Estimations of microbial mortality in Lake Erie

Four stations (23, 84, and 357; Fig. 1) were occupied in Lake Erie during early August 2005 on RV the Lake Guardian and during late August during the the MELEE X (Microbial Ecology of the Lake Erie Ecosystem) cruise on the CCGS ‘Limnos’. Data from both ship’s automated water column profilers were used to collect temperature and oxygen profiles to model the water column structure. Dissolved oxygen measurements were subsequently confirmed with a hand-held YSI BOD probe 58. Water from stations was collected from within (5 to 10 m) and below (12 – 18 m) epilimnion via a rosette cast. Size-fractionated chl a was determined from parallel triplicate filtration of samples collected on 0.2, 2 , and 20 µm pore-size polycarbonate filters (47 mm diameter; Millipore), after extraction (ca. 24 h, -20°C) in 90% acetone. Chl a retained on the different size class filters was quantified with a Turner designs TD-700 fluorometer using the non-acidification protocol (Welschmeyer 1994). Phytoplankton communities and heterotrophic bacteria were analyzed by flow cytometry. Samples were preserved in 1% formalin and flash frozen in liquid N until analysis. Densities of heterotrophic bacteria (stained with Hoechst-dye), phycoerthrin-containing picocyanobacteria and photosynthetic picoeukaryotes were determined using a FACScaliber (BD®) flow cytometer to determine fluorescence patterns and particle size from forward angle light scatter for samples (Jochem et al 20??). Whole water samples (40 mL) were collected and preserved with glutaraldehyde (2 % v/v, final), and stored in the dark in sterile polypropylene tubes at 4°C.

Duplicate microplankton samples were analyzed according to Hasle (1978) to identify and quantify the major taxonomic categories of microzooplankton and phytoplankton present. Samples (180 ml) were preserved with acid Lugol’s solution (final concentration 10%) and counted using an inverted microscope. Organisms were identified to the major taxonomic category and abundance was established. A minimum of 200 organisms or 100 grids (microplankton enumerations) were counted per sample (Omori and Ikeda 1984).

The microzooplankton grazing/seawater dilution experiments were performed to determine the impact of zooplankton grazing on algal mortality. These experiments involved four treatments (100%, 70%, 40%, and 15% whole lake water) each performed in triplicate 1.2 L bottles. Whole water collected for microzooplankton grazing experiments was gravity filtered in-line through acid-cleaned 200 µm screening (Nitex) whereas filtered water was gravity filtered through a 0.2 µm filter capsules (Pall). An additional bottle was filled with 0.2 µm filtered seawater to verify that the 0.2 µm filters removed all particles. All bottles were amended with nitrate (20 µM), silicon (20 µM) orthophosphate (1.25 µM) in order to assure nutrient replete growth of phytoplankton (Landry et al. 1995). An additional triplicate set of experimental bottles of whole seawater without nutrients was used to examine the effect of nutrient additions (Landry et al. 1995). Sealed bottles were placed in on-deck incubators maintained at ambient surface-water temperature during the period of incubation. Incubators were shielded with neutral density screening to reduce light levels to ca. 37% of total surface solar radiation. Samples for chlorophyll a analysis, A. anophagefferens abundance, and flow cytometric analysis were obtained from each of the experimental bottles at the end of the 24 h incubations.

Net growth rates of heterotrophic bacteria, picocyanobacteria (i.e. Synechococcus sp.), photosynthetic picoeukaryotes, and the total phytoplankton community (TPC; based on chlorophyll a) were determined using initial and final cell densities or chlorophyll a concentrations. Growth rates were calculated using the formula: k = [ln (Bt/B0)]/t where k is the net growth rate, Bt is the biomass or cell density at t = 1 d, B0 is the biomass or cell density at t = 0 d, and t is the length of the experiment (Gobler et al. 2002). Mortality rates in the dilution experiments were determined from linear regressions of net (apparent) growth rate versus the proportion of whole seawater (0.15, 0.40, 0.70, 1.0). The slope of this regression yielded the mortality rate, which we assumed was predominantly due to microzooplankton grazing, and the y-intercept, adjusted for the nutrient addition, was equivalent to the theoretical growth rate in the absence of predators (Landry et al. 1995).

Transmission Electron Microscopy (TEM) was employed to examine the percentage of visibly infected cells (FVIC) in the native bacterial community, as well as to estimate the abundance of viruses released per bacterium lysed (average burst size). Preserved samples were collected onto carbon-coated collodion films atop 400-mesh electron microscope grids by centrifugation (1 hour, 15 minutes at 16,600 x g). Grids were subsequently rinsed, stained with 0.75% uranyl formate, and rinsed again. The FVIC and burst size were determined by TEM as previously described (Weinbauer and Suttle, 1996). Samples were viewed At the University of Tennessee Microscopic Analysis Facility with a Hitachi H-800 TEM at an accelerating voltage of 100 KeV. For each sample, two grids were prepared, and 1,000 bacterial cells examined. Burst size was defined as the average number of viral particles in all visibly infected cells. This represent the minimum burst size, as cells may have developed further particles prior to lysis (Weinbauer et al., 2002).

Viruses inside bacterial cells have been estimated to only be seen during the last ~10% of the lytic cycle (Proctor et al., 1993). To account for this, conversion factors (3.7 to 7.17) are multiplied by the FVIC to estimate the percentage of infected cells (FIC). This provides a range (as a percentage) of the entire population that are infected cells. The fraction of bacterial cell mortality attributable to viral lysis (FMVL) has previously been found using the factor-of-two rule (Proctor et al., 1993). However, since a proportion of the infected population is also removed by grazers, FIC and FMVL were determined according to Binder (1999), where bacterial infection (also as percentages of the total bacterial population) is derived from the following: (a.) FIC = 7.1 • FVIC - 22.5 • FVIC2 (b.) FVML = (FIC + 0.6 • FIC2) / (1 - 1.2 • FIC)

For more information, contact:

Christopher J. Gobler
Marine Sciences Research Center
Stony Brook University
Stony Brook, N.Y. 11794
(631)287-8397

Steve Wilhelm
University of Tennessee
Knoxville, TN 37966
phone: 865-974-0665