MEDIA

PAULJ. HARRISON AMCE Program, Hong Kong University of Science and Technology

JOHN A. BERGES Depament of Biological Sciences, University of Wisconsin-~Milwaukee

1 .O. Introduction Natural seawater (NW) is a complex medium contain- I. I. Historical Perspective on Previous Media and ing more than 50 known elements and a large and vari- ~ecentAdvances able number of organic compounds. For algal culture, 2.0. Materials Required direct use of NW is seldom acceptable. Without the 2.1. Chemicals addition of further nutrients and trace metals, the yield 2.2. Equipment, Glassware, and Tubing of algae is usually too low for culture maintenance or 2.3. Water Sources. Treatment, and Storage laboratory experiments, and thus enrichment is nor- 3.0. Stock Solutions mally required. In addition, variations in the quality of 3.1. Macronutrients (Nitrogen, Phosphorus, and Silicon) NW throughout the year, the need to control nutrient 3.2. Trace Metals and trace element concentrations, and the limited avail- 3.3. Vitamins ability of seawater at inland locations make the option 3.4. pH Buffers of artificial seawater (AW) attractive (see Section 4.2). 3.5. Chelators The preparation of NW with an enrichment solution 3.6. Soil Extracts of nutrients, trace metals, and vitamins and the prepa- 3.7. Germanium Dioxide ration of AW are described in this chapter. For clarity, 4.0. General Methods of Media Preparation we use the term natural seawater to refer to unenriched 4.1. Natural Seawater NW, and artijicial seawater for unenriched AW. The 4.2. Artificial Seawater term enrichment solution refers to the macronutrients, 5.0. Media trace elements, and vitamins that must be added to both 5.1. Artificial Seawater Media NW and AW to produce a substantial algal yield. The 5.2. Natural Seawater Media materials required for preparation of seawater media 6.0. Comparing Recipes Based On Algal Response and the main recipes of stock solutions of macro- 7.0. References nutrients, trace elements, and vitamins are described. Methods and precautions that are required in media Key Index Words: Marine Phytoplankton, Culture preparation are covered. Recipes for certain algal Media, Artificial Seawater, Nutrient Enrichments, groups and various natural and synthetic seawater Trace Metals, Vitamins recipes are compared. The recipes have been tested for

Copyright O 2005 by Academic Press Algal Cuhrcring Tcrbniques All rights of reproduction in any form resewed. CHAPTER 3

PAULJ. HARRISON AMCE 1?rogram, Hong Kong University of Science and Technology

JOHNA. BERGES Depal-tment of Biological Sc iences, University of Wisconsin-Milwaukee

CONTENTS 1.0. INTRODUCTION

1 .O. Introduction Natural seawater (NW) is a complex medium contain- I.I. Historical Perspective on Previous Media and ing more than 50 known elements and a large and vari- ~ece't Advances able number of organic compounds. For algal culture, 2.0. Materials Required direct use of NW is seldom acceptable. Without the 2. I. Chemicals addition of further nutrients and trace metals, the yield 2.2. Equipment, Glassware, and Tubing of algae is usually too low for culture maintenance or 2.3. Water Sources, Treatment, and Storage laboratory experiments, and thus enrichment is nor- 3.0. Stock Solutions mally required. In addition, variations in the quality of 3.1. Macronutrients (Nitrogen, Phosphorus, and Silicon) NW throughout the year, the need to control nutrient 3.2. Trace Metals and trace element concentrations, and the limited avail- 3.3. Vitamins ability of seawater at inland locations make the option 3.4. pH Buffers of artificial seawater (AW) attractive (see Section 4.2). 3.5. Chelators The preparation of NW with an enrichment solution 3.6. Soil Extracts of nutrients, trace metals, and vitamins and the prepa- 3.7. Germanium Dioxide ration of AW are described in this chapter. For clarity, 4.0. General Methods of Media Preparation we use the term natural seawater to refer to unenriched 4. I. Natural Seawater NW,and altificial seawater for unenriched AW. The 4.2. Artificial Seawater term e7zrichment solzdtion refers to the macronutrients, 5.0. Media trace elements, and vitamins that must be added to both 5.1. Artificial Seawater Media NW and AW to produce a substantial algal yield. The 5.2. Natural Seawater Media materials required for preparation of seawater media 6.0. Comparing Recipes Based On Algal Response and the main recipes of stock solutions of macro- 7.0. References nutrients, trace elements, and vitamins are described. Methods and precautions that are required in media Key Index Words: Marine Phytoplankton, Culture preparation are covered. Recipes for certain algal Media, Artificial Seawater, Nutrient Enrichments, groups and various natural and synthetic seawater Trace Metals, Vitamins recipes are compared. The recipes have been tested for

Copyright O ZOOS by Academic Press Algal Cnltco-ing Techniqzrer All rights of reproduction in any form reserved. planktonic algae, but reports indicate that the com- organic compounds that sequester potentially toxic monly used recipes are also suitable for benthic diatoms metals, and organic compounds that keep iron in solu- and some seaweeds. tion. In replacing soil extracts, numerous trace elements Comparing today's marine culture media with those and vitamins are usually added to culture media. These of 30 years ago (McLachlan 1973), it is interesting to include iron, manganese, zinc, cobalt, copper, molybde- see the progress that has been made and that several num, vitamin B12,thiamine, and biotin. Artificial chela- media are quite broad-spectrum, indicating that most tors such as EDTA (ethylenediaminetetraacetic acid) are culturable algae can be grown by using only a few dif- added to keep iron in solution and to keep free ionic ferent media. The most challenging phytoplankton to metal concentrations at nontoxic levels. culture are still the oceanic species. The history of the development of defined media was largely learning how to dispense with soil extract. The historical development of artificial media has been thor- oughly reviewed (Provasoli et al. 1957, Kinne 1976). 1.1. Historical Perspective on Previous One of the first attempts to design an AW medium for Media and Recent Advances algae began about 90 years ago (Allen and Nelson 1910). More extensive analyses of NW (Lyman and Many basic media concepts that are used today were Fleming 1940) stimulated the development of new developed in the late 1800s and early 1900s (for a review recipes (Chu 1946, Levring 1946) that attempted to see Allen and Nelson 1910; Allen 1914). Early workers imitate NW precisely, but they were frequently consid- soon learned that the ratios of chemicals were not ered too complex. Because these recipes were similar to always critical, and subsequently various media recipes NW, they had the same defect, namely the formation were developed with only slight modifications. It was of a precipitate during autoclaving when they were well known that chemicals that were added to seawater enriched. Autoclaving drives carbon dioxide out of the contained impurities such as trace elements, and these seawater, causing a shift in the carbonate buffer system. often improved growth (Allen and Nelson 1910). The The resulting pH of around 10 causes the precipitation importance of culture pH, iron, vitamins for culture of ferric phosphate and ferric hydroxides. The amount growth, and the avoidance of metal toxicity and impu- and composition of the precipitate varies, often leading rities in distilled water also were established very early to inconsistent growth in the medium. Preoccupation (Allen and Nelson 1910; Allen 1914). with making a complete medium autoclavable without NWmay be the preferred seawater base if large quan- precipitation led to the following extensive modifica- tities are required, if a good source is really available, or tions in recipes: if open ocean species are being cultured in the laboratory. However, for near-shore sources, the salinity may vary 1. Addition of synthetic metal chelators such as seasonally and large phytoplankton blooms may alter the EDTA or nitrilotriacetic acid (NTA) to decrease organic compounds in the seawater. To enhance the algal metal precipitates yield, various additions must be made to the NW. In the 2. Addition of a pH buffer such as Tris or early 1900s, boiling water was used to extract unknown, glycylglycine (7 .O-8.5 range), because the amount variable amounts of inorganic and organic compounds of precipitate increased as the pH rose during from soil and when it was added it produced good autoclaving growth with few morphological changes during the 3. Reduction in salinity, thereby reducing the amount long-term maintenance of algal cul~res.Allen (1914) of salts available for precipitation concluded that organic substances were required in 4. Replacement of Mg2+and Ca2+with more soluble trace amounts. Soil extract, originally introduced by univalent salts Pringsheim (1912), was established in the marine 5. Replacement of inorganic phosphorus with an culture methodology by Foyn's (1934) now famous organic source (e.g., sodium glycerophosphate) to "Erdschreiber" medium (note that this name was origi- avoid the precipitation of Ca3(P04)2(Droop 1969) nally written as Erd-Schreiber and that the original 6. Introduction of weak solubilizers, which are acids recipe was derived from Schreiber [1927]). We now (e.g., citric acid) having highly soluble salts with know that soil extract performs numerous functions in calcium culture media, and it has largely been replaced by specific compounds. Soil extract provides various elements and As a result of these extensive chemical modifications in vitamins needed for plant growth, metal complexing by ion ratios (Provasoli et al. 1957), few early recipes bore much resemblance to NW. Various simplified AW on 83 strains (Harrison et al. 1980). The AW base was recipes were developed (Provasoli et al. 1957, taken from Kester et al. (1967), and the ratios of the McLachlan 1959, 1964), but many of these recipes major ions closely match those found within NW. could grow only a few species or favored a particular The enrichment solution was a modified enrichment group of algae (Provasoli et al. 1957, Kinne 1976). More solution originally developed by Provasoli (1968). The recently, several recipes such as the one by Kester et al. modifications were the omission of Tris (the pH buffer) (1967) and one commercial one, Instant Ocean QGng and the addition of silicate. The omission of Tris and Spotte 1974), have ion ratios of the major con- was compensated for by adding equimolar amounts of stituents that are very similar to NW. NaHC03 and HC1 to prevent precipitation during Although one would expect artificial media to be autoclaving. During autoclaving COz is lost, and COz much more chemically defined than NW, in some can be added indirectly before autoclaving by adding respects, artificial media are not completely defined due NaHC03 or by directly bubbling COz through the to the contaminants in reagent grade salts. Because of medium. Ths medium has a 2.3 : 1 chelator to trace the large amounts of major salts that must be added, metal ratio, compared to 1 : 1 in 'f' medium (Guillard trace contaminants (e.g., copper, zinc, iron) in these and Ryther 1962), which may reduce the tendency to salts can result in higher concentrations of some metals form metal precipitates and metal toxicity (Harrison in AW than those naturally present in oceanic surface et al. 1980). Over the past 2 decades, further changes water. The use of ion exchange columns to remove were made to ESAW that significantly improved the these contaminants, as described by Morel et al. (1979), medium. The forms of phosphate, iron, and silicate can greatly reduce this problem (note, however, that were changed and the trace element mixture was altered Chelex 100 removes only cations), but it is a time- to include nickel, molybdenum, and selenium (Berges consuming process (see Chapter 4). With the interest et al. 2001). in trace metal availability and trace metal toxicity, the development of Aquil by Morel et al. (1979) permitted, for the first time, a complete definition of chemical speciation of various components, as calculated from 2.0. MATERIALSREQUIRED thermodynamic equilibria, by controlling trace element contamination and precipitate formation. The medium Aquil is useful for trace metal studies of copper, zinc, 2.1. Chemicals nickel, cobalt, lead, and cadmium, because they remain in cationic form in seawater. More recent changes in the Most chemicals required to make marine media are preparation of Aquil have been the purification of the available from various chemical suppliers. Reagent Chelex column to avoid contamination by the chelating grade salts (e.g., American Chemical Society grade) agents, use of alternative sterilization procedures, and should be used if possible. The organic chemicals such an increase in the concentration of trace metal buffers as vitamins, buffer, and chelators are available from (Price et al. 1988/89). Sigma Chemical Company. If brands of a chemical are Medium K (Keller et al. 1987) was developed from cul- changed, this should be noted, because different brands turing fastidious oceanic phytoplankton, and it has been are likely to have different amounts of contaminants or tested on 200 ultraplankton clones representing seven impurities. algal classes. It is not recommended for coastal species. This medium includes selenium, both nitrate and ammo- nium, increased chelation, reduced copper, and a moder- ate level of pH buffering. The chelation to total metal 2.2. Equipment, Glassware, and Tubing ratio is 10 : 1, and the EDTA concentration is 104 M. There is also a synthetic counterpart. The recently for- Most required equipment comprises standard items in mulated MNK medium and Pro99 medium (see later laboratories: analytical and top-loading balances, pH discussion and Appendix A) are also formulated for open meter, hot-plate-magnetic stirrer, and so on. Boro- ocean phytoplankton, and preliminary results suggest silicate glassware should be used exclusively for all that they are superior to Kmedium for certain algae (coc- glassware, including stock bottles, beakers, and cultural colithophores and Prochlorococcl~s,respectively). tubes and flasks (examples of brand names are Pyrex and A broad-spectrum AW medium (enrichment solution Kimax). Teflon or plastics are recommended, because with artificial water [ESAW) was developed and tested they reduce breakage. Check the mimufacturer's specifications for usage, such as autoclaving and storage All containers and tubing used for cultures and media of concentrated chemicals. stocks should be carefully selected to avoid toxic com- Keep the glassware and plasticware to be used in media pounds. For general purpose culturing, we recommend preparation separate from general purpose laboratory flasks and test tubes made of borosilicate glass and tissue use. Washing protocols vary, depending on the experi- culture-grade polycarbonate or polystyrene plasticware. ments planned, but in general it is important to be aware Teflon-lined caps are recommended for screw-top glass that tap water often contains high amounts of nutrients, test tubes, and black caps should be autoclaved several trace metals, and heavy metals. Therefore, if tap water is times in changes of seawater, because new caps may used for washing and rinsing, then make sure that deion- release toxic phenolics when heated (McLachlan 1973). ized water is used for the final rinse. Furthermore, For studies on silicon limitation, polycarbonate is domestic detergents leave a residual film on glassware. recommended. However, borosilicate glassware may be The detergents from most large chemical supply com- used as long as it has not been rinsed with any acid that panies are satisfactory, but labels should be read causes severe leaching of silicate from the glass. to determine if the contents meet your requirements. Likewise, rubber stoppers (or anythmg that releases New glassware and plasticware should be degreased in volatile compounds when heated) should be autoclaved dilute NaOH, soaked in dilute HC1, and then soaked in separately from media. Older autoclaves with copper deionized water for several days before use. Glassware tubing should be avoided, because excess copper is toxic should not be cleaned in chromic acid, because to algae. The autoclave steam may be contaminated chromium is toxic to many phytoplankters (McLachlan with metals or chemicals used to inhibit corrosion of the 1973). Teflon is useful only for stock bottles and is not autoclave. See Chapter 5 for further information on suitable for culture vessels because of its reduced light- sterilization procedures. transmission properties. Polycarbonate is good for culture vessels, especially for experiments involving trace-metal limitation. Polypropylene may yield toxins from stocks, notably silicate stocks (Brand et al. 1981). 2.3. Water Sources, Treatment, and Storage More information on culture vessels is provided in Chapters 2, 4, and 5. Glassware should be autoclaved, The source of seawater may determine one's success in clean glassware and plasticware should be stored in culturing certain species. To obtain NW free from pol- closed cupboards, and open vessels should be covered. lution, it may be necessary to collect offshore water. One should also be aware of potential toxicity from Oligotrophic open ocean water is ideal, because it is low tubing and other materials. Bernhard was one of the in nutrients and trace metals, and these components first to call attention to the inhibitory effects of some can be added in required amounts in an enrichment culture materials by testing more than 50 types on solution. In addition, this water contains less sediment phytoplankton and zooplankton (Bernhard and Zattera and possibly less phytoplankton, making it easier to 1970, Bernhard 1977). A later study examined latex filter. tubings of rubber, polyvinyl chloride (Tygon), and sili- Nearshore water may be seasonally variable due to cone, and Price et al. (1986) found that latex tubing was rainfall and runoff inputs, which may have elevated surprisingly toxic to phytoplankton, zooplankton, and nutrients and sediments and decreased salinity. If bacteria. Even using latex tubing to siphon water from inshore water is used, then water below the photic one bottle to another one rendered the water toxic for zone or pycnocline is likely to have less sediments and phytoplankton growth. The toxic compound was not algal biomass to remove by filtration. Water should identified, but preliminary results indicated that it may not be collected during blooms, especially when noxious be pentachlorophenols and tetrachlorophenols used to species are present. Various pumps or large water preserve the latex tubing (Price et al. 1986). Tygon bottles may be used to obtain the water. Water should tubing was generally safe, provided that the powder not be collected with a water bottle that uses latex inside the new tubing was carefully removed by rinsing tubing for the rubber spring that closes off the ends of before use. Silicone tubing was completely safe to use. the water bottle, because the latex rubber tubing Colored or black rubber stoppers may be toxic, and renders the water toxic for some algae (Price et al. therefore silicone stoppers are recommended, especially 1986). Because filtration may be a slow process, plastic the ones from Cole-Palmer that are made by injecting containers or carboys are usually filled and brought back small air-bubbles into the polymer and are thus lighter to the laboratory for filtration. Usually a large-scale fil- and easier to work with than the solid silicone stoppers. tration apparatus is used, such as 147- or.-293-mm- diameter membrane filters contained in a plastic holder if possible) and in the dark (or covered with black (e.g., Millipore or Pall-Gelman). A prefilter may be plastic). placed on top of the membrane filter to slow down the Filtered seawater is traditionally sterilized by steam cloggng of the membrane filter. Alternatively, filter car- autoclaving for 15 minutes at 12 1°C and 15 lb in-2 or tridges (e.g., Pall-Gelman Acropak capsules) are rela- longer, depending on the volume. After autoclaving, tively inexpensive, do not require any special filter leave the media for 24 hours to equilibrate, so that gases holders, and do not clog as readily as membrane filters. such as CO2 are allowed to diffuse into the medium. To The choice of filter pore size is determined by the avoid formation of a precipitate during autoclaving, the source of the seawater, its intended use, and the volume following treatments are helpful: needed. Normally, water should be filtered to 0.45 pm with membrane filters or, in special cases, down to 0.2 1. Adding 1.44 mL of 1N HC1 and 0.12 g of pm. If glass fiber filters are used, which are much faster NaHC03 per liter. These additions indirectly add and clog less quickly, then a GF/F filter (e.g., Whatman) COZand lower the pH, which helps to reduce the with a nominal pore size of 0.7 pm is recommended. formation of a precipitate during autoclaving Occasionally, dissolved organic matter removal may (Harrison et al. 1980). Carbon dioxide may be be required for special projects or because of suspected added directly by bubbling the medium before contamination. Dissolved organics may be removed by autoclaving (Morel, personal communication). adsorption onto activated charcoal. The charcoal is 2. Cooling the seawater quickly after autoclaving by prepared by washing with benzene, methanol, or 50% standing it in cold tap water in a sink helps to ethanol and distilled water (Craigie and McLachlan prevent precipitation. 1964). Dissolved organics in small volumes of seawater 3. Sterilizing by filtration with a 0.22-pm membrane may be removed by adding 2 g of powdered, washed filter. charcoal per liter of seawater, stirring for 1 hour and 4. Pasteurizing by heating the seawater to 90°C-95°C then filtering. Large volumes of seawater are passed for 24 hours. Sometimes this heating is done for a through charcoal in a glass column. The charcoal may shorter time but repeated twice or three times, be washed as described previously, but it must not be with cooling between heating periods allowed to dry before the addition of seawater. There is (tpdallization). no "best method" for cleaning charcoal, and often a 5. Adding pH buffers such as 4-5 rnM Tris or simple washing with seawater or distilled water may be glycylglycine. Note, however, that these buffers are acceptable (Guillard, personal communication). One organic compounds and may encourage bacterial should adjust the cleaning to suit the organism and growth (Fabregas et al. 1993). In addition, Tris , purpose of the experiments planned. Dissolved organ- may be toxic to some phytoplankton species ics may also be removed by exposing the seawater to (McLachlan 1973, Blankley 1973). high-intensity ultraviolet light (Armstrong et al. 1966). 6. Adding high concentrations of EDTA (e.g., lo4 M This destroys most dissolved organics and sterilizes the in medium K; Keller et al. 1988) for algae that seawater. tolerate it. Depending on where the seawater is collected, the 7. Sterilizing smaller volumes of seawater with a salinity varies, especially with different seasons. The microwave (Keller et al. 1988). salinity should be noted for each collection. Offshore 8. Lowering the concentration of iron. seawater salinity normally ranges 32 to 35 psu, whereas inshore water may often be <30 psu. Most algae grow well between 30 and 35 psu, but some species do not tolerate reduced salinities. If a lower salinity is desired, 3.0. STOCK SOLUTIONS the salinity must be decreased by adding deionized water before any nutrients, trace metals, or vitamins are added, to avoid dilution of these components. 3.1. Macronutrients (Nitrogen, Filtered seawater can be stored in either glass or Phosphorus, and Silicon) plastic carboys, often 20 liters for ease of handling. Rec- tangular containers require minimal storage and can be Macronutrients are generally considered to be nitrogen, stacked. New containers should be leached for several phosphorus, and silicon. However, silicon is required days with diluted (i.e., 10%) HCI and then rinsed thor- only for diatoms, silicoflagellates, and some chryso- oughly. The seawater should be kept cool (refrigerated phytes. These macronutrients are generallyrequired in a ratio of 16N:16Si:l.P (Parsons et al. 1984, Brzezinski Ammonium may be an alternative nitrogen source 1985), and the ambient ratio in NW is often similar to and may be added as NH4C1. At the typical pH of the ratio required by the algae, except in some es- seawater (8.2), there is about 90% NH4 and 10% tuaries where there are large inputs of nitrogen and NH3 (ammonia). Because considerable quantities of phosphorus. Most media do not balance the relative ammonia may be lost from the medium through concentrations of macronutrients needed for algal volatilization during autoclaving, ammonium should be growth. Several popular media (e.g., f/2 medium) have added aseptically after autoclaving. As the pH of the nitrogen : phosphorus ratios >16 : 1, indicating that the culture medium increases during algal growth, the ratio phytoplankton would be phosphorus-limited in senes- of NIX, : NH? increases and reaches 1 : 1 at a pH 9.3. cent phase (Berges et al. 2001). Therefore, substantial amounts of ammonium may be Unfortunately, experimentalists usually pay little lost from the culture if the algal culture is kept mixed attention to the nitrogen : phosphorus or nitrogen : by bubbling with air. Ammonium, at concentrations of silicon ratios in the medium that they are using, which 100 to 250 pM, may be inhibitory to some coastal will ultimately determine which nutrient limits growth species, but most coastal species tolerate concentrations and influences the chemical composition and physio- as high as 1,000 @I (McLachlan 1973). Similarly, logical rates when the cells become senescent. Similarly, some oceanic species show toxicity to ammonium at carbon concentrations and carbon : nitrogen ratios are only 25 pM (Keller et al. 1987), whereas others rarely considered. Many media have a bicarbonate con- (e.g., Prochlorococczls, Bolidomonas) tolerate concentra- centration of about 2 rnM and nitrogen (nitrate) of tions >SO0 pM. In some special cases, urea is another about 500 pM or higher, which yields a carbon : nitro- form of nitrogen to consider, but it decomposes gen ratio of about 4 : 1. According to the Redfield ratio, when heated (McLachlan 1973). In experiments in the chemical composition of the average phytoplankter which different forms of nitrogen are compared, it is is 106C : 16N : lP, or 6.7C : IN. Therefore, most important to note that 1 pM urea provides 2 pM media are nitrogen-rich relative to carbon, and carbon N . L-', because the urea molecule contains two atoms could become limiting, depending on the growth rate of nitrogen. of the phytoplankton and the surface area of the Silicate is added as Na2Si03. 9H,O. Because silicic medium through which atmospheric C02can diffuse acid enhances precipitation, it is useful to omit it from (Riebesell et al. 1993). When culture pH rises quickly the medium if one is culturing species that do not to 9 or higher, this may be an indication that carbon require silicate (e.g., most flagellates). If concentrated - may be limiting. Species that can readily use bicarbon- stock solutions (e.g., 100 mM) are prepared in deion- ate may be able to grow, whereas other species that are ized water and acidified to pH 2 (McLachlan 1973), sil- more dependent on C02may exhibit a reduced growth icate polymerizes. When this stock is added to seawater, rate or cell yield. Depending on the use of the seawa- it may take several days before the entire concentration ter, one may consider bubbling with C02or adding of silicon that was added is available for uptake and more bicarbonate in late exponential phase to ensure growth (Suttle et al. 1986). It is recommended to store that carbon is not limiting algal growth. This is espe- the 100-mM stock solution of Na2Si03at its pH of dis- cially important in physiological experiments where it solution in deionized water (pH = 12.6) at 4°C in the is essential to know which nutrient is limiting during dark (Suttle et al. 1986). When the silicon stock is added senescent phase. to seawater, it should be added slowly with rapid stir- To simplify routine media preparation, recipes are ring. Autoclaving the Na2Si03stock solution in a glass usually divided into working stock solutions. Direct container may result in etching of the glass and precip- combinations of several stock solutions without dilution itation, and therefore it is recommended to prepare it in water may result in undesirable precipitation. To in a Teflon-coated bottle (Suttle et al. 1986). make a working solution of several stocks, add one stock To help control contamination with bacteria or fungi, to a certain volume of water and mix thoroughly before autoclave all nutrient stocks and then use sterile tech- adding the next stock solution. Nitrate and phosphate niques subsequently. Another possibility is to sterilize are normally added as NaN03 and NaHP04 H20. the stocks through a 0.2-~mfilter. Generally, nutrient In some media, phosphate is added as sodium glyc- enrichments should be added aseptically after autoclav- erophosphate to make the trace metal salts less prone to ing, but they may also be added before autoclaving, precipitation; however, sodium glycerophosphate may except in the case of ammonium. precipitate as a calcium salt at elevated temperatures For concentrations of the various macronutrient (Provasoli 197 1). stock solutions for the various media, see Appendix A. 3.2. Trace Metals 3.4. pH Buffers

Trace metals and vitamins are usually prepared as Two common pH buffers are used to prevent or reduce "primary" stocks of high concentrations to permit precipitation: Tris (2 -amino-2 - [hydroxymethyl]- 1-3- weighing of reasonable amounts. These are used to propanediol) and glycylglycine (McLachlan 1973). Mix make "working" solutions from which the final medium Tris base and Tris : HCI as per Sigma instructions and is made (see Appendix A for examples). Because some make a stock to be added as desired; for example, 1 mg primary or working solutions are kept for very long L-' for a Tris concentration of lO"M. Adjust the pH periods, evaporation through ground glass stoppers, with concentrated HC1 to obtain the desired pH of the screw caps, or plastic bottles may be significant. Mark medium. Glycylglycine is readily soluble in water, and the liquid level on the bottle and keep it cold or wrapped the powder can be added directly to seawater. It is in laboratory film. slightly acidic, and it may be necessary to make a small Typical trace metal stock solutions may consist of adjustment to the pH with several drops of 1N NaOH. chloride or sulphate salts of zinc, cobalt, manganese, Tris may be toxic to some species (McLachlan 1973; use selenium, and nickel, and they are kept in a solution no more than 1-5 rnM), whereas glycylglycine is non- containing the chelator EDTA. Iron is usually kept as a toxic. Apparently, neither Tris nor glycylglycine can separate solution, and it should be chelated or kept in serve as a nitrogen source for algal growth, but they can M HCl to avoid precipitation. It may be added as serve as a carbon source for bacteria (Fabregas et al. ferric chloride, ferrous sulphate, or ferrous ammonium 1993). They may also interfere with the analysis of dis- sulphate, but the latter compound contains ammonium, solved organic nitrogen and ammonium. and this may be a problem if one is conducting nitro- HEPES (N-[2 -hydroxyethyl] piperazine-N'- [2 - gen uptake studies. There is sufficient boron in NW ethanesulphonic acid]) and MOPS (3-N-morpholino and therefore it is not necessary to add it, but boron propane sulfonic acid) are used extensively in fresh- should be added to AW. The stock solutions for various water media (McFadden and Melkonian 1986), but they recipes are given in Appendix A. For a more detailed are not commonly used in marine media. Loeblich discussion of trace metals, see Chapter 4. (1975) compared the growth of a marine dinoflagellate in several buffers (including MOPS, HEPES, Tris, glycylglycine, and TAPS N-Tris [hydroxymethyl] methyl-3 -aminopropanesulfonic acid), and concluded , 3.3. Vitamins that Tris and TAPS provided maximal growth with minimal pH change. Usually three vitamins-vitamin B12(cyanocobalamin), thiamine, and biotin-are added, but very few algae need all three vitamins (Provasoli and Carlucci 1974). 3.5. Chelators The general order of vitamin requirements for algae is vitamin Biz > thiamine > biotin. If large-scale culturing Chelate : metal ratios of 1.5 : 1 to 3 : 1 are commonly of a single species is the goal, check what vitamins this used. EDTA is the most common chelator and is usually species requires. It may be possible to omit the addition purchased as the disodium salt (Na2EDTA.2H20)that of two of the three vitamins, because most species is readily soluble in water. However, EDTA has been require only one or two. Vitamins are normally added noted to inhibit the growth of some oceanic species aseptically (through a 0.2-pm filter) after the medium (Muggli and Harrison 1996). For routine use in culture has been autoclaved. Vitamins maintain maximum media, nitrilotriacetic acid (NTA) and citric acid are less potency if they are filter sterilized (0.2-pm filtration) effective than EDTA, but they are used sometimes in rather than autoclaved. Autoclaving may cause decom- experimental work (Brand et al. 1986). Chelators are position of some vitamins, but it is thought that discussed extensively in Chapter 4. some algae may be able to use some of these decompo- sition p;oducts (Provasoli and Carlucci 1974). vitamin stocks may be frozen for long periods without noticeable degradation, and the stocks may be refrozen 3.6. Soil Extracts after each use. These three vitamin stocks may be com- bined into a single working solution for a 1,000-fold In the simplest media, only nitrogen and phosphorus dilution. and soil extract are added to NW. Erdschreiber is an example of such a medium, and it has been used suc- 4.2. Artificial Seawater cessfully to grow various planktonic and benthic species (McLachlan 1973). Some culture collections still use Artificial or synthetic seawater consists of two parts: the soil extract to maintain some species (see Plymouth basal (main) salts that form the "basal seawater" and the Erdscheiber in Appendix A), but it is seldom used in enrichment solution (often the same as the enrichment physiological experiments when a defined medium is solution that is added to NW). In the early AW recipes, preferred. calcium and magnesium salts were reduced to avoid pre- cipitation. Because precipitation can now be avoided (Harrison et al. 1980), the more recent recipes ensure that the ratio of the major ions is identical to NW. For 3.7. Germanium Dioxide example, in ESAW (see Appendix A), the basic salts (usually 10 or 11) are divided into the anhydrous sodium In special cases, germanium dioxide may be added to or potassium salts (i.e., NaCl, Na2S04,KCI, NaHC03, prevent the growth of diatoms (Lewin 1966), but other KBr, NaF) and the hydrated chloride or sulphate salts algae may also be affected. For example, the addition of (i.e., MgC12 . 6H20, CaClz H20,and SrC12 6H20). 100 mg L-I of Ge02prevents diatom growth in macro- . - The anhydrous salts must be dissolved separately from algal cultures (Markham and Hagmeier 1982), but the hydrated salts, and then the two solutions can be McLachlan et al. (1971) found that GeOL inhibited mixed together and the enrichment solution compo- brown algal growth. Thomas et al. (1978) added ger- nents (macronutrient, trace metals, and vitamins) can be manium dioxide to natural phytoplankton samples in an added after the salt solution is autoclaved. . attempt to separate flagellate productivity from diatom Studies of the physiology of marine phytoplankton productivity and noted that dinoflagellate photo- have been greatly facilitated by the ability to culture synthesis was inhibited as well. Therefore, it seems these species in a defined medium (i.e., AW). One of the unwise to add germanium dioxide during physiological great advantages of AW is that the composition of the experiments. seawater is relatively constant over several decades, unless the impurities in the major salts (e.g., NaCl, Na2S04,and KCl) change either by a change in brands or a change in the manufacturing process. In 1985, we suddenly were unable to grow the common diatom, Thalassiosira pseudonana (Hust.) Hasle et Heimdal, despite the fact that we were using the same ESAW recipe that we used for the previous 7 years. We later The various aspects and precautions involved in media discovered that selenium was previously an impurity preparation have already been covered in the sections in sufficient quantity in our basal salts, and therefore it on materials required (Section 2) and stock solutions had not been necessary to add it (Price et al. 1987, (Section 3). Harrison et al. 1988). We assume that a change in the process of producing one of the basal salts reduced selenium contamination. When we added selenium to 4.1. Natural Seawater the new enrichment solution, T. pseudonana grew well. Therefore, selenium is routinely added to our me- The general steps are as follows: dium now (Berges et al. 2001). Another advantage of AW for nutrient limitation 1. Obtain good-quality NW with salinity >30, if studies is that one can control the amount of the limit- possible. ing nutrient because there is little or none in the AW, 2. Filter as soon as possible, and store the seawater in unlike NW. Similarly, one can precisely control the the dark and cold (4°C). nutrient ratios. However, cleanly collected oligotrophic 3. Sterilize as needed by filtration (0.2 pm), ocean water may also be used, because nutrients and autoclaving, ultraviolet light treatment, or trace metals are also very low. pasteurization. Commercial preparations are synthetic mixes and can 4. Add macronutrients, Fe-EDTA, trace metals, and be purchased in various amounts. One of the best is vitamins aseptically after autoclaving. Mix Instant Ocean, produced by Aquarium Systems, Inc. thoroughly after the addition of each stock. (King and Spotte 1974). The ion ratio of the irrajor salts is very close to NW. Macronutrients, trace metals, and culture collections). When compiling the recipes in vitamins must be added. If large-scale preparation is Appendix A, we used both original recipes and those required, this may be an economical alternative to provided by major culture collection Web sites. The buylng individual salts and weighing and mixing the reader is cautioned that the Web site recipes are subject salts. to change; indeed, Berges et al. (2001) recommended For all the AW basal salt recipes, if contamination is that Web-based recipes may have real advantages for a concern, then we recommend that you analyze the phycological research. basal medium for copper, zinc, cobalt, lead, manganese, In both AW and NW recipes, there is some variation nickel, chromium, etc. For example, we measured 2.5, in salinity. McLachlan (1973) considered that salinity 4.0, and 0.13 pM lead, copper, and cadmium, respec- was not an "inherent feature" of media recipes. Although tively, in ESAW, which uses reagent grade salts. This salinities of 35 are often considered normal, most artifi- concentration of copper is sufficient for growth, cial media tend to produce somewhat lower salinities (27 depending on the concentration of the other nutrients, to 30). Many coastal phytoplankton species can be cul- trace metals, and chelation. Brand et al. (1986) found tured at a much lower salinity (cf. McLachlan 1973). that 4.0 pM copper is toxic in the absence of a chelator Sometimes it is necessary to grow axenic cultures. for some species. Details for preparing test media for bacteria and fungal contamination are provided in Chapter 8.

5.1. Artificial Seawater Media

Relatively few artificial water media are commonly Comparisons between media are complicated by used. Most are based on the ASM and ASP formulations many factors. McLachlan (1973) observed: "Numerous of Provasoli and McLachlan, with minor variations (the enriched and synthetic media have been formulated, AWs described by Goldman and McCarthy [I9781 and which together with generally trivial modifications, Keller et al. [I9871 both fall into this category). There almost equal the number of investigators." This has cer- are numerous species-specific artificial media that have tainly remained true. Many modifications result from a been developed; for example, YBC-11, designed for desire to increase the flexibility of a medium (i.e., nitrogen-fixing strains of Trichodesmium (Chen et al. creating multiple nutrient stocks so that individual 1996), was derived from an earlier medium by Ohki et macronutrient and micronutrient concentrations may al. (1992). As noted previously, the ion ratios of be manipulated) or to reduce the number of stocks nec- these media have diverged from that of NW (Table 3.I), essary in cases where various different media are being particularly with respect to sulfate and magnesium used in a single laboratory. (In Appendix A note the dif- (Kester et al. 1967). ESAW was formulated on the ferent recipes that incorporate f/2 trace metals stock.) basis of Kester et al. (1967) and is thus quite similar to In many cases, minor modifications to the orignal NW. recipes have been made (e.g., changing a nitrogen source or adding a single trace metal) and an entirely new name has been given to the medium with this minor modification. In other cases, fairly extensive TABLE 3.1 Comparison of the ratio (normalized to modifications have been made, yet a medium name has K) of selected major ions in different artificial seawater been retained or simply designated as "modified." In recipes and natural seawater. still other cases, subsequent modifications have been made in addition to a name change to honor the origi- nator (e.g., "Grund" versus "von Stosch"; von Stosch Recipe No+ Cl- Mg2+ so42- K' 1963, Guiry and Cunningham 1984). More commonly used media such as f/2 and ESAWESNW have not Aquil 60 5 1 7.0 3.1 I .O only evolved over time (Berges et al. 2001), but they can ASNlll 64 69 3.6 2.0 I .O change substantially in different publications (e.g., ASP-M 40 47 4.0 2.0 I .O ESAW 52 59 5.1 3.1 I .O compare the recipe for f medium given in Guillard and YBC-II 42 45 5.0 2.5 I .O Ryther [I9621 with that for f/2 medium in Guillard NW 47 55 5.3 2.8 I .o [I9751 and with those given on the Web sites of major - -- Several commercially available AWs are also avail- Web sites). A variety of detailed recipes are now avail- able. Instant Ocean is one of the oldest and best- able on culture collection Web sites, but the reader is evaluated (discussed previously), but Tropic Marin Sea cautioned that there is considerable variation in recipe Salt (Tropic Marin) is also well-regarded by European details and even in the names used to describe recipes aquarists, and Aus Aqua Pty Ltd (www.algaboost.com) between sites. However, some generalizations about sells various AW formulations, including ESAW, and media recipes can be made. enrichment solutions such as f/2. In published research, In terms of vitamins there is remarkable consistency it appears that relatively few phycologists use ready- in media: thiamine, biotin, and B12 all are normally made salts, but at least one recipe available on the added, at quite comparable concentrations, although Culture Collection of Algae and Protozoa (CCAP) Web they may not be required (see Appendix A). Some media site uses a commercial sea salt preparation (Ultramarine specify that autoclaving should be avoided, because it Synthetics, Waterlife Research Industries Ltd.). More can lead to decomposition of vitamins, but species that recently, Sigma-Aldrich has begun producing a dry sea require vitamins are usually able to grow well on the salt mixture (S9883). When using these preparations, it decomposition products (see McLachlan 1973). is important to note that the dry mixture may not be Trace metals stock solutions are more variable, but it homogenous; subsamples from a large package may vary appears that most recipes have basic similarities (see considerably, especially if the salts have hydrated during Appendix A). Elements such as iron, zinc, manganese, storage. cobalt, copper, and molybdenum are almost always included. Less commonly found, but critical to certain species, is selenium (Harrison et al. 1988). Nickel is required by most algae if urea is the nitrogen source, 5.2. Natural Seawater Media because nickel is a component of the enzyme unease (Syrett 1981). For oceanic species, a widely variable set Among the major NW enrichment media recom- of metals has been added, including the potentially toxic mended for a broad spectrum of algae, f/2 medium and vanadium and chromium. There are also numerous variations, different versions of Erdschreiber medium metals found in some of Provasoli7searly media, such as (e.g., Plymouth Erdschreiber medium), and ESNW aluminum, ruthenium, lithium, and iodine (Provasoli et appear to dominate citations (Berges et al. 2001, see al. 1957), that are rarely found in media currently used. Appendix A). The K-medium family has been specifi- As noted earlier, the trace metal mixture is more criti- cally developed for oceanic species (Keller et al. 1987), cal when AW is used, particularly if water of very high and two new oceanic media, MNK and Pro99 media, purity (i.e., >18.2 Mi2 water, such as that provided by offer promising improvements. More species-specific MilliQ systems) is used. media that are frequently used include the L-medium Concentrations and forms of macronutrients (e.g., f, family (Guillard and Hargraves 1993; Guillard 1995), f/2, and f/50) in media commonly vary. Typically, GPM and variants (Sweeney et al. 1959; modified by macronutrients are in great excess in comparison with Loeblich 1975 and Blackburn et al. 1989), and numer- natural concentrations, particularly in the case of ous media for oceanic cyanobacteria, including SN media intended for aquaculture. For example, Walne's (Waterbury et al. 1986), PC (Keller in Andersen et al. medium (Appendix A) contains >1 mM nitrate, or about 1997), PRO99 (Chisholm, unpublished), and ASNIII 40 times the maximum levels found in coastal waters. (Rippka et al. 1979); BG medium (Rippka et al. 1979) As noted above, the ratios of nutrients relative to algal and variations are also recommended for marine requirements often appear to make little sense (Berges cyanobacteria but are less commonly used with oceanic et al. 2001). Lower nutrients have been found to species. For macroalgal cultures, enrichments based support growth of oceanic species, but often it is only on the "Grund" medium of von Stosch (1963) are the major nutrients that have been reduced in the most commonly used. Provasoli's ES medium and its recipe. McLachlan (1973) noted that ammonium can modifications are also used (McLachlin 1973, West and become toxic to some species of phytoplankton at con- McBride 1999). centrations of 100 p&t or greater, and Berges et al. It is difficult to provide a comprehensive set of rec- (2001) speculated that this may be the reason that dilu- ommendations as to which media are best for certain tions of media in which ammonium is included may species. A good starting point is the media in which the improve growth of some species. species are being maintained in the culture collections It is also worth noting that there are commercially from which they are obtained (see culture collection available premade enrichment stocks. Some major culture collections sell nutrient stock solutions for found that assessment based on growth rate' and various culture media as well as premixed culture media final biomass did not always agree. For example, and seawater (e.g., CCAP, at www.ife.ac.uk/ccap, and Phaeocystis pouchetti (Hariot) Lagerh. and Karlodinium CCMP, at http://ccmp.bigelow.org). Sigma-Aldrich micrum (Leadbeater et Dodge) Larsen (Gymnodinium sells f/2 medium (dry salt mixture G1775 or liquid galatheanum [Lohm.] Kofoid & Swezy) both grew at G9903), and AusAqua Pty. Ltd. sells f/2 medium under equal rates (p) in ESAW and ESNW, but whereas l? the name AlgaBoost and ES enrichment stocks. pouchetti achieved a threefold higher biomass in ESAW versus ESNW, K. micrum cultures were almost twice as dense in stationary phase in ESNW as in ESAW. Growth rates are relatively straightforward to 6.0. COMPARINGRECIPES BASED measure by performing cell counts, but this is quite labor-intensive, even when using electronic particle ON ALGAL RESPONSE counters (see Chapters 16-18). Measurements of fluo- rescence are much quicker and agree very well with cell Growing phytoplankton remains an art as well as a numbers in most species, so long as cultures remain in science, and in our experience, most investigators tend exponential growth phase and are measured at the same to settle on media that "work" for the species they are time of day, if grown on a light : dark cycle. If culturing growing, rather than engage in wholesale comparisons is done in 25 x 150-mm screw-capped culture tubes, to determine which media are the best. McLachlan then repeated measurements of cultures can easily be (1973) observed that media preferences were "rarely made with a Turner Designs fluorometer. For further supported by comprehensive, qualitative comparisons." information on measuring growth rates, see Chapter 18. Any media comparison should be based on the use of Determining the biomass of stationary phase cultures the medium by the investigator. There are three broad requires cell counts, and it is probably wise to measure categories of general use: culture maintenance, algal cell volumes as well; in this case, fluorescence is unsuit- biomass yield, and physiological (growth rate) experi- able (see Berges et al. 2001). Although such compar- ments. It is strongly advised that the growth rate in NW isons are rarely done quantitatively, assessing media on and the medium be compared, because growth rate is a the basis of the length of time a culture can remain at a general index of algal health in the medium. Some stationary biomass is a criterion that is probably quite species grow better if there is a large surface relevant to culture collections. area/volume ratio to allow the diffusion of gases into the Another criterion for media evaluation is whether the medium. original morphology of the cells is maintained, but this In principle, the simplest comparison to make is is seldom evaluated (Harrison et al. 1980) and can be whether the algae grow. Consult the culture collection impractical for small species. Many recipes that include Web sites for the most recent information on recipes soil water extract justify the addition on the basis that and also to find the most appropriate medium for a it maintains the original cell morphology during long- particular strain. In practice, this is considerably term culturing. complicated by the culture methods. For example, some investigators may maintain their culture collections quite conscientiously, transferring cultures weekly or more often. In this case, species that grow quicldy (i.e., a high exponential growth rate, p) could be favored. Under these conditions, a medium that was relatively poorly buffered or contained relatively low levels of Allen, E. J. 1914. On the culture of the plankton diatom Tha- macronutrients might be regarded as superior. On the lassiosira gravida Cleve, in artificial sea-water. 3.Ma?: Biol. other hand, cultures are often transferred far less fre- Assoc. U.K. 10:417-39. quently, and consequently they can persist in stationary Allen, E. J., and Nelson, E. W. 1910. On the artificial culture phase for especially long periods if they are kept in dim of marine plankton organisms. 3. Ma?: Biol. Assoc. U.K. 8: light. In these cases, a medium that supports a higher 42 1-74. growth rate might not be regarded as superior, but Armstrong, F. A. J., Williams, P. M., and Strickland, J. D. H. media that are more strongly buffered or have nutrients 1966. 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