Biotechnology Techniques, Vol 12, No 11, November 1998, pp. 847–850

Alginate-entrapped Haslea ostrearia asinoculum for the greening of oystersThierry Lebeau*, Richard Moan, Vincent Turpin and Jean-Michel RobertLaboratoire de Biologie Marine, Institut des Substances et des Organismes de la Mer (ISOmer), Université deNantes, 2, rue de la Houssinière, BP 92208, 44322 Nantes cedex 3, Francefax: 00.33.2.51.12.56.12., e-mail: jmrobert@nat.svt.sciences.univ-nantes.fr

Entrapment in calcium alginate beads of the marine diatom, Haslea ostrearia, was successfully used for stock-culturemanagment and afterwards the sowing of ponds for the greening of oysters. After storage during almost 2 months,viable and cultivable cells were recovered from beads by dissolving alginate matrix but an original way lies in directlyintroducing beads in ponds and promoting natural cell leakage.

Keywords: immobilization, alginate beads, microalgae, Haslea ostrearia, diatom, stock-culture management, sowingof oyster ponds

IntroductionThe pennate diatom, Haslea ostrearia, synthesizes andexcretes an hydrosoluble, blue-green pigment, named mar-ennine, responsible for the greening of the oyster gills butfavourable results depend on natural environmental condi-tions. In order to promote this greening in the Bourgneufbay (France), cell cultures are currently produced at theindustrial level (SOPROMA, Bouin, Vendée, France). Thisproduction is performed in batch free-cell cultures, using6 m3 tanks under carefully controlled incubation condi-tions. Breeders of oysters put out their oysters to nurse forintensive greening. Nevertheless, this system presentssome disadvantages: contamination risks during fosterageand transport, freight transport and large greening demandcompared with supply (storage is not alloweed with free-cell cultures), particularly during new year feasts whenmost of oysters are sold. Immobilization offers a practicalalternative to free-cell cultures of microalgae (Lewin,1990). Most publications deal with entrapped microalgaefor exometabolite productions, extraction of heavy metalsfrom aqueous solution (Wilde and Benemann, 1993) andwaste water treatment (Vilchez et al., 1997) but littleinterest is taken in microalgal stock culture management,provided by the increase of cell viability when microalgaeare immobilized (Hertzberg and Jensen, 1989; Kaya andPicard, 1995; Ma and Zhang, 1996; Kannapiran et al.,1997) and few studies deal with culturability of releasedcells (Lukavsky et al., 1986; Hertzberg and Jensen, 1989;Kaya and Picard, 1995).

The aim of our experiments was to place inocula of H.ostrearia with easy and long-term storage without loss ofviability at breeders of oysters’s disposal with the intention

© 1998 Chapman & Hall

of inoculating oyster ponds at the right moment withcalibrated inocula. In a previous work (Lebeau et al. 1998),H. ostrearia was successfully entrapped in agar-gel discs formarennine production and a long-term operational stabil-ity of immobilized microalgal cultures was shown. Never-theless, it was impossible to recover microalgae entrappedin agar matrices. Therefore, entrapment in alginate gelappeared to allow the cells to be recovered after thedissolution of the matrix.

In this work, we tested algal cell maintenance of viabilityin calcium alginate beads at two algal cell concentrationsand culture conditions, and studied cell culturability aftergel dissolution (EDTA and citrate) in two media andnatural cell leakage from alginate matrix during incuba-tion.

Materials and methodsAlgal strain and maintenance mediumThe study was performed using an axenic strain of Hasleaostrearia Simonsen isolated from oyster-pond waters of theBouin district (Vendée, France). The cells were charac-terized by an average modal length of 70 mm, 60 mmcorresponding to the critical length below which algalgrowth is disturbed and the ratio of deformed cellsbecomes high (Robert, 1978). The algal cultures weremaintained by weekly transfer to fresh ES 1/3 medium(Lebeau et al., 1998): 288.2 mM NO3

2, 6.26 mM NH41,

100 mM SiO32, 307.4 mM dissolved organic nitrogen,

16.2 mM dissolved organic phosphorus.

Preparation of inoculaAlgal precultures were performed applying a two-stepprocedure. Cells from the clone pool were first precultured

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for about 6 days in 250-ml Erlenmeyer flasks filled with150 ml maintenance (ES 1/3) medium. The incubation wasat 15°C and light was at 3 3 1016 quanta cm22 s21 witha 14/10h light/dark cycle. Then the flask contents wereinoculated in 2-l Erlenmeyer flasks containing 1 liter of(ES 1/3) medium. Algal inocula were collected by cen-trifugation (4000g, 6 min, 15°C) from cultures in theexponential growth stage after incubation of these largerflasks for c. 1 week in identical temperature and lightingconditions. Cell suspensions with concentrations rangingbetween 107 and 108 cells ml21 (7.3–73 mg dry wt. ml21)were obtained.

Cell immobilizationAlgal cells were entrapped in calcium alginate as followed:sodium-alginate (Prolabo, 1.5 g) was dissolved in distilledwater (65 ml) by slow stirring for 1.5 h and a solution ofNaCl (2.8 g) in distilled water (31 ml) was added. pH wasadjusted to 7.8 with 0.1 M NaOH. Alginate powder wassterilized under UV radiation for 16 h while other com-pounds and the vessel were autoclaved at 120°C for 20 minbefore use. The sterile solution was mixed thoroughly withcell suspension until desired cell concentration i.e. 5 3 105

or 107 cells ml21 gel (0.4–7.3 mg dry wt. ml21) wasreached. Alginate was at 1.5 % (w/w). Beads of about3 mm diam were obtained by dropping the alginate cellmixture into a solution of 0.07 M CaCl2 (salinity and pHwere respectively adjusted to 28 g l21 and 7.8) for less than20 min using a peristaltic pump supplied with a calibratedneedle.

Growth and storage experimentsCells entrapped in alginate beads were distributed amongsttest-tubes: each one was holding 1 ml beads (5 3 105 or107 cells ml21 gel) with 25 ml liquid (ES 1/3) medium.For standard experiments (growth conditions currentlyused), temperature and illumination conditions were thoseof precultures. For storage experiments, the temperatureand light intensity were, respectively, 4°C and 2 3 1015

quanta cm22 s21 with a 14/10h light/dark cycle.

All experiments were performed in duplicate and resultswere expressed as mean.

Dissolution of beads and determination ofculturable cells1 ml beads was recovered and calcium alginate matrixdissolved with 10 mM EDTA for experiments in growthconditions, or 5 mM EDTA or 10 mM citrate for storageexperiments.

In this latter case, cells released from the beads wereinoculated (2 3 103 cells ml21 medium) into 250-ml

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Erlenmeyer flasks filled with 100 ml of ES 1/3 medium orenriched sea water [“NPSi” medium, Turpin (pers.comm.)]. This medium, enriched only with 87.9 mMNaNO3, 7.14 mM K2HPO4 and 100 mM Na2SiO3 iscurrently used for intensive greening of the oyster ponds asfertilizers in agriculture. Temperature and illuminationconditions were those of precultures.

Analytical proceduresThe algal population of immobilized-cell cultures wasassessed by direct method (cell counts using a Nageotte-type haematocymeter) and indirect method to estimate thenumber of immobilized viable cells by chlorophyll adetermination: the pigments were extracted from the beadswith acetone 90 % (v/v) at 4°C in obscurity for 24 hours.After centrifugation (1500g, 15 min), the absorbance at665 nm before and after acidification with HCl 2 M wasdetermined in the supernatant according to Lorenzenmethod (1967).

Results and discussionFigure 1 shows growth of immobilized algal cells at 4°Cand low-light intensity (storage conditions, Figure 1a), andat 15°C (growth conditions, Figure 1b). For standardexperiments (at 15°C), immobilized algal cultures grewduring incubation, whatever initial cell concentration,without lag time, which revealed no toxicity of the matrixand no cellular stress. Maximum growth rate was aboutthose of free cells (data not shown) with 5 3 105 cells ml21

gel, i.e. 1.1 day21, but only 0.2 day21 with 107 cells ml21

gel. In this latter case, quick substrate depletion (particu-larly nitrogen), enhanced resistance to substrate diffusion(Radovich, 1985) and self-shading (Vilchez et al., 1997)may explain the poor growth rate. After 4 and 7 days with,respectively, 107 and 5 3 105 cells ml21 gel, the decreaseof chlorophyll a concentration – which indicates algal cellviability – essentially expressed substrate depletion becauseof batch incubation.

During storage conditions, the initial cell concentrationdid not significantly influenced slow algal growth rate dueto the low temperature (4°C) and low light intensity (2 31015 quanta cm22 s21). In these conditions, cell viabilitywas maintained for almost two months (constant chloro-phyll a/cell concentration ratio) and could have beenextended as has been shown with other marine diatoms,e.g. Phaeodactylum tricornutum (Hertzberg and Jensen, 1989)and Navicula dissipata Hastelt (Ma and Zhang, 1996)immobilized in alginate beads for one year. With thisresult, it is possible to inoculate oyster ponds at the rightmoment – free-cell cultures can not be stored – withcalibrated inocula (practically no growth during storageespecially with the highest cell concentration tested).

Figure 1 Growth of immobilized H. ostrearia cellsexpressed as mean cell count (filled symbol) and chloro-phyl a in cells (void symbol). Initial cell loading: (j, h),5 3 105 ml21 gel; (d, s), 107 cells ml21 gel. (a) storageconditions at 4°C; (b) growth conditions at 15°C.

Figure 2 Cell leakage in the medium expressed asreleased/immobilized cells ratio at two initial loading: j,5 3 105 ml21 gel; d, 107 cells ml21 gel; ——, storageconditions (4°C); – - – - –, growth conditions (15°C). Con-centrations of released cells (3 106 cells ml21 medium)are given in parentheses (underlined numbers refer to thelowest initial cell concentration).

Greening of oysters

In order to inoculate ponds, two strategies may beemployed. As a consequence of algal growth, cell leakagefrom gel matrices used for entrapment is a well-knownphenomenon (Bailliez et al., 1988; Vilchez et al., 1997)which can be applied to the sowing of ponds as done inagriculture. In this way, inoculated beads may be directlydistributed in ponds after period of storage. This phenom-enon, medium-dependant, was already shown with H.ostrearia cells entrapped in agar sheet (Lebeau et al., 1998)and occured also in alginate beads (Figure 2).

In this latter case, leakage was favoured more by thetemperature (greater at 15°C) than by the initial cellconcentration in beads. If we consider the released/immobilized cells ratio, leakage, which includes releasedcells from alginate beads and cell multiplication intomedium, became significantly only after about 10 and 15days at 15°C and 4°C respectively. At 4°C, the amount of

free cells increased with initial cell concentration while thebest result, obtained at 15°C with 5 3 105 cells ml21 gel(1.8 106 cells ml21, 1200%), was due to active cell divisioninside the beads. Moreover, in the case of Haslea ostrearia,cell leakage seems to be more important in alginate gelcompared with agar gel (Lebeau et al., 1998) although thecontrary was shown by Leon and Galvan (1995) withChlamydomonas reinhardtii, and leakage depends on alginateconcentration and type of multivalent cation (Dainty et al.,1986).

In the second way, after various periods of storage at 4°Cand low light intensity, calcium alginate beads were dis-solved in EDTA or citrate to obtain suspension and theculturability of cells was estimated (Table 1). Whateverculture conditions, culturability of cells was maintainedafter 12 days storage and at least two months with ES 1/3medium (Table 1a) without important change in themaximum growth rate and maximum cell biomass. It didnot exceed about two weeks storage with “NPSi” medium(Table 1b). This result may be explained by possiblechemical changes (e.g. complexation) of the medium dueto sterilization (Fiala, 1987; Lukavsky, 1992). Whatevermedium was used, the lag time which is a consequence ofcellular stress increased with duration of storage but didnot reach three weeks as observed by Kaya and Picard(1995) with Scenedesmus bicellularis. On the other hand, noappreciable differences were observed in relation to initialcell concentration or dissolving agent althought citrateassociated with “NPSi” medium seems to procurate lesscellular stress and better growth of cells (Table 1b).

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Table 1 Culturability of cells previously immobilized at 5 3 105 cells ml21 gel and 107 cells ml21 gel (values givenin parentheses) and stored at 4°C for various periods. Calcium alginate beads were dissolved in citrate or EDTAand released cells were cultivated in (a) ES 1/3 medium or (b) “NPSi” medium.

[a] ES 1/3 medium [b] “NPSi” medium

Period of storage (day) 2 12 30 60 2 12*

Na2-citrateMaximum growth rate (day21) 1.31 (1.20) 0.90 (0.78) 1.43 (1.53) 1.56 (1.13) 1.17 (1.27) 0.68 (0.50)Maximum cell concentration (Log10 cells ml21) 5.64 (5.58) 5.51 (5.55) 5.48 (5.40) 5.66 (5.50) 5.25 (4.81) 4.91 (4.34)Lag time (day) 1 (1) 2 (1) 3 (3) 5 (5) 1 (1) 1 (1)

EDTAMaximum growth rate (day21) 1.17 (1.36) 0.86 (0.58) 1.33 (1.69) – 0.49 (0.41) 0.34 (0.26)Maximum cell concentration (Log10 cells ml21) 5.45 (5.60) 5.94 (5.39) 5.47 (5.39) – 4.90 (4.43) 3.35 (3.90)Lag time (day) 1 (2) 2.5 (2.5) 3 (3) – 1.5 (3) 10 (2)

*: cells were no longer viable.

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Chemical process permitted immediatly and massive cellleakage but contact between cells and citrate or EDTA maynot exceed 45 minutes after which viability fall (data notshown) and an additional step is required. This way setconsequently a problem for large-scale process. Conse-quently, natural leakage due to cell multiplication insidebeads, seems the best way.

These first results on immobilized-cell storage are promis-ing because of long-term storage permitted by immobiliza-tion and two possible ways of cell recover in order toinoculate ponds for the greening of oysters: chemical usingEDTA or citrate, and natural due to active cell mutiplica-tion inside gel matrix which seems the most promisingone. We are now testing the sowing of ponds with cells ofH. ostrearia immobilized in calcium alginate beads usingthe second way. Algal cells/oysters amount ratio will bedefined to optimize greening.

AcknowledgmentWe are particularly grateful for the technical assistance toMr P. Gaudin. This work was supported by the CountyCommission of Loire-Atlantique and the “Pays de la Loire”Region.

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Received: 27 July 1998Revisions requested: 20 August 1998

Revisions received: 7 October 1998Accepted: 7 October 1998