Immobilized microalga Scenedesmus quadricauda (Chlorophyta, Chlorococcales) for long-term storage and for application in fish culture water quality control

Yean-Chang Chen

Department of Aquaculture

National Taiwan Ocean University

Keelung, Taiwan

(Aquaculture. 2001. 195(1-2):71-80)

 

(FIGURE 7,8 and 9)

Abstract

The green microalga Scenedesmus quadricauda was cultivated and entrapped into alginate-beads for long-term storage. The entrapped algal cells were alive and maintained their physiological activities after three years of storage in absolute darkness, at 4ºC without the liquid medium. The number of coenobia in the beads increased more than ca. 40 times after they were re-cultured in an aqueous medium for four weeks. TEM observations showed that the organelle, pyrenoids of entrapped S. quadricauda cells disappeared after long-term storage. However, the pyrenoids were reconstructed when the algal cells were re-cultured into an aqueous medium with light. These results indicated that the entrapped algal cells remained alive by consuming the reserve caps of pyrenoids. The algal beads were also applied to fish culture water quality control. In those cultures with algal beads, the ammonium concentrations decreased obviously. Immobilized microalga in alginate-beads, particularly the species Scenedesmus quadricauda, can be used as algal stock for long-term algal species preservation in a laboratory and subsequently can be applied for controlling the water quality in fish cultures.

 

Keywords: Immobilization; microalga; Scenedesmus quadricauda; SEM; TEM; water quality.

1. Introduction.

Numerous reports on immobilized cells concerning algae, bacteria, in-vitro plant cell cultures etc, have supported the view that cell metabolic activities and efficiencies may remain as they are in normal conditions. During immobilization, algal cells maintain their respiratory and photosynthetic activities. Immobilization prevents algal cells from being washed out or grazed by herbivores. 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 of immobilized algae include nutrient and heavy metal removal 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 into alginate-beads are useful in stock culture management. The preparation of alginate-bead is easier, cheaper and more readily available than other methods, such as cryopreservation (Romo and Pérez-Martínez, 1997).

As mentioned above, immobilized algae are applied primarily in wastewater treatment. There have been few studies that reported on immobilized algal cells for application in aquaculture, such as controlling fish culture water quality. The aim of this study was to test the feasibility of using the algal immobilization technique to preserve the microalgal species, Scenedesmus quadricauda, and determining the algal viability after long-term storage. The immobilized algal beads were also used to control the fish culture water quality.

 

2. Materials and Methods.

2.1 The microalga culture.

Pure Scenedesmus quadricauda de Bréb cultures were isolated from the fishpond at the Department of Aquaculture, National Taiwan Ocean University, in Keelung, Taiwan. The S. quadricauda were incubated in a 1000-ml flask containing 600 ml liquid medium (Provasoli's Enriched Freshwater, PEF medium, pH7.0) (Provasoli and Pintner, 1960) at 100 µmol photons.m^-2.s^-1 at 24ºC and 12:12, dark: light photoperiods. The media were replaced weekly.

2.2 Immobilization of the microalga.

Microalga entrapment 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 S. quadricauda strain was concentrated using centrifugation (10 min, 1000xg r.c.f.), and then mixed homogeneously into the sodium alginate solution to make the beads. The numbers of algal coenobia per ml of mixture were counted (ca. 2-30000 coenobia per ml) using a hemacytometer (Bright-Line, improved Neubauer, 0.1 mm deep) under a light microscope (Zeiss, Axioskop). Beads, about 4 mm in diameter, were formed by dropping the alginate-algal mixture into a solution of 0.03 M CaCl₂ at room temperature using a burette (Sibata, Taiwan). In this experiment, 1 ml of the mixture could drop just 10 drops (beads). The beads were allowed to harden in the CaCl₂ solution for 30 min. Subsequently, they were washed several times in autoclaved distilled water to remove excess CaCl₂. The wet beads were then directly and immediately stored in a well sealed flask without any liquid medium addition, the flask was wrapper with aluminum foil, and then maintained in absolute darkness at 4ºC until used, preventing algal growth. The number of re-cultured cells was also counted, one hundred beads were dissolved in a solution of 6 ml of 5% sodium hexametaphosphate and 24 ml of PEF culture medium (Romo and Pérez-Martínez, 1997). The dissolved solution was then used for counting by a hemacytometer.

2.3 Fixation of algal materials for electron microcopy studies.

The freshly made algal beads, the stored algal beads and the re-cultured algal beads were gently crushed into fractions using the forceps individually. These fractions and normal culture (free-living) Scenedesmus quadricauda cells were collected in 15-ml centrifuge tubes followed by separate fixation in 0.1 M sucrose solution containing 4% glutaraldehyde and 0.1 M sodium cacodylate buffer (pH 7.0) at 4ºC for 2 h. They were then rinsed twice with a 0.1 M sodium cacodylate buffer containing 10 mM CaCl₂, and sucrose concentration successively reduced to 0.05 M. This treatment was followed by two rinses by a pure (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.

Thereafter, all materials were 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, some 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, some 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.

2.4 Algal bead application for controlling fish culture water quality.

Four glass jars (60x55x33 cm), each containing 100 L of freshwater, were assigned as groups 1, 2, 3, and 4. Ten male tilapia, Oreochromis mossambicus (average body length 14 cm) were cultured in each jar. Groups 1 and 2 were cultured under continuous light of 100 µmol photons.m^-2.s^-1 irradiance at 24ºC. Groups 3 and 4 were cultured under the same irradiance but with 12:12, dark: light photoperiods (light at 8:00 and dark at 20:00) at 24ºC.

Freshly made algal beads (ca. 2-3000 coenobia in each beads) (Fig. 1) were cultured in PEF medium for two weeks. The beads were subsequently packaged into three nylon net bags (pore size was ca. 2x3 mm in diameter) (Fig. 2), each containing 4000 algal beads. They were then hung in groups 2, 3 and 4, respectively. For group 4, the nylon bag was removed from the jar when the lights turned off at 20:00 (the initial darkness), and replaced until the light is turned on at 8:00 (the initial light). Group 1 was the control group, which only contained the fish. The tilapia in each jars were fed with 2.5 grams of commercial feed (Abon, Taiwan) everyday. The feeds contained 45% of protein, 5% fat, 3% crude cellulose, 10% ash and 10% water. A pH meter (Suntex, Taiwan), a digital oxygen meter (Lutron DO-5510, Taiwan) and colorimetric test kits (Aquamerck Ammonium, Merck, Germany; detail methods also see the instructions for use that described in the test kits of Aquamerck ammonium) 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 everyday. However, groups 3 and 4 were tested twice daily (at 8:00 and 20:00) as the light came on at 8:00 and turned off at 20:00. All cultures were adequately aerated during the period between day 1 to day 13. Aeration ceased from day 14 to day 18.

 

3. Results:

3.1 The storage, culture and electron microscopy studies of the immobilized algal cells.

Freshly made algal beads (Fig. 1) contained an average 2470±460 Scenedesmus quadricauda coenobia in each bead. The wet beads without liquid medium were stored in darkness at 4ºC for three years. The algal coenobia did not lose their reproduction ability within the beads after long-term storage, and the number of coenobia was the same as those of fresh algal beads. The amount of coenobia increased tremendously to average 9X10⁴±2300 coenobia in each stored bead (Fig. 1) following culture in PEF medium for 4 weeks.

Thin-sections of freshly made algal beads (Fig. 3) showed that a large chloroplast, filling most of the cell, was the predominant organelle. The chloroplasts were typically composed of stacks of thylakoids. The simple internal suspended pyrenoids were normally closely surrounded by caps reserve materials. That is, the ultrastructures of freshly immobilized algal cells were the same as those of the free-living algal cells. In contrast, the thin-sections of the long-term stored of immobilized algal cells, the chloroplast pyrenoids had disappeared, although a chloroplast filled most of the cell (Fig. 4). However, the pyrenoids were reconstructed within a week after those cells were re-cultured in PEF medium at 100 µmol photons.m^-2.s^-1 irradiance and 12:12, dark: light photoperiods at 24ºC.

The SEM observations of the fractions of extended period stored algal beads showed that the morphology of the immobilized Scenedesmus quadricauda coenobia (Fig. 5) was nearly the same as normal coenobia (Fig. 6), except that coenobia spines were modified into bent shapes.

3.2 Application of algal alginate-beads for fish culture water quality control.

3.2.1 The NH₄⁺-N concentrations of fish cultures.

As shown in figure 7, the NH₄⁺-N concentration of group 1 increased quickly to 17 mg/l within a week. The freshwater in group 1 was replaced on day 7, 20:00. However, the NH₄⁺-N concentration still showed an increasing trend from 1.5 to 15 mg/l during the period from day 8 to day 18 after the water was replaced.

In contrast, groups 2, 3 and 4 showed low concentration levels of NH₄⁺-N (total average was 5.01 mg/l) during the period from day 1 to day 13. The average NH₄⁺-N concentrations of groups 2, 3 and 4 were 4.85, 4.96, and 5.24 mg/l, respectively. After aeration ceased during the period between day 14 to day 18, the average NH₄⁺-N concentrations of groups 2, 3 and 4 were 0.66, 2.25, and 2.38 mg/l, respectively.

3.2.2 The fish culture DO levels.

As shown in figure 8, the DO levels of all culture groups were around 7 mg/l when aerated. However, there were obvious differences in DO levels between the four culture groups when aeration ceased. After ceasing aeration (from day 14 to day 18), group 2 showed the highest DO level of 4.8 mg/l, (4.16 mg/l on average). The DO levels of group 3 varied (1.7±0.8 mg/l). The DO levels of group 4 were stable, 2.2 mg/l on average. The DO levels of group 1, the control group, were the lowest, with only 0.96 mg/l as an average. It is obvious that the alga could affect the DO level when no aeration was provided.

3.2.3 The fish culture pH values.

As shown in figure 9, the pH values of group 3 were the most changed when aerated. However, the average pH values of the four groups were very close. The average pH values of groups 1, 2, 3, and 4 were 7.72, 7.78, 7,70 and 7.71, respectively. In contrast, after aeration was suspended, the pH values of all groups decreased. The average pH value of group 1 was 7.46. In contrast, the pH values of culture groups with algal beads were near to pH 7.0, in which, groups 2, 3 and 4 were 7.16, 6.96 and 7.07, respectively. This result showed that alga controlled water quality to neutral when aeration ceased.

3.2.4 The body length of tilapia (The growth of tilapia).

After an 18-day of cultures, the average body length of group 1 was 14.5±0.3 cm, was the poorer in growth. Group 2 was 16.3±0.2 cm, was the best in growth. Group 3 was 15.1±0.3 cm and group 4 was 15.5±0.2 cm.

 

4. Discussion:

The alginate-beads method has the following advantages: (1) bead preparation is easier, cheaper than cryopreservation and ready to use, (2) the beads can be stored in refrigerators, (3) immobilized cells can be rapidly introduced into liquid cultures. Romo and Pérez-Martínez (1997) reported that they found similar satisfactory results when this technique was used with the chlorophyte Scenedesmus obliquus (Turp) Kütz. The present study also confirmed that the technique is suitable for S. quadricauda. Moreover, algal alginate-beads without liquid medium directly kept in darkness at 4ºC for more than three years were capable of growing and initiated new cultures when transferred into a fresh medium. The amount of S. quadricauda coenobia was 9X10⁴ per bead. This was the same amount as found in a normal culture. Tamponnet et al. (1985) had similar results. They reported that immobilized Euglena gracilis alga was kept for more than two years in alginate-beads. It is important to point out that growth is possible within the alginate-beads under suitable culture, which reveals that the algal viability is maintained within the beads.

The reason that S. quadricauda remained alive within the beads in the dark at low temperatures for an extended period may be due to the caps reserve of the pyrenoids that present materials for maintaining their basic requirements. TEM observations showed that the ultrastructure of S. quadricauda cells lost the pyrenoids after extended storage. The lost pyrenoids were reconstructed when the algae were re-cultured in an aqueous medium with light. However, SEM observations showed that the morphology of S. quadricauda cells were not altered extensively when they were entrapped in the alginate-bead with the exception of bent spines in contrast to the straight spines of normal free-living S. quadricauda cells.

The present study, ammonium (NH₄⁺-N) in fish cultures with algal beads was utilized by the immobilized microalga as nutrient and resulted in lower concentrations (ca. 5 mg/l). The biomass of the immobilized algae increased ca. 15 times in 18 days of culture in this experiment. This proved that the physiological conditions of the immobilized algal cells were not interrupted by the immobilization. Thus, the immobilized algae can be used successfully to control the water quality in fish cultures. Chevalier and de la Noüe (1985) also reported that Scenedesmus cells immobilized on κ-carrageenan beads are as efficient as free cells in taking up ammonium.

It was found that aeration could maintain the DO and pH at acceptable levels for fish. When aeration was suspended, the algal beads were able to increase the DO and kept pH levels near to 7.0 in water. In contrast, ammonium concentrations of fish cultures with algal beads were at low levels (i.e. 1.76~5.01 mg/l) regardless if the cultures were aerated. However, when aeration ceased, the ammonium concentration of the fish cultures with algal beads showed much lower concentrations than those cultures with aeration. This may be a response of tilapia with decreased metabolism and decreased excretion when they suddenly suffered the low levels of DO.

Although the average pH values of culture groups with algal beads were near 7.0, in which the pH values of group 3 were the most affected. In contrast to the pH values of group 4 that was more stable than those of group 3 when aeration ceased. These two groups were cultured under the same photoperiods, 12:12, dark:light. However, the algal beads of group 4 were taken away from the jar following darkness. In this study, all immobilized algal cells could leave the water easily and immediately, just by taking away the algal containers (the nylon net bags), to avoid the algal respiratory effects. In contrast, it is impossible to remove all the free-living and suspended microalgae from the water of normal cultures.

By judging the data of ammonium, DO and pH value (Fig. 7, 8 and 9) and the results of fish growth clearly indicated that the water quality in cultures with algal beads (groups 2, 3 and 4) were better than the culture without algal beads (group 1). That is, the algal alginate-beads could control the water quality to be suitable for tilapia growing.

In conclusion, the microalga Scenedesmus quadricauda previously entrapped into alginate-beads maintained their normal physiological activities and grew within the beads, just like free-living algal cells. Therefore, this technique is very suitable for preserving microalgal species and the application of microalga in aquaculture for controlling water quality.

 

ACKNOWLEDGEMENT:

Financial support from NSC grants 88-2313-B-019-04; 89-2313-B-019-032 and COA grants 88-AST-1.4-FID-02 (12-2); 89-AST-1.2-FID-05 (06) of the Republic of China is greatly appreciated.

 

REFERENCES:

 

Chevalier, P., de la Noüe, J., 1985. Wastewater nutrient removal with microalgae immobilized in carrageenan. Enzyme Microb. Techol. 7:621-624.

Faafeng, B. A., von Donk, E., Källqvist, S. T., 1994. In situ measurement of algal growth potential in aquatic ecosystems by immobilized algae. J. Appl. Phycol. 6:301-308.

Garbisu, C., Gil, J.M., Bazin, M. J., Hall, D. O., Serra, J. L., 1991. Removal of nitrate from water by foam-immobilized Phormidium laminosum in batch and continuous-flow bioreactors. J. Appl. Phycol. 3:221-234.

Proulx, D., de la Noüe, J., 1988. Removal of macronutrients form wastewater by immobilized algae. In: Moo-young M. (ed.), Bioreactor immobilized enzymes and cells: Fundamental and application. Elsevier applied science, London, pp:301-310.

Provasoli, L., Pintner, I. J. 1960. Artificial media for freshwater algae: problems and suggestions. In: Tryon Jr., C. A. and Hartman, R. T. (Eds.), The Ecology of Algae Pymatuning Laboratory (Univ. Pittsburg) Special Publ. 2. pp. 84-96.

Romo, S., Pérez-Martínez, C., 1997. The use of immobilization in alginate beads for long-term storage of Pseudanabaena galeata (Cyanobacteria) in the laboratory. J. Phycol. 33:1073-1076.

Spurr, A. R. 1969. A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26:31-43.

Smith, M., Croft, S. 1991. Embedding and thin section preparation. In: Harris, I. R. (Ed.), Electron Microscopy in Biology. IRL press, Oxford, New York, Tokyo. PP.17-37.

Tamponnet, C., Costantino, F., Barbotin, J. -N., Clavayrac. R. 1985. Cytological and physiological behaviour of Euglena gracilis cells entrapped in a calcium alginate gel. Physiol. Plant. 63: 277-283.

Wilkinson, C. S., Goulding, K. H., Robinson, P.K., 1990. Mercury removal by immobilized algae in batch culture systems. J. Appl. Phycol. 2:223-230.

Figure legends:

Figs. 1-2: the algal alginate-beads. Fig. 1, (a): the long-term stored algal bead that contained ca. 2,500 algal coenobia. (b): the stored bead that contained ca. 20,000 algal coenobia had been re-cultured in PEF medium with light for two weeks. (c): the stored bead that contained ca. 90,000 algal coenobia had been re-cultured in PEF medium with light for four weeks. Fig. 2, the algal beads were packaged in a nylon-net bag for subsequently hanging in the water of fish culture.

Figs. 3-6: the ultrastructure of algal cells. Figs.3-4: observations under TEM. Fig. 3, the thin-section of algal cell that showed a thylakoids (TH) composed chloroplast (CH) fills most of the cell, in which a pyrenoid (P) is normally suspended. CW: cell wall. SC: starch cap. Fig. 4, thin-section of long-term stored algal cell. The chloroplast (CH) was simply composed of thylakoids (TH) without the pyrenoid. CW: cell wall. Figs. 5-6: observation under SEM. Fig. 5, Scenedesmus quadricauda coenobium located within the fractions (F) of alginate-bead. The spines (arrow heads) were modified into bent shapes. Fig. 6, free-living S. quadricauda coenobium with straight spines.

Figs.7-9: total NH₄⁺-N, Dissolved Oxygen and pH values of the four groups of fish cultures of eighteen days culture. Groups 1 and 2 were cultured under continuous light of 100 µmol photons.m^-2.s^-1 irradiance at 24ºC. Groups 3 and 4 were cultured under the same irradiance but with 12:12, dark: light photoperiods at 24ºC. Group 1 only contained fish, however, groups 2, 3 and 4 contained fished and the algal beads. The algal beads of group 4 were removed following darkness and replaced until the light came on. Fig.7, total NH₄⁺-N concentrations of the fish culture in all the groups. Fig.8, Dissolved Oxygen of the fish culture in all the groups. Fig. 9, pH values of the fish culture in all the groups.