Immobilized Isochrysis galbana (Haptophyta) for long-term storage and applications for feed and water quality control in clam (Meretrix lusoria) cultures

 

Yean-Chang Chen

Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan

email: ycchen@mail.ntou.edu.tw; b0232@ind.ntou.edu.tw

 

Received 22 April 2003;

Key words: Haptophyta; Immobilization; microalga; Isochrysis galbana; SEM; TEM; water quality.

 

Abstract

The marine microalga Isochrysis galbana was cultivated and entrapped in alginate beads for long-term storage. The entrapped cells were alive and maintained their physiological activities after one year of storage in absolute darkness at 4ºC without a liquid medium. The number of cells in the beads increased more than 32 times when they were subsequently re-cultured in an aqueous medium for five weeks, showing that they had remained alive during storage. TEM observations showed that the entrapped cells reduced their cell covering and pyrenoid size compared with the normal free-living cells after long-term storage.  The algal beads were also applied to feed and water quality control in clam cultures' leading to a marked decrease in ammonium concentrations. Algal cells escaped from the beads provided a food source for the clams. This might reduce the cost of clam culture compared to traditional culture methods. Therefore, immobilized I. galbana can be used for long-term preservation of algal stock in the laboratory and applied practically to clam cultures.

 

 

Introduction:

Isochrysis galbana Parke (Haptophyta) primarily occurs as a unicellular flagellate, but also as palmelloid stages in brackish marine environments. Two long isokont flagella emerge from a gullet-like structure, but no haptonema is present.  I. galbana, because of its good nutrititional qualities (i.e. highly unsaturated fatty acids) and small cell size, is widely used in aquaculture, principally as feed in the early stages of growth of mollusk larvae, fish and crustaceans. One of the principal problems that face laboratories producing mollusks and crustaceans in the larval stages is the high cost of producing live food (microalgae), which contributes up to 30 % of the total cost of production (Valenzuela-Espinoza et al. 1999). One of the alternatives for reducing this cost is to use immobilized microalgae, which are cheaper than the algae produced by traditional methods and are ready for use.

Entrapment of cells within Ca-alginate spheres is a simple and non-destructive method that preserves their metabolic and physiological properties and has become the most widely used technique for immobilizing living cells (Hertzberg and Jensen 1989; Romo et al. 1997). Numerous reports on immobilized cells involving algae, bacteria, in-vitro plant cell cultures etc., have supported the view that cell metabolic activities and the efficiencies may remain as they are under conventional culture conditions.

During immobilization, algal cells maintain their respiratory and photosynthetic activities. (Romo et al. 1997; Chen 2001). Immobilization prevents algal cells from being washed out of the culture or grazed by herbivores. Some immobilized algae can, when stored at low temperatures (4ºC) in darkness, resume normal growth after more than 12 months of immobilization (Faafent et al. 1994). Practical applications include removal of nutrients and heavy metals from wastewater (Chevalier and de la Noüe 1985; Wilkinson et al. 1990; Proulx and de la Noüe 1988; Garbisu et al. 1991). Entrapment, storage and processing algal cells into alginate-beads are useful in stock culture management. The preparation of alginate-beads is easier, cheaper and more convenient than other methods such as cryopreservation (Romo and Pérez-Martínez 1997).

A previous study by the author (Chen 2001) showed that immobilized cells of the freshwater alga Scenedesmus quadricauda could be kept in storage for more than three years and retained their normal physiological activities. These alginate-beads can be used to decrease the ammonium concentration in fish cultures.

The aims of the study reported here was to test the feasibility of using the technique to preserve Isochrysis galbana, and to determine its viability after long-term storage. If successful, it was also planned to test the use of the immobilized alga for culture of the clam (Meretrix lusoria).

 

Materials and methods

Culture and preparation of alginate beads

Isochrysis galbana cultures were isolated from the marine fishpond at the Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan. I. galbana was cultivated in 1000-mL flasks containing 600 mL liquid medium (modified Provasoli's Enriched Seawater, PES medium, salinity=20‰) (Provasoli and Pintner 1960) at 100 µmol photon m-2.s-1 at 24ºC and a 12:12, dark: light photoperiod. The medium was replaced weekly.

Entrapment of I. galbana in alginate-beads was performed as follows: A 3% (v/v) sodium alginate (Sigma A-7128) solution was autoclaved for 20 min at 121ºC. The Isochrysis galbana strain was concentrated by centrifugation (1000 x g, 20 min), and then mixed into the sodium alginate solution to make homogeneous beads.  Cell density was counted (ca. 6-7 x 106 cells mL-1) using a hemacytometer (Bright-Line, improved Neubauer, 0.1 mm deep) under a light microscope (Zeiss, Axioskop). Beads, about 4-5 mm diameter, were formed by dropping the alginate-algal mixture into a solution of modified PES medium (20‰ salinity) containing 0.03 M CaCl2 at room temperature using a burette.  1 mL of the mixture yielded 10 drops (beads). The beads were allowed to harden in the CaCl2 solution for 30 min. Subsequently, they were washed several times in autoclaved modified PES medium (20‰ salinity) to remove excess CaCl2. The wet beads were then immediately stored in a well-sealed flask without any liquid medium addition, the flask was wrapped with aluminum foil, and then maintained in absolute darkness at 4ºC until used, preventing algal growth. The number of re-cultured cells was counted, by dissolving one hundred beads in a solution of 6 mL of 5% sodium hexametaphosphate and 24 mL of modified PES culture medium (20‰ salinity)(Chen 2001). The resulting solution was then used for counting algal cells by hemacytometer.

The freshly made algal beads and the re-cultured long-term storage algal beads were gently crushed into fragments individually using forceps. These fragments and normal culture (free-living) Isochrysis galbana cells were collected in 15-mL centrifuge tubes followed by separate pre-fixation in modified PES medium (20‰ salinity) with 2% glutaraldehyde at 4ºC for 2 h, and then fixed in a 0.1 M sucrose solution containing 4% glutaraldehyde and 0.1 M sodium cacodylate buffer (pH 7.0) at 4ºC for 4 h. They were then rinsed twice with a 0.1 M sodium cacodylate buffer containing 10 mM CaCl2, and the sucrose concentration was successively reduced to 0.05 M. This was followed by two rinses in sucrose-free 0.1 M sodium cacodylate buffer containing 10 mM CaCl2.

Post-fixation was performed with 2% OsO4 in 0.1 M sodium cacodylate buffer containing 10 mM CaCl2 for 1 h at 4ºC.

All materials were then rinsed four times with a sodium cacodylate buffer containing 10 mM CaCl2, three times with aqueous ethanol (50%) and gradually dehydrated in ethanol (50, 70, 85, 95, 100%). Dehydrated materials were prepared for transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

For TEM, dehydrated materials were rinsed in propylene oxide (three times, 30 min each), followed by infiltration in propylene oxide-Spurr's resin in a decreasing ratio from 2:1 (2 parts propylene oxide: 1 part Spurr's resin) to 1:1, each for 4 h. Samples were then suspended in pure Spurr's resin for two days at 4ºC in darkness before embedding in Spurr's resin (Spurr 1969). The thin-sections were stained with uranyl acetate and lead citrate according to Smith and Croft (1991).

For SEM, dehydrated materials were dropped onto specimen holders and then dried with a critical-point-drying machine (Hitachi-HCP-1). Finally, they were coated onto an ion coater (Joel, JCF-1100E) for 220s.

 

Use of algal beads for controlling clam culture water quality and for feed.

Four glass jars (30 x 30 x 25 cm), each containing 20 L modified PES medium (20‰ salinity), were assigned as groups 1, 2, 3 and 4. Twenty clams (Meretrix lusoria) (average shell length 2.5 cm) were cultured in each jar. Groups 1 and 2 were cultured under 100 µmol photon m-2 s-1 continuous light irradiance at 24ºC. Groups 3 and 4 were cultured under the same conditions but with 12:12, dark: light photoperiods (light at 0800 and dark at 2000).

Freshly made algal beads (Figure 1) were cultured in modified PES medium (20‰ salinity) for five weeks. The beads were subsequently packaged into two nylon net bags (mesh opening ca. 2x3 mm) (Figure 2), each containing 2000 beads. The bags were then hung in groups 2, 3 and 4, respectively. For group 3, the nylon bag was removed from the jar when the lights turned off at 20:00 (the initial darkness) and replaced until the light was turned on at 8:00 (the initial light). Group 1 was the control group, which only contained the clams fed with free-living (normal) Isochrysis galbana at 6-7 X 104 cells mL-1 every two days. In contrast, groups 2, 3 and 4 were not fed normal free-living I. galbana. A pH meter (Suntex, Taiwan), a digital oxygen meter (Lutron DO-5510, Taiwan) and colorimetric test kits (Aquamerck Ammonium, Merck, Germany) were used to test the pH value, dissolved oxygen level (DO) and NH4+-N concentration, respectively. The water quality of groups 1 and 2 was tested once each day. Groups 3 and 4 were tested twice daily, when the light came on at 8:00 and off at 20:00. All cultures were adequately aerated during the experimental period.

 

Results:

Storage, culture and electron microscopy studies of immobilized cells.

Freshly made algal beads (Figure 1) contained an average 6.8 x 105 ± 2.2 x 104 cells per bead. The wet beads without liquid medium were stored in darkness at 4ºC for a year. The immobilized cells did not lose their ability to grow after long-term storage, and cell number in the beads was the same as that of fresh algal beads.  Cell number rose to 2.1 x 107 ± 5.4 x 106 cells in each stored bead (Figure 1) following culture in modified PES medium for 5 weeks.

Thin sections of freshly made beads (Figure 3) showed that a large chloroplast filled most of the cell.  The conspicuous chloroplasts were typically composed of stacks of thylakoids in groups of three. The multilayered cell coverings (organic scales) (Figures 3, 4), which were separated by amorphous electron-dense material were laid outside the distinct cytoplasmic membrane . The ultrastructure of freshly immobilized cells was the same as that of free-living Isochrysis (Figure 4). However, the cell covering in the latter was more developed. By contrast, after long-term storage of immobilized cells, the organic scales were minimal or had disappeared (Figure 5), the cytoplasmic membrane was less distinct and the pyrenoids were smaller than those of normal cells. However, the organic scales and pyrenoids appeared normal again within a week after cells had been cultured in modified PES medium at 100 µmol photon m-2 s-1 irradiance and 12:12, dark: light photoperiods at 24ºC.

 SEM sections of immobilized cells, which had been stored for an extended period (1 year) and then re-cultured for five weeks, showed inconspicuous gullet-like structures (Figure 6). Flagella protruded from these structures, and were lost when the organism was no longer alive. The morphology was nearly the same as that of normal cells (Figure 7). In addition, cells located at the surface of the bead showed two long flagella (Figure 8). These cells escaped from the beads into the culture water after 5~6 weeks of re-culture. Because of the small size of these cells (ca. 3~5 µm diameter), the flagella of most I. galbana were “burned” away by the high voltage electron beam during SEM observation. However, some flagella on the immobilized beads were protected by the alginate, and could therefore be observed (Figure 8).

 

Use of algal alginate-beads for clam culture

The ammonium concentration of group 1 increased to 9.5 mg L-1 at day 30 of culture and remained at this high level. During the 30 days of culture the average ammonium concentration was 5.92±3.1 mg L-1, the highest among all groups.

In contrast, groups 2, 3 and 4 showed low concentrations of ammonium during the 30 days of culture. The ammonium concentration of group 2 increased to 3.5 mg L-1 at day 15, then remained around at 3.3 to 3.5 mg L-1 from days 15 to 30 of culture. The ammonium concentration of group 3 fluctuated with a slow trend toward increase, but remained between 4.3 to 4.5 mg L-1 during light periods and between 5 to 5.3 mg L-1 during dark periods after day 19 of culture. The ammonium concentrations of group 4 fluctuated with a trend towards increase, from 0.3 to 5.1 mg L-1 during light periods and from 0.1 to 6.3 mg L-1 during dark periods during the 30 days of culture. The average ammonium concentrations of groups 2, 3 and 4 were 2.75±1.1, 3.65±1.5 and 3.94±1.7 mg L-1, respectively. However, there was no significant difference in ammonium concentration between groups 2, 3 and 4 during light periods.

There were obvious differences in DO concentrations among the four culture groups during the 30 days of culture. The DO concentrations of group 1 showed a decreasing trend, from 7.2 to 6.2 mg L-1   The DO concentrations of group 2 showed the highest values, from 7.3 increasing to 8.7 mg L-1. Groups 3 and 4 (especially the latter) showed fluctuating values.  The average DO concentration for groups 1, 2, 3, and 4 was 6.8±0.36, 8.2±0.5, 7.5±0.58 and 6.6±0.77 mg L-1, respectively.

The pH value for group 1 was 6.5, the lowest among all groups. The pH values for group 2 were steady at around 8.4±0.07. The pH values for group 3 showed some changes. However, the pH values for group 4 showed the most change. The  pH values for groups 3 and 4 were around 8.0±0.5 and 7.7±0.5, respectively.

Initially, algal cells were rare in the water of groups 2, 3 and 4. After 1 week of culture some cells escaped from the alginate-beads, becoming active and swimming freely in the water. The cell number in groups 2, 3 and 4 ranged between 8 x 102 and 3 x 103 cells mL-1 during the 30 days of culture. In group 2 some escaped cells assumed the palmelloid morphology and adhered to the wall of the jar in brown film-like layers (the biomass of those palmelloid cells was not assessed). In the algal bead re-culture experiments, the concentration of escaped cells was ca. 5 x 104 cells mL-1 at week 6 in a 5-L flask containing 3 L modified PES medium. This value continued to rise until reaching 5.6 x 107 cells mL-1.

After 30 days of culture, the average clam length for groups 1, 2, 3 and 4 was 2.65 cm, 3.0 cm, 2.9 cm and 2.8cm, respectively. The clam survival rate for groups 1, 2, 3 and 4 was 25%, 85%, 80% and 70%, respectively.

 

Discussion:

This study confirmed that the technique reported by Romo and Pérez-Martínez (1997) and Chen (2001) with the non-motile freshwater green alga Scenedesmus is also suitable for the motile marine alga, Isochrysis galbana.  Moreover, those algal alginate-beads without liquid medium kept in the dark at 4ºC for more than one year were capable of growth and initiated new cultures when transferred to fresh medium and suitable growth conditions. The number of cells was ca. (2.18X107) per bead after re-culture was similar to that found in a normal culture. Hertzberg and Jensen (1989) immobilized the marine diatom Phaeodactylum tricornutum in alginate beads and had similar results. However, the present study required only 5 weeks to reach that amount of cells in contrast to the study by Hertzberg and Jensen, which required 5½ months. It is important to point out that growth is possible within the alginate-beads under suitable culture conditions.

TEM showed that the pyrenoid size was reduced after extended storage. The cell coverings component was also reduced, perhaps to save and store materials for cell survival. However, ultrastructural observations showed that the structures of I. galbana cells were not altered extensively when they were entrapped in the alginate-beads.

In the present study ammonium of the clam cultures was utilized as a nutrient by the immobilized alga, which resulted in lower concentrations (average 2.75 mg L-1). The cell numbers of the immobilized alga increased ca. 32 times in 35 days of culture in this experiment. This proved that the physiological capabilities of the immobilized algal cells were not excessively disrupted by the immobilization. Thus, the immobilized algae can be used successfully to control the water quality in clam cultures. Chen (2001) and Chevalier and de la Noüe (1985) also reported that Scenedesmus cells immobilized in beads were as efficient as free cells in taking up ammonium.

The pH values of culture groups 2, 3 and 4 with algal beads were around 7.7 to 8.4. The pH values of group 4 were affected the most. The pH values of group 2 were much more stable than those of groups 3 and 4, which were cultured under the same irradiance and photoperiods. The algal beads of group 3 were removed from the jar during darkness to avoid the algal respiratory effects. Therefore, the slight pH value fluctuations might be due to the clam respiration during the periods without the alga, as well as effects of DO levels. In contrast, the DO levels of group 2 were always higher than those of the other groups. In addition, the film-like palmelloid Isochrysis galbana layer in group 2 may have also aided in maintaining good quality culture water. In group 3 the algal beads were removed during darkness. The algal beads of group 4 were not removed during darkness, consequently, the pH values and DO levels of group 4 were the lowest, because of the combined algal and clam respiration. 

Judging from the results ammonium, DO and pH values and the clam growth and survival rates, the water quality in cultures with algal beads (groups 2, 3 and 4) was better than the cultures without algal beads (group 1). The algal alginate-beads could maintain the water quality suitable for clam culture.

The biomass of the escaped algae varied, possibly due to the physiology of the clams that ate the microalgae, as well as the environmental conditions affecting microalgal growth, such as the concentration of  ammonium (as nutrient), DO levels and pH values. Certainly consumption by the clams reduced algal biomass. Microalgal cells probably escaped from the surface of the beads due to continuous cell division inside the beads. The space inside the beads is limited, therefore the cell volume expanded and extruded excess cells out from the surface of the beads into the culture water. The escaped Isochrysis galbana cells seemed not to affect the water quality according the study results. This finding provides a sustainable clam culture system without adding microalgae (live feeds). This method should reduce the cost of clam culture tremendously compared to traditional culture methods, because the microalgal beads can act as a convenient “auto-feed” system to reduce the cost of clam culture.

 

Acknowledgements

Financial support from NSC grants 88-2313-B-019-04; 89-2313-B-019-032: 90-2313-B-019-031 and COA grants 88-AST-1.4-FID-02 (12-2); 89-AST-1.2-FID-05(06): 90AS-1.3.1-FA-F1(4); 91-AS-2.1.5-FA-F1(4) of the Republic of China is greatly appreciated.

 

References

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Figure legends:

Figure 1: (a) Algal beads after long-term storage, containing ca. 6.81 x 105 Isochrysis galbana cells. (b) Stored beads containing ca. 2.18 x 107 I. galbana cells, after re-cultured in PEF medium with light for five weeks. Unit of rule = 1 mm.

Figure 2: Clam culture with algal beads. Arrow indicates the nylon net bag with algal beads inside. Arrowhead indicates the aeration system.

Figure 3: Thin-section of freshly immobilized Isochrysis galbana cell. A chloroplast (C) with prominent thylakoids in which a pyrenoid (P) is normally suspended, fills most of the cell. Thylakoids are typically grouped as threes. a: amorphous electron-dense material.  ML: multi-layers cell covering. V: vacuole. PL: Plasmalemma. Nu: nucleus.

Figure 4: Thin-section of free living algal cell. The chloroplast again composed of thylakoids and a prominent pyrenoid. The cell has obvious PL and cell covering (ML). GU: gullet-like structure.

Figure 5: Thin-section of Isochrysis galbana from a long-term storage bead. The pyrenoid is reduced. The cell has covering (ML) disappeared and PL were not conspicuous.

Figure 6: Isochrysis galbana on  and within surface of a fragment (F) of a re-cultured long-term storage algal bead. O: Newly extruded I. galbana on the surface of the bead. W: entrapped I. galbana protruding from the surface.

Figure 7: Free-living I. galbana. GU: gullet-like structure. c: cell covering (organic scale).

Figure 8: Flagella (FL) of I. galbana on the algal bead surface.