DIATOM MEDIA: CHRONOLOGY OF EVOLUTION

“For microbes everything is everywhere, but the environment selects” (Patterson, 2009) and the environment being either natural or artificial. The preceding section, explains the artificial selectable environment. For a better understanding of the contributions during previous years, the historical development towards revolutionizing the diatom marine culture media is divided into three centuries (19^th, 20^th and 21^st Centuries).
 
Miquel (1892) in 19^th century suggested media recipe which is a stepping stone towards the success in further developments in diatom seawater media.  Table 1 provides media recipes which showed evolution in the true sense in chronological order.

Table 1: Molar concentrations of the nutrients found in different marine diatom medium

(1) Miquel, 1892-93 (2) van Heurck, 1893-96 (3) Allen and Nelson, 1910 (4) Ketchum and Redfield, 1938 (5) Matudaira, 1942 (6) Hunter, 1948 (7) Provosaliet al., 1957 (8) Provasoli, 1968 (9) McLachlan 1964, Goldman and McCarthy, 1978 (10) Guillard and Hargraves, 1993 (11) Keller et al., 1987 (12) Bergeset al., 2001 (13) Gagneux-Moreauxet al., 2007

Miquel (1892) observed that the water samples (of lakes, ponds and sea) could not sustain luxuriant growth of algae in controlled conditions of the laboratory environment. Analysis showed that, natural water requires artificial enrichment of mineral salts like nitrogen, phosphorous, sulphur, potassium, calcium, magnesium, iron, silicon, sodium, bromine and iodine (Miquel, 1892).  This led to the in situ culture of diatoms (freshwater and marine) with nutrient elements (Peach and Drummond, 1924). Miquel formulated a nutrient media (Miquel, 1890-93) for freshwater diatoms which subsequently tried for marine benthic diatoms (Allen and Nelson, 1910). Miquel also distinguished between “ordinary cultivations” in which one or more species are cultivated together and “pure cultivations” where a single species is made to pass through all the phases of its existence in order to follow every modification. Pure cultivations were found viable for artificial culture of diatoms and also for a number of microscopic observations (van Heurck, 1893-96). Macchiati (1892a, b, c) published theoretical data based on the experiments with the cultivation of diatoms. Further, Gill H. (van Heurck, 1893-96), also designed a media for the growth of diatoms where the salts were added into the sterilized seawater. Miquel points out the harmful effects in exposure of diatom cultivation to direct light (van Heurck, 1886). Flasks were exposed to the direct sunlight on a board, close to some glass windows which were situated facing north direction, at the same time care was taken to place between the glass and the flasks a plate of pale green glass of the height of the flask and a wooden board slightly higher than the liquid (van Heurck, 1893-96). Diatoms cultured were Pleurosigma angulatum W.Sm., Cymatopleura solea (Brѐb) W.Sm. various Nitzschia, Cymbella and Navicula species (van Heurck, 1893-96). Subsequent contributions by Allen EJ, Nelson EW, Guillard RRL, Provasali L and coworkers paved way for the success in seawater media. The major contribution in the artificial seawater media by Allen and Nelson (1910) were done with the intention of having a suitable and a stable food in the form of diatoms to rear marine larvae.

Grave (1902) cultivated diatoms as food for larvae of marine origin. He obtained diatoms by placing sand collected from the sea bottom, in aquaria. This was the first attempt to try artificial sea water for diatoms, which was a solution based on the molecular concentrations of sea water (van’t Hoff, 1905). After some preliminary experiments on Miquel’s media, Allen and Nelson (1910) found that; potassium nitrate, sodium nitrate and ammonium nitrate are the most important elements, resulting in the omission of potassium bromide and potassium iodide which did not affect much. They realized that silica was important for diatoms, and found potassium silicate was not a satisfactory source of silica (https://ccmp.bigelow.org/ accessed on 20^th June 2011). They persistently grew many of the ecologically important diatoms Asterionella japonica Cl., Biddulphia mobiliensis (Bail.) Grun., Biddulphia regia (Schultze), Chaetoceros densum Cl., Chaetoceros decipiens Cl., Chaetoceros constrictum Grun, Cocconeis scutellum Ehr. var. minutissima Grun, Coscinodiscus excentricus Ehr, Coscinodiscus granii Gough, Ditylium brightwellii (West) Grun., Lauderia borealis Grun., Nitzschia closterium W.Sm., Phaeodactylum tricornutum, Nitzschia seriata Cl., Rhizosolenia stolterfothii Perag, Skeletonema costatum, Streptotheca thamensis Shrubs., Thalassiosira decipiens Grun) but were contaminated with bacteria(Allen and Nelson, 1910). They also reported that “Miquel’s Sea-water" in addition to the growth of diatoms also supports several other unidentified species of Rhodophyceae, Myxophyceae, filamentous Chlorophyceae (Enteromorpha, Vaucheria, etc.) and even young plants of Laminaria (Provasali et al., 1957).

Foyn's Erd-Schreiber medium (1934) is a combination of Schreiber's medium (Schreiber, 1927) and soil extract. Gross in the early 1930's used the modified media (Foyn's) to cultivate pure cultures of marine diatoms - Biddulphia mobiliensis (Bail.) Grun., Chaetoceros didymus Ehr., Chaetoceros pseudocritinus Ostenfeld, Coscinodiscus excentricus, Coscinodiscus granii Gough, Coscinodiscus radiatus Ehr., Coscinodiscus sub-bulliens Jörgensen, Coscinodiscus obscurus (?), Coscinodiscus sp., Ditylium brightwelli (West), Melosira borreri Grev., Rhiziosolenia alata Brightw. f. indica (Pérag.), Skeletonema costatum, Streptotheca thamensis and Thalassiosira sp.

Ketchum and Redfield’s media a modified variant of Allen and Nelson’s media with MgSO₄ to enrich media to culture Navicula closterium and produceda continuous supply of axenic culture. This media was subsequently used to culture other unicellular organisms which require physiological research (Ketchum and Redfield, 1938). Matudaira in 1942 modified Miquel’s solution with compounds like sodium bicarbonate, sodium silicate, manganese chloride, boric acid and copper sulphate to obtain the effects of inorganic sulphides on Skeletonema costatum.

Comprehensive review of Provasali et al., (1957) on the development of marine media to culture marine diatoms gave a host of new recipes. This also showed that sea water substitutes based on analyses of sea water retain the defects of the former and are unsuitable for most species even when enriched with essential trace elements. Small additions of extracts of natural substances improve the media. The ASP – 2 Medium, an artificial seawater medium designed by Provasali et al., (1957), was to serve both for bacterized and pure cultures of photosynthetic marine algae. The media had a lower value of nitrate and phosphate to suppress excessive bacterial growth. As the isolated marine algae are being strict phototrophs, no carbon sources were added. Due to these, there was no bacterial growth in the media. The most prominent element in their S3 vitamin mixture was Cyanacobalamin (B₁₂) followed by thiamine and biotin. Other elements like Nicotinic acid, Thymine, Inositol, Ca pantothenate, p-Aminobenzoic acid and Folic acid were added as a precautionary measure. It allowed the growth of several diatoms, chrysomonads, cryptomonads, dinoflagellates, blue-green algae and chlorophytes and was a very good medium for Phormidium persicinum Gomont., Gyrodinium californicum Bursa.and two other species of Gyrodinium, Amphidinium klebsii Carter, Prymnesium parvum Carter, Rhodomonas lens Pascher & Ruttner, Stephanopyxis turris (Grev.) Ralfs. and Pilinia sp. It has been found, however, that some organisms may require more trace metals or more metal chelators or both. Their further modification of parent media by the addition of Nitrilotriacetic acid (ASP- 2 NTA) (Provasali et al., 1957) was found to be useful for growing diatoms like Chaetoceros ceratosporus Ostenfeld (Yamaguchi et al., 2005). The media has separate component for vitamins, while Tris and Nitrilotriacetic acid played a role as buffers.

ASP-M media (McLachlan 1964, Goldman and McCarthy 1978) an artificial enriched sea water medium was derived from the Provasoli’s earlier ASP Medium series for culturing marine macro and micro algae.  The trace metal solutions (TMS II) were derived from the S1 metal solution Provasali and Pintner (1953) alongwith a complex vitamin solution.

The ES1/3 enrichment solution results in a third of the ES enrichment (Provasoli, 1968) for a main part of elements and the vitamin solution is that described by Guillard and Ryther (1962). ES1/3 appeared to be more suitable for H. ostrearia. Robert (1983) obtained long-term productive cultures of H. ostrearia after modifying the enriched seawater medium ES (Provasoli, 1968).

The original artificial Aquil medium (Morel et al., 1979) was modified by Price et al., (1989). The modifications are as follows:

Major nutrient solutions:

• The major nutrients PO₄^3-, NO₃^- and SiO₃^2- are prepared as concentrated stock solutions.
• The concentration of SiO₃^2Π and NO₃^- is increased to 10^-4 and 3 Χ 10^-4 respectively.
• The nutrient solutions are diluted with Q-H₂O to a get a final concentration twice that of Aquil and then chelaxed together.

Trace metal enrichment:

• The concentration of Molybdenum (Mo) is increased from 1.5 to 100 nM.
• NaMoO₄ is used in place of (NH₄)₆.Mo7O₂₄. 4H₂O.
• Na₂SeO₃ is included in the composition at a concentration of 10nM.
• The concentration of EDTA is increased from 5µM to 10 or 100µM to minimize the effects of contaminating metals.

Thalassiosira pseudonana (clone CCMP 1335), Thalassiosira weissflogii (Grunow) Fryxell & Hasle (clone ACTIN, CCMP 1336) (Roberts et al., 2007), Emiliania huxleyi (Lohmann) Hay & Mohler (Dupont et al., 2004), Thalassiosira oceanica Hasle (Granger et al., 2004) were cultured in this media. Aquil media is best suited to study the physiological studies related to trace metal metabolism (Gagneux-Moreaux et al., 2007).  Moreau (1996) used the artificial Aquil medium (Morel et al., 1979; Price et al., 1989) and f/50 medium derived from the enriched seawater f/2 medium (Guillard, 1982) for experiments involving cultures of Haslea ostrearia (Gallion) Simonsen.

The L1 medium (Guillard and Hargraves, 1993) a natural seawater enriched media is a modification of the f/2 medium. . The difference is a broader trace metal composition in L1. L1-trace metal solution is used also in many other media like the Ostreococcus Medium Brian Palenik (https://ccmp.bigelow.org/ accessed on 20^th June 2011). Culture collection like Scandinavian Culture Collection of Algae and Protozoa use this media as a standard medium for marine diatoms (http://www.sccap.dk/media/marine/2.asp accessed on 20^th June 2011). Thalassiosira pseudonana,  Phaeodactylum tricornutum (Ast et al., 2009). Chaetoceros elmorei Boyer, Cyclotella quillensis Bailey, Cymbella pusilla Grun. and Anomoeoneis costata (Kütz.) Hust. Trace elements, vitamins and silica were added according to the `L1' medium (Saros and Fritz, 2002).

K medium (Keller et al., 1987) was developed for oligotrophic marine phytoplanktons. The prominent feature of this medium is that it uses 10-fold higher EDTA chelation than most common marine media, and hence availability of trace metals, thereby reducing the possibility of metal toxicity. Drawback lies in the high macronutrient for some ocean organisms and the precipitation of silica. Algal culturing book prescribes of using natural oligotrophic ocean water rather than coastal seawater for the base. Diatoms used were Pseudonitzschia pungens (Grun. ex Cl.) Hasle., Pseudonitzschia fraudulenta (Cl.) Hasle., Pseudonitzschia pungens v. pungens and Pseudonitzschia pungens v. multiseries Hasle (Hargraves et al., 1993).

The artificial medium ESAW (Berges et al., 2001) is a modified media of the Harrison et al., (1980). The artificial medium, ESAW (Harrison et al., 1980) based on artificial seawater medium was similar to the ionic composition of sea water (Kester et al., 1967) enriched with Provasoli’s ES solution to balance the macronutrient and chelate concentrations. After the proposed ESAW medium by Harrison et al., 1980, numerous minor changes led to a modified ESAW medium (Berges et al., 2001). Berges et al., 2001 found that the modified media has improved the older one significantly. The only modifications lie in the:

• Addition of borate in the salt solution (Original: Addition of borate in trace metals)
• Inorganic phosphate (Original: Glycerophosphate)
• Preparing silicate stock solution at half strength without acidification.
• Additional trace elements like Na₂MoO₄. 2H₂O, Na₂SeO₃ and NiCl₂. 6H₂O.
• Iron added as chloride (to remove ammonium) from a separate stock with equimolar EDTA.
• Filter sterilization (Berges et al., 2001)

Diatom artificial medium (DAM) was developed based on the Aquil model (Gagneux-Moreaux et al., 2007). The diatom artificial medium DAM allows long-term and productive culturing of Haslea ostrearia in controlled conditions. DAM contains the various elements in sufficient amounts for the optimal development of this diatom. This medium would allow the study of the potential bioaccumulation of metals in H. ostrearia (absorption and adsorption of metals, kinetics) and to evaluate their impact on the growth and the culture quality under controlled conditions. Consequently, DAM was considered as a well-adapted artificial medium for H. ostrearia culture. Amphora hyaline Kütz., Bacillaria paradoxa Gmelin., Chaetoceros sp. Coscinodiscus granii, Haslea crucigera (W.Sm.) Simonsen, Navicula ramosissima (Ag.) Cl., Nitzschia compressa (Bailey) Boyer, Odontella aurita (Lyngbye) Ag., Phaeodactylum tricornutum, Pleurosigma intermedium W.Sm. and Skeletonema costatum, Thalassionema sp.