DOC and TDN measurements. Samples were acidified to pH 2 using concentrated HCl (Sigma Aldrich 32 %, pura) and analyzed for dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) concentrations using a Shimadzu TOC-V. Ultrapure water was used as a DOC/TDN blank and potassium hydrogen phthalate and potassium nitrate were used for DOC and TDN standards, respectively.
Optical properties. For fluorescence and absorbance measurements, samples were transferred to 1 cm quartz fluorescence cuvette and analyzed using a Horiba Aqualog spectrofluorometer. For all time points collected during exudation experiments, raw absorbance spectra (A(λ)) were collected using a Horiba Aqualog at 3 nm intervals between 240 and 600 nm. A(λ) were then converted to Naperian absorption coefficient spectra (a(λ)) using the equation below.
a(λ) = 2.303×A(λ)/L (1)
where L (m) is the pathlength of the spectrophotometer cell (0.01 m).
Spectral slope coefficients from 300 to 500 nm (S300-500, nm-1) were determined by fitting a(λ) spectra to the equation
a(λ) = a300*EXP(-S300-500(λ – 300)) (2)
using a nonlinear curve fitting routine in Matlab 2015a®. A(λ) at various wavelengths was normalized to DOC concentration and the pathlength of the cell to determine changes in the carbon-normalized A(λ) during incubation experiments.
Anorm(λ) = A(λ)/([DOC]×L) (3)
For instance, specific UV absorbance at 254 nm (SUVA254; m-1 L mg-1), or A(254) normalized to pathlength (m) and DOC concentration (mg L-1).
Fluorescence was recorded from excitation wavelengths 240 to 600 at 3 nm intervals. Emission was recorded from ~243 to 297 nm at fixed ~3.3 nm intervals to created excitation-emission matrix (EEM) spectra. Integration time = 2s. Ultrapure water used as the fluorescence blank and was subtracted from all EEM spectra. Blank corrected EEM spectra were corrected for any inner filter effects using the Aqualog software. All spectra were normalized to the water Raman scattering signal so that all fluorescence data is reported in Raman units (RU). Rayleigh scattering signals were removed from EEM spectra using the Matlab ® (version 2015a) routine outlined previously (Zepp et al. 2004 doi: 10.1016/j.marchem.2004.02.006).
The following additional parameters were also calculated:
Apeak: intensity and location of maximum in the "A" region (ex/em <260 nm/400 – 460 nm) in (intensity x ex. location x em. location) (Coble et al. 1996)
Cpeak: intensity and location of maximum in the "C" region (ex/em 320 – 360 nm/420 – 460 nm) in (intensity x ex. location x em. location) (Coble et al. 1996)
Tpeak: intensity and location of maximum in the "T" region (ex/em <260 nm /320 – 350 nm) in (intensity x ex. location x em. location) (Coble et al. 1996)
These parameters (A, C, T peaks) were normalized to tank volume and Sargassum biomass for every time point. Therefore they are reported in RUxL/g.