Metadata

Description
Surface water samples were collected monthly in 1-L and 30-mL brown high density polyethylene bottles from all LTER sites and stored on ice. The 1-L samples were refrigerated upon receipt and filtered as soon as possible through pre-combusted 0.7um GF/F glass fiber filters and 0.2um Nylon filters, sequentially.After filtration, a 30 mL portion was saved and stored frozen for total carbohydrate analysis. The remaining sample was acidified with 3 mL of concentrated hydrochloric acid for subsequent analyses. Optical measurements performed using Fluorescence and UV-VIS spectroscopy include Maximum Wavelength, Maximum Intensity, Fluorescence Index, Percent 285, Peak 1, and A-254. Maximum Wavelength is the emission wavelength that gives maximum intensity at a fixed excitation of 313nm and is used to differentiate between carbon sources (terrestrial vs. marine/microbial). Since it measures aromaticity and conjugation, a high Maximum wavelength corresponds to material of terrestrial origin while low values correspond to material of marine or microbial origin. Maximum Intensity is the maximum emission intensity obtained at a fixed excitation wavelength of 313 nm. Fluorescnce Index is a ratio of emission intensities at 450 and 500nm measured at a fixed excitation of 370nm and is also used to measure conjugation in molecules.Percent 285 is obtained by performing a synchronous scan to determine the amount of proteins in a water sample. A synchronous scan is obtained by scanning a range of excitation and emission wavelengths with an offset between them (typically 30 nm). This produces four peaks which can be used to characterize organic matter. Peak 1 is also used for the determination of proteins but it is an absolute intensity at 285nm. A-254 is used as a proxy for aromatics and conjugation. SUVA at 254nm is defined as the Specific UV Absorbance and it is obtained by measuring the UV absorbance at 254nm and normalizing it for the DOC concentration. Milli-Q water is used as a blank and subtracted from all samples. Measurements are corrected for lamp scattering. Filtered and acidified samples were submitted for organic carbon and total nitrogen analysis. Filtered and acidified samples were separated into humic and non-humic fractions by passing through polyvinylpyrrolidinone (PVP). PVP separates dissolved organic matter into humic and non-humic fractions based on hydrogen bonding interactions with vicinal hydroxyl groups as are prevalent in humic substances. The PVP was washed thoroughly with acid and base prior to use. The PVP powder was washed with acid solution by gently shaking in dilute acid solution for roughly 24 hours. After settling, the acid solution was decanted. Then the PVP was rinsed with water and decanted three times. The PVP was then similarly washed with basic solution and afterwards rinsed with water as above. The sequence of acid washing, rinsing, base washing, and rinsing was performed a total of three times. After washing, the PVP is stored in Milli-Q water and from this point on the PVP should not be allowed to dry. A plug of combusted glass wool is added to the columns to prevent the passage of PVP through the columns. Then a slurry of PVP is placed in 250 mL Kontes glass separation columns. Next, a height of around 5 to 7 cm of PVP is added to the columns. Once in the columns, the PVP is further washed with around 30 mL of 0.1 N sodium hydroxide (NaOH), around 30 mL of Milli-Q water, and around 30 mL of 0.1N hydrochloric acid (HCl). The separation is then carried out by passing 250 mL of filtered and acidified sample water through the column at a rate no faster than approximately 1 drop per second. The first 100 mL through the column is discarded and then around 30 mL is collected for subsequent protein analysis and around 20 mL is collected for total organic carbon (TOC). The fraction that passes through the column is the non-humic fraction. At this point, the humic fraction is retained on the column. The humic fraction is eluted from the column using around 10 mL portions of 0.01 N NaOH and is collected into 50 mL volumetric flasks. Within about 5 mL of the 50 mL mark, 1 mL of 2N HCl is added to the flask to neutralize the base, then the final volume is adjusted to 50 mL. The humic fraction is then submitted for TOC and TN. Total carbohydrates were measured on filtered (0.2 um) water samples that have been frozen prior to analysis. The samples were analyzed according to the TPTZ method (Myklestad et al., 1997). This method consists of sulfuric acid hydrolysis to convert carbohydrates to reducing sugars with subsequent color formation based on the reduction of iron.

Citation
Myklestad, S M 1997. Asensitive and rapid method for analysis of dissolved mono- and polysaccharides in seawater. Marine Chemistry, 56: 279-286.

Instrumentation
Whatman 0.7um glass fiber filters Millipore 0.2um hydrophilic membranes Nalgene 1-L and 30-mL brown polyethylene bottles Jobin Yvon Horiba Fluoromax 3 Shimadzu 2101PC Spectrophotometer Carlo Erba NA 1500 Nitrogen/Carbon Analyzer