Lake water samples were collected at specific depths with a five-liter Niskin bottle during normal LTER limnological sampling. Sub-samples were decanted into three-1 L Nalgene bottles (2-light and 1-amber), two-500 mL amber Nalgene bottles, three-150 mL borosilicate glass bottles, two-20 mL scintillation vials, and one-30 mL serum vial. The two-one liter clear Nalgene bottles were used for the ETS experiment. Depending on the lake and depth at which each analysis was performed, 1000-2000 mL of lake water was filtered through a Whatman 47 mm GF/F filter. The filter was folded in half (organic material inside), placed in a glassine envelope, and stored at 0 degrees Celsius until analysis (<30 min). In an ice bath, the filter was combined with 3 mL of homogenization buffer and homogenized for 90 seconds with a glass/teflon tissue grinder. The mixture was decanted into a cone centrifuge tube; it was centrifuged in the cold for 3 minutes, vortexed for 30 seconds, and centrifuged for another 15 minutes. 0.5 mL of the extract was pipetted supernatant into three 1-cm quartz cuvettes (2 replicate, 1 control) and placed in an ice bath. The control sample was boiled for 10 minutes and cooled in an ice bath. 1.5 mL of substrate solution and 0.5 mL of INT solution was added to each cuvette, vortexed for 30 seconds, and incubated at 1-4C for one hour. The reaction was terminated in the cuvette with 0.5 mL of termination solution.
The absorbance was measured at 490 nm with a spectrophotometer. Light absorption by the sample idirectly proportional to the moles of electrons transferred through the electron transport system (ETS). Community ETS (umol O2 L-1 hr-1) was calculated using the following equation:
ETS = (AbsR - AbsC) a * b c * t
where AbsR is the average absorbance of the replicate samples, AbsC is the absorbance of the control sample, a is ratio of the volume of homogenization buffer to the volume of lake water filtered, b is the ratio of the final volume of reaction mixture in each cuvette to the volume of extract supernatant, c is the extinction coefficient for formazan (31.8 Abs cm-1 umol O2-1), and t is the incubation period.
Community ETS was adjusted to ambient lake temperature using the Arrhenius equation:
ETSadj = ETS * e^(Ea (( 1 / (CI + 273 K)) - ( 1 / (CA + 273 K))) / R )
where Ea is the energy of activation (15,000 cal mol-1, Q10 = 2.66), CI is the incubation temperature (C), CA is the ambient lake water temperature at specific depth, and R is a gas constant (1.987 cal mol-1 K-1).
A first order relationship exists between ETS activity and respiratory capacity in aquatic microorganisms (e.g., Kenner and Ahmed 1975, Christiansen et al. 1980). Our studies have revealed that 44% and 56% of measured ETS activity is from bacterioplankton and phytoplankton, respectively, in Lake Bonney (Takacs and Priscu, unpublished data). Using these relationships, in concert with published respiration:ETS ratios (Packard 1985), we derived a community respiration:ETS ratio of 0.61 for the water column of Lake Bonney. Individual respiration:ETS ratios for bacteria and phytoplankton were computed as 0.513 and 0.097, respectively.