Growth rates NE Pacific and Atlantic diatom isolates under various Zn and Co additions in experiments with cultures collected from R/V Thomas G. Thompson cruise TN280, along Line P in the NE Pacific, in May of 2012 (NCEI Accession 0277723)

Acquisition Description:
Location: Northeast Pacific Line P Transect 48.8167 N 128.667 W

Media and culturing techniques Northwest Atlantic Thalassiosira pseudonana CCMP1335 cultures were maintained in a 24°C incubator under constant fluorescent lighting (65 µmol quanta m-2 s-1 PAR). Northeast Atlantic Phaeodactylum tricornutum CCMP632 cultures were maintained in an 18°C incubator under constant fluorescent lighting (90 µmol quanta m-2 s-1 PAR). Both Pacific Pseudo-nitzschia delicatissima UNC1205 and Pacific Thalassiosira sp. UNC1203 were grown in an 18°C incubator under constant fluorescent lighting (85 µmol quanta m-2 s-1 PAR). All cultures were randomly repositioned each day to avoid any effect of subtle variation in light intensity on growth. T. pseudonana CCMP1335 and P. tricornutum CCMP632 (Bigelow Laboratory, East Boothbay, ME) were obtained from the Mincer and Saito laboratory culture collections at the Woods Hole Oceanographic Institution, respectively. Pacific P. delicatissima UNC1205 and Thalassiosira sp. UNC1203 were obtained from the Marchetti laboratory at the University of North Carolina. Both Pacific isolates were collected at station P8 of Line P, a Northeast Pacific ocean transect located off of Vancouver Island comprising 26 sampling stations and ending at Ocean Station Papa at 50°N 145°W (Fig. 1a). All cultures were axenic and maintained by sterile technique until needed. Polycarbonate and plastic bottles were cleaned to remove trace metal contaminants before use. This procedure involved, at minimum, a 72h soak in <1% Citranox detergent, five rinses in mili-Q water, a 7d soak in 10% HCl, and five rinses with dilute acid (HCl, pH 2). Cultures were grown in microwave sterilized 28 mL polycarbonate centrifuge tubes, and all solutions were pipetted only after a tip rinse procedure consisting of three rinses with 10% HCl followed by three rinses with sterile dilute HCl (pH 2). All culture work was conducted in a Class 100 clean room. Culture media was prepared after that used by Sunda and Huntsman for trace metal experimentation (Sunda and Huntsman 1992, 1995b). Microwave sterilized, 0.2µm-filtered Pacific seawater from the North Pacific station “Aloha” (22° 45'N, 158° 00'W) was used as the media base. Macronutrients were added to this sterile base to a final concentration of 88.2µM NaNO3, 41.5µM NaH2PO4, and 106µM Na2SiO3 and were chelexed before use. Added vitamins included 2 nM biotin, 0.37 nM B12 as cyanocobalamin, and 300 nM thiamine and were also chelexed before use. Trace metals were added to final media concentrations of 10-7 M FeCl3, 4.8 x 10-8 M MnCl2, 4.0 x 10-8 M CuSO4, 10-7 M NiCl2, and 10-8 M Na2O3Se within a 10-4 M ethylenediamine tetraacetic acid disodium salt (EDTA, Acros Organics, C10H14N2Na2O8) metal ion buffer system. All media amendments were sterile filtered through acid rinsed 0.2µm filters before addition to final media, and final media equilibrated for at least 12h before inoculation. Established cultures of each diatom were first acclimated in low-metal media containing 1 nM total Zn or less for at least three transfers. These acclimated cultures were used to initially inoculate transfer 1 (T1) cultures at 1% volume. For all diatoms, Zn or Co limitation experiments were first performed using a range of Zn concentrations with Co omitted and vice versa. We refer to experiments using media amended with Zn or Co (while omitting the other) as “simple limitation” experiments. Experiments varying concentrations of both Zn and Co were also conducted, allowing for three-dimensional visualizations of growth rates as used previously (Saito et al. 2002; Saito and Goepfert 2008). We refer to experiments using media amended with both Zn and Co as “matrix” experiments. In vivo fluorescence (Turner Instruments TD-700) of simple and matrix experiment cultures was measured on a near-daily basis as a proxy for chlorophyll a and is reported as relative fluorescence units (RFU). Growth rates were calculated from the exponential portion of each culture’s growth curve. Computed ratios of [Zn2+] and [Co2+] to total concentrations, whose values are 10-3.99 and 10-3.63 respectively, were used to convert added metal concentrations to free ion concentrations and are the same as those used by Sunda and Huntsman (Sunda and Huntsman 1995).

Isolation sources and locations: * Pseudonitzschia delicatissima UNC1205 and Thalassiosira UNC1203 were isolated from station P8 of the Line P transect, 48.817°N 128.666°W * Phaeodactylum tricornutum CCMP632 was ordered from Bigelow, the strains original location of isolation was 54°N 4°W * Thalassiosira pseudonana CCMP1335 was also from Bigelow, original location of isolation was 40.756° N 72.82° W