The Geosiphon symbiosis has been cultured in our laboratory since 1994. The crucial factor for successful cultures seems to be phosphate limitation. We use three different substrates for the cultures, a slightly brownish 'aquarium quartz sand', chemically clean fine white quartz sand (Sigma), or sterilized soil from the natural habitat. The latter is still the most reliable way to grow the symbiosis, and these cultures usually can be maintained for 6-12 months. The substrate can be recycled several times. In the sand system, the symbiosis grows more slowly, but some cultures have been maintained for several years.
The culture microcosm is composed of a polypropylene flower pot with filter paper strips (or a roll) running through holes in the bottom (Fig. 13). For soil cultures, dry soil is sieved through a 2-mm sieve, washed three times with distilled water, dried, sieved again, and mixed with charcoal powder (1-2 gkg-1). A layer of coarse sand overlaid by fine sand is filled into the pot, and the soil added on top. The substrate is soaked with distilled water from below, and put into the glass beaker containing distilled water.
pelri dish parafilm soil substrate polyethylene pot fine sand coarse sand filter paper glass beaker distilled water
Fig. 13. Culture system for the Geosiphon symbiosis
A thin, irregular charcoal layer is scattered on the substrate surface. The setup is covered with aluminium foil and autoclaved three times at 2-day intervals. The pot is then covered with a 9-cm Petri plate lid and sealed with parafilm.
For the quartz sand cultures, the brownish 'aquarium sand' is acid-washed before use. Sand culture pots are soaked with a nutrient-poor 'Geosiphon medium' (GM32): 0.1 mM MgSO4, 0.2 mM Ca(NO3)2, 0.1 mM KCl, 1 pM K2HPO4,12 pM FeCl3,12 pM Na2-EDTA, 0.08 pM ZnSO4,0.9 pM MnCl2,16.2 pM H3BO3,0.04 pM CoCl2,0.08 pM Na2MoO4,0.01 pM CuSO4, 0.01 pM NaHSeO3, 0.01 pM NiCl2, 0.01 pM Na3VO4, 0.005 pM K2Cr2O7, 0.5 mM MES, pH 6.0 (KOH-titrated). We currently also add KBr (0.1 pM; 'GM33'). The sand cultures do not work without a thin charcoal layer scattered at the surface (Figs. 1,2 and 3).
To inoculate the cultures, one to several clusters of 10-20 Geosiphon bladders from an established culture are placed onto the substrate. This always carries enough free-living cyanobacteria. Alternatively, Geosiphon spores isolated from cultures under sterile conditions are put on the surface, slightly covered with substrate, and a few 1-mm pieces of a Nostoc thallus are placed nearby. We normally use Nostoc punctiforme strain 1:126 (originally isolated from Geosiphon by D. Mollenhauer; =SAG 69.79, =PCC 9503), which is cultured on BG11 medium solidified with 2% agar and overlaid with cellophane foil. The spores formed are globose in fine substrates (Schufiler et al. 1994), but may be 'deformed' in coarse sand (Fig. 7). The cultures are grown at 20 °C with a 14-h light/10-h dark rhythm and illuminated with a photosynthetic photon flux density (PPFD) of 80160 pmol m-2 s-1 (fluorescenttubes TDL58W/25CEand L58W/77Fluora).
The procedures above work well, but we sometimes modify details. e.g. some cultures may grow better when the pot in the beaker has direct contact with the water. However, by using the filter paper 'bridges', moisture conditions are more stable and culture success more predictable. Cultures on soil are possible, but less reliable, without the addition of charcoal. Some lots of the natural substrate lead to excessive growth of Nostoc. This might be due to higher phosphate content or accessibility (see Fig. 4), e.g. because of a higher pH. The substrate may be buffered to a pH of 5-6 but, when using organic buffers, this can have unforeseen effects on nutrient availability. Our methods can therefore probably be refined.
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