Mangroves are one of the most productive natural ecosystems and form an important part of the coastal and estuarine ecosystem and are nursery ground for many organisms'. Phenols are one of the major groups of secondary metabolites in plants. Phenolic acids in soil are naturally formed during humic acid breakdown. Oilier sources in sediment are flavanoids leached from plant debris, those formed during lignin decomposition and those synthesized by soil microorganism

Imelda, Joseph

Kishore Chandra, P

Panigrahi, A

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Indian Journal o f  M arine Sciences Vol. 29, Septem ber 2000, pp. 243-247Tolerance of three marine microalgae to cryoprotectants dimethyl sulfoxide,methanol and glycerolIm elda  Joseph* , A . Pan igrah i &  P. K ishore C hand ra  Central Institute o f  Brackish water Aquaculture, 101/B, Mahalingapuram, Chennai- 600 034, IndiaReceived 28 January 1999, revised 28 June 2000concentrations for DMSO were at 5% and lower Methanni • k V . Were d Wlthln 60 min- Safermethanol was almost negligent for all the three straps 6 CaSeS> P° St thaW viabi,ity in 5%W ithout loss o f viability, cryopreserved samples of biological m aterials have to be stored in liquid nitrogen at -196°C . M uch work on cryopreservation in aquatic species is involved in milt samples of various fishes '. C ryopreservation o f fresh water phytoplankton has also been studied more than that of m arine species2'7. S ince not much information is available on tolerance o f cryoprotectants, cryogenic m ethods used for other species have been tried without detailed studies on their optimization. In the process o f cryopreservation, pre-incubation o f cells in cryoprotectants is very im portant in preventing cell dam age during freezing. Though, these chemicals are toxic beyond certain levels, their mechanisms are poorly understood8. The m aximum tolerable limit of cryoprotectant with highest post-thaw viability is used in norm al cryopreservation experim ents9.C ryoprotectants are divided into those that are perm eable and non-perm eable to cell membrane. The perm eable ones reduce the formation o f ice and cell volume rem ains unchanged while freezing as well as thawing. Non perm eable cryoprotectants will m inim ize dehydration o f cells during freezing by converting extracellular solution into a gas. M arine micro-algae, living in an environment o f high*Present address: NRC ot CIBA, Narakkal, Cochin-682 505 Kerala, India.osmonality with high concentrations o f non- permeable solutes have a rigid cell wall. This characteristic may result in less biological recovery after shrinking7. Therefore, three permeable cryoprotectants, dim ethyl sulfoxide (DMSO), methanol and glycerol were selected for the present tolerance of study on three species o f marine m icro­algae, Chaetoceros• calcitrans, Tetraselmis gracilis and Chlorella marina. The study could help in improving the efficiency o f cryopreservation of marine micro-algae and m ore species could be preserved by this method.Materials and MethodsThree marine microalgae, Tetraselmis gracilis (Prasinophyceae), Chaetoceros calcitrans (Bacillario- phyceae) and Chlorella marina (Chlorophyceae) were selected from the cultures.Cultures were routinely m aintained in F/2 medium in 10 ml test tubes and 100 to 250 ml conical flasks at a tem perature o f 22 to 25°C and illumination o f 800 to 1000 lux on a 12 h: 12 h light and dark cycle. Sub culturing was done in every 10 d. Algae were harvested on 10th day o f growth. Harvested algae were centrifuged at 4000 rpm for 5 min. The resulting concentrates were immediately used for the cryoprotectant exposure experiments.244 INDIAN J. MAR SCI., VOL. 29, SEPTEMBER 2000T he salinity  o f algal concentrates and cryoprotectants were m aintained at 30%o. The algae were exposed to 0, 5, 10, 15, 20, 25, 30 and 40%  (v/v) concentrations o f DM SO, m ethanol and glycerol. Exposure times were studied by incubating the cultures for 1, 5, 15, 30, 60 and 120 min. All incubations were performed within a tem perature range o f 22 to 25°C using duplicate test tubes with 1ml o f algal suspension and 4 ml o f  cryoprotectant solution. After each incubation time, 0.1 ml o f suspension was pipetted out and used to inoculate 10 ml o f fresh sterilized F/2 medium in test tubes, achieving 100 times dilution for algae as well as cryoprotectant.The cells in 10ml test tubes were allowed to grow in the culture conditions described, for 8 to 10 d. The final cell concentration was taken by counting using a hem ocytom eter' and expressed as cells/ml. The sam ples with different levels o f  cryoprotectants were observed after 24 h o f freezing to record the cell recovery as whole cells rem aining after thawing. The viability was calculated from  the growth rate achieved in cultures after thawing. For T. gracilis and C. marina, the determ ination o f cell recovery was difficult, since the non-viable cells did not undergo cel 1-lysis during freezing. The instantaneous growth rates w ere calculated as follows:Instantaneous _  Final count (C t)-In itia l count (Co) growth rate/d (r )“  Duration (t)For a better and * m ore homogenous graphic com parison o f the responses by different algae, the growth rates were expressed as relative growth rates with respect to the unexposed controls.D . . . .  Final co u n t-In itia l count _ _Relative growth ra te= ------------ :----------------------xlOOInitial countThe significance o f the results obtained in the present study has been validated by two-way ANOVA treatm ents between concentrations of cryoprotectants and strains.ResultsAlgal cultures obtained after exposure to DMSO, methanol and glycerol dilutions showed different responses depending on the species and the concentration o f each cryoprotectant (Table 1). The response o f T. gracilis, C. calcitrans and C. marina to different concentrations o f glycerol, methanol andDMSO are shown in Fig. 1. Lethal doses of methanol and DMSO took effect mainly between 15 and 30 min. Generally the algae tolerated ascending levels of cryoprotectants up to the level at which viability drastically reduced. M yxotrophic growth was exhibited in some cases where the experimental treatment produced growth responses exceeding the growth of controls (for C. Calcitrans, Table IB), thus showing that some cryoprotectants could enhance growth of algae when present in very little concentrations. Tetraselmis gracilis was more tolerant to all the three cryoprotectants at all concentrations (Fig 1). On exposure to DMSO and glycerol, growth was not greater than that for controls even for T. gracilis. The highest concentration of DMSO and methanol where cells were viable was 30%, and at 40% it was non-viable. Whereas, C. calcitrans at 25% and C. marina at 30% cells were non viable in DM SO and methanol. In glycerol, all the three strains were viable up to 30% concentration. W hile C. calcitrans alone was non viable at 40% glycerol. For C. calcitrans, rapid mortality was observed from 25 to 40% of methanol and DMSO. The mortality of T. gracilis was affected above 10% concentration of methanol. The surviving cells of T. gracilis and C. marina sedimented at the bottom of the test tube after 24 h incubation (in methanol from 25%). The C. marina cells showed a colour change at higher concentrations (20% and above) in DM SO and methanol (bleached). The samples with different levels of cryoprotectants were observed after 24h of freezing. The percentage o f cell recovery and growth rate per day after thawing is presented in Table 2. In the present study 5% DM SO and 10% glycerol had enhanced post-thaw viability of cryopreserved m icro­algae. On statistical analysis using two way ANOVA, significance in variation was found between three different algal strains over treatments of DMSO, glycerol and methanol at 1% level.DiscussionThe methodology followed in the present study is very much reliable in determining the tolerance of marine micro algae to different cryoprotectants. The methodology followed by Canavate7 to determine the viability of algal cells after cryopreservation was almost similar to the present one. The cell viability in control after post thaw is less than 0.01% for all the three algal strains. W hereas, the cell recovery andJOSEPH et al.\ TOLERANCE OF MICROALGAE TO CRYOPROTECTANTSFig. 1 Relative growth rate o f C. calcitrans, T  gracilis and C. marina on using cryoprotectants, DMSO, glycerol and methanol.T gracilis (Glycerol)C. calcitrans (Glycerol) [SoT. gracilis /DMSO)C. calcitrans (DMSO)120 T - gracilis (Methanol)100  -  01 *C. calcitrans (Methanol)C. marina (DMSO)C. marina (Glycerol)C. marina (Methanol)Time of exposure (min)246 INDIAN J. MAR SCI., VOL. 29, SEPTEMBER 2000Table 1— Final cell density (106cells/ml) and standard deviation (in parentheses) in DMSO, methanoland glycerol concentrations% Concentrations (v/v)Algal strain 0 5 10 15 20 25 30 40DMSOTetraselmis gracilis 1.0(0.08)0.80(0.03)0.80(0.01)0.81(0.02)0.78(0.05)0.70(0.05)0.30(0.03)00Chaetoceros calcitrans . 4.52(0.28)4.68(0.15)4.30(0.21)4.20(0.12)1.80(0.08)00 00 00Chlorella marina ■ 8.50(0.37)8.30(0.42)8.00(0.28)7.00(0.25)4.0(0.18)2.8(0.08)00 00MethanolTetraselmis gracilis 1.13(0.16)0.95(0.14)0.82(0.18)0.76(0.20)0.72(0.14)0.56(0.15)0.18(0.18)00Chaetoceros calcitrans 4.50(0.76)4.90(0.78)5.00(0.65)4.80(0.50)2.50(0.48)0.85(0.43)00 00Chlorella marina 9.35(0.80)8.00(0.75)7.20(0.70)6.80(0.60)4.50(0.52)00 00 00GlycerolTetraselmis gracilis 1.20(0.82)1.20(0.58)1.00(0.63)0.98(0.71)0.85(0.80)0.80(1.00)0.40(0.92)0.28 (0.11)Chaetoceros calcitrans 4.50(0.18)4.36(0.16)4.20 ' (1.00)4.00(0.85)3.50(0.90)2.50(0.19)1.00(0.90)00Chlorella marina 9.50(0.95)9.40(1.20)9.00(1.13)8.60(0.85)8.00(0.99)7.50(1.5)5.00(1.4)2.00(1.8)growth rate for C. calcitrans by using 5% DM SO and 10% glycerol was 10.2 t.o 12.8% and 0.45 to 0.62% respectively (Table 2). N o recovery rate was determ ined for T. gracilis and C. marina. The growth rate varied from  0.20 to 0.25% in the above treatm ents o f D M SO  and glycerol for T. gracilis. For C. marina, the growth rate ranged from 0.15 to 0.35. V iable cells o f  C. calcitrans could be detected easily after thaw ing since the dam age caused during freezing o f cells has reflected in rapid cell lysis, whereas, it was difficult for T. gracilis and C. marina. For T. gracilis, the viable cells were actively moving after 24h o f post thaw.As given in Table 1 (A, B and C), for C. calcitrans, T. gracilis and C. marina 5% DMSO was effective. G lycerol at 5% was good for C. calcitrans, T. gracilis and C. marina, whereas, methanol at 10% was effective only for C. calcitrans.The use o f 5% DM SO found to be useful for cryopreservation in the present study as reported by T suru11, whereas, studies by Canavate7 has shown that algae could grow well after cryopreserving in 15% DMSO. Me Lellan4 also had described a 5% (v/v) DM SO concentration as the effective dose for cryopreservation o f marine diatom s. Generally DM SO was less effective than glycerol when used as a cryoprotectant at higher concentrations in the present study. Day & Fenw ick5 have also reported a sim ilar observation on cryopreservation o f T. gracilis.The loss o f cell viability in cells during cryopreservation could be attributed to factors like increase in cryoprotectant level in cells and osmotic changes during ice formations4,12. Therefore to assess good cryopreservation o f microalgae, its ability to withstand hyper toxicity o f cryoprotectants is very im portant13,7.JOSEPH et al.\ TOLERANCE OF MICROALGAE TO CRYOPROTECTANTS 247Table 2— Cell recovery and growth rate/d of dilution cultures after thawing from -196°C  algal concentrates ..previously incubated with 5% DMSO, 5% methanol and 10% glyceroi and control (without cryoprotectant)Algal strain% Recovery Growth rate%  Recovery Growth rate% Recovery Growth rate5% DMSONd0.2010.200.45Nd0.155% MethanolT. gracilisNd0.00C. calcitrans0.000.00C. marina0.000.0010% GlycerolNd0.2512.800.62Nd0.35ControlNd0.000.000.00Nd0.01Nd=not determinedThe cell structure o f T. gracilis and C. marina rem ained intact even after 100 fold dilution, whereas, C. calcitrans cells were disintegrated on post thaw dilution at lethal levels o f cryoprotectants. It was found that methanol is an ineffective cryoprotectant. M e L ellan4 has reported a sim ilar observation for diatoms.An evaluation o f algal tolerance to cryoprotectants at controlled room  tem peratures had not given any indication on loss o f viability during the process. The m echanism s o f sub-lethal and lethal damage, and the d ifference in viability am ong different strains of micro algae has to be investigated further for better understanding o f the m echanisms o f cryopreservation. As reported in earlier studies, factors such as cooling rate and salinity could also be responsible for the low recovery o f C. calcitrans com pared to T. gracilis and• 5,14C. marinaAcknowledgementW e are thankful to D r G R M Rao, Director, Central Institute o f Brackishw ater Aquaculture (CIBA), Chennai and D r L. H. Rao, Head o f Division, Crustacean Culture Division, for the constantencouragem ent during the present study.References1 Leung L K P & Jamieson B G M, Live preservation o f fishgamates. In: Fish evolution and  systematics: Evidence fo r  spermatozoa, edited by B.G.M. Jamieson (CambridgeUniversity Press, Cambridge) 1991, pp. 245-265.2 Beaty M. H. & Parker B C, Investigations ofcryopreservation and storage o f eukaryotic algae andprotozoa, J  Phycol, 26 (Suppl) (1990) 5.3 Morris G M, Cryopreservation o f 250 strains of Chlorococcales by the method o f  two-step cooling, Brit Phycol J, 13 (1978) 15-24.4 Me Lellan M R, Cryopreservation o f diatoms, Diatom Res, 4 (1989)301-318.5 Day J G & Fenwick C, Cryopreservation o f members o f the genus Tetraselmis used in aquaculture, Aquaculture, 118 (1993) 151-160.6 Grima E M, Sanchez Perez J A, Camacho F  G, Acien Fernandez F  G, Lopez Alonso D & Segura del Caslillo C I, Preservation o f the marine alga, Isochrysis galbana: Influence on the fatty acid profile, Aquaculture, 123 (1994) 377-385.7 Canavate J P  and Lubian L M, Tolerance o f six marine micro algae to the cryoprotectants dimethyl sulfoxide and methanol, J Phycol, 30(1994) 559-565.8 Fahy G M, Lilley T  H Lindsell H, Douglas M J & Meryman H T, Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search o f molecular mechanisms, Cryobiology, 27(1990)247-268.9 Morris G J & Farrant T, Interactions o f cooling rate and protective additive on the survival o f washed human erythrocytes frozen to -196°C , Cryobiology, 9 (1972) 173-181.10 Guillard R R L  & Ryther J H, Studies on marine planktonic diatoms:I. Cyclotella nana Hustedt and Detinula conferacea (Cleve) Gran, Canadian J  Microbiol, 8 (1962) 229-239.11 Tsuru S, Preservation o f marine and freshwater algae by means o f freezing and freeze-drying, Cryobiology, 10 (1973) 445-452.12 Morris G M, The cryopreservation o f Chlorella. I. Interactions o f rate o f cooling, protective additive and warming rate, Arch Microbiol, 107 (1976) 57-62.13 Morris G M, in: Cryopreservation (Institute o f Terrestrial Ecology, Cambridge) 1981, pp 1-27.14 Montaini E, Zittelli G C, Tredici M R, Grima E M, Fernandez Sevilla J M & Sanchez Perez J A, Long term preservation o f Tetraselmis suecica: Influence o f storage on viability and fatty acid profile, Aquaculture, 134 (1995) 81-90.

Tolerance of three marine microalgae to cryoprotectants dimethyl sulfoxide, methanol and glycerol

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