Metadata

Description
The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. The freshwater marsh site (TS/PH2) and mangrove site (TS/PH6) are sampled semi-continuously for TN and TP as part of the FCE LTER Program (see http://fcelter.fiu.edu). The Florida Bay site (TS/PH9) was sampled monthly as part of the SERC Water Quality Monitoring Network (http://serc.fiu.edu/wqmnetwork/). Water samples were collected on August 4, 2003 from three sites in the Everglades hydroscape. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The three sampling sites selected for this study were distributed along a transect extending from the freshwater marsh of Taylor Slough through the mangrove fringe and into Florida Bay. Water quality parameters such as total nitrogen (TN), dissolved organic nitrogen (DON), dissolved organic carbon (DOC) and other basic water quality parameters were determined in monthly grab samples taken from all sites. Four experiments were initiated to examine the physical, chemical, biological, and combined physical/biological effects on DOM and DON from the Everglades. Water samples were collected at 10 cm depth using acid washed, autoclaved distilled water (ADW) rinsed 8 l brown Nalgene bottles. Sample bottles were rinsed three times with sample water prior to collection. A 1 l brown Nalgene bottle was collected from the TS/PH9 site for a bacteria inoculum. The first experiment was designed to test the effect of salinity on the flocculation of DOM. The idea was to quantify the amount of DOM from the Everglades that flocculates and/or precipitates out of solution upon transport to the higher salinity waters of Florida Bay. Low salinity water samples (200 ml) from TS/PH 2 and TS/PH 6 were placed in individual 250 ml Nalgene brown bottles to which varying amounts of artificial sea salt crystals were added to produce salinity levels of 0, 5, 15, 25, and 35 ppm. The bottles were placed on a shaker table at 200 rpm for 1 h and then stood for 24 h at 22 degrees C. Dissolved organic carbon (DOC) concentration was measured after filtration through 0.2.um Durapore membrane filters to estimate flocculation. Filtered samples were also analyzed for fluorescence properties. The experiments were conducted in duplicate.Subsamples (150 ml) of filtered (less than 0.2 um) water (TS/PH 2, 6a, and 9) were irradiated using a solar simulator at 765 W m2 (corresponding to solar noon at mid-latitude). After irradiation of designed period (0, 0.5, 1, 2, 4 and 7 days of continuous irradiation; 24 hour day periods of simulated sunlight), samples were filtered (less than 0.2um) and analyzed for DOC concentration and UV absorption and fluorescence emission properties. The experiment was conducted in duplicate. Upon return to the laboratory, water samples were immediately passed through a GF/F filter, then through a sterile 0.2 um cartridge filter, and stored in brown Nalgene sample bottles. The 1 l water sample collected from TS/PH9 was filtered through a sterile 0.8 um filter and stored at 4 degrees C to use as inoculum. 2 l of the 0.2 um filtered water were placed into each of the twelve, 2.5 l polycarbonate bottles that had been acid washed and ADW rinsed. The salinity of each of the bottles was adjusted to that of TS/PH9 (34.5) with NaCl. Nutrient additions were made to effect final concentrations of 5 uM P as H3PO4 and 100 uM C as glucose. The P and C additions assured that N limiting conditions were maintained in the bottles (Stephanauskas et al., 1999). A 10 ml bacterial inoculum of GF/F filtered water from TS/PH9 was added at the initiation of the experiment. Duplicate bottles were covered with aluminum tape and incubated in the dark at 30 degrees C. DON bioavailability was quantified using 16 day incubations (Servais et al., 1989; Boyer & Groffman, 1996) during which we measured loss of the DON fraction and any change in nutrients and bacterial N. Bottles were sampled at 0, 2, 4, 8, and 16 days by collecting a 250 ml volume of water from each of the bottles. Unfiltered samples were analyzed for total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), alkaline phosphatase activity (APA), leucine aminopepdidase (LAP), bacterial numbers (BACT), and bacterial production (BP). Subsamples were filtered through Whatman GF/F filters prior to analysis for nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), soluble reactive phosphorus (SRP), maximum fluorescence intensity (Fmax), wavelength of maximum fluorescence intensity (fmax), fluorescence index (FI), and synchronous fluorescence (Peak I).Fluorescence spectra were measured with a Jobin-Yvon-Horiba Spex Fluoromax-3 fluorometer equipped with a 150W continuous output Xenon arc lamp. Two fluorescence indices were obtained by single emission scan measurements at excitation wavelengths of 313 nm and 370 nm. For each scan, fluorescence intensity was measured at emission wavelengths ranging from 330 to 500 nm and from 385 to 550 nm, respectively, with a 5 nm bandpass for excitation and emission wavelengths. From the 313 nm scan, the maximum intensity (Fmax) and maximum wavelength (Fmax) were determined (De Souza Sierra et al. 1994, 1997). From the 370 nm scan, a fluorescence index (FI) was calculated (McKnight et al., 2001). These two indices have been used to distinguish the source of DOM (marine (microbial) vs. terrestrial (higher plant)). Furthermore, synchronous excitation-emission fluorescence scan was obtained at a constant offset value between excitation and emission wavelengths. All spectra were recorded at an offset value of 30 nm. From the synchronous scan, the fluorescence peak intensity at an excitation wavelength around 280 nm was measured as a proxy for proteinaceous materials concentration (Lu et al., 2003; Jaffe et al., 2004). The inner filter effect of measured fluorescence spectra were collected using UV-visible (UV-Vis) absorption spectra of each sample (McKnight et al. 2001). The UV-Vis absorption spectra were collected using a Shimadzu UV-2102PC spectrophotometer. Absorption scans were collected between 250 and 800 nm in a 1cm quartz cuvette using Milli-Q water as the blank. Two optical parameters were determined: (1) the specific UV absorbance at 254 nm (SUVA254) and (2) the slope (S) parameter. The SUVA254 parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m-1) divided by the DOC concentration measured in mg L-1 (Weishaar et al., 2003). The slope of the UV-Vis spectrum, the S parameter was obtained (Battin, 1998 and references therein). Unfiltered water samples were analyzed for total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), alkaline phosphatase activity (APA), and turbidity (NTU). TOC was measured by direct injection onto hot platinum catalyst in a Shimadzu TOC-5000 after first acidifying to pH;less than 2 and purging with CO2-free air. TN was measured using an ANTEK 7000N Nitrogen Analyzer using O2 as carrier gas instead of argon to promote complete recovery of the nitrogen in the water samples (Frankovich & Jones, 1998). TP was determined using a dry ashing, acid hydrolysis technique. Filtrates were analyzed for soluble reactive phosphorus (SRP), nitrate + nitrite (NOX-), nitrite (NO2-), ammonium (NH4+), and silicate (Si(OH)4) by flow injection analysis (Alpkem model RFA 300). TON was determined by difference of TN and DIN. We used an alkaline phosphatase activity assay (APA) to determine the production of extracellular enzymes to mineralize phosphate from organic material (Hashimoto et al., 1985). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding o-methylfluorescein phosphate. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 430 nm, emission = 507 nm). The increase in fluorescence over the incubation period determined APA (uM h-1). We used a similar method to determine the leucine aminopeptidase activity (LAP) to estimate production of extracelluar enzymes which mineralize nitrogen from organic material (Hoppe 1983). Duplicate unfiltered 3 ml samples were incubated for 2 hours after adding L-leucine-7-amido-4-methylcourmarine. Initial and 2 hour readings were measured using a Gilford Fluoro IV Spectrophotometer (excitation = 380 nm, emission = 440 nm) and used the change over the incubation period to determine LAP activity (uM h-1). Bacterial counts (BACT) were determined using epifluorescence microscopy with DAPI staining technique (Coleman, 1980; Porter & Feig, 1980). Samples were collected and Formalin buffered with phosphate solution to a final concentration of 2%. Aliquots were incubated at a final concentration of 25 ug ml-1 DAPI in a filtration tower for 20 minutes prior to filtration onto a 0.2 um black polycarbonate filter. The filter was then mounted onto a slide with low fluorescence immersion oil and examined under a 100W Hg bulb. Bacterial enumeration was performed by counting 10 sampling fields of a known size per slide, with a minimum of 300 cells per slide counted. Bacteria production (BP) was determined using 3H-thymidine incorporation incubations (Bell, 1993) using triplicates of each sample and a 4% final concentration formalin blank for each. With each 3H-thymidine incubation experiment we ran a blank sample for specific activity of the 3H-thymidine. Filters were placed in scintillation vials to which 10 ml of Ultima Gold fluor was added and counted in a Beckman liquid scintillation counter using external channels ratio correction. Some parameters were not measured directly, but were calculated by difference. Nitrate (NO3-) was calculated as NOX- - NO2-. Dissolved inorganic nitrogen (DIN) was calculated as NOX- + NH4+. Total organic nitrogen (TON) was defined as TN - DIN. Concentrations for all of these water quality variables are reported in uM, except where noted. All N:P ratios discussed are calculated on a molar basis. The total dissolved nitrogen pool (TDN) in a sample is made up of DIN and DON. At 0, 4, and 16 days, bacteria were classified as either spheres or rods and their diameters and lengths measured using a Whippled grid with smaller division of 2 nm. We assumed that half the bacteria were spheres, the other half rods. Bacterial cell volume (BV, um3) was calculated using standard equations. The C:V used was for bacteria grown under N limitation and should therefore be applicable to this experiment.

References:
Battin, T J 1998. Dissolved organic materials and its optical properties in a blackwater tributary of the upper Orinoco River, Venezuela. Organic Geochemistry, 28: 561-569.

Bell, R T 1993. Estimating production of heterotrophic bacterioplankton via incorporation of tritated thymidine. Lewis Publishers, Boca Raton, Florida, 8 pp.

Boyer, J N 1996. Bioavailability of water extractable organic carbon fractions in forest and agricultural soil profiles. Soil Biology and Biochemistry, 28: 783-790.

Coleman, A W 1980. Enhanced detection of bacteria in natural environments by fluorochrome staining of DNA. Limnology and Oceanography, 25: 948-951 .

De Souza Sierra, M M 1994. Fluorescence spectroscopy of coastal and marine waters. Marine Chemistry, 47: 127-144.

De Souza Sierra, M M 1997. Spectral identification and behavior of dissolved organic fluorescent materials during estuarine mixing processes. Marine Chemistry, 58: 51-58.

Frankovich, T A 1998. A rapid, precise, and sensitive method for the determination of total nitrogen in natural waters. Marine Chemistry, 60: 227-234.

Hashimoto, S K 1985. Relationship between alkaline phosphatase activity and orthophosphate in the present Tokyo Bay. Journal of Environmental Science and Health, Part A 20A: 781-908.

Hoppe, H G 1983. Significance of exoenxymatic activities in the ecology of brackish water: measurements by means of methyllumbeliferyl-substrates. Marine Ecology Progress Series, 11: 299-308.

Jaffe, R 2004. Source characterization of dissolved organic matter in a subtropical mangrove-dominated estuary by fluorescence analysis. Marine Chemistry, 84: 195-210.

Lu, X 2003. Molecular characterization of dissolved organic matter in freshwater wetlands of the Florida Everglades. Water Research, 37: 2599-2606.

McKnight, D M 2001. Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity. Limnology and Oceanography, 46: 38-48.

Porter, K G 1980. The use of DAPI for identifying and counting aquatic microflora. Limnology and Oceanography, 25: 943-948.

Servais, P 1989. Simple method for determination of biodegradable dissolved organic carbon in water. Applied Environmental Microbiology, 55: 2732-2734.

Stepanauskas, R 1999. Bioavailability of wetland-derived DON to freshwater and marine bacterioplankton. Limnology and Oceanography, 44: 1477-1485.

Weishaar, J L 2003. Evaluation of specific ultraviolet absorbance as an indicator of the chemical composition and reactivity of dissolved organic carbon. Environmental Science and Technology, 37: 4702-4708.

Instrumentation
Whatman 0.7um glass fiber filers, Millipore 0.2um hydrophilic membranes (Millipore Co.), Nalgene 1-L, 8-Land 250-mL brown polyethylene bottles (Nalge Co.), Jobin Yvon Horiba Fluoromax 3, Shimadzu 2101PC Spectrophotometer, Solar simulator (Suntest XLS+, ATLAS Material Testing Technology LLC), Shimadzu TOC 5000 Analyzer, ANTEK 7000N Nitrogen Analyzer, Gilford Fluoro IV Spectrophotometer