Experimental design The experiment was carried out for eighteen days in the Live Food Culture Laboratory of the Department of Aquaculture, Faculty of Fisheries, Bangabandhu Sheihk Mujibur Rahman Agricultural University. The experiment was conducted in completely randomized design with five treatments. Kosaric medium was used as a control medium for treatment T1. In treatments T2 to T5, sodium nitrate of (NaNO3) of Kosaric media was replaced with urea.
Collection and maintenance of S. platensis Pure stock sample of S. platensis was collected from Department of Aquaculture, Bangladesh Agricultural University, Mymensingh. After collecting the stock, it was maintained in the Live Food Culture Laboratory of the Department of Aquaculture, Faculty of Fisheries, Bangabandhu Sheikh Mujibur Rahman Agricultural University. Pure stock culture of S. platensis was maintained in Kosaric medium (KM), modified after Zarrouk (1996). Growth of S. platensis was monitored at every alternative day and was checked under microscope to confirm its purity. Preparation of Kosaric medium Kosaric medium is widely used as the standard medium for S. platensis culture.
For the preparation of Kosaric medium, the above mentioned amount of chemicals from no. 1 to 8 was weighted by the help of electric balance and took in a 1.0 L conical flask. Then 0.5 ml micronutrient solution was pipetted in the flask and distilled water was added to make the volume 1.0 L. pH adjustment of all media Prior to culture initiation of S. platensis pH of all media were adjusted at 9.0 by incorporating either 0.1 N HCl or 0.1 N NaOH depending on pH condition of the media. When it was found that the pH value of one media was above 9.0 then 0.1 N HCl was added and when the pH value was found below 9.0, 0.1 N NaOH was added until pH becomes stable at 9.0. Experimental culture of S. platensis Five treatments with three replication was used to culture microalgae S. platensis in 1.0 L conical flask. S. platensis were inoculated into each culture flask to produce a culture containing 10% suspension (OD at 620 nm=0.20) (Habib, 1998). The flasks were kept under fluorescent light (TFC, FL-40, SD/38 day light) in light: dark (12h: 12h) conditions in Live Food Culture Laboratory. These culture flasks were continuously aerated using electric aerators (Sobo, Aquarium pump SB-348A). Samplings were performed at every three alternative days for each flask to observe the optical density (OD), physico-chemical properties, dry cell weight and chlorophyll a content.
Estimation of S. platensis cell weight (dry basis) Sample containing 50 ml S. platensis suspension was filtered through a Whatman GF/C filter paper of 0.45 µm mesh size and 47 mm diameter, which was dried in an oven for 24 hrs at 70 °C and weighed prior to the filtration. When the sample was being filtered it was washed with 20 ml acidified water (pH = 4) in order to remove insoluble salts. After that the filter papers were put in a glass Petridis and kept in the oven Estimation of S. platensis cell weight (dry basis) Sample containing 50 ml S. platensis suspension was filtered through a Whatman GF/C filter paper of 0.45 µm mesh size and 47 mm diameter, which was dried in an oven for 24 hrs at 70 °C and weighed prior to the filtration. When the sample was being filtered it was washed with 20 ml acidified water (pH = 4) in order to remove insoluble salts. After that the filter papers were put in a glass Petridis and kept in the oven.
Estimation of chlorophyll a S. platensis sample were collected in order to estimate chlorophyll a content. Collected sample (10 ml) was filtered with an electric filtration unit using filter papers (Whatman GF/C of 0.45 µm mesh size and 47 mm diameter). These filtered samples together with filter paper was taken into a test tube and ground with a glass rod and finally mixed with 10 ml of 100% redistilled acetone. Each of the test tubes was wrapped with foil papers to inhibit the contact of light. The wrapped test tubes were kept into a refrigerator overnight. Then the refrigerated samples were homogenized for 2 minutes followed by centrifugation at 4000 rpm for 10 minutes. After centrifugation, the supernatant was isolated and taken for chlorophyll determination. Optical densities of the samples were determined at 664 nm, 647 nm and 630 nm by using UV spectrophotometer (DR 5000). A blank with 100% acetone was run simultaneously. Chlorophyll a was calculated using the following formula (Habib, 1998): Chlorophyll a (mg/L) = 11.85 (OD 664) - 1.54 (OD 647) - 0.08 (OD 630).
Measurement of optical density: Optical density was measured during the time of sampling at 620 nm, by using UV spectrophotometer (DR 5000). The sample of S. platensis grown in different treatments were taken in puvet and placed in a spectrophotometer. Then the OD of the samples was recorded. Determination of Physico-chemical properties of the culture media Water temperature (ºC) of the culture media was measured on the sampling day by a Maximum- Minimum thermometer (push button reset). Light intensity (lux/m2 /s) was measured during sampling days by using a lux-meter (LX-9621). Dissolved oxygen (mg/L) of the culture media was measured by using a dissolved oxygen meter (HQ40d multi) (Figure 5). pH of the culture media was measured by an electric pH meter (sensIONTM+ PH3).
3.7 Statistical analysis One way analysis of variance (ANOVA) in completely randomized design of mean of cell weight, chlorophyll a content, and optical density of S. platensis cultured under different treatments was done to find out whether there was any significant difference among treatment means, while LSD test was used to compare the treatment means (Hofmann, 2008)