Evolution of an Artificial Seawater Medium: Improvements in Enriched Seawater, Artificial Water Over the Last Two Decades 1

J. Phycol. 37, 1138–1145 (2001)

EVOLUTION OF AN ARTIFICIAL SEAWATER MEDIUM: IMPROVEMENTS IN ENRICHED SEAWATER, ARTIFICIAL WATER OVER THE LAST TWO DECADES 1

John A. Berges,2 Daniel J. Franklin School of Biology and Biochemistry, Queen’s University, Belfast BT9 7BL, Northern Ireland, United Kingdom and Paul J. Harrison Department of Earth and Ocean Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada

Although most phycologists use natural seawater Most phycologists who culture algae find it conve- for culturing marine species, artificial media con- nient to work with natural seawater (NW) as a base for tinue to play important roles in overcoming prob- their media. Nonetheless, issues of the varying quality lems of supply and seasonal variability in the quality of water through the year, the need to control nutri- of natural seawater and also for experiments involv- ent and trace element concentrations, and simply the ing manipulation of micro- and macronutrients. Sev- limited availability of seawater at inland locations eral artificial media have been developed over the make artificial seawater (AW) alternatives attractive. last 90 years; enriched seawater, artificial water Even where these limitations are not at issue, it is in- (ESAW) is among the more popular recipes. ESAW trinsically more satisfying to many researchers to ex- has the advantage of an ionic balance that is some- plicitly define the composition of their media so that what closer to that of normal seawater. The original experiments may be repeated. paper compared the growth of 83 strains of microal- The distinction between AW and NW media is not gae in natural seawater (ESNW) versus ESAW and straightforward. In theory, the difference simply de- determined that 23% grew more poorly in the artifi- pends on whether one starts with seawater from the cial water. Since 1980, however, the composition of field or distilled water plus mineral salts, but in prac- ESAW, as used by the original authors, has changed tice these criteria are difficult to apply (cf. McLachlan considerably. In particular, the added forms of phos- 1973). Both types of media share similar problems in phate, iron, and silicate have been changed and the that with respect to culturing requirements there are trace metal mixture has been altered to include scarcities of some elements (e.g. nutrients like N and nickel, molybdenum, and selenium. We tested P) and overabundances of others (e.g. heavy metals); whether these changes improved the ability of the ar- because of the measures taken to overcome these tificial medium to grow previously difficult to grow problems, none of the formulations used for culturing phytoplankton species. To test this, we selected eight can truly be considered “natural.” For seawater from species that had been shown to grow better in ESNW the field, addition of N, P, and Si are almost always than in ESAW and compared their growth again, us- necessary to prevent limitation in cultures, whereas ing the currently used recipe with all the above mod- nutrient-rich deep ocean waters can have imbalances ifications. For all but one species (Apedinella spinifera), between toxic and essential elements (e.g. Cu and Mn, growth rate and final yield was no different between see Sunda et al. 1981). In the case of ASW, there can the media but in one case (Emiliania huxleyi) was be shortages of essential elements in the basal salt mix- slightly higher in ESAW. No differences in cell mor- ture, omission of minor elements present in natural phology or volume were found in any case. We con- seawater, or contamination of reagent grade salts. For clude that changes to the enrichment portion of the both ASW and seawater from the field, the problem is recipe have significantly improved this artificial sea- to provide algae with levels of trace elements needed water medium and that it can be used to grow an to match major nutrients without causing toxicity. So- even wider range of coastal and open ocean species. lutions to this problem include addition of ingredients Key index words: artificial seawater; marine phyto- such as soil extracts or trace metals and vitamin mix- plankton; nutrient enrichment tures complemented with artificial chelators (e.g. EDTA) and organic buffers (e.g. Tris). Abbreviations: AW, artificial seawater; CS, citrate An enormous number of seawater media recipes substituted; EE, EDTA enhanced; ES, enriched sea- have been published. Their historical development water; LN, low nutrient; NW, natural seawater has already been well reviewed (see Harrison et al. 1980 and references therein). We did not consider those media that had been optimized for single species or particular growth phases (e.g. Ace 25 medium for Acetabularia acetabulum, Hunt and Mandoli 1996). 1Received 15 March 2001. Accepted 11 September 2001 We tried to determine how widely different media (in- 2Author for correspondence: e-mail [email protected]. cluding more recent modifications) have been used in

1138

CHANGES TO ARTIFICAL SEAWATER MEDIA 1139 i 0.01 0.001 0.01 0.01 0.01 (AK) L1 11.7 h 1000 (ESNW) K 0.05 0.05 2.42 5.97 g ESAW t al. (1957)]. ublished from 1981 to present. tions are given in brackets). Because some original e. f ASW 0.05–0.075 0.05 e 1.978 0.201 0.002 0.56 200 61.46 0.0005 Yes GPM d M). 0.248 0.021 0.01 Modified VS c VS (Grund) b f/2 a 1.765 0.883 0.50.08 5 0.040.7 0.55 0.3 0.9 0.04 — 0.883 2.9 0.7 0.01 0.01 0.3 1.5 0.4 0.4 f 1575 506 21 81 75[74] 293[121] 258 156 20 M) O 2 6H ) O 11.7 100 0.103 14.9 100 11.7 O 0.05 0.03 0.03 0.03 0.09 4 4 2 2 O 0.072 0.036 0.03 0.015 0.03 O 0.16 0.05–0.1 0.1 0.105 0.05 0.105 2 2 O O O 0.15 0.08 0.08 0.25 0.08 0.08 2 O 1.8 0.9 0.1 100 0.02 0.9 0.9 0.9 O110 O 2H 2 2 O 2H H 2 2 2 O 11.7 0.005 11.7 0.59 11.7 2 (SO O 0.08 0.05 0.05 0.05 2 9H 2 4 4 2 4H ) 7H 4 7H 3 4H 4 7H 6H 4 3 4 j H 6H 3 4 6H 3 4 4 4 2 PO 4 4 3 2 2 2 2 3 Cl 1. Characteristics of commonly used seawater media for algae. 4 glyceroPO SiO MoO VO EDTA 2 2 2 3 2 BO SeO elements (mM) chelators, and buffers ( HPO CrO 3 2 2 2 12 Keller et al. (1987) (AK salt base described in the same paper). Guiry and Cunningham (1984). Guillard (1975). Harrison et al. (1980). General Purpose Media, Sweeney et al. (1959) [Loeblich (1975); Blackburn (1989); uses the metal solution of Provasoli e Guillard and Ryther (1962). von Stosch (1963). Artificial Seawater Medium, McLachlan (1964) [Goldman and McCarthy (1978)] (also includes recipe for salt base). Guillard and Hargraves (1993); L2 (Guillard 1995) (same as L1 except EDTA raised to 90 From the Institute for Scientific Information Web of Science database, which records citations from over 5700 major journals p Fe(NH Thiamine-HClBiotinB 593 4 296 — 2 5929 — 2965 4093 296 8.2 297 2 300 4.1 300 2.1 2.1 MnSO NaNO MnCl K CuSO Na NiSO CoCl H NaH ZnCl FeSO Na NH Na CoCl H FeCl K FeEDTAEDTANa 23.3 10 CoSO KNO ZnSO Na Citations are based on the number listed in ISI Web of Science citations as February 2001 (citations popular modifica a b c d e f g h i j manuscripts present data other than the description of medium itself, this represents a potential overestimate true usag Citations Soil extract Vitamins (nM) Tris Table Intended useSeawater sourceMajor nutrients and Broad spectrum Broad spectrum Macroalgae Macroalgae N Dinoflagellates Broad spectrum Broad spectrum Oceanic species Diatoms N N N N A N (A) N (A) N Metals, minor elements,

1140 JOHN A. BERGES ET AL. the past 20 years by using a database (Institute for Sci- compiled. Bearing in mind these limitations and fo- entific Information Web of Science, Thomson Scien- cusing on the more recent literature, it is clear that rel- tific, accessed through www.mimas.ac.uk) to search for atively few media are widely used and the composi- citations of the major seawater media papers. Weak- tions are similar (Table 1). nesses in this analysis include tendencies for authors However, beyond problems in correct attribution, to cite papers that make minor modifications to older there is a more serious problem with media: each re- papers (e.g. Guiry and Cunningham 1984 vs. von search group is likely to make numerous substitutions Stosch 1963), to cite their own works which in turn cite and modifications to media based on their own pref- the original papers, to miscite papers (e.g. McLachlan erences and experiences. This can be illustrated for is regularly miscited as “MacLachlan”), or to cite reci- the Harrison et al. (1980) medium. The medium was pes compiled in secondary sources (e.g. McLachlan in based on an AW that closely matched the ionic com- Stein 1973 or Guillard in Smith and Chanley 1975) position of seawater (Kester et al. 1967) and an en- and so lose the original citations. As just one example, richment (Provasoli 1958) that the authors believed Provasoli’s most important media-related publications offered more balanced macronutrient concentrations (Provasoli et al. 1957, describing the second of his ASP and chelation than other published alternatives. The media; Provasoli 1958, in which enriched seawater original paper showed that for 64 of 83 species tested, [ES] medium is described, and Provasoli 1968, which growth was as good in ESAW as it was in ESNW. Over reviews the two media recipes) have received over 600 the 20 years since its publication, numerous minor citations, but this dropped drastically after publication changes to the medium have been made; Table 2 of McLachlan (1973), in which the three recipes were summarizes these changes. The original AW recipe

Table 2. ESAW medium as modified from Harrison et al. (1980) and ESAW medium over the past two decades. Footnotes highlight the changes from the original recipe.

gL1 stock solution Final concentration in media Salt solution I—anhydrous salts a NaCl 21.19 363 mM Na2SO4 3.55 25.0 mM KCl 0.599 8.04 mM NaHCO3 0.174 2.07 mM KBr 0.0863 725 M b H3BO3 0.0230 372 M NaF 0.0028 65.7 M Salt solution II—hydrated salts a MgCl2 6H2O 9.592 41.2 mM CaCl 2H O 1.344 9.14 mM 2 2 SrCl2 6H2O 0.0218 82 M Major nutrient I—nitrate [1 mLL1] NaNO3 46.7 549 M Major nutrient II—phosphate [1 mLL1]c NaH2PO4 H2O 3.09 21 M Major nutrient III—silicate [2 mLL1]d Na2SiO3 9H2O 15 105 M Metals stock I—iron [1 mLL1]e FeCl 6H O 1.77 6.56 M 3 2 Na2EDTA 2H2O 3.09 6.56 m Metals stock II—trace metals [1 mLL1]f ZnSO4 7H2O 0.073 254 nM CoSO 7H O 0.016 5.69 nM 4 2 MnSO4 4H2O 0.54 2.42 M g 3 Na2MoO4 2H2O 1.48 10 6.1 nM g 4 Na2SeO3 1.73 10 1 nM NiCl 6H Og 1.49 103 6.3 nM 2 2 Na2EDTA 2H2O 2.44 8.29 M Vitamin stock [1 mLL1]h Thiamine-HCl 0.1 297 nM Biotin 0.002 4.09 nM B12 0.001 1.47 nM a Anyhydrous and hydrated salts must be dissolved separately; masses assume specific gravity 1.021 at 20C. b Borate now added only in salt solution, not in trace metals. c New phosphate source removes the organic glycerophosphate. d Silicate stock solution is not acidified and is made at half previous strength to facilitate dissolution. e Iron now added solely as chloride (removes ammonium) in a separate stock with equimolar EDTA. f Trace metal stock now made 10 original concentration. EDTA must be dissolved first. For convenience, each metal can be added at 10 mLL1 from individual 100 concentrated stocks. g New addition to recipe. h Filter sterilize; store frozen. CHANGES TO ARTIFICAL SEAWATER MEDIA 1141 has been maintained exactly as in the original paper, had previously been shown to grow well on both NW and AW. but the nutrient stocks have changed substantially, as Since 1980, all of the NEPCC clones used have been maintained on ESNW. noted in the footnotes. Silicate stocks are now pre- Culture conditions. Growth experiments were all performed pared and added without acidification to prevent po- between May and December 1992. Cultures were grown in 50-mL lymerization, which would tend to make the silicate borosilicate screw-cap glass tubes with Teflon cap liners. Condi- less available for phytoplankton (Suttle et al. 1986). tions were maintained as in Harrison et al. (1980), that is, cultures were grown at 16 1 C under 50 5 mol quanta Other additions have become necessary because the m 2s 1 irradiance, provided on a 14:10 light:dark cycle. Cul- quality of reagent grade salts has steadily improved to tures were gently inverted to mix cells twice daily. Cultures were the point where trace contamination is no longer suf- acclimated for a minimum of eight generations in NW (see be- ficient: nickel (essential for the metabolism of urea, low) before experiments were performed. Price and Morel 1991), molybdate, and selenium (re- Growth and cell size measurements. Growth rates were moni- tored daily by in vivo fluorescence and measured directly in quired by a wide variety of species; Price et al. 1987, 50-mL tubes, using a Turner Designs Model 10-AU (Turner De- Harrison et al. 1988) are now added. The combined signs, Sunnyvale, CA) fluorometer. Preliminary experiments effects of these changes and additions to the original on the species selected showed that exponential growth rates recipe on the overall quality of the medium is un- (, d 1) calculated from fluorescence measurements were iden- tested, and their usefulness in culturing species from tical to those based on cell counts (see below) during the loga- rithmic growth phase. Cell counts and cell volume determina- various taxa has not been assessed. tions were performed using a model TAII Coulter Counter In this study we evaluate the effects of the modifica- (Beckman Coulter, Palo Alto, CA) equipped with a population tions and additions. Rather than simply testing the accessory. Depending on the size of the species used, either a newer ESAW against the original ESAW, we chose to 70-m or a 200-m aperture was used; the instrument was cali- brated with 5-m or 40-m latex microspheres, respectively. In compare the AW with NW using the new modifica- cases where measurements were made on samples preserved tions in both cases. This allowed us to test some of the with acid Lugol’s iodine, volumes were corrected for shrinkage same species that more poorly in their original ESAW according to Montagnes et al. (1994). versus ESNW (Harrison et al. 1980) and thus address Seawater treatments. Seawaters were prepared from either the issue of AW versus NW more directly. NW or AW with identical enrichments (ES). Natural seawater was obtained from a deep (50 m) intake at the Department of We also explored two other issues raised more re- Fisheries and Oceans Laboratory, West Vancouver. The water cently in the literature: the effects of chelation and was aged in linear polyethylene containers for several weeks be- nutrient concentrations. Conflicting concerns suggest fore use; this is the water normally used for maintenance of the that EDTA concentrations might not be optimal. The NEPCC. AW was prepared as described in Harrison et al. (1980), with the modifications described in Table 2. Chemicals chelator is toxic to some species (see Harrison et al. were American Chemical Society reagent grade and were ob- 1980), whereas others actually benefit from addition of tained from BDH Ltd. (Poole, England), Fisher Scientific (Chi- EDTA at up to 10:1 (mol:mol) with metals (see Keller cago, IL), or Sigma Chemical Co. (St. Louis, MO). et al. 1987). To examine this, we used media with ei- Three additional seawater treatments were prepared and ther 10-fold greater EDTA or with EDTA replaced with tested on all species. These were identical to ESAW except (1) nutrients were lowered 25-fold, except for P and Si, which were citrate. The nutrient concentrations typically used in adjusted to 2 M and 22 M, respectively (low nutrient [LN]AW); enriched media are orders of magnitude greater than (2) EDTA was substituted with equimolar citrate (added as a so- those found in nature, and there is evidence that this dium salt; citrate substituted [CS]AW); or (3) EDTA was in- may affect growth of some species (see Keller et al. creased 10-fold (EDTA enhanced [EE]AW). All media were sterilized by filtration through a 147-mm Mil- 1987). To test this, we also used a medium with 25-fold lipore GS filter (Millipore, Corp., Bedford, MA) (pore size 0.22 lower nutrients than what is normally found in ESAW. m), with a Gelman A/E prefilter (Pall Corp., Ann Arbor, MI). Experimental design and statistical analyses. Three replicate cul- materials and methods tures of each species were inoculated from logarithmically grow- Species selected. Based on the results of Harrison et al. (1980), ing cultures acclimated to light and temperature conditions. In- nine species were selected for experiments. All species were ob- oculations were made by adding 1 mL of culture into 40 mL of tained from the Northeast Pacific Culture Collection (NEPCC, each of the five media. Cultures were allowed to grow until a pla- University of British Columbia, Vancouver, www.ocgy.ubc.ca/ teau in fluorescence was reached, diluted 1:40 into fresh me- projects/nepcc/index.html; clones are designated in Table 2) dium, and allowed to grow to a plateau once again. At the sec- and where possible are the same clones used by Harrison et al. ond plateau, samples were taken for cell counts and volume (1980). Six species were chosen that had previously been shown determinations. Cultures assigned different seawater treatments to grow more poorly in AW than in NW. Of these, Apedinella were grown simultaneously, and the position of cultures with re- spinifera had shown deterioration of cells (loss of motility, spect to banks of lights was assigned randomly. shape, and color) in AW versus NW, whereas Phaeocystis pouchetti, Analyses of variance were performed on growth rates (calcu- Chrysochromulina ericina, Imantonia rotunda, Gymnodinium sim- lated based on changes in fluorescence over the period of loga- plex, and Scrippsiella trochoidea had shown 70%–90% reduction rithmic growth), the final fluorescence reached at the plateaus, in final cell yield in AW versus NW (Harrison et al. 1980). All and on the cell counts and cell volumes found at the second these species were isolated from Station P (4926N, 13640W) plateau. The two growth curves for each replicate were treated in March 1976, except S. trochoidea, which was isolated from En- as blocks in the analysis. Where significant differences were glish Bay, British Columbia in August 1972 (see Harrison et al. found (P 0.05), they were examined using Tukey’s multiple 1980). Two additional species that isolated more recently from comparison techniques (SYSTAT, Wilkinson 1990). oceanic sites and that had not been grown extensively in AW previously were used: Emiliania huxleyi (isolated from Station P results in June 1991) and Gyrodinium galatheanum (isolated from Sta- tion P in August 1984). The diatom Thalassiosira pseudonana (iso- All species grew in all types of media tested. When lated in September 1958) was included as a control, because it cultures were examined microscopically, no obvious 1142 JOHN A. BERGES ET AL.

For the other media treatments, there were no significant differences detected between EEAW and ESAW in any parameter for any species (Fig. 2, illustrative data shown only for A. spinifera). Growth rates for CSAW were not significantly different from ESAW in any case, but maximum fluorescence and maximum cell numbers reached in stationary phase were significantly lower in CSAW than in ESAW for T. pseudonana, A. spinifera, and I. rotunda (Fig. 2, data shown only for A. spinifera). In these three species, significantly larger cell volumes were also found in CSAW versus ESAW, suggesting that clumping may have occurred. LNAW gave the lowest maxima in fluorescence and cell numbers in all cases; however, there were no differences in growth rates over the exponential phase between ESAW and LNAW (Fig. 2, data shown only for A. spinifera). Based on cell numbers, yields were typically two to five times lower in LNAW than in ESAW (Fig. 2, data shown only for A. spinifera), and nitrate was undetectable in all LNAW cultures.

discussion Effects of modifications since initial publication. From our results, it appears that the changes made to ESAW following the original publication have significantly improved the medium; only one of the species that was previously shown to grow more poorly in ESAW than ESNW showed this in the new ESAW. It is impossible to say which of the changes (switch to inorganic P, alter- Fig. 1. Growth curves for three species of marine phytoplankton, (A) Chrysochromulina ericina, (B) Apedinella spinifera, ation of iron form, stopping acidification of Si stocks, and (C) Emiliania huxleyi, grown in ES with a natural (NW, ) increased chelation with EDTA, or addition of Ni, Mo, or an artificial water (AW, ) base. Cultures were grown at a and Se) have produced better growth in the species mean irradiance of 50 mol quantam 2s 1 and 16 C. Fluores- that grew poorly in the original medium; indeed, it cence was measured in a Turner Model 10 fluorometer. Each point represents the mean of three replicate cultures. Error may even depend on which species is considered. bars represent standard errors of the mean of three cultures. The history of ESAW is just one example of how The drop in fluorescence between days 10 and 15 corresponds changes to a medium can accumulate over many to a 1:40 dilution of the cultures with fresh medium. years. How can researchers be kept informed of such changes? One obvious method is through continual on-line updates of recipes, maintained by major culture collections. Several culture collections already ef- differences were seen between cells grown on any of fectively do this (e.g. NEPCC, the Culture Collection the five media, although there was some tendency for of Algae and Protozoa, and the Provasoli-Guillard Na- cells to clump in CSAW treatments. tional Center for Culture of Marine Phytoplankton). Typical growth curves are shown for three species It then falls to individual researchers to carefully spec- in ESNW versus ESAW (Fig. 1). Comparing ESNW ify which variations in the medium they have used. Ul- and ESAW, there were few differences in growth rate: timately, culture collections could consider creating growth rates of A. spinifera were significantly higher in databases that record which medium variations work ESNW, whereas E. huxleyi grew significantly faster in best for a certain species, and they could also allow re- ESAW (Fig. 1, B and C; Table 3). In terms of culture searchers to record their own observations on-line. yield at the plateau, fluorescence and cell number Nutrient concentrations and culture limitation. From Ta- measurements correlated well, but variations in cell ble 1, it is clear that artificial and enriched media numbers were usually greater than those of fluores- frequently have much higher nutrient concentrations cence, because of more limited replication (Table 3). than are typically found in marine environments. For eight of the nine species, fluorescence and cell There is speculation that such high nutrient levels are yields were the same or greater in ESAW than for detrimental to some species, particularly those iso- ESNW (Table 3, Fig. 1). In one species, Gyrodinium lated from oceanic environments (e.g. Keller et al. galatheanum, cell yield was significantly lower in ESAW 1987); these species may benefit from reduced nutri- versus ESNW. No differences in cell volumes were ent and trace metal concentrations. However, this found for any species. does not seem to be the case for the two more recent CHANGES TO ARTIFICAL SEAWATER MEDIA 1143

Table 3. Comparison of growth rate (), the maximum cell number and fluorescence achieved in stationary phase, and the cell volume in stationary phase of nine species of marine phytoplankton in ES with a natural or artificial seawater base.

Species Clone Seawater base (d1) Maximum fluorescence Maximum cell number (105) Cell volume (m3) Phaeocystis pouchetti PM225 Natural 0.72 0.01 76 15 1.69 0.60 27.9 1.4 (Hariot) Lagerh. Artificial 0.70 0.01 105 18 5.45 0.99 24.7 0.4 Gymnodinium simplex D119 Natural 0.42 0.02 25 6 0.31 0.02 187 3.8 (Lohm.) Kofoid & Swezy Artificial 0.44 0.01 30 5 0.44 0.06 199 5.2 Chrysochromulina ericina PM109 Natural 0.77 0.02 54 5 3.16 0.70 22.4 4.4 Parke & Manton Artificial 0.82 0.03 92 7 3.51 0.27 20.7 1.9 Imantonia rotunda PM226 Natural 0.58 0.01 179 15 27.1 2.6 8.5 0.2 Reynolds Artificial 0.65 0.01 258 18 68.4 4.9 8.6 0.4 Emiliania hyxleyi (Lohm.) PM732 Natural 0.56 0.03 93 17 10.3 3.1 15 0.4 Hay & Mohler in Hay et al. Artificial 0.62 0.02 168 17 20.1 3.7 14.2 0.2 Apedinella spinifera CR451 Natural 0.59 0.01 72 3 2.5 0.1 83.4 1.8 Throndsen Artificial 0.49 0.03 58 9 2.47 0.2 92.5 0.8 Gyrodinium galatheanum D555 Natural 0.33 0.02 45 4 0.46 0.04 950 28 Kofoid & Swezey Artificial 0.31 0.02 37 6 0.26 0.04 888 24 Scripsiella trochoidea D15 Natural 0.30 0.02 26 7 0.18 0.06 2681 94 (Stein) Loeblich II Artificial 0.32 0.02 35 6 0.22 0.03 2506 125 Thalassiosira pseudonana B58 Natural 1.05 0.02 97 6 14.8 2.5 20.9 0.4 (Hust.) Hasle & Heimdal Artificial 1.09 0.01 125 4 18.8 1.6 25.9 1.6 Clone numbers refer to isolates held in the North East Pacific Culture Collection. Values presented are means standard error of measurements in three independent cultures over two growth cycles. Figures in bold indicate where statistically significant differences (P 0.05) exist between seawater bases. oceanic isolates we have examined in our study; we sities achieved in ESAW and ESNW were high enough found no differences between ESAW and LNAW, ex- to suggest that light limitation may have been involved, cept in final yields. Interestingly, Keller et al. (1987) and pH in selected measured cultures approached chose to use both ammonium and nitrate in their K 9.0, which would indicate potential CO2 limitation media for oceanic species and also noted that some (see Riebesell et al. 1993). We have observed in an ex- species show toxic effects if ammonium is greater traordinary number of published studies in which than 25 M. It is possible that part of the reason why cells are sampled in the plateau phase that the limit- some species appear to prefer lower nutrients may ing factor is not specified. Clearly, this makes compar- simply relate to ammonium toxicity in those media to isons of measurements taken in the plateau phase which ammonium has been added. nearly impossible because the type of nutrient limita- The advantage of AW is that one can precisely con- tion/starvation (N, P, Si, or some trace metal) will in- trol nutrient ratios and therefore the type of nutrient fluence the chemical composition and the physiology limitation/starvation at the end of the growth phase of the cells in the plateau (early senescence) phase. in batch cultures. Unfortunately, experimentalists usu- Water sources and reagent grade salts. It is beyond the ally pay little attention to the N:P or N:Si ratios in the scope of this article to give a detailed review of culture medium they are using, which will ultimately deter- methods, but some reminders of good practice are mine which nutrient will limit growth and influence useful. The source of distilled water is a critical factor; chemical composition and physiological rates when there are many variations in distillation and deioniza- the cells reach senescence. Based on the Redfield N:P tion procedures. Between our laboratories, we have ratio of 16 for the average phytoplankter, P limitation had good success in growing marine species using ei- should occur first in normal ESAW, F and K media ther a combination of glass distillation and resin-bed (Table 1, Fig. 3). ASW and GPM should exhaust N deionization or a MilliQ system (Millipore Corp.). Some first, whereas VS medium is the only one listed that ac- vigilance is necessary because column life can vary tually balances N and P in a 16:1 ratio. However, for considerably, depending on fluctuations in the quality diatoms one must also consider the N:Si ratio. Brzez- of the source water. inski (1985) demonstrated a wide range of N:Si ratios, Use of reagent grade salts is important; general but often the N:Si ratio is near 1:1. If this is true, then purpose grade salts contain significant levels of poten- Si would be exhausted before N in all Si-containing tially toxic heavy metals. Trace metal analysis of the media except ASW, and this is only because of the AW base (performed using column-stripping and in- rather low N specified in the recipe (Table 1, Fig. 3). duction-coupled plasma mass-spectrometry; see Ori- For LNAW cultures with nutrients 1/25th of normal ans and Boyle 1993) used in these experiments re- and P and Si adjusted upward, N would be expected vealed 2.5 M Pb and 0.13 M Cd was added in the to be exhausted first, and this was indeed observed. salts themselves. Although this can be of some benefit However, yields (in terms of cell numbers) were only to the medium (e.g. it is not necessary to add Cu be- two to five times lower than full enrichments. This cause a background level of 4–5 M is added with the clearly suggests that factors other than N or P were salts), it can also cause difficulties; Se additions only limiting in the fully enriched (ES) cultures. The den- became necessary when the purity of salts changed 1144 JOHN A. BERGES ET AL.

Fig. 3. Ratios of nitrogen to phosphorus (A) and nitrogen to silica (B) (by atoms) found in six commonly used seawater media (described in Table 1). Abbreviations are as in Table 1. Dotted lines represent typical N:P ratios for most phytoplankton species (16:1) and N:Si ratios commonly found in planktonic marine diatoms (1:1).

grade tubing such as latex and Tygon tubing can be toxic to some phytoplankton (see Price et al. 1986). Prospects for further improvements to seawater media. One difficulty in trying to refine and improve seawater media is that many critical elements in seawater remain difficult to measure. This includes not only things like trace metals but also organic compounds. Such com- Fig. 2. Rates of growth (A), maximum fluorescence in station- pounds are abundant in ocean waters, and they are ary phase (B), maximum cell numbers in stationary phase (C), only beginning to be quantified (e.g. McCarthy et al. and cell volume in stationary phase (D) for cultures of Apedinella 1997); it is already clear that they are reactive in such spinifera grown in ESNW or ESAW or in LNAW, CSAW, or 10-fold environments (Amon and Benner 1994). Organics re- higher EEAW (see Materials and Methods). Other conditions are as described in Fig. 1. Bars represent means of three replicate cul- main the significant “mystery” ingredient in media us- tures and error bars represent standard errors of means. ing “soil extract” (see McLachlan 1973). In this study, we used reference species in culture and found clear improvement of an artificial me- due to changes in the manufacturing process or dium. It is clear, however, that the best test of an artifi- changes in suppliers (cf. Provasoli and Pintner 1960). cial medium will be its ability to isolate and maintain For these reasons, it is particularly important that new species. We are just beginning to appreciate the well-defined media such as Aquil (Price et al. 1988) physiological diversity of phytoplankton from diverse are used for trace-metal limitation experiments. marine environments, and versatile media such as Sterilization and culture materials. In this study, we ESAW will help us to explore this diversity in the labo- elected to sterilize our media using filtration. This ratory. avoids certain issues of coprecipitation of ions due to pasteurization or autoclaving, but there are also meth- Supported by the Natural Sciences and Engineering Research ods available to lower the pH during the autoclaving Council, Canada. We thank Steve Ruskie for technical assis- process to prevent precipitation (see Harrison et al. tance and Maureen Soon and Dr. Kristin Orians (Earth and 1980). Use of buffers such as Tris may also help to Ocean Sciences, UBC) for metals analysis of artificial seawaters. control the pH and thus prevent precipitation (see Keller et al. 1987), but Tris may be toxic to some spe- Amon, R.M.W. & Benner, R. 1994. Rapid cycling of high-molecu- cies (see Harrison et al. 1980) and can serve as an or- lar-weight dissolved organic matter in the ocean. Nature ganic substrate for bacteria (e.g. Fabregas et al. 1993). 369:549–52. Furthermore, it is important to note that many Blackburn, S., Hallegraeff, G., & Bolch, C. H. J.1989. Vegetative re- common materials in the laboratory can be toxic to production and sexual life cycle of the toxic dinoflagellate Gymnodinium catenatum from Tasmania, Australia. J. Phycol. phytoplankton species. The problems associated with 25:577–90. various rubbers and plastics have been long appreci- Blankley, W. 1973. Toxic and inhibitory materials associated with ated (see Bold 1942, Blankley 1973), but even food- culturing. In Stein, J. [Ed.] Handbook of Phycological Methods: CHANGES TO ARTIFICAL SEAWATER MEDIA 1145

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