Quantifying Algal Growth and Nutrient Dynamics

Lugol’s iodine preserved cells were enumerated using a Beckman Coulter Multisizer™ 3 Coulter Counter® with a 50 µm aperture which allowed cell densities to be quantified with a relative standard deviation of 3%. Cell densities of selected samples were verified microscopically with a hemacytometer. Growth rates were calculated for each day of the experiment based upon changes in cell abundance according to the equation µ = Ln(N₂/N₁)/(t₂–t₁) where N₁ and N₂ equal the biomass at time 1 (t₁) and time 2 (t₂) respectively [54] . Nitrate was analyzed by reducing the nitrate to nitrite using spongy cadmium as per Jones [55] . Ammonium and phosphate were analyzed using techniques modified from Parsons et al[56] . Total dissolved N and P were analyzed using persulfate digestion techniques from Valderrama [57] . Urea was analyzed according to Price and Harrison [58] . These nutrient analyses provided 100±10% recovery of standard reference material (SPEX CertiPrep™) for nitrate, ammonium, phosphate, total dissolved N, and total dissolved P. Whole water samples were analyzed for the hepatoxin microcystin by first freezing samples at −80°C for 24 h and then lysing the cells using an Abraxis QuikLyse™ Cell Lysis kit for Microcystins/Nodularins ELISA Microtiter Plate according to the manufacturer’s instructions. Lysed samples were then analyzed with a colorimetric immunoassay using an Abraxis Microcystins/Nodularins (ADDA) ELISA Kit according to the manufacturer’s instructions [59] (link). This method provided an analytical precision of ±2% and a 96±2% recovery of spiked samples. Bulk alkaline phosphatase activity was measured for each replicate experimental sample on a Turner Designs TD-700 fluorometer (EM filter of 410–600 nm and EX filter of 300–400 nm) using 4-Methylumbelliferone phosphate (250-µM concentration) as the substrate [60] . Alkaline phosphatase activity measured by this assay has been shown to be significantly correlated with the expression of the gene encoding for alkaline phosphatase (phoX) in Microcystis aeruginosa LE-3 (p<0.005) [34] (link) and provided an analytical precision of ±4%. Maximum quantum efficiency of photosystem II (PSII) was estimated from in vivo (F_i) and DCMU (3,4-dichlorophenyl-1,1-dimethylurea)-enhanced in vivo fluorescence (F_m) of each replicate experimental sample on a Turner Designs TD-700 fluorometer (EM filter of >665 nm and EX filter of 340–500 nm). All readings were blank corrected using BG-11 media. DCMU blocks electron transfer between PSII and PSI and yields maximal fluorescence and previous studies have demonstrated that F_v/F_m can be a sensitive diagnostic of nutrient limitation, reaching a maximal value of ∼ 0.65 under nutrient replete conditions, and decreasing to less than half of that under nutrient limitation [61] (link).