Experimental flasks were maintained under a photon density of 150 ± 50 μmol ·
m−2 · s−1 using a combination of halogen selleck kinase inhibitor and fluorescent lights for a 12:12 light:dark (L:D) photoperiod. To prevent phosphorus and carbon limitation within the cultures, phosphorus and carbon were added as NaH2PO4 and NaHCO3 to maintain concentrations of 10 μM and 3 μM, respectively. The flasks were aerated to ensure water movement and the maintenance of aerobic conditions. The pH of all flasks was monitored daily and maintained between 8.1 and 8.3. To maintain treatment conditions, water was exchanged every second day over the 8 d experiment. After 8 d, algal samples were spun dry in a salad spinner (80 revolutions) to remove excess water before being weighed. The changes in biomass (wet weight)
of algal tissue during the experimental period were measured to estimate growth. Mean relative growth rates (RGR), expressed as mg · g−1 · d−1, were calculated according to the following equation, assuming exponential growth: (2) The samples were then divided into new and older tissue. New tissue was defined as the tissue developed during experimental culture, and older tissue was the initial tissue added to the culture. After being separated, tissue samples were oven-dried for 48 h at 60°C before being ground to a fine powder using a mortar and pestle. These samples were analyzed for phlorotannin, N, and C tissue content using NIRS. All experimental samples were scanned using an NIR spectrophotometer following the same protocol used for the calibration PI3K inhibitor samples. The concentration of phlorotannin, nitrogen, and carbon in the experimental samples was then estimated by the newly developed NIRS calibration equations (described above) using the PREDICT algorithm within the VISION software package. Statistical analyses. All data were analyzed with the statistical package STATISTICA 8 (StatSoft Inc., Tulsa, OK, USA). Cochran’s test was used to test data for homogeneity of variances, and data were transformed
where necessary [log (phlorotannin) and 1/(carbon)2] to meet the assumptions of normality for analyses of variance (ANOVA). medchemexpress Two-way ANOVA was used to determine the effects of ammonium and temperature on growth. To account for the nonindependence of the measurements of new apical and older basal tissue from each thallus, repeated measures ANOVA was used to determine the effects of temperature and ammonium availability on N, C, C:N, and phlorotannin content of Sargassum tissue. Age of tissue was treated as the within effect, and temperature and ammonium as the between effects. NIRS calibration models. PLS regression between laboratory values and NIRS spectra produced calibration equations for phlorotannin, nitrogen, and carbon content in Sargassum tissue with high coefficient R2 values and low standard errors of calibration and cross-validation (Table 1 and Fig. 1).
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