**2. System construction and strategies for culture plan**

### **2.1. The production site**

bioremediation [3–6]. *Spirulina* that highly contains various functional materials has been focused as an important nutrition source for the future of humans and animal feeds, so that related industry has been gradually growing [2, 7, 8, 12]. In addition, the biomass plays an important role in broad ranges of industries [9, 10] and functional foods using proteins [3, 11]. In particular, *S. platensis* has been mostly studied for industrial production, in which the most important factors for an increase of production could be nutrient concentration, light intensity, and optimal water temperature [12]. *Spirulina* sp. including other microalgae are cultured on a large scale worldwide for industrial use [13–17]. Proteins and phytopigments (phycocyanin and β-carotene) along with polyunsaturated fatty acids from *Spirulina* are one of the high

Nowadays, microalgae are mostly cultured in photobioreactors (PBRs) and open raceway systems (ORSs) for industrial production [2, 19, 20]. Advantages of PBR include applicability of various designs in production, easy control of growth condition, prevention of biological contamination, and high productivity. On the contrary, it requires high expenses in initial investments, device maintenance, and expansion of mass production facility [20–22]. In contrast, ORS

and it is also advantageous due to inexpensive materials for facility (PVC, FRP, concrete, plastic, and soil) and easy to scale up structure. In respect of commercialization, ORS needs a low initial investment cost, while it has a highly efficient productivity, so that ORS has been attracting more interest [15, 20, 23–30]. However, there are still steps that need to be taken for the commercialization of ORS, which includes a control of water temperature and light intensity depending on season, elevation of aerial productivity, development of highly efficient microalgae species, development of low cost and highly efficient culture medium, technology of contamination improvement, and establishment of protocol for year-round culture and harvest [19, 20, 31–33].

Culture conditions of microalgae in ORS and designing of system are closely related with environment of selected area. *Spirulina* was the most widely used in outdoor cultivation trials and interests for commercial production have been increasing in many places based on the studies of intensive ecological and physiological research and development over four decades. However, this cyanobacterium needs high temperatures for optimal growth, thus commercial production has been limited until subtropical areas. In temperate regions, high temperatures are recorded in summer season, while the temperature is certainly low in fall and winter seasons. The overall temperature range should not be a suitable range for *Spirulina* growth in a year. Consequently, little information of commercial production has been reported in temperate areas [32, 34, 35]. Thus, an environmental analysis needs to be carried out on selected area for a culture system with maximal productivity based on which culture system needs to be constructed. Recently, the concept of building information modeling (BIM) developed in construction field was first introduced by the National Institute of Standards and Technology (NIST) of the Department of Commerce in the USA in 2007. BIM refers to a recent construction process that analyzes data with 3D modeling method using big data and information of the past and then applies it from designing to installation process, avoiding existing 2-D design. Therefore, BIM technology is advantageous due to cost saving for installation operation and prediction of future [36–38]. As shown in success cases such as the Disney Concert Hall of the US, the Olympic Main Stadium of Beijing, and Melbourne Recital Center, BIM technology has emerged as an important issue in architectural industry, so that it is considered beneficial to apply it to a microalgae production facility.

in the air though it has a low aerial productivity than PBR,

value-added materials that bring health to humans [2, 13, 18].

can directly use solar energy and CO2

62 Cyanobacteria

The city of Ansan was selected for the pilot study for production of *Spirulina*. Ansan is located in Western South Korea (37.287°N, 126.833°E), about 30 km south of Seoul, on the coast of the Yellow Sea. Recent average temperatures generally exceed 30°C in summer, and in winter are above −5°C. The average temperature is 12.5°C, and number of sunny days per year generally ranged from 86 to 124, with >10 h of sun per day for the whole year. Mean monthly temperatures are shown in **Figure 1A**. **Figure 1B** shows that weekly average solar radiation ranged from 1721 to 5671 Wh/m2 . From 2006 to 2010, the number of rainy days per year ranged from 99 to 138, and average annual rainfall was about 1150 mm in the locality of the cultivation area.

#### **2.2. Construction of culture system and its structure**

**Figure 2** shows schematic processes for planning and construction of *Spirulina* culture system using BIM technology. A pilot system for microalgae production was constructed in the Korea Institute of Ocean Science and Technology based on BIM technology which analyzes atmospheric environment data (temperature, solar radiation, and shadow effect etc., **Figure 3**) of the past for a long time, and predicts the future based on the data in order to design an optimal eco-friendly structure.

**Figure 4A** is a vertical section of microalgae pilot system constructed based on BIM, in which the roof and side windows were designed with a maximal consideration of natural ventilation, and optimal construction cost and efficiency was realized by a four-way slide window at the side and the introduction of automatic opening and shutting system on the roof. **Figure 4B** is a horizontal section of the modified ORS culture facility. Each size of the culture facility was 10,000 (W) × 3250 (L) × 550 (H) mm, and the culture raceway was finished with concrete after vertical excavation of the ground as deep as 600 mm for the purpose of using geothermal heat as shown in **Figure 4**. Depth of medium for the culture was maintained as 400 mm. Boiler pipes were buried in the concrete floor of the modified ORS for maintenance of culture temperature in the winter.

Computational fluid dynamics (CFD) was conducted to analyze and optimize the mixing (e.g., water flow and paddle rotational speed) of medium with *S. maxima* cells in the ORSs. The main purpose is to find the suitable rotational speed of the paddle to maximize mixing in the flow field.

**Figure 1.** Mean monthly temperature (A) and integrated weekly solar radiation (Wh/m2 ) (B) at the location of the study for past 5 years during 2006–2010.

The commercial software Flow-3D (version 10.0) was chosen for this analysis. The simulation was performed by ARA Consulting & Technology Co., by applying two rotational speeds (15 and 30 rpm) (**Figure 5**). As a result, rotational speed was selected as 15 rpm in the ORSs. Simplified structures of the ORS and the greenhouse for *S. maxima* cultivation were shown in **Figure 6**.

raceway on the 10th day when the culture concentration of *S. maxima* reached to 0.5– 0.8 g/L. Once concentration of secondary culture reached to 0.5–0.8 g/L on the 17th day, 20 tons of each culture medium with the same composition was made and added to the modified ORS. After 5 h, 300 L of sample from the secondary culture was inoculated. The rotational speed of the paddle wheel that circulates and mixes the culture medium was kept at 15 rpm. Batch culture was performed in the ORS for about 1 year from April 4, 2011 to March 16, 2012.

Cultivating *Spirulina maxima*: Innovative Approaches http://dx.doi.org/10.5772/intechopen.74265 65

**Figure 2.** Schematic processes for planning and construction of *Spirulina* culture system using BIM technology.

The electric boiler that was installed for maintenance of temperature in the winter was operated from late October to early April to heat the system electrically and to maintain optimal

**2.4. Maintaining culture system**

#### **2.3. The microorganism, experimental design, and culture conditions**

The axenic culture of *S. maxima* Cy-23, was obtained from Korea Marine Microalgae Culture Center (Pukyong National University, Busan, Korea) and was initially inoculated in a 5 L conical flask containing modified *Spirulina* medium [42] at plant room temperature (23–30°C). Thereafter, secondary culture with scale up was performed in a 300-L PE circular cylinder

**Figure 2.** Schematic processes for planning and construction of *Spirulina* culture system using BIM technology.

raceway on the 10th day when the culture concentration of *S. maxima* reached to 0.5– 0.8 g/L. Once concentration of secondary culture reached to 0.5–0.8 g/L on the 17th day, 20 tons of each culture medium with the same composition was made and added to the modified ORS. After 5 h, 300 L of sample from the secondary culture was inoculated. The rotational speed of the paddle wheel that circulates and mixes the culture medium was kept at 15 rpm. Batch culture was performed in the ORS for about 1 year from April 4, 2011 to March 16, 2012.

#### **2.4. Maintaining culture system**

The commercial software Flow-3D (version 10.0) was chosen for this analysis. The simulation was performed by ARA Consulting & Technology Co., by applying two rotational speeds (15 and 30 rpm) (**Figure 5**). As a result, rotational speed was selected as 15 rpm in the ORSs. Simplified structures of the ORS and the greenhouse for *S. maxima* cultivation were shown in **Figure 6**.

) (B) at the location of the study

The axenic culture of *S. maxima* Cy-23, was obtained from Korea Marine Microalgae Culture Center (Pukyong National University, Busan, Korea) and was initially inoculated in a 5 L conical flask containing modified *Spirulina* medium [42] at plant room temperature (23–30°C). Thereafter, secondary culture with scale up was performed in a 300-L PE circular cylinder

**2.3. The microorganism, experimental design, and culture conditions**

**Figure 1.** Mean monthly temperature (A) and integrated weekly solar radiation (Wh/m2

for past 5 years during 2006–2010.

64 Cyanobacteria

The electric boiler that was installed for maintenance of temperature in the winter was operated from late October to early April to heat the system electrically and to maintain optimal

**Figure 3.** The retrospective analysis of BIM based 3D modeling for visualization of shadow area at summer (A) and winter (B) solstices and estimation of monthly solar radiation of two raceway pond (C) at the location of the study for past 5 years during 2006–2010. Arrows indicates the location of culture area.

water temperature. The temperature of the culture medium was maintained between 20 and 25°C, and the water temperature for October when its operation was initially started was in the range of 21–23°C. Total electricity consumption during the period of boiler operation was measured by using an electronic watt-hour meter (LD3410CT-005Te). The boiler operation was set as a variable type depending on variation in water temperature (on 30 min, off 30 min), and the maximum operation rate (on 50 min, off 10 min) was used to minimize fluctuation of water temperature during the period of rapid drop in room temperature in the winter. Daily measurement values were summed every week and expressed in mean value ± standard deviation for an analysis of each environment and electricity consumption data.

**2.5. Biomass concentration, harvesting, and productivity**

Before harvesting, the biomass concentration was estimated by sampling and filtering 20 mL of each culture using a vacuum pump and GF/C filter paper (Whatman), and the filter paper was dried in a dry oven (65°C) for 24 h, followed by the measurement of biomass. Biomass was measured two times a week. For harvesting, cultured *S. maxima* samples in 3 tons of culture medium were harvested by using a Tubular Separator (GQLY series) at 15,000 rpm for 3 h in an interval of a week. Harvested samples were kept in a −50°C deep freezer (DEASAN)

**Figure 4.** A cross sectional view of the designed open raceway system for pilot production of *Spirulina maxima* using BIM analysis (patent no. 10–1,142,358, 10–1,142,359, 10–1,110,068). A: Vertical view of the system; a: Automatically opening and shutting window, b: 4-way sliding windows including anti-insect nets, c: Raceway pond, d: Paddle-wheel, e: Electronic boiler pipe, f: Concrete structure. B: Horizontal view of the system; main gate (g) has located to the south.

Cultivating *Spirulina maxima*: Innovative Approaches http://dx.doi.org/10.5772/intechopen.74265 67

for a biochemical analysis and then freeze-dried by using a freeze dryer (ILSHIN).

**Figure 4.** A cross sectional view of the designed open raceway system for pilot production of *Spirulina maxima* using BIM analysis (patent no. 10–1,142,358, 10–1,142,359, 10–1,110,068). A: Vertical view of the system; a: Automatically opening and shutting window, b: 4-way sliding windows including anti-insect nets, c: Raceway pond, d: Paddle-wheel, e: Electronic boiler pipe, f: Concrete structure. B: Horizontal view of the system; main gate (g) has located to the south.

#### **2.5. Biomass concentration, harvesting, and productivity**

water temperature. The temperature of the culture medium was maintained between 20 and 25°C, and the water temperature for October when its operation was initially started was in the range of 21–23°C. Total electricity consumption during the period of boiler operation was measured by using an electronic watt-hour meter (LD3410CT-005Te). The boiler operation was set as a variable type depending on variation in water temperature (on 30 min, off 30 min), and the maximum operation rate (on 50 min, off 10 min) was used to minimize fluctuation of water temperature during the period of rapid drop in room temperature in the winter. Daily measurement values were summed every week and expressed in mean value ± standard deviation for an analysis of each environment and electricity

**Figure 3.** The retrospective analysis of BIM based 3D modeling for visualization of shadow area at summer (A) and winter (B) solstices and estimation of monthly solar radiation of two raceway pond (C) at the location of the study for

past 5 years during 2006–2010. Arrows indicates the location of culture area.

consumption data.

66 Cyanobacteria

Before harvesting, the biomass concentration was estimated by sampling and filtering 20 mL of each culture using a vacuum pump and GF/C filter paper (Whatman), and the filter paper was dried in a dry oven (65°C) for 24 h, followed by the measurement of biomass. Biomass was measured two times a week. For harvesting, cultured *S. maxima* samples in 3 tons of culture medium were harvested by using a Tubular Separator (GQLY series) at 15,000 rpm for 3 h in an interval of a week. Harvested samples were kept in a −50°C deep freezer (DEASAN) for a biochemical analysis and then freeze-dried by using a freeze dryer (ILSHIN).

Areal productivity was calculated by using the following equation. Data used for analysis were monthly mean value (mean value ± standard deviation) by adding the weekly measurement values.

Areal productivity (AP, g/m2 /d) = Volumetric productivity (VP) × (Odepth × 1000) (1)

where Odepth is the pond operating depth.

### **2.6. Analysis of biochemical components and pigments**

The crude biochemical composition of cultured *S. maxima* was determined according to the AOAC method [43]. Crude carbohydrate was determined by the phenol–sulfuric acid reaction, crude lipid was extracted by the Soxhlet method, and crude ash was prepared at 550°C

in the dry-type furnace. The moisture was determined by keeping the sample in a dry oven at 105°C for 24 h and the crude protein was determined by the Kjeldahl method. Unfortunately, the samples of November and December 2011 were denatured by sudden trouble of refrigera-

**Figure 6.** Simplified inner structure of ORS and the greenhouse for *S. maxima* cultivation at Ansan City, KIOST. The number of 20 was harvesting time and the number of 167 was the days after inoculation. Photographed by Taeho Kim in 2011.

For phycocyanin analysis, dried *S. maxima* powder was weighted accurately to 50 mg, and then mixed with 10 mL sodium phosphate buffer (pH 7.0) in centrifuge tube. The mixed sample was sonicated for 1 min and stored in refrigerator overnight maintained at 20°C. The sample was mixed well using vortex and then was centrifuged for 20 minutes at 10°C at 3000 rpm. The supernatants were repeatedly collected until the color came off. After collecting all the phycocyanin, the supernatant was filtered using GF/C filter, and the red absorbency of each replicate was at 620 and 652 nm using phosphate blank buffer with spectrophotometer (Optizen POP

C − PC (mg/L) = (A620 − 0.474 × A652)/5.34 (2)

mg \_\_\_

For chlorophyll-*a* analysis, dried *A. maxima* powder was weighted accurately at 50 mg and then mixed with 5 mL-90% acetone. The mixed sample was sonicated for 1 min and then stored in refrigerator for 30 min maintained at 4°C. The pigment extracts have to be kept on ice and in the dark. The sample was mixed well using vortex and then was centrifuged

<sup>L</sup> ) <sup>×</sup> buffer volume (mL) \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

sample weight (g) <sup>×</sup> \_\_\_\_\_\_\_\_\_\_\_\_ <sup>1</sup> <sup>L</sup>

Cultivating *Spirulina maxima*: Innovative Approaches http://dx.doi.org/10.5772/intechopen.74265 69

<sup>1000</sup> mL (3)

bio). Concentration of phycocyanin was then calculated using below formula [44].

tor for biochemical and pigments analysis.

phycocyanin (mg/g) <sup>=</sup> <sup>C</sup> <sup>−</sup> PC(

**Figure 5.** Summarized results on mixing simulation of medium with *S. maxima* cells in the ORSs by applying CFD of flow-3D software. A: 3D applied raceway pond (10 m in length, 3.25 m in width and 0.6 m in depth) with paddle, B: Interface condition of boundary line by interactive boundary layer modeling techniques, C and D: Velocity magnitude and particles maps at 60 second after operation for various aspect of particles with different rotational speeds (C: 15 rpm, D: 30 rpm).

Areal productivity was calculated by using the following equation. Data used for analysis were monthly mean value (mean value ± standard deviation) by adding the weekly measure-

Areal productivity (AP, g/m2 /d) = Volumetric productivity (VP) × (Odepth × 1000) (1)

The crude biochemical composition of cultured *S. maxima* was determined according to the AOAC method [43]. Crude carbohydrate was determined by the phenol–sulfuric acid reaction, crude lipid was extracted by the Soxhlet method, and crude ash was prepared at 550°C

**Figure 5.** Summarized results on mixing simulation of medium with *S. maxima* cells in the ORSs by applying CFD of flow-3D software. A: 3D applied raceway pond (10 m in length, 3.25 m in width and 0.6 m in depth) with paddle, B: Interface condition of boundary line by interactive boundary layer modeling techniques, C and D: Velocity magnitude and particles maps at 60 second after operation for various aspect of particles with different rotational speeds (C: 15 rpm,

ment values.

68 Cyanobacteria

D: 30 rpm).

where Odepth is the pond operating depth.

**2.6. Analysis of biochemical components and pigments**

**Figure 6.** Simplified inner structure of ORS and the greenhouse for *S. maxima* cultivation at Ansan City, KIOST. The number of 20 was harvesting time and the number of 167 was the days after inoculation. Photographed by Taeho Kim in 2011.

in the dry-type furnace. The moisture was determined by keeping the sample in a dry oven at 105°C for 24 h and the crude protein was determined by the Kjeldahl method. Unfortunately, the samples of November and December 2011 were denatured by sudden trouble of refrigerator for biochemical and pigments analysis.

For phycocyanin analysis, dried *S. maxima* powder was weighted accurately to 50 mg, and then mixed with 10 mL sodium phosphate buffer (pH 7.0) in centrifuge tube. The mixed sample was sonicated for 1 min and stored in refrigerator overnight maintained at 20°C. The sample was mixed well using vortex and then was centrifuged for 20 minutes at 10°C at 3000 rpm. The supernatants were repeatedly collected until the color came off. After collecting all the phycocyanin, the supernatant was filtered using GF/C filter, and the red absorbency of each replicate was at 620 and 652 nm using phosphate blank buffer with spectrophotometer (Optizen POP bio). Concentration of phycocyanin was then calculated using below formula [44].

$$\mathbf{C} - \text{PC (mg/L)} = \left< \mathbf{A}\_{\text{s20}} - 0.474 \times \mathbf{A}\_{\text{s22}} \right> \left< 5.34 \right> \tag{2}$$

$$
\mathbf{C} - \mathbf{r}C \text{ (mg/L)} = \left(\mathbf{A}\_{\text{e20}} - 0.474 \times \mathbf{A}\_{\text{e32}}\right) \text{C.S4} \tag{2}
$$

$$
\text{physccyanin (mg/g)} = \frac{\mathbf{C} - \mathbf{P}C \left(\frac{\text{mg}}{\text{L}}\right) \times \text{buffer volume (mL)}}{\text{sample weight (g)}} \times \frac{1 \,\text{L}}{1000 \,\text{mL}} \tag{3}
$$

For chlorophyll-*a* analysis, dried *A. maxima* powder was weighted accurately at 50 mg and then mixed with 5 mL-90% acetone. The mixed sample was sonicated for 1 min and then stored in refrigerator for 30 min maintained at 4°C. The pigment extracts have to be kept on ice and in the dark. The sample was mixed well using vortex and then was centrifuged for 20 minutes at 10°C at 3000 RPM. The supernatants were repeatedly collected until the color came off. After collecting all the chlorophyll, the supernatant was filtered using a 0.2 μm syringe filter, and the absorbency of each replicate was at 625, 647, and 664 nm using phosphate blank buffer with spectrophotometer (Optizen POP bio). Concentration of chlorophyll was then calculated using below formula [45].

$$\mathbf{C\_{s}(mg/L)} = \left(12.62 \times \mathbf{A\_{\\_64}}\right) - \left(2.99 \times \mathbf{A\_{\\_67}}\right) - \left(0.04 \times \mathbf{A\_{\\_625}}\right) \tag{4}$$

The maximum daily medium temperature of the culture was over 20°C and up to 30°C during the period from January to August. **Table 1** and **Figure 7** present year-round change data of

and electricity consumption during the culture of *S. maxima* from April 2011 to March 2012. As for illumination intensity during the study period, the maximum value was 1590 μmol/m<sup>2</sup>

Temperature of culture medium showed a change in a range of 20.2–26.8°C from April 4, 2011 to May 31. The highest water temperature of the year was 33.0°C on July 26 as an effect of increase in outer temperature. In addition, the average evaporation rate in August 2011 was

during the second week of December and recorded the lowest water temperature of the year at 16.0°C on February 23, 2012. Culture medium temperature for optimal growth of *Spirulina* was between 35 and 37°C, and should be kept at least at 15–20°C [2]. Since severe air temperature in this area was recorded from −18 to 36°C, and it was below 10°C on average in the winter, the ORS with glass greenhouse was electrically heated by the boiler during the period from late October to early April to maintain at least 15°C, the lowest temperature for *Spirulina* culture. These results should serve as fundamental data for setting temperature and running

The mean initial salinity of May and June was 16.6 ± 0.9 psu due to the effect of added tap water. As culture days increase, salinity concentration showed a range of change between 13.1 and 18.4 psu during the year due to effects of evaporation of culture medium and supplementation of freshwater. In addition, the mean salinity concentration during the entire culture period was 16.5 ± 1.3 psu. The pH change during the culture period showed a relatively small variation between 9.9 and 11.9. Variation of pH from May 4, 2011 to September 9 was between 9.9 and 10.97, pH change between September 14 and September 30 was ranging between 11.15 and 11.90, and then it went down to below 11.0 from October 4. The ending pH on March 16 was 9.97. Internal room temperature of the plant during the culture period was in the range of 3.2–55.0°C, and the mean room temperature was 24.3 ± 10.5°C. The mean total electricity consumption (kWh) of the microalgae pilot plant was 10.3 ± 1.1 kWh per day during the initial culture between April 4, 2011 and May 4, during which the boiler was operated for maintenance of optimal medium temperature (20.2–26.1°C). Boiler operation was stopped between May 6, 2011 and October 16, 2011 for optimal temperature, during which 1.3 ± 0.6 kWh of electricity was used on average per day. The range of biomass of *S. maxima* that was produced year-round using batch culture method in the raceway system with glass greenhouse structure in the present study was 5.68–37.67 g/m2

*S. maxima* was between 16.0 and 33.0°C, and mean water temperature was 23.6 ± 3.2°C.

a boiler in order to maintain optimal medium temperature in the winter.

in which the highest productivity of the year was recorded as 37.67 g/m2

(750 L) by Richmond et al. [46], productivity was 15–27 g/m2

when temperature increased. According to a study on *S. platensis* culture in a raceway system

Considering that limiting factors important for growth and productivity of microalgae are solar radiation, carbon supply, water temperature, and dissolved oxygen [5, 46, 48], a control system on these factors are also critical. BIM designing technology that was introduced in the present study to predict these effects had advantages including investigation of spatiotemporal

of *S. platensis* in a raceway system (135,000 L) conducted in Spain showed 2–17 g/m2

/s), temperature of culture medium, room temperature, salinity, pH,

/h. Water temperature gradually decreased from October, went below 20°C

/s. Year-round water temperature of culture raceway for

Cultivating *Spirulina maxima*: Innovative Approaches http://dx.doi.org/10.5772/intechopen.74265

/s,

71

/d,

/d [47].

/d in the summer

/d, whereas a study on culture

radiation amount (μmol/m<sup>2</sup>

701 ± 136.4 ml/m2

and the minimum value was 7 μmol/m<sup>2</sup>

$$\begin{aligned} \text{Cylorophyl} &= \text{g/m} \text{ (mg/g)} \\\\ \text{Chlorophyll} &= \text{a (mg/g)} = \frac{\text{C}\_{\text{s}} \text{(mg)} \times \text{acetone volume (mL)}}{\text{sample weight (g)}} \times \frac{1 \text{ L}}{1000 \text{ mL}} \end{aligned} \tag{5}$$

#### **2.7. Measurement of climatic and culture conditions**

Various parameters of the system were measured on a daily basis including room temperature (TENMARS) and light intensity (Lux Meter TM-205), water temperature (UNIS thermometer), pH (pH METER D-51, HORIBA), and salinity (SALT MATER YK-31SA). Although humidity was not a variable of interest, it seems the humidity was dropping in the plant as the level of medium kept in the plant was consistently lowering by evaporation. Evaporation amount was measured during August when culture medium was highly evaporated. To specify the amount of water being evaporated each day per unit area (m2 ), daily evaporation rate was measured and then averaged as ml/m2 /h. The amount of evaporation was then supplemented daily with underground tap water in KIOST (HCO3 4 6.4 mg/L, Ca 20 mg/L, Cl 13.6 mg/L, SO4 2− 11.4 mg/L, Na 8.64 mg/L, Mg 3.99 mg/L, K 1.97 mg/L, T-N 1.66 mg/L, NO3 -N 1.61 mg/L, T-P 0.02 mg/L, Co, Mo, and B 0 mg/L while Fe, Zn, Cu and Mn were not detected, and pH was 7.3).

A statistical program (IBM SPSS, NY, USA) was used for statistical analysis in order to test significance of environmental factors and pigments and biomass of *S. maxima* (one way ANOVA, Tukey test, *P* values <0.05). Monthly variations among climatic and culture conditions, biomass, biochemical components, and pigment concentrations were statistically analyzed by one way ANOVA with Pearson's multiple range tests at p < 0.05 (SPSS version 12.0, NY, USA) for the identification of significant seasonal differences during the study period. All analyses were performed on triplicate samples.
