Flow cytometry data was collected in September 2015, in waters off South Australia.
The general purpose of the study is to be able to establish background knowledge on the ecosystem on the continental shelf of South Australia and the impact of upwelling/saline outflow events on microbial communities to ultimately develop a biogeochemical model of the region. Sampling was carried out during cruises conducted on board the RV Ngerin as part of the Southern Australian Integrated Marine System (SAIMOS). During each cruise, the physical, chemical and biological properties of the chlorophyll fluorescence maximum (DCM) layer were investigated. Flow cytometry data has been collected for picophytoplankton, bacteria and viruses.
Six main stations have been sampled over the course of the study, five are located on the 100 m isobath, i.e. RS (35.508S, 136.278E), B2 (35.418S, 136.148E), B3 (35.258S, 136.048E), SAM2CP/B4 (35.168S, 135.418E) and SAM5CB/B5 (35.008S, 135.198E), and one from an offshore station (B1; 36.188S, 136.178E) located southwest of Kangaroo Island. Note that combining the distances between stations (14–25 nautical miles), the average component of the current velocity at middepth along the shelf (0.01 m s21) and the average speed of the vessel (i.e. 9 knots) indicate that different water masses were sampled at each station. Additional samples have on occasion been collected from the National Reference Station (NRS) at Kangaroo Island (35.832S, 136.447E) and the SA Spencer Gulf Mouth Mooring (SAM8SG, 35.25S, 136.690E), where the saline outflow occurs.
Since May 2014 water samples were taken at each station at three depths; 5m, DCM and 10m below DCM. In vivo CTD profiles were used to identify the temperature, salinity and fluorescence profiles of the water column, ultimately determining the depth of the Deep Chlorophyll Maximum (DCM). Niskin bottles were used for seawater sampling for nutrients, chlorophyll a, Pico phytoplankton, bacteria and virus analysis and Particulate Inorganic Matter, Particulate Organic Matter, Total Suspended Sediments, and phytoplankton community compositions and abundances. If no DCM could be identified, seawater sampling was done at or near the thermocline interface.
Seawater samples of 50-70 mL were filtered through bonnet syringe filters (0.45 um porosity, Micro Analytix Pty Ltd) and stored at –20oC for nutrient analysis. Dissolved ammonium (NH3, detection limit 0.071 µM), oxides of nitrogen (NOx (NO2 + NO3), detection limit 0.071 µM), phosphate (PO4, detection limit 0.032 µM) and silicate (SiO2, detection limit 0.333 µM), were determined by flow injection analysis with a QuickChem 8500 Automated Ion Analyser.
Chlorophyll a concentrations were determined by filtering 2L seawater samples through stacked 5um mesh and pre-combusted glass fibre filter (Whatman GF/F, nominal pore size 0.7 um porosity). Filterswere stored in cryovials and frozen in liquid nitrogen in the field. Samples were stored at -80oC until analysis. Chlorophyll a was analysed via High Performance Liquid Chromatography using anAgilent LC1260 HPLC with a photodiode array detector and a refrigerated autosampler. Since May 2014 SAIMOS has analysed chlorophyll pigments using HPLC techniques. The data being supplied is slightly processed (reformatted really) to show similar results to the previous spectrophotometry analyse method.
For picophytoplankton, triplicate 1 ml samples were added to cryovials which were pre-spiked with 10 ul glutaraldehyde (25% EM grade). For bacteria and viruses, triplicate 1 ml samples were added to cryovials which were pre-spiked with 20 ul glutaraldehyde (25% EM grade). For bacteria and viruses, triplicateSamples were fixed for 10-15 minutes in the dark in the fridge (4oC) before being submerged in liquid nitrogen. Samples were then stored at -80oC in the laboratory until analysed by flow cytometry.
Picophytoplankton and bacteria and viruses samples were analysed using flow cytometry. Picophytoplantkon samples were thawed at 37oC, 1 um beads (Polysciences) added as an internal reference and analysed using a FACSVerse (Becton Dickenson) flow cytometer fitted with a 488 nm laser. For picophytoplankton, acquisition was run for 3 minutes on a medium flow rate (~67 ul min-1). Samples for bacteria and viruses were thawed as above, diluted 10 fold in Tris EDTA (pH = 8.0, Sigma-Aldrich), stained with SYBR I green (0.5 x 10-4 final concentration, Molecular Probes) in the dark at 80oC for 10 minutes and then 1.0 um fluorescent beads (Polysciences) added an internal reference (Brussaard 2004). Bacteria and viruses were analysed using the same flow cytometer as above, with acquisition run for 2 minutes on a low flow rate (~25 ul min-1). Data were analysed with FlowJo software (Tree Star(R)). Picophytoplankton groups were differentiated based on their scattering and fluorescence signals (Marie et al. 2000a, b). Bacteria and viruses were separated on plots of side scatter (SSC) and green (SYBR) fluorescence and SSC and red (Chl a) fluorescence. In deep (D3) samples, Prochlorococcus could usually be discriminated from bacteria in SSC and red fluorescence plots (and SSC and green fluorescence), however at 5 and the DCM, Prochlorococcus coincided with bacteria. To correct for this in the stained samples, Prochlorococcus were included within the bacterial group for all depths in the analysis. Bacteria counts were then corrected for by subtracting total counts of Prochlorococcus (obtained from non-stained picophytoplankton samples) from the bacteria group.
Australia’s Integrated Marine Observing System (IMOS) is enabled by the National Collaborative Research Infrastructure Strategy (NCRIS). It is operated by a consortium of institutions as an unincorporated joint venture, with the University of Tasmania as Lead Agent.
South Australian Research and Development Institute (SARDI)