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Study on the medium effect for the growth rate of Spirulina, Arthrospira platensis in natural seawater

Spirulina, Arthrospira platensis, is the most important microalgae for the production of biomass as health food for human and animal food. In this experiment, the growth rates of A. platensis were tested at seven different media (F-2 with CaCO3, F-2 with NaHCO3, Z-1 with CaCO3, Z-1 with NaHCO3, Z-2 with NaHCO3, urea and T-super with NaHCO3, and PES with NaHCO3) at pH 9 to 10 and salinity 30%0. The culture was started at the optical density (OD) 0.20 and the experimental period was ended after 10 days in the laboratory room. Among the media urea and T-super with sodium bicarbonate were recorded the best. The optimum growth of A. platensis (OD 0.69) was observed in urea and T-super with sodium bicarbonate. In addition, urea and T-super with sodium bicarbonate gave rise to the maximum growth of A. platensis and then followed by Z-1 medium with NaHCO3 and PES medium with NaHCO3. The minimum OD 0.44 was recorded in F-2 medium by using CaCO3. The results of present investigation revealed the potential use of seawater with some nutrients for laboratory culture of A. platensis.


Introduction
Spirulina, A. platensis, was the dominance of life on earth for more than 3.5 billion years ago. The name "Spirulina" is derived from the Latin word for "helix" or "spiral" denoting the physical configuration of the organism when it forms swirling, microscopic stands [1].
Spirulina is not a nitrogen-fixing cyanobacteria (blue-green algae) [2]. It has a long history of safe human consumption, known to be safe and nutritious. It can play an important role in human and animal nutrition, environmental protection through wastewater recycling and energy conservation [2,3]. Spirulina is rich in proteins (60-70%), vitamins and minerals used as protein supplement in diets of undernourished poor children in developing countries [2]. One gram of Spirulina protein is equivalent to one kilogram of assorted vegetables [4].
The length of cell is 125-225 μm and the cell's width is 25-30 μm. Ends of trichomes are not or very slightly attenuated, terminal cells broadly rounded. Cells of trichomes are 10-12 μm in diameter and 2-5 μm long. The filaments are motile, gliding along their axis and have no heterocyst. Reproduction is accomplished by fragmentation of a mature trichome into many shorter segments through destruction of specialized multiple intercalary cells.
There are about 1.386 x 10 12 (one trillion three hundred eighty-six billion) cubic meters of water on Earth. About 97% of it is in the oceans and seas. Of the remaining water, about 3% is freshwater. Nearly 70% of that freshwater is frozen in icecaps of Antarctica and Greenland. Most of the remainder is present as soil moisture or lies in deep underground aquifers not accessible to human use. Of the 1%  [5]. So, we will have to use seawater for agriculture.
Spirulina media contain generally high concentrations of nutrients that impact their cost [6]. The cost of nutrients is considered to be a major factor for A. platensis biomass production [7]. The advantages of A. platensis production in seawater medium are: (i) lower fertilizer cost; (ii) saving farmland by using waste sea beach; (iii) seawater culture is not easily polluted by heavy metals and contaminations. The utilization of seawater media for the cultivation of A. platensis will be reduced the production cost considerably [8].
High pH and temperature are the key factors for large scale A. platensis culture indoors. The optimum temperature for A. platensis culture is in the range of (28-33°C) [9]. A. platensis is a cyanobacterium growing at elevated pH. In addition A. platensis requires relatively high pH values (9.5 to 11.0 with an optimum at 10.5) and is, therefore, less subject to contaminations [10]. A. platensis is the edible microorganisms which grow naturally in tropical regions inhabiting alkaline lakes containing sodium carbonate or sodium bicarbonate other materials and a source of fixed nitrogen of tropical and subtropical regions. These lakes are found near volcanoes or even within the caldera of the volcano [11].
In nature lakes of the world, the limited supply of nutrients usually regulates growth cycles. New nutrients come from an upwelling from inside the earth, when rains wash soils into the lakes, or from pollution. The alga grows rapidly and reaches a maximum density, and then dies off when nutrients are exhausted. A new seasonal cycle begins when decomposed algae release their nutrients or when more nutrients flow into the lake [12,13]. Zarrouk (1966) introduced the famous alkaline medium to enable the analysis of natural habitat [14]. The nutrient source for A. platensis is nitrate. However, Stanca and Popovici [5] proved that the use of urea as source of nutrient was increased the A. platensis biomass production.

Materials and methods
A. platensis cultivation was studied in the laboratory of Marine Science Department of Pathein University. The strain which has 20 ‰ of salinity and 10.3 of pH value was collected from Yay Hkar Innn (Lat. 22˚ 02.598ˊ Nand Long. 95˚ 54.545ˊ E) in Sagaing Township, Sagaing Region. In the laboratory, the stock algal culture was maintained in one litter plastic bottles using urea and T-super. The culture bottles were placed on the shelf and illuminated with 40-watt fluorescent lamps. For the culture experiments, the natural seawater was collected from Ngwe Saung or Chaung Tha coast. Seawater was boiled and then filtered with Whatman No. 540 filter paper. During the experimental period, the laboratory apparatus (pipettes, conical flasks, beakers, drinking plastic bottles), refractometer, pH meter, aerators, digital balance, and digital photo colorimeter was used for culture. A. platensis were cultivated in three different pH values (9.0, 9.5 and 10.0) which were adjusted by using calcium carbonate and sodium bicarbonate with modified F-2 medium (Table 1), two of modified Zarrouk's media [14]: Z-1 medium (Table 2) and Z-2 medium (Table 3), Provasoli's enriched seawater (PES) medium [6] (Table 4) and urea and T-super medium. The nutrient urea and T-super medium was prepared as the ratio 5:1 that 10 g of urea and 2 g of T-super were separately solute with each one litre of distil water which 10 ml of each nutrient solution was daily feed into the culture bottles. Other nutrients were fed once when the culture was started.

Effects of F-2 medium with CaCO 3
Growth rates of A. platensis in different pH values with F-2 medium were illustrated in Figure 1. Increasing or decreasing cell densities were found within age for all different pH values at salinity 30‰ of initial Optical Density (OD) 0.2 by using F-2 medium with CaCO 3 .
In pH value 9.0, the growth was gradually increased within the experiment and then gradually reduced to OD 0.31 at the end of the experiment. The maximum OD 0.40 was reached 4 or 5 days of the experiment and the color was yellowish-green.
In pH value 9.5, the growth was gradually increased within the experiment for six days and then gradually reduced to OD 0.33 at the end of the experiment. The maximum OD 0.41 was obtained on 6 th day of the experimental period. The color was pale-green at the end of the culture.
In pH value 10.0, the growth was gradually increased within the experiment for six days and then gradually reduced after to reach the maximum point of OD 0.44. It was reduced to nearly double time of the initial OD 0.20 at the experimental period end.
In this experiment, the maximum growths were occurred on days 5 and 6. Doubling times occurred on day 5 in all pH. The reducing time was found on day 7 in pH 9.5 and 10, and day 6 in pH 9.0. If the pH values were increased, the growth of A. platensis was good. This result suggested that A. platensis preferred to grow in high pH values and the best pH was 10.

Effects of F-2 medium with NaHCO 3
Growth rates of A. platensis in F-2 medium with NaHCO 3 at different pH values were illustrated in Figure 2. Cell densities both increased and decreased within age for all different pH values at salinity 30‰ with initial OD 0.20 in F-2 medium with NaHCO 3 .
The cells were gradually grown for seven days in pH value 9.0 and nearly steadied at maximum OD 0.40 and then slowly reduced to OD 0.32.
The growth of A. platensis was gradually increased in pH 9.5 and steadied at 6 th and 7 th days, and then slowly reduced at the end of the experiment. In pH 9.5, the maximum OD was 0.43 and the color was pale-green.
The A. platensis growth was increased within the experiment of pH 10.0 for eight days and then slowly reduced in last two days. Double time of cell growth was observed within 4 th day. The culture of A. platensis was light-green in color and the maximum OD was 0.47.
In this medium, the maximum growth rates were found on 7 th and 8 th days. Doubling times of cell growth occurred on day 7 in pH 9.0 and day 6 in another pH. The reducing time occurred on 8 th day in pH 9.0 and on 9 th day in other pH. If the pH value was increased, the growth of A. platensis was good and the best pH was 10.0. This result suggested that A. platensis preferred to survive in high pH values.

Effects of Z-1 medium with CaCO 3
Growth rates of A. platensis in Z-1 medium with CaCO 3 at different pH values were illustrated in Figure 3. Increasing or decreasing cell densities were found within age for all different pH values at salinity 30‰ of initial OD 0.20 in Z-1 medium with CaCO 3 .
In this medium, the best growth rate (OD 0.54) was observed in pH value 9.5. The cells were gradually grown for one week and then rapidly reduced after the nutritional discomfort. Over doubling, times occurred in all pH values. The reducing date occurred on 9 th day in pH 9 and others on 8 th day. According to this result, A. platensis do not prefer higher and lower pH values to grow.

Effects of Z-1 medium with NaHCO 3
Growth rates of A. platensis in Z-1 medium with NaHCO 3 at different pH were shown in Figure 4. Cell densities increased with age for all pH values with initial optical density 0.20 in Z-1 medium with NaHCO 3 .
The growth of A. platensis was gradually increased in all pH using Z-1 medium with NaHCO 3 . Cell decreasing was occurred only in pH 9.0 on day 7. The best growth rates in each pH value were OD 0.55, OD 0.57 and OD 0.62 respectively which were found at the end day of the culture. Over doubling, times were observed on 5 th day in all pH values. Tripling time (OD 0.6) was found on 9 th day in only pH 10. In this medium, A. platensis grew up until the culture end. The color was darkgreen at the end of the culture. This experiment suggested that A. platensis preferred to survive in high pH values.

Effects of Z-2 medium with NaHCO 3
Growth rates of A. platensis in Z-2 medium with NaHCO 3 at different pH values were as shown in Figure 5. Cell densities increased with age for all different pH values at salinity 30‰ with initial optical density 0.20 in Z-2 medium. The culture of A. platensis was dark-green in color at the end of the experiment. For three different pH tested, pH 10 in Z-2 medium with NaHCO 3 was found doubling time superior to other pH values (pH 9.0 and pH 9.5). The doubling times were observed on 4 th day of the experimental period in pH 10.0 and 5 th day in other pH. The maximum OD 0.61 was reached in pH 10.0 within 10 days of the experimental period.
In this experiment, the reducing time did not occur during the culture period. The growth increased and doubling time was observed faster with increasing pH. From this result, pH 10.0 was suitable for production of A. platensis in small volume culture.

Effects of Urea and T-super medium with NaHCO 3
Growth rates of A. platensis in urea and T-super medium with NaHCO 3 at different pH values were as shown in Figure 6. Cell densities increased with age for all different pH values with initial optical density 0.20 in urea and T-super medium. The culture of A. platensis was dark-green in color at the end of the experiment.
In pH value 9.0, the growth of A. platensis was gradually increased and maximum point of OD was three times the initial. The culture of A. platensis was dark-green in color. In pH value 9.5, A. platensis growth was gradually increased within the experiment and nearly double time was observed 4 th day of the experiment. The maximum OD 0.67 was reached within 10 days of the experiment. The color was dark-green at the end of the culture.
In pH value 10.0, the growth of A. platensis was gradually increased and over doubling time was observed 5 th day of the experiment. The color was dark-green and the maximum OD 0.69 was reached within 10 days of the experiment.
In this experiment, A. platensis grew up the culture end. Over doubling times started from day 5. If the pH value was increased, the growth of A. platensis was also increased. This experiment suggested that A. platensis may be to survive in high pH values.

Effects of PES medium with NaHCO 3
Growth rates of A. platensis in PES medium with NaHCO 3 at different pH values were as shown in Figure 7. Cell densities increased with age for all different pH values at salinity 30‰ with initial OD 0.20 PES medium. The culture of A. platensis was dark-green in color at the end of the experiment.
In this culture, the growth of A. platensis was gradually increased to maximum points of OD which were OD 0.56 in pH 9.0, OD 0.62 in pH 9.5 and OD 0.61 in pH 10.0. The doubling times were occurred at 5 th day in all pH tested except in pH 9.0.
During this experiment, there was no found cell growth reduced within the culture period. This result suggests that A. platensis survive in high pH values. If the pH value was increased, the growth of A. platensis was good.    The effect of media and pH on growth of A. platensis at the end of the culture was shown in Figure  8. A. platensis survived in various pH and media. The highest growth was found at pH 10 and urea and T-super medium. Therefore, the yield of A. platensis is influenced by pH and medium.
Richmond and Grobbelaar [10] observed that the optimal pH for the growth of A. platensis was 10.5. Mahadevaswamy [16] said that the yield of A. platensis could decrease when pH values were decreased.
Florian et al. [17] and Yean-Chang C [18] reported that the best medium to reduce the cultural cost and increase the biomass of A. platensis cultures was modified by Zarrouk's medium. Dineshkumar  al. [19] stated the highest yields of A. platensis culture by using Zarrouk's medium and F-2 medium were found on 30 th day and 21 st day as 1.86 dw/L and 0.52 dw/L, respectively.