NOAA - Great Lakes Environmental Research Laboratory
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Trophic state index (TSI) was calculated from chlorophyll a concentrations (µg L–1) according to Carlson (1977). Chlorophyll a concentrations were determined using the Welschmeyer method (see Twiss and Wilhelm data). The TSI was calculated from chlorophyll a concentration using the formula TSI (chl a) = 10 (6–((2.04–0.68 LN (chl a))/LN (2)).
Bacterial Abundance (BA) (cells ml–1) was obtained from triplicate formalin–fixed samples stored shipboard at 4°C. Cells were collected on a 0.2 µm black polycarbonate filters (Osmonics) and stained using 4’,6’–– diamidino–2–phenylindole (DAPI) following the method described by Porter and Feig (1980). Cells were observed under epifluorescence microscopy using a Zeiss Axioskop with a DAPI fluorescence emission filter. Cells were counted for 10 fields and 200+ total cells.
Bacterial Cellular Biovolume (CBV) (µm3 cell–1). Over 200 bacterial cells were photographed with an R–T Spot camera (Diagnostic Instruments, Inc.) and sized using Metamorph Image Analysis software (Universal Imaging Corp.) with halo correction to determine cellular biovolume. Size was calibrated by using fluorescent polystyrene spheres (PolySciences, Inc.) ranging in size from 0.1 – 5.0 µm in diameter. Cells were identified as cylinders + spheres (for bacillus shapes) and spheres (for coccus shapes). CBV was calculated from the following formula: ?*(w/2)^2*(l–w)+4/3*?*(w/2)^3.
Total bacterial biovolume (TBV) (µm3 ml–1)was calculated as the product of cell number and cellular biovolume.
Bacterial productivity (BP) (µg C ml–1 hr–1) was estimated by 3H–leucine incorporation into bacterial proteins, according to the method described by Jørgensen (1992). 3H–leucine (Perkin Elmer NET 135H ) was added to triplicate samples plus a formalin–fixed control in 100 µL portions of a preparation containing 50 µCi. Following a 60 minute incubation period at ambient temperature, samples were filtered onto 0.2 µm cellulosic filters (Osmonics). Filters were frozen aboard the ship at –20 °C for a week and processed upon return to the laboratory. The protein fraction was precipitated with 5% trichloracetic acid (Sigma 490–10). The soluble nucleic acid portion was collected in disposable test tubes beneath the filtration manifold. Filters with protein were dried for several hours. Scintilene (Fisher SX2–4) and Scintiverse (SX2–17) were added to protein and nucleic acid fractions, respectively. The radioactivity of 3H–leucine (dpm) was measured using a calibrated Beckman 6500 liquid scintillation counter.
Bacterial Respiration (BR) (µg C ml–1 hr–1) was estimated from a five day change in oxygen concentration in biochemical oxygen demand (BOD) bottles. Algal and grazing particles were removed from whole water using a 1.0 µm filtration capsule (Whatman). The 1.0 µm filtered water was placed into six 300 ml BOD bottles. The initial oxygen concentration for the first three bottles was determined using Winkler titration with azide modification (APHA 1995). The remaining three bottles were incubated at ambient temperature and in the dark (covered with aluminum foil) for five days (120 hours). The final oxygen concentration was determined from the remaining three bottles. The difference between initial and final oxygen concentration was converted to moles of carbon dioxide respired using a respiratory coefficient of 0.82 ( Søndergaard et al. 1995)
Bacterial growth Efficiency (BGE) (%) was calculated as the ratio of productivity to assimilation using the formula described by del Giorgio and Cole (2000). BGE = BP (BP+BR)–1.
Particulate Phosphorus: Triplicate portions of lake water were filtered through 1.0 µm polycarbonate filters to capture algal particles (algal particulate phosphorus or APP). Triplicate portions of the remaining filtrate were then filtered through 0.2 µm polycarbonate filters to trap bacterial particles (bacterial particulate phosphorus or BPP). The remaining filtrate represented the dissolved fraction or total soluble phosphorus (TSP). Filters and filtrate were frozen in the shipboard freezer, transported via cooler, and stored in the laboratory freezer until analysis. Upon return to the laboratory, the phosphorus content of the samples was determined using the method of Murphy and Riley (1962) with persulfate digestion. The absorbances of the samples at 885 nm were read in a Spectro Genesys 5 spectrophotometer. Triplicate standards and triplicate reagent blanks were run in parallel throughout the analysis. Total phosphorus (TP) was calculated as the sum: APP + BPP + TSP.
Bacterial phosphorus quota (P quota) (nM cell–1) was calculated from the bacterial particulate phosphorus (BPP) / bacterial abundance, BA
Labile dissolved organic carbon (LDOC) concentration (µM) was estimated using the method of Søndergaard et al. (1995). Whole water (950 mL) filtered through 0.2 µm filtration capsule (Whatman) to remove bacteria, algae, and bacterivores. This filtrate was inoculated with 50 mL (5%) of water passed through a 1.0 µm filtration capsule (Whatman) to remove algae and bacterivores. This inoculum contained about 95 percent of the bacteria found in whole water samples. Samples were amended with NH4Cl and Na2HPO4 were added to give final concentrations of 5.6 µM and 1.4 µM respectively, to ensure that bacterial cells were not growth limited by N and P and to maximize carbon utilization. The mixture was added to six BOD bottles. Oxygen concentrations were determined initially on three BOD bottles to estimate the amount of oxygen respired. The remaining three BOD bottles were incubated in the dark at ambient temperature for approximately 30 days. Oxygen concentrations were determined using the Winkler method with the alkaline azide modification as described in APHA (1995). Oxygen consumed was converted to moles of carbon dioxide released by bacteria, assuming a respiratory quotient of 0.82 (Søndergaard et al. 1995).
Bacterial growth was estimated by measuring the increase in total bacterial biovolume (bacterial abundance (cells ml–1) * bacterial cellular biovolume (µm3 cell–1)) over the 30 day interval. To determine bacterial cellular biovolume, cells were stained with 4’,6’–diamidino–2–phenylindole (DAPI) using the method of Porter and Feig (1980), photographed with an RT Spot camera (Diagnostics, Inc.) attached to a Zeiss Akioskop, and sized (initially and at 30 days) using Metamorph Image analysis software (Universal Imaging Corp.) with halo correction. Cell size was calibrated by using fluorescent polystyrene spheres (PolySciences, Inc.) ranging in size from 0.1 – 5.0 µm in diameter.
LDOC was determined as the sum of the carbon respired plus the increase in total bacterial carbon over 30 days. We assumed that all available LDOC had been consumed by bacteria during the 30 day incubation period.
Total Dissolved Organic Carbon (TDOC) (µM): Whole water was filtered through pre–combusted GF/F filters and placed into duplicate pre– combusted glass ampules. The ampules were sealed immediately and frozen in the shipboard freezer (–5 °C) for further analysis. Samples were analyzed, with parallel standard, for TDOC using a Shimadzu Total Organic Carbon Analyzer TOC–VCPN.
Tracey T. Meilander, Ph.D. ()
Curriculum Coordinator
Center for Public Health Preparedness
Department of Biological Sciences
Kent State University
Robert T. Heath ()
Dept. Biol. Sciences
Kent State University
