“Starter cultures” in 250 ml flasks of the microalgae species Chaetoceros muelleri (CS-176/6) and Skeletonema tropicum (CS-604/6) are obtained from the CSIRO National Algae Culture Collection in Hobart. C.muelleri is predominantly a single celled diatom with a cell diameter of 8-10 microns (Kipp, 2010). S.tropicum is a colony forming diatom of variable length with a cell diameter of 5.3-23 microns (Louisiana Universities Marine Consortium, 2009; Sarno et al, 2005). The Algae Laboratory is fully equipped for axenic culture applications with autoclavable borosilicate glassware: volumetric flasks, pipettes, erlenmayer flasks, graduated cylinders and petri dishes. Working stock solutions and primary stock solutions are routinely composed (Figure 14). Sterilized seawater from the autoclave (Siltex) (Figure 15) is prepared in two litre flasks with culture medium F2. This medium is made up into stock solutions of the following chemicals:
Ø Sodium/Potassium Nitrate
Ø Sodium Di-Hydrogen Orthophosphate
Ø Sodium Silicate
Ø Ferric Chloride and Trace Elements
Ø Ethylenediaminetetraacetic Acid Di-Sodium Salt (EDTA)
Ø Stock Solution of Vitamins B6; B12 and Thiamine
|Figure 14 Algal Laboratory|
Algal cell densities are monitored using a haemacytometer (Neubauer Brand) which consists of two chambers each with a volume of 0.1mm3 containing a marked counting grid 1mm2 in area.
|Figure 15 Autoclave|
The haemacytometer grid is divided by triple lines into 9 large squares, each 1mm x 1mm. Each large square is divided into 25 medium squares each 0.23mm on a side and each medium square is further divided into 16 small squares, each 0.05mm on a side. The measurement is the average number of algal cells per 1mm square, so the centre large square is counted. To obtain the total number of cells in the large square, the number of cells in each of the 25 medium squares are counted, recorded then added. After counting each of the two chambers, the haemacytomer and coverslip is rinsed in distilled water and the procedure repeated twice more to give a total of 6 counts. To obtain the cell density, calculate the average cell count and multiply by the conversion factor 1 x 104.
The laboratory microscope is an Olympus Compound CH2 binocular. Two litre flasks are inoculated with algae culture in the Exponential Phase of growth and provided with gentle aeration and a bank of fluorescent lighting (Figure 16). The Innoculation rate is 0.5% to 1% of the volume of the new culture, i.e. 5 to 10mLs into one litre. There are five phases of algal growth in batch culture: Lag Phase, Exponential Phase, Declining Growth Phase, Stationary Phase and Death Phase (Figure 17). The intention is to keep cells healthy and increasing in biomass, therefore transfer to newly prepared culture medium is undertaken in Exponential Phase. The duration of the Exponential Phase depends on the size of the innoculum, the growth rate, the capacity of the medium and the capacity of the culture conditions to support algae growth (CSIRO Australian National Algal Collection, 2010).
Algae will reach optimum growth levels in three days and is subsequently transferred to several prepared 20 litre clear polycarbonate carboys (Figure 18). The Mass Algal Culture Room contains 4 x 10 tonne and 4 x 5 tonne circular tanks. While a tank is filling with seawater, 75 grams of potassium nitrate powder is dissolved through a 50 micron screen. Sodium silicate and Phosphoric acid are added in a measure of 20mLs each. When maximum depth is reached, four carboys of algae, either C.muelleri or S.costatum are inoculated. C.muelleri is a single celled diatom and is grown in the 10 tonne tanks, where density is maintained at one million cells per ml. S.costatum is a chain forming diatom and is grown in the 5 tonne tanks.
|Figure 17 Pattern of microalgal growth in batch culture|
|Figure 18 Polycarbonate carboys|