Introduction :

Azolla is a water fern that floats on the surface of fresh water ponds, rivers, and flooded fields in the tropics and subtropics of both the New and Old Worlds. Its size is typically 1-2.5 cm in diameter, but some species can achieve the size of 15 cm or more. It belongs to a derived group of ferns Azollaceae and is closely related to several other aquatic taxa such as Salvinia and Marsilea

Biology of azolla: 

  •  It is one of the few pteridophyte lineages (any of a division (Pteridophyta) of vascular plants (such as a fern) that have roots, stems, and leaves but lack flowers or seeds) that have evolved heterospory (with male and female spores in different sizes), a condition that precedes evolution of the seed. 
  • Traditionally, seven species are recognized and they are divided into three series, with  
  1. A. filiculoides, A. rubra, A. caroliniana, A. microphylla, and A. mexicana in section Azolla (found in tropical areas of the Americas, New Zealand, and Australia), 
  2. A. pinnata in section Rhizosperma (occurring in Africa, Australia, and Asia), 
  3. And A. nilotica in section Tetrasporocarpia (distributed only in Africa)
  • A molecular phylogenetic study has now more or less confirmed this taxonomic scheme with the only change that A. nilotica and A. pinnata form a monophyletic instead of a paraphyletic group as in the morphological cladistic analysis .
  • The cyanobacteria are housed in a cavity of dorsal lobe of the leaf.
  •  All of the strains isolated from these seven Azolla species belong to Anabaena azollae

Reproduction / Multiplication of azolla: 

  • Azolla has both sexual and asexual reproduction.
  • During sexual reproduction, each (female) megaspore contains a small colony of Anabaena filaments bearing akinetes (spores of the cyanobacterium), and so the bacterium is passed down to the next generation of the water fern.
  •  Its asexual reproduction is achieved simply by multiplication of fragmented fronds (fern leaves). 
  • Under ideal growth conditions, the water fern grows and reproduces (asexually) extremely fast, often able to double its weight in 2-5 d. 

   Azolla (mosquito fern, duckweed fern, fairy moss, water fern) is a genus of seven species of aquatic ferns in the family Salviniaceae. They are extremely reduced in form and specialized. It can fix atomospheric nitrogen, carry out photosynthesis and uptake nutrients from its surrounding environment through its root system. It has wide range of use including fodder for dairy cattle, pigs, chicken, ducks and fish, used as a biofertilizer. 

Need of project :

     In Vigyan ashram, we are facing problem in multiplication of azolla,this could be due to diferent factors such as temperature and humidity, deficeincy of NPK, deficeincy of micronutrint, pH of water etc. Primary use of azolla rightnow in may 2020, is fish feed for Tilapia.Otherthan this we require azolla as a nitrogen source.

Objectives:

  1. To study the effect of :
    1. Temperature
    2. Humidity
    3. pH
    4. Micronutrient
    5. NPK on azolla growth patteren.
  2. To boost availability of azolla for composting.
  3. Use of azolla as a feed for fish and animals.

Trail : 01

Protocol:

  • Existing four beds converted into five bed with different chemical composition ( 01/05/20 ). 
  • Then beds were refilled with water (700 L/bed ) and treated with 5 gm bleaching powder.  ( 02/05/20). 
Image 1: Reconstruction and refilled beds with water.
  • Equidistant foggers were fixed and labelled beds ( 03/05/20 )
  • Composition of each bed is as follows:
Azolla Bed No ChemicalFull formConcentration
Bed No :10:52:34Potassium phosphate100 gm
Bed No :2MgSO4Magnesium sulphate50 gm
Bed No :3SSPSingle super phosphate100 gm
Bed No :40:52:34 + MgSO4Potassium phosphate + magnesium sulphate100 gm + 50 gm
Bed No :5Soil + Slurry + 0:52:34 + SSP + MgSO4 + Mineral MixturePotassium phosphate + magnesium sulphate + Single super phosphate5 kg + 3 L+ 100 gm +100 gm+50 gm +20 gm
  • Addion of azolla i.e. 1.8 kg into each bed.
Image 2: Addtion of azolla into beds

 Now, The structural details of these azolla beds are as follows:

Image 4: Dimension and structure of azolla beds.
Image 5 : 3D drawing of azolla beds.
  • Dome is called as azolla dome. This dome is divided into five equal parts.
  • Depth = 0.6 ft, Length = 8 ft, Breadth = 5 ft
  • Total amount of water required foe each bed = 700 L.
  • Foggers are fixed to maintain humidity.
  • Total no of foggers : 14

Roll of chemicals is as follows :

  1. 0:52:34 : Formulated using Phosphorous 52% and Potassium 34%, Special NPK (00-52-34) is a 100% water soluble fertilizer which promotes the soil fertility. It is a great source of P & K soil nutrients for plants, specially useful during early and end stages of crop growth.
  2. MgSO4 : Mg involved in photosynthesis and sulfur activate plant enzyme      system. Deficiency of mg affect plant colour. In short Mg required for plant to give green colour.
  3. SSP : Ensures a large number of nodules on the roots, which fix atmospheric Nitrogen directly into the soil and also increase Nitrogen uptake.
  4. Mineral mixture : 
  5. Bleaching powder : To resist/avoid the growth of algae. Beacause algae has symbiotic relationship with azolla.
  6. Soil : To provide microbes and micronutrients.

Parameters considered during Azolla reading are categorized into 3 main parts:

  • Water Testing :
    • Includes pH, EC, TDS, Hardness, Potassium (K), water temperature.Water temperature, pH, EC and TDS has been taken at 4:00 pm daily. And hardness and potassium content calculated after 2 days.
  • Climatic conditions : 
    •   Includes Temperature and Humidity. Temperature and humidity readings has been taken  four times i.e. 8:00 am, 12:00 pm, 3:00 pm and 6:00 pm daily. Both inner and outer temperature and humidity reading has been taken.
  • Azolla Harvesting : 
    •   Includes Date, weight and use of harvested azolla.

Air flow within dome: 

         Hot air will rise up to the top of the dome. A very simple solution to implement a ventilation system will be to install vents on the top of the dome, e.g., a window. The hot air will be able to escape on the top. Thereby, the air pressure inside the dome will be lower than outside. If we want to let the cold air go inside of the dome, it should also contain vents on the bottom of the structure.If this solution is implemented in the dome, there should also be a (automatic) system which can open and close the vents when it is needed. This system has a low energy consumption if we compare it with other possible ventilation systems.

Another solution to ventilate a dome is to use cooling fans. The problem with this solution is that you need a lot of energy in order to power the cooling fans. It is possible to power the cooling fans with solar panels. Since this dome will only use a 12 V battery, it’s almost impossible to use it in this case.

Readings of azolla beds (05/05/20 – 23/5/20)

  1. Water Testing :  https://docs.google.com/spreadsheets/d/1bKUKhH1xpqBjJsAmc_433kefZC10CjeF9-hRCCSh24U/edit?ts=5eafc1e1#gid=0
  2. Climatic conditions :  https://docs.google.com/spreadsheets/d/1bKUKhH1xpqBjJsAmc_433kefZC10CjeF9-hRCCSh24U/edit?ts=5eafc1e1#gid=583583872

Observation :

  • Growth of azolla after 6 days:
Image 6 : Growth of azolla after 6 days.

According to the observation and study, in first 6 days azolla seems green in first bed ( potassium phosphate) , second (magnesium sulphate) and third ( single super phosphate) bed. And it becomes yellow in fourth ( Potassium phosphate + magnesium sulphate ) and fifth bed ( Potassium phosphate + magnesium sulphate + Single super phosphate ).

Azolla bed NoCauses
Bed No : 1Observe green due to presence of phosphate.
Bed No : 2Observe green due to presence of magnesium.
Bed No : 3Observe green due to presence of phosphate.
Bed No : 4Observe yellow 
Bed No : 5 Observe yellow

Azolla weight is calculated after 6 days to observe growth changes, which is as follows :

Azolla Bed No Weight in gm (after 6 days) weight in gm (after 6 days) Weight increased by percent/day
Bed No: 01potassium Phosphate4254.1
Bed No: 02Magnesium sulphate 5685.2
Bed No: 03Calcium dihydrogen phosphate1651.5
Bed No: 04 potassium Phosphate +
Magnesium sulfate
1050.94
Bed No: 05Soil Slurry + all 3+ mineral mixture 4023.7
  • Growth of azolla after 12 days :

Azolla in Bed No. 4 , has lowest weight gain, this is due to precipitation of magnesium phosphate. So non availability of phospharus as well magnesium. Bed No. 2 has highest weight gain may be due to availability of magnesium. Bed No.1 is runner up in the competition where the availability of phosphorus is key factor. If we compare bed 1 with bed 4 potassium availability is same but deficiency is phosphorus.

Image 7 : Growth of azolla after 12 days.

There is no significant change in colour of azolla as compared to first six days, but size of azolla become small. And weight also decreased in first,second and fifth bed. But weight is increased in third and fouth bed which were lowest wt. gain in first six days. This is may be because of precipitation of magnesium phosphate, potassium sulphate is solubalize in water and that potassium sulfate used by azolla for growth.

Azolla weight is calculated after 12days to observe growth changes, which is as follows :


Azolla Bed No
Intial weight of Azollaweight after 12 days ) Wt. loss/gain in gm wt. loss (%) wt. increased or decreased per day (%)
Bed No: 01425 277 – 148 34 (loss)2.8
(loss)
Bed No: 02568 68 -500 88
(loss)
7.3
(loss)
Bed No: 03165 354 189 114
(Gain)
9.5
(gain)
Bed No: 04105 512 407 387
(Gain)
32.2
(gain)
Bed No: 05402 446 -44 10.9
(loss)
0.9
(loss)

3. Growth of azolla after 19 days :

Image 8 : Growth of azolla after 19 days.
Image 9 : Browning of azolla after 19 days.

Azolla weight is calculated after 19 days to observe growth changes, which is as follows :

Azolla Bed No Initial wt.(wt after 12 days)Increased / decreased weightof azolla after19 days.(gm)Wt. loss/gain in gm Wt. loss/gain in % Wt. loss/gain per day (after 19 days.)
Bed No: 01277155-12255.9
(loss)
2.94
(loss)
Bed No: 02689123133.8
(gain)
7.04
(gain)
Bed No: 03354277-7778.24
(loss)
4.11
(loss)
Bed No: 04512272-24053.12
(loss)
2.79
(loss)
Bed No: 05446210-23647.08
(loss)
2.47
(loss)

Weight and size of azolla decreases day by day. And colour also changes from green to red and then brown . Its due to degradation of chlorophyll. In a way this reduce photosynthetic activity means reduce growth .

Trial : 01 (In tray)

  • Five trays with dimensions 41 cm length, 30 cm breadth and 7 cm height used to conduct experiment.
  • Trays were filled with 4.5 L water each (4/06/20) .
  • Trays with chemical composition is as follows:
Tray No Chemical compositionTreatmentInitial wt. of azolla. (gm)
Tray No 10:52:340.55 gm20
Tray No 2MgSO40.29 gm20
Tray No 3SSP0.55 gm20
Tray No 40:52:34 + MgSO40.55 gm + 0.29 gm20
Tray No 5Soil + Slurry 0.28 gm + 170 ml20
Image 01 : After additon of 20 gram azolla.

Observation after 6 days (10/06/20) :

Image No 02 : Azolla growth after 6 days.
Tray No        Chemical compositionInitial wt. of azollaWt. of sample after 6 days. (gm)Increase % wt. after 6 days.Increase % wt. Per day
Tray No 10:52:3420 gm5025041.6
Tray No 2MgSO420 gm3919532.5
Tray No 3SSP20 gm5527545.8
Tray No 40:52:34 + MgSO420 gm6532554.1
Tray No 5Soil + Slurry 20 gm2311519.1

Observations after 12 days : (16/06/20)

Tray No         Chemical compositionInitial wt. of azolla (after 6 days)Wt. of sample after 12 days. (gm)Increase % wt. after 12 days.Increase % wt. Per day
Tray No 10:52:345028564.6
Tray No 2MgSO43946117.99.8
Tray No 3SSP5557103.638.6
Tray No 40:52:34 + MgSO4655483.076.9
Tray No 5Soil + Slurry 231878.266.5

Trial : 02

Protocol :

  • Existing azolla was harvested and beds were refilled with water 700 lit/bed.
  • Bubblers were fixed in each bed .
    • To study the effect of bubblers on azolla growth, bubblers were fixed in each bed.
Image 01 : Bubblers are fixed into beds.
  • 2 kg azolla was added in each bed.
  • Timer is installed for automization of foggers.
Image 02 : After addition of 2 kg azolla in each bed.
  • Chemical composition of Azolla beds is as follows:
Azolla Bed No CompositionTreatment
Bed No : 1 Jeevamrut  9 L
Bed No : 2Nitrate Culture6 L
Bed No : 3Vermiwash25 L
Bed No : 4Fertilizers (Ca(NO3)2, 13:0:45, Iron, zinc, copper, molybdenum, 0:52:34, 0:0:50, KNO3, MgSO4  )400 gm, 50 gm, 1 gm, 1 gm, 1 gm, 1 gm, 1 gm, 50 gm, 50 gm, 50 gm, 100 g .
Bed No : 5Slurry10 L

Formulas :

  1. % wt. gain =  wt. gain / initial wt. * 100
  2. % wt.gain per day = % wt. gain / 6

Observations after 6 days :

Image 03: Growth of azolla after 6 days.
Azolla Bed No CompositionInitial wt. per bed (kg)Increase weight after 6 days (kg)% wt. Gain% wt. gain per day
Bed No : 1 Jeevamrut20.0592.950.491
Bed No : 2 Nirtrate culture22.55127.521.25
Bed No : 3 Vermiwash21.12656.39.38
Bed No : 4 Fertilizers21.52676.312.71
Bed No : 5 slurry21.57278.613.1

Azolla harvesting details:

DateTimeWeight (kg)Total azolla (kg)
01/06/206:00 PM1kg/bed5 kg
03/06/2010:00 AM1kg/bed(except bed 1)4 kg
04/06/2010:00 AM1kg/bed(except bed 1)4 kg

Observations after 12 days:

Image No 02 : Azolla growth after 12 days.
Azolla Bed No CompositionInitial wt. of azolla (kg)Wt. after 12 days% wt. gain% wt. gain per day.
Bed No : 1 Jivammrut22.42341.066.84
Bed No : 2Nitrate Culture24.677233.60.305
Bed No : 3Vermiwash23.9031950.57
Bed No : 4Fertilizers (Ca(NO3)2, 13:0:45, Iron, zinc, copper, bronze, molybdenum, 0:52:34, 0:0:50, KNO3, MgSO4  )23.598179.90.39
Bed No : 5Slurry23.0001500.31

Observation after 18 days: (12/06/20)

Image No 03 : Azolla growth after 18 days.
Image No 04 : Azolla becomes small and turn into brown colour.
Azolla Bed No       CompositionWt. Of azolla after 12 days (kg)Wt. after 18 days (kg)% wt. gain/loss% wt. gain per day.
Bed No :   1Jivammrut2.4232.6631.09
( gain)
0.18
Bed No : 2Nitrate Culture4.6776.531.39 (gain)0.23
Bed No : 3Vermiwash3.9032.830.72 (loss)0.12
Bed No : 4Fertilizers (Ca(NO3)2, 13:0:45, Iron, zinc, copper, bronze, molybdenum, 0:52:34, 0:0:50, KNO3, MgSO4  )3.5985.9771.66 (gain)0.27
Bed No : 5Slurry3.0003.9961.33 (gain)0.22

Trial : 02 (first repetition)
Protocol :

  • Existing azolla was harvested and beds were refilled with water 700 lit/bed for repetation of second trial to determine effect of compositions mention above.
  • Treatment of bleaching powder given 2 days before addition of azolla.

Observation after 6 days : (28/06/20)

Azolla Bed No 
CompositionInitial wt. of azolla (kg)Weight after 6 days (kg)% wt.Gain% wt.gain per day
Bed No: 1 Jeevamrut21.19059.59.91
Bed No: 2Nirtrate culture22.75137.522.91
Bed No: 3Vermiwash21.62581.2513.54
Bed No: 4Fertilizers22.485124.2520.7
Bed No: 5slurry21.9609816.33

Observations after 12 days : (3/07/20)

Azolla Bed No  
CompositionInitial wt. of azolla (kg)weight after 12 days (kg)% wt.Gain% wt.gain per day
Bed No : 1Jeevamrut25.531276.5546.09
Bed No : 2Nirtrate culture27.182359.159.85
Bed No : 3Vermiwash25.017250.8541.80
Bed No : 4Fertilizers26.101305.0550.84
Bed No : 5slurry25.187259.3543.225
Image No 01 : Growth of azolla after 12 days.

Observations after 18 days : (9/07/20)

Azolla Bed No  CompositionInitial wt. og azolla (kg)weight after 18 days (kg)% wt. Gain% wt.gain per day
Bed No :1 Jeevamrut22.497124.8520.8
Bed No : 2Nirtrate culture22.17108.518.08
Bed No : 3Vermiwash20.98449.28.2
Bed No : 4Fertilizers22.887144.3524.05
Bed No : 5slurry23.298164.927.48
Image No 02 : Growth of azolla after 18 days.

Trial : 02 (second repetition)

  • Existing azolla was harvested and beds were refilled with water 700 lit/bed for repetation of second trial to determine effect of compositions mention above.
  • Treatment of bleaching powder given 2 days before addition of azolla.

Observations after 06 days: 

Bed No CompositionInitial wt. Of azollaWeight after 6 days% wt.Gain% wt.gain per day
B1Jeevamrut1 kg0.68568.511.41
B2Nirtrate culture1 kg1.211121.120.1
B3Vermiwash1 kg0.9009015
B4Fertilizers1 kg1.20012020.18
B5slurry1 kg0.89489.414.9

Observations after 12 days: 

Bed NoCompositionInitial wt. Of azollaweight after 12 days (kg)% wt.Gain% wt.gain per day
B1Jeevamrut1 kg2.6526544.16
B2Nirtrate culture1 kg3.452345.257.5
B3Vermiwash1 kg2.391239.139.85
B4Fertilizers1 kg2.98029849.66
B5slurry1 kg2.563256.342.7

Comparison :

Wt. gain after 6 daysWt. gain after 6 days% Wt. gain after 6 days% Wt. gain after 6 days% wt.gain per day% wt.gain per day
Bed No CompositionFirst repetitionSecond repetitionFirst repetitionSecond repetitionFirst repetitionSecond repetition
B1Jeevamrut59.568.559.568.59.9111.41
B2Nitrate culture137.5121.1137.5121.122.9120.1
B3Vermiwash81.259081.259013.5415
B4Fertilizers124.25120124.2512020.720.18
B5slurry9889.4
89.416.3314.9

Comparison :

Wt. gain after 12 daysWt. gain after 12 days% Wt. gain after 12 days% Wt. gain after 12 days% wt.gain per day% wt.gain per day
Bed NoCompositionFirst repetitionSecond repetitionFirst repetitionSecond repetitionFirst repetitionSecond repetition
B1Jeevamrut5.532.65276.52654644.16
B2Nitrate culture7.183.452359345.259.857.5
B3Vermiwash5.02.391250239.141.839.85
B4Fertilizers6.12.98030529850.849.66

Comparision between three repetition :

Conclusion :

From above data and graphs we can say that azolla growth best in B1 (Nitrate culture) and B4 (Fertilizer), good in B5 (Biogas slurry) and B1 (Jeevmrut). But discussion with J.B.Joshi sir and Dixit sir we come to know that beds were in circular dome so didn’t exactly know what is affecting the growth of azolla. So we had decided that we were going with single composition in all beds to know what affects growth of azolla either composition or temp and humidity.

According to above discussion we set new experiment which is as follows :

Azolla Bed No  CompositionAmount of compositionInitial wt. of azolla (kg)
1Biogas Slurry6 L1
2Biogas Slurry6 L1
3Biogas Slurry6 L1
4Biogas Slurry6 L1
5Biogas Slurry6 L1

Wt. of azolla after 6 days :

Azolla Bed No   CompositionAmount of compositionInitial wt. of azolla (kg) Wt. of azolla after 6 days (gm) % wt. gain per day
1Biogas Slurry6 L16510.8
2Biogas Slurry6 L129348.8
3Biogas Slurry6 L113021.6
4Biogas Slurry6 L1698116.33
5Biogas Slurry6 L135358.83

NOTE :

To achieve objectives of these project various trials has been given. But there is a need to work on temperature, Humidity, and light intensity.