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

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Nickel limits Trichodesmium N 2 fixation 115conversion of acetylene reduction to dinitrogen reduction(Capone and Montoya 2001). The Bunsen coefficient forC 2 H 2 under growth conditions is 0.086 and is negligible.The results were calculated and presented as N 2 pmol h 2lcell 21 (Capone and Montoya 2001).ResultsUsing the traditional unchelexed YBCII medium (Chenet al. 1996), I observed normal growth of Trichodesmiumwithout adding Ni or bioavailable nitrogen. I thenmeasured Ni concentrations in the unchelexed mediumusing the automated flow injection ion chromatographypretreatment method coupled with HR-ICPMS (Ho et al.2010). The background concentrations of Ni in theunchelexed medium prepared in our laboratory generallyranged from 50 to 150 nmol L 21 (Table 1), showing that Niwas present as a contaminant in the medium and thatremoving trace metal impurities in the medium wasrequired to study trace metal nutrition for Trichodesmium.We then prepared trace metal–defined culture medium bypassing the YBCII seawater through chelating resins toremove trace metal impurities as described in the Methodssection (Table 1). The background Ni concentrations in thechelated seawater was , 0.1 nmol L 21 . Using trace metal–defined YBCII culture medium, we observed that themaximum sustainable biomass of Trichodesmium wassignificantly higher in the presence than in the absence ofadded Ni (Fig. 1). Using the conversion factor of 250 mm 3per cell, the total maximum sustainable biomass orestimated cell numbers of Trichodesmium in the Ni-repletemedium was approximately 64,000 cells per mL of seawater(Fig. 1). This value is 10 times greater than for thetreatment without Ni. The growth rates were 0.19, 0.30,and 0.31 d 21 for 0, 4, and 40 nmol L 21 Ni treatments,respectively (Fig. 1A). After adjusting the concentrationsof EDTA and Fe in the medium from 2 mmol L 21 and400 nmol L 21 to 20 mmol L 21 and 100 nmol L 21 , a similarNi limitation result was observed. Trichodesmium barelygrew in the medium without Ni (Fig. 1B). The growth rateswere 0.05, 0.16, and 0.16 d 21 for 0, 40, and 400 nmol L 21Ni treatments, respectively (Fig. 1B). We observed that thelowest dissolved Ni concentration in the culture mediumrequired to maintain optimal growth rates and biomass wasaround 50 nmol L 21 . The intracellular Ni quotas rangedfrom 0.50 to 1.0 mmol mol 21 PinTrichodesmium growingin the chelated medium with sufficient Ni.For Ni enrichment experiments in natural seawater, thegrowth of T. erythraeum IMS101 was significantly slowerin the natural seawater without the addition of 10 nmol L 21Ni (Fig. 2). Additionally, the results of the preliminary fieldaddition experiment in the South China Sea also suggestthat Ni addition could enhance Trichodesmium growth inthe ocean. We observed 5, 64, and 66 trichomes L 21 in thecontrol, Ni-only, and Ni-Fe-P–treated bottles, respectively,at a station (117u519E; 20u039N) and observed 22, 46, and35 trichomes L 21 in the control, Ni-only, and Ni-Fe-P–treated bottles, respectively, at the other station (118u309E;21u289N). However, because this field experiment wascarried out with the use of a single bottle with longFig. 1. Comparison of the sustainable cellular biomass or thegrowth curves of Trichodesmium grown in trace metal–definedYBCII media with different Ni concentrations. Cell numbers wereestimated by the conversion of total cell volume measured. (A)The total Ni concentrations in the media were 0, 4, and40 nmol L 21 . The concentrations of EDTA and Fe in the mediawere 2 mmol L 21 and 400 nmol L 21 , respectively. Initialphosphate concentration in the medium was 50 mmol L 21 ;bioavailable nitrogen concentration was below detection limit.The growth rates were 0.19, 0.30, and 0.31 d 21 for the treatmentswith the addition of 0, 4, and 40 nmol L 21 Ni, respectively. Therates were calculated during the log-linear portion (r 2 . 0.96) ofthe curve between days 8 and 16 for the treatments with Ni addedand between days 8 and 14 for the treatment without Ni added.(B) The experiment was carried out with six different Niconcentrations: 0, 40, 80, 100, 200, and 400 nmol L 21 . Thegrowth rates of the five treatments with Ni added were all similar.Only the growth curves of the treatments with 0, 40, and400 nmol L 21 Ni added are shown here. Cell counting ended onday 20. The EDTA and Fe concentrations in the media were20 mmol L 21 and 100 nmol L 21 , respectively. The growth ratesduring the exponential growth period were 0.05, 0.17, and 0.16 d 21for 0, 40, and 400 nmol L 21 Ni treatments, respectively. Thegrowth rates were calculated during the log-linear portion (r 2 .0.99) of the curve between days 6 and 20.
116 HoFig. 2. Comparison of the growth curves and sustainablecellular biomass of Trichodesmium grown in natural surfaceseawater with and without Ni addition. Cell numbers wereestimated by the conversion of total cell volume. The controltreatments (solid circle, n 5 2) were not spiked with extra Ni; theenriched treatments (open circle, n 5 4) were spiked with10 nmol L 21 Ni. The growth rates of Trichodesmium were 0.14and 0.22 d 21 for the treatments in which Ni was not added andadded, respectively. The errors were propagated with the errors ofthe growth rates in each culture. The seawater was the surfacewater of the SCS, in which the original Ni concentrations were2 nmol L 21 . The bioavailable nitrogen concentrations in theseawater were below detection limit, and the seawater wasenriched with 50 mmol L 21 phosphate and 100 nmol L 21 Fe.incubation time, the results should be considered preliminary,and they were consistent with the laboratory findingsreported in this study.The results in Fig. 3 show that Ni availability canregulate both SOD activities and nitrogen fixation activitiesin Trichodesmium. The cell-normalized SOD activities ofTrichodesmium were positively correlated with Ni concentrationin the artificial medium at Ni concentrations ,50 nmol L 21 , and a fivefold difference in activity existedbetween the lowest and highest Ni treatments (Fig. 3).Elevated Ni availability also significantly enhanced nitrogenfixation rates in Trichodesmium. Cell volume–normalizednitrogen fixation activities increased linearly withincreasing Ni concentrations in Trichodesmium at Niconcentrations , 50 nmol L 21 and saturate at Niconcentrations . 50 nmol L 21 under the experimentalconditions (Fig. 3). Overall, the nitrogen fixation ratesranged from 0.08 to 0.48 pmol N 2 production h 21trichome 21 , with Ni concentrations ranging from 10 to100 nmol L 21 . The nitrogen fixation rates were comparableto the value observed in the field studies, which mostlyranged from 0.10 to 2.0 pmol N h 21 trichome 21 (Caponeet al. 2005).DiscussionNi limitation in Trichodesmium—The results of Figs. 1,2 indicate that Trichodesmium grows much more quickly inseawater enriched with Ni and that the 2 nmol L 21 Ni inthe natural surface seawater might not be enough toFig. 3. The SOD activities and nitrogen fixation rates ofTrichodesmium grown in media with various Ni concentrations.Total cell numbers were estimated by the conversion of total cellvolume. The culture media were prepared by trace metal–definedmedium with 20 mmol L 21 EDTA and 100 nmol L 21 total Fe.Total dissolved Ni concentrations were 10, 20, 50, 100, and200 nmol L 21 , which were equivalent to 6.7, 13, 33, 67, and133 pmol L 21 of inorganic Ni, respectively (Westall et al. 1976;Price and Morel 1991). The range of these Ni concentrations waschosen to correspond to the result observed in Fig. 1B. Thegrowth rates did not increase once total dissolved Ni concentrationswere above 40 nmol L 21 in the culture medium. The growthrates were 0.14 and 0.11 d 21 for the treatments with Niconcentrations of 10 and 20 nmol 21 , respectively. The growthrates for the treatments with Ni concentrations . 50 nmol L 21ranged from 0.18 to 0.22 d 21 with no correlation to Niconcentration. The SOD activities were obtained from the averageof three to four replicate samples of a single-culture bottle(Table 2). The error bars show the standard deviation of thereplicate analysis. Analyses of total dissolved Ni concentrationsfor nitrogen fixation rates were 10, 20, 50, and 100 nmol L 21 . Theunit of the nitrogen fixation rates was shown by using theconversion factor of 50 cells per trichome for rate comparisonwith previous field studies.support its maximum biochemical demand for optimalgrowth in seawater with otherwise sufficient nutrientconcentrations. During the exponential growth period,the growth rates and the increase in total cell volume orbiomass yield of the 4 and 40 nmol L 21 Ni treatments seemto be similar in the early growing stage, which may be dueto Ni carry-over effect in the cells or a sufficient Ni uptakein the early stage (Fig. 1A). The growth rates observed inthe modified low-Fe, high-EDTA culture medium wererelatively lower than the rates observed in high-Fe, low-EDTA medium (Fig. 1B), possibly because of relativelylow Fe availability. Overall, these results indicate thatoptimal growth of Trichodesmium requires an ample supplyof Ni. A recent field study observed that Ni can be alimiting factor for nondiazotrophic cyanobacteria (Dupontet al. 2010). Dupont et al. (2010) found that cyanobacteriagrowth can be enhanced with Ni addition in the Gulf ofCalifornia. Their radioactive tracer experiments alsosuggested that much of natural dissolved Ni might not bebioavailable to small phytoplankton in seawater. Thesefindings and my study both suggest that more extensive Nienrichment experiments in the tropical and subtropical
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Ho, Tung-Yuan. Nickel limitation of nitrogen ... - Academia Sinica

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