Coale: Methodology for Enrichment and Incubation Experiments

Six enrichment experiments were conducted on three cruises to the Southern Ocean during the US JGOFS program. One of these cruises was in the Ross Sea (PII-3 and PII-4) during the austral summer. The other two cruises were conducted in waters immediately north and south of the Antarctic Polar Front Zone in fall (SI-1,SI-2) and spring (SII-3, SII-4).

Seawater was collected using acid cleaned, teflon-coated, 30-L Go-Flo bottles suspended on nonmetallic kevlar line at mid mixed layer depths (Bruland et al., 1979). The Go-Flo bottles were placed in a rack on the portable clean laboratory that was accessible from the inside. New 22 liter polycarbonate carboys were used for the enrichment experiments. They were equipped with Nalgene filling/venting closures with 3 tubing ports. A 6mm ID Bev-A-line tube ran from one of the two large tubing ports to the bottom of the bottle (Coale, 1991). All parts were cleaned according to the protocol described in Martin et al.(1991).

Eight carboys were rinsed and filled with raw seawater, with its resident phytoplankton population, in the clean laboratory van at each enrichment site. Incubation carboys were filled serially from each Go-Flo bottle to assure homogeneity. Four casts of 2 Go-Flo bottles were required to fill all the enrichment carboys. The carboys were spiked with 1000 ppm Fe(NO₃)₃ (Fisher iron reference solution) producing approximate concentrations of 0.2, 0.5, 1.0 and 2.5 nM Fe. Two more carboys were spiked to ~5 nM Zn, one with 2.5 nM Fe and one without Fe. Two replicate carboys with nothing added served as controls.

Initial samples for chlorophyll a, nutrients, particulate organic carbon (POC), total organic carbon(TOC), dissolved organic carbon (DOC), and phytoplankton species counts were drawn directly from the Go-Flo bottles to save enrichment water for future sampling. An initial trace metal sample for ambient iron and zinc was drawn from each enrichment carboy directly. These were immediately acidified for future analysis.

The enrichment bottles were placed in deckboard incubators with ambient flowing surface seawater for temperature control. Carboys were incubated for 7–16 days. Sub-samples were taken by attaching an acid cleaned Bev-A-line tube from an air tank to the tubing ports of the caps. Air was used first to mix the carboys by bubbling, then pressurizing the carboy to expel the required volume of sample (Coale, 1991). Chlorophyll a, TOC and nutrient samples were taken every day or every other day. POC samples were drawn daily once the experiment started to show a significant change in the chlorophyll a and nutrient values. Samples for epifluorescent microscopy were taken initially and again, near the end of the experiment.

Trace metal samples were analyzed in the laboratory using organic extraction with APDC/DDDC into chloroform (Bruland et al., 1979). Nitrate, nitrite, soluble reactive phosphate and silicic acid were analyzed by standard autoanalytical methods immediately onboard the ship (Gordon et al., 2000). Chlorophyll a samples were concentrated onto a 25mmGF/F Whatman filter and pigments were extracted in 90% cold acetone for 24 hrs. The concentration of chlorophyll a was measured using a Turner Design fluorometer. Samples for epifluorescent microscopy were collected on 25 mm polycarbonate filters (0.8 mm pore size) and fixed with gluteraldehyde, then stained with diamidino phenylindole dihidrochloride (DAPI). These samples were then enumerated according to Chavez et al. (1991).

POC samples (< 800mL) were pressure filtered with compressed breathing air onto a pre charred 25mm GF/F Whatman filter. Filters were stored on pre-charred aluminum foil in petri dishes and analyzed in the laboratory on a Control Equipment Corporation 440 Elemental analyzer. Unfiltered TOC samples were collected in triple rinsed charred glass 40ml vials and stored frozen. These were thawed in the laboratory and 5 mL of 50% phosphoric acid was added per 1mL of sample. The samples were bubbled with ultra pure grade oxygen (grade 5) and TOC was measured in filtered and unfiltered samples on a Dohrmann DC-190. DOC was obtained by subtracting POC from the unfiltered measurement. Protocols for sampling and analysis followed Sharp (1993) and Sharp et al. (1995).

References:

Bruland, K.W., Franks, R.P., Knauer, G.A., Martin, J.H., 1979. Sampling and analytical methods for the determination of copper, cadmium, zinc and nickel at nanogram per liter level in seawater. Analytical Chemistry Acta 105, 223-245

Chavez, F.P., Buck, K.R., Coale, K.H., Martin, J.H., DiTullio, G.R., Welschmeyer, N.A., Jacobson, A.J., Barber, R.T., 1991. Growth and grazing, sinking and iron limitation of equatorial Pacific phytoplankton. Limnology & Oceanography 36, 1816-1833.

Coale, K.H., 1991. Effects of iron, manganese, copper and zinc enrichments on productivity and biomass in the subarctic Pacific. Limnology & Oceanography 36, 1851-1864

Martin, J.H., Gordon, M.R., Fitzwater, S.E., 1991. The case of iron. Limnology & Oceanography 36, 1793-1802.

Sharp, J.H., 1993. The dissolved organic carbon controversy: an update. Oceanography 6, 45-50.

Sharp, J.H., Benner, R., Bennett, L., Carlson, C.A., Fitzwater, S.E., Peltzer, E.T., Tupas, L.M., 1995. Anaslysis of dissolved organic carbon in seawater: the JGOFS EqPac methods comparison. Marine Chemistry 48, 91-108