Calcium alginate immobilized marine microalgae: Experiments on growth and short-term heavy metal accumulation

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Marine Pollution Bulletin 51 (2005) 823–829

Calcium alginate immobilized marine microalgae: Experimentson growth and short-term heavy metal accumulation

I. Moreno-Garrido *, O. Campana, L.M. Lubian, J. Blasco

Institute of Marine Sciences of Andalucia (CSIC), Campus Rıo San Pedro, 11510 Puerto Real, Cadiz (Espana), Spain

Abstract

Growth of 11 calcium alginate immobilized marine microalgal species belonging to eight taxonomical groups has been checked inthe present work. Cellular densities inside the calcium alginate beads were monitored during 17 days. Good growth and maintenanceof the structure of the beads were both found for some of the assayed species. One of those species (Tetraselmis chui, Prasinophy-ceae) was selected in order to perform a short term (up to 24 h) heavy metal accumulation experiment. Beads of calcium alginatecontaining (or not) cells of T. chui were exposed to 820 lg L�1 Cu and 870 lg L�1 Cd separately during a 24 h period, and accu-mulation of heavy metals in the beads was measured after this time and compared. Concentration of each metal in the supernatantswas monitored at 5, 10, 60 min and 24 h from the beginning of the experiment. After 24 h, practically all Cu was removed by thebeads. Beads with immobilized algae removed around 20% of total Cd, while beads without algae removed half of that percentage.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Immobilization; Phytoplankton; Copper; Cadmium; Accumulation

1. Introduction

Immobilization of microorganisms is a current topicin biotechnology. Among the different immobilizationtechniques, calcium alginate matrix is one of the mostused (Smidsrød and Skjak-Braek, 1990). In contrast toother matrices, such as polyurethane foams (Thepenieret al., 1985), calcium alginate is nontoxic and let differ-ent type of microorganisms to grow inside. The trans-parency of small calcium-alginate beads is enough topermit the growth of immobilized microalgae (Codd,1987; Hertzberg and Jensen, 1989). Additionally it isan easy, cheap and feasible technique to be used in re-search laboratories (Papagregoriou, 1987).

0025-326X/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.marpolbul.2005.06.008

* Corresponding author. Tel.: +34 956 832 612; fax: +34 956 834701.

E-mail address: [email protected] (I. Moreno-Gar-rido).

Unfortunately, not all microalgal species present agood growth when immobilized. In this work, a 17 dayexperiment was carried out in order to determine whichspecies are more adequate for developing different typesof experiments: related to measuring toxicity or relatedto measuring accumulation of potentially toxic sub-stances. Stability (maintenance of the structure) of thebeads as function of the immobilized species has beenalso checked.

Biomass from freshwater (Brady et al., 1994; Guan-zon et al., 1995; Maeda et al., 1990) and marine (Burdinand Bird, 1994; Chen et al., 1998; Fisher, 1985; Garn-ham et al., 1992a; Davis et al., 2003) micro and macro-algae is known to adsorb different pollutants, such asheavy metals. This capacity can be exploited for differ-ent purposes. In some freshwater environments it wouldbe possible to design devices to remove pollutantsfrom liquid media, but these designs are not feasible inopen waters due to the high aquatic volumes and (nor-mally) low levels of dissolved toxicants, as dilution or

824 I. Moreno-Garrido et al. / Marine Pollution Bulletin 51 (2005) 823–829

precipitation processes in marine environments are fre-quently quick and effective. In spite of this, the use ofimmobilized microalgae in marine environments can re-port information about toxic events by the observationof the immobilized cells or by bulk chemical analysisof the substances accumulated in the beads.

2. Materials and methods

2.1. Experimental organisms

Microalgal inocula were obtained from exponentiallygrowing cultures. All species were obtained from theMarine Microalgae Culture Collection of the ICMAN(MMCC-ICMAN, included in the BIOCISE). Thespecies used wereNannochloropsis gaditana (Eustigmato-phyceae); Heterocapsa sp. (Dynophyceae); Rhodomonas

salina (Cryptophyceae); Isochrysis aff. galbana (Prym-nesiophyceae); Thalassiosira pseudonana, Chaetoceros

gracilis, Phaeodactylum tricornutum and Skeletonema

costatum (Bacillariophyceae); Tetraselmis chui (Prasino-phyceae); Porphyridium cruentum (Rhodophyceae) andDunaliella salina (Chlorophyceae).

2.2. Media

For stock cultures and growing experiments, naturalfiltered (0.45 lm) and sterilized seawater was used, en-riched with f/2 medium (Guillard and Ryther, 1962).50 mg L�1 SiO2 was added in the case of bacillariophy-ceae. For the accumulation experiments, artificial sea-water (ASTM, 1975) was used, and low levels ofnutrients added (Moreno-Garrido et al., 2003a,b). Thismedia has been successfully used for culturing microal-gae for short periods of time, and contains only 6 lg L�1

of NO3 (added as NaNO3) and 6 lg L�1 of PO4 (addedas Na2HPO4). The absence of trace elements and, overall, EDTA in this medium ensures lack of interferencesby these substances in the results of the assay. Cells werecultured under continuous zenital light (white light,around 300 lE m�2 s�1) in a temperature-controlledchamber (20 ± 1 �C).

2.3. Immobilization technique

Cells were immobilized in calcium-alginate beads fol-lowingMoreno-Garrido (1997). For growth experiments,beads were kept in 250 mL spherical flasks containing100 mL of f/2 mediumwith around 50 mL of beads. Sam-ples were taken regularly till day 17 after the beginning ofthe test and fixedwith formalin. Known volumes of beadswere dissolved by soft sonication with known volumes oftri-sodium citrate (3%, w/v) and cellular densitiescounted. All counts were performed in quadruplicate.For metal accumulation experiments beads containingan initial cellular density of 3.3 · 106 cells mL�1 of the

Prasinophyceae T. chui were added to 45 mL of culturein 100 mL capacity conical flasks.

2.4. Metals

Cu was added as sulphate and Cd was added as chlo-ride. All reagents were analytical grade and supplied byMerck. Initial metal concentrations were certified byAAS analysis at 5 min after the beginning of the tests,being 0.037 mg L�1 for Cu and, 0.039 mg L�1 for Cd.

2.5. Sampling and analysis procedures

Samples from the supernatants were taken at 5, 10,60 min and 24 h, stabilized with Suprapur nitric acid(Merck) and measured in a graphite-furnace AAS withZeeman correction (Perkin–Elmer 4100 ZL) after dilu-tion with de-ionized (Milli-Q) water. All beads fromeach flask were taken 24 hours after the beginning ofthe accumulation experiments and freeze dried for48 h. Weighed sub-samples were digested following Lor-ing and Rantala (1992) and accumulated metal mea-sured in the same way as supernatants. All glasswarewas previously washed with diluted nitric acid, all exper-iments were carried out in triplicates. Basal levels of Cuand Cd in solution (with or without cells) were mea-sured. Possible adsorption of metals to glassware wasalso measured.

3. Results and discussion

Growth curves for the 11 microalgal species during 17days (cultured with f/2 medium) are shown in Fig. 1.

Growth of the Eustigmatophyceae Nannochloropsis

gaditana is not shown because after day 3 calcium algi-nate beads containing cells of this species began to losestructure under the experimental conditions. As cellularcounts were based on measuring a known volume ofbeads, when calcium alginate became less solid, countsbecame impossible.

Changes in the stability of the beads could be due todifferent causes. First, all cultures used in the experi-ments were monoalgal but not axenic. In any case, ster-ilization of sodium alginate is possible but difficult, andcan alter the gelation strength properties of the resultingcalcium alginate (unpublished data). On the other hand,sterilization has not much sense if beads are going to beused in the field. Bacterial populations in exponentiallygrowing microalgal cultures does not surpass 3% bio-mass of existing algae (Gonzalez-DelValle, 1997), butalginate can act as a carbon source and thus acceleratethe bacterial growth. Each monoalgal culture is knownto keep particular assemblages of bacteria (Gonzalez-DelValle et al., 1994), and differences between bacterialpopulations could explain differences in the maintenance

C. gracilis

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P. tricornutum

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Heterocapsa sp.

0 3 6 9 12 15 180.00.20.40.60.81.01.21.41.61.8

Th. pseudonana

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020406080

100120140160180200

R. salina

0 3 6 9 12 15 184

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D. salina

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P. cruentum

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020406080

100120140160180200

I. galbana

0 3 6 9 12 15 180

50100150200250300350

S. costatum

0 3 6 9 12 15 180

5

10

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T. chui

Time (Days)0 3 6 9 12 15 18

010203040506070

x10

6 cells

mL-1

Fig. 1. Growth curves for the calcium alginate marine microalgal species assayed. Error bars represent standard deviation of four counts.

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of the calcium alginate bead structure, related with thecapacity of different bacterial strains to attack calciumalginate. Second, immobilized microalgae can sequesteror give off substances to their immediate environment

changing the viscosity of the beads. None of these twoexplanations has been proved or investigated.

Heterocapsa sp. did not grow well within the beads.This was expected because of the difficulties in culturing

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dinoflagellates in solid media. Many species among theClass Dynophyceae (=Pyrrophyceae) show facultativeor obligate heterotrophy and require special media fortheir culturing. Although Heterocapsa sp. seems to bea strict heterotrophic species, possibly reduced accessto nutrients or just immobilization imply the deteriora-tion of the cells. Marine dinoflagellates could be suitableorganisms to detect toxicity, as some species present lowtoxicity thresholds for certain toxicants (Anderson andMorel, 1978; Saifullah, 1978; Blasco et al., 2003). Addi-tionally, this is one of the main phytoplanktonic groupsin ocean and coastal waters. But difficulties in handlingmost of the cultured species and low division rates pre-vent their use for ecotoxicological bioassays.

Among the assayed diatoms (Bacillariophyceae),Skel-etonema costatum does not grow adequately in the exper-imental conditions. Centric diatoms are the mostimportant group (from an ecological point of view) inoceanic phytoplankton, but organisms forming chains(such as S. costatum) can generate problems whencounted by electronic devices (Coulter counter or flowcytometry), if responses of free population cells are in-tended to be compared with responses of immobilizedcells. Additionally, under light microcopy, cells ofchain-forming cells liberated from calcium alginate beadsare not easy to count. This is the reason for discarding T.pseudonana, although this species present a good growthunder experimental conditions for more than ten days.On the other hand,Chaetoceros gracilis presents a contin-uous growth under the experimental conditions and keepsthe structure of beads. This species is cosmopolitan, donot form chains and demonstrated to be sensitive to dif-ferent toxicants. The use of this species as a tool for mar-ine ecotoxicologists has been proposed (Moreno-Garridoet al., 2001). P. tricornutum is not a centric diatom, butpennate. This species also present a good growth withincalcium alginate beads. P. tricornutum can be found incoastal waters and estuaries, as stands certain salinityvariations. All this advantages have been taken into ac-count and mark this species as a good target for ecotoxi-cology bioassays, as can be noted by several authors (Cidet al., 1995; ISO, 1995; Mayasich et al., 1986; Morelli andScarano, 2001;Wiegman et al., 2002). In situ toxicity bio-assays have been recently successfully developed by usingcalcium alginate immobilized populations of this species(Moreira dos Santos et al., 2002).

D. salina is a green flagellate (Chlorophyceae) presentin coastal waters. The high tolerance to salinity of micro-algae belonging to this species let them colonize saltpans, and it is the only microalgal species able to resistsalinities to saturation. But resistance of D. salina isnot only to salt. In a mixing experiment, this species tendto dominate over others when subjected to metal stress(Moreno-Garrido et al., 1999b). Abalde et al. (1995) re-ported that populations ofD. salina exposed to very highcopper concentration (8 mg Cu2+ L�1) was unaffected,

while exposition to even higher copper concentrations(12 mg Cu2+ L�1) only slightly affected the growth. Inspite of this, some guidelines for measuring toxicity rec-ommend this species as adequate for toxicity bioassays(APHA, 1989). There are some references about thecapacity of this species to accumulate nutrients or poten-tially toxic substances (Rebhun and Ben-Amotz, 1986;Saez et al., 2001; Thakur and Kumar, 1999). Calciumalginate beads containing cells of D. salina in the exper-imental conditions began to lose structure after day 10.

Isochrysis aff. galbana is a Primnesiophyceae. Thisclass includes coccolithophorids (Chretiennot-dinet,1990), a very ecologically important group in oceanicsystems, although the order Isochrysidales does not car-ry coccolithus (calcium-carbonate scales over the cells).I. aff. galbana grows very well within calcium alginatebeads and demonstrated to be very sensitive, at leastto some toxicants (Moreno-Garrido et al., 2000), but itis not frequent to find references about this species re-lated to ecotoxicology, but aquaculture, as it seems tobe a good source of essential lipids.

P. cruentum is one of the few unicellular generaamong the red algae. There are no data about the sensi-tivity of this species to toxicants, but this species hasbeen proposed as a source of sulphated exopolysaccha-rides. The presence of these substances laying over thecell walls (and dropped to the media: old cultures of thisspecies trend to increase viscosity) could interfere in tox-icity experiments, but the use of this species in accumu-lation bioassays could be a very interesting topic toexplore. After a good growth during the first five dayswithin the calcium alginate beads, cellular density ofpopulations of P. cruentum stabilizes (but do not suffera decrease, as D. salina or R. salina does).

R. salina belongs to the Class Cryptophyta. Potentialheterotrophy of the complex organisms included in thisclass has not been completely elucidated. After few days,cells of this species began to die when immobilized incalcium alginate beads, under the experimental condi-tions. This species demonstrated to be very sensitive tocopper (Moreno-Garrido et al., 1999a), but seems tobe more resistant to other potential toxicants, such assurfactants (Blasco et al., 2003).

T. chui (Prasinophyceae) is one of the most resistantspecies among known microalgae to a wide variety ofstressors, including toxicants (Hampel et al., 2001;Blasco et al., 2003) or even UV radiation (Monteroet al., 2002). T. chui is able to resist in some cases stressinglevels of more than one order of magnitude higher thanother species. Resistance mechanisms of this tetraflagel-late are not clear, but the use of this species in potentiallypolluted environments should be considered. Within thecalcium alginate beads, cells present a good growth ratetill day 5, and after that time population stabilizes.

All these reasons tend us to select this latter species forthe following accumulation experiment. This is the first

Cu Cd

% A

ccum

ulat

ed M

etal

0

20

40

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80

100

Fig. 3. Percentage of total added metal removed by calcium alginatebeads immobilizing 3.3 · 106 cells mL�1 T. chui (grey bars) andcalcium alginate beads (white bars). Error bars represent standarddeviation between replicates (n = 3).

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report, as far as we know, about the design of a metalaccumulation experiment involving immobilized T. chui.

It is known that products obtained from macroalgae(as alginate is) tend to accumulate certain substances(Burdin and Bird, 1994; Crist et al., 1994; Greene andBedell, 1990; Jang et al., 1995; Nestle and Kimmich,1996), over all cations. Increase of accumulation capac-ity by immobilization of living algae in these matriceshas also been demonstrated (Garnham et al., 1992a;Moreno-Garrido et al., 2002). Thus, metal accumulationcapacity of calcium alginate beads with and withoutcells of T. chui was compared. Concentrations of copper(left) and cadmium (right) in the liquid media after 5, 10,60 min and 24 h are shown in Fig. 2. Metal concentra-tion in beads was determined after 24 h. A volume of5 mL of calcium-alginate beads mean a dry weight of0.32 ± 0.03 g (standard deviation). Knowing the finaldry weight of all the beads in each flask, it is easy to cal-culate the percentage of total initial metal that has beenremoved by the beads (Fig. 3). Flasks with metals andno beads were examined in order to determinate if somemetal was lost by adsorption to the glassware. In thecase of the copper, final concentration in these flaskswas near 25 lg L�1 lower than the initial (820 lg L�1),but for cadmium, no significant variations were found.As shown, beads with and without algae accumulatedmost of the copper in the media (Fig. 3, left). High dilu-tion of samples (needed for a good measure of metalsdissolved in seawater in the graphite-furnace AAS) canexplain disagreement between Figs. 1 and 2 for beadswithout immobilized algae respect to copper. In the caseof beads containing immobilized microalgae, the agree-ment between both data for copper is complete: levels ofcopper in media after 24 h are below 5% of the initialconcentrations. Respect to cadmium, when measuredin supernatants, there is a trend of higher metal accumu-lations for beads with immobilized algae, but differencesbetween replicates do not permit to establish statisticaldifferences. In the opposite, in the case of cadmiumaccumulated in beads (Fig. 3, right), there are statisticaldifferences (ANOVA, P < 0.05, method of the 95% Fish-

5' 10' 60' 24 h0

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-1C

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Fig. 2. Concentration of Cu (left) and Cd (right) measured insupernatants as a function of time (5, 10, 60 min and 24 h). Greybars mean flasks containing calcium alginate beads immobilizing3.3 · 106 cells mL�1 T. chui. White bars mean flasks with calciumalginate beads. Error bars represent standard deviation of replicates(n = 3).

er�s least significant difference) between beads with andwithout immobilized algae within: beads with algaeaccumulated near double amount of cadmium (20% oftotal Cd) than beads without algae.

Accumulation of cations by algae (such as Cu or Cd)seems to be two stage process. There is a first, fast accu-mulation phase where metabolism of the cell does notplay any role (adsorption due to chemical processes)and a second, slower accumulation phase where cellularbiological mechanisms drives the entering of the metalinto the cells (absorption) (Garnham et al., 1992b;Moreno-Garrido et al., 2002). In this respect, the amountof metal able to be removed by washing with EDTA (ametal chelator) will be different for living and dead cells(Moreno-Garrido et al., 1998). In spite of this second pro-cess, variations of pH due to photosynthesis (performedby cells) can improve the capacity of adsorption of metalsin the inner space of the beads, permeable to water butwith restricted water circulation, as higher values of pHdecreased the solubility of divalent cations. Variationsof pH were found for different treatments. Artificial seawater employed in the bioassays had an initial pH valueof 8.2 that keeps constant during all experimental timein flasks with metals but without beads and flasks withbeads not containing immobilized cells within (8.17 ±0.04, standard deviation). However, final pH values ofmedia in flask containing cells were higher: 8.83 ± 0.2for Cu and 9.42 ± 0.02 for Cd. Values for flasks withbeads containing algae but not metals (used for checkingthe background levels of metals, which were undetectableby our techniques) were slightly (around 0.2 values of pH)higher. Thus, metabolism of photosynthetic organismscould have a double positive effect related to the accumu-lation of metals: by absorption in the inner cellular spaceand by the increase of pH values in the closest environ-ment of the cell, which facilitates precipitation.

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Alginate compounds demonstrated to have highaffinity (and even selectivity: Jang, 1995) for copper. Infact, copper is one of the triggers for alginate productionby some terrestrial phytopathogens (Kidambi et al.,1995), response developed in order to inactivate in thisway the toxicity of this metal used in agriculture as fun-gicide and bactericide.

In contrast to freshwater environments, bioremedia-tion in marine environments is not an easy topic. Highwater volumes and very low concentrations, added tothe difficulties of engineering in coastal and open oceanenvironments pose problems to design effective strate-gies for sequestering dissolved pollutants in an efficientmode. But immobilization of marine microalgae couldovercome these problems.

Recent efforts from ecotoxicologists trend to carry theexperimental organisms to actual locations, avoiding inthis way manipulation of the samples or lack of ecologi-cal relevance which is produced when samples are carriedto the laboratory conditions. In situ toxicity tests are asignificant advance in ecotoxicology, in spite of the greatdifficulties that these experimental designs pose. Someexperiments with semi-permeable membranes can befound in the literature (Munawar and Munawar, 1987),but we believe that gel immobilization is a better solutionfor transporting microalgae to the field and recover themfor laboratory analysis (Moreira dos Santos et al., 2002).

On the other hand, a problem difficult to overcome forecotoxicologists is the possibility of episodic pollutionevents (McCahon and Pascoe, 1990) in environmentssuch as coastal and open seas. High levels of pollutantscan occur during short periods of time, affecting faunaand flora, and disappear in few days or even hours. Ifaccumulation of pollutants in calcium alginate beadswere stable in time, could act as a register for recentevents. Additionally, the great accumulation capacityof beads with living microalgal cells within, could revelthe occurrence of chronic low level contamination. Allthese open questions offers a wide field of investigation.

Acknowledgement

This work has been granted by the Spanish NationalPlan of Scientific and Technique Investigation, project‘‘Aplicacion de sistemas inmovilizados de microalgasmarinas en la incorporacion y evaluacion de la toxicidadde substancias en ecosistemas marinos’’ (REN2001-2095/MAR).

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