are difficult, due to the fact that sudden ice formation may occur at any time and at any given undercooling temperature. What could be evaluated in the future is the tolerance of microalgae to temperatures slightly above the freezing point in relation to the storage time. The effects of such temperatures on cell suspensions were denominated as indirect chilling injury by Morris (1981) and were thought to be responsible for cell damage after periods of days of exposure. This type of
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study would help to advance the knowledge of marine microalgae resistance to low temperatures, more than cryopreservation trials in which the occurrence of ice formation makes osmotic events principally responsible for lack of viability (Grout and Morris, 1987). Lowering the freezing point could be safely achieved with the use of cryoprotectants at concentrations below toxic thresholds for algae. Marine microalgae have been demonstrated to tolerate quite high DMSO and methanol concentrations when incubated at room temperature (Cafiavate and Lubian, 1994); but nothing is known at reduced temperatures. This tolerance is in contrast to high cryoprotectant toxicities described for Chlorella (Morris, 1976), diatoms (McLellan, 1989) and Tetraselmis suecica (Fenwick and Day, 1992). Flassch et al. ( 1975) reported on high viabilities obtained for frozen T. suecica with the use of lO-20% glycerol or 15% DMSO. It is likely that the different methods employed to estimate viability among studies might be the reason for part of such differences in observing tolerance. Motility does not seem to be a reliable method to assess Tetraselmis viability. This species was shown to require variable times to recover its normal swimming activity after cryoprotectant exposure (Cahavate and Lubian, 1994) and after cryopreservation. Inducing ice formation was found to have no effect on the viability of any cryopreserved species. This is the first report in which a comparative study is described between seeded and unseeded algal samples. Most of the work on the cryopreservation of microalgae has been done without the use of ice nucleation induction (Morris, 1978; Saks, 1978; BenAmotz and Gilboa, 1980; McLellan, 1989; Chevalier, 1991). The correct application of the seeding mechanism avoids undercooling (Fig. 3) and thus the effects of temperature increase after the sudden release of the latent heat of fusion. Therefore, freezing after undercooling causes samples to deviate from the programmed temperature of the freezing chamber. This results in a faster cooling rate to that pre-established (Fig. 1) and is due to the tendency of straws to rapidly equilibrate temperatures with their environment. This fact is of special importance if considering its occurrence during ice formation, when the use of a rapid drop in temperature may result in lethal intracellular ice formation (Morris, 198 1) . It seems that seeding ice formation may play a more important role in organisms which are optimally cryopreserved at slow cooling rates. Seeding of ice was used in the cryopreservation of the rotifer Brachionus plicatilis, a species that showed optimum viabilities at cooling rates of -0.2 and -0.3”C min-‘, and did not survive at cooling rates above -05°C min-’ (Ok amoto et al., 1987). The lack of significance for seeding ice formation in the algae under study is in agreement with the better results obtained using faster cooling rates. Seeding for 10 s effectively avoided undercooling, whereas longer seeding periods produced some undercooling. This period is shorter than that of 30 s used by Toledo and Kurokura ( 1990) to cryopreserve B. plicatilis in a medium of a similar composition ( 10% DMSO in seawater) to ours. A possible improvement in the cryopreservation of rotifers could be suggested for seeding times shorter than 30 s. The cooling rates used to evaluate the effects of seeding ice formation may be considered as slow to medium rates. Therefore, species such as Ch. gracilis which have been described to be optimally cryopreserved at 15°C min-’ (McLellan, 1989) could have been suboptimally cooled in our work. Nannochloris atomus tolerated very well a wide range of cryopreservation stressing conditions. It is possible to achieve high viability of Nannochloris species even without the participation of any cryoprotectant (Ben-Amotz and Gilboa, 1980; Chevalier, 1991; Cafiavate and Lubian, 1994). The lack of significance for the cooling rate
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in the cryopreservation of R. baltica, T. chuii, I. galbana and N. gaditana is likely to be related to the use of a cryoprotectant concentration as high as 15% DMSO. The inverse relationship between cryoprotectant concentration and dependence on the cooling rate was described a long time ago (Morris and Farrant, 1972). Similar viabilities were found among cooling rates from 0.5 to 5°C min- ’ by Fenwick and Day ( 1992) when cryopreserving Tetraselmis suecica with 10% glycerol as cryoprotective agent. Our results show that three species (T. chuii, N. gaditana and N. atomus) of the six tested can be cryopreserved achieving viabilities in excess of 50% following simple procedures available in routine hatchery facilities. Low viabilities obtained for R. baltica and I. galbana and the very limited literature available on their cryopreservation suggest that more research is needed in the future on both species. Zsochrysis galbana was the only species which significantly lost viability when cooled in two steps to - 196°C in comparison to the single cooling step to - 50°C. Such a difference could indicate its need for a longer stabilization period after ice formation in order to achieve osmotic equilibration at subfreezing temperatures and avoid crystallization during quenching in LN (McFarlane, 1987). A longer equilibration time at a lower temperature ( - 60°C) was found by Meyer ( 1985) to produce the best results in the cryopreservation of Thalassiosira weisflogii. Cooling in a - 20°C freezer gave, in general, similar results to those obtained with the use of - 80°C as a first cooling step. Rhodomonas baltica and Ch. gracilis were the only species where the use of - 80°C resulted in better survival rates than exposure to - 20°C. It is likely that faster cooling rates during the change from liquid to solid phase are more efficient than slow or steady rates for cryopreserving five of the algae species studied. No clear conclusion, however, can be reached for I. galbana. Cooling to - 80°C has the advantage of providing faster cooling rates if larger volumes of algal concentrates are considered for cryopreservation. However, such cooling rates will never be as fast as those described in our work for 0.5 ml straws, due to obvious differences in volume. The short-term storage of viable algal biomass at temperatures within the - 20 to - 80°C range, offered by laboratory freezers, could be helpful for hatchery operations. However, since - 139°C is the limit for ice crystal growth (Morris, 198 1) , such practices will need detailed investigations on the rate at which viability is lost in relation to the duration of storage. One should also bear in mind that, if large numbers of cells are to be cryopreserved, complementary techniques for harvesting and concentrating algal cultures are needed. As a possible alternative to continuous centrifugation, microalgae flocculation with chitosan could be used (Morales et al., 1985; Lubian, 1989).
Acknowledgements
This work was supported by the Regional Agriculture and Fisheries Council (Junta de Andalucia) through a collaboration programme with the National Council Research (CSIC) . Special thanks are due to J.C. Sacristan (Carburos Metalicos) for his assistance with the cryogenic equipment.
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