In this course you will learn introductory terms and concepts related to biofertilizers and their production
- Course Structure: Contents and Assessment Pattern
- General Instructions for the course
- Module 1
- Lesson 1: Introduction to Biofertilizer Production
- Quiz on: Lesson 1: Introduction to Biofertilizer Production
- Lesson 2: Nitrogen Fixing Microorganisms
- Lesson 3: Video Demonstration- Biofertilizer production using Nitrogen fixing Microorganisms
- Quiz on Lessons 2 and 3: Nitrogen Fixing Microorganisms and Biofertilizer Production
- Module 1: INQUIRY BASED LEARNING (IBL) Problem I
- Project component 1
- Module 2
- Lesson: Phosphate Solubilising Microorganisms
- Quiz on: Lesson: Phosphate Solubilising Microorganisms
- Module 2: Inquiry Based Learning (IBL) Problem II
- Project component 2
- Module 3
- Lesson: Indole-3-Acetic Acid Producing Microorganisms
- Quiz on Lesson: Indole-3-Acetic Acid Producing Microorganisms
- Module 3: Inquiry Based Learning (IBL) Problem III
- Project component 3
- Module 4
- Lesson: Siderophore Producing Microorganisms
- Quiz on Lesson: Siderophore Producing Microorganisms
- Module 4: Inquiry Based Learning (IBL) Problem IV
- Project component 4
Lesson 2: Nitrogen Fixing Microorganisms
Lesson 2: Nitrogen fixing Microorganisms
Nitrogen fixing Microorganisms:
- What are they?
- Nitrogen fixing microorganisms are organisms which fix atmospheric (gaseous form) nitrogen, reducing it to ammonia and make it available to the plants. Such organisms are also called diazotrophs. The bacteria produce Nitrogenase enzyme, which is sensitive to the presence of oxygen and responsible for this process.
- What is their importance?
- Nitrogen is an important component of the cell. It is a constituent of nucleic acids (DNA and RNA), ATP (the energy currency of the cell) and proteins. Proteins are components of cell membranes and other structural components of the cell. Enzymes which are the work horses of the cell are also proteins. Availability of fixed nitrogen is important for plant growth. Inoculation of seeds or soil with Nitrogen fixing microorganisms increases the crop yield significantly.
- What are the examples of Nitrogen fixing Microorganisms?
Nitrogen fixing microorganisms are of the following types:
- Free living Bacteria: e.g. Azotobacter, cyanobacteria/ BGA (Blue Green Algae which are also photoautotrophic); Azospirillum sp.
- Endophytic Bacteria: Rhizobium sp., the microaerobically nitrogen-fixing bacteria, Acetobacter diazotrophicus, Herbaspirillum seropedicae and Azoarcus spp.; Azospirillum sp.
- Azorhizobium, Bradyrhizobium, Mesorhizhobium, Rhizobium and Sinorhizobium are collectively called rhizobia. Endophytes like Rhizobium sp. live and fix nitrogen within specialized structures (called root nodules) of leguminous plants. Some endophytes, though living within plant parts, do not form specialized structures.
Image credits: Ninjatacoshell licensed underCC BY-SA 3.0
4. On which nutrient media would you grow them?
They can be enriched and isolated on the following media:
- For free-living nitrogen fixing bacteria such as Azotobacter sp.: Nitrogen- free media such as Ashby’s medium or Jensen’s medium is used.
- For endophytic bacteria such as Rhizobium sp.: CRYEMA (Congo Red Yeast Extract Mannitol Agar) medium is used.
- How would you detect their presence?
- Nitrogen fixing bacteria (e.g. Azotobacter and Rhizobium sp.) grow on agar media as colourless or whitish mucoid colonies.
- Here are the links for images of Azotobacter colonies:
- IMG_7343 licensed under CC BY-NC
- Details of isolation, purification of Rhizobium sp. and appearance/ images of colonies on CRYEMA medium can be found at : http://www.ijiras.com/2017/Vol_4-Issue_5/paper_64.pdf 
- What is the composition of Ashby’s Mannitol Agar medium?
- Ingredients Gms / Litre: Mannitol 20.000, Dipotassium phosphate 0.200, Magnesium sulphate 0.200, Sodium chloride 0.200, Potassium sulphate 0.100, Calcium carbonate 5.000, Agar 30.000. Final pH ( at 25°C) 7.4±0.2.
- Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Mix well and pour into sterile Petri plates when they are moderately hot ie 55-60°C. Allow the media to solidify or set.
- Note: Due to presence of calcium carbonate, the prepared medium forms opalescent solution with white precipitate
- What is the composition of CRYEMA medium ?:
- Composition of CRYEMA medium:
Ingredients Gms / Litre: Yeast extract 1.000, Mannitol 10.000, Dipotassium phosphate 0.500, Magnesium sulphate 0.200, Sodium chloride 0.100, Congo red 0.025, Agar 30.000. Final pH ( at 25°C) 6.8±0.2
- Autoclave the media at 15 PSI (ie lbs/ in2), at 121.6°C for 15-20 min. Cool the media to room temperature before use. Pour agar containing media into sterile petri plates when they are moderately hot ie 55-60°C. Allow the media to solidify or set.
- How do you measure/ determine the activity of nitrogenase enzyme responsible for Nitrogen fixation? It is possible to estimate nitrogenase activity by measuring the rate of conversion of the substrate (N2) to the product (NH3). Since NH3 is involved in other reactions in the cell, it is often desirable to label the substrate with 15N to provide accounting or “mass balance” of the added substrate. A more common assay, the acetylene reduction assay or ARA, estimates the activity of nitrogenase by taking advantage of the ability of the enzyme to reduce acetylene gas to ethylene gas. These gases are easily quantified using gas chromatography.
Acetylene Reduction Assay: The activity of nitrogenase enzyme responsible for Nitrogen fixation is elucidated by the method of ARA (Acetylene Reduction Assay). The Acetylene reduction assay is carried out as per the method of Hardy et al (1973). . The principle of the method is that acetylene is reduced to ethylene catalyzed by enzyme nitrogenase; the process is assessed by gas chromatography.
Quantification of biological nitrogen fixation is also carried out by using 15N-isotope techniques.
15N-enrichment approach, natural abundance technique, and the ureide assay represent the most useful techniques to assess biological N2 fixation .
Process of biofertilizer production: using nitrogen fixing organisms
Since soil has an abundance of a variety of microorganisms and nitrogen fixing bacteria such as Azotobacter sp. grow slowly, at first we need to follow enrichment culture technique where we increase their numbers in comparison with the rest of the organisms. So, we inoculate suitable soil sample into Ashby’s nitrogen free medium and incubate it for 2 -4 days, so that only the nitrogen fixing organisms can grow and multiply. Then we isolate the Azotobacter sp. from this enrichment medium by streaking on Ashby’s agar medium and incubating further. The isolated culture is then further purified by repeating the steps of streaking on Ashby’s medium and incubating the streaked plates. The purified culture is then suspended in sterile saline and mixed with sterile carrier material such as charcoal/ cocopeat and the suspension dried to get the biofertilizer.
- Oda Steenhoudt, Jos Vanderleyden, Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects, FEMS Microbiology Reviews, Volume 24, Issue 4, October 2000, Pages 487–506, https://doi.org/10.1111/j.1574-6976.2000.tb00552.x
- Sneha Ogale, Karan Singh Yadav and Shrutika Navale. Screening of endophytic bacteria from the pharmacologically important medicinal plant Gloriosa superba for their multiple plant growth promoting properties. The Pharma Innovation. 2018; 7(1): 208-214.
- Ogale S., Kulkarni A., Nichit S.and Shah D. ‘Screening Of Endophytes Of Gloriosa superba For Their Plant Growth Promoting Potential And Optimization Of Indole-3-Acetic Acid Production. World Journal of Pharmaceutical Research’. 2018. 7 (4): 145-155.
- Hardy, R.W.F., Burns, R.C., Holsten, R.D. (1973). Application of the acetylene-ethylene reduction assay for measurement of nitrogen fixation. Soil Biol Biochem 5:47–81.
- Rao Subba N S. Soil microorganisms and plant growth. Oxford & IBH Pub. Co. ©1977 ,New Delhi.