Quantifying algal biomass (N.

oculata)

A. DRY WEIGHT 1. For dry weight determination, triplicate culture samples (2 5 ml) were diluted (1:10) with distilled water and filtered through pre-weighed 1.2-mm membrane filters (Sartorius, Goettingen, Germany). Filtered cells were then quickly washed with 25 ml of distilled water and dried to a constant weight at 105C.

2. After growth, biomass was separated from the medium by centrifugation at 5000 rpm for 15 min, using a centrifuge model 42426 (ALC, Milan, Italy). Biomass was dried at 105 C for 48 h, pulverized in a mortar and stored at 20 C for later analysis.

3. The biomass dry weight was obtained after filtration with a 0.2 mm membrane average pore and dried in an oven until constant weight.

4. Microalgal dry weight per liter (g /L) was measured according to the method previously reported (American Public Health Association, 1998). Microalgal cells were collected by centrifugation and washed twice with deionized water. Microalgal pellet was dried at 105 C for 16 h for dry weight measurement (Takagi et al., 2006).

B. Metode Lain

1. Cell density was measured turbidimetrically at 665 nm and correlated to the dry weight by a standard graph. Cell density was measured turbidimetrically at 665 nm and converted from an appropriate calibration curve to dry mass (DW). All cultures were initiated with an OD665 of about 0.1. OD665 was monitored every 24 h until the stationary phase was reached.

2. The biomass concentration is determined by measuring the optical density which is the absorbance value of the culture suspension at 680 nm. The correlation of optical density (OD680) and biomass concentration was previously established by the regression equation Y =0.44X+0.03 (P<0.03); where Y is the biomass concentration measured in g/l and X is the OD680.

3. For cell density evaluation, the number of cells was quantified by using a counting chamber (haemocytometer). The biomass dry weight was obtained after filtration with a 0.2 mm membrane average pore and dried in an oven until constant weight. The optical density of the culture was evaluated by using a spectrophotometer, at wavelength /540 nm.

4. direct microscopic count was performed with Brightline Hemocytometer (BOECO, Hamburg, Germany) and a Nikon Eclipse

TS100 inverted metallurgical microscope (Nikon Corporation, To- kyo, Japan). Cell density (cells mL1) of N. oculata was measured by an Ultrospec 3300 pro UV/Visible spectrophotometer (Amersham Biosciences, Cambridge, UK) at the absorbance of 682 nm (A682). Each sample was diluted to give an absorbance in the range 0.11.0 if optical density was greater than 1.0.

5. The microalgae (N. oculata) concentration was determined daily by optical density measurements at 625 nm by a UVvis spectrophotometer, model Lambda 25 (PerkinElmer, Milan, Italy).