In this study, growth of C. vulgaris in batch culture was investigated in relation to some physical and chemical factors. For this purpose, culture medium for C. vulgaris was prepared in sea water and the culture was kept under four combinations of light regime and carbondioxide supply. Three salinity levels, 25, 30 and 35 S were employed. Growth of the alga in culture varied with respect to number of individuals and size of maxima under different culture conditions. C. vulgaris showed better growth in culture that was kept in 25 salinity under 24 h illumination and 24 h of carbondioxide supply.
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Use of microalgae as food sources in both freshwater and marine fish culture has now become very common. The commercial utilization of microalgae has especially gained attention with the discovery that some algae are they best natural source of β-carotene. Further interest in the microalgae as food organisms has been particularly generated in recent years since microalgae has now extensively used as live food for commercially important molluscs, fish and crustaceans during at least part of their life cycles. In fact, aquaculture is now one of the most rapidly growing areas in the field of food production all over the world.
Microalgal culture is one of the modern biotechnologies and Chlorella has been one of most commonly used algae in algal biotechnology. Chlorella vulgaris (Chlorophyta) is also one of the most significant algae as a food organism for fish larvae.
The first unialgal cultures were achieved by Beijerink 1980 with Chlorella vulgaris and Chlorella was also one of the first algae to be isolated as a pure culture. In addition commercial large-scale culture of microalgae commenced in the early 1960s in Japan with the culture of Chlorella. By 1980 there were 46 large-scale factories in Asia producing more than 1000 kg of microalgae (mainly Chlorella) per month and 1996 about 2000 t of Chlorella were traded in Japan alone. Later Chlorella was also cultured as health foods for human consumption or as by-products in waste water treatment.
Cultural characteristics of Chlorella were investigated by several researchers since the late 1950s[7-11]. The present study was aimed to investigate the influence of light duration and CO2 on growth of C. vulgaris together with salinity.
MATERIALS AND METHODS
C. vulgaris was obtained from the a culture center and culture medium of C. vulgaris was prepared in sea water filtered through 55-60 μm diatomite filter. Sea water was then subjected to UV and autoclaved at 170°C 4 h. The culture medium was prepared as in Table 1. Experiments were carried out in erlenmayer flasks (500 mL) with 250 mL of medium and maintained in a controlled culture room. All glassware was washed in HCl 10% and autoclaved at 170°C before use. 250,000 cells mL-1 was inoculated into each erlenmayer and closed with aluminium foil. Burker counting chamber was used for cell counts.
Four combinations of light regimes and CO2 supply were employed.
|24 h light phase and 24 h CO2 supply
|12 h light phase and 24 h CO2 supply
|24 h light phase and 12 h CO2 supply
|24 h light phase and no CO2 supply
Light intensity was adjusted at 2000-3000 luxes from flourescent lamps. CO2 was supplied to the cultures by connecting an industrial CO2 tube to the air blower of the culture room. All cultures were kept at a constant temperature of 24°C and pH of the culture medium was maintained at 7.5 since the rate of the growth was inhibited at higher pH levels. Three salinity levels (25, 30, 35 S) were used and salinity of sea water was reduced with distilled freshwater.
|Preparation of enriched sea water medium
|All solutions exclusive of vitamin solution were autoclaved at 120°C for 30 min. 1 mL from each solution is added to 1 L culture medium.
As seen from the Fig. 1-4 the alga displayed similar growth patterns in all cultures kept under different conditions. Cell numbers of the alga remained almost unchanged during 1-3 days (lag phase). The alga then started to increase in cell numbers and continued to increase until a maximum was reached. This active multiplication phase usually started on 5th or 6th day and lasted for 17-33 days according to the culture conditions. Cell numbers always decreased rapidly after all maxima.
C. vulgaris reached to a maximum of 10000x106 cells mL-1 on 21st day in culture under continuous illumination and CO2 supply at 25 S. A maximum of 5500x106 cells mL-1 at 30 S and a maximum of 4700x106 cells mL-1 at 35 S occurred on 17th an 19th days, respectively (Fig. 1).
In cultures with 24 h illumination and 12 h CO2 supply maximum cell densities for 25, 30 and 35 S were 5100x106, 4400x106 and 3350x106 cells mL-1, respectively (Fig. 2).
Cultures kept under 12 h illumination and 24 h CO2 supply, maximum cell numbers of 1400x106, 600x106 and 1800x106 cells mL-1 were recorded at 25, 30 and 35 S, respectively (Fig. 3).
In culture with 24 h illumination and without CO2 maxima for 25, 30 and 35 S occurred in the order of 275x106, 800x106 and 900x106 cells mL-1 (Fig. 4).
Of all, best growth of C. vulgaris occurred in the culture subjected to 24 h illumination and 24 h CO2 supply at 25 S since maximum cell number was reached in a shorter time and the size of the maximum was by far greater than those recorded in other cultures with different conditions.
|Growth of C. vulgaris in culture with 24 h illumination and 24 h CO2 supply at 25, 30 and 35 S
|Growth of C. vulgaris in culture with 24 h illumination and 12 h CO2 supply at 25, 30 and 35 S
|Growth of C. vulgaris in culture with 12 h illumination and 24 h CO2 supply at 25, 30 and 35 S
This may indicate that continuous illumination strongly supported the growth of the alga in this study. However, the photosynthetic processes of Chlorella was reported to become saturated at relatively low illumination, ranging from 4000-30000 lux depending on the strain.
|Growth of C. vulgaris in culture with 24 h illumination with no CO2 supply at 25, 30 and 35 S
In addition, 25 and 30 S levels appeared to be more suitable for the growth of the alga than 35 S when culture was kept under 24 h illumination accompanied with 12-24 h CO2 supply. This finding is in harmony with the study of Fabregas et al. who found salinity and nutrient concentration to be closely related to the growth of marine food algae.
It has been shown that 1 mL of pure CO2 is needed for the production of 1 mg (dry weight) of Chlorella cells. Thus, the most important problem in liquid algal cultures seems to be an adequate carbon supply. The rate of CO2 transfer into liquid phase was reported to decrease in parallel with the elevation of temperature. The present study was in harmony with the study of Oh-Hama and Miyachi since the growth of the alga was observed to be poorest in the culture with no CO2 supply despite continuous illumination. In contrast, the best growth was achieved under continuous CO2 supply. This finding may also show that CO2 supply can strongly affect the growth of Chlorella under the culture conditions employed in this study. Fabregas et al. also reported that an increase in the nutrient concentration did not produce an increase in biomass production but CO2 added to the cultures increased the final biomass production of the alga in the culture of another marine alga Tetraselmis suecica. This also holds true for Chlorella in this study. In addition, Chlorella was reported to show excellent stabilities to high concentrations of CO2 and high growth rates of Chlorella species in up to 50% CO2 were emphasized.