- Email: [email protected]
Viability of mass algal cultures preserved by freezing and freeze-drying
aquacultural engineering ELSEVIER
Aquacultural
Engineering
16 (1997) 205-211
Viability of mass algal cultures preserved by freezing and freeze-drying Beatriz Corderoa *, Domenico Voltolinah “Centro de Investigucicin Cientifica y de Educacicin Superior de Ensenada (CICESE), Departamento de Acuicultura, Ave. Espinoza 843, Apartado Postal 2732, Ensenada, Baja California, M&co “Facultad de Ciencias Marinas. Universidad Autdnoma de Sinalou, Paseo Claussen s/n, Apartado Postal 610, Mazatlbn, Sinaloa, Mixico Received
22 January
1995; accepted
4 December
1996
Abstract The microalgae Chaetoceros sp. and Phaeodactylum tricomutum were grown in semicontinuous 15 I cultures, a volume commonly used for mid-scale inocula in aquacultural practices. The daily yields were concentrated and stored by freezing and freeze-drying for up to 1 month. After slow or fast freezing without the protective agents glycerol or Me7S0, neither of the microalgae was capable of active growth, while with the cryoprotectants slow freezing gave good results after 7 days of storage at -20°C. After 15 and 30 days, however, microalgae growth was poor, probably due to bacterial contamination. Freeze-drying gave even worse results, which we attribute to severe losses of storage products after this treatment. 0 1997 Elsevier Science B.V. Keywords:
Microalgae; Freeze-drying;
Preservation
1. Introduction
One of the main tasks of microalgae collections research and for commercial purposes. In Mexico, Investigacibn Cientifica y de Educaci6n Superior several commercial hatcheries, some of which have “Corresponding USA.
author: CICESE, Department
0144-X609/97/$17.00 Q 1997 Elsevier PII s0l44-8609(97)00001-0
of Aquaculture,
is to provide viable inocula for the collection of the Centro de de Ensenada is the choice for limited laboratory facilities and
P.O. Box 434844. San Diego. CA 92143.4844.
Science B.V. All rights reserved
206
B. Cordero, D. VoltolinalAquacultural Engineering 16 (1997) 205-211
are unable to maintain their stock strains or even their mid-scale cultures (from 15 to 50-60 I), which are used as inocula for the final steps of their microalgal production line. In addition, some facilities have not been designed with the necessary redundancy, so that failures in the production line are not uncommon. Sometimes, nearby hatcheries are able to provide some of their extra production, and in several cases we have been able to supply up to 100 1 of emergency inocula of at least one working strain, so that production may be re-established with little delay. However, when neither option is available, the resulting delay is likely to cause severe losses of the hatches involved. There are several alternatives to the use of live microalgae. However, the review by Coutteau and Sorgeloos (1992) reveals that algal substitutes are not popular in hatcheries because of their poor performance in hatchery conditions and high cost. These factors and scarce availability were also mentioned as major objections to their use, in several personal interviews with hatchery operators on the Pacific Mexican coast. On the other hand, there is sufficient evidence that frozen or freeze-dried microorganisms retain their viability for long periods of time (Holm-Hansen, 1973; Ben-Amotz and Gilboa, 1980; Buitrago, 1992). For this reason, we looked into the feasibility of maintaining large-scale inocula in a viable form using simple and inexpensive techniques, which would allow instant shipping, at an affordable cost, of some of the microalgae strains we supply to Mexican commercial hatcheries.
2. Materials and methods The microalgae
used were strains CH-X-1 and PH-T-1 (Chaetoceros sp. and from CICESE’s collection. Both are widely used in Mexican commercial aquaculture for feeding penaeid and bivalve larvae (Trujillo-Valle, 1993). They were kept in several 15 1 semicontinuous cultures with 50 and 75% daily dilutions for CH-X-1 and PH-T-1 respectively, in f medium (Guillard and Ryther, 1962). Other culture conditions were: salinity 32%; temperature 21 fl”C; pH between 7.5 and 8.2, kept within these limits by unmetered CO2 injection. Stirring was by air bubbling and lighting was continuous, provided by white fluorescent lamps giving an irradiance of 120 pmol mA2 s-i at the culture surface. Daily production was concentrated by centrifugation at approximately 12OOg for 15 min and divided into aliquots, in order to evaluate the dietary value and the changes in biochemical composition resulting from preservation and storage (Cordero-Esquivel et al., 1993; Cordero-Esquivel and Voltolina, 1996). In addition, several 1 ml aliquots with cell concentrations close to 1 x lo9 cells ml-’ were used to check whether centrifugation caused initial cell damage; others were either frozen and stored in a commercial freezer at -20°C or dipped in liquid nitrogen until frozen solid (15-30 s) and stored in an Ultra Low freezer at -60°C. In both cases, aliquots were also freeze-dried in a Labconco freeze-drier and stored in a
Phaeoductylum tricomutum Bohlin, respectively)
B. Cordero,
D. VoltolinalAquacultural
Engineering
16 (1997)
207
205-211
desiccator. In the case of samples frozen at -20°C some were untreated and others had added glycerol or Me*SO (dimethyl sulfoxide) in concentrations of 1, 5 and 10% (v/v). Viability was measured as the presence or absence of growth in triplicate 1:lO serial dilutions, from an original 1 x IO’ cells ml -’ subsample down to 1 x 10” cells ml ‘. Growth was measured daily as optical density increases at 550 nm in a Hach DR 2000 spectrophotometer. Observations at the microscope confirmed whether the increases were due to microalgae or to bacterial growth. For the preserved samples, these trials were performed after 7, 15 and 30 days from harvest.
3. Results The dilutions of higher order (10’ and lo4 cells ml-‘) prepared with samples taken before and after centrifugation had the same growth rate with no undue lag, which we assumed to mean that centrifugation at low gravity did not cause cell damage. Slow or fast freezing without glycerol or Me$O, resulted in complete loss of viability of Chaetoceros and PhaeoductyZum after 1 week of storage, since no growth or viable cells were observed even after 1 week of incubation. However, when protective agents were used in combination with slow freezing and storage at -20°C survival was high in all cases: Chaetoceros grew well from 2 days after thawing; Phaeodactylum preserved with glycerol showed no lag phase, and this was only 2 days with Me2S0 (Tables 1 and 2). Survival was much poorer after 15 days of storage. Two to three days after thawing, bacterial growth was very high in the samples with glycerol, and it was only slightly lower in those with Me,SO. Washing with sterile seawater and resus-
Table I Viability of triplicate serial dilutions of Chartoceros sp. frozen different concentrations, after different times of storage I days
at -20°C
with glycerol
15 days
and Me,SO
in
30 days
I x 10’
1x 10”1 x 10 1 x lo4 1 X 10’ I x 10” 1 x 105 1 X IO” 1 x IO’ 1 x 10” 1 x 1oi 1 X lOA
I 5
+ +
+++z +++ +++” +++
-
_
_
_
_
+
_ +++? +++
-
IO
+
+++:++-
+-
+
+-
-
_
+
+++?
+-
-~
+ + +
+f+L ++tz ++f2
_ f-+-
+ + +
f-3 +++’ ff+‘-
_ _ -
_ + +
_ -
_ -
_ -
Glycerol
(96)
MezSO (96)
I 5 10
+++++
+. growth; -. no growth after 7 days. Exponents indicate When not indicated, first growth was 24 h from inoculum.
the delay
(in days) to obtain
first growth.
208
B. Cordero, D. VoltolinalAquacultural Engineering 16 (1997) 205-211
Table 2 Viability of triplicate serial dilutions of Phaeodactylum tricomutum frozen at -20°C with glycerol and Me,SO in different concentrations after different times of storage 7 days
15 days
30 days
1 x 10’ 1 x 10” 1 x 105 1 x lo4 1 x 10’ 1 x lo6 1 x 10s 1 x 104 1 x 10’ 1 x lo6 1 x 105 1 x lo4 Glycerol (%) + 1 + 5 10 +
+-+++ +++
MezSO (%) 1 5 10
+++‘+++ f--2 f+++*-
+ + +
--cc+ +-
+ -
+__A--_ -
--
+ + +
_
_
+++’ -
_
+++ -
+ -
_
-
+ + +
-
-
--+++ ++-
-
_
-
+, growth; -, no growth after 7 days. Exponents indicate the delay (in days) to obtain first growth. When not indicated, first growth was 24 h from inoculum.
pending in new medium allowed only partial recovery, after a long lag phase. For this reason, the samples used after 30 days to obtain the initial 1 x 10’ cells ml-’ dilution were washed twice with sterile seawater and reconcentrated at 65Og only. Most of the bacteria were eliminated and we obtained fair survival and good growth with 5% and 10% glycerol-protected Chaetoceros (Table 1) and with 10% in the case of Phaeodactylum (Table 2). With 1% Me,SO a few live cells were observed in the initial dilutions, but recovery and growth were slow for both species (Table 1). Microscopic examination of freeze-dried algae after rehydration showed that only a few cells were apparently viable, but incubation of the serial dilutions showed some growth only in the initial 1 x 10’ test tubes (Tables 3 and 4).
Table 3 Viability of triplicate serial dilutions of freeze-dried Chaetoceros sp. after slow freezing with glycerol and Me-$0 in different concentrations after different times of storage 30 days
15 days
Glycerol (%) 1 5 10 MezSO (%) 1 5 10
1 x 10’
1 x 10”
1 x lo”
1 x lo4
1 x 10’
1 x lo6
1 x lo-5
1 x lo4
+ + +
_ -
-
_ -
_ +
-
_ -
-
+ + +
-
-
_ _
+ -
-
_ -
-
+, growth; -, no growth, after 7 days.
209
B. Cordero, D. VoltolinalAquacultural Engineering 16 (1997) 205-211
Table 4 Viability of triplicate serial dilutions of freeze-dried Phaeodactylum tricornutum after slow freezing with glycerol and MezSO in different concentrations after different times of storage 30 days
I5 days
Glycerol (96) 1 5 10 Me,SO (96) I 5 10
1 x10’
1x 10”
1 x lo5
1 x lo4
1 x 10’
1 x 10”
1x 1v
1 x 10”
+ + _
_ _ _
_ _ _
_ _ _
+ + _
_
_ _ _
_ _
+ + _
_ _ _
_ _ _
_ _ _
_ _ _
_ _ _
_
_ _ _
_
_
+, growth; -, no growth, after 7 days.
4. Discussion
and conclusions
Preservation of live microalgae by freezing and freeze-drying is still an open held for investigation, more so when the microalgae are of marine origin. Techniques used successfully with freshwater algae (Holm-Hansen, 1963; Holm-Hansen, 1973; Takano et al., 1973; Morris, 1976a,b) are not effective with marine ones, probably because of the interactions of ice crystal formation and salt saturation on protein denaturation and lipoprotein solubilization (Ben-Amotz and Gilboa, 1980). In the case of diatoms, however, McLellan (1989) pointed out that several marine species are amenable to cryopreservation at -196°C while those of freshwater origin suffer irreversible cell injury, either due to intracellular ice formation or to freezing-induced leakage of vacuolar contents into the cell cytoplasm. Protective agents such as glycerol and Me$SO have colligative properties: they form hydrogen bonds which prevent the diffusion of water molecules and the formation of an ice front, which are responsible for the denaturation of the intracellular biochemical fractions (Withers, 1985). Our data confirm the protective role of these substances, but show that the initial good viability declines with time. They also show that these treatments are not effective when the cells are vacuumdried after freezing, probably because this process causes a loss of cell metabolites, especially important in the case of lipids (Cordero-Esquivel et al., 1993). Similar results were obtained by Molina-Grima et al. (1994) with lsochrysis galbanu, which showed only a 2% survival after freeze-drying and a decrease of viability over time. Cells frozen without protection do not undergo initial losses, but storage at -20°C causes a loss of organic cell contents, of only 5% after 2 months in the cast of Chaetoceros, but of more than 10% for Phaeodactylum, mostly explained by a decrease in proteins and carbohydrates (Cordero-Esquivel et al., 1993). The presence of a good bacterial substrate susch as glycerol (Bianchi, 1976) probably favours an increase in bacterial concentration even at that temperature and enhances those changes, which would explain the storage-related loss of viability of
210
B. Cordero, D. Voltolina/Aquacultural
Engineering 16 (1997) 205-211
glycerol-preserved microalgae. MezSO is more likely to be directly responsible for the loss of microalgae viability, since it affects the permeability of cell membranes (Shoaf and Lium, 1976), causing an osmotic cell imbalance at the time of thawing. Our data are only in partial disagreement with McLellan’s claim (McLellan, 1989) that marine diatoms are amenable to cryopreservation, since his purpose was to obtain at least 50% survival, and the time of recovery of growth was not an issue. For our practical purposes, the percentage of survival must be much higher, as indicated by our dilution procedure, designed to provide > 90% viability. The delay in recovery of growth was also considered essential, since we aimed to obtain mass inocula for emergency use. The data we obtained indicate that fast freezing and freeze-drying are not adequate for this purpose and that slow freezing at high temperatures, as suggested by others authors (Millamena et al., 1990; Buitrago, 1992), is more likely to be a viable solution, after finding the proper balance between rates of freezing and thawing temperatures for storage and concentration of the cryoprotector. Until then, our standard procedure of keeping algal concentrates in a refrigerator will only solve the problems of short-term storage and shipping, but not that of being able to provide large-scale inocula for instant shipping, which prompted this investigation.
References Ben-Amotz, A., Gilboa, A., 1980. Cryopreservation of marine unicellular algae. 1. A survey of algae with regard to size, culture age. photosynthetic activity and chlorophyll-to-cell ratio. Marine Ecology Progress Series, 2, 157-161. Bianchi, M., 1976. Etude taxonomique et distribution Ccologique des bactCries vibrioides du milieu marine. These de Doctorat, Universitt D’Aix-Marseille II. Buitrago, E.B., 1992. Concentracibn y presetvacibn de microalgas coma reserva de aliment0 de organismos marinos cultivados. Prog. Iberoam. Cienc. Tee. Subprog. II - Acuicultura, CYTED-D, 1, 47-53. Cordero-Esquivel, B., Voltolina, D., Correa-Sandoval, F., 1993. The biochemical composition of two diatoms after different preservation techniques. Comparative Biochemistry and Physiology 105B, 369-373. Cordero-Esquivel, B., Voltolina, D., 1996. Nutritional value of preserved microalgae for subadult My&s galloprovincialis. Journal of the World Aquaculture Society, 27, 113-118. Coutteau, P., Sorgeloos, P., 1992. The use of algal substitutes and the requirement for live algae in the hatchery and nursery rearing of bivalve molluscs: an international survey. Journal of Shellfish Research, 11, 467-476. Guillard, R.R.L., Ryther, J.H., 1962. Studies on marine planktonic diatoms. Cyclofella nana Hustedt and Detonula confewacea (Cleve) Gran. Canadian Journal of Microbiology, 8,229-239. Holm-Hansen, O., 1963. Viability of blue-green and green algae after freezing. Physiologia Plantarum, 16,530-540. Helm-Hansen, O., 1973. Preservation by freezing and freeze-drying. In: Stein, J.R. (Ed.), Handbook of Phycological Methods. Cambridge University Press, Cambridge, pp. 195-205. M&&an, M.R., 1989. Crytopreservation of diatoms. Diatoms Research, 4,301-318. Millamena, O.M., Aujero, E.J., Borlonga, LG., 1990. Techniques on algae harvesting and preservation for use in culture and as larval food. Aquatic Engineering, 9, 295-304. Molina-Grima, E., Srinchez-Perez, J.A., Garcia-Camacho, F., AciCn-FernBndez, F.G., Lbpez-Alonso, D.,
B. Cordero, D. VoltolinaiAquacultural Engineering 16 (1997) 205-211
211
Segura del C&Ho, C.I., 1994. Preservation of the marine microalga, Isochtysis galhana: influence on the fatty acid profile. Aquaculture, 123, 377-385. Morris, G.J., 1976. The cryopreservation of Chlorella. 1. Interactions of rate of cooling, protective addition and warming rate. Archives of Microbiology, 107, 57-62. Morris, G.J., 1976. The cryopreservation of Chlorella. 2. Effect of growth temperature on freezing tolerance. Archives of Microbiology 107, 309-312. Sheaf, T.W., Lium, B.W., 1976. Improved extraction of chlorophyll a and b from algae using dimethyl sulfoxide. Limnology and Oceanography, 21, 926-928. Takano, M., Sado, J.I., Ogawa, T., Terui, G.. 1973. Freezing and freeze-drying of Spin&a platensis. Cryobiology, 10, 440-444. Trujillo-Valle, M.L., 1993. La coleccibn de microalgas del CICESE. Comunicaciones Academicas. Serie Acuicultura, CICESE, CIACT9301, p. 103. Withers, L.A., 1985. Cryopreservation of cultured plant and protoplasts. In: Kartha, K.K. (Ed.), Cryoprotection of Plant Cells and Organs. CRC Press, Boca Raton, pp. 243-265.
Aquacultural
Engineering
16 (1997) 205-211
Viability of mass algal cultures preserved by freezing and freeze-drying Beatriz Corderoa *, Domenico Voltolinah “Centro de Investigucicin Cientifica y de Educacicin Superior de Ensenada (CICESE), Departamento de Acuicultura, Ave. Espinoza 843, Apartado Postal 2732, Ensenada, Baja California, M&co “Facultad de Ciencias Marinas. Universidad Autdnoma de Sinalou, Paseo Claussen s/n, Apartado Postal 610, Mazatlbn, Sinaloa, Mixico Received
22 January
1995; accepted
4 December
1996
Abstract The microalgae Chaetoceros sp. and Phaeodactylum tricomutum were grown in semicontinuous 15 I cultures, a volume commonly used for mid-scale inocula in aquacultural practices. The daily yields were concentrated and stored by freezing and freeze-drying for up to 1 month. After slow or fast freezing without the protective agents glycerol or Me7S0, neither of the microalgae was capable of active growth, while with the cryoprotectants slow freezing gave good results after 7 days of storage at -20°C. After 15 and 30 days, however, microalgae growth was poor, probably due to bacterial contamination. Freeze-drying gave even worse results, which we attribute to severe losses of storage products after this treatment. 0 1997 Elsevier Science B.V. Keywords:
Microalgae; Freeze-drying;
Preservation
1. Introduction
One of the main tasks of microalgae collections research and for commercial purposes. In Mexico, Investigacibn Cientifica y de Educaci6n Superior several commercial hatcheries, some of which have “Corresponding USA.
author: CICESE, Department
0144-X609/97/$17.00 Q 1997 Elsevier PII s0l44-8609(97)00001-0
of Aquaculture,
is to provide viable inocula for the collection of the Centro de de Ensenada is the choice for limited laboratory facilities and
P.O. Box 434844. San Diego. CA 92143.4844.
Science B.V. All rights reserved
206
B. Cordero, D. VoltolinalAquacultural Engineering 16 (1997) 205-211
are unable to maintain their stock strains or even their mid-scale cultures (from 15 to 50-60 I), which are used as inocula for the final steps of their microalgal production line. In addition, some facilities have not been designed with the necessary redundancy, so that failures in the production line are not uncommon. Sometimes, nearby hatcheries are able to provide some of their extra production, and in several cases we have been able to supply up to 100 1 of emergency inocula of at least one working strain, so that production may be re-established with little delay. However, when neither option is available, the resulting delay is likely to cause severe losses of the hatches involved. There are several alternatives to the use of live microalgae. However, the review by Coutteau and Sorgeloos (1992) reveals that algal substitutes are not popular in hatcheries because of their poor performance in hatchery conditions and high cost. These factors and scarce availability were also mentioned as major objections to their use, in several personal interviews with hatchery operators on the Pacific Mexican coast. On the other hand, there is sufficient evidence that frozen or freeze-dried microorganisms retain their viability for long periods of time (Holm-Hansen, 1973; Ben-Amotz and Gilboa, 1980; Buitrago, 1992). For this reason, we looked into the feasibility of maintaining large-scale inocula in a viable form using simple and inexpensive techniques, which would allow instant shipping, at an affordable cost, of some of the microalgae strains we supply to Mexican commercial hatcheries.
2. Materials and methods The microalgae
used were strains CH-X-1 and PH-T-1 (Chaetoceros sp. and from CICESE’s collection. Both are widely used in Mexican commercial aquaculture for feeding penaeid and bivalve larvae (Trujillo-Valle, 1993). They were kept in several 15 1 semicontinuous cultures with 50 and 75% daily dilutions for CH-X-1 and PH-T-1 respectively, in f medium (Guillard and Ryther, 1962). Other culture conditions were: salinity 32%; temperature 21 fl”C; pH between 7.5 and 8.2, kept within these limits by unmetered CO2 injection. Stirring was by air bubbling and lighting was continuous, provided by white fluorescent lamps giving an irradiance of 120 pmol mA2 s-i at the culture surface. Daily production was concentrated by centrifugation at approximately 12OOg for 15 min and divided into aliquots, in order to evaluate the dietary value and the changes in biochemical composition resulting from preservation and storage (Cordero-Esquivel et al., 1993; Cordero-Esquivel and Voltolina, 1996). In addition, several 1 ml aliquots with cell concentrations close to 1 x lo9 cells ml-’ were used to check whether centrifugation caused initial cell damage; others were either frozen and stored in a commercial freezer at -20°C or dipped in liquid nitrogen until frozen solid (15-30 s) and stored in an Ultra Low freezer at -60°C. In both cases, aliquots were also freeze-dried in a Labconco freeze-drier and stored in a
Phaeoductylum tricomutum Bohlin, respectively)
B. Cordero,
D. VoltolinalAquacultural
Engineering
16 (1997)
207
205-211
desiccator. In the case of samples frozen at -20°C some were untreated and others had added glycerol or Me*SO (dimethyl sulfoxide) in concentrations of 1, 5 and 10% (v/v). Viability was measured as the presence or absence of growth in triplicate 1:lO serial dilutions, from an original 1 x IO’ cells ml -’ subsample down to 1 x 10” cells ml ‘. Growth was measured daily as optical density increases at 550 nm in a Hach DR 2000 spectrophotometer. Observations at the microscope confirmed whether the increases were due to microalgae or to bacterial growth. For the preserved samples, these trials were performed after 7, 15 and 30 days from harvest.
3. Results The dilutions of higher order (10’ and lo4 cells ml-‘) prepared with samples taken before and after centrifugation had the same growth rate with no undue lag, which we assumed to mean that centrifugation at low gravity did not cause cell damage. Slow or fast freezing without glycerol or Me$O, resulted in complete loss of viability of Chaetoceros and PhaeoductyZum after 1 week of storage, since no growth or viable cells were observed even after 1 week of incubation. However, when protective agents were used in combination with slow freezing and storage at -20°C survival was high in all cases: Chaetoceros grew well from 2 days after thawing; Phaeodactylum preserved with glycerol showed no lag phase, and this was only 2 days with Me2S0 (Tables 1 and 2). Survival was much poorer after 15 days of storage. Two to three days after thawing, bacterial growth was very high in the samples with glycerol, and it was only slightly lower in those with Me,SO. Washing with sterile seawater and resus-
Table I Viability of triplicate serial dilutions of Chartoceros sp. frozen different concentrations, after different times of storage I days
at -20°C
with glycerol
15 days
and Me,SO
in
30 days
I x 10’
1x 10”1 x 10 1 x lo4 1 X 10’ I x 10” 1 x 105 1 X IO” 1 x IO’ 1 x 10” 1 x 1oi 1 X lOA
I 5
+ +
+++z +++ +++” +++
-
_
_
_
_
+
_ +++? +++
-
IO
+
+++:++-
+-
+
+-
-
_
+
+++?
+-
-~
+ + +
+f+L ++tz ++f2
_ f-+-
+ + +
f-3 +++’ ff+‘-
_ _ -
_ + +
_ -
_ -
_ -
Glycerol
(96)
MezSO (96)
I 5 10
+++++
+. growth; -. no growth after 7 days. Exponents indicate When not indicated, first growth was 24 h from inoculum.
the delay
(in days) to obtain
first growth.
208
B. Cordero, D. VoltolinalAquacultural Engineering 16 (1997) 205-211
Table 2 Viability of triplicate serial dilutions of Phaeodactylum tricomutum frozen at -20°C with glycerol and Me,SO in different concentrations after different times of storage 7 days
15 days
30 days
1 x 10’ 1 x 10” 1 x 105 1 x lo4 1 x 10’ 1 x lo6 1 x 10s 1 x 104 1 x 10’ 1 x lo6 1 x 105 1 x lo4 Glycerol (%) + 1 + 5 10 +
+-+++ +++
MezSO (%) 1 5 10
+++‘+++ f--2 f+++*-
+ + +
--cc+ +-
+ -
+__A--_ -
--
+ + +
_
_
+++’ -
_
+++ -
+ -
_
-
+ + +
-
-
--+++ ++-
-
_
-
+, growth; -, no growth after 7 days. Exponents indicate the delay (in days) to obtain first growth. When not indicated, first growth was 24 h from inoculum.
pending in new medium allowed only partial recovery, after a long lag phase. For this reason, the samples used after 30 days to obtain the initial 1 x 10’ cells ml-’ dilution were washed twice with sterile seawater and reconcentrated at 65Og only. Most of the bacteria were eliminated and we obtained fair survival and good growth with 5% and 10% glycerol-protected Chaetoceros (Table 1) and with 10% in the case of Phaeodactylum (Table 2). With 1% Me,SO a few live cells were observed in the initial dilutions, but recovery and growth were slow for both species (Table 1). Microscopic examination of freeze-dried algae after rehydration showed that only a few cells were apparently viable, but incubation of the serial dilutions showed some growth only in the initial 1 x 10’ test tubes (Tables 3 and 4).
Table 3 Viability of triplicate serial dilutions of freeze-dried Chaetoceros sp. after slow freezing with glycerol and Me-$0 in different concentrations after different times of storage 30 days
15 days
Glycerol (%) 1 5 10 MezSO (%) 1 5 10
1 x 10’
1 x 10”
1 x lo”
1 x lo4
1 x 10’
1 x lo6
1 x lo-5
1 x lo4
+ + +
_ -
-
_ -
_ +
-
_ -
-
+ + +
-
-
_ _
+ -
-
_ -
-
+, growth; -, no growth, after 7 days.
209
B. Cordero, D. VoltolinalAquacultural Engineering 16 (1997) 205-211
Table 4 Viability of triplicate serial dilutions of freeze-dried Phaeodactylum tricornutum after slow freezing with glycerol and MezSO in different concentrations after different times of storage 30 days
I5 days
Glycerol (96) 1 5 10 Me,SO (96) I 5 10
1 x10’
1x 10”
1 x lo5
1 x lo4
1 x 10’
1 x 10”
1x 1v
1 x 10”
+ + _
_ _ _
_ _ _
_ _ _
+ + _
_
_ _ _
_ _
+ + _
_ _ _
_ _ _
_ _ _
_ _ _
_ _ _
_
_ _ _
_
_
+, growth; -, no growth, after 7 days.
4. Discussion
and conclusions
Preservation of live microalgae by freezing and freeze-drying is still an open held for investigation, more so when the microalgae are of marine origin. Techniques used successfully with freshwater algae (Holm-Hansen, 1963; Holm-Hansen, 1973; Takano et al., 1973; Morris, 1976a,b) are not effective with marine ones, probably because of the interactions of ice crystal formation and salt saturation on protein denaturation and lipoprotein solubilization (Ben-Amotz and Gilboa, 1980). In the case of diatoms, however, McLellan (1989) pointed out that several marine species are amenable to cryopreservation at -196°C while those of freshwater origin suffer irreversible cell injury, either due to intracellular ice formation or to freezing-induced leakage of vacuolar contents into the cell cytoplasm. Protective agents such as glycerol and Me$SO have colligative properties: they form hydrogen bonds which prevent the diffusion of water molecules and the formation of an ice front, which are responsible for the denaturation of the intracellular biochemical fractions (Withers, 1985). Our data confirm the protective role of these substances, but show that the initial good viability declines with time. They also show that these treatments are not effective when the cells are vacuumdried after freezing, probably because this process causes a loss of cell metabolites, especially important in the case of lipids (Cordero-Esquivel et al., 1993). Similar results were obtained by Molina-Grima et al. (1994) with lsochrysis galbanu, which showed only a 2% survival after freeze-drying and a decrease of viability over time. Cells frozen without protection do not undergo initial losses, but storage at -20°C causes a loss of organic cell contents, of only 5% after 2 months in the cast of Chaetoceros, but of more than 10% for Phaeodactylum, mostly explained by a decrease in proteins and carbohydrates (Cordero-Esquivel et al., 1993). The presence of a good bacterial substrate susch as glycerol (Bianchi, 1976) probably favours an increase in bacterial concentration even at that temperature and enhances those changes, which would explain the storage-related loss of viability of
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glycerol-preserved microalgae. MezSO is more likely to be directly responsible for the loss of microalgae viability, since it affects the permeability of cell membranes (Shoaf and Lium, 1976), causing an osmotic cell imbalance at the time of thawing. Our data are only in partial disagreement with McLellan’s claim (McLellan, 1989) that marine diatoms are amenable to cryopreservation, since his purpose was to obtain at least 50% survival, and the time of recovery of growth was not an issue. For our practical purposes, the percentage of survival must be much higher, as indicated by our dilution procedure, designed to provide > 90% viability. The delay in recovery of growth was also considered essential, since we aimed to obtain mass inocula for emergency use. The data we obtained indicate that fast freezing and freeze-drying are not adequate for this purpose and that slow freezing at high temperatures, as suggested by others authors (Millamena et al., 1990; Buitrago, 1992), is more likely to be a viable solution, after finding the proper balance between rates of freezing and thawing temperatures for storage and concentration of the cryoprotector. Until then, our standard procedure of keeping algal concentrates in a refrigerator will only solve the problems of short-term storage and shipping, but not that of being able to provide large-scale inocula for instant shipping, which prompted this investigation.
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