Samples of different tissue types were also integrated into the collection regime, including samples of exclusively young tissue, old tissue, reproductive tissue, blades, and stipes from different individuals. To create NIRS calibration equations

for nitrogen (N) and carbon (C), 75 samples were collected for total nitrogen and total carbon analysis from different Sargassum individuals. To capture a wide a range of total nitrogen and carbon variation found in Sargassum, tissue was collected over a 6-month period (November 2007–April 2008). Samples collected from the field were augmented with samples from laboratory and field experiments where nutrient availability was enriched. Sample preparation. 

All samples from each calibration set (phlorotannin, N, and C) were freeze-dried in the condition they were collected MK-1775 nmr and, after 48 h, removed from the freeze dryer and stored in sealed containers at room temperature. Samples were ground to a fine powder using a ball grinder and returned to sealed containers until further analysis. In addition to the phlorotannin calibration samples from the field, a set of “spiked” samples was created to extend the range of the calibration equation to encompass higher phlorotannin concentrations found in winter months. This sample set was created from one large sample, which had been ground to a homogenous powder. The sample was split into 11 subsamples, and phloroglucinol (Sigma-Aldrich Pty. Ltd., Sydney, Australia), the base unit of phlorotannin, was added to each Copanlisib cell line of these subsamples Etofibrate to create a range of different dry

weight percentages of phloroglucinol. The percentages of phloroglucinol per dry weight of tissue of these subsamples were 1, 2, 3, 4, 6, 7, 8, 10, 12, 14, and 16%.These spiked samples were vigorously shaken to ensure that the added phloroglucinol was evenly mixed within each sample. The spiked samples were stored in sealed containers and added to the main phlorotannin calibration set (n = 96) for NIRS scanning and traditional phlorotannin analysis. NIRS scanning.  To obtain spectra for each calibration sample, samples were scanned using a near infrared reflectance spectrophotometer (Model 6500; NIR Systems, Silver Springs, MD, USA) (Horn et al. 1999, Andre and Lawler 2003). Spectral data were generated by flashing each sample with monochromatic light at 2 nm intervals across a range from 1,100 to 2,500 nm. Reflectance across this range was measured and stored using VISION software (Version 1.0; FOSS NIRSystems, Laurel, MD, USA). The software converted reflectance (R) readings to absorbance (A) values using the equation, (1) Absorbance values were used for all analyses and calibration development. Chemical analyses.