IRRI2 GROWING MEDIUM
We are grateful to Azolla Foundation Associates Francisco Carrapiço and Ana Pereira for providing the information on this page.
Pereira & Carrapiço (2009)* compared the relative growth rate, duplication time and production of biomass of Azolla filiculoides grown in artificial conditions using four different culture media (H-40, IRRI1, IRRI1 Fe 10x, and IRRI2).
(* Pereira, A. L. & Carrapiço, F., 2009. Culture of Azolla filiculoides in artificial conditions. Plant Biosystems vol. 143, pp. 431-434.)
Based on their study, Pereira & Carrapiço identified the IRRI2 medium, which was developed by the International Rice Research Institute (IRRI), as the best nutrient medium for promoting azolla’s growth. They published the following table of nutrient concentrations in micromoles (µM) for the IRRI2 medium:
Macronutrients: µM
CaCl₂.2H₂O: 40 µM
MgSO₄7H₂O: 40 µM
K₂SO₄: 40 µM
NaH₂PO₄.H₂O: 20 µM
Micronutrients µM
CuSO₄.5H₂O: 0.01 µM
H₃BO₃: 0.2 µM
Na₂MoO₄.2H₂O: 0.15 µM
ZnSO₄.7H₂O 0.01: µM
CoCl₂.6H₂O 0.01: µM
MnCl₂4H₂O 0.5: µM
Fe- EDTANa₂: 0.5 µM
Ana Pereira also provided the following information to J. Bujak (personal communication, September 2015). This includes conversion from micromoles to grammes, plus the details needed to make the H-40 medium.
| Concentration | For 1 L | Stock solution | |
| Macronutrients | 1000x | ||
| CaCl2.2H2O | 40 µM | 0.00588 g | 5.88 g |
| MgSO4.7H2O | 40 µM | 0.00985 g | 9.86 g |
| K2SO4 | 40 µM | 0.00697 g | 6.97 g |
| NaH2PO4.H2O | 20 µM | 0.00276 g | 2.76 g |
| Micronutrients | 1000x | ||
| CuSO4.5H2O | 0.01 μM | 0.0000025 g | 0.0025 g |
| H3BO3 | 0.20 μM | 0.000012 g | 0.012 g |
| Na2MoO4.2H2O | 0.15 μM | 0.000036 g | 0.036 g |
| ZnSO4.7H2O | 0.01 μM | 0.0000029 g | 0.0029 g |
| CoCl2.6H2O | 0.01 μM | 0.0000024 g | 0.0024 g |
| MnCl2.4H2O | 0.50 μM | 0.000099 g | 0.099 g |
| Iron source | 1000x | ||
| EDTANa2.2H2O | 0.50 μM | 0.00019 g | 0.19 g |
| FeSO4.7H2O | 0.50 μM | 0.00014 g | 0.14 g |
Ana Pereira noted that there are two ways to prepare the IRRI medium.
1) Preparation of 1 litre (L) of IRRI medium
In 500 ml of distilled water add the macronutrients.
Dissolve the micronutrients in 100 ml of distilled water and to avoid precipitation add 0.01 ml of 6N H2SO4 (1.6 ml H2SO4 in 10 ml of distilled H2O). Add the micronutrients to the macronutrients solution and mix.
For the iron source, dissolve the EDTANa22H2O in 0.268 ml of 1N KOH (add 56.11 g of KOH to 1000 ml of distilled water). Add the FeSO4.7H2O and 100 ml of distilled water. Agitate. Allow the solution to stand until the formation of a red complex.
Then, add to the iron solution to the macronutrients plus micronutrients solution. Add more distilled water to obtain 1 L of medium. Agitate and measure pH. Adjust the pH to 6-6.5 with 1M NaOH.
2) However, due to the low amount of chemicals it is necessary to make stock solutions.
Stock solutions:
Macronutrients – prepare 1 L of each chemical is separate bottles in distilled.
Micronutrients – Dissolve the chemicals compounds in 100 ml of distilled water. To avoid precipitation add 10 ml of 6N H2SO4 (1.6 ml H2SO4 in 10 ml of distilled H2O). Add more distilled water until 1000 ml. Shake the solution to dissolve.
Iron – Dissolve the EDTANa22H2O in 268 ml of 1N KOH (add 56.11 g of KOH to 1000 ml of distilled water). Add the FeSO4.7H2O and add distilled water until 1000 ml. Allow the solution to stand until the formation of a red complex.
For 1 L in distilled water of IRRI2 medium add the following amounts from the stock solutions:
1 ml of stock of micronutrients
1 ml of each of the stock solution of the macronutrients
0.1 ml of the stock solution of the iron source
Adjust the pH to 6-6.5 with 1M NaOH
H-40 GROWING MEDIUM
The H-40 medium is easier to make and by my extensive experience in azolla growth it works also very well to growth azolla in large quantities.
| Macronutrients | Quantity | Stock 20x |
| g/L | g/L | |
| CaCl2. 2H2O | 0.222 | 4.44 |
| MgSO4.7H2O | 0.197 | 3.94 |
NaCl |
0.028 | 0.56 |
| KH2PO4 | 0.048 | 0.96 |
| K2HPO4.3H2O | 0.011 | 0.22 |
| KCl | 0.149 | 2.98 |
| Micronutrients | Stock 1000x | |
| g/L | g/L | |
| CuSO4.5H2O | 0.00008 | 0.08 |
| MnSO4.H2O | 0.00154 | 1.54 |
| H3BO3 | 0.00286 | 2.86 |
| Na2MoO4.2H2O | 0.00025 | 0.25 |
| ZnSO4.7H2O | 0.00022 | 0.22 |
| CoCl2.6H2O | 0.0004 | 0.4 |
| Iron source | g/L | Stock 1000x |
| g/L | ||
| EDTANa2.2H2O | 0.00268 | 2.68 |
| FeSO4.7H2O | 0.002 | 2 |
There are two ways to prepare the H-40 medium.
1) Preparation of 1 L of H-40 medium
Add all the chemicals to 1 L of distilled water and mix. Measure pH and adjust the pH to 6-6.5 with 1M NaOH.
2) However, due to the low amount of chemicals it is necessary to make stock solutions.
For 1 L in distilled water of H-40 medium add the following amounts from the stock solutions:
50 mL of macronutrient stock solution
1 mL of micronutrient stock solution
1 mL of Fe-EDTA (iron source) stock solution
Adjust the pH to 6-6.5 with 1M NaOH
Additional note from Alexandra and Jonathan Bujak
Easy, domestic cultivation of azolla
Please note that azolla is designated as an ‘invasive species’ in some countries, so check this first, and never dispose of azolla in a way that could add it to the local water system.
Although you can use the listed growing medium ingredients, you can also use some shallow water (although deeper water is fine) and add a little soil to a container, which can be as shallow a 3cm, or deeper if you prefer.
The soil provides many of the nutrients that azolla needs. It isn’t used to root the azolla, which is free-floating, so just add a little soil which doesn’t need to cover the bottom of the container. It is essential to NOT add any fertilizers used for plants and vegetables, as these contain large amounts of nitrogen compounds, which azolla does not need, and which will promote the growth of unwanted fungi which will eventually kill the azolla plants. However, azolla does need phosphate fertilizer and this makes a huge difference in its health and growth. We searched the internet for a suitable source of phosphates and found Monosodium Phosphate 300 Grams: http://www.amazon.co.uk/s/ref=nb_sb_noss?url=search-alias%3Dkitchen&field-keywords=Monosodium%20Phosphate%20300%20Grams
….. but similar products should also work well.
After we added a little, there was a massive increase in azolla’s growth.
Remember that sub-zero temperatures will kill azolla and that it is a shade plant, so avoid direct sunlight.