Metadata

Description
Ten grams of biomass sample (20 g of periphyton) were placed into 250-ml Nalgene brown bottles. The bottles were filled with 200-250 ml of Milli-Q water and incubated in the dark. Milli-Q water was used as the extractant to avoid (1) post-leaching processing of DOM such as complexation/precipitation and oxidation/polymerization, which affect the quantitative analyses of leachate, and (2) contamination of natural water UDOM in biomass leached UDOM. Furthermore, polyphenols especially leached from mangrove leaves are unstable under high salinity conditions (unpublished data); we therefore used Milli-Q water to investigate the potential leaching amount of DOC from biomass. To unify the experimental conditions, seagrass was also immersed into Milli-Q water. We added 1 mg ml-1 of NaN3 as a bacteriostat to half of the bottles (referred as to w/ NaN3, and w/o NaN3 treatments, respectively) to test the role of microbial activity on the leaching rates and composition of leachate. The incubations ran for 36 days, and every three days, the water was decanted from each bottle and replaced with fresh Milli-Q water (w/ or w/o NaN3). The decanted samples were first filtered through pre-combusted (470 degrees C for 4h) GF-F glass fiber filters (nominal pore size, 0.7 um), and then through a 0.22 um Durapore membrane filter. Water samples decanted from the periphyton treatments were centrifuged to remove suspended solids at 3,000 rpm for 10 min before filtration. The precipitation obtained by centrifugation was returned to the sample bottle. The filtered water samples were stored at 4 degrees C for no more than 1 week before analyses. The DOC content of the water samples was analyzed using a total organic carbon analyzer after acidifying the sample (pH less than 2) with HCl and purging with N2 gas for 5 min. Although some mangrove leachate produced a fine particulate upon acidification, probably due to aggregation of polyphenols, they were uniformly dispersed through purging with N2 gas, and the average of standard deviation (SD) of measurements was less than 3%. The sugar content of the water samples was analyzed colorimetrically using the Phenol-Sulfuric Acid Method (Dubois et al. 1956; Liu et al. 1973). Briefly, 1 ml of water sample and 1 ml of 5% phenol aqueous solution (w/v) was pipetted into a test tube, and then 5 ml of concentrated sulfuric acid was added. After mixing vigorously with a vortex mixer, the solution was shaken on a reciprocating shaker for 30 min and the absorbance at 490 nm was measured using a UV-Vis scanning spectrophotometer. Glucose solutions, of concentrations ranging from 4 to 40 mg C L-1, were used as calibration standards. The detection limit of this method (absorption = 0.01) was around 0.4 mg C L-1 with a 1-cm pathlength quartz cuvette. Total phenol content was measured colorimetrically using Folin-Denis Method (Waterman & Mole 1994). Briefly, a 0.2-3.4 ml aliquot of water sample was pipetted into a test tube and the volume was increased to 3.4 ml by adding Milli-Q water if necessary. Then 0.2 ml of Folin-Denis reagent and 0.4 ml of saturated sodium carbonate solution were added in sequence. After standing for 30 min, the absorbance at 760 nm was measured on a Shimadzu UV-2101PC UV-visible spectrophotometer. Tannic acid solutions, of concentrations ranging from 1 to 5 mg C L-1, were used as calibration standards. The detection limit of this method (absorbance = 0.01) was around 0.25 mg C L-1 with a 1-cm pathlength quartz cuvette.

References:
Dubois, M 1956-01-01. Colorimetric method for determination of sugar and related substances.. Analytical Chemistry, 28: 350-356.

Liu, D 1973-01-01. Determination of carbohydrates in lake sediment by a modified phenol-sulfuric acid method.. Water Research, 7: 741-746.

Waterman, P G 1994-01-01. Analysis of phenolic plant metabolites.. Blackwell Scientific, Oxford, London, 238 pp.

Instrumentation
Whatman GF/F glass fiber filters, Nalgene polyethylene bottles, Shimadzu 2101PC Spectrophotometer, Carlo Erba NA 1500 Nitrogen/Carbon Analyzer (Carlo Erba, Milan, Italy).