J Phycol. U, 168-171 (1985) NOTE SILICON UPTAKE BY ALGAE WITH NO KNOWN Si REQUIREMENT. II. STRONG pH DEPENDENCE OF UPTAKE KINETIC PARAMETERS IN PHAEODACITLUM TRICORNUTUM (BACILLARIOPHYCEAE)1 Gerhardt F. Riedel2 and David M. Nelson College of Oceanography. Oregon Statr:- University. Corvallis, Oregon 97331 In the pH ranges and disso[?ved Si (oncentralions of our ex? periments undissociated silicic add (Si(OH).) and the first con. jugate base (SiO(OH). -) together are predicted to make up more than 99% of the total Si. Silica polymers are nOI thermodynam? ically stable at these 10.... dissolved Si concentrations (Stumm and Morgan 1970). In a few high-pH. high-Si culturCll sepiolite (Mg.Si,O,) saturation is predicted (Woll;m et aJ. 1968). but our previous experiments (Nelson et al. 1984) have shown evidence of actual precipitation only at higher concentrations. The cell-specific Tate of Si uptake (p) is presented in Fig. 1A for both pH series as a function of total (initial + added) dissolved Si concentration. The de? pendence of p upon the total dissol ....ed Si concen? tration can be approximated by hyperbolic satura? tion kinetics as described by the Michaelis-Menten equation (Dugdale 1967): P",.. ?S (3) p= K + S > where S is the concentration of the substrate under consideration and K, is the concentration of that substrate at which P = p"",~/2. The results were fit? ted by iterative nonlinear regression (Wilkinson 1961) to determine K, and Pfllil~ and their associated confidence limits. The kinetic parameters obtained by the direct fit were used to define the solid lines in Fig. 1A-C as well. In both series P. tricomutum exhibited hyperbolic saturation kinetics for Si uptake. In the high pH (I) (2)[SiO,(OH).?- Ja,,> = 10 -'fro[SiO(OH).-1 pie preparation and '?Sianalysis were as desc:ribed b~' Nelson and Goering (1977). The pH of the cultures was measured at the beginning and end of the incubations with an Orion model 80 I pH meter, equipped with a Corning 476050 glass/AgiAgel elec? trade, standardized at pH 7..0 and 10.0. A slight increase in the pH was observed over the duration of the incubations. a.oout 0.1 unit in the low series. and 0~05 unit in the high series. due to photosynthesis. A trend of increasing pH with the amount of '?Si added, about 0.3 unit in the low pH lierie~ and 0.1 pH unit in the high serie~. ""as due to the alkalinity of the isotopr:- solution. The dissoh'ed S.i concentration of the medium prior to the '?Si addition was determined by the method of Strickland and Parsons (1972) on replicate samples. The concentrations of the various species of silicic add in the experimental flasks were calculated from the mean pH over the incubation period, the measured concentration of dissolved 5i and the dissociation constants (Sjoberg et aL 1981): [Si(OR), 'laB' = 10-"'7 ISi(OH).J In a previous paper (Nelson et a1. 1984) we re? ported on aspects of Si uptake by two algae with no known Si requirement. the diatom Phaeodart)'lum tri? cornutum (Bohlin) and the prasinophyte Platym,mas sp. Both species demonstrated hyperbolic saturation kinetics for Si uptake with half-saturation constants considerably higher than those previously found for Si-requiring diatoms (e.g. Paasche 1973, Nelson et al. 1976, Kilham et a1. 1977). In this paper we report on the Si uptake kinetics of Phaeodact)'lum triwrnu? tum as a function of pH over a pH range that alters the chemical speciation of dissolved Si 10 a large degree. ABSTRACT Silicon uptake kijll'lits oj the diatom Phaeodactylum tricornutum (Bohlin) wert' examined at pH 8.8 ? 0.1 and pH 9.7 ? 0.1. Uptake folloU's hyperbolic saiUratiQPI kineticJ at both pH's, but at the higher pH tht' halJsat? uration constant for uptake is 11.8 j,LM, as 0IJposrd to 54.8 p.;.Wal thl' lower pH. When the uplahf rate is ex? mnined as a function oj Ihe mlculaled concentration of the m071ol'alent conjugatl' basi', SiO(OH)J-, thl! halfmt" uration comtanl Jor uptake is 6.6 ",M at either pH. Ke)1 index words: diatoms; pH; Phaeodactylum tri? cornutum; siliric acid: uptake kinnics I .4cupted: 16 AugUJt 1984. I Present Address: Harbor Branch 1nstitut.ion 1m;., R. R. # I, Box: 196-A, Fort Pierce, Florida 33450. Pho.~odaclylum Iricomulum (done Pet Pd. obtaioed from R. R. L. Guillard, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts) wasgrowlI to 6 >( 10' cells'mL" in fj2 medium (Guillardand Ryther 1962) with 60,uM added Si at a temperature of 200 C and under cominuous illumination of 170 /.IE?m-~?sec?', The cells wert' centrifuged at 1000 >( g for 15 min and resus? pended in 1'/2 without added Si (background lSi I= 1.5 jlM). The cells were then centrifuged again and resuspended in two 2 L Rasks off/2 medium without added Si, one with the pH adjusted to 8.5 and one to pH 9.6. Before the uptake experiments were begun these cultures were left undisturbed at least I h. thus allowing any rapidly dissolvinginorgank silicate phases to dissolve (Nelson el a!. 1984). For uptake experiments 250 mL aliquots were taken from the cell suspension and inoculated with '?Si? labeled silicic acid solution (95.2 atom W. '"Si) to the deJ;ired levr:-[s. The cultures were then incubated at 170 /iE'm-"sec-J at 20? C for 2 h. Atthe end of the incubations the samples were lilr.ered through 0.8 lim polycarbonate membrane filters (Nucleopore Inc.). Sam- J68 pH DEPENDENCE OF 51 UPTAKE 169 T\I\1 f 1. lillfJ-mlllrutillll m'HUml~ (KJJi'T 11{j(~'IIIPI(/hl' IIsillg ltI/al SI, (11111 m/rlllaird tOl/fmtrll/iml\ of Imdjj.m,;tl.l"d .~ilj(i( (Ind. mill/hI' lint ,wd ;f(lllld nilljil{:all' b'lir' 11\ mb,llrall'l (?951)( fiJl/fidmft' jl/ln? Fa/). Sl SilOlll, sio/Oll/, S,ll,IOII!,' I'U~?rir--.. (pM} (p\t! ("MI !.n~!) LlIw 54.8 ? 12.7 50.1 ? 12.G 6.65 ? 1.04 0.81 ? 0.13 High 11.8 ? 7.0 5.36.:t 3.56 6.62 ? 3,69 6.8 ? 3,43 50 200 (5) (6) ~, E + Si(OH)~ ... E-SjO(OH)~? (external) k, in which: + H+ ..... E + Si(OH)~ (4) (internal) (where E represents a Si transport protein unaf~ fected by pH over the range of consideration, E-5iO(OHh- the transport protein-substrate com? plex, and kit k~ and k~ the rate constants for the individual reactions), can be shown to lollow the rate law: K, values are 54.8 ~M in the low pH series, and 11.8 in the high pH series, Considering the calculated SiO(OHh- concentration as the substrate the esti? mated K. values are 6.65 and 6.62 ILM for the high and low pH series, respectively. The uptake rates versUs the calculated concentration of SiO(OHh? are shown in Fig. lB. These results are also shown as Hanes-Woolf plots (Fig. lC). which linearize the Michaelis-Menten expression and set the x intercept equal to - K?? the y intercept equal to KJPmu. and the slope equal to Pm?? - As further evidence of the relationship between the K, and the concentration of SiO(OHh- we have estimated the K, values for Si uptake using the ca1culatedconcentrations offour possible substrates. total dissolved Si, undissociated Si(OH)4' SiO(OH)~- and SiO,z(OH)22.. (Table 1). Only for SiO(OHh- is there agreement of the K. values for the twO pH ranges. Dependence of enzyme ki? netics on the add/base chemistry of the substrate is well known (Segel 1976). The near equality of K. for two differem pH val? ues only when SiO(OH),- is considered the substrate could be interpreted to suggest that SiO(OHh- rath? er than Si(OH)~, is the substrate that binds to the Si-transport protein. However. on closer examina? tion a number of mechanisms not requiring initial binding of SiO(OHh- can be constructed which pre? dict the same experimental result. For example. when analyzed analogously to the original Michaelis-Men? ten derivation, the reactions: 50 40 30 '~ U) 20 10 0 -10 0 10 20 30 40 50 SiOIOH)3- (jJMI series both the K. and the Pm.. are lower than in the low pH series when total dissolved Si is considered as the substrate. Thus, at the high pH 5i uptake proceeds more rapidly at low concentrations. but less rapidly at high concentrations than at the lower pH. The differences between the K. in the two series are considerable. Using total Si as the substrate, the FIG. 1A-C. The ralt" of up~ke of Si by Phaf(ldflr()'{um (,.iro;-? nullll1l liS a function of substTlIlc:c:oncentration al two differellt pH r,mges. 8.6 (0) and 9.5 (,c,.). A) UpLake rate \'t'Tsu5lotal tf'".u.:tive silica. B) uptake rate versus calculated concentration of SiO(OH)~' ion. C) Hanes-Woolf plOI with SiO(OH}," ion as the suhsITatt'. 170 GERHARDT F. RIEDEL AND DAVID M. NELSON (7) Km is a dimensionless quantity analogous to the Mi? chaelis-Menten constant in the more simple Mi? chaelis-Ment~n derivation. and is by definition in? dependent of pH. The product (K,.,[H+]) has units of concentration and comparison ofequations 3 and 5 show that this product is equal to the experimen? tally obtained half-saturation constant (K.) when un? dissociated silicic acid is considered as the substrate. Thus, the observed K, must decrease with increasing pH. However, using the equilibrium relation be? tween 5i(OH)., 5iO(OHh- and H+ (eq. 1), this rate law can be rearranged to: P = 10-\147 K m + lSiO(OHh-J which has a half-saturation concentration for SiO(OHh- that does not vary with pH. Si uptake mechanisms that predict constant K, with varying pH when SiO(OHh- is considered the sub? strate can be constructed using any species of dis? solved 5i as the reacting substrate. However, they aU have in common the formation ofan E-5iO(OH)5? complex. Thus, SiO(OH)5- is not necessarily the species in solution that reacts with the Si transport protein, but our results indicate it may be the species transported across the cell membrane. Recent models for the observed dependence of metal uptake by phytoplank.ton on free ion activity also invoke mech? anisms in which, through ligand exchange, various organic and inorganic speCies of metals may actually react with the metal binding site, but only the metal ion is actually bound and/or transported (Anderson and Morel 1982). The advantage of such a mecha? nism to an organism is that it allows an uptake system to operate on substrates at far higher concentrations than would be the case if the mechanism used the apparent substrate. The hypothesis that 5iO(OHh- is the true sub? strate for 5i uptake depends on the assumption that the K. of the 5i-transport protein remains constant over the pH range S.8-9.7. While changing pH can change the K. of an enzyme by altering the active site or the tertiary structure, it is by no means certain over any particular pH range. For example, the digestive enzyme chymotrypsin exhibits a constant K. for the substrate acetyl-L tryptophane amide over the pH range 6.6 to 8.0, while the Vm.~ increases with pH due to noncompetitive inhibition by H+ (Bernhard 1968). In our case, the observed fit be? tween the change in the K. with pH and the disso? ciation of silicic acid could arise from coincidental changes in the K. with pH. However, we believe the probabilit~?ofsuch a fortuitous match to be far small? er than the probability of the K. for the true sub? strate remaining constant over a 1 pH unit interval. The Pm>i>< for 5i uptake is somewhat higher for the low pH series, The observed decrease in Pm?? at the higher pH could be interpreted in terms of a non? competitive inhibition by hydroxyl ion, once it is assumed that the K, for the true substrate (SiO(OHh-) is constant over the pH range (Segel 1976). However, Phaeodact.vlum tricornulunP responds to elevated pH with reduced growth (Hayward 1968), so the observed reduction in the maximum Si uptake rate may simply be t.he result ofa lower rate imposed on all assimilatory pathways at. high pH. Phaeodactylwntril.'ornutum is a diatom, albeit a very unusual one in that it does not require Si for growth, and its Si uptake system may reasonably be suspected to be evolutionarily related to that of Si-requiring diatoms. This would imply that Si-requiring diatoms may also show dependence of Si uptake parameters on pH. Azam et al. (1974) and Bhattacharyya and Volcani (1980) have shown that the rate of silicon uptake by Nituchia alba at fixed total Si concentra? tions increases with increasing pH over the pH range 4-9. Although kinetic parameters in their experi? ments cannot be determined at different pH values due to the experimental design. these results are consistent with a decrease of K. for 5i(OH)4 or total dissolved Si with pH as in our experiments. In the surface waters of the open ocean the ex? treme pH range is between 7.5-8.5 (Parsons and Takahashi 1973), while the total 5i Content of sur? face seawater is often dose to the measured K, for Si uptake for marine diatoms (Bainbridge I9S0, Nel? son et a1. 1981). If the 5i uptake systems of 5i-re? quiring diatoms have K, values that vary with pH as does that ofPhaeodactylum I'riconwluln, the variations of pH alone could have significant impact on the uptake rate of 5i and the growth rate of diatoms in the oceans. In freshwater, where pH ranges are greater, the pH dependence of Si uptake kinetics could have even more profound effects. If the K, of 5i uptake is proven to vary with pH for diatoms in general, this varia.ble must be included in future studies of the 5i uptake parameters of diatom pop? ulations in both the laboratory and the field. The authors thank Dr. J. R. Lara-Lara and R. Millan-Nune~ tor their help in earlier phases of this study. M. Brzezinski for the nonlinear kinetic curve fitting. and Drs. R. R. L. Guillard. F, M. M. Morel and P, A. Wheeler lor valuable discussions during manuscript preparation. HBF cOlltribution no. 434. Anderson, M. A. & Morel, F. M. M. 1982. The influence of aqueous iron chemistry on the uptake of iron by the coastal diatom TJU1.1as.no,lira U'i'usflogii. Limnol. Ouarwgr. 27:779-813. Azam, F., Hemmingsen, B. D, &: Volcani, B. E. [974. Role of silicon in diatom metabolism \'. Silicic add transporl and metabolism in the heterotrophic diatom .Vllzschia alba.?1rch. Mim,,10(.. 97:103-14. Bainbridge. A. E. 1980. GEOSECS Atlantic Expedition Volume 2: Senions and Profiles. National Science Foundation, U.S. Government Priming Office, Washington D.C. 198 pp. Bernhard, S. A. 1968. TIuI Structure and Fwutum of Enzymn. Benjamin. New York, 324 pp. Bhauachar)'Ya, P. & Volcani, D. E. 1980. Sodium dependent sili!:ate transport in th", apochlorotic marine diatom Nituchia alba. Prot. Nat.?'!.cad. Sci, USA. 77:6386-90. Dugdale, R.C. 1967. Nutrient limitation in the sea: dynamics. identification, and significance. Llmnl1/. Ouanogr. 12:685-95. Guillard, R. R. L. &: Ryther, J. H. 1962. Studies of marine phy- pH DEPENDENCE OF SI UPTAKE 171 loplanktonic diatoms t. CycloUl/alllll/lJ Hustedt, and Dt'lonu{1l corl/m.'Qcta,e (Cleve) Gran. Can. j. ,\fjerobiol. 8:229-39. Hayward.], 1968. Studies on the growth of Phatodactylurn lri? wYnutum. IV. Comparisons of different isolates. j. Mar. BiQI. Assoc. U.K. 48:657-66. Kilham, S. S., Kolt. C. L. &. Tilman. D. 1977. Phosphate and silicate kinetics for the Lake Mkhigan diatom Dilltoll/a pion? gatulll. Guat LaMs Rn3:93-9. Nelson, D. M. & Goering,]. J. 1977. A stable isotope method to measure silicic acid uptake by marine phytoplankton. :l.nll.l. Biochern.. 78:139-47, Nelson, D. M .? Goering, J. J. &. Boisseau, D. W. 1981. Con? sumption and regeneration of silicic acid in l.hree coastal upwelling s~'slems> In Richarrli, F. A. [Ed.] CCJaSlai UplJli'l/? mg. American Geophysical Union. Washington, D.C.? pp. 242-6. Nelson, D. M.. Goering. J. j., Kilham, S. S. Be GuiUard, R. R. L. . 1976. Kinetics of silicic acid uptake and rates of silica dis? solution in the marine diatom Tha/ajjiosim p....lldonana. j. Phycol. 12:246-52. Nelson, D. M., Riedel, G. F.. Millan-Nunez. R. &. Lara-Lara, 1. R. 1984. Silicon uptake by algae with no known 5i require? ment. 1. True cellular uplake and pH-induced precipitation j. Ph.w:o/. 21, 171-175 (1985) by PlIa~oducl)'iulll Irim1'ltutum (Bacillarjoph~:ceae) and PilIly? 1II0ll/B sp. (prasinophyccae).j. Phywt. 20: 141-7. Paasche, E. 1973. Silicon and the ecology of marine plankl.Onic diatoms 11. Silicon-uptake kinetics of five diatom species. Mar. Bioi. 19:262-9. Parsons, T. &. Takahashi, M. 1973. Biologlcal Orl'allogmph.( Pro? UHf5. Pergamon Press, New York. 186 pp. Segel. I. H. 1976. Biochemical Clllrulati01lJ. 2nd edition. John Wiley and Sons, New York. 441 pp. Sjoberg. S" Norden. A. & Nils,!. 19B\. Equilibrium and struc? tural studies of silicon(1 V) and a1umillum(IlI) in aqueous 50? lution.Mar. Chern. 10:521-32. StrkkJand,j. D. H. & Parsons. T. R. 1972. A Prar.lual Harldbooh ofStau'u tn A Ilaiym. 2nd ed. Fisheries Research Board of Can? ada, Ottawa, 310 pp, Stumm, W. & Mor!fomugh, Ontario Kg] 7B8 and Dat}id R. S. Lean National Water Research Institute, Box 5050, Burlington, Ontario L7R 4A6 ABSTRACT Chroococcoid Cl'tlnobac/erio. (0.7-1.3 IJ,rn in diamelfr) weT/, disco't'erfd to be a significan.t componen.t of thl' Lakl' Onto. rio plankton. lh'ing epifiuoresanu microscopy, the dmsities of these microorganisms wert found to vary b.v four orders ofmagnitude with a singll' largl' peak in abun? dance (6.5 x 10' cells mL -') corresponding to the time of maximum water temperature. The morphology and abun? dance of these cyanobacteria were similar to thos" previ? ously found in oceanic $)'5tl'ms. The)' constituted 10% of the bacterial numbers in the epilimnion during this period. approximatel)' 40% ofthe biomass ojprokaryotes les's than 2.0 IJ,m, a71d 30% of the bioma.u of all microorganisms less than 20 IJ,m in siu. Siufractionation studies indicated that the)' were mpomible for approximately 38% of the I Accepted: 29 Septnnber 1984. ? Address for reprint requests. total primary' production during lime~'lJfpeak abundancr. and u'ere imporlm~i i'n phosplron4j uptake. Cyanobacteria obsen1ed in the food l'acuolej of heterotrophic microfla? gellates and in the guts oj rotifen suggest that the latter organisms lila)' be important rom-Ulnl'TS 0/ this prokaryote population. Key index word.\: Lake Ontario plrmkton; cya flohacteria; microbial abundance Chroococcoid cyanobacteria (0.5-2,0 Ilm) are photosynthetic, prokaryotic microorganisms that have only recently been shown to be an important component of oceanic plankton communities Uohn? son and Sieburth 1979, Waterbury et al. 1979. Krempin and Sullivan 1981). They were overlooked in earlier studies because the conventional Uter? moh] settling technique for counting phytoplankton