MATERIALS AND METHODS

The epiphytic (collected from Eicchornia sp. and Hydrilla sp.) diatom samples were collected and stored in non-reactive plastic bottles using the procedure followed by (Kelly et al., 2008)

ISOLATION: ‘The collected samples were then isolated by using modified Pasteur pipette (see Andersen and Kawachi, 2005) under the inverted microscope (40X magnification). The isolated cells were grown in WCg medium (Wright 1964, Guillard and Lorenzen 1972, Guillard 1975; Guillard, unpublished) with 16:8 light/dark photoperiod for a period of 7 days. Diatom identification was carried out by observing the isolated sample under the light microscope (40X magnification). The culture conditions were maintained at 26-270C with a light intensity of 28-32 µmol m-2 s-1.

CULTURE CONDITIONS: The different nitrogen sources used to check the response of sources on Nitzschia sp. cell as well as lipid quantity were sodium nitrate and urea.To study the effect of both the stresses, experiments were carried out in 250 ml Erlenmeyer flasks each containing 100 ml of appropriate medium. Control cultures were also maintained under similar culture conditions. The experiments were set up for 7 days. The cell count and extraction of total lipid for the two microalgae were determined every day. All the experiments were carried out in triplicate.

CELL COUNT ESTIMATION: Growth rate was determined using cell counting method throughout the study period. 1 ml of the culture was taken onto a slide and counted for growth rate measurement. Diatom frustules devoid of chloroplasts were not included during the counts. The growth rate calculation is based on the fact that during exponential growth, the rate of increase in cells/unit time is proportional to the number of cells present in the culture (Anderson, 2005). The specific growth rate (μ) is defined as the increase in cell density/unit time (Garcia et al., 2007) and formulated.

LIPID ANALYSIS: Lipid was extracted using Bligh and Dyer, 1959 method. 25 ml of the sample was sonicated for 1 hour at room temperature to disrupt the cell wall. This was done with the addition of the chloroform: methanol (2:1 v/v) solvent. Chloroform layer was extracted since the lipid from the algae dissolves in chloroform layer. This layer was further evaporated using rotary evaporator to obtain lipids. The total lipid content was determined gravimetrically.