Ho, Tung-Yuan. Nickel limitation of nitrogen ... - Academia Sinica

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Nickel limits Trichodesmium N 2 fixation 113Table 1. The concentrations (nmol L 21 ) of dissolved trace metals in unchelated YBCII medium, chelated YBCII medium (beforeadding trace metals), and the surface seawater collected in the SCS. It should be noted that the background concentrations of trace metalsin the unchelexed artificial culture medium can vary from time to time and from laboratory to laboratory because the culture mediumprepared in different laboratories by different people with the use of different brands of chemicals would cause different levels ofcontamination through the preparation procedures.Concentration (n 5 3)Culture mediumNi Fe Mn Zn Cu Co CdUnchelated YBC II 13064 8.960.3 7.060.6 1361 3.160.2 0.1160.004 0.8060.04Chelated YBC II 0.0460.01 0.1560.01 0.0260.02 0.1560.3 0.1060.2 0.00560.001 0.01060.001SCS surface water 2.060.01 0.1460.06 3.060.05 0.7460.3 1.360.06 0.02760.005 0.1060.005generally follows the procedures for preparing trace metal–defined culture media (Price et al. 1989; Ho et al. 2003).Previous studies showed that cell volume was closelycorrelated with carbon content in various marine phytoplankton(Thompson et al. 1991; Verity et al. 1992;Montagnes et al. 1994), indicating that cell volume is areliable parameter for the estimate of biomass or growthrates in phytoplankton, including Trichodesmium (Tuit et al.2004). Because the Trichodesmium strain used in this studyexisted in a single trichome state during the growth period,the total cell volume of Trichodesmium samples can bedetermined by a particle counter. I made an extra effort inthis study to evaluate the accuracy of total cell volumeobtained by Coulter Counter (Beckman Coulter, Multisizer3). The total cellular volume of the same Trichodesmiumculture samples were repeatedly determined simultaneouslyby Coulter Counter and a microscope (Carl Zeiss, AxioImager A2) with hemocytometer (Marienfeld, Neubaueer).Using the microscope and hemocytometer, the lengths andwidths of all trichomes per unit sample volume weremeasured, and the total cell volume was estimated bymultiplying the total length of trichomes with the crosssectionalarea. Overall, the precision of the replicateanalysis of the total cell volume obtained by the CoulterCounter was much better than the microscope – 1–5% and30–60%, respectively. The total cellular volume obtained bythe particle counter was comparable or slightly lower thanthe value obtained by the microscope with hemocytometer,indicating that the particle counter can be a reliable methodof estimating the total cell volume of Trichodesmium.We observed by microscope that individual cell volumevaried significantly from cell to cell in each trichome.However, the variations of the averaged cell volume of theTrichodesmium grown in various Ni treatments wereanalytically insignificant because the individual variationswere mostly canceled out by dividing total cell volume bytotal cell numbers. Additionally, we observed consistenttrichome size during growth periods in different Nitreatments, suggesting that average cell size was comparableduring the growth period in this study. Thus, theincrease in total cellular volume can be a good estimate ofthe increase in total cell numbers or the increase in growthrates. Total cell numbers can be estimated by using theconversion factor of average cell volume per cell, whichmostly ranged from 200 to 250 mm 3 per cell in this study.We also observed that chlorophyll a (Chl a) concentrationsby fluorometric analysis (Turner Designs) exhibited stronglinear correlations with total cell volume measured, withcorrelation coefficients (r 2 ) up to 0.99 (p , 0.01) during theexponential phase. The conversion factor of cell volume toChl a was about 0.10 ng chlorophyll per 10 6 mm 3 of cellvolume.To determine intracellular trace metal quotas accurately,we modified the concentrations of Fe and ethylenediaminetetraaceticacid (EDTA) in the original YBCII culturemedium (Chen et al. 1996). It is likely that high levels of Feadded to the culture medium would be precipitated andadsorbed on the algal surface (Ho et al. 2003). Weincreased the EDTA concentration from 2 to 20 mmol L 21and decreased the total Fe concentration from 400 to100 nmol L 21 in YBCII medium so that the inorganic Feconcentration was about 500 pmol L 21 (Ho et al. 2003).Unless otherwise noted, Trichodesmium was grown underthe modified trace metal–defined medium. Initial phosphateconcentration in the medium was 50 mmol L 21 ;bioavailable nitrogen was not added, and the backgroundconcentrations of nitrate and nitrite were below thedetection limit in the culture medium. Other trace metalconcentrations in the medium were 20, 4, 1, 2.5, and11 nmol L 21 for Mn, Zn, Cu, Co, and Mo, respectively.Cells were grown twice for at least 10 generations in thetrace metal–defined culture medium with a 50 nmol L 21 Niconcentration and then transferred to new media enrichedwith total dissolved Ni concentrations varying from 0 to400 nmol L 21 .Trace metal concentrations in natural seawater andculture media were measured by an automated pretreatmentmethod with a high-resolution inductively coupledplasma spectrometer (HR-ICPMS) (Ho et al. 2010). Thenatural seawater used in this study was collected at anoffshore basin site in the northern South China Sea (SCS)by using trace metal–clean techniques and using TefloncoatedGo Flo bottles (General Oceanics) mounted on aTeflon-coated rosette equipped with Kevlar wire. The totaldissolved Fe and Ni concentrations in the natural waterwere 0.14 and 2.0 nmol L 21 , respectively (Table 1).To determine intracellular trace metal quotas, culturedcells were harvested onto acid-washed 25 mm polycarbonatefilters with a 10 mm pore size in a class 100 trace metal–clean laboratory during the light portion of the light–darkcycle. The filtered cells were rinsed with trace metal–cleanseawater, the chelexed seawater, to remove the culturemedium residue. The trace metal–clean seawater is theseawater passed through a column filled with chelex 100
114 Horesins, in which trace metal concentrations are extremelylow (Table 1). Trace metal quotas in the phytoplanktonand trace metal concentrations in the seawater were thendetermined using a HR-ICPMS (Thermo Scientific ElementXR) fitted with a desolvation system (ElementalScientific). The Fe quotas in the cells we have observedwere around 10 mmol mol 21 P, which was slightly higherthan the Fe quotas observed in other marine phytoplanktongrown in a low-Fe culture medium (Ho et al. 2003),thus indicating that extracellular Fe adsorption on Trichodesmiumwas insignificant. The details of the analyticalprecision, accuracy, and detection limits of the ICPMSmethod for seawater and phytoplankton were described inour previous studies (Ho et al. 2003, 2007, 2010).Two Ni enrichment experiments in natural seawater werecarried out in this study. First, 10 mL of the culturedspecies Trichodesmium IMS 101, maintained in themodified YBCII medium enriched with 50 nmol L 21 Ni,was inoculated in 1 liter of natural seawater collected fromthe surface water of the SCS. The background concentrationsof bioavailable nitrogen in the surface water werebelow detection limits, and the background-dissolvedconcentrations of phosphate, Ni, and Fe were 20, 2.0,and 0.14 nmol L 21 , respectively. The natural seawater wassterilized by filtering the water through 0.2 mm acid-washedpolycarbonate filters and was amended with 50 mmol L 21phosphate, 100 nmol L 21 Fe, and other trace metals—except Ni.A preliminary Ni enrichment experiment was alsocarried out in the field by using natural surface seawatercollected in the northern SCS during 02–12 June 2010. Thesurface seawater was collected at depths of 20–30 m, wherenitrate and nitrite concentrations in the seawater werebelow detection limits. Seawater samples were then filteredthrough 200 mm acid-washed polycarbonate filters toremove large zooplankton in trace metal–clean laminarflow benches on board. Three 1 liter trace metal–cleanedpolycarbonate bottles amended with different concentrationsof Ni, Fe, and P were used. No nutrients or tracemetals were added to the control sample bottle. The Nionlybottle was enriched with 10 nmol L 21 Ni, and thethird bottle was enriched with 10 nmol L 21 Ni,100 nmol L 21 Fe, and 5 mmol L 21 phosphate. The totalincubation time was 13 days, including 9 days on board and4 days in laboratory. The culture was maintained on boardwith a light intensity of 30 mmol quanta m 22 s 21 under a12 : 12 light : dark cycle at 27uC. After being brought backto the laboratory, the samples were maintained in growthchambers at 90 mmol quanta m 22 s 21 under a 12 : 12 hlight : dark cycle at 26uC. We filtered 500 to 1000 mL of thesamples and resuspended the filtered trichomes in 5 mL oforiginal filtered seawater. The total numbers of trichomesin the suspended samples were then counted by using afluorescence microscope with green excitation (Carl Zeiss,Axio Imager A2).SOD activities were determined using a cytochrome c(Cyt c) with xanthine-xanthine oxidase method based onthe ability of SOD to inhibit the reduction of Cyt c byscavenging superoxide anion radicals produced by thexanthine xanthine oxidase system (Flohe and Otting 1984).One unit of SOD activity is defined as the amount ofenzyme that produced a 50% inhibition of Cyt c reduction.The calibration curve, with an r 2 of . 0.99 (p , 0.01), wasfirst established between the reciprocal of the absorbance offerrous Cyt c and the enzymatic activity by using sevenSOD standards. For each bottle, a linear correlation wasobserved between biomass and SOD activities. The SODactivity of each sample was obtained using the SOD assayfor four different sample volumes. I then collectedTrichodesmium samples through filtration, resuspendedthe cells in 1 mL of seawater, and determined the cellvolume. The cells were broken with sonication under anice-water slurry. After observing pigment release, Icentrifuged the samples and removed the particulatematerial. Then, 100 mL of cell sample was added into a3 mL quartz cuvette prefilled with 2.89 mL of phosphatebuffer solution, which contained xanthine and Cyt c. Then,10 mL of xanthine oxidase was added to the cuvette foranalysis (Flohe and Otting 1984).To determine nitrogen fixation activity, 20 mL Agilentflat-bottom headspace vials fitted with Teflon-coatedsilicone septa were used. Both the vials and the septa werecleaned rigorously using Micro-90 (International ProductsCorporation) cleaning solution and 1 mol L 21 superpureHCl (Seastar) to remove trace metal impurities. The 20 mLvials were first soaked in 2% Micro-90 overnight and thencleaned with Milli-Q water. They were then filled with 10%superpure HCl overnight and rinsed thoroughly withElement-grade Milli-Q water immediately before use. Wethen determined nitrogen fixation rates with the acetylenereduction method (Capone and Montoya 2001). TheTrichodesmium samples grown under treatments rangingfrom 10 to 100 nmol L 21 Ni were added into each vial withan upper gas volume of 10 mL. Right before the lights wenton, 10 mL aliquots of Trichodesmium culture were added tothe 20 mL vials with sterile, acid-washed tips, which werethen sealed using Al caps and acid-washed septa with theTeflon end toward to the samples. The vials were thenincubated in the original incubator for 6 to 12 h. Fourpointtime courses from 6 to 12 h were prepared for eachindividual treatment. Two milliliters of the 10 mL headspacein the vials was removed and replaced with 2 mL ofacetylene generated from carbide (Capone and Montoya2001). Then, gas-tight plastic needles were used to add 2 mLof 99.9% acetylene to the serum sample vials to reach a20% final concentration in the upper gas phase. The vialswere then gently shaken to mix the seawater containingTrichodesmium in the vials. The subsamples were incubatedin the original growth chamber for 6–12 h under the samegrowth irradiance and temperature as the original cultures.We then sampled 1 mL of the headspace gas from the vialsand injected it into clean, empty 10 mL serum vials forethylene analysis. The ethylene production rate wasmeasured using an Agilent 7890A gas chromatograph witha Poropak N column (Agilent, HP-PLOT Al 2 O 3 S) and aflame ionization detector. The ethylene concentrations werequantified using the calibration curve obtained from theethylene standard. Nitrogen fixation rates for Trichodesmiumin the Ni treatments were estimated by using theacetylene reduction method with a ratio of 4 : 1 for the
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Ho, Tung-Yuan. Nickel limitation of nitrogen ... - Academia Sinica

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